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PMID:30552

Angiotensin I-converting enzyme in human urine.

It was demonstrated that angiotensin I-converting enzyme was excreted in human urine. The mean activity of the enzyme in normal urine was found to be 0.38 +/- 0.04 (S.E.M.) units/day (n = 18) and the enzymic activity correlated well with the concentration of the excreted sodium (r = 0.76, p less than 0.005). Urinary angiotensin I-converting enzyme was partially purified. Three different molecular weights of enzyme (greater than 400 000, 290 000 and 140 000) were demonstrated by Sephadex G-200 gel filtration. The enzymic properties of these three enzymes were identical with those of angiotensin I-converting enzyme from human lung with regard to inhibitory effects (bradykinin potentiator c and Arg-Pro-Pro), Cl- dependency, pH optimum and KM value.

Angiotensin I-converting enzyme in human urine. It was demonstrated that angiotensin I-converting enzyme was excreted in human urine. The mean activity of the enzyme in normal urine was found to be 0.38 +/- 0.04 (S.E.M.) units/day (n = 18) and the enzymic activity correlated well with the concentration of the excreted sodium (r = 0.76, p less than 0.005). Urinary angiotensin I-converting enzyme was partially purified. Three different molecular weights of enzyme (greater than 400 000, 290 000 and 140 000) were demonstrated by Sephadex G-200 gel filtration. The enzymic properties of these three enzymes were identical with those of angiotensin I-converting enzyme from human lung with regard to inhibitory effects (bradykinin potentiator c and Arg-Pro-Pro), Cl- dependency, pH optimum and KM value.

PMID:30554

The effect of bile on the crystallisation of calcium carbonate, a constituent of gallstones.

When calcium and bicarbonate ions were mixed at room temperature (approximately 20 degrees C) to give concentrations of 4 mmol/1 and 21 mmol/1 respectively and the pH of the solution was kept at 8.3, vaterite, a form of calcium carbonate, was precipitated almost immediately as spheres of diameter 45 micron. The crystallisation of this material could be slowed down by adding to the crystallising medium small amounts of pyrophosphate or citrate which often inhibit crystal growth. High concentrations of sodium chloride (90 mmol/1) did not, however, affect the reaction. Very small amounts of gallbladder bile from patients with only cholesterol on the surface of their gallstones inhibited the crystallisation of calcium carbonate, and the size of the spheres was only 0.37 times those produced in water. The activity of the bile could be attributed to material with a molecular weight greater than 10 000. On the other hand, bile from patients having some calcium carbonate on the gallstone surface had less activity than comparable amounts of bile from patients with only cholesterol in this area. The active material may, therefore, play a part in preventing the deposition of calcium carbonate in gallstones.

The effect of bile on the crystallisation of calcium carbonate, a constituent of gallstones. When calcium and bicarbonate ions were mixed at room temperature (approximately 20 degrees C) to give concentrations of 4 mmol/1 and 21 mmol/1 respectively and the pH of the solution was kept at 8.3, vaterite, a form of calcium carbonate, was precipitated almost immediately as spheres of diameter 45 micron. The crystallisation of this material could be slowed down by adding to the crystallising medium small amounts of pyrophosphate or citrate which often inhibit crystal growth. High concentrations of sodium chloride (90 mmol/1) did not, however, affect the reaction. Very small amounts of gallbladder bile from patients with only cholesterol on the surface of their gallstones inhibited the crystallisation of calcium carbonate, and the size of the spheres was only 0.37 times those produced in water. The activity of the bile could be attributed to material with a molecular weight greater than 10 000. On the other hand, bile from patients having some calcium carbonate on the gallstone surface had less activity than comparable amounts of bile from patients with only cholesterol in this area. The active material may, therefore, play a part in preventing the deposition of calcium carbonate in gallstones.

PMID:30555

Determination of blood ionized calcium in a large segment of the normal adult population.

In two different laboratories ionized calcium was determined by use of a calcium selective electrode system of recent design in specimens of whole blood drawn from a total of 100 volunteers. Identical mean values were obtained in each laboratory. A small standard deviation was found supporting the view that [Ca2+] is normally maintained within a narrow range. Ancillary factors in [Ca2+] determination were evaluated, including effects of in-vivo produced changes in pH, and effects of addition of small amounts of heparin to the whole blood sample. A veno-arterial difference in [Ca2+] was insignificant.

Determination of blood ionized calcium in a large segment of the normal adult population. In two different laboratories ionized calcium was determined by use of a calcium selective electrode system of recent design in specimens of whole blood drawn from a total of 100 volunteers. Identical mean values were obtained in each laboratory. A small standard deviation was found supporting the view that [Ca2+] is normally maintained within a narrow range. Ancillary factors in [Ca2+] determination were evaluated, including effects of in-vivo produced changes in pH, and effects of addition of small amounts of heparin to the whole blood sample. A veno-arterial difference in [Ca2+] was insignificant.

PMID:30556

Neuroendocrine control of prolactin in experimental animals.

Hypothalamic regulation of prolactin secretion in animals (mammals) and man appears to be similar, and no significant differences have yet been demonstrated. The hypothalamus contains neurotransmitters and polypeptides that can either inhibit or stimulate prolactin release, although the predominant influence under basal conditions is to inhibit prolactin release. Thus pituitary stalk section or placement of lesions in the basal tuberal region of the hypothalamus results in increased prolactin release and sometimes in initiation of lactation. Among agents in the hypothalamus that can inhibit prolactin release, the most important appear to be an as yet unidentified polypeptide prolactin release inhibiting factor (PIF) and dopamine. There is some evidence that dopamine may account for most, if not all, of the prolactin release inhibiting activity of the hypothalamus. Agents that increase dopamine activity, i.e. L-dopa, monoamine oxidase inhibitors, etc., depress prolactin release. Acetylcholine also can inhibit prolactin release, but it appears to act via the catecholamines. Of the agents in the hypothalamus that stimulate prolactin release, the most important appear to be an as yet uncharacterized polypeptide prolactin releasing factor (PRF), thyrotropin releasing hormone (TRH) and serotonin. TRH is as effective in releasing prolactin as in releasing TSH, but under most physiological states, TSH and prolactin release do not occur together. Serotonin and its precursors, tryptophan and 5-hydroxytryptophan, are powerful releasors of prolactin and have been shown to be involved in some physiological states in which prolactin is released, i.e. during suckling, stress, etc. Other agents in the hypothalamus that can stimulate prolactin release include GABA and some prostaglandins, but these have not yet been shown to be involved in physiological control of prolactin secretion. Exteroceptive stimuli that alter prolactin release act through the CNS and hypothalamus, but some hormones and drugs also can act directly on the pituitary to promote or depress prolactin release.

Neuroendocrine control of prolactin in experimental animals. Hypothalamic regulation of prolactin secretion in animals (mammals) and man appears to be similar, and no significant differences have yet been demonstrated. The hypothalamus contains neurotransmitters and polypeptides that can either inhibit or stimulate prolactin release, although the predominant influence under basal conditions is to inhibit prolactin release. Thus pituitary stalk section or placement of lesions in the basal tuberal region of the hypothalamus results in increased prolactin release and sometimes in initiation of lactation. Among agents in the hypothalamus that can inhibit prolactin release, the most important appear to be an as yet unidentified polypeptide prolactin release inhibiting factor (PIF) and dopamine. There is some evidence that dopamine may account for most, if not all, of the prolactin release inhibiting activity of the hypothalamus. Agents that increase dopamine activity, i.e. L-dopa, monoamine oxidase inhibitors, etc., depress prolactin release. Acetylcholine also can inhibit prolactin release, but it appears to act via the catecholamines. Of the agents in the hypothalamus that stimulate prolactin release, the most important appear to be an as yet uncharacterized polypeptide prolactin releasing factor (PRF), thyrotropin releasing hormone (TRH) and serotonin. TRH is as effective in releasing prolactin as in releasing TSH, but under most physiological states, TSH and prolactin release do not occur together. Serotonin and its precursors, tryptophan and 5-hydroxytryptophan, are powerful releasors of prolactin and have been shown to be involved in some physiological states in which prolactin is released, i.e. during suckling, stress, etc. Other agents in the hypothalamus that can stimulate prolactin release include GABA and some prostaglandins, but these have not yet been shown to be involved in physiological control of prolactin secretion. Exteroceptive stimuli that alter prolactin release act through the CNS and hypothalamus, but some hormones and drugs also can act directly on the pituitary to promote or depress prolactin release.

PMID:30563

Serum and urine phosphate during short-term beta-adrenergic blockade in healthy men.

Treatment of 6 healthy adult men with a beta-blocker, timolol, resulted in a rise in serum phosphate which was maintained for the 5 days of therapy. This rise was accompanied by a transient fall in urine phosphate and a rise in the tubular maximum for phosphate reabsorption per unit glomerular filtration rate (TmPO4/GFR). No change in circulating parathyroid hormone or growth hormone could be demonstrated.

Serum and urine phosphate during short-term beta-adrenergic blockade in healthy men. Treatment of 6 healthy adult men with a beta-blocker, timolol, resulted in a rise in serum phosphate which was maintained for the 5 days of therapy. This rise was accompanied by a transient fall in urine phosphate and a rise in the tubular maximum for phosphate reabsorption per unit glomerular filtration rate (TmPO4/GFR). No change in circulating parathyroid hormone or growth hormone could be demonstrated.

PMID:30564

Physostigmine for cardiac and neurologic manifestations of phenothiazine poisoning.

Psychotropic drugs such as the phenothiazine neuroleptics and tricyclic antidepressants are known to cause electrocardiographic abnormalities as well as a central anticholinergic syndrome. Physostigmine is known to reverse the central muscarinic anticholinergic manifestations by inhibition of the enzyme cholinesterase. An unusual case of trifluoperazine overdose, in which the patient presented with cardiac arrhythmias and a central anticholinergic syndrome, is presented. Treatment with physostigmine reversed the central anticholinergic syndrome as well as the electrocardiographic abnormalities. Effects of phenothiazines on altering cardiac status are also discussed.

Physostigmine for cardiac and neurologic manifestations of phenothiazine poisoning. Psychotropic drugs such as the phenothiazine neuroleptics and tricyclic antidepressants are known to cause electrocardiographic abnormalities as well as a central anticholinergic syndrome. Physostigmine is known to reverse the central muscarinic anticholinergic manifestations by inhibition of the enzyme cholinesterase. An unusual case of trifluoperazine overdose, in which the patient presented with cardiac arrhythmias and a central anticholinergic syndrome, is presented. Treatment with physostigmine reversed the central anticholinergic syndrome as well as the electrocardiographic abnormalities. Effects of phenothiazines on altering cardiac status are also discussed.

PMID:30596

Parenteral treatment of acute psychotic patients with agitation: a review.

Representative studies which elucidate present treatment principles regarding parenteral administration of neuroleptics for acute psychoses with agitation are reviewed. "Rapid tranquillization" with drugs such as haloperidol generally appears preferable, but controlled comparisons with more conservative types of treatment are lacking. It is suggested that parenteral chlorpromazine should be avoided because of its tendency to provoke severe hypotension, whereas loxapine apparently is a valuable drug if strong sedation is required for behavioural control. Possible advantages of ultra-high-dose therapy need to be proved in controlled trials, and the occurrence of toxic side-effects requires further evaluation. From an ethical and psychological point of view, it is recommended that antiparkinsonian medication should be administered simultaneously with neuroleptics which induce a high incidence of acute dystonia. Several types of acute psychosis with agitation which do not require treatment with a neuroleptic as drug treatment of first choice are briefly mentioned.

Parenteral treatment of acute psychotic patients with agitation: a review. Representative studies which elucidate present treatment principles regarding parenteral administration of neuroleptics for acute psychoses with agitation are reviewed. "Rapid tranquillization" with drugs such as haloperidol generally appears preferable, but controlled comparisons with more conservative types of treatment are lacking. It is suggested that parenteral chlorpromazine should be avoided because of its tendency to provoke severe hypotension, whereas loxapine apparently is a valuable drug if strong sedation is required for behavioural control. Possible advantages of ultra-high-dose therapy need to be proved in controlled trials, and the occurrence of toxic side-effects requires further evaluation. From an ethical and psychological point of view, it is recommended that antiparkinsonian medication should be administered simultaneously with neuroleptics which induce a high incidence of acute dystonia. Several types of acute psychosis with agitation which do not require treatment with a neuroleptic as drug treatment of first choice are briefly mentioned.

PMID:30597

Intramuscular/oral lorazepam in acute alcohol withdrawal and incipient delirium tremens.

An open study was carried out in 21 chronic alcoholics with severe withdrawal symptoms and incipient delirium tremens to evaluate the efficacy of adjuvant treatment with intramuscular lorazepam (5mg). All symptoms subsided within 2 hours after a single injection and remained under control with oral lorazepam (mean daily dose 7 mg). No adverse reactions attributable to lorazepam were observed.

Intramuscular/oral lorazepam in acute alcohol withdrawal and incipient delirium tremens. An open study was carried out in 21 chronic alcoholics with severe withdrawal symptoms and incipient delirium tremens to evaluate the efficacy of adjuvant treatment with intramuscular lorazepam (5mg). All symptoms subsided within 2 hours after a single injection and remained under control with oral lorazepam (mean daily dose 7 mg). No adverse reactions attributable to lorazepam were observed.

PMID:30598

The use of a kymograph in a comparative trial of flunitrazepam and meprobamate in elderly patients.

A double-blind crossover trial was carried out in 31 hospitalized elderly patients receiving night-time sedation to compare the effects of flunitrazepam (0.5 mg) and meprobamate (200 mg). After 1 week on placebo, patients received 1-week's treatment with each drug in random order. Quality of sleep was assessed by a nurse at hourly intervals over 8 hours each night. In 11 patients, the results were compared with those from kymographic recordings measuring patient restlessness (motility index). No statistically significant difference was found between the two active drug treatments, and there was a close correlation between the two methods of assessment. It is suggested that the kymograph may well be a useful and economic method of evaluating the effectiveness of hypnotics.

The use of a kymograph in a comparative trial of flunitrazepam and meprobamate in elderly patients. A double-blind crossover trial was carried out in 31 hospitalized elderly patients receiving night-time sedation to compare the effects of flunitrazepam (0.5 mg) and meprobamate (200 mg). After 1 week on placebo, patients received 1-week's treatment with each drug in random order. Quality of sleep was assessed by a nurse at hourly intervals over 8 hours each night. In 11 patients, the results were compared with those from kymographic recordings measuring patient restlessness (motility index). No statistically significant difference was found between the two active drug treatments, and there was a close correlation between the two methods of assessment. It is suggested that the kymograph may well be a useful and economic method of evaluating the effectiveness of hypnotics.

PMID:30599

3,4,5-Trimethoxybenzoic acid, a new mescaline metabolite in humans.

After ingestion of 400 mg of mescaline sulfate by human volunteers, 3,4,5-trimethoxybenzoic acid was isolated from urine and identified by gas chromatography-mass spectrometry. The amount of this anionic mescaline metabolite was found to be very low as compared with that of the well-konwn 3,4,5-trimethoxyphenylacetic acid. The significance of this finding is discussed.

3,4,5-Trimethoxybenzoic acid, a new mescaline metabolite in humans. After ingestion of 400 mg of mescaline sulfate by human volunteers, 3,4,5-trimethoxybenzoic acid was isolated from urine and identified by gas chromatography-mass spectrometry. The amount of this anionic mescaline metabolite was found to be very low as compared with that of the well-konwn 3,4,5-trimethoxyphenylacetic acid. The significance of this finding is discussed.

PMID:30600

Species differences in the disposition and metabolism of 6,11-dihydro-11-oxodibenz[be]oxepin-2-acetic acid (isoxepac) in rat, rabbit, dog, rhesus monkey, and man.

The disposition and metabolism of 6,11-dihydro-11-oxodibenz[be]oxepin-2-acetic acid (isoxepac), a new nonsteroidal anti-inflammatory agent, has been studied in rat, rabbit, dog, rhesus monkey, and man. Animals were given single oral or parenteral doses of 5 or 50 mg/kg; man received approximately 3 mg/kg orally. Fecal excretion of radioactivity occurred in the rat (26--37%) and dog (33--49%), whereas in the other species elimination was mainly urinary (less than 83%). Biliary excretion accounted for 18--52% of the dose in the rat and dog. Enterohepatic circulation was demonstrated in both species. Plasma of all species was found to contain mainly unchanged isoxepac. The compound was rapidly eliminated from plasma of dog, rhesus monkey and man, but was more slowly eliminated in rat and rabbit. In the rabbit and dog the principal metabolites were the glycine and taurine conjugates of isoxepac, respectively, whereas in the rhesus monkey and man, isoxepac was excreted unchanged or as the glucuronide.

Species differences in the disposition and metabolism of 6,11-dihydro-11-oxodibenz[be]oxepin-2-acetic acid (isoxepac) in rat, rabbit, dog, rhesus monkey, and man. The disposition and metabolism of 6,11-dihydro-11-oxodibenz[be]oxepin-2-acetic acid (isoxepac), a new nonsteroidal anti-inflammatory agent, has been studied in rat, rabbit, dog, rhesus monkey, and man. Animals were given single oral or parenteral doses of 5 or 50 mg/kg; man received approximately 3 mg/kg orally. Fecal excretion of radioactivity occurred in the rat (26--37%) and dog (33--49%), whereas in the other species elimination was mainly urinary (less than 83%). Biliary excretion accounted for 18--52% of the dose in the rat and dog. Enterohepatic circulation was demonstrated in both species. Plasma of all species was found to contain mainly unchanged isoxepac. The compound was rapidly eliminated from plasma of dog, rhesus monkey and man, but was more slowly eliminated in rat and rabbit. In the rabbit and dog the principal metabolites were the glycine and taurine conjugates of isoxepac, respectively, whereas in the rhesus monkey and man, isoxepac was excreted unchanged or as the glucuronide.

PMID:30602

Comparative physiological disposition of ellipticine in several animal species after intravenous administration.

The physiological dispositon of ellipticine (NSC 71795) has been studied in the mouse, rat, dog and monkey after administration of [1-14C]ellipticine at 6 mg/kg iv (3 mg/kg to monkey). Ellipticine was very rapidly distributed from the blood of all species and was deposited in tissues. The rate of elimination of ellipticine from blood was species-dependent, half-times ranging from 22 min in mouse to 210 min in rat, and probably reflected the rate of metabolism of the drug. The rate of elimination of metabolites from blood was also species-dependent, half-times ranging from 140 min in mouse to 380 min in rat, and probably reflected the rate of biliary secretion of the metabolites. Ellipticine was widely but not uniformly distributed throughout the tissues including brain, and some of the highest concentrations of drug and metabolites were in liver, which is probably the primary site of metabolism. The concentrations of ellipticine and metabolites in tissues were species-dependent, correlating with species differences in rates of metabolism and excretion. All species excreted 80% of the dose via the fecal route and 10% via the urinary route, primarily as metabolites during the first 24 hr after dosing. Metabolites entered the gastrointestinal tract by biliary secretion and ellipticine entered by an ion-trapping mechanism. Evidence is presented that the major pathway for ellipticine metabolism in rat was to 9-hydroxyellipticine, which did not accumulate in liver but was conjugated to its glucuronide and sulfate, which were secreted in bile. Other pathways involved hydroxylation and glucuronide conjugation. The pharmacokinetics of ellipticine are correlated with its toxic side effects, such as acute hypotention and neurological symptoms. They are also correlated with its potential as an antitumor agent, such as its ability to achieve values for the area under the curve of concentration vs. time (CXt) in tumors, which would be adequate for therapy. Based upon these correlations, the drug should be administered in the clinic by iv infusion, or, provided its bioavailability is found to be satisfactory, by the oral route.

Comparative physiological disposition of ellipticine in several animal species after intravenous administration. The physiological dispositon of ellipticine (NSC 71795) has been studied in the mouse, rat, dog and monkey after administration of [1-14C]ellipticine at 6 mg/kg iv (3 mg/kg to monkey). Ellipticine was very rapidly distributed from the blood of all species and was deposited in tissues. The rate of elimination of ellipticine from blood was species-dependent, half-times ranging from 22 min in mouse to 210 min in rat, and probably reflected the rate of metabolism of the drug. The rate of elimination of metabolites from blood was also species-dependent, half-times ranging from 140 min in mouse to 380 min in rat, and probably reflected the rate of biliary secretion of the metabolites. Ellipticine was widely but not uniformly distributed throughout the tissues including brain, and some of the highest concentrations of drug and metabolites were in liver, which is probably the primary site of metabolism. The concentrations of ellipticine and metabolites in tissues were species-dependent, correlating with species differences in rates of metabolism and excretion. All species excreted 80% of the dose via the fecal route and 10% via the urinary route, primarily as metabolites during the first 24 hr after dosing. Metabolites entered the gastrointestinal tract by biliary secretion and ellipticine entered by an ion-trapping mechanism. Evidence is presented that the major pathway for ellipticine metabolism in rat was to 9-hydroxyellipticine, which did not accumulate in liver but was conjugated to its glucuronide and sulfate, which were secreted in bile. Other pathways involved hydroxylation and glucuronide conjugation. The pharmacokinetics of ellipticine are correlated with its toxic side effects, such as acute hypotention and neurological symptoms. They are also correlated with its potential as an antitumor agent, such as its ability to achieve values for the area under the curve of concentration vs. time (CXt) in tumors, which would be adequate for therapy. Based upon these correlations, the drug should be administered in the clinic by iv infusion, or, provided its bioavailability is found to be satisfactory, by the oral route.

PMID:30601

Biotransformation of methyl 5-cyclopropylcarbonyl-2-benzimidazolecarbamate (ciclobendazole) in rats and dogs.

A major metabolite of ciclobendazole (methyl 5-cyclopropylcarbonyl-2-benzimidazolecarbamate) excreted in the urine, bile, and feces of rats was methyl 5-cyclopropylcarbonyl-6-hydroxy-benzimidazolecarbamate, established by comparison of the proton magnetic resonance and mass spectra with that of the authentic compound. This compound represented 8.2% and 7.1% of the dose, respectively, in extracts of 24-hr urine and 48-hr feces samples of rats, but was only a minor metabolite in dog urine (1% of the dose). The unchanged drug was only detected in dog feces, the major route of excretion of radioactivity in the dog. 5-Cyclopropylcarbonyl-2-amino-benzimidazole was present in rat urine (2.5% of the dose). A major metabolite in dog bile was probably 5-cyclopropylcarbinol-2-aminobenzimidazole, formed by loss of the methoxycarbonyl group and reduction of the carbonyl function in the 5-position.

Biotransformation of methyl 5-cyclopropylcarbonyl-2-benzimidazolecarbamate (ciclobendazole) in rats and dogs. A major metabolite of ciclobendazole (methyl 5-cyclopropylcarbonyl-2-benzimidazolecarbamate) excreted in the urine, bile, and feces of rats was methyl 5-cyclopropylcarbonyl-6-hydroxy-benzimidazolecarbamate, established by comparison of the proton magnetic resonance and mass spectra with that of the authentic compound. This compound represented 8.2% and 7.1% of the dose, respectively, in extracts of 24-hr urine and 48-hr feces samples of rats, but was only a minor metabolite in dog urine (1% of the dose). The unchanged drug was only detected in dog feces, the major route of excretion of radioactivity in the dog. 5-Cyclopropylcarbonyl-2-amino-benzimidazole was present in rat urine (2.5% of the dose). A major metabolite in dog bile was probably 5-cyclopropylcarbinol-2-aminobenzimidazole, formed by loss of the methoxycarbonyl group and reduction of the carbonyl function in the 5-position.

PMID:30605

Metabolism of haloforms to carbon monoxide. II. In vivo studies.

Administration of haloforms (trihalomethanes) to rats led to substantial elevations in blood carbon monoxide levels. The administration of 13C-bromoform led to the formation of similarly enriched 13CO. A dose-dependent relationship between bromoform dose and CO production was observed. It was found that phenobarbital, but not 3-methylcholanthrene, treatment increased the blood CO levels seen after the administration of bromoform as compared to saline-treated controls. Lower blood CO levels were found in rats given 2H-bromoform as compared to rats given bromoform. Furthermore, SKF 525-A significantly inhibited the in vivo metabolism of bromoform to CO. Administration of either diethyl maleate or D-penicillamine did not alter the blood CO levels produced in response to bromoform administration. The in vivo metabolism of haloforms to CO followed the halide order; thus, administration of iodoform yielded the highest blood CO levels, whereas chloroform yielded the lowest levels.

Metabolism of haloforms to carbon monoxide. II. In vivo studies. Administration of haloforms (trihalomethanes) to rats led to substantial elevations in blood carbon monoxide levels. The administration of 13C-bromoform led to the formation of similarly enriched 13CO. A dose-dependent relationship between bromoform dose and CO production was observed. It was found that phenobarbital, but not 3-methylcholanthrene, treatment increased the blood CO levels seen after the administration of bromoform as compared to saline-treated controls. Lower blood CO levels were found in rats given 2H-bromoform as compared to rats given bromoform. Furthermore, SKF 525-A significantly inhibited the in vivo metabolism of bromoform to CO. Administration of either diethyl maleate or D-penicillamine did not alter the blood CO levels produced in response to bromoform administration. The in vivo metabolism of haloforms to CO followed the halide order; thus, administration of iodoform yielded the highest blood CO levels, whereas chloroform yielded the lowest levels.

PMID:30603

Characterization of the metabolites of ellipticine in rat bile.

The two major metabolites of ellipticine (NSC 71795) were isolated from rat bile by a combination of solvent extraction, partition column chromatography, and reverse phase high-performance liquid chromatography. Purification and structural elucidation of the bile products were aided by administration of the drug with a dual label (14C and 2H). The two metabolites were shown to be the sulfate and glucuronide conjugates of 9-hydroxyellipticine by chemical, enzymatic, and mass-spectral fragmentation comparison with synthetic and enzymatically prepared reference compounds.

Characterization of the metabolites of ellipticine in rat bile. The two major metabolites of ellipticine (NSC 71795) were isolated from rat bile by a combination of solvent extraction, partition column chromatography, and reverse phase high-performance liquid chromatography. Purification and structural elucidation of the bile products were aided by administration of the drug with a dual label (14C and 2H). The two metabolites were shown to be the sulfate and glucuronide conjugates of 9-hydroxyellipticine by chemical, enzymatic, and mass-spectral fragmentation comparison with synthetic and enzymatically prepared reference compounds.

PMID:30607

Complications in the estimation of hepatic blood flow in vivo by pharmacokinetic parameters. The area under the curve after the concomitant intravenous and intraperitoneal (or intraportal) administration of acetaminophen in the rat.

Hepatic blood flow can be estimated from the area under the blood or plasma concentration-time curve of a drug following an intravenous dose and either an oral or intraperitoneal dose. The validity of the method depends on several factors which alter the area under the curve, that is, hemodynamic interactions due to the drug or its metabolites, the use of plasma concentration as opposed to blood concentration data, incomplete absorption on oral or intraperitoneal administration, enterohepatic recycling of the drug or its metabolite, and the presence of extrahepatic metabolism. The validity of the method was tested in rats with tracer doses of acetaminophen. After the simultaneous administration of an iv dose of 14C-acetaminophen and an intraportal dose of 3H-acetaminophen to rats, the availability after the first-pass hepatic extraction of acetaminophen in rats was 0.56 +/- 0.05. Hepatic blood flow estimated by the area under the curve for the respective routes of administration was 78.1 +/- 16.1 ml/min/kg. However, after the simultaneous administration of an iv tracer dose of 14C-acetaminophen and an ip dose of 3H-acetaminophen, the apparent availabilities calculated from the areas under the curve were highly variable and tended to be greater (0.73 +/- 0.11). Thus the estimates of the hepatic blood flow also tended to be higher (159 +/- 65 ml/min/kg).

Complications in the estimation of hepatic blood flow in vivo by pharmacokinetic parameters. The area under the curve after the concomitant intravenous and intraperitoneal (or intraportal) administration of acetaminophen in the rat. Hepatic blood flow can be estimated from the area under the blood or plasma concentration-time curve of a drug following an intravenous dose and either an oral or intraperitoneal dose. The validity of the method depends on several factors which alter the area under the curve, that is, hemodynamic interactions due to the drug or its metabolites, the use of plasma concentration as opposed to blood concentration data, incomplete absorption on oral or intraperitoneal administration, enterohepatic recycling of the drug or its metabolite, and the presence of extrahepatic metabolism. The validity of the method was tested in rats with tracer doses of acetaminophen. After the simultaneous administration of an iv dose of 14C-acetaminophen and an intraportal dose of 3H-acetaminophen to rats, the availability after the first-pass hepatic extraction of acetaminophen in rats was 0.56 +/- 0.05. Hepatic blood flow estimated by the area under the curve for the respective routes of administration was 78.1 +/- 16.1 ml/min/kg. However, after the simultaneous administration of an iv tracer dose of 14C-acetaminophen and an ip dose of 3H-acetaminophen, the apparent availabilities calculated from the areas under the curve were highly variable and tended to be greater (0.73 +/- 0.11). Thus the estimates of the hepatic blood flow also tended to be higher (159 +/- 65 ml/min/kg).

PMID:30613

[Drug prevention of bronchial asthma: inhibition of histamine and exercise-induced asthma by a new anti-anaphylactic oral preparation (ketotifen) (author's transl)].

The protective anti-asthmatic effect of a new anti-allergic drug with histaminolytic and anti-anaphylactic properties was tested in various controlled clinical studies of induced bronchospasm in asthmatic patients (provocation tests using a histamine aerosol, and exercise-induced asthma using a bicycle ergometer). The drug, ketotifen (a cycloheptathiophene derivative) was compared with a classical antihistaminic (clemastine) and a known cell stabiliser (disodium cromoglycate). In both models ketotifen provided significant protection.

[Drug prevention of bronchial asthma: inhibition of histamine and exercise-induced asthma by a new anti-anaphylactic oral preparation (ketotifen) (author's transl)]. The protective anti-asthmatic effect of a new anti-allergic drug with histaminolytic and anti-anaphylactic properties was tested in various controlled clinical studies of induced bronchospasm in asthmatic patients (provocation tests using a histamine aerosol, and exercise-induced asthma using a bicycle ergometer). The drug, ketotifen (a cycloheptathiophene derivative) was compared with a classical antihistaminic (clemastine) and a known cell stabiliser (disodium cromoglycate). In both models ketotifen provided significant protection.

PMID:30606

Factors affecting the metabolism of [14C]acetylhydrazine in rats.

Some factors affecting the metabolism of the potent hepatotoxin, acetylhydrazine, were studied in rats. After ip administration of [14C]acetylhydrazine, 36% and 38% of the dose was recovered in the urine and as 14CO2, respectively. The major urinary metabolites were diacetylhydrazine and the pyruvic acid and alpha-oxoglutaric acid acetylhydrazones. The acetylation of acetylhydrazine to diacetylhydrazine was found to be dose-dependent and to be inhibited by coadministered isoniazid and p-aminosalicylic acid. Coadministered acetylisoniazid had no effect on acetylation. The proportion of acetylhydrazine recovered as 14CO2 presumably reflects the amount metabolized by the microsomal oxidation pathway, thought to be responsible for the toxicity, and also possibly by hydrolysis to acetate and hydrazine. This latter pathway could not be confirmed, as only a small proportion of hydrazine administered to rats was recovered. The inhibition of acetylation by p-aminosalicyclic acid, but not isoniazid, significantly increased the excretion of 14CO2, suggesting that isoniazid may also inhibit a pathway resulting in the production of 14CO2. These results indicate that the metabolism and hepatotoxicity of acetylhydrazine may be different when it is produced as a metabolite of isoniazid than when it is given alone.

Factors affecting the metabolism of [14C]acetylhydrazine in rats. Some factors affecting the metabolism of the potent hepatotoxin, acetylhydrazine, were studied in rats. After ip administration of [14C]acetylhydrazine, 36% and 38% of the dose was recovered in the urine and as 14CO2, respectively. The major urinary metabolites were diacetylhydrazine and the pyruvic acid and alpha-oxoglutaric acid acetylhydrazones. The acetylation of acetylhydrazine to diacetylhydrazine was found to be dose-dependent and to be inhibited by coadministered isoniazid and p-aminosalicylic acid. Coadministered acetylisoniazid had no effect on acetylation. The proportion of acetylhydrazine recovered as 14CO2 presumably reflects the amount metabolized by the microsomal oxidation pathway, thought to be responsible for the toxicity, and also possibly by hydrolysis to acetate and hydrazine. This latter pathway could not be confirmed, as only a small proportion of hydrazine administered to rats was recovered. The inhibition of acetylation by p-aminosalicyclic acid, but not isoniazid, significantly increased the excretion of 14CO2, suggesting that isoniazid may also inhibit a pathway resulting in the production of 14CO2. These results indicate that the metabolism and hepatotoxicity of acetylhydrazine may be different when it is produced as a metabolite of isoniazid than when it is given alone.

PMID:30610

Metabolism of methimazole by rat liver cytochrome P-450-containing monoxygenases.

The incubation of methimazole (1-methyl-2-thioimidazole, MMI) with rat hepatic microsomes led to the formation of 3-methyl-2-thiohydantoin and N-methylimidazole. In addition, an NADPH-stimulated binding of 14C and 35S from [14C]- and [35s]MMI to microsomal macromolecules was seen. Both the NADPH-stimulated N-methylimidazole formation and binding of radioactivity from [14C]- and [35S]MMI to microsomal macromolecules appeared to be catalyzed largely by the cytocrhome P-450 to monoxygenase systems of rat hepatic microsomes. A portion of the radioactivity bound to microsomes incubated with [14C]- and [35S]MMI was released as unchanged MMI on prolonged incubation under acid conditions; this suggests that strong binding of MMI to microsomes occurred. A portion of the 35S bound to microsomes incubated with [35S]MMI can be released as 35SCN- on incubation of the 35S-labeled microsomes with CN-. These data suggest that a portion of the sulfur released in the metabolism of MMI to N-methylimidazole is in the form of atomic sulfur (S), which binds to cysteine sulfhydryl groups (R-S-H) in microsomal proteins to form a hydrodisulfide (R-S-S-H).

Metabolism of methimazole by rat liver cytochrome P-450-containing monoxygenases. The incubation of methimazole (1-methyl-2-thioimidazole, MMI) with rat hepatic microsomes led to the formation of 3-methyl-2-thiohydantoin and N-methylimidazole. In addition, an NADPH-stimulated binding of 14C and 35S from [14C]- and [35s]MMI to microsomal macromolecules was seen. Both the NADPH-stimulated N-methylimidazole formation and binding of radioactivity from [14C]- and [35S]MMI to microsomal macromolecules appeared to be catalyzed largely by the cytocrhome P-450 to monoxygenase systems of rat hepatic microsomes. A portion of the radioactivity bound to microsomes incubated with [14C]- and [35S]MMI was released as unchanged MMI on prolonged incubation under acid conditions; this suggests that strong binding of MMI to microsomes occurred. A portion of the 35S bound to microsomes incubated with [35S]MMI can be released as 35SCN- on incubation of the 35S-labeled microsomes with CN-. These data suggest that a portion of the sulfur released in the metabolism of MMI to N-methylimidazole is in the form of atomic sulfur (S), which binds to cysteine sulfhydryl groups (R-S-H) in microsomal proteins to form a hydrodisulfide (R-S-S-H).

PMID:30616

[Biochemistry of depression. Literature analysis].

The author goes briefly over the metabolism of the main cerebral monoamines, the functioning of synapses, as well as the methods used in studying the biochemistry of depression. Beyond all existing contradictory results, a review of the main works in this field enables us to point out some leading ideas:--Depression would be due to and/or accompanied by a monoaminergic deficiency: some authors emphasize the serotonin one, others the noradrenaline one.--The regulation of mood most probably finds its origin in the monoaminergic balance, rather than in the gross rates of any particular monoamine.--Disturbances are to be found on all metabolic levels: monoaminergic, hydroelectrolytic, hormonal, glucidic, lipidic, lipidic... Close intrication exists between those different metabolisms.--The interaction between the different aminergic systems and the metabolic ways, as well as the dispersion of the acknowledged results, impose more and more the necessity of a biochemical typology of depression, which would lead to a predictive approach to the evolution and treatment of depressive illness.

[Biochemistry of depression. Literature analysis]. The author goes briefly over the metabolism of the main cerebral monoamines, the functioning of synapses, as well as the methods used in studying the biochemistry of depression. Beyond all existing contradictory results, a review of the main works in this field enables us to point out some leading ideas:--Depression would be due to and/or accompanied by a monoaminergic deficiency: some authors emphasize the serotonin one, others the noradrenaline one.--The regulation of mood most probably finds its origin in the monoaminergic balance, rather than in the gross rates of any particular monoamine.--Disturbances are to be found on all metabolic levels: monoaminergic, hydroelectrolytic, hormonal, glucidic, lipidic, lipidic... Close intrication exists between those different metabolisms.--The interaction between the different aminergic systems and the metabolic ways, as well as the dispersion of the acknowledged results, impose more and more the necessity of a biochemical typology of depression, which would lead to a predictive approach to the evolution and treatment of depressive illness.

PMID:30611

Partial purification and characterization of dihydropyrimidinases from calf and rat liver.

The partial purification of rat and calf liver dihydropyrimidinase (EC 3.5.2.2) is described. Molecular weights of the native calf and rat liver enzymes were estimated by gel-filtration chromatography to be 252,000 and 266,000 daltons, respectively. Subunit molecular weights of the calf and rat liver enzyme were estimated by SDS-gel electrophoresis to be 59,000 and 62,000 daltons, respectively. The native enzyme in both species is thought to comprise four subunits. The purified enzyme from both species was capable of catalyzing the hydrolytic ring opening of dihydrouracil, 5-phenylhydantoin, hydantoin, and alpha-phenylsuccinimide.

Partial purification and characterization of dihydropyrimidinases from calf and rat liver. The partial purification of rat and calf liver dihydropyrimidinase (EC 3.5.2.2) is described. Molecular weights of the native calf and rat liver enzymes were estimated by gel-filtration chromatography to be 252,000 and 266,000 daltons, respectively. Subunit molecular weights of the calf and rat liver enzyme were estimated by SDS-gel electrophoresis to be 59,000 and 62,000 daltons, respectively. The native enzyme in both species is thought to comprise four subunits. The purified enzyme from both species was capable of catalyzing the hydrolytic ring opening of dihydrouracil, 5-phenylhydantoin, hydantoin, and alpha-phenylsuccinimide.

PMID:30617

Effects of adrenergic blocking agents on lipolysis and adenyl cyclase activity induced by vasoactive intestinal polypeptide (VIP).

VIP stimulates lipolysis and adenyl cyclase activity in the rat adipose tissue. VIP-induced lipolysis and adenyl cyclase activity are not affected by phenoxybenzamine. VIP-induced lipolysis is inhibited by propranolol but VIP-induced adenyl cyclase activity is not.

Effects of adrenergic blocking agents on lipolysis and adenyl cyclase activity induced by vasoactive intestinal polypeptide (VIP). VIP stimulates lipolysis and adenyl cyclase activity in the rat adipose tissue. VIP-induced lipolysis and adenyl cyclase activity are not affected by phenoxybenzamine. VIP-induced lipolysis is inhibited by propranolol but VIP-induced adenyl cyclase activity is not.

PMID:30619

Effect of hydrazine, isonicotinic acid hydrazide, hydrazine sulfate, and dimethylhydrazine on guanylate cyclase activity.

The chemical carcinogen hydrazine is a potent stimulator of guanylate cyclase. In the present investigation we found that three chemical carcinogens structurally related to hydrazine, isonicotinic acid hydrazide, hydrazine sulfate, and dimethylhydrazine, decreased guanylate cyclase activity. It is of interest that hydrazine has been shown to increase DNA synthesis whereas isonicotinic acid hydrazide, hydrazine sulfate, and dimethylhydrazine decrease DNA synthesis. The relationship, if any, linking the guanylate cyclase-cyclic GMP system to DNA synthesis and carcinogenesis remains to be explored.

Effect of hydrazine, isonicotinic acid hydrazide, hydrazine sulfate, and dimethylhydrazine on guanylate cyclase activity. The chemical carcinogen hydrazine is a potent stimulator of guanylate cyclase. In the present investigation we found that three chemical carcinogens structurally related to hydrazine, isonicotinic acid hydrazide, hydrazine sulfate, and dimethylhydrazine, decreased guanylate cyclase activity. It is of interest that hydrazine has been shown to increase DNA synthesis whereas isonicotinic acid hydrazide, hydrazine sulfate, and dimethylhydrazine decrease DNA synthesis. The relationship, if any, linking the guanylate cyclase-cyclic GMP system to DNA synthesis and carcinogenesis remains to be explored.

PMID:30620

Purification and properties of hexoasmine isomerase (EC5.3.1.19) from pig intestinal mucosa.

Hexosamine isomerase from pig intestinal mucosa was purified about 70-fold. The aminosugar product of the enzymatic reaction was identified as glucosamine 6-phosphate and amino acid product as glutamic acid. The pH optimum was 7.1 Km for fructose 6-phosphate was 6.3 X 10(-4) mol/1 and for L-glutamine 6.5 X 10(-4) mol/1.

Purification and properties of hexoasmine isomerase (EC5.3.1.19) from pig intestinal mucosa. Hexosamine isomerase from pig intestinal mucosa was purified about 70-fold. The aminosugar product of the enzymatic reaction was identified as glucosamine 6-phosphate and amino acid product as glutamic acid. The pH optimum was 7.1 Km for fructose 6-phosphate was 6.3 X 10(-4) mol/1 and for L-glutamine 6.5 X 10(-4) mol/1.

PMID:30621

Modulation of rat guanylate cyclase activity in vitro by chemical carcinogens.

The nucleotide cyclic GMP has been reported to be involved in cell proliferation and malignant transformation. Nitroso chemical carcinogens activate the enzyme guanylate cyclase (EC 4.6.1.2) which catalyzes the production of cyclic GMP. The present investigation demonstrates that compounds from other major classes of carcinogens including (1) alpha-halo ethers (chloromethyl methyl ether); (2) aromatic amines (benzidine and B-naphthylamine); (3) polycyclic hydrocarbons (1,2-benzanthracene and acridine); (4) azo dyes (p-dimethylaminoazobenzene), and (5) aflatoxins (B1, B2, G1, G2) produced a striking and significant inhibition of guanylate cyclase over a general concentration range of 0.5-13 mmol/1 in a variety of tissues. Some of the nitrosamides which increase guanylate cyclase activity, increase DNA synthesis whereas carcinogens which decrease guanylate cyclase activity inhibit DNA or RNA synthesis suggesting a relationship between cyclic GMP, DNA synthesis, and chemical carcinogenesis.

Modulation of rat guanylate cyclase activity in vitro by chemical carcinogens. The nucleotide cyclic GMP has been reported to be involved in cell proliferation and malignant transformation. Nitroso chemical carcinogens activate the enzyme guanylate cyclase (EC 4.6.1.2) which catalyzes the production of cyclic GMP. The present investigation demonstrates that compounds from other major classes of carcinogens including (1) alpha-halo ethers (chloromethyl methyl ether); (2) aromatic amines (benzidine and B-naphthylamine); (3) polycyclic hydrocarbons (1,2-benzanthracene and acridine); (4) azo dyes (p-dimethylaminoazobenzene), and (5) aflatoxins (B1, B2, G1, G2) produced a striking and significant inhibition of guanylate cyclase over a general concentration range of 0.5-13 mmol/1 in a variety of tissues. Some of the nitrosamides which increase guanylate cyclase activity, increase DNA synthesis whereas carcinogens which decrease guanylate cyclase activity inhibit DNA or RNA synthesis suggesting a relationship between cyclic GMP, DNA synthesis, and chemical carcinogenesis.

PMID:30623

CNS and pituitary effects of hypothalamic peptides and MSH.

The first part of this essay discussed the CNS effects of the pituitary peptide MSH. These investigations anticipated studies of the CNS effects of peptides found in the hypothalamus which were discussed in the second part of the essay. Some of the endocrine actions of the hypothalamic peptides upon the release of pituitary hormones were discussed in the last part of the essay. Together, the CNS and pituitary effects of hypothalamic peptides and MSH provide intriguing examples of areas of investigation which can be expected to be greatly expanded in the future.

CNS and pituitary effects of hypothalamic peptides and MSH. The first part of this essay discussed the CNS effects of the pituitary peptide MSH. These investigations anticipated studies of the CNS effects of peptides found in the hypothalamus which were discussed in the second part of the essay. Some of the endocrine actions of the hypothalamic peptides upon the release of pituitary hormones were discussed in the last part of the essay. Together, the CNS and pituitary effects of hypothalamic peptides and MSH provide intriguing examples of areas of investigation which can be expected to be greatly expanded in the future.

PMID:30630

Slow association-dissociation equilibrium of NADP-linked isocitrate dehydrogenase from beef liver in relation to catalytic activity.

In this paper experiments are reported which show evidence for a relation between quaternary structure and catalytic activity of cytoplasmic NADP-linked isocitrate dehydrogenase from beef liver. The inactivation of the enzyme occurring upon dilution and the plots of the catalytic activity versus the enzyme concentration indicate that the monomeric species is catalytically inactive and that the monomer-dimer equilibrium is shifted towards the dimer upon binding of the substrate magnesium isocitrate complex. The association of the enzyme following binding of the substrate takes place at a rate comparable with that of the enzymatic reaction, which results in a 'hysteretic' behaviour of the enzyme. The possibility is discussed that slow changes in quaternary structure can give rise to a physiological regulation of the enzymatic activity.

Slow association-dissociation equilibrium of NADP-linked isocitrate dehydrogenase from beef liver in relation to catalytic activity. In this paper experiments are reported which show evidence for a relation between quaternary structure and catalytic activity of cytoplasmic NADP-linked isocitrate dehydrogenase from beef liver. The inactivation of the enzyme occurring upon dilution and the plots of the catalytic activity versus the enzyme concentration indicate that the monomeric species is catalytically inactive and that the monomer-dimer equilibrium is shifted towards the dimer upon binding of the substrate magnesium isocitrate complex. The association of the enzyme following binding of the substrate takes place at a rate comparable with that of the enzymatic reaction, which results in a 'hysteretic' behaviour of the enzyme. The possibility is discussed that slow changes in quaternary structure can give rise to a physiological regulation of the enzymatic activity.

PMID:30631

Nuclear-magnetic-resonance study of aggregations and conformations of melanostatin and related peptides.

The 1H and 13C nuclear magnetic resonance spectra of melanostatin (Pro-Leu-Gly-NH2) and related peptides (Pro-Leu-Gly, Z-Pro-Leu-Gly, Z-Pro-Leu-Gly-NH2 and Z-Pro-Leu-Gly-OCH3, where Z = benzyloxycarbonyl) were analysed in a variety of solvents. At physiological pH, the melanostatin molecule is N-protonated in aqueous solution. The concentration dependences of the chemical shifts of amide-proton and carbonyl-carbon resonances and of proton spin-lattice relaxation times were observed in relation to molecular aggregations. In dimethylsulfoxide solution, aggregations were observed for N-protonated melanostatin and Pro-Leu-Gly prepared with HCl and for the Na salt of Z-Pro-Leu-Gly but not for N-protonated melanostatin prepared with HClO4 or HNO3, unprotonated melanostatin, Z-Pro-Leu-Gly-NH2, or Z-Pro-Leu-Gly-OCH3. The leucine NH and glycine CO groups of N-protonated melanostatin are involved in the intermolecular hydrogen bonds of aggregates. The leucine NH group of N-protonated Pro-Leu-Gly also forms the intermolecular hydrogen bond. The solvent and temperature dependences of the chemical shifts of amide-proton and carbonyl-carbon resonances were measured to determine intramolecular hydrogen bonding. In dimethylsulfoxide solution, N-protonated melanostatin molecules in part take the beta-turn structure and the trans carboxamide NH proton and carbonyl oxygen of the proline residue form an intramolecular hydrogen bond.

Nuclear-magnetic-resonance study of aggregations and conformations of melanostatin and related peptides. The 1H and 13C nuclear magnetic resonance spectra of melanostatin (Pro-Leu-Gly-NH2) and related peptides (Pro-Leu-Gly, Z-Pro-Leu-Gly, Z-Pro-Leu-Gly-NH2 and Z-Pro-Leu-Gly-OCH3, where Z = benzyloxycarbonyl) were analysed in a variety of solvents. At physiological pH, the melanostatin molecule is N-protonated in aqueous solution. The concentration dependences of the chemical shifts of amide-proton and carbonyl-carbon resonances and of proton spin-lattice relaxation times were observed in relation to molecular aggregations. In dimethylsulfoxide solution, aggregations were observed for N-protonated melanostatin and Pro-Leu-Gly prepared with HCl and for the Na salt of Z-Pro-Leu-Gly but not for N-protonated melanostatin prepared with HClO4 or HNO3, unprotonated melanostatin, Z-Pro-Leu-Gly-NH2, or Z-Pro-Leu-Gly-OCH3. The leucine NH and glycine CO groups of N-protonated melanostatin are involved in the intermolecular hydrogen bonds of aggregates. The leucine NH group of N-protonated Pro-Leu-Gly also forms the intermolecular hydrogen bond. The solvent and temperature dependences of the chemical shifts of amide-proton and carbonyl-carbon resonances were measured to determine intramolecular hydrogen bonding. In dimethylsulfoxide solution, N-protonated melanostatin molecules in part take the beta-turn structure and the trans carboxamide NH proton and carbonyl oxygen of the proline residue form an intramolecular hydrogen bond.

PMID:30632

Histidine in the nucleotide-binding site of NADP-linked isocitrate dehydrogenase from pig heart.

Incubation of pig heart NADP-dependent isocitrate dehydrogenase with ethoxyformic anhydride (diethylpyrocarbonate) at pH 6.2 results in a 9-fold greater rate of loss of dehydrogenase than of oxalosuccinate decarboxylase activity. The rate constants for loss of dehydrogenase and decarboxylase activities depend on the basic form of ionizable groups with pK values of 5.67 and 7.05, respectively, suggesting that inactivation of the two catalytic functions results from reaction with different amino acid residues. The rate of loss of dehydrogenase activity is decreased only slightly in the presence of manganous isocitrate, but is reduced up to 10-fold by addition of the coenzymes or coenzyme analogues, such as 2'-phosphoadenosine 5'-diphosphoribose (Rib-P2-Ado-P). Enzyme modified at pH 5.8 fails to bind NADPH, but exhibits manganese-enhanced isocitrate binding typical of native enzyme, indicating that reaction takes place in the region of the nucleotide binding site. Dissociation constants for enzyme . coenzyme-analogue complexes have been calculated from the decrease in the rate of inactivation as a function of analogue concentration. In the presence of isocitrate, activating metals (Mn2+, Mg2+, Zn2+) decrease the Kd value for enzyme . Rib-P2-Ado-P, while the inhibitor Ca2+ increases Kd. The strengthened binding of nucleotide produced by activating metal-isocitrate complexes may be essential for the catalytic reaction, reflecting an optimal orientation of NADP+ to facilitate hydride transfer. Measurements of ethoxyformyl-histidine formation at 240 nm and of incorporation of [14C]ethoxy groups in the presence and absence of Rib-P2-Ado-P indicate that loss of activity may be related to modification of approximately one histidine. The critical histidine appears to be located in the nucleotide binding site in a region distal from the substrate binding site.

Histidine in the nucleotide-binding site of NADP-linked isocitrate dehydrogenase from pig heart. Incubation of pig heart NADP-dependent isocitrate dehydrogenase with ethoxyformic anhydride (diethylpyrocarbonate) at pH 6.2 results in a 9-fold greater rate of loss of dehydrogenase than of oxalosuccinate decarboxylase activity. The rate constants for loss of dehydrogenase and decarboxylase activities depend on the basic form of ionizable groups with pK values of 5.67 and 7.05, respectively, suggesting that inactivation of the two catalytic functions results from reaction with different amino acid residues. The rate of loss of dehydrogenase activity is decreased only slightly in the presence of manganous isocitrate, but is reduced up to 10-fold by addition of the coenzymes or coenzyme analogues, such as 2'-phosphoadenosine 5'-diphosphoribose (Rib-P2-Ado-P). Enzyme modified at pH 5.8 fails to bind NADPH, but exhibits manganese-enhanced isocitrate binding typical of native enzyme, indicating that reaction takes place in the region of the nucleotide binding site. Dissociation constants for enzyme . coenzyme-analogue complexes have been calculated from the decrease in the rate of inactivation as a function of analogue concentration. In the presence of isocitrate, activating metals (Mn2+, Mg2+, Zn2+) decrease the Kd value for enzyme . Rib-P2-Ado-P, while the inhibitor Ca2+ increases Kd. The strengthened binding of nucleotide produced by activating metal-isocitrate complexes may be essential for the catalytic reaction, reflecting an optimal orientation of NADP+ to facilitate hydride transfer. Measurements of ethoxyformyl-histidine formation at 240 nm and of incorporation of [14C]ethoxy groups in the presence and absence of Rib-P2-Ado-P indicate that loss of activity may be related to modification of approximately one histidine. The critical histidine appears to be located in the nucleotide binding site in a region distal from the substrate binding site.

PMID:30633

Plasma hypoxanthine levels in pigs during acute hypoxemia. A correlation between lactate and base deficit concentrations.

Tissue hypoxia was induced in pigs by artificial ventilation with 6% O2 in N2 for 18 min. Base deficit, lactate and hypoxanthine increased linearily during this period, and were significantly higher than initial values in the course of 6 min of hypoxemia. High correlation coefficients between hypoxanthine and lactate (mean 0.98) and between hypoxanthine and base deficit (mean 0.98) were found. During the recovery period when the animals were ventilated with air, high correlation coefficients between lactate and hypoxanthine (mean 0.75) and between base deficit and hypoxanthine (mean 0.83) were also found. It is concluded that plasma hypoxanthine concentrations should be used to assess tissue hypoxia also in clinical situations.

Plasma hypoxanthine levels in pigs during acute hypoxemia. A correlation between lactate and base deficit concentrations. Tissue hypoxia was induced in pigs by artificial ventilation with 6% O2 in N2 for 18 min. Base deficit, lactate and hypoxanthine increased linearily during this period, and were significantly higher than initial values in the course of 6 min of hypoxemia. High correlation coefficients between hypoxanthine and lactate (mean 0.98) and between hypoxanthine and base deficit (mean 0.98) were found. During the recovery period when the animals were ventilated with air, high correlation coefficients between lactate and hypoxanthine (mean 0.75) and between base deficit and hypoxanthine (mean 0.83) were also found. It is concluded that plasma hypoxanthine concentrations should be used to assess tissue hypoxia also in clinical situations.

PMID:30635

Stimulation of immune reactivity by methoxy-substituted glycerol ethers incorporated into the feed.

In mice, the plaque-forming cell response to sheep red blood cells was stimulated by a mixture of methoxy-substituted glycerol ethers isolated from Greenland shark liver oil and by synthetic 1-0-(2-methoxyhexadecyl)-glycerol, given in the diet. In preliminary experiments, this synthetic compound also increased the ability of parental spleen cells to induce graft-vs.-host reactions in F1 hybrid mice. Glycerol ethers occur in the bone marrow fat of mammals and in the membrane phospholipids. It is postulated that the methoxy-substituted glycerol ethers supplied in the diet may stimulate the bone marrow and/or may be incorporated into membrane lipids, thereby changing the structure and function of the membranes.

Stimulation of immune reactivity by methoxy-substituted glycerol ethers incorporated into the feed. In mice, the plaque-forming cell response to sheep red blood cells was stimulated by a mixture of methoxy-substituted glycerol ethers isolated from Greenland shark liver oil and by synthetic 1-0-(2-methoxyhexadecyl)-glycerol, given in the diet. In preliminary experiments, this synthetic compound also increased the ability of parental spleen cells to induce graft-vs.-host reactions in F1 hybrid mice. Glycerol ethers occur in the bone marrow fat of mammals and in the membrane phospholipids. It is postulated that the methoxy-substituted glycerol ethers supplied in the diet may stimulate the bone marrow and/or may be incorporated into membrane lipids, thereby changing the structure and function of the membranes.

PMID:30636

Effect of chronic clonidine treatment and withdrawal on tyrosine hydroxylase activity in peripheral ganglia and the locus coeruleus.

As is observed clinically, cessation of chronic clonidine treatment in the rat results in a syndrome characterized by sympathetic hyperactivity. After three weeks of chronic oral administration of clonidine, tyrosine hydroxylase (TOH) activity was unchanged in superior cervical ganglia and locus coeruleus, but was reduced (45%) in the celiac ganglia. Abrupt cessation of treatment resulted in increases in TOH activity in superior cervical and celiac ganglia (to 135 and 250% of controls) and in the locus coeruleus (170% of control). These data suggest a selective effect of clonidine treatment and withdrawal on vasomotor fibers. A mechanism explaining physical dependence on clonidine is proposed.

Effect of chronic clonidine treatment and withdrawal on tyrosine hydroxylase activity in peripheral ganglia and the locus coeruleus. As is observed clinically, cessation of chronic clonidine treatment in the rat results in a syndrome characterized by sympathetic hyperactivity. After three weeks of chronic oral administration of clonidine, tyrosine hydroxylase (TOH) activity was unchanged in superior cervical ganglia and locus coeruleus, but was reduced (45%) in the celiac ganglia. Abrupt cessation of treatment resulted in increases in TOH activity in superior cervical and celiac ganglia (to 135 and 250% of controls) and in the locus coeruleus (170% of control). These data suggest a selective effect of clonidine treatment and withdrawal on vasomotor fibers. A mechanism explaining physical dependence on clonidine is proposed.

PMID:30638

Effects of alpha-agonists on circulatory responses to somatic afferent nerve stimulation.

The effect of intracisternal (i.c.m.) injection of clonidine, noradrenaline, and piperoxane on the pressor response to electrical stimulation of a peripheral somatic afferent nerve was investigated using anaesthetized cats. It was found that noradrenaline caused a depression of the magnitude of the pressor response and this effect was antagonized by subsequent injection of piperoxane i.c.m. In contrast, clonidine had no significant effect on the magnitude of the somatic pressor reflex but caused a dose-dependent prolongation of the reflex after cessation of nerve stimulation. This prolongation was antagonized by piperoxane which also caused an increase in the magnitude of the reflex. Piperoxane alone had no significant effect on the magnitude or duration of the reflex. Neither magnitude nor duration of the somatic pressor reflex was influenced significantly by a reduction in resting blood pressure. It is suggested that clonidine and noradrenaline act at different sites within the central nervous system to produce qualitatively different changes in the efferent sympathetic discharge pattern modulating the somatic pressor reflex.

Effects of alpha-agonists on circulatory responses to somatic afferent nerve stimulation. The effect of intracisternal (i.c.m.) injection of clonidine, noradrenaline, and piperoxane on the pressor response to electrical stimulation of a peripheral somatic afferent nerve was investigated using anaesthetized cats. It was found that noradrenaline caused a depression of the magnitude of the pressor response and this effect was antagonized by subsequent injection of piperoxane i.c.m. In contrast, clonidine had no significant effect on the magnitude of the somatic pressor reflex but caused a dose-dependent prolongation of the reflex after cessation of nerve stimulation. This prolongation was antagonized by piperoxane which also caused an increase in the magnitude of the reflex. Piperoxane alone had no significant effect on the magnitude or duration of the reflex. Neither magnitude nor duration of the somatic pressor reflex was influenced significantly by a reduction in resting blood pressure. It is suggested that clonidine and noradrenaline act at different sites within the central nervous system to produce qualitatively different changes in the efferent sympathetic discharge pattern modulating the somatic pressor reflex.

PMID:30639

Behavioural evidence for GABAergic activity of the benzodiazepine flurazepam.

Following reports that unilateral intranigral injections of putative GABAergic drugs induce contralateral rotational behaviour in rats, the effects of similar injections of the benzodiazepine flurazepam have been studied. Flurazepam mimicked the effects of the GABA agonist muscimol and the GABA analogue baclofen by inducing a dose-related contralateral rotation. This response was anatomically associated with the GABA-rich zona reticulata of the substantia nigra and was attenuated by the GABA antagonist picrotoxin but not by the dopamine antagonist haloperidol or by destruction of the ipsilateral nigrostriatal dopamine pathway with 6-hydroxydopamine. These results suggest that in this behavioural model flurazepam may show GABAergic activity by indirectly enhancing GABA transmission at synapses with receptors located on nigral non-dopaminergic neurons controlling postural asymmetry.

Behavioural evidence for GABAergic activity of the benzodiazepine flurazepam. Following reports that unilateral intranigral injections of putative GABAergic drugs induce contralateral rotational behaviour in rats, the effects of similar injections of the benzodiazepine flurazepam have been studied. Flurazepam mimicked the effects of the GABA agonist muscimol and the GABA analogue baclofen by inducing a dose-related contralateral rotation. This response was anatomically associated with the GABA-rich zona reticulata of the substantia nigra and was attenuated by the GABA antagonist picrotoxin but not by the dopamine antagonist haloperidol or by destruction of the ipsilateral nigrostriatal dopamine pathway with 6-hydroxydopamine. These results suggest that in this behavioural model flurazepam may show GABAergic activity by indirectly enhancing GABA transmission at synapses with receptors located on nigral non-dopaminergic neurons controlling postural asymmetry.

PMID:30640

The effects of HGCl2 and mersalyl on mechanisms regulating intracellular calcium and transmitter release.

HgCl2 and mersalyl increased and later decreased both the spontaneous and evoked transmitter liberation at the frog neuromuscular junction. Lower concentration of HgCl2 exhibited only an inhibitory effect on transmitter release. These mercurials inhibited calcium transport of mitochondria and synaptosomal vesicles. Lower concentrations of HgCl2 showed a stimulatory effect on mitochondrial calcium uptake. It is suggested that the effect of mercurials on transmitter release is mediated via changes of the intracellular calcium ion concentration.

The effects of HGCl2 and mersalyl on mechanisms regulating intracellular calcium and transmitter release. HgCl2 and mersalyl increased and later decreased both the spontaneous and evoked transmitter liberation at the frog neuromuscular junction. Lower concentration of HgCl2 exhibited only an inhibitory effect on transmitter release. These mercurials inhibited calcium transport of mitochondria and synaptosomal vesicles. Lower concentrations of HgCl2 showed a stimulatory effect on mitochondrial calcium uptake. It is suggested that the effect of mercurials on transmitter release is mediated via changes of the intracellular calcium ion concentration.

PMID:30650

Angiotensin, thirst, and sodium appetite: retrospect and prospect.

The fact that drinking in response to some hypovolemic stimuli was attenuated by nephrectomy but not by ureteric ligation led to the suggestion that the renal renin-angiotensin system may play a role in hypovolemic thirst. The isolation of a thirst factor from the kidney and the demonstration that this substance was renin supported the hypothesis. Subsequently, it was shown that the effects of renin on drinking were mediated through angiotensin II, which proved to be a potent dipsogenic substance when administered systemically or injected directly into the brain. Recently, it has been shown that angiotensin II, infused intravenously or through the carotid artery at rates that produce increases in plasma angiotensin II levels similar to those that occur in mild sodium depletion, causes the water-replete animal to drink. This discovery establishes that angiotensin is a physiological stimulus to drinking but it leaves open the question of the extent of the involvement of renal renin in normal thirst. Other unsolved problems are the role of cerebral isorenin in angiotensin thirst and its relationship with renal renin, and in view of its stimulating action on sodium intake when infused into the brain, whether angiotensin plays a significant role in sodium appetite.

Angiotensin, thirst, and sodium appetite: retrospect and prospect. The fact that drinking in response to some hypovolemic stimuli was attenuated by nephrectomy but not by ureteric ligation led to the suggestion that the renal renin-angiotensin system may play a role in hypovolemic thirst. The isolation of a thirst factor from the kidney and the demonstration that this substance was renin supported the hypothesis. Subsequently, it was shown that the effects of renin on drinking were mediated through angiotensin II, which proved to be a potent dipsogenic substance when administered systemically or injected directly into the brain. Recently, it has been shown that angiotensin II, infused intravenously or through the carotid artery at rates that produce increases in plasma angiotensin II levels similar to those that occur in mild sodium depletion, causes the water-replete animal to drink. This discovery establishes that angiotensin is a physiological stimulus to drinking but it leaves open the question of the extent of the involvement of renal renin in normal thirst. Other unsolved problems are the role of cerebral isorenin in angiotensin thirst and its relationship with renal renin, and in view of its stimulating action on sodium intake when infused into the brain, whether angiotensin plays a significant role in sodium appetite.

PMID:30653

The effect of temperature on sperm motility. II. Is bacterial growth a factor?

The previous demonstration that sperm kept at body temperature (37 degrees C) had a marked deterioration in motility accompanied by an overgrowth of bacteria in the semen and a concomitant decrease in pH led to this study to test the hypothesis that the decrease in motility was caused by the bacteria or by bacterial alteration of seminal pH. Semen specimens from fertile prevasectomy patients with and without added antibiotics were maintained at 20 degrees C and 37 degrees C and evaluated at 3, 12, and 18 hours after collection. There was still a significant deterioration in spermatozoal motility in the samples kept at 37 degrees C even when bacterial growth and change in pH were prevented by buffered antibiotics. Although the decrease in spermatozoal motility at body temperature may in part be attributed to bacterial growth or the products of bacterial metabolism, clearly another factor is present related to time and temperature and independent of the presence of bacteria.

The effect of temperature on sperm motility. II. Is bacterial growth a factor? The previous demonstration that sperm kept at body temperature (37 degrees C) had a marked deterioration in motility accompanied by an overgrowth of bacteria in the semen and a concomitant decrease in pH led to this study to test the hypothesis that the decrease in motility was caused by the bacteria or by bacterial alteration of seminal pH. Semen specimens from fertile prevasectomy patients with and without added antibiotics were maintained at 20 degrees C and 37 degrees C and evaluated at 3, 12, and 18 hours after collection. There was still a significant deterioration in spermatozoal motility in the samples kept at 37 degrees C even when bacterial growth and change in pH were prevented by buffered antibiotics. Although the decrease in spermatozoal motility at body temperature may in part be attributed to bacterial growth or the products of bacterial metabolism, clearly another factor is present related to time and temperature and independent of the presence of bacteria.

PMID:30654

[Endocrine apparatus of human gastric mucosa].

Endocrine cells of the gastric fundal and pyloric mucosa in patients suffering from gastric and duodenal ulcers with different gastric juice acidity, were studied. At hyperacid ulcers, the number of argyrhilic endocrine cells of gastric fundus was increased to 218 +/- 21.1 as compared with the normal acidity 164 +/- 14.8, while it was decreased to 97 +/- 15.1 as compared with hypoacidity. Argentaffinic cells were found in patients with hypo- and unacid gastric juice only. The electron microscopy revealed six types of endocrine cells (A-like, ECL, G, D, D1, EC). The ultrastructural changes of A-like, ECL, and G cells in patients with hyperacid gastric juice indicated their high functional activity. A balance seems to exist between activating and inhibiting systems of the endocrine apparatus of gastric mucosa which accounts for the hyperacidity of the gastric juice.

[Endocrine apparatus of human gastric mucosa]. Endocrine cells of the gastric fundal and pyloric mucosa in patients suffering from gastric and duodenal ulcers with different gastric juice acidity, were studied. At hyperacid ulcers, the number of argyrhilic endocrine cells of gastric fundus was increased to 218 +/- 21.1 as compared with the normal acidity 164 +/- 14.8, while it was decreased to 97 +/- 15.1 as compared with hypoacidity. Argentaffinic cells were found in patients with hypo- and unacid gastric juice only. The electron microscopy revealed six types of endocrine cells (A-like, ECL, G, D, D1, EC). The ultrastructural changes of A-like, ECL, and G cells in patients with hyperacid gastric juice indicated their high functional activity. A balance seems to exist between activating and inhibiting systems of the endocrine apparatus of gastric mucosa which accounts for the hyperacidity of the gastric juice.

PMID:30655

[Gastric secretion in rats during long-term histamine administration].

The acid--forming, acid--neutralizing functions of the stomach, morphological state of its walls and level of histamine in the blood were studied in control rats and in rats subjected to continuous histamine administration. In the latter, intensive elevation of intragastric acidity in empty stomach, an increase in the histamine contents in the blood, and distrophic processes in the wall of stomach occurred. The functional--morphological data obtained suggest remarkable adaptational abilities of the stomach.

[Gastric secretion in rats during long-term histamine administration]. The acid--forming, acid--neutralizing functions of the stomach, morphological state of its walls and level of histamine in the blood were studied in control rats and in rats subjected to continuous histamine administration. In the latter, intensive elevation of intragastric acidity in empty stomach, an increase in the histamine contents in the blood, and distrophic processes in the wall of stomach occurred. The functional--morphological data obtained suggest remarkable adaptational abilities of the stomach.

PMID:30666

Hemolytic anemia in patients receiving sulfasalazine.

Hemolytic anemia is a well-recognized complication of sulfasalazine treatment. 17 of 40 (43%) patients with inflammatory bowel disease receiving sulfasalazine had evidence of hemolysis as detected by starch gel electrophoresis. Only 47% (8) of patients with hemolysis had Heinz body formation. The hemoglobin was significantly reduced in patients with hemolysis and 53% had a reticulocyte count of greater than 5%. A significant correlation was noted between hemolysis and serum sulfapyridine level, but no correlation was seen with serum sulfasalazine level. There was no significant difference in disease extent or activity in patients with hemolysis compared to those without hemolysis. Hemolysis is not a rare side-effect of sulfasalazine therapy. Heinz body formation is not invariably found in sulfasalazine-induced hemolysis.

Hemolytic anemia in patients receiving sulfasalazine. Hemolytic anemia is a well-recognized complication of sulfasalazine treatment. 17 of 40 (43%) patients with inflammatory bowel disease receiving sulfasalazine had evidence of hemolysis as detected by starch gel electrophoresis. Only 47% (8) of patients with hemolysis had Heinz body formation. The hemoglobin was significantly reduced in patients with hemolysis and 53% had a reticulocyte count of greater than 5%. A significant correlation was noted between hemolysis and serum sulfapyridine level, but no correlation was seen with serum sulfasalazine level. There was no significant difference in disease extent or activity in patients with hemolysis compared to those without hemolysis. Hemolysis is not a rare side-effect of sulfasalazine therapy. Heinz body formation is not invariably found in sulfasalazine-induced hemolysis.

PMID:30668

In vitro binding of 2-acetaminofluorene to RNA and protein with rat liver microsomes.

Covalent binding of 2-acetaminofluorene[9-14C] with exogenous Torula yeast RNA and endogenous protein was investigated in liver microsome system in vitro. The binding to protein was 100 times higher than that to RNA. Requirement of NADPH, effectiveness of methylcholanthrene treatment, and inhibition by 7,8-benzoflavone suggest possible involvement of mixed-function oxidases in this binding. The binding was not due to contaminated cytosol in the microsome fraction. Addition of cytosol, sulfate ion, and ATP diminished the binding. Parallel experiments using N-hydroxy-2-acetaminofluorene[9-14C] denied major contribution of this metabolite to the binding of 2-acetaminofluorene in the microsome system. Ring-hydroxylated product was suggested as a possible metabolite for the binding.

In vitro binding of 2-acetaminofluorene to RNA and protein with rat liver microsomes. Covalent binding of 2-acetaminofluorene[9-14C] with exogenous Torula yeast RNA and endogenous protein was investigated in liver microsome system in vitro. The binding to protein was 100 times higher than that to RNA. Requirement of NADPH, effectiveness of methylcholanthrene treatment, and inhibition by 7,8-benzoflavone suggest possible involvement of mixed-function oxidases in this binding. The binding was not due to contaminated cytosol in the microsome fraction. Addition of cytosol, sulfate ion, and ATP diminished the binding. Parallel experiments using N-hydroxy-2-acetaminofluorene[9-14C] denied major contribution of this metabolite to the binding of 2-acetaminofluorene in the microsome system. Ring-hydroxylated product was suggested as a possible metabolite for the binding.

PMID:30669

Role of prostaglandins in ulcerative colitis. Enhanced production during active disease and inhibition by sulfasalazine.

Prostaglandin E2 (PGE2) level in rectal mucosa excised from 17 patients suffering from ulcerative colitis was 2-fold higher than that found in rectal mucosa of 17 normal subjects: 2.0 +/- 0.4 and 0.9 +/- 0.2 ng per mg of wet tissue, respectively. Accumulation of PGE 2 in 24-hr cultures of rectal mucosa specimens obtained from patients with ulcerative colitis was 112% higher than that observed in cultures from control subjects. Addition of sulfasalazine, sulfapyridine, and 5-aminosalicylic, acid to the culture medium of ulcerative colitis mucosa resulted in inhibition of PGE2 production by 34, 32, and 62%, respectively, compared to rectal specimens cultured in drug-free medium. These results suggest that PGE may mediate the inflammatory response in ulcerative colitis and that some of the therapeutic effect of sulfasalazine and its constituents are related to the inhibition of PGE synthesis.

Role of prostaglandins in ulcerative colitis. Enhanced production during active disease and inhibition by sulfasalazine. Prostaglandin E2 (PGE2) level in rectal mucosa excised from 17 patients suffering from ulcerative colitis was 2-fold higher than that found in rectal mucosa of 17 normal subjects: 2.0 +/- 0.4 and 0.9 +/- 0.2 ng per mg of wet tissue, respectively. Accumulation of PGE 2 in 24-hr cultures of rectal mucosa specimens obtained from patients with ulcerative colitis was 112% higher than that observed in cultures from control subjects. Addition of sulfasalazine, sulfapyridine, and 5-aminosalicylic, acid to the culture medium of ulcerative colitis mucosa resulted in inhibition of PGE2 production by 34, 32, and 62%, respectively, compared to rectal specimens cultured in drug-free medium. These results suggest that PGE may mediate the inflammatory response in ulcerative colitis and that some of the therapeutic effect of sulfasalazine and its constituents are related to the inhibition of PGE synthesis.

PMID:30670

Effect of the acid secretory state on intramural pH of rabbit gastric mucosa.

Intramural pH of the gastric mucosa was measured using a microelectrode technique in rabbit gastric pouches under different secretory conditions and luminal acidity. Exposure of spontaneously secreting or metiamide-treated fundic pouches to a relatively high concentration of luminal acid. HCl 120 mM, for 60 min, led to a marked net loss of luminal H+ which was associated with a significant decrease in the intramural pH (7.28 +/- 0.09 to 6.88 +/- 0.10 and 7.23 +/- 0.07 to 6.99 +/- 0.09, respectively). A linear relationship was observed between the rates of net disappearance of luminal acid and the intramural pH. All 10 spontaneously secreting and five metiamide-treated pouches had superficial mucosal erosions. In contrast, when fundic pouches were exposed to luminal acid in histamine-treated animals, the net loss of luminal H+ was negligible and the intramural pH remained at its base-line level (7.25 +/- 0.07). Histamine stimulation without acid in the lumen caused a small but insignificant increase in the intramural pH (7.27 +/- 0.03 to 7.39 +/- 0.05). Only three of the eight histamine-treated fundic pouches had lesions. In the antral pouches the intramural pH changes in response to exposure to luminal acid were smaller and histamine treatment did not influence the intramural pH. None of the antral pouches had lesions. The results suggest that acidification of the tissue by the diffusion of luminal acid may be an important factor in the pathogenesis of acute gastric ulceration. The acid secretory state of the gastric mucosa can significantly influence the acid-base balance in the mucosa and thus modify its response to acid diffusing from the lumen. Histamine stimulation protected the gastric mucosa by improving its buffering capacity and/or otherwise decreasing the diffusion of H+ from the lumen into the mucosa.

Effect of the acid secretory state on intramural pH of rabbit gastric mucosa. Intramural pH of the gastric mucosa was measured using a microelectrode technique in rabbit gastric pouches under different secretory conditions and luminal acidity. Exposure of spontaneously secreting or metiamide-treated fundic pouches to a relatively high concentration of luminal acid. HCl 120 mM, for 60 min, led to a marked net loss of luminal H+ which was associated with a significant decrease in the intramural pH (7.28 +/- 0.09 to 6.88 +/- 0.10 and 7.23 +/- 0.07 to 6.99 +/- 0.09, respectively). A linear relationship was observed between the rates of net disappearance of luminal acid and the intramural pH. All 10 spontaneously secreting and five metiamide-treated pouches had superficial mucosal erosions. In contrast, when fundic pouches were exposed to luminal acid in histamine-treated animals, the net loss of luminal H+ was negligible and the intramural pH remained at its base-line level (7.25 +/- 0.07). Histamine stimulation without acid in the lumen caused a small but insignificant increase in the intramural pH (7.27 +/- 0.03 to 7.39 +/- 0.05). Only three of the eight histamine-treated fundic pouches had lesions. In the antral pouches the intramural pH changes in response to exposure to luminal acid were smaller and histamine treatment did not influence the intramural pH. None of the antral pouches had lesions. The results suggest that acidification of the tissue by the diffusion of luminal acid may be an important factor in the pathogenesis of acute gastric ulceration. The acid secretory state of the gastric mucosa can significantly influence the acid-base balance in the mucosa and thus modify its response to acid diffusing from the lumen. Histamine stimulation protected the gastric mucosa by improving its buffering capacity and/or otherwise decreasing the diffusion of H+ from the lumen into the mucosa.

PMID:30676

Uneven and transient secretin release after a liquid test meal.

In order to test the hypothesis that secretin may be released intermittently after feeding, the plasma secretin concentration was measured at short intervals, and the duodenal pH continuously recorded, after a liquid test meal in 10 human subjects. Secretin was measured with a radioimmunoassay in which the entire molecule is specifically recognized and the immunoreactivities of human secretin and of the porcine peptide used as standard are similar. In the total group, no significant variation of the mean plasma secretin concentration above basal was observed within 45 min after feeding. Analysis of the individual patterns revealed that secretin was released transiently in 5 of the 10 subjects, namely those showing variations of the duodenal pH down to or below 4.0 at some time after the meal. The mean peak delta secretin in these subjects was 7.2 pg ml-1 of plasma. In 7 additional subjects, bolus intravenous injections of 0.005 and 0.01 clinical units kg-1 of porcine secretin resulted in peak delta secretin concentrations of 5.5 and 10.5 pg ml-1, respectively, and were followed by a significant increase of bicarbonate output in the duodenal aspirate. These results indicate that secretin is released unevenly and intermittently in the early period after a liquid meal in man, in amounts that seem sufficient for the initiation of a significant bicarbonate response.

Uneven and transient secretin release after a liquid test meal. In order to test the hypothesis that secretin may be released intermittently after feeding, the plasma secretin concentration was measured at short intervals, and the duodenal pH continuously recorded, after a liquid test meal in 10 human subjects. Secretin was measured with a radioimmunoassay in which the entire molecule is specifically recognized and the immunoreactivities of human secretin and of the porcine peptide used as standard are similar. In the total group, no significant variation of the mean plasma secretin concentration above basal was observed within 45 min after feeding. Analysis of the individual patterns revealed that secretin was released transiently in 5 of the 10 subjects, namely those showing variations of the duodenal pH down to or below 4.0 at some time after the meal. The mean peak delta secretin in these subjects was 7.2 pg ml-1 of plasma. In 7 additional subjects, bolus intravenous injections of 0.005 and 0.01 clinical units kg-1 of porcine secretin resulted in peak delta secretin concentrations of 5.5 and 10.5 pg ml-1, respectively, and were followed by a significant increase of bicarbonate output in the duodenal aspirate. These results indicate that secretin is released unevenly and intermittently in the early period after a liquid meal in man, in amounts that seem sufficient for the initiation of a significant bicarbonate response.

PMID:30679

Gastroesophageal reflux and bleeding esophageal varices.

The incidence of gastroesophageal reflux was evaluated with the use of a pH probe in 12 patients with cirrhosis and recent variceal hemorrhage and in 15 healthy control subjects. Short episodes of reflux occurred in 42% of the patients and in 47% of the controls. During an observation period of 1 hr, the cumulative duration of reflux was similar in patients (2.5 +/- 1.3 min) and controls (3.1 +/- 1.4 min). Mean lower esophageal sphincter pressures were normal in both groups but did not show a significant correlation with the duration of reflux. These data support previous observations that gastroesophageal reflux dose not appear to be a contributing factor in the development of variceal hemorrhage.

Gastroesophageal reflux and bleeding esophageal varices. The incidence of gastroesophageal reflux was evaluated with the use of a pH probe in 12 patients with cirrhosis and recent variceal hemorrhage and in 15 healthy control subjects. Short episodes of reflux occurred in 42% of the patients and in 47% of the controls. During an observation period of 1 hr, the cumulative duration of reflux was similar in patients (2.5 +/- 1.3 min) and controls (3.1 +/- 1.4 min). Mean lower esophageal sphincter pressures were normal in both groups but did not show a significant correlation with the duration of reflux. These data support previous observations that gastroesophageal reflux dose not appear to be a contributing factor in the development of variceal hemorrhage.

PMID:30680

Topical effects of 16,16 dimethyl prostaglandin E2 on gastric acid secretion and mucosal permeability to hydrogen ions in dogs.

The effects of luminal instillation of 16,16 dimethyl PGE2 (dmPGE2) on gastric acid secretion and back diffusion of H+ were studied in anaesthetised dogs which were prepared with a segment of the greater curvature of the stomach mounted in a double lumen chamber. This model permitted simultaneous evaluation of two segments of mucosa, one control and the other test, supplied by the same vascular pedicle. Imfusion of histamine (1.0 microgram/kg/min, intravenously) stimulated brisk acid secretion in both chambers. Topical application of 25 microgram dmPGE2 in 20 ml 0.3 M HC1 to the test chamber for 30 minutes prevented acid secretion from the test mucosa during a second histamine infusion. Since the control chamber showed no evidence of inhibition this indicates that dmPGE2 acted directly on the secretory cells, rather than after absorption from the bloodstream. This observation, however, does not exclude a possible local effect on mucosal blood flow. Direct exposure of the gastric mucosa to dmPGE2 increased the rate of back diffusion of H+ because of disruption of the permeability barrier, indicated by increased H+ back diffusion, Na+ efflux, and a reduction in potential difference. However, H+ loss was small compared to the reduction in acid output.

Topical effects of 16,16 dimethyl prostaglandin E2 on gastric acid secretion and mucosal permeability to hydrogen ions in dogs. The effects of luminal instillation of 16,16 dimethyl PGE2 (dmPGE2) on gastric acid secretion and back diffusion of H+ were studied in anaesthetised dogs which were prepared with a segment of the greater curvature of the stomach mounted in a double lumen chamber. This model permitted simultaneous evaluation of two segments of mucosa, one control and the other test, supplied by the same vascular pedicle. Imfusion of histamine (1.0 microgram/kg/min, intravenously) stimulated brisk acid secretion in both chambers. Topical application of 25 microgram dmPGE2 in 20 ml 0.3 M HC1 to the test chamber for 30 minutes prevented acid secretion from the test mucosa during a second histamine infusion. Since the control chamber showed no evidence of inhibition this indicates that dmPGE2 acted directly on the secretory cells, rather than after absorption from the bloodstream. This observation, however, does not exclude a possible local effect on mucosal blood flow. Direct exposure of the gastric mucosa to dmPGE2 increased the rate of back diffusion of H+ because of disruption of the permeability barrier, indicated by increased H+ back diffusion, Na+ efflux, and a reduction in potential difference. However, H+ loss was small compared to the reduction in acid output.

PMID:30681

Gastric secretion and basal gastrin concentration in bilharzial hepatic fibrosis.

Gastric secretion and fasting plasma gastrin levels were investigated in 26 patients with bilharzial hepatic fibrosis and 26 controls. The groups did not differ in their basal secretion. When stimulated by intravenous infusion of histamine the maximal acid output in patients with bilharzial hepatic fibrosis was significantly less than in the control group. This was unlikely to be a result of neutralisation by reflux of alkaline duodenal contents as the volumes of reflux were not different from control subjects, but was compatible with a true reduction in gastric secretion as assessed by two-component hypothesis. Neither the lowered gastric acidity nor the liver damage in patients with bilharzial hepatic fibrosis correlated with circulating gastrin. The fasting levels of plasma gastrin in these patients were not different from controls. As in other liver diseases the cause of diminished gastric secretion remains unclear.

Gastric secretion and basal gastrin concentration in bilharzial hepatic fibrosis. Gastric secretion and fasting plasma gastrin levels were investigated in 26 patients with bilharzial hepatic fibrosis and 26 controls. The groups did not differ in their basal secretion. When stimulated by intravenous infusion of histamine the maximal acid output in patients with bilharzial hepatic fibrosis was significantly less than in the control group. This was unlikely to be a result of neutralisation by reflux of alkaline duodenal contents as the volumes of reflux were not different from control subjects, but was compatible with a true reduction in gastric secretion as assessed by two-component hypothesis. Neither the lowered gastric acidity nor the liver damage in patients with bilharzial hepatic fibrosis correlated with circulating gastrin. The fasting levels of plasma gastrin in these patients were not different from controls. As in other liver diseases the cause of diminished gastric secretion remains unclear.

PMID:30682

Secretion pattern of secretin in man: regulation by gastric acid.

Median concentration of plasma secretin in the fasting state in 11 achlorhydria patients, 17 normal subjects, eight duodenal ulcer patients, and 11 Zollinger-Ellison patients was 0.3, 1.2, 2.5, and 5.9 pmol x 1(-1), respectively. Aspiration of gastric acid normal subjects and duodenal ulcer patients was followed by a significant lowering of the plasma secretin concentration. In normal subjects insulin-induced hypoglycaemia resulted in increased secretin levels when gastric acid was allowed to enter the duodenum, whereas no changes were observed when gastric acid was aspirated. Simultaneous measurements of intraduodenal pH and plasma secretin concentration in the fasting state and in response to a meal showed that rapid falls in intraduodenal pH were followed by short-lived increments in plasma secretin concentration. These changes in pH and in secretin levels were diminished after cimetidine. It is concluded that gastric acid in man does trigger release of secretin and that secretin is secreted intermittently both in the fasting state and in response to a meal when boluses of acid enter the duodenum.

Secretion pattern of secretin in man: regulation by gastric acid. Median concentration of plasma secretin in the fasting state in 11 achlorhydria patients, 17 normal subjects, eight duodenal ulcer patients, and 11 Zollinger-Ellison patients was 0.3, 1.2, 2.5, and 5.9 pmol x 1(-1), respectively. Aspiration of gastric acid normal subjects and duodenal ulcer patients was followed by a significant lowering of the plasma secretin concentration. In normal subjects insulin-induced hypoglycaemia resulted in increased secretin levels when gastric acid was allowed to enter the duodenum, whereas no changes were observed when gastric acid was aspirated. Simultaneous measurements of intraduodenal pH and plasma secretin concentration in the fasting state and in response to a meal showed that rapid falls in intraduodenal pH were followed by short-lived increments in plasma secretin concentration. These changes in pH and in secretin levels were diminished after cimetidine. It is concluded that gastric acid in man does trigger release of secretin and that secretin is secreted intermittently both in the fasting state and in response to a meal when boluses of acid enter the duodenum.

PMID:30683

Short chain fatty acid absorption by the human large intestine.

Short chain fatty acid absorption from the human rectum has been studied in 46 subjects attending an obesity clinic, using a dialysis bag technique. From a mixed electrolyte solution, acetate concentrations fell from 97.0 to 64.2 mmol/l, and sodium from 97.8 to 85.1 mmol/l with respective net absorption rates of 8.1 and 5.2 mumol/cm2/h. From a solution with mixed short chain fatty acids acetate concentration fell from 62.3 to 37.6 mmol/l, propionate from 20.2 to 11.5 mmol/l, and butyrate from 25.7 to 17.3 mmol/l with absorption rates of 5.2, 1.8, and 1.9 mumol/cm2/h. Lowering pH from 7.2 to 5.5, to test the possibility that absorption occurred by passive non-ionic diffusion, had no effect on absorption rates, although pH rose rapidly in the dialysis fluid. These results are comparable with rates of acetate absorption from the animal large intestine. The hypothesis that short chain fatty acids are not absorbed from the large gut and therefore contribute to faecal bulk by retaining water in the bowel lumen may need revision.

Short chain fatty acid absorption by the human large intestine. Short chain fatty acid absorption from the human rectum has been studied in 46 subjects attending an obesity clinic, using a dialysis bag technique. From a mixed electrolyte solution, acetate concentrations fell from 97.0 to 64.2 mmol/l, and sodium from 97.8 to 85.1 mmol/l with respective net absorption rates of 8.1 and 5.2 mumol/cm2/h. From a solution with mixed short chain fatty acids acetate concentration fell from 62.3 to 37.6 mmol/l, propionate from 20.2 to 11.5 mmol/l, and butyrate from 25.7 to 17.3 mmol/l with absorption rates of 5.2, 1.8, and 1.9 mumol/cm2/h. Lowering pH from 7.2 to 5.5, to test the possibility that absorption occurred by passive non-ionic diffusion, had no effect on absorption rates, although pH rose rapidly in the dialysis fluid. These results are comparable with rates of acetate absorption from the animal large intestine. The hypothesis that short chain fatty acids are not absorbed from the large gut and therefore contribute to faecal bulk by retaining water in the bowel lumen may need revision.

PMID:30684

The effect of streptozotocin analogues on guanylate cyclase activity.

Streptozotocin, a nitrosamide carcinogen, enhances the activity of guanylate cyclase. Six analogues of streptozotocin were studied in order to elucidate critical structure-activity relationships pertaining to the activation of guanylate cyclase. Analogue 1, known as chlorozotocin, has a nitroso group and increased guanylate cyclase activity 17 to 28-fold. Analogue III, which also has a nitroso group, but greater structural modifications with 4 acetate groups extending off of the glucose moiety, activated guanylate cyclase in colon but not in kidney. The other analogues (II,IV,VI, and VIII) lacking nitroso groups, either had no effect or produced mild decreases in guanylate cyclase activity.

The effect of streptozotocin analogues on guanylate cyclase activity. Streptozotocin, a nitrosamide carcinogen, enhances the activity of guanylate cyclase. Six analogues of streptozotocin were studied in order to elucidate critical structure-activity relationships pertaining to the activation of guanylate cyclase. Analogue 1, known as chlorozotocin, has a nitroso group and increased guanylate cyclase activity 17 to 28-fold. Analogue III, which also has a nitroso group, but greater structural modifications with 4 acetate groups extending off of the glucose moiety, activated guanylate cyclase in colon but not in kidney. The other analogues (II,IV,VI, and VIII) lacking nitroso groups, either had no effect or produced mild decreases in guanylate cyclase activity.

PMID:30685

Reconstitution of rabbit muscle aldolase after dissociation and denaturation at alkaline pH.

Tetrameric rabbit muscle aldolase is dissociated to the inactive monomer at strongly alkaline pH (pH greater than or equal to 12). As shown by sedimentation velocity, fluorescence emission, and specific activity, the final profiles of dissociation, denaturation, and deactivation run parallel. Increasing incubation time proves the enzyme to be metastable in the pH range of deactivation. At 10 less than pH less than 12 "hysteresis" of the deactivation-reactivation reaction is observed. Short incubation at pH greater than or equal to 12 leads to high yields of reactivation (greater than or equal to 60%), while irreversibly denatured enzyme protein is the final product after long incubation. The kinetics of reconstitution under essentially irreversible conditions (pH 7.6) can be described by a sequential uni-bimolecular mechanism, assuming partial activity of the isolated subunits. The kinetic constants correspond to those observed for the reactivation after denaturation at acid pH or in 6M guanidine. HCl. Obviously the pH-dependent deactivation and reactivation of aldolase at alkaline pH obeys the general transconformation/association model which has been previously reported to hold for the reconstitution of numerous oligomeric enzymes after denaturation in various denaturants.

Reconstitution of rabbit muscle aldolase after dissociation and denaturation at alkaline pH. Tetrameric rabbit muscle aldolase is dissociated to the inactive monomer at strongly alkaline pH (pH greater than or equal to 12). As shown by sedimentation velocity, fluorescence emission, and specific activity, the final profiles of dissociation, denaturation, and deactivation run parallel. Increasing incubation time proves the enzyme to be metastable in the pH range of deactivation. At 10 less than pH less than 12 "hysteresis" of the deactivation-reactivation reaction is observed. Short incubation at pH greater than or equal to 12 leads to high yields of reactivation (greater than or equal to 60%), while irreversibly denatured enzyme protein is the final product after long incubation. The kinetics of reconstitution under essentially irreversible conditions (pH 7.6) can be described by a sequential uni-bimolecular mechanism, assuming partial activity of the isolated subunits. The kinetic constants correspond to those observed for the reactivation after denaturation at acid pH or in 6M guanidine. HCl. Obviously the pH-dependent deactivation and reactivation of aldolase at alkaline pH obeys the general transconformation/association model which has been previously reported to hold for the reconstitution of numerous oligomeric enzymes after denaturation in various denaturants.

PMID:30690

Nucleotides and coenzymes in nuclei isolated from rat liver.

In rat-liver nuclei, isolated by the non-aqueous technique, the concentrations and labelling rates of the purine moiety of acid-soluble nucleotides were determined and compared with corresponding data for non-fractionated tissue and nuclei-free cytoplasm. Livers were used from untreated rats, from rats with a highly stimulated synthesis of NAD and from rats following a heavy metabolic load with adenosine. Under all circumstances, the nuclear and cytoplasmic concentrations of nucleotides (e.g. ATP and its dephosphorylated forms, pyridine nucleotides) and of free glucose were practically identical. Specific radioactivities after a pulse with formate also indicated a nucleo-cytoplasmic equilibrium for purine-containing nucleotides. It is concluded that precursor pools for nuclear biosyntheses as well as energy supply for other nuclear activities may be determined by an analysis of the non-fractionated tissue.

Nucleotides and coenzymes in nuclei isolated from rat liver. In rat-liver nuclei, isolated by the non-aqueous technique, the concentrations and labelling rates of the purine moiety of acid-soluble nucleotides were determined and compared with corresponding data for non-fractionated tissue and nuclei-free cytoplasm. Livers were used from untreated rats, from rats with a highly stimulated synthesis of NAD and from rats following a heavy metabolic load with adenosine. Under all circumstances, the nuclear and cytoplasmic concentrations of nucleotides (e.g. ATP and its dephosphorylated forms, pyridine nucleotides) and of free glucose were practically identical. Specific radioactivities after a pulse with formate also indicated a nucleo-cytoplasmic equilibrium for purine-containing nucleotides. It is concluded that precursor pools for nuclear biosyntheses as well as energy supply for other nuclear activities may be determined by an analysis of the non-fractionated tissue.

PMID:30691

Kawasaki disease: a 'new' pediatric enigma.

More than 250 cases of this puzzling illness, first described in Japan a decade ago, have now been reported in the United States. The etiology, epidemiology, and true incidence remain to be determined. But since some 2% of victims develop severe cardiac complications and some may die suddenly months after apparent complete recovery, pediatricians must now consider this entity in the differential diagnosis of protracted, febrile illness.

Kawasaki disease: a 'new' pediatric enigma. More than 250 cases of this puzzling illness, first described in Japan a decade ago, have now been reported in the United States. The etiology, epidemiology, and true incidence remain to be determined. But since some 2% of victims develop severe cardiac complications and some may die suddenly months after apparent complete recovery, pediatricians must now consider this entity in the differential diagnosis of protracted, febrile illness.

PMID:30692

Endocrine pancreatic tumors.

The pathology and cell biology of endocrine pancreatic tumors are reviewed. It is probable that all these tumors are "functioning" in the sense that they elaborate hormones that cause more or less conspicuous clinical syndromes. Identification of such secretory products is essential for an optimal diagnosis, localization, treatment, and follow-up. Recent data indicate that endocrine pancreatic tumors evolve from progenitor cells of ducts. This histogenetic mechanism may explain the occurrence not only of mixed or multihormonal tumors but also of tumors producing hormones that are absent from the adult human pancreas. In addition to their clinically apparent effects, many endocrine pancreatic tumors affect the surrounding endocrine pancreas in a characteristic way. The mechanisms behind and the potential diagnostic usefulness of these changes are discussed.

Endocrine pancreatic tumors. The pathology and cell biology of endocrine pancreatic tumors are reviewed. It is probable that all these tumors are "functioning" in the sense that they elaborate hormones that cause more or less conspicuous clinical syndromes. Identification of such secretory products is essential for an optimal diagnosis, localization, treatment, and follow-up. Recent data indicate that endocrine pancreatic tumors evolve from progenitor cells of ducts. This histogenetic mechanism may explain the occurrence not only of mixed or multihormonal tumors but also of tumors producing hormones that are absent from the adult human pancreas. In addition to their clinically apparent effects, many endocrine pancreatic tumors affect the surrounding endocrine pancreas in a characteristic way. The mechanisms behind and the potential diagnostic usefulness of these changes are discussed.

PMID:30694

Further characterization of Mycobacterium ulcerans toxin.

Mycobacterium ulcerans produces an exotoxin in culture which, when inoculated into guinea pig skin, causes inflammation, necrosis, edema, and other histopathological changes resembling those in infections of humans. The toxin was resistant to heat and to alkalies and was moderately acid labile. Toxic activity was destroyed by Pronase, phospholipase, lipase, amylase, and glucosidase but not by trypsin, collagenase, cellulase, lysozyme, hyaluronidase, or neuraminidase. Toxic activity was resistant to treatment with 2-mercaptoethanol, urea, guanidine hydrochloride, p-chloromercuribenzoate, ethylenediaminetetraacetate, and sodium deoxycholate but was destroyed by sodium m-periodate and sodium dodecyl sulfate. The toxin was precipitated by a wide range of ammonium sulfate concentrations. Extraction with chlorofrom-methanol or petroleum ether destroyed its activity. Isopycnic density gradient ultracentrifugation in KBr produced a high-density lipoprotein layer with a 24-fold increase in specific activity. The results indicate that this toxin is a high-molecular-weight phospholipoprotein-polysaccharide complex.

Further characterization of Mycobacterium ulcerans toxin. Mycobacterium ulcerans produces an exotoxin in culture which, when inoculated into guinea pig skin, causes inflammation, necrosis, edema, and other histopathological changes resembling those in infections of humans. The toxin was resistant to heat and to alkalies and was moderately acid labile. Toxic activity was destroyed by Pronase, phospholipase, lipase, amylase, and glucosidase but not by trypsin, collagenase, cellulase, lysozyme, hyaluronidase, or neuraminidase. Toxic activity was resistant to treatment with 2-mercaptoethanol, urea, guanidine hydrochloride, p-chloromercuribenzoate, ethylenediaminetetraacetate, and sodium deoxycholate but was destroyed by sodium m-periodate and sodium dodecyl sulfate. The toxin was precipitated by a wide range of ammonium sulfate concentrations. Extraction with chlorofrom-methanol or petroleum ether destroyed its activity. Isopycnic density gradient ultracentrifugation in KBr produced a high-density lipoprotein layer with a 24-fold increase in specific activity. The results indicate that this toxin is a high-molecular-weight phospholipoprotein-polysaccharide complex.

PMID:30695

Lactate dehydrogenase mutants of Streptococcus mutans: isolation and preliminary characterization.

Mutants of Streptococcus mutans were isolated which lack the enzyme activity L (+)-lactate dehydrogenase. Reversion studies indicate that the genetic defects are in the structural gene for the enzyme. The mutants produce less titratable acid from glucose, adhere better to hydroxyapatite, and accumulate more plaque when grown in the presence of sucrose than does the parent strain. These findings suggest a possible use for the mutants as effector strains in the replacement therapy of dental caries.

Lactate dehydrogenase mutants of Streptococcus mutans: isolation and preliminary characterization. Mutants of Streptococcus mutans were isolated which lack the enzyme activity L (+)-lactate dehydrogenase. Reversion studies indicate that the genetic defects are in the structural gene for the enzyme. The mutants produce less titratable acid from glucose, adhere better to hydroxyapatite, and accumulate more plaque when grown in the presence of sucrose than does the parent strain. These findings suggest a possible use for the mutants as effector strains in the replacement therapy of dental caries.

PMID:30696

Effects of surface-active agents on neutrophil receptors.

An easily performed assay to identify the C3b and Fc receptors on human neutrophils was developed. Salmonella typhimurium were treated with fluorescein and then incubated in nonimmune fresh human serum, which led to C3b fixation via activation of the alternative pathway. Similarly, type II pneumococci were treated with fluorescein and opsonized with type-specific rabbit antiserum. Neutrophils bearing C3b and Fc receptors formed rosettes with the respective bacteria, which were easily readable because of their bright fluorescence. Incubation of neutrophils at 37 degrees C with C3-coated bacteria generated 54 +/ 4% C3b rosettes, whereas neutrophils incubated with immunoglobulin G-coated bacteria yielded 75 +/ 7% rosettes. Incubation at 4 degrees C inhibited the formation of C3b rosettes but not Fc rosettes. Heat inactivation of the fresh human serum at 56 degrees C for 30 min completely inhibited the formation of the C3b rosettes, and addition of heat-aggregated immunoglobulin G to the polymorphonuclear leukocyte blocked the ability of the polymorphonuclear leukocyte to bind immunoglobulin G-coated bacteria. Addition of 1.0 mM N-ethylmaleimide, 0.1 mg of trypsin per ml, 10 mM H2O2, O2- generated by xanthine-xanthine oxidase, and 8 times 10(-4) M hydrocortisone inhibited the C3b receptor, but did not inhibit the Fc receptor. In neutrophils, the selective effect of the various inhibitors suggests that the Fc and C3b receptors are distinct entities.

Effects of surface-active agents on neutrophil receptors. An easily performed assay to identify the C3b and Fc receptors on human neutrophils was developed. Salmonella typhimurium were treated with fluorescein and then incubated in nonimmune fresh human serum, which led to C3b fixation via activation of the alternative pathway. Similarly, type II pneumococci were treated with fluorescein and opsonized with type-specific rabbit antiserum. Neutrophils bearing C3b and Fc receptors formed rosettes with the respective bacteria, which were easily readable because of their bright fluorescence. Incubation of neutrophils at 37 degrees C with C3-coated bacteria generated 54 +/ 4% C3b rosettes, whereas neutrophils incubated with immunoglobulin G-coated bacteria yielded 75 +/ 7% rosettes. Incubation at 4 degrees C inhibited the formation of C3b rosettes but not Fc rosettes. Heat inactivation of the fresh human serum at 56 degrees C for 30 min completely inhibited the formation of the C3b rosettes, and addition of heat-aggregated immunoglobulin G to the polymorphonuclear leukocyte blocked the ability of the polymorphonuclear leukocyte to bind immunoglobulin G-coated bacteria. Addition of 1.0 mM N-ethylmaleimide, 0.1 mg of trypsin per ml, 10 mM H2O2, O2- generated by xanthine-xanthine oxidase, and 8 times 10(-4) M hydrocortisone inhibited the C3b receptor, but did not inhibit the Fc receptor. In neutrophils, the selective effect of the various inhibitors suggests that the Fc and C3b receptors are distinct entities.

PMID:30697

Protective ability of Salmonella ribosomal protein and RNA in inbred mice.

Ribosomal vaccines prepared from Salmonella typhimurium were effective immunogens in A/J, C3H/HeDub, and C3H/HeJ mice. Purified ribosomal components were also tested as immunogens in the inbred mice. Protein isolated from a Salmonella ribosomal fraction could protect all three mouse strains. Although purified RNA was shown to be protective for A/J and C3H/HeDub mice, it was not protective for C3H/HeJ mice. Protective immunity could be induced in A/J and C3H/HeDub mice by various immunostimulants. Immunity in C3H/HeJ mice, however, could only be induced by Salmonella ribosomes or protein isolated from the Salmonella ribosomal fraction.

Protective ability of Salmonella ribosomal protein and RNA in inbred mice. Ribosomal vaccines prepared from Salmonella typhimurium were effective immunogens in A/J, C3H/HeDub, and C3H/HeJ mice. Purified ribosomal components were also tested as immunogens in the inbred mice. Protein isolated from a Salmonella ribosomal fraction could protect all three mouse strains. Although purified RNA was shown to be protective for A/J and C3H/HeDub mice, it was not protective for C3H/HeJ mice. Protective immunity could be induced in A/J and C3H/HeDub mice by various immunostimulants. Immunity in C3H/HeJ mice, however, could only be induced by Salmonella ribosomes or protein isolated from the Salmonella ribosomal fraction.

PMID:30698

Characteristics of an adenovirus type 19 conjunctivitis isolate and evidence for a subgroup associated with epidemic conjunctivitis.

Although adneovirus type 19 (Ad19) was first described in 1955, this virus was not associated with disease until its isolation from outbreaks of conjunctivitis in 1973. A strain of Ad19 isolated from a case of conjunctivitis in Seattle in 1974 was compared with the reference strain (3911). Plaque number and size were enhanced by 30 mM MgCl2. Low pH and chloroform treatment had no effect on either strain's activity, but the two strains were sensitive to pH 8. Growth curves were characteristic of adenoviruses, but differences were seen in the amount of virus released. The ratios of particles to plaque-forming units (approximately 10,000:1) were similar for both. Both virus preparations contained high concentrations of group-specific complement-fixing antigen. Cross-reactions were seen by hemagglutination inhibition and immunoelectron microscopy between antisera to Ad8, Ad9, and Ad10 versus both strains of Ad19, but were not seen by neutralization. We would like to suggest, based on exclusive conjunctivitis association and cross-reactions, that the four cross-reacting serotypes, Ad8, Ad9, Ad10, and Ad19, represent a subgroup of adenoviruses specfically associated with conjunctivitis.

Characteristics of an adenovirus type 19 conjunctivitis isolate and evidence for a subgroup associated with epidemic conjunctivitis. Although adneovirus type 19 (Ad19) was first described in 1955, this virus was not associated with disease until its isolation from outbreaks of conjunctivitis in 1973. A strain of Ad19 isolated from a case of conjunctivitis in Seattle in 1974 was compared with the reference strain (3911). Plaque number and size were enhanced by 30 mM MgCl2. Low pH and chloroform treatment had no effect on either strain's activity, but the two strains were sensitive to pH 8. Growth curves were characteristic of adenoviruses, but differences were seen in the amount of virus released. The ratios of particles to plaque-forming units (approximately 10,000:1) were similar for both. Both virus preparations contained high concentrations of group-specific complement-fixing antigen. Cross-reactions were seen by hemagglutination inhibition and immunoelectron microscopy between antisera to Ad8, Ad9, and Ad10 versus both strains of Ad19, but were not seen by neutralization. We would like to suggest, based on exclusive conjunctivitis association and cross-reactions, that the four cross-reacting serotypes, Ad8, Ad9, Ad10, and Ad19, represent a subgroup of adenoviruses specfically associated with conjunctivitis.

PMID:30699

Effect of pH on the growth and glucose metabolism of Neisseria gonorrhoeae.

This study examined the effect of pH on the metabolism of glucose by Neisseria gonorrhoeae. Radiorespirometric studies revealed that cells growing at pH 7.2 or 8.0 metabolized glucose primarily (ca. 80%) via the Entner-Doudoroff pathway. The remainder of the glucose was metabolized via the pentose phosphate pathway (ca. 20%). The tricarboxylic acid cycle was not active during glucose catabolism at either pH 7.2 or 8.0, and acetate accumulated in the medium. Cells growing at pH 6.0 had markedly increased pentose phosphate pathway activity (ca. 50%) and a functioning tricarboxylic acid cycle. The alteration in pathways was not due to differences in growth rate, but to the pH of the medium. Chemical fractionation of labeled cells and total hexose analyses revealed that growth pH markedly affected the composition of the gonococcus.

Effect of pH on the growth and glucose metabolism of Neisseria gonorrhoeae. This study examined the effect of pH on the metabolism of glucose by Neisseria gonorrhoeae. Radiorespirometric studies revealed that cells growing at pH 7.2 or 8.0 metabolized glucose primarily (ca. 80%) via the Entner-Doudoroff pathway. The remainder of the glucose was metabolized via the pentose phosphate pathway (ca. 20%). The tricarboxylic acid cycle was not active during glucose catabolism at either pH 7.2 or 8.0, and acetate accumulated in the medium. Cells growing at pH 6.0 had markedly increased pentose phosphate pathway activity (ca. 50%) and a functioning tricarboxylic acid cycle. The alteration in pathways was not due to differences in growth rate, but to the pH of the medium. Chemical fractionation of labeled cells and total hexose analyses revealed that growth pH markedly affected the composition of the gonococcus.

PMID:30700

Factors affecting the aggregation of Actinomyces naeslundii during growth and in washed cell suspensions.

Various factors affecting the aggregation of Actinomyces naeslundii strain 12104 were studied. When the pH of glucose-supplemented growth medium fell below 5.5, the cells aggregated and formed microbial masses which tenaciously adhered to the culture vessels. When the organism was cultured in the same medium in the absence of glucose, maximum growth was reduced and the final culture pH values remained above 6.5, but the cells were more dispersed and nonadherent. Adjusting the final pH of these cultures to below 5.5 with HCl caused the cells to aggregate. Cells from unsupplemented cultures with final pH values of 6.7 were washed by centrifugation, dispersed by vigorous shaking, and suspended in buffer at pH values ranging from 4.5 to 8.0. Aggregation (expressed as the percent reduction of optical density at 520 nm after incubation at 37 degrees C) occurred rapidly at pH values below 6.0 but did not readily occur at higher pH values. Aggregation of strain 12104 in washed cell suspensions was induced by low pH and influenced by cell concentration and ionic strength of the environment. Low pH values also induced aggregation in washed cell suspensions of Streptococcus mutans, Streptococcus sanguis, Streptococcus salivarius, and Actinomyces viscosus.

Factors affecting the aggregation of Actinomyces naeslundii during growth and in washed cell suspensions. Various factors affecting the aggregation of Actinomyces naeslundii strain 12104 were studied. When the pH of glucose-supplemented growth medium fell below 5.5, the cells aggregated and formed microbial masses which tenaciously adhered to the culture vessels. When the organism was cultured in the same medium in the absence of glucose, maximum growth was reduced and the final culture pH values remained above 6.5, but the cells were more dispersed and nonadherent. Adjusting the final pH of these cultures to below 5.5 with HCl caused the cells to aggregate. Cells from unsupplemented cultures with final pH values of 6.7 were washed by centrifugation, dispersed by vigorous shaking, and suspended in buffer at pH values ranging from 4.5 to 8.0. Aggregation (expressed as the percent reduction of optical density at 520 nm after incubation at 37 degrees C) occurred rapidly at pH values below 6.0 but did not readily occur at higher pH values. Aggregation of strain 12104 in washed cell suspensions was induced by low pH and influenced by cell concentration and ionic strength of the environment. Low pH values also induced aggregation in washed cell suspensions of Streptococcus mutans, Streptococcus sanguis, Streptococcus salivarius, and Actinomyces viscosus.

PMID:30701

Mechanism of coaggregation between Actinomyces viscosus T14V and Streptococcus sanguis 34.

Actinomyces viscosus T14V and Streptococcus sanguis 34 coaggregate by a mechanism which is not inhibited by 1 M NaCl, is dextran independent, requires calcium, is pH dependent with an optimum at pH 8.0 to 8.5, and appears to require the interaction of a protein or glycoprotein on A. viscosus with a carbohydrate on S. sanguis. The coaggregation is inhibited more than 80% by 0.01 M lactose, 0.02 M beta-methyl-D-galactoside, or 0.05 M D-galactose; inhibition of coaggregation was less than 10% in 0.1 M alpha-methyl-D-galactoside, melibiose, maltose, cellobiose, sucrose, and a number of monosaccharides. At very high concentrations of enzyme, protease from S. griseus destroyed the reactive site on A. viscosus but not on S. sanguis. Both were totally resistant to dextranase. Periodate (0.01 M; pH 4) inactivated both bacteria. The ability of S. sanguis to coaggregate with A. viscosus was not destroyed by phenol-water extraction at 65 degrees C for 15 min. When the bacteria were cultured under specified conditions, the coaggregation was highly reproducible. Under the same conditions, T14AV, the avirulent mutant of A. viscosus T14V, did not coaggregate with S. sanguis 34. Electron microscopic studies of coaggregates, labeled immunochemically with antibody to A. viscosus, indicated that fibrils on A. viscosus may be involved in the coaggregation.

Mechanism of coaggregation between Actinomyces viscosus T14V and Streptococcus sanguis 34. Actinomyces viscosus T14V and Streptococcus sanguis 34 coaggregate by a mechanism which is not inhibited by 1 M NaCl, is dextran independent, requires calcium, is pH dependent with an optimum at pH 8.0 to 8.5, and appears to require the interaction of a protein or glycoprotein on A. viscosus with a carbohydrate on S. sanguis. The coaggregation is inhibited more than 80% by 0.01 M lactose, 0.02 M beta-methyl-D-galactoside, or 0.05 M D-galactose; inhibition of coaggregation was less than 10% in 0.1 M alpha-methyl-D-galactoside, melibiose, maltose, cellobiose, sucrose, and a number of monosaccharides. At very high concentrations of enzyme, protease from S. griseus destroyed the reactive site on A. viscosus but not on S. sanguis. Both were totally resistant to dextranase. Periodate (0.01 M; pH 4) inactivated both bacteria. The ability of S. sanguis to coaggregate with A. viscosus was not destroyed by phenol-water extraction at 65 degrees C for 15 min. When the bacteria were cultured under specified conditions, the coaggregation was highly reproducible. Under the same conditions, T14AV, the avirulent mutant of A. viscosus T14V, did not coaggregate with S. sanguis 34. Electron microscopic studies of coaggregates, labeled immunochemically with antibody to A. viscosus, indicated that fibrils on A. viscosus may be involved in the coaggregation.

PMID:30704

A simple method of screening for antisperm antibodies in the human male. Detection of spermatozoal surface IgG with the direct mixed antiglobulin reaction carried out on untreated fresh human semen.

A simple and rapid test for the detection of antisperm antibodies of the IgG class on freely swimming spermatozoa in fresh human semen is described. The test is based on the formation of motile mixed agglutinates between erythrocytes sensitized with incomplete anti-Rh-antibodies and freely swimming spermatozoa with surface antisperm antibodies, after mixing both cell types together with anti-IgG antiserum. Agglutination of the red blood cells serves as an internal control. The test can be applied on ejaculates with spermatozoa concentrations down to one million per ml, provided the motility is sufficient. The percentage of motile spermatozoa found to be coated with antisperm antibodies of the IgG class, and the extent of the coating, proved to be correlated with the agglutination titer of circulating antisperm antibodies and with the inhibition of sperm penetration into cervical mucus. The test can be used as a screening for the presence of antisperm autoantibodies in serum and semen.

A simple method of screening for antisperm antibodies in the human male. Detection of spermatozoal surface IgG with the direct mixed antiglobulin reaction carried out on untreated fresh human semen. A simple and rapid test for the detection of antisperm antibodies of the IgG class on freely swimming spermatozoa in fresh human semen is described. The test is based on the formation of motile mixed agglutinates between erythrocytes sensitized with incomplete anti-Rh-antibodies and freely swimming spermatozoa with surface antisperm antibodies, after mixing both cell types together with anti-IgG antiserum. Agglutination of the red blood cells serves as an internal control. The test can be applied on ejaculates with spermatozoa concentrations down to one million per ml, provided the motility is sufficient. The percentage of motile spermatozoa found to be coated with antisperm antibodies of the IgG class, and the extent of the coating, proved to be correlated with the agglutination titer of circulating antisperm antibodies and with the inhibition of sperm penetration into cervical mucus. The test can be used as a screening for the presence of antisperm autoantibodies in serum and semen.

PMID:30706

Effect of endogenous and exogenous progesterone on human endometrial enzymes.

Activities of 9 enzymes were determined biochemically in the endometrium. In Trial I (five women) 25 mg progesterone were injected i.m. on day 9 of the cycle; and endometrial biopsy taken 24 hours later was compared with endometrium from day 10 and day 21, taken in two untreated cycles from the same volunteers. Similarly, in Trial II (five women) 50 mg progesterone were injected on day 9, biopsy taken on day 11 and compared with days 11 and 21 from untreated cycles. The specific activites of lactate dehydrogenase, isocitrate dehydrogenase (ICDH), malate dehydrogenase, glutamate dehydrogenase, beta-glucuronidase, acid phosphatase (ACP) and alkaline phosphatase (AP) were significantly higher in the secretory phase. Twenty-five milligrams progesterone (after 24 hours) caused increases of some enzymes, significant only for AP. Fifty milligrams (after 48 hours) increased significantly the activity of ICDH and ACP. Biochemical changes, especially increase of ICDH, can be used for detection of the effect of progesterone on the endometrium.

Effect of endogenous and exogenous progesterone on human endometrial enzymes. Activities of 9 enzymes were determined biochemically in the endometrium. In Trial I (five women) 25 mg progesterone were injected i.m. on day 9 of the cycle; and endometrial biopsy taken 24 hours later was compared with endometrium from day 10 and day 21, taken in two untreated cycles from the same volunteers. Similarly, in Trial II (five women) 50 mg progesterone were injected on day 9, biopsy taken on day 11 and compared with days 11 and 21 from untreated cycles. The specific activites of lactate dehydrogenase, isocitrate dehydrogenase (ICDH), malate dehydrogenase, glutamate dehydrogenase, beta-glucuronidase, acid phosphatase (ACP) and alkaline phosphatase (AP) were significantly higher in the secretory phase. Twenty-five milligrams progesterone (after 24 hours) caused increases of some enzymes, significant only for AP. Fifty milligrams (after 48 hours) increased significantly the activity of ICDH and ACP. Biochemical changes, especially increase of ICDH, can be used for detection of the effect of progesterone on the endometrium.

PMID:30707

The effect of antibody against a purified sperm-coating antigen on human sperm.

A purified fraction of human seminal plasma containing a sperm-coating antigen and two minor contaminants was used to immunize rabbits by intravenous route. An antiserum containing only the antibody against the sperm-coating antigen as examined by immunoelectrophoresis was obtained from one rabbit. The effect of this antiserum on human sperm was examined by sperm-immobilization and sperm-agglutination tests. The results revealed that the antibody against the purified sperm-coating antigen was incapable of immobilzing or agglutinating human sperm. This indicates that the purified sperm-coating antigen is unlikely to be useful as an antifertility antigen for immunologic fertility control.

The effect of antibody against a purified sperm-coating antigen on human sperm. A purified fraction of human seminal plasma containing a sperm-coating antigen and two minor contaminants was used to immunize rabbits by intravenous route. An antiserum containing only the antibody against the sperm-coating antigen as examined by immunoelectrophoresis was obtained from one rabbit. The effect of this antiserum on human sperm was examined by sperm-immobilization and sperm-agglutination tests. The results revealed that the antibody against the purified sperm-coating antigen was incapable of immobilzing or agglutinating human sperm. This indicates that the purified sperm-coating antigen is unlikely to be useful as an antifertility antigen for immunologic fertility control.

PMID:30708

An improved assay technique for the proteolytic activity of individual human spermatozoa.

An improved substrate-film technique has been developed for the assay of released proteinase from individual human spermatozoa. During the preparation of the thin gelatin membrane, it is pretreated with formaldehyde and NaOH. These agents alter the plasma membrane and the outer acrosomal membrane of the spermatozoon, facilitating the release of acrosomal enzymes. This effect is further enhanced by the addition of albumin to the incubation mixture. More than 90% of the spermatozoa in a normal ejaculate give a reaction by this method. It reaches a maximum after 4 hours of incubation and does not increase further even up to 12 hours. No difference in the reaction between washed and unwashed ejaculated spermatozoa can be found. The nature of the proteolytic activity and its possible significance in infertility are discussed.

An improved assay technique for the proteolytic activity of individual human spermatozoa. An improved substrate-film technique has been developed for the assay of released proteinase from individual human spermatozoa. During the preparation of the thin gelatin membrane, it is pretreated with formaldehyde and NaOH. These agents alter the plasma membrane and the outer acrosomal membrane of the spermatozoon, facilitating the release of acrosomal enzymes. This effect is further enhanced by the addition of albumin to the incubation mixture. More than 90% of the spermatozoa in a normal ejaculate give a reaction by this method. It reaches a maximum after 4 hours of incubation and does not increase further even up to 12 hours. No difference in the reaction between washed and unwashed ejaculated spermatozoa can be found. The nature of the proteolytic activity and its possible significance in infertility are discussed.

PMID:30709

Effect of medroxyprogesterone acetate contraception on cytoplasmic estrogen receptor content of the human cervix uteri.

Specimens of cervical tissue from five women each on 150 mg and 450 mg regimens of medroxyprogesterone acetate (MPA) for contraceptive purposes, were obtained through punch biopsy 15 days after injection. In another group of five women each on the same contraceptive regimens, punch biopsies of the cervix uteri were obtained 30 days after injection. These times corresponded to maximum and optimum blood levels of MPA respectively. Corresponding tissue from the same anatomical position in patients matched, where possible, for age and parity was obtained from hysterectomy specimens to serve as controls. Quantification of estrogen receptor content in the cytoplasm of these tissues was achieved through standard procedures. Analysis of data showed that MPA suppressed estrogen receptor content significantly compared to controls, but that there were no differences in this effect between the two dosages or time of biopsy.

Effect of medroxyprogesterone acetate contraception on cytoplasmic estrogen receptor content of the human cervix uteri. Specimens of cervical tissue from five women each on 150 mg and 450 mg regimens of medroxyprogesterone acetate (MPA) for contraceptive purposes, were obtained through punch biopsy 15 days after injection. In another group of five women each on the same contraceptive regimens, punch biopsies of the cervix uteri were obtained 30 days after injection. These times corresponded to maximum and optimum blood levels of MPA respectively. Corresponding tissue from the same anatomical position in patients matched, where possible, for age and parity was obtained from hysterectomy specimens to serve as controls. Quantification of estrogen receptor content in the cytoplasm of these tissues was achieved through standard procedures. Analysis of data showed that MPA suppressed estrogen receptor content significantly compared to controls, but that there were no differences in this effect between the two dosages or time of biopsy.

PMID:30711

The effect of norgestrel and clogestone on the spontaneous motility of the human fallopian tube.

Spontaneous isometric contractions in vitro of both isthmic and ampullary parts of the human fallopian tube were studied. The tubes were obtained during the early luteal phase of the menstrual cycle from patients undergoing abdominal hysterectomy and who had ingested clogestone acetate or norgestrel, either 1 or 12 hours prior to the experiment. A dose of 150 microgram dl-norgestrel given orally 12 hours preoperatively caused a significant increase in the frequency of spontaneous isometric contractions together with a significant reduction in the area under the curve for each contraction. The possible effect of this altered pattern of motility is discussed.

The effect of norgestrel and clogestone on the spontaneous motility of the human fallopian tube. Spontaneous isometric contractions in vitro of both isthmic and ampullary parts of the human fallopian tube were studied. The tubes were obtained during the early luteal phase of the menstrual cycle from patients undergoing abdominal hysterectomy and who had ingested clogestone acetate or norgestrel, either 1 or 12 hours prior to the experiment. A dose of 150 microgram dl-norgestrel given orally 12 hours preoperatively caused a significant increase in the frequency of spontaneous isometric contractions together with a significant reduction in the area under the curve for each contraction. The possible effect of this altered pattern of motility is discussed.

PMID:30712

Lactate and pyruvate utilization by the spermatozoa of infertile human males.

In studying the metabolic pathway of lactate and pyruvate in human spermatozoa from fertile and infertile subjects, pyruvate 1-C14 and lactate 1-C14 were used as substrates in a radiorespirometry system. Spermatozoa from patients with 20 +/- 2.0/10(6) spz/ml and decreased motility (group B) showed a more active decarboxylation of both pyruvate 1-C14 and lactate 1-C14 with a higher production of lactate C14, oxaloacetate, citrate, and isocitrate than those from fertile normal subjects (group A). Spermatozoa from fertile patients with normal counts but low motility (35 +/- 2.0% +++ motility) showed a similar rate of decarboxtlation of pyruvate 1-C14 but a greater rate of decarboxylation of lactate 1-C14 than those from fertile subjects. The low utilization of pyruvate by the spermatozoa from group C infertile patients, with intermediate counts and motility could be explained by a metabolic failure similar to that produced by some inhibitors of the respiratory chain. We do not have an explanation for the metabolic behavior observed in spermatozoa from group B patients, and further research on this point is desirable.

Lactate and pyruvate utilization by the spermatozoa of infertile human males. In studying the metabolic pathway of lactate and pyruvate in human spermatozoa from fertile and infertile subjects, pyruvate 1-C14 and lactate 1-C14 were used as substrates in a radiorespirometry system. Spermatozoa from patients with 20 +/- 2.0/10(6) spz/ml and decreased motility (group B) showed a more active decarboxylation of both pyruvate 1-C14 and lactate 1-C14 with a higher production of lactate C14, oxaloacetate, citrate, and isocitrate than those from fertile normal subjects (group A). Spermatozoa from fertile patients with normal counts but low motility (35 +/- 2.0% +++ motility) showed a similar rate of decarboxtlation of pyruvate 1-C14 but a greater rate of decarboxylation of lactate 1-C14 than those from fertile subjects. The low utilization of pyruvate by the spermatozoa from group C infertile patients, with intermediate counts and motility could be explained by a metabolic failure similar to that produced by some inhibitors of the respiratory chain. We do not have an explanation for the metabolic behavior observed in spermatozoa from group B patients, and further research on this point is desirable.

PMID:30713

Intrauterine device and pelvic inflammatory disease.

Using a retrospective case control design on 101 women with a first episode of acute pelvic inflammatory disease (PID), it was found that 15% were wearing an intrauterine device, as compared to 7% out of a control group of 101 women matched for age, marital status, and interval since their last pregnancy termination. No statistically significant correlation between IUD usage and PID was demonstrated. A significant correlation (P less than 0.01) between previous induced abortion and subsequent PID was found. In the PID group, a significantly higher proportion of previous abdominal and pelvic operations (P less than 0.005) was found as compared to the control group, but the numbers were small. In the absence of a higher frequency of IUD wearers among PID patients as compared with matched controls, we do not believe that there is an increased risk of pelvic inflammatory disease.

Intrauterine device and pelvic inflammatory disease. Using a retrospective case control design on 101 women with a first episode of acute pelvic inflammatory disease (PID), it was found that 15% were wearing an intrauterine device, as compared to 7% out of a control group of 101 women matched for age, marital status, and interval since their last pregnancy termination. No statistically significant correlation between IUD usage and PID was demonstrated. A significant correlation (P less than 0.01) between previous induced abortion and subsequent PID was found. In the PID group, a significantly higher proportion of previous abdominal and pelvic operations (P less than 0.005) was found as compared to the control group, but the numbers were small. In the absence of a higher frequency of IUD wearers among PID patients as compared with matched controls, we do not believe that there is an increased risk of pelvic inflammatory disease.

PMID:30714

Effect of kallikrein on bull sperm motility in vitro.

The application of kallikrein--a kinin-releasing porcine pancreatic kininogenase--exerted considerable stimulation of bull sperm motility in vitro. The kallikrein was used in two concentrations: 1 and 100 KU/ml semen. Both concentrations gave similar effect: at the 4th hour of incubation at room temperature the motility rate of spermatozoa exceeded five and four times, respectively, that of the control samples. Higher penetrating ability of bull spermatozoa in cow's estral secretion was also recorded in the experimental samples treated with kallikrein in both concentrations at the 2nd and 4th hour of incubation in comparison with the controls.

Effect of kallikrein on bull sperm motility in vitro. The application of kallikrein--a kinin-releasing porcine pancreatic kininogenase--exerted considerable stimulation of bull sperm motility in vitro. The kallikrein was used in two concentrations: 1 and 100 KU/ml semen. Both concentrations gave similar effect: at the 4th hour of incubation at room temperature the motility rate of spermatozoa exceeded five and four times, respectively, that of the control samples. Higher penetrating ability of bull spermatozoa in cow's estral secretion was also recorded in the experimental samples treated with kallikrein in both concentrations at the 2nd and 4th hour of incubation in comparison with the controls.

PMID:30715

Serum and seminal plasma prolactin levels in oligospermia.

Prolactin concentration was estimated by radioimmunoassay in seminal plasma and serum of 26 oligospermic and 23 normospermic men. No significant difference was found in the mean serum prolactin concentration between normospermic and oligospermic subjects, but there was an inverse correlation between serum prolactin level and sperm count within the group of oligospermic patients. Although the mean prolactin concentration in seminal plasma of oligospermic group was somewhat lower than in the normospermic group the difference did not reach statistical significance, and no correlation was found between seminal plasma prolactin concentration and sperm count within either group.

Serum and seminal plasma prolactin levels in oligospermia. Prolactin concentration was estimated by radioimmunoassay in seminal plasma and serum of 26 oligospermic and 23 normospermic men. No significant difference was found in the mean serum prolactin concentration between normospermic and oligospermic subjects, but there was an inverse correlation between serum prolactin level and sperm count within the group of oligospermic patients. Although the mean prolactin concentration in seminal plasma of oligospermic group was somewhat lower than in the normospermic group the difference did not reach statistical significance, and no correlation was found between seminal plasma prolactin concentration and sperm count within either group.

PMID:30716

A simple ambulatory method for removal of occult IUDs.

With the increased popularity of the IUD as a contraceptive method, there is a rise in the cases of occult IUDs as a result of the disappearance of the threads from the vagina. Our experience has shown that in the cases where the marker is missing, but the IUD is in the uterine cavity, it can be easily removed by using the Novak curette. This procedure spares the patients general anesthesia, D & C, and unnecessary hospitalization with its expenses.

A simple ambulatory method for removal of occult IUDs. With the increased popularity of the IUD as a contraceptive method, there is a rise in the cases of occult IUDs as a result of the disappearance of the threads from the vagina. Our experience has shown that in the cases where the marker is missing, but the IUD is in the uterine cavity, it can be easily removed by using the Novak curette. This procedure spares the patients general anesthesia, D & C, and unnecessary hospitalization with its expenses.

PMID:30717

Pure crystalline estradiol pellet implantation for contraception.

The subcutaneous implantation of estradiol pellets was found to be a simple and effective contraceptive method with good patient acceptance and minimal untoward effects. The pellets (25 mg each) were implanted through a Kearn's trocar into the abdominal wall, 2.5 to 5 cm above and parallel to Poupart's ligament. The regimen began with four pellets, and the dose was maintained or decreased by one pellet every 6 months (four, three, two, one). A potent progestogen was utilized monthly for induction of withdrawal bleeding. Altogether, 236 patients were followed for a total of 1,060 courses in 6,360 cycles (489,02 woman-years). Two pregnancies occurred during therapy. Pearl's index was 0.37. No significant alterations occurred in body weight and blood pressure. Glucose tolerance test, standard blood profiles, and Papanicolaou smears were normal during therapy. No cases of thrombophlebitis, blurred vision, headaches, gastric symptoms, or amenorrhea-galactorrhea were observed. The suppression of ovulation was confirmed by endometrial biopsies, basal body temperature, and serum follicle-stimulating hormone, luteinizing hormone, estradiol, and progesterone in a selected group of patients.

Pure crystalline estradiol pellet implantation for contraception. The subcutaneous implantation of estradiol pellets was found to be a simple and effective contraceptive method with good patient acceptance and minimal untoward effects. The pellets (25 mg each) were implanted through a Kearn's trocar into the abdominal wall, 2.5 to 5 cm above and parallel to Poupart's ligament. The regimen began with four pellets, and the dose was maintained or decreased by one pellet every 6 months (four, three, two, one). A potent progestogen was utilized monthly for induction of withdrawal bleeding. Altogether, 236 patients were followed for a total of 1,060 courses in 6,360 cycles (489,02 woman-years). Two pregnancies occurred during therapy. Pearl's index was 0.37. No significant alterations occurred in body weight and blood pressure. Glucose tolerance test, standard blood profiles, and Papanicolaou smears were normal during therapy. No cases of thrombophlebitis, blurred vision, headaches, gastric symptoms, or amenorrhea-galactorrhea were observed. The suppression of ovulation was confirmed by endometrial biopsies, basal body temperature, and serum follicle-stimulating hormone, luteinizing hormone, estradiol, and progesterone in a selected group of patients.

PMID:30718

Definition of period of induction of deciduoma in the rat using ornithine decarboxylase as a marker of growth onset.

The activity of uterine ornithine decarboxylase (ODC) was measured during the 24 hours after systemic induction of decidualization. Following a latent period of 2.5 hours, activity rose and reached a peak of 5.6 +/- 1.1 fold at 5 hours after induction. The activity then declined and a second, lower peak was seen at 21 hours. The increase in ODC activity was suppressed by treatment with cycloheximide (50 mg/kg) and dactinomycin, 500 microgram/rat, confirming that the enzyme response reflects protein synthesis. The increase and the base-line level of ODC activity were suppressed by ergocornine pretreatment (1 mg/rat) which disturbs the endocrine balance of pseudopregnancy. It was concluded that the growth and differentiation of decidual tissue began 2.5 hours after application of the induction stimulus.

Definition of period of induction of deciduoma in the rat using ornithine decarboxylase as a marker of growth onset. The activity of uterine ornithine decarboxylase (ODC) was measured during the 24 hours after systemic induction of decidualization. Following a latent period of 2.5 hours, activity rose and reached a peak of 5.6 +/- 1.1 fold at 5 hours after induction. The activity then declined and a second, lower peak was seen at 21 hours. The increase in ODC activity was suppressed by treatment with cycloheximide (50 mg/kg) and dactinomycin, 500 microgram/rat, confirming that the enzyme response reflects protein synthesis. The increase and the base-line level of ODC activity were suppressed by ergocornine pretreatment (1 mg/rat) which disturbs the endocrine balance of pseudopregnancy. It was concluded that the growth and differentiation of decidual tissue began 2.5 hours after application of the induction stimulus.

PMID:30719

Electrophoretic patterns of total, nuclear, and flagellar proteins from ejaculated human spermatozoa.

By means of selective solubilization methods and slab gel electrophoresis, reproducible patterns of 19, 37, and 56 protein bands were found to be associated with nuclear, "flagellar," and total human spermatozoa, respectively. Forty protein bands were found between the molecular weight of 12,400 to 160,000 daltons. Twelve bands were associated with values lower than 12,400 daltons. The nuclear major bands were located in a low molecular weight zone, while "flagellar" ones were located in a high molecular weight zone. None of these bands represents degradation products since a) in the solubilized samples neither acrosin, chymotrypsin, nor trypsin activities were present, b) in the presence of two protease inhibitors the same electrophoretic patterns were observed, and c) labelled globins added during sample manipulation were quantitatively recovered without degradation.

Electrophoretic patterns of total, nuclear, and flagellar proteins from ejaculated human spermatozoa. By means of selective solubilization methods and slab gel electrophoresis, reproducible patterns of 19, 37, and 56 protein bands were found to be associated with nuclear, "flagellar," and total human spermatozoa, respectively. Forty protein bands were found between the molecular weight of 12,400 to 160,000 daltons. Twelve bands were associated with values lower than 12,400 daltons. The nuclear major bands were located in a low molecular weight zone, while "flagellar" ones were located in a high molecular weight zone. None of these bands represents degradation products since a) in the solubilized samples neither acrosin, chymotrypsin, nor trypsin activities were present, b) in the presence of two protease inhibitors the same electrophoretic patterns were observed, and c) labelled globins added during sample manipulation were quantitatively recovered without degradation.

PMID:30720

Conservative correction of uterine anomalies in cases of congenital and posttraumatic infertility.

Uterine anomalies are due either to primary congenital malformations, or to secondary traumatic lesions of the intrauterine cavity as well as to pathology of the endometrium. The latter two etiologic factors create difficulties in the correct diagnosis of a congenital malformation and despite the convincing hysterosalpingographic findings a false diagnosis of a congenital malformation and despite the convincing hysterosalpingographic findings a false diagnosis is frequent. On the other hand the various degrees of uterine anomalies cannot always convince the gynecologist to undertake a plastic operation where the results for future fertility are doubtful. In our experience the extensive beneficial use of a variety of selected IUDs for the correction of intrauterine lesions also resulted in the correction of the size and shape of the uteri, previously diagnosed as malformed. The preliminary results of treatment in 110 cases of uterine anomalies after the application of a selected IUD combined with the administration of high doses of gestagens, showed an overall satisfactory improvement or complete reconstruction to a normal uterus in 86 (78%) of the cases. Higher fertility rate, better pregnancy outcome, correct diagnosis of the existing malformation, and safer decisions for further correction have also been attributed to the beneficial effects of the above treatment.

Conservative correction of uterine anomalies in cases of congenital and posttraumatic infertility. Uterine anomalies are due either to primary congenital malformations, or to secondary traumatic lesions of the intrauterine cavity as well as to pathology of the endometrium. The latter two etiologic factors create difficulties in the correct diagnosis of a congenital malformation and despite the convincing hysterosalpingographic findings a false diagnosis of a congenital malformation and despite the convincing hysterosalpingographic findings a false diagnosis is frequent. On the other hand the various degrees of uterine anomalies cannot always convince the gynecologist to undertake a plastic operation where the results for future fertility are doubtful. In our experience the extensive beneficial use of a variety of selected IUDs for the correction of intrauterine lesions also resulted in the correction of the size and shape of the uteri, previously diagnosed as malformed. The preliminary results of treatment in 110 cases of uterine anomalies after the application of a selected IUD combined with the administration of high doses of gestagens, showed an overall satisfactory improvement or complete reconstruction to a normal uterus in 86 (78%) of the cases. Higher fertility rate, better pregnancy outcome, correct diagnosis of the existing malformation, and safer decisions for further correction have also been attributed to the beneficial effects of the above treatment.

PMID:30721

Studies on biochemical characteristics of an early decidual protein.

Previous work in our laboratory has demonstrated the specific synthesis of a soluble protein (A) in the rat endometrium on day L4 of pseudopregnancy 1 h after the decidual induction; the A protein synthesis being higher in the antimesometrial region (AMR) than in the mesometrial one. We have now examined in the AMR of rat endometria on day L4 of pseudopregnancy: 1) the effect of cycloheximide (500 microgram, i.p. 30 min before decidual induction) on the biosynthesis of the A protein, and 2) the effect of actinomycin D (Act. D) (5 microgram/uterine horn 60 min before the decidual stimulation) on the induction of A protein synthesis. The synthesis of the A protein was determined by acrylamide gel electrophoresis of endometrial soluble proteins after double-labeling incorporation of amino acids. Results showed that: 1) the cycloheximide inhibited the synthesis of the A protein which indicates a de novo protein synthesis; 2) the Act. D inhibited the A synthesis suggesting that synthesis of an Act. D-sensitive product is one of the earliest macromolecular synthetic events after decidual induction.

Studies on biochemical characteristics of an early decidual protein. Previous work in our laboratory has demonstrated the specific synthesis of a soluble protein (A) in the rat endometrium on day L4 of pseudopregnancy 1 h after the decidual induction; the A protein synthesis being higher in the antimesometrial region (AMR) than in the mesometrial one. We have now examined in the AMR of rat endometria on day L4 of pseudopregnancy: 1) the effect of cycloheximide (500 microgram, i.p. 30 min before decidual induction) on the biosynthesis of the A protein, and 2) the effect of actinomycin D (Act. D) (5 microgram/uterine horn 60 min before the decidual stimulation) on the induction of A protein synthesis. The synthesis of the A protein was determined by acrylamide gel electrophoresis of endometrial soluble proteins after double-labeling incorporation of amino acids. Results showed that: 1) the cycloheximide inhibited the synthesis of the A protein which indicates a de novo protein synthesis; 2) the Act. D inhibited the A synthesis suggesting that synthesis of an Act. D-sensitive product is one of the earliest macromolecular synthetic events after decidual induction.

PMID:30722

Release rate of testosterone and estrogens from polydimethylsiloxane implants for extended periods in vivo compared with loss in vitro.

Release rates of testosterone, estrone, and estradiol placed in chambers made from polydimethylsiloxane (PDS) tubing (Dow Corning "Silastic," 3.35 mm ID x 4.65 mm OD) were studied in 14 freemartin cattle with minimal or non-detectable endogenous hormone secretion, and in 0.9% saline:methanol (1:1) baths shaken at 38 degree C. Eighty-seven implants, varying in length from 2 to 10 cm, were placed in 14 animals for 27 to 235 days. The average release rates +/- standard errors, in microgram/cm/day, were testosterone, 55.9 +/- 2.4, estrone, 12.6 +/- 1.8, and estradiol, 11.1 +/- 1.1. A relatively constant release rate was found over the period of time studied and sufficient steroid remained for potential release over periods exceeding 1 year. The dose of hormone delivered was sufficient to increase mounting activity in testosterone-treated animals and estrual activity in those receiving estrogens. Corresponding release rates in vitro for four 10-cm implants containing either testosterone, estrone, or estradiol were 94.3 +/- 1.9, 15.5 +/- 0.7, and 12.7 +/- 0.6 microgram/cm/day, respectively. The general magnitude of release rate in animals could be predicted from laboratory tests.

Release rate of testosterone and estrogens from polydimethylsiloxane implants for extended periods in vivo compared with loss in vitro. Release rates of testosterone, estrone, and estradiol placed in chambers made from polydimethylsiloxane (PDS) tubing (Dow Corning "Silastic," 3.35 mm ID x 4.65 mm OD) were studied in 14 freemartin cattle with minimal or non-detectable endogenous hormone secretion, and in 0.9% saline:methanol (1:1) baths shaken at 38 degree C. Eighty-seven implants, varying in length from 2 to 10 cm, were placed in 14 animals for 27 to 235 days. The average release rates +/- standard errors, in microgram/cm/day, were testosterone, 55.9 +/- 2.4, estrone, 12.6 +/- 1.8, and estradiol, 11.1 +/- 1.1. A relatively constant release rate was found over the period of time studied and sufficient steroid remained for potential release over periods exceeding 1 year. The dose of hormone delivered was sufficient to increase mounting activity in testosterone-treated animals and estrual activity in those receiving estrogens. Corresponding release rates in vitro for four 10-cm implants containing either testosterone, estrone, or estradiol were 94.3 +/- 1.9, 15.5 +/- 0.7, and 12.7 +/- 0.6 microgram/cm/day, respectively. The general magnitude of release rate in animals could be predicted from laboratory tests.

PMID:30727

Reversible sterilization.

The present techniques for sterilization of women consist of different methods to block the passage through the oviducts. The disadvantage of all techniques is that the possiblity of restoring fertility is very limited. The aim of the present study is to demonstrate a method of sterilization which is reversible.

Reversible sterilization. The present techniques for sterilization of women consist of different methods to block the passage through the oviducts. The disadvantage of all techniques is that the possiblity of restoring fertility is very limited. The aim of the present study is to demonstrate a method of sterilization which is reversible.

PMID:30728

The characterisation of thawed semen, and the timing and route of insemination associated with conception in the human.

The temporal and qualitative aspects of the conceptual process of a small number of pregnancies achieved by donor insemination have been analysed to understand the semen values required and the optimal insemination timing and route.

The characterisation of thawed semen, and the timing and route of insemination associated with conception in the human. The temporal and qualitative aspects of the conceptual process of a small number of pregnancies achieved by donor insemination have been analysed to understand the semen values required and the optimal insemination timing and route.

PMID:30729

The anti-reproductive pharmacology of LH-RH and agonistic analogues.

LH-RH and three particular ("super") analogues were evaluated for agonistic (ovulation-induction and short-term uterotrophic properties) and postcoital contraceptive activity in rodents. Additionally, LH-RH and/or a representative analogue (D-Ala6-des Gly10-Pro9-LH-RH ethylamide) were tested for postcoital contraceptive/vaginal smear/return to fertility effects, precoital contraceptive activity, and effects on puberty in the immature female. All compounds induced ovulation and uterotrophic effects and terminated pregnancy when administered either pre- or post-implantation. LH-RH and the representative analogue, while terminating pregnancy postcoitally, produced an associated break in the characteristic leucocytic vaginal smear of pregnancy to one of cornification by day 12; at this time mating and insemination were reestablished and all rats carried to normal term. Precoitally, LH-RH administered to nembutalized (but not to unblocked) rats produced a 50% reduction in the pregnancy rate and a 38% decrease in the number of viable pups delivered. In immature rats, the representative analogue delayed puberty (i.e. vaginal canalization) and retarded the growth of the ovaries, uteri, and anterior pituitary gland. The collective data strongly support the concept that LH-RH and agonistic derivatives, in spite of their putative pro-fertility classification, are characteristically antifertility by nature. Since the latter effect appears to be the paradoxically dominant one, it is suggested that LH-RH agonism is synonymous with contraception. Furthermore, such peptides may represent a new potential approach to fertility control.

The anti-reproductive pharmacology of LH-RH and agonistic analogues. LH-RH and three particular ("super") analogues were evaluated for agonistic (ovulation-induction and short-term uterotrophic properties) and postcoital contraceptive activity in rodents. Additionally, LH-RH and/or a representative analogue (D-Ala6-des Gly10-Pro9-LH-RH ethylamide) were tested for postcoital contraceptive/vaginal smear/return to fertility effects, precoital contraceptive activity, and effects on puberty in the immature female. All compounds induced ovulation and uterotrophic effects and terminated pregnancy when administered either pre- or post-implantation. LH-RH and the representative analogue, while terminating pregnancy postcoitally, produced an associated break in the characteristic leucocytic vaginal smear of pregnancy to one of cornification by day 12; at this time mating and insemination were reestablished and all rats carried to normal term. Precoitally, LH-RH administered to nembutalized (but not to unblocked) rats produced a 50% reduction in the pregnancy rate and a 38% decrease in the number of viable pups delivered. In immature rats, the representative analogue delayed puberty (i.e. vaginal canalization) and retarded the growth of the ovaries, uteri, and anterior pituitary gland. The collective data strongly support the concept that LH-RH and agonistic derivatives, in spite of their putative pro-fertility classification, are characteristically antifertility by nature. Since the latter effect appears to be the paradoxically dominant one, it is suggested that LH-RH agonism is synonymous with contraception. Furthermore, such peptides may represent a new potential approach to fertility control.

PMID:30730

The effect of ovarian wedge resection and incision on circulating gonadotropin in patients with polycystic ovarian disease.

Wedge resection (WR) was performed in 12 women with polycystic ovarian disease (PCOD), and Incision was done in 4 PCOD patients without any resection of ovarian tissue. Serum LH, FSH, estradiol-17beta (E2), progesterone, and urinary 17 ketosteroid (17KS) were measured serially before and after surgery. Neither WR nor Incision had any effect on FSH levels. Serum LH levels which had been hypergonadotropic preoperatively, became markedly lower 7--14 days after surgery in 12 wedge-resected and 2 incised patients. Within 7 days after WR there was a significant fall of E2 and a decrease of 17KS. In addition to those hormonal changes observed after WR, BBT charts turned out to be diphasic after the oral administration of dydrogesterone (Duphaston) in 12 out of 17 PCOD patients. The present data suggest that the reduction of the serum LH, induced by an interaction between the ovarian steroidogenesis and the suprapituitary mechanisms, might be involved in the occurrence of ovulation after WR in PCOD patients.

The effect of ovarian wedge resection and incision on circulating gonadotropin in patients with polycystic ovarian disease. Wedge resection (WR) was performed in 12 women with polycystic ovarian disease (PCOD), and Incision was done in 4 PCOD patients without any resection of ovarian tissue. Serum LH, FSH, estradiol-17beta (E2), progesterone, and urinary 17 ketosteroid (17KS) were measured serially before and after surgery. Neither WR nor Incision had any effect on FSH levels. Serum LH levels which had been hypergonadotropic preoperatively, became markedly lower 7--14 days after surgery in 12 wedge-resected and 2 incised patients. Within 7 days after WR there was a significant fall of E2 and a decrease of 17KS. In addition to those hormonal changes observed after WR, BBT charts turned out to be diphasic after the oral administration of dydrogesterone (Duphaston) in 12 out of 17 PCOD patients. The present data suggest that the reduction of the serum LH, induced by an interaction between the ovarian steroidogenesis and the suprapituitary mechanisms, might be involved in the occurrence of ovulation after WR in PCOD patients.

PMID:30732

Pharmacological treatment of obesity.

Obesity results when the ingestion of energy exceeds its utilization, leading to an excessive expansion of the adipose tissue mass. Current pharmacological therapy for the obese patient focuses primarily on reducing energy intake. Anorectic agents reduce food consumption by modifying central systems in the brain which are involved in appetite regulation. These agents are reviewed in terms of mechanism of action, and clinical safety and efficacy in suppressing appetite and promoting weight loss. Newer anorectic agents which are being evaluated currently in clinical and animal studies are described. Clinical assessments of therapeutic regimens utilizing the thyroid hormones and human chorionic gonadotropin are evaluated. Finally, an overview of novel pharmacological approaches to the treatment of obesity is presented.

Pharmacological treatment of obesity. Obesity results when the ingestion of energy exceeds its utilization, leading to an excessive expansion of the adipose tissue mass. Current pharmacological therapy for the obese patient focuses primarily on reducing energy intake. Anorectic agents reduce food consumption by modifying central systems in the brain which are involved in appetite regulation. These agents are reviewed in terms of mechanism of action, and clinical safety and efficacy in suppressing appetite and promoting weight loss. Newer anorectic agents which are being evaluated currently in clinical and animal studies are described. Clinical assessments of therapeutic regimens utilizing the thyroid hormones and human chorionic gonadotropin are evaluated. Finally, an overview of novel pharmacological approaches to the treatment of obesity is presented.

PMID:30734

The antimicrobial activity of urine of paraplegic patients receiving methenamine mandelate.

The antimicrobial activity of urine collected from adult male paraplegics ingesting methenamine mandelate (MM) was evaluated. The in vitro bacterial growth in urine from these patients was inhibited when the free formaldehyde (HCHO) concentration was 10 to 22 microgram per ml. When the HCHO concentration was in the region of 28 microgram per ml or greater, bactericidal effect became apparent. Urine containing 1323 microgram of MM per ml with a pH of 5.9 when freshly voided had sufficient HCHO to be bacteriostatic. Urine containing at least 1740 microgram of MM per ml with a pH of 5.1 or less when freshly voided was bactericidal. The latter concentration of MM in urine was usually achieved when the patient ingested 4 g of MM per day in divided doses and the intake of fluid was not excessive. Under some circumstances an individual receiving MM without an additional acidifying agent may produce urine with a pH low enough to release sufficient HCHO to exert a useful antibacterial effect. However, supplementary acidification with ammonium chloride produced marked lowering of the urinary pH in all patients receiving MM, resulting in bactericidal levels of HCHO.

The antimicrobial activity of urine of paraplegic patients receiving methenamine mandelate. The antimicrobial activity of urine collected from adult male paraplegics ingesting methenamine mandelate (MM) was evaluated. The in vitro bacterial growth in urine from these patients was inhibited when the free formaldehyde (HCHO) concentration was 10 to 22 microgram per ml. When the HCHO concentration was in the region of 28 microgram per ml or greater, bactericidal effect became apparent. Urine containing 1323 microgram of MM per ml with a pH of 5.9 when freshly voided had sufficient HCHO to be bacteriostatic. Urine containing at least 1740 microgram of MM per ml with a pH of 5.1 or less when freshly voided was bactericidal. The latter concentration of MM in urine was usually achieved when the patient ingested 4 g of MM per day in divided doses and the intake of fluid was not excessive. Under some circumstances an individual receiving MM without an additional acidifying agent may produce urine with a pH low enough to release sufficient HCHO to exert a useful antibacterial effect. However, supplementary acidification with ammonium chloride produced marked lowering of the urinary pH in all patients receiving MM, resulting in bactericidal levels of HCHO.

PMID:30735

Bladder surface mucin. Examination of possible mechanisms for its antibacterial effect.

We have previusly provided physiologic and histochemical data implicating the bladder surface mucin layer as an important new antibacterial defense mechanism. This mucin or its contents seems to act as an "antiadherence factor", inhibiting bacterial adherence to the bladder mucosa and thereby facilitating the removal of bacteria by the voiding process. The present study was designed to investigate three mechanisms by which the mucin might repel bacterial attachment. Our data suggest that neither IgA nor a chelating agent are anti-adherence factors. We did find, however, that pH had a significant effect on the adherence of bacteria to mucosal cells stripped of their mucin layer. This result suggest that electrochemical charge is important in bacterial adherence. We beleive that the mucin layer both provides an electrochemical coat on the bladder surface that is a poor substrate for bacterial adherence and blocks the receptor sites of the transitional cells to which the microbes might adhere.

Bladder surface mucin. Examination of possible mechanisms for its antibacterial effect. We have previusly provided physiologic and histochemical data implicating the bladder surface mucin layer as an important new antibacterial defense mechanism. This mucin or its contents seems to act as an "antiadherence factor", inhibiting bacterial adherence to the bladder mucosa and thereby facilitating the removal of bacteria by the voiding process. The present study was designed to investigate three mechanisms by which the mucin might repel bacterial attachment. Our data suggest that neither IgA nor a chelating agent are anti-adherence factors. We did find, however, that pH had a significant effect on the adherence of bacteria to mucosal cells stripped of their mucin layer. This result suggest that electrochemical charge is important in bacterial adherence. We beleive that the mucin layer both provides an electrochemical coat on the bladder surface that is a poor substrate for bacterial adherence and blocks the receptor sites of the transitional cells to which the microbes might adhere.

PMID:30733

Direct and reflex myocardial effects of intracoronary administered contrast materials in the anesthetized and conscious dog: comparison of standard and newer contrast materials.

The direct and indirect actions on left ventricular dynamics of contrast material (sodium meglumine diatrizoate) currently used for coronary arteriography, modified ionic material (sodium meglumine calcium metrizoate) and non-ionic material (metrizamide) were assessed in conscious and anesthetized dogs. In both anesthetized and conscious animals, the diatrizoate compound caused an early (3--10 sec after injection) decrease in peak dp/dt and dp/dt/LVP40, followed by late (10--20 sec after injection) increases in these variables. The predominant early and later effects of the calcium metrizoate compound were increases in parameters of LV contractile state. Metrizamide produced no significant early alterations, but later induced a small increase in these variables. The positive inotropic actions of each of the contrast materials were attenuated by beta adrenergic blockade. The early effects of the contrast materials were similar in the presence of segmental ischemia. The late positive inotropic effects in response to the diatrizoate compound and metrizamide were not observed in the ischemic state, while the positive inotropic response induced by the calcium metrizoate compound was significantly reduced. Thus intracoronary administration of sodium meglumine diatrizoate produced direct myocardial depression, followed by adrenergically mediated myocardial stimulation. Calcium metrizoate caused prominent direct and adrenergically mediated augmentation in contractile state. Metrizamide induced the least alteration in LV contractile state.

Direct and reflex myocardial effects of intracoronary administered contrast materials in the anesthetized and conscious dog: comparison of standard and newer contrast materials. The direct and indirect actions on left ventricular dynamics of contrast material (sodium meglumine diatrizoate) currently used for coronary arteriography, modified ionic material (sodium meglumine calcium metrizoate) and non-ionic material (metrizamide) were assessed in conscious and anesthetized dogs. In both anesthetized and conscious animals, the diatrizoate compound caused an early (3--10 sec after injection) decrease in peak dp/dt and dp/dt/LVP40, followed by late (10--20 sec after injection) increases in these variables. The predominant early and later effects of the calcium metrizoate compound were increases in parameters of LV contractile state. Metrizamide produced no significant early alterations, but later induced a small increase in these variables. The positive inotropic actions of each of the contrast materials were attenuated by beta adrenergic blockade. The early effects of the contrast materials were similar in the presence of segmental ischemia. The late positive inotropic effects in response to the diatrizoate compound and metrizamide were not observed in the ischemic state, while the positive inotropic response induced by the calcium metrizoate compound was significantly reduced. Thus intracoronary administration of sodium meglumine diatrizoate produced direct myocardial depression, followed by adrenergically mediated myocardial stimulation. Calcium metrizoate caused prominent direct and adrenergically mediated augmentation in contractile state. Metrizamide induced the least alteration in LV contractile state.

PMID:30738

[The hypertrophic obstructive cardiomyopathy (HOCM) of the newborn].

A case of a newborn infant with clinical and angiocardiographic signs of hypertrophic obstructive cardiomyopathy (HOCM) is presented. The baby died after a short therapy with beta-blockers. Light- and electron-microscopic investigations showed severe disorganization of muscular cellular arrangement and disturbances of intracellular structures of the interventricular septum. HOCM is a genetically determined disease which can present clinically in the newborn period and may simulate congenital cardiac malformations.

[The hypertrophic obstructive cardiomyopathy (HOCM) of the newborn]. A case of a newborn infant with clinical and angiocardiographic signs of hypertrophic obstructive cardiomyopathy (HOCM) is presented. The baby died after a short therapy with beta-blockers. Light- and electron-microscopic investigations showed severe disorganization of muscular cellular arrangement and disturbances of intracellular structures of the interventricular septum. HOCM is a genetically determined disease which can present clinically in the newborn period and may simulate congenital cardiac malformations.

PMID:30739

The effect of atebrine and an acridine analog (BCMA) on the coenzyme fluorescence spectra of cultured melanoma and Ehrlich ascites (EL2) cells.

Coenzyme fluorescence spectra of single living cells are due to free pyridine nucleotides (folded configuration), bound pyridine nucleotides (unfolded configuration) and a third component, possibly a mixture or flavins. Such spectra can be used to recognize possible differences in coenzyme composition between cell lines or changes of metabolic pathways due to chemicals acting at levels below or above cytotoxicity, by high resolution spectrofluorometry. A study of spectra recorded from cultured Ehrlich ascites (EL2), and Harding Passey melanoma cells (HPM-67 and HPM-73 line) grown under comparable conditions, shows that free NAD(P)H predominates in HPM-67 and EL2, while this coenzyme is bound in HPM-73. The free/bound ratio may be profoundly modifed by chemicals, e.g. in the HPM-73 increase of free and decrease of bound NAD(P)H occurred upon treatment with 10(-6) oligomycin. When atebrine at levels (10(-6) M) below cytotoxicity was added, there was a decrease of the free NAD(P)H spectrum possibly through energy transfer from NAD(P)H to atebrine. Consideration of long range energy transfer i.e., excitation of atebrine by fluorescence of NAD(P)H vs. short range transfer of excitation energy from free NAD(P)H to atebrine, favors the latter mechanism. A transient (reversible) increase in atebrine fluorescence is seen following intracellular microinjection of substrate (e.g. glucose-6-P) leading to an increase in free NAD(P)H. At cytotoxic levels of atebrine (e.g 2 x 10(-5) M) an irreversible increase of atebrine fluorescence is seen. The microspectrofluorometric technique appears therefore well suited to study physiological processes at the level of intracellular coenzymes, as well as possible processes of intermolecular energy transfer in the microenvironment.

The effect of atebrine and an acridine analog (BCMA) on the coenzyme fluorescence spectra of cultured melanoma and Ehrlich ascites (EL2) cells. Coenzyme fluorescence spectra of single living cells are due to free pyridine nucleotides (folded configuration), bound pyridine nucleotides (unfolded configuration) and a third component, possibly a mixture or flavins. Such spectra can be used to recognize possible differences in coenzyme composition between cell lines or changes of metabolic pathways due to chemicals acting at levels below or above cytotoxicity, by high resolution spectrofluorometry. A study of spectra recorded from cultured Ehrlich ascites (EL2), and Harding Passey melanoma cells (HPM-67 and HPM-73 line) grown under comparable conditions, shows that free NAD(P)H predominates in HPM-67 and EL2, while this coenzyme is bound in HPM-73. The free/bound ratio may be profoundly modifed by chemicals, e.g. in the HPM-73 increase of free and decrease of bound NAD(P)H occurred upon treatment with 10(-6) oligomycin. When atebrine at levels (10(-6) M) below cytotoxicity was added, there was a decrease of the free NAD(P)H spectrum possibly through energy transfer from NAD(P)H to atebrine. Consideration of long range energy transfer i.e., excitation of atebrine by fluorescence of NAD(P)H vs. short range transfer of excitation energy from free NAD(P)H to atebrine, favors the latter mechanism. A transient (reversible) increase in atebrine fluorescence is seen following intracellular microinjection of substrate (e.g. glucose-6-P) leading to an increase in free NAD(P)H. At cytotoxic levels of atebrine (e.g 2 x 10(-5) M) an irreversible increase of atebrine fluorescence is seen. The microspectrofluorometric technique appears therefore well suited to study physiological processes at the level of intracellular coenzymes, as well as possible processes of intermolecular energy transfer in the microenvironment.

PMID:30740

Dynamics of respiratory VT response to isocapnic pHa forcing in chemodenervated cats.

Arterial blood pH (pHa) was continuously monitored in decerebrate or pentobarbital-anesthetized cats with a rapidly responding hydrogen ion sensor inserted into the aorta. Alveolar carbon dioxide partial pressure and pHa were controlled independently during infusions of 1 N NaHCO3 or 0.5 N HCl into the inferior vena cava. Shifts in pHa up to 0.3 unit were effected isocapnically within 2.5-20 s over a working pHa range of 6.9-7.7. Before carotid sinus neurotomy, average onset latency of the tidal volume (VT) response to acid and alkaline pHa shifts was less than 5 S and the average VT response half time was less than 8.5 s regardless of whether the vagus nerves had been interrupted. After carotid sinus neurotomy, the deltaVT onset latency was approximately doubled, whereas the response half time was prolonged about eightfold on the average. Subsequent vagotomy tended further to increase the responding time lag. Nevertheless, the minimum response latency after peripheral chemodenervation was less than the deltapHa forcing rise time. It is concluded that the central chemoreceptors promptly sense pH change in the arterial blood and that neural processes adjust the time course of the respiratory response through the VT controller.

Dynamics of respiratory VT response to isocapnic pHa forcing in chemodenervated cats. Arterial blood pH (pHa) was continuously monitored in decerebrate or pentobarbital-anesthetized cats with a rapidly responding hydrogen ion sensor inserted into the aorta. Alveolar carbon dioxide partial pressure and pHa were controlled independently during infusions of 1 N NaHCO3 or 0.5 N HCl into the inferior vena cava. Shifts in pHa up to 0.3 unit were effected isocapnically within 2.5-20 s over a working pHa range of 6.9-7.7. Before carotid sinus neurotomy, average onset latency of the tidal volume (VT) response to acid and alkaline pHa shifts was less than 5 S and the average VT response half time was less than 8.5 s regardless of whether the vagus nerves had been interrupted. After carotid sinus neurotomy, the deltaVT onset latency was approximately doubled, whereas the response half time was prolonged about eightfold on the average. Subsequent vagotomy tended further to increase the responding time lag. Nevertheless, the minimum response latency after peripheral chemodenervation was less than the deltapHa forcing rise time. It is concluded that the central chemoreceptors promptly sense pH change in the arterial blood and that neural processes adjust the time course of the respiratory response through the VT controller.

PMID:30741

Slow postcapillary changes in blood pH in vivo: titration with acetazolamide.

A stopped-flow pH electrode apparatus was used to investigate the mechanisms underlying slow changes in plasma pH (pHO) after blood leaves the pulmonary capillaries in carbonic anhydrase-inhibited animals. After acetazolamide was administered to an anesthetized dog or cat, arterial blood was withdrawn through the electrode apparatus into a syringe. Syringe movement was then suddenly stopped. Temperature and pHO of the blood in the electrode chamber were monitored both before and after blood withdrawal ceased. After stopping flow, pHO of the blood in the electrode chamber a) rose 0.02 after a dose of about 1 mg/kg acetazolamide; b) did not change after a dose of about 2 mg/kg acetazolamide; and c) fell 0.10 after a dose greater than about 5 mg/kg acetazolamide. With reasonable red cell and plasma carbonic anhydrase activities assumed for each dose level of acetazolamide, a computer model of the reaction and transport processes occurring in blood after gas exchange in the lung yielded predicted time courses of pHo that were in good agreement with the experimental results. The observed slow pHo changes are largely a result of disequilibrium of [H+] between red blood cells and plasma as blood leaves the pulmonary capillaries.

Slow postcapillary changes in blood pH in vivo: titration with acetazolamide. A stopped-flow pH electrode apparatus was used to investigate the mechanisms underlying slow changes in plasma pH (pHO) after blood leaves the pulmonary capillaries in carbonic anhydrase-inhibited animals. After acetazolamide was administered to an anesthetized dog or cat, arterial blood was withdrawn through the electrode apparatus into a syringe. Syringe movement was then suddenly stopped. Temperature and pHO of the blood in the electrode chamber were monitored both before and after blood withdrawal ceased. After stopping flow, pHO of the blood in the electrode chamber a) rose 0.02 after a dose of about 1 mg/kg acetazolamide; b) did not change after a dose of about 2 mg/kg acetazolamide; and c) fell 0.10 after a dose greater than about 5 mg/kg acetazolamide. With reasonable red cell and plasma carbonic anhydrase activities assumed for each dose level of acetazolamide, a computer model of the reaction and transport processes occurring in blood after gas exchange in the lung yielded predicted time courses of pHo that were in good agreement with the experimental results. The observed slow pHo changes are largely a result of disequilibrium of [H+] between red blood cells and plasma as blood leaves the pulmonary capillaries.

PMID:30747

ATP hydrolysis and synthesis by the membrane-bound ATP synthetase complex of Methanobacterium thermoautotrophicum.

The membrane-bound ATP synthetase complex of Methanobacterium thermoautotrophicum showed maximum activity for ATP hydrolysis at pH 8, at temperatures between 65 and 70 degrees C, and at an ATP-Mg2+ ratio of 0.5. Anaerobic conditions were not prerequisite for enzyme activity. The enzyme showed a Km value for ATP of 2 mM, and activity was Mg2+ dependent; Mn2+, Co2+, Ca2+, and Zn2+ could replace Mg2+ to some extent. Other nucleoside triphosphates could be hydrolyzed. N,N'-dicyclohexylcarbodiimide inhibited ATP hydrolysis. A proton-motive force, artificially imposed by a pH shift or valinomycin, resulted in ATP synthesis in whole cells. The ATP synthetase complex of the thermophilic methanogenic bacterium is similar to those described in aerobic and anaerobic microorganisms.

ATP hydrolysis and synthesis by the membrane-bound ATP synthetase complex of Methanobacterium thermoautotrophicum. The membrane-bound ATP synthetase complex of Methanobacterium thermoautotrophicum showed maximum activity for ATP hydrolysis at pH 8, at temperatures between 65 and 70 degrees C, and at an ATP-Mg2+ ratio of 0.5. Anaerobic conditions were not prerequisite for enzyme activity. The enzyme showed a Km value for ATP of 2 mM, and activity was Mg2+ dependent; Mn2+, Co2+, Ca2+, and Zn2+ could replace Mg2+ to some extent. Other nucleoside triphosphates could be hydrolyzed. N,N'-dicyclohexylcarbodiimide inhibited ATP hydrolysis. A proton-motive force, artificially imposed by a pH shift or valinomycin, resulted in ATP synthesis in whole cells. The ATP synthetase complex of the thermophilic methanogenic bacterium is similar to those described in aerobic and anaerobic microorganisms.

PMID:30748

Enchancement of streptococcal transformation yield by proteolytic enzymes.

Trypsin and other proteolytic enzymes, added together with transforming DNA or during cell-DNA contact to competent cultures of several streptococcal strains, enchanced (10 to 600%) the yield of genetic transformation (stimulation). With few exceptions, the level of stimulation was high (over 100%) when competence was low (below 2%). Stimulation was caused by the action of an enzyme on competent cells and not on any other component of transformation mixture. The phenomenon occurred when the enzyme was added to the culture not earlier than 7 min before and not later than 5 min after the period of cell-DNA contact. The presence of trypsin during cell-DNA contact caused: (i) the alterations at cell surface, demonstrated by electron microscopy, increased release of 3H-amino acid-labeled material, and higher cell susceptibility to autolysis; (ii) the increase of both total and irreversible binding of DNA by the cells; and (iii) the decrease of early nucleolytic degradation of DNA by cells. These and other data point to the importance of a delicate balance of recipient cell's surface nuclease activities in the effectiveness of transformation process. It is also possible that trypsin eliminates an unknown cellular factor which obstructs DNA-cell receptors interaction.

Enchancement of streptococcal transformation yield by proteolytic enzymes. Trypsin and other proteolytic enzymes, added together with transforming DNA or during cell-DNA contact to competent cultures of several streptococcal strains, enchanced (10 to 600%) the yield of genetic transformation (stimulation). With few exceptions, the level of stimulation was high (over 100%) when competence was low (below 2%). Stimulation was caused by the action of an enzyme on competent cells and not on any other component of transformation mixture. The phenomenon occurred when the enzyme was added to the culture not earlier than 7 min before and not later than 5 min after the period of cell-DNA contact. The presence of trypsin during cell-DNA contact caused: (i) the alterations at cell surface, demonstrated by electron microscopy, increased release of 3H-amino acid-labeled material, and higher cell susceptibility to autolysis; (ii) the increase of both total and irreversible binding of DNA by the cells; and (iii) the decrease of early nucleolytic degradation of DNA by cells. These and other data point to the importance of a delicate balance of recipient cell's surface nuclease activities in the effectiveness of transformation process. It is also possible that trypsin eliminates an unknown cellular factor which obstructs DNA-cell receptors interaction.

PMID:30749

Degradation of 4-hydroxyphenylacetic acid by Trichosporon cutaneum.

Trichosporon cutaneum degraded 4-hydroxyphenylacetic acid to acetoacetic and malic acids. 3.4-Dihydroxyphenylacetic acid, an intermediate in the reaction sequence, underwent hydroxylation before the benzene ring was cleaved.

Degradation of 4-hydroxyphenylacetic acid by Trichosporon cutaneum. Trichosporon cutaneum degraded 4-hydroxyphenylacetic acid to acetoacetic and malic acids. 3.4-Dihydroxyphenylacetic acid, an intermediate in the reaction sequence, underwent hydroxylation before the benzene ring was cleaved.

PMID:30750

Coordination chemistry of microbial iron transport compounds: rhodotorulic acid and iron uptake in Rhodotorula pilimanae.

The mechanism by which iron uptake is facilitated by the siderophore rhodotorulic acid (RA) in the yeast Rhodotorula pilimanae was investigated with radioactively labeled Fe and RA and kinetically inert, chromic-substituted RA complexes. The weight of the evidence supports a model in which RA mediates iron transport to the cell but does not actually transport iron into the cell. It is proposed that RA exchanges the ferric ion at the cell surface with a membrane-bound chelating agent that completes the active transport of iron into the cell. Uptake of 55Fe in ferric rhodotorulate was much more rapid than uptake of RA itself. Two exchange-inert chromic complexes of RA showed no uptake.

Coordination chemistry of microbial iron transport compounds: rhodotorulic acid and iron uptake in Rhodotorula pilimanae. The mechanism by which iron uptake is facilitated by the siderophore rhodotorulic acid (RA) in the yeast Rhodotorula pilimanae was investigated with radioactively labeled Fe and RA and kinetically inert, chromic-substituted RA complexes. The weight of the evidence supports a model in which RA mediates iron transport to the cell but does not actually transport iron into the cell. It is proposed that RA exchanges the ferric ion at the cell surface with a membrane-bound chelating agent that completes the active transport of iron into the cell. Uptake of 55Fe in ferric rhodotorulate was much more rapid than uptake of RA itself. Two exchange-inert chromic complexes of RA showed no uptake.

PMID:30751

Partial purification and properties of CTP:phosphatidic acid cytidylyltransferase from membranes of Escherichia coli.

The cytosine liponucleotides CDP-diglyceride and dCDP-diglyceride are key intermediates in phospholipid biosynthesis in Escherichia coli (C. R. H. Raetz and E. P. Kennedy, J. Biol. Chem. 248:1098--1105, 1973). The enzyme responsible for their synthesis, CTP:phosphatidic acid cytidylytransferase, was solubilized from the cell envelope by a differential extraction procedure involving the detergent digitonin and was purified about 70-fold (relative to cell-free extracts) in the presence of detergent. In studies of the heat stability of the enzyme, activity decayed slowly at 63 degrees C. Initial velocity kinetic experiments suggested a sequential, rather than ping-pong, reaction mechanism; isotopic exchange reaction studies supported this conclusion and indicated that inorganic pyrophosphate is released before CDP-diglyceride in the reaction sequence. The enzyme utilized both CTP and dCTP as nucleotide substrate for the synthesis of CDP-diglyceride and dCDP-diglyceride, respectively. No distinction was observed between CTP and dCTP utilization in any of the purification, heat stability, and reaction mechanism studies. In addition, CTP and dCTP were competitive substrates for the partially purified enzyme. It therefore appears that a single enzyme catalyzes synthesis of both CDP-diglyceride and dCDP-diglyceride in E. coli. The enzyme also catalyzes a pyrophosphorolysis of CDP-diglyceride, i.e., the reverse of its physiologically important catalysis.

Partial purification and properties of CTP:phosphatidic acid cytidylyltransferase from membranes of Escherichia coli. The cytosine liponucleotides CDP-diglyceride and dCDP-diglyceride are key intermediates in phospholipid biosynthesis in Escherichia coli (C. R. H. Raetz and E. P. Kennedy, J. Biol. Chem. 248:1098--1105, 1973). The enzyme responsible for their synthesis, CTP:phosphatidic acid cytidylytransferase, was solubilized from the cell envelope by a differential extraction procedure involving the detergent digitonin and was purified about 70-fold (relative to cell-free extracts) in the presence of detergent. In studies of the heat stability of the enzyme, activity decayed slowly at 63 degrees C. Initial velocity kinetic experiments suggested a sequential, rather than ping-pong, reaction mechanism; isotopic exchange reaction studies supported this conclusion and indicated that inorganic pyrophosphate is released before CDP-diglyceride in the reaction sequence. The enzyme utilized both CTP and dCTP as nucleotide substrate for the synthesis of CDP-diglyceride and dCDP-diglyceride, respectively. No distinction was observed between CTP and dCTP utilization in any of the purification, heat stability, and reaction mechanism studies. In addition, CTP and dCTP were competitive substrates for the partially purified enzyme. It therefore appears that a single enzyme catalyzes synthesis of both CDP-diglyceride and dCDP-diglyceride in E. coli. The enzyme also catalyzes a pyrophosphorolysis of CDP-diglyceride, i.e., the reverse of its physiologically important catalysis.

PMID:30752

Effects of temperature on the autolytic enzyme system of Streptococcus faecalis.

The cellular autolytic reaction system in Streptococcus faecalis ATCC 9790 was analyzed for relative increases in reaction rates with increasing temperature by determination of Arrhenius activation energies (E). The systems examined were: (i) an isolated wall-enzyme complex in 0.01 M sodium phosphate, pH 6.9; (ii) exponential-phase cells suspended in 0.01 or o.3 M sodium phosphate pH 6.8, or in 0.04 M ammonium acetate, pH 6.8, (iii) growing cultures deprived of glucose or lysine; and (iv) cultures treated in growth media with the nonionic detergent, Triton X-100. For detergent-treated cells, E values were between 23.9 and 27.4 kcal/mol (ca. 100.1 to 174.7 kJ/mol) at concentrations of Triton X-100 between about 0.03 and 0.072 mg/ml. E values dropped sharply to 11.5 to 13.0 kcal/m-l (ca. 48.2 to 54.4 kJ/mol) at Triton X-100 concentrations of 0.12 mg/ml or higher. For the remaining systems, E values ranged from 16 to 20 kcal/mol (ca. 67.0 to 83.7 kJ/mol) (wall lysis, cellular autolysis in 0.01 M sodium phosphate or in 0.04 M ammonium acetate, and autolysis of glucose-starved cells) to 31 to 38 kcal/mol (ca 129.8 to 159.1 kJ/mol) (cellular autolysis in 0.3 M sodium phosphate or autolysis of lysine-starved cells). High concentrations of Triton X-100 appear to lower the E values below the 16 to 20 kcal/mol observed for the autolysis of isolated walls. This effect may be related to disruption by the detergent of a hydrophobic complex regulating cellular autolysis in vivo.

Effects of temperature on the autolytic enzyme system of Streptococcus faecalis. The cellular autolytic reaction system in Streptococcus faecalis ATCC 9790 was analyzed for relative increases in reaction rates with increasing temperature by determination of Arrhenius activation energies (E). The systems examined were: (i) an isolated wall-enzyme complex in 0.01 M sodium phosphate, pH 6.9; (ii) exponential-phase cells suspended in 0.01 or o.3 M sodium phosphate pH 6.8, or in 0.04 M ammonium acetate, pH 6.8, (iii) growing cultures deprived of glucose or lysine; and (iv) cultures treated in growth media with the nonionic detergent, Triton X-100. For detergent-treated cells, E values were between 23.9 and 27.4 kcal/mol (ca. 100.1 to 174.7 kJ/mol) at concentrations of Triton X-100 between about 0.03 and 0.072 mg/ml. E values dropped sharply to 11.5 to 13.0 kcal/m-l (ca. 48.2 to 54.4 kJ/mol) at Triton X-100 concentrations of 0.12 mg/ml or higher. For the remaining systems, E values ranged from 16 to 20 kcal/mol (ca. 67.0 to 83.7 kJ/mol) (wall lysis, cellular autolysis in 0.01 M sodium phosphate or in 0.04 M ammonium acetate, and autolysis of glucose-starved cells) to 31 to 38 kcal/mol (ca 129.8 to 159.1 kJ/mol) (cellular autolysis in 0.3 M sodium phosphate or autolysis of lysine-starved cells). High concentrations of Triton X-100 appear to lower the E values below the 16 to 20 kcal/mol observed for the autolysis of isolated walls. This effect may be related to disruption by the detergent of a hydrophobic complex regulating cellular autolysis in vivo.

PMID:30753

Transformation in pneumococcus: protein content of eclipse complex.

A two-step purification of pneumococcal eclipse complex is described, which uses sucrose gradient sedimentation followed by agarose gel permeation chromatography. Purified complex contains, in addition to donor DNA single strands, macromolecular material that can be labeled with methionine or leucine during development of competence. This material co-chromatographed with eclipse complex DNA on hydroxylapatite, was dissociated from the DNA by sodium dodecyl sulfate, and was completely digested by Pronase. The sodium dodecyl sulfate-released material eluted as a single peak in sodium dodecyl sulfate chromatography. These properties were consistent with the noncovalent association with eclipse complex of a protein or class of proteins with a narrow range of polypeptide sizes. Evidence for the specific association of this protein with transforming DNA is eclipse was also obtained from parallel purification from 35S-labeled nontransformed cells; the amount of methionine label in the corresponding fractions in such cells was only 5% of that in transformed cells.

Transformation in pneumococcus: protein content of eclipse complex. A two-step purification of pneumococcal eclipse complex is described, which uses sucrose gradient sedimentation followed by agarose gel permeation chromatography. Purified complex contains, in addition to donor DNA single strands, macromolecular material that can be labeled with methionine or leucine during development of competence. This material co-chromatographed with eclipse complex DNA on hydroxylapatite, was dissociated from the DNA by sodium dodecyl sulfate, and was completely digested by Pronase. The sodium dodecyl sulfate-released material eluted as a single peak in sodium dodecyl sulfate chromatography. These properties were consistent with the noncovalent association with eclipse complex of a protein or class of proteins with a narrow range of polypeptide sizes. Evidence for the specific association of this protein with transforming DNA is eclipse was also obtained from parallel purification from 35S-labeled nontransformed cells; the amount of methionine label in the corresponding fractions in such cells was only 5% of that in transformed cells.

PMID:30754

Salmonella typhimurium LT-2 mutants with altered glutamine synthetase levels and amino acid uptake activities.

To determine whether Salmonella typhimurium has a nitrogen control response, we have examined the regulation of nitrogen utilization in two mutants with fivefold and threefold elevations in their glutamine synthetase activities. The mutants do not require glutamine for growth on glucose--ammonia medium but do have altered growth on other nitrogen sources. They grow better than an isogenic control on media containing arginine or asparate, but more slowly with proline or alanine as nitrogen sources. This unusual growth pattern is not due to altered regulation of the ammonia assimilatory enzymes, glutamate dehydrogenase and glutamate synthase, or to changes in the enzymes for aspartate degradation. However, transport for several amino acids may be affected. Measurement of amino acid uptake show that the mutants with high glutamine synthetase levels have increased rates for glutamine, arginine, aspartate, and lysine, but a decreased rate for proline. The relationship between glutamine synthetase levels and uptake was examined in two mutants with reduced, rather than increased, glutamine synthetase production. The uptake rates for glutamine and lysine were lower in these two glutamine auxotrophs than in the Gln+ controls. These results show a correlation between the glutamine synthetase levels and the uptake rates for several amino acids. In addition, the pleiotropic growth of the mutants with elevated glutamine synthetase activities suggests that a nitrogen control response exists for S. typhimurium and that it can be altered by mutations affecting glutamine synthetase regulation.

Salmonella typhimurium LT-2 mutants with altered glutamine synthetase levels and amino acid uptake activities. To determine whether Salmonella typhimurium has a nitrogen control response, we have examined the regulation of nitrogen utilization in two mutants with fivefold and threefold elevations in their glutamine synthetase activities. The mutants do not require glutamine for growth on glucose--ammonia medium but do have altered growth on other nitrogen sources. They grow better than an isogenic control on media containing arginine or asparate, but more slowly with proline or alanine as nitrogen sources. This unusual growth pattern is not due to altered regulation of the ammonia assimilatory enzymes, glutamate dehydrogenase and glutamate synthase, or to changes in the enzymes for aspartate degradation. However, transport for several amino acids may be affected. Measurement of amino acid uptake show that the mutants with high glutamine synthetase levels have increased rates for glutamine, arginine, aspartate, and lysine, but a decreased rate for proline. The relationship between glutamine synthetase levels and uptake was examined in two mutants with reduced, rather than increased, glutamine synthetase production. The uptake rates for glutamine and lysine were lower in these two glutamine auxotrophs than in the Gln+ controls. These results show a correlation between the glutamine synthetase levels and the uptake rates for several amino acids. In addition, the pleiotropic growth of the mutants with elevated glutamine synthetase activities suggests that a nitrogen control response exists for S. typhimurium and that it can be altered by mutations affecting glutamine synthetase regulation.

PMID:30755

Benzeneboronic acid selectively inhibits sporulation of Bacillis subtilis.

m-Aminobenzeneboronic acid at levels of 0.2 mM in nutrient broth medium selectively inhibited sporulation without appreciably altering vegetative growth. Significant inhibitory effects were seen even when it was added as late as 6 h after the end of logarithmic growth. The pH changes associated with growth and sporulation of Bacillus subtilis in nutrient broth were not significantly altered by the inhibitor. When it was present in cultures of actively growing cells, its inhibitory effect could not be reversed by simple dilution. The compound caused extensive clumping, of cells, which appeared not to be related to the ability of boronates to esterify to diols.

Benzeneboronic acid selectively inhibits sporulation of Bacillis subtilis. m-Aminobenzeneboronic acid at levels of 0.2 mM in nutrient broth medium selectively inhibited sporulation without appreciably altering vegetative growth. Significant inhibitory effects were seen even when it was added as late as 6 h after the end of logarithmic growth. The pH changes associated with growth and sporulation of Bacillus subtilis in nutrient broth were not significantly altered by the inhibitor. When it was present in cultures of actively growing cells, its inhibitory effect could not be reversed by simple dilution. The compound caused extensive clumping, of cells, which appeared not to be related to the ability of boronates to esterify to diols.