Datasets:

andersonbcdefg

/

pubmed_pairs_part2

like 0

[Left ventricular function in hypertension treated with beta blockers].

In 14 patients with essential hypertension, left ventricular function was assessed echocardiographically before and after 4 and 8 weeks of treatment with the betablocking agent atenolol. Atenolol was given orally in a dose of 100 mg/day. After 4 weeks of treatment systolic blood pressure decreased from 160 to 138 mm Hg(p less than 0.001) and diastolic pressure from 105 to 91 mm Hg(p less than 0.001). Heart rate decreased from 76 to 64 beats/min (p less than 0.05). Systolic shortening of the left ventricular transverse diameter declined from 41 to 36% (p less than 0.01), though in no instance did it fall below the lower limit of normality (30%). After 8 weeks of betablocking therapy, blood pressure and heart rate remained essentially unchanged. Systolic shortening increased slightly but insignificantly to 38%. The left ventricular enddiastolic diameter did not change throughout the study. It is concluded that longterm betablocking therapy is associated with a significant reduction of left ventricular function which improves in the later stage of treatment. Since the diminution of left ventricular function is slight, the induction of left heart decompensation is unlikely, at any rate in patients with initially normal left ventricular function.

The preparation of pregnancy urine for an estrogen profile.

A method is described for purifying the estrogen content of pregnancy urine with little loss of the labile estrogens. The procedure makes use of the initial 50-fold purification effected by their precipitation whith ammonium sulphate, with simultaneous elimination of most urinary corticosteroids and 50--60% of urinary ketosteroids. It also employs the antioxident ascorbic acid as an additive in most stages of the procedure. The mild organic-solvent-HIO partition system of Brown is used for separating the strongly polar, 2including all "labile" estrogens, and of the weakly polar estrogens, from neutral steroids. The remaining neutral steroid still interfering with the assays were removed by an ascorbic acid treated ion exchange resin (AG 1). The final residues were revealed by mass-spectroscopy to consist almost solely of estrogens. Gas-liquid chromatography in which just 2 chromatograms are required yields a total of 12 "estrogen" peaks (for 12 estrogens which are excreted in amounts greater than 0.1 mg/day) in normal pregnancy urine, including all the known labile estrogens. Identification as estrogen for all but a few minor peaks of the gas chromatogram was obtained by mass-spectroscopy. The practical significance of the method lies in the fact that some labile estrogens are much more important in the estrogen metabolism of pregnant and nonpregnant women than heretofore generally thought.

[Abiotic synthesis of peptides containing a dicarboxylic acid and their catalytic properties].

Studies have been made on thermal synthesis of peptides containing amino acids (glycine, alanine, leucine, threonine, and histidine) and succinic acid. The synthetic products were found to be heterogeneous, comprising mostly linear polymers. In the peptides obtained, the ratio between individual amino acids differed from the initial one. It was demonstrated that glycine exhibits high capacity for polymerization. At the same time, most polymerized peptides contained large amounts of leucine. In hydrolyzates of some of the peptides, a new non-identified amino acid was found which was formed from threonine. The peptides obtained exhibited catalytic properties: they hydrolyzed-p-nitrophenyl-phosphate, increased the breakdown of ascorbic acid by hydrogen peroxide and splitted inorganic phosphate from ATP. These activities depended on PH of the medium, the duration of action and substrate concentration. Synthetic products which contained threonine residues, exhibited higher activities as compared to other peptides. The data obtained reveal a possibility of incorporating the organic acids into polypeptide chains during abiogenic synthesis of biologically active substances. Such compounds with non-specific catalytic properties could serve as one of the steps in evolution of biocatalyzers.

Anti-inflammatory drug actions on allergic responses in guinea-pig skin.

Five non-steroidal anti-inflammatory drugs (indomethacin, naproxen, meclofenamic acid, feprazone and phenylbutazone: NSAIDs) and three glucocorticosteroids (dexamethasone, hydrocortisone and prednisolone) have been tested as local inhibitors of increased vascular permeability in guinea-pig skin. Lesions were induced by histamine or by antigen to evoke type I (passive cutaneous anaphylaxis), type III (reverse passive Arthus) and type IV (delayed hypersensitivity) allergic reactions. NSAIDs and glucocorticosteroids caused either weak, inconsistent inhibition or slight, high-dose inhibition of the response to histamine. None of the drugs tested showed significant inhibition of the type IV response. The NSAIDs caused dose-related inhibition of both type I and type III responses whereas glucocorticosteroids were ineffective. Maximum inhibition with the NSAIDs was never greater than 50--60% Feprazone, meclofenamic acid and indomethacin were the most potent inhibitors of histamine, PCA and Arthus responses respectively. The possible significance of the effects of these anti-inflammatory agents on vascular permeability is discussed.

Thymic epithelial injury in graft-versus-host reactions following adrenalectomy.

In four separate experiments 140 adults A(H-2a) x C57BL/6(H-2b) F1hybrid mice were surgically adrenalectomized and divided into three experimental groups. Seventy-one additional adult F1hybrids (AXC57BL/6) which had not been adrenalectomized were divided into three similar groups. In Group 1 (GvH group), GvH reactions were induced by the injection of 50 x 106 pooled parental lymphoid cells intravenously. The second group (syngeneic group) received 50 x 106 pooled F1 hybrid lymphoid cells intravenously. The third group (uninoculated group) received no lymphoid inoculum. At regular intervals the animals were killed, autopsied, and histologically studied. Visceral alterations of GvH reaction were recorded in the thymus, lymph nodes, spleen, and liver in the GvH groups; none was present in the other groups. The thymuses in the nonadrenalectomized GvH group underwent prompt involution characterized by size reduction and cortical lymphoid cell depletion. These changes were not apparent in the GvH adrenalectomized group. Both GvH groups, however, demonstrated an effacement of the medulla, lymphocyte incursion into the medulla, lymphocyte emperipolesis of medullary epithelial cells, gradual disappearance of Hassall's corpuscles, epithelial cell injury, and an ingress of macrophages laden with nuclear and cellular debris. This study suggests that the stress and corticosteroid response which accompany a GvH reaction account for the reduction in the thymic size and cortical lymphoid cell mass. The medullary alterations, therefore, would appear to be initiated by the GvH reaction per se.

[Moderate doses of urokinase (UK) in the treatment of myocardial infarct and pulmonary embolism].

Since 1972, UK in moderate doses have been used in the treatment of severe or massive pulmonary emboli (PE) and of myocardial infarction (MI) present for less than 24 hours. The standard dose is 2,700,000 CTA units per 24 hours administered as a continuous infusion, in association with appropriate heparin therapy and a platelet anti-aggregant agent in order to palliate the hyperagregant effects of thrombolytic drugs. Laboratory surveillance has now been greatly simplified and is limited to that of the associated heparin therapy. In the acute phase of myocardial infarction, a personal randomised study of 120 cases consisting of 60 treated with heparin + UK and 60 with heparin alone showed that UK decreased mortality, cardiac arrhythmias and cardiac failure. Comparative studies at lower doses have failed to show any significant difference between the two groups of patients treated and the authors feel that the use of UK should be reserved for very recent infarctions in young subjects. In PE, the effectiveness of UK was assessed in 180 severe cases. It depended upon the length of time for which the thrombus had been present. Before the 5th day, there was early average revascularisation of 40 p. 100 of the avascular territory. Mortality was reduced to 15 p. 100 and at the 3rd week 32 p. 100 of the survivors had complete revascularisation, and 68 p. 100 partial but adequate revascularisation. Adjuvant therapy such as a combination of Lysil Plasminogen and/or defibrinating agent currently make it possible to reinforce therapeutic thrombolysis.

Niemann-Pick disease type D: lipid analyses and studies on sphingomyelinases.

Lipids and sphingomyelinase activity were studied in spleen, liver, and brain tissues of a 13-year-old boy with Niemann-Pick disease type D (NPD-D). The greatest lipid changes occurred in spleen; cholesterol, cholesterol esters, total phospholipids, sphingomyelin, and bis-(monoacylglyceryl)phosphate were increased above normal range. In liver, striking increases were observed in cholesterol and bis-(monoacylglyceryl)phosphate. Minor changes in neutral and acidic glycolipid patterns occurred in liver, spleen, and brain. Sphingomyelinase activity (optimal at pH 5.0) was elevated above mean control levels in liver and spleen, but not in brain, kidney, or leukocytes. Enzyme properties were generally normal. Activity of NPD-D liver crude homogenate, but not that of normal liver homogenates, was inhibited at high protein concentrations. Activity levels of a second sphingomyelinase, optimal at pH 7.4, in NPD-D brain were apparently normal. These findings are generally consistent with the classification of NPD-D as a sphingomyelin lipidosis.

Inosine nucleosidase from Azotobacter vinelandii. Purification and properties.

An enzyme catalyzing the hydrolysis of purine nucleosides was found to occur in the extract of Azotobacter vinelandii, strain O, and was highly purified by ammonium sulfate fractionation, DEAE-cellulose chromatography, hydroxylapatite chromatography and gel filtration on Sephadex G-150. A strict substrate specificity of the purified enzyme was shown with respect to the base components. The enzyme specifically attacked the nucleosides without amino groups in the purine moiety: inosine gave the maximum rate of hydrolysis and xanthosine was hydrolyzed to a lesser extent. The pH optimum of inosine hydrolysis was observed from pH 7 to 9, while xanthosine was hydrolyzed maximally at pH 7. The Km values of the enzyme for inosine were 0.65 and 0.85 mM at pH 7.1 and 9.0, respectively, and the value for xanthosine was 1.2 mM at pH 7.1. Several nucleotides inhibited the enzyme: the phosphate portions of the nucleotides were suggested to be responsible for the inhibition by nucleotides. Although the inhibition of the enzyme by nucleotides was apparently non-competitive type with respect to inosine, allosteric (cooperative) binding of the substrate was suggested in the presence of the inhibitor. The physiological significance of the enzyme was discussed in connection with the degradation and salvage pathways of purine nucleotides.

Premedication with lorazepam for bronchoscopy under general anaesthesia.

Lorazepam 3 or 4 mg i.m. was given to 100 patients as premedication before bronchoscopy under thiopentone-suxamethonium anaesthesia. Forty-nine of the patients assessed as anxious received oral lorazepam as preoperative night sedation also. Lorazepam was an effective night sedative. Forty-two of the 49 patients slept well and were calm and co-operative in the morning. Following the i.m. injection of lorazepam, 64% of patients had complete lack of recall for 4--10 h following premedication. Only 5% recalled correctly a simple objective test of memory initiated in the anaesthetic room. The frequency of recall was higher in those who consumed alcohol regularly and in females. There was one case of awareness during bronchoscopy in a patient who received only a small dose of lorazepam (2.8 mg per 70 kg). Side-effects were minimal and patient acceptance was impressive. These results show an advance on previous studies using pethidine and diazepam. Further improvement is needed, particularly in adjusting the dose of lorazepam to body weight and to factors such as age, sex and alcohol intake.

Interaction of pyridoxal 5-phosphate with apo-serine hydroxymethyltransferase.

The interaction of pyridoxal 5-phosphate with beef liver serine hydroxymethyltransferase (5,10-methylenetetrahydrofolate:glycine hydroxymethyltransferase, EC 2.1.2.1) has been investigated using sedimentation velocity, kinetic and equilibrium techniques. No evidence for an aggregating system could be found in sedimentation velocity experiments in the presence or absence of pyridoxal 5-phosphate. Reassociation of pyridoxal 5-phosphate with apoenzyme and reacquisition of enzymic activity follow identical kinetics. An initial fast step is followed by a second order process with a rate constant of 66 M-1. s-1. A dissociation constant of 27.5 micrometer was obtained from equilibrium studies. No interaction of binding sites was exposed by altering pH or in the presence of glycine or folate. Maxima observed in pH profiles with both binding and reactivation are interpreted as the composite fo two overlapping processes, one of which is ionization of the pyridinium nitrogen of pyridoxal 5-phosphate and the other a functional group on the apoenzyme. Evidence is presented to indicate the necessity for the formation of an enzyme . pyridoxal 5-phosphate Schiff's base complex during catalytic turnover.

Purification and comparative properties of the glycoprotein nicotinamide adenine dinucleotide glycohydrolase from rat liver microsomal and plasma membranes.

NAD glycohydrolase, or NADase (NAD+ glycohydrolase, EC 3.2.2.5) was solubilized with porcine pancreatic lipase from isolated fractions of microsomes and plasma membranes obtained from rat livers. The enzyme from each organelle was further purified by DEAE-cellulose chromatography, gel filtration and isoelectric focusing. The solubilized, partially purified enzymes had similar molecular weights, pH-activity profiles and Km values. Marked charge heterogeneity was observed for the microsomal enzyme on isoelectric focusing between pH 6 and 8 with maximum activity focusing at pH 8.0. Plasma membrane NADase displayed a single peak at pH 6.7. Treatment of the partially purified microsomal or plasma membrane enzyme with neuraminidase resulted in a single peak of activity on isoelectric focusing (pH 3.5--10) with a pI of 9.2. Polyacrylamide gel electrophoresis of either NADase revealed a periodate-Schiff positive band which was coincident with enzyme activity. Compositional analyses of the microsomal enzyme focusing at pH 8.0 confirmed the presence of hexoses, hexosamines and sialic acid. Differences in carbohydrate composition might be important in determining the subcellular distribution of this enzyme.

Effects of glutamine, methionine sulfone and dexamethasone on rates of synthesis of glutamine synthetase in cultured hepatoma cells.

Glutamine synthetase (EC 6.3.1.2) activity of hepatoma tissue culture cells is elevated by corticosteroids and depressed by glutamine (Kulka, R.G., Tomkins, G.M. and Crook, R.B. (1972) J. Cell Biol., 54, 175--179). The transfer of cells from high (1--5 mM) to low (0.2--0.4 mM) concentrations of glutamine causes a marked increase in glutamine synthetase activity. The addition of a glutamine antagonist, methionine sulfone (1 mM) to cells suspended in high (1 mM) concentrations of glutamine also causes an increase of glutamine synthetase activity which is greater than that elicited by the transfer of cells to low concentrations of glutamine. Rates of synthesis of glutamine synthetase have been measured by radioimmunoprecipitation in hepatoma tissue culture cells incubated under various conditions. Incubation of cells with the synthetic corticosteroid hormone, dexamethasone, markedly stimulates the relative rate of glutamine synthetase biosynthesis. Glutamine, or its analogue, methionine sulfone, have no effect on the relative rate of synthesis of the enzyme. However, total protein and RNA synthesis increase markedly with increasing external glutamine concentration in the range 0--1 mM. Methionine sulfone (1 mM) inhibits the degradation of glutamine synthetase in the presence of 1 mM glutamine. The data are consistent with the conclusion that the corticosteroid, dexamethasone, elevates glutamine synthetase activity by stimulating its rate of synthesis, whereas methionine sulfone elevates glutamine synthetase activity by inhibiting the glutamine-stimulated degradation of preformed enzyme.

Conversion of T3 and rT3 to 3,3'-T2: pH dependency.

In the extrathyroidal deiodination of T4 the importance of T3 and rT3 for the peripheral action of thyroid hormones is well documented. With the development of a specific radioimmunoassay for 3,3'-T2, a deiodination product of both T3 and rT3, we were able to characterize these subsequent enzymatic reactions as well as the degradation of 3,3'-T2 in rat liver homogenate. It was found that the reaction T3 leads to 3,3'-T2 is slow compared to the conversion of T4 to T3. The pH activity profile shows a peak at 8.4. The reaction rT3 leads to 3,3'-T2 is very fast, with an apparent KM of 4 x 10(-7) M. The reaction velocity is significantly higher in acid than in alkaline pH. The deiodination of 3,3'-T2 is also faster in the acid than in the alkaline range. It is concluded that the outer ring of T4 is more readily deiodinated in acid and the inner ring in alkaline media, and that 3,3'-T2 is mainly degraded to 3-T1. In the acid pH range T3 may accumulate and in the alkaline range rT3 by the pH characteristics of these ractions. Therefore small shifts in pH can enhance the potent inhibitory action of rT3 on the 5'-deiodination of T4 adding another mechanism to the peripheral regulation of thyroid hormone activity.

Drug kinetics and alcohol ingestion.

Acute and chronic ethanol ingestion can alter both the pharmacodynamics and pharmacokinetics of other drugs. For psychotherapeutic drugs, modification of drug action by alcohol is much more important than kinetic interaction, such as ethanol induced drug metabolism. In contrast, the importance of the effects of alcohol on the kinetics of other classes of drug is incomplete. The probability and mechanism of alcohol kinetic interactions with other drugs can nevertheless be anticipated, in part, on the basis of the extent of binding of the drug to plasma proteins, the capacity of the liver for extracting the drug from blood passing through the liver and the true distribution space of the drug. Highly bound drugs with low intrinsic hepatic clearance are among the most commonly reported to have their kinetics altered by ethanol (e.g. benzodiazepines, phenytoin, tolbutamide and warfarin). Less highly bound drugs are less consistently affected (e.g. meprobamate, glutethimide, pentobarbitone and phenobarbitone). Acute administration of ethanol to laboratory animals or incubation of microsomal preparations with ethanol inhibits the mixed function oxidase activity. In the human, the elimination half-life of meprobamate, pentobarbitone and tolbutamide is increased by acute ethanol administration. Chronic administration of ethanol to rats and humans causes proliferation of the smooth endoplasmic reticulum, increase in microsomal protein content and cytochrome P450 and results in an augmentation in drug metabolising ability of the microsomes in vitro. Even though the plasma half-life of some drugs is decreased by chronic ethanol ingestion, the clinical determination of the mechanism is incomplete because few studies have measured drug metabolite levels. In addition, alcohol effects on drug distribution have not been studied very extensively. The effects of chronic alcohol ingestion on drugs with low and high hepatic extraction, high and low binding, important tissue localisation and microsomal and non-microsomal metabolism will be quite different. Systematic studies of the mechanism of alcohol kinetic interactions are needed. Such kinetic studies should be combined with pharmacodynamic measures in order to establish the clinical importance of changes in drug kinetics.

Ultrastructural findings in the mouse exocrine pancreas during graft-versus host reaction.

12- to 36-hours-old newborn CBA mice were intraperitoneally injected each with 10 X 10(6) allogeneic spleen cells of adult C57Bl mice. Control mice received syngeneic spleen cells of adult CBA mice either in the same way or remained untreated. The animals were killed 1, 3, 7, 14 or 21 days after the spleen cell injection. The pancreas was studied histologically and electron microscopically by common methods. Together with an interstitial lymphohistiocytic infiltration of the pancreas different acinar cell alterations were observed in the mice treated with allogeneic spleen cells: 1. Acute lethal pancreatic cell damages on the day after the intraperitoneal injection of allogeneic spleen cells. 2. Membrane-lined inclusion vacuoles between the 7th and 21st experimental day. 3. Atrophy of pancreatic acinar cells in the terminal stage of a severe graft-versus-host disease. The pathogenesis of the observed pancreatic changes seems to be due to nonimmunological and immunological processes. The membrane-lined inclusion vacuoles of the acinar cells could be the consequence of a cell-mediated immune process in the graft-versus-host reaction.

Physiological role and membrane lipid modulation of the membrane-bound (Mg2+, na+)-adenosine triphosphatase activity in Acholeplasma laidlawii.

The membrane-bound adenosine triphosphatase (ATPase) activity of Acholeplasma laidlawii B differs in many respects from the common (Mg2+, Ca2+)-ATPase activity of higher bacteria, most notably in that it is specifically activated by Mg2+ and strongly and specifically stimulated by Na+ (or Li+). Various inhibitors diminish the ATPase activity with a concentration dependence which suggests that a single enzyme species is responsible for all of the observed ATP hydrolytic activity (both basal and Na+ stimulated). The Km for ATP is influenced by temperature but not by membrane lipid fatty acid composition. Vmax is influenced by both of these factors, showing a break in Arrhenius plots which falls below the lipid phase transition midpoint but well above the lower boundary when a phase transition occurs within the temperature range studied. The apparent energy of activation for Vmax is strongly influenced by lipid fatty acid composition both above and below the break. When whole cells of A. laidlawii B are incubated in KCl or NaCl buffers, they rapidly swell and lyse if deprived of an energy source or treated with ATPase inhibitors at concentrations which significantly inhibit enzyme activity in isolated membranes, whereas in sucrose or MgSO4 buffers of equal osmolarity, the cells are stable under these conditions. These results suggest that the membrane ATPase of A. laidlawii B is intimately associated with the membrane lipids and that it functions as a monovalent cation pump which regulates intracellular osmolarity as the (Na+, K+)-ATPase does in eucaryotes.

Studies on the mechanism of the malate dehydrogenase reaction.

The stereospecificity of the chicken heart mitochondrial malate dehydrogenase as well as the ability of this enzyme to form various abortive complexes has been further investigated. The enzyme was found to be specific for the A-hydrogen of NADH. Complex formation of the enzyme with oxalacetate and oxidized coenzymes is pH-dependent and is promoted at alkaline pH values. The enol form of oxalacetate appears to be the species that participates in the formation of the complexes. The binding of L-malate, D-malate, or hydroxymalonate to the enzyme. NADH complex is also pH-dependent, and involves a group on the enzyme with a pK of 7.5. The binding of L-malate is promoted at alkaline pH values, whereas the binding of D-malate and hydroxymalonate is favored at acidic pH values. These results indicate that L-malate and enol-oxalacetate preferentially or exclusively bind to the nonprotonated form of the enzyme, whereas keto-oxalactate, hydroxymalonate, and D-malate only bind to the protonated form of the enzyme. Based on this conclusion, a detailed chemical mechanism for the malate dehydrogenase reaction has been postulated and a schematic illustration of the transition state of the enzyme is presented.

An experimental study on "replantation toxemia". The effect of hypothermia on an amputated limb.

Hind legs of dogs were amputated at the middle of the thigh and preserved in three different conditions: in ice water, in a refrigerator, and at room temperature. After 6 or 12 hours of ischemia, recirculation was established. The survival rate of the animals was observed and measurement of limb edema, potassium, pH, and lactate in the blood was performed to study the effects of hypothermia on prevention of "replantation toxemia." Cooling of the amputated limb was effective for prevention of toxemia, and the cooling effect was greater in ice water than in a refrigerator. However, when cooled in ice water, some animals died due to toxemia when the time of ischemia was prolonged to 12 hours. In the dead animals, a close relationship was observed between the developement of toxemia and metabolic acidosis due to the increase in lactate.

Enteroendocrine APUD cells in the digestive tract of larval Barbus conchonius (Teleostei, Cyprinidae).

The development of Barbus conchonius is described with special attention to the differentiation of the gut. Amine precursor uptake and decarboxylation (APUD) are present in enteroendocrine cells during development, whereas these processes are lacking in adult specimens. The first APUD cells originate on the fourth day of development in the anterior part of the gut and on the fifth day in the caudal areas. The APUD facility of the cells disappears within 2 days, and after the 6th day APUD cells can no longer be distinguished in the intestinal epithelium. The first APUD cells were obsserved when four types of enteroendocrine cells were recognized with the electron microscope. These enteroendocrine cells contain granules of different electron densities, and microtubules and cilia can be observed. Some enteroendocrine-like cells are found below the basement membrane of the intestinal epithelium, indicating a possible extra-endodermal origin. APUD cells, except melanoblasts, have not been found migrating from the neural crest in ventral direction. The origin of the enteroendocrine cells of B. conchonius is discussed.

Dopamine antagonistic effects of a series of analogues of oxiperomide and spiroxatrine measured behaviourally in the rodent.

The activity spectra of oxiperomide, spiroxatrine and analogues were determined in two experimental models of abnormal peri-oral movements (induced by intrastriatal dopamine and subcutaneously administered 2-(NN-dipropyl)amino-5,6-dihydroxy-1,2,3,4-tetrahydronaphthalene (NN-diPr-5,6-diOHATN) in the guinea-pig), and in a stereotypy test (induced by subcutaneous apomorphine in the guinea-pig); the ability of the test compounds to induce catalepsy or catatonia in the rat was also determined. The parent compounds oxiperomide and spiroxatrine possessed optimal activity in all tests, although responses to the series of compounds allowed clear differentiation between an ability to antagonize the peri-oral movements (dopamine- or NN-di Pr-5,6-diOHATN induced) and an ability to antagonize apomorphine stereotypy. However, all compounds that antagonized the abnormal peri-oral movements also caused catalepsy/catatonia. The results are considered in terms of the selection of suitable agent(s) for the treatment of peri-oral dyskinesias.

Role of membrane-bound calcium in taste reception of the frog.

1. The frog gustatory responses to various salt stimuli and distilled water were greatly enhanced after the tongue was treated with an alkaline solution containing salts of low concentration. The incubation of the alkali-treated tongue in solution of pH 6.0 containing Ca2+ restored reversibly the behaviour of the gustatory receptor to that before the treatment, while Mg2+ had no ability to do this. 2. The responses to salt stimuli and distilled water were greatly decreased after the tongue was incubated in solutions of pH 5.3 containing Ca2+. 3. On piece of tongue incubated in a solution of pH 5.3 containing 45Ca released a larger amount of 45Ca by the alkali treatment than another piece incubated in pH 7.0. It was concluded that removal of Ca2+ from the gustatory receptor membrane by the alkali treatment led to enhancement of the responses and binding of extra Ca2+ to the membrane by the incubation in acidic CaCl2 solution led to suppression of the responses. We emphasized that a conformational change of the receptor domains plays an important role in the transduction process of the gustatory response.

Adrenergic and 'non-adrenergic' components in the contractile response of the vas deferens to a single indirect stimulus.

1. The mechanical response of the longitudinal smooth muscle of the rat vas deferens to stimulation of its motor nerves by a single pulse has been examined. The motor nerves were stimulated in vivo via the spinal outflows in the pithed rat or in vitro by field stimulation. 2. The contraction in the whole vas consisted of two components, an initial, rapid, brief contraction reaching a maximum at 300 msec and a second, slower and more prolonged contraction reaching its maximum at 600 msec. When the isolated vas was divided into prostatic and epididymal halves the contribution of these two components varied. The initial rapid component was more prominent in the prostatic half and the slower, second component more prominent in the epididymal half. Lowering the bath temperature caused, in both halves, these two components to merge into a single, slow, prolonged response. Both components were more rapid and briefer than the equivalent response of rat anococcygeus. 3. The second, slow component was abolished by alpha-adrenoceptor antagonist drugs, potentiated and prolonged by drugs which inhibit the neuronal uptake of noradrenaline and absent from tissues taken from rats pre-treated with reserpine, suggesting that the neurotransmitter for this component is noradrenaline. 4. These experiments were extended to the mouse or guinea-pig vas deferens. Both showed the same two component mechanical response as the rat vas and in both the second, slow component was preferentially inhibited by alpha-adrenoceptor antagonists and potentiated by drugs blocking noradrenaline uptake. 5. Drugs known to reduce the response to repetitive nerve stimulation of the vas were examined for their effect on the response to a single stimulus. Lysergic acid diethylamide preferentially inhibited the second, slow phase of contraction whereas apomorphine preferentially inhibited the first rapid phase. Guanethidine inhibited responses but any differential effects could not be analysed due to its stimulant properties. 6. These results show that there are two components even to the response to a single stimulus. The second of these appears to be adrenergic while the transmitter responsible for the first remains to be determined.

Self-poisoning in Auckland reconsidered.

Data is presented describing 345 self-poisoners presenting at the accident and emergency department and 211 referred to the psychiatric liaison service at Auckland Hospital during the 12 month period between August 1976 and July 1977. Both groups show the age and sex distribution commonly seen in self-poisoning, with a preponderance of young females. Referral rates increased with age and with the implication of anti-depressants, neuroleptics and anticonvulsants. Only 20 percent of the referred patients had a serious psychiatric disorder. Personality disorder, depressive neurosis and transient situational disturbance accounted for 80 percent. The benzodiazepine group of anxiolytics was implicated in 40 percent of incidents and the use of this class of medication is discussed in relation to the problem of self-poisoning. It is suggested that if all Auckland doctors made a decision not to prescribe oral benzodiazepines, the incidence of self-poisoning in the city could be reduced to a half to two thirds of its present proportions.

Fluorescent complexes of DNA with DAPI 4',6-diamidine-2-phenyl indole.2HCl or DCI 4',6-dicarboxyamide-2-phenyl indole.

4',6-Dioarboxyamide-2-phenyl indole (DCI), a non-ionic structural analogue of 4',6-diamidine-2-phenyl indole.2HCl (DAPI), was synthesized in order to verify the hypothesis of intercalation of both dyes into the DNA double helix. The influence of pH, viscosity, and different concentrations of SDS (sodium dodecylsulphate) or NaCl on the optical and fluorescent properties and the changes in thermal transition of both dye complexes with DNA confirm the affinity of the dyes to the double helix as well as their stabilizing influence on the secondary DNA structure. The results of binding studies, carried out by fluorescent methods have shown that the dyes are strongly bound to DNA, though the number of binding sites is small. According to the experimental data, the fluorescent properties of DAPI and DCI complexes with DNA are connected with the intercalating binding mechanism of these dyes. On the other hand, the eventual ionic or hydrogen bonds of dyes outside the DNA helix do not change noticeably their fluorescent properties.

Stoichiometry of vectorial H+ movements coupled to electron transport and to ATP synthesis in mitochondria.

In order to verify more directly our earlier measurements showing that, on the average, close to four vectorial H(+) are rejected per pair of electrons passing each of the three energy-conserving sites of the mitochondrial electron transport chain, direct tests of the H(+)/2e(-) ratio for sites 2 and 3 were carried out in the presence of permeant charge-compensating cations. Site 2 was examined by utilizing succinate as electron donor and ferricyanide as electron acceptor from mitochondrial cytochrome c; the directly measured H(+)/2e(-) ratio was close to 4. Energy-conserving site 3 was isolated for study with ferrocyanide or ascorbate plus tetramethylphenylenediamine as electron donors to cytochrome c and with oxygen as electron acceptor. The directly measured H(+)/2e(-) ratio for site 3 was close to 4. The H(+)/ATP ratio (number of vectorial H(+) ejected per ATP hydrolyzed) was determined with a new method in which the steady-state rates of both H(+) ejection and ATP hydrolysis were measured in the presence of K(+) + valinomycin. The H(+)/ATP ratio was found to approach 3.0. A proton cycle for oxidative phosphorylation is proposed, in which four electrochemical H(+) equivalents are ejected per pair of electrons passing each energy-conserving site; three of the H(+) equivalents pass inward to derive ATP synthesis from ADP and phosphate and the fourth H(+) is used to bring about the energy-requiring electrogenic expulsion of ATP(4-) in exchange for extramitochondrial ADP(3-), via the H(+)/H(2)PO(4) (-) symporter.

Some physiological effects of ketamine in sheep.

The effects of intravenous injections of ketamine in sheep on the cardiovascular and respiratory systems are compared with the effects of intracerebroventricular injections of the same drug and also with the effects of intravenous barbiturate and steriod anaesthetics. Intravenously administered ketamine caused an initial fall in arterial blood pressure the extent of which was dose dependent. This depression was short lived and was occasionally followed by a mild pressor phase. Intracerebroventricular injection of the drug provoked only a mild transient rise in mean arterial blood pressure. The intravenous injection of ketamine gave a brief period of respiratory depression which was mirrored in the PaO2 and PaCO2 levels followed by a period of respiratory stimulation with elevated PaO2 levels. The comparison of the three injection anaesthetics showed that the blood gas tensions with ketamine showed there was a brief period of respiratory depression similar to that seen with the steroid anaesthetic but that the barbiturate caused a much longer depression similar to that seen with the steroid anaesthetic but that the barbiturate caused a much longer depression. The blood gas tensions following the steroid anaesthetic soon returned to normal while the tensions following ketamine indicated an elevated PaO2 after the initial depression. The blood gas tensions following intracerebroventricular injection of ketamine were difficult to evaluate due to the variable period of apnoea which followed the injection.

The electron microscopy of "fibrinoid necrosis" in pulmonary arteries.

Fibrinoid necrosis was induced in the pulmonary arteries of five male Wistar albino rats by feeding them on a diet adulterated by the addition of 0.07% ground Crotalaria spectabilis seeds by weight. Electron microscopy of the arteries affected by the process showed fibrin in the thrombus occluding their lumens and in the arterial intima, held up from further penetration of the media by the inner elastic lamina. Naturally occurring gaps in this lamina were found, and it is postulated that they determine the characteristic histological configuration of fibrinous vasculosis. The smooth muscle cells of the media of the pulmonary veins showed clear evaginations, devoid of myofilaments and organells, indicative of sustained constriction compatible with viability of the cells. In contrast, the smooth muscle cells of the media of the pulmonary arteries showed loss of myofilaments leading to frank necrosis. Other cells seen in the media include fibrocytes and "vasoformative reserve cells." The authors consider that the latter have considerable and varied potential. Once liberated during the process of fibrinoid necrosis in the arterial media they may play an important part in pulmonary vascular pathology as, for example, in the formation of the plexiform lesion.

Effect of generalized graft-versus-host-reaction on B- and T-lymphocytes and a benzpyrene-induced murine sarcoma.

The investigations of 400 adult hybrids from A/Jax fem. BALB/c male were aimed at answering the following questions: 1. What is the influence of a generalized graft-versus-host-reaction (GVHR) on the induction and growth of benzpyren-induced sarcoma? 2. What is the behavior of B- and T-lymphocytes during GVHR, and during induction and growth of benzpyren-induced sarcoma? 3. Which are the deductions to be drawn with regard to the effect of B- and T-lymphocytes during tumorigenesis and tumor growth?

Influence of morphine anaesthesia on the endocrine-metabolic response to open-heart surgery.

Twelve patients scheduled for aortic valve replacement during extracorporal circulation were randomly allocated to either morphine anaesthesia or fluroxene anaesthesia. Morphine in a total dose of 4 mg/kg was administered before skin incision. At the start of extracorporal circulation all patients received 25 g glucose intravascularly. The endocrine-metabolic response to surgery, as expressed by changes in plasma ACTH, cortisol, insulin, growth hormone, cyclic adenosine-3',-5'-monophosphate (cyclic AMP), glucose, free fatty acids, blood b-hydroxybutyrate and cumulative nitrogen balance was measured before and during anaesthesia and surgery, and on the first five post-operative days. It was found that morphine anaesthesia blocked the increase in ACTH, cortisol, growth hormone, cyclic AMP, and glucose during surgery. However, after initiation of extracorporal circulation only ACTH, cortisol, and, to a lesser degree, the glucose and insulin response to glucose were lowered by morphine anaesthesia. From the first to the fifth days after operation no differences between the two groups could be demonstrated in any parameter. Cumulative nitrogen balance was similar in the two groups. It is concluded that morphine in large doses administered before skin incision inhibits the initial endocrine-metabolic response to open-heart surgery, but that the effect is short-lasting and without effect on overall postoperative protein catabolism.

Cottonseed protein derivatives as nutritional and functional supplements in food formulations.

Cottonseeds contain protein with desirable food functional and nutritional properties. Storage globulins make up most of the protein stored in cottonseed and can be separated into five fractions by gel filtration chromatography. Each fraction is distinguishable from the other by its amino acid and polyacrylamide gel electrophoretic properties. Proteins of cottonseed contribute greatly to the functional properties of emulsions, co-isolates, and texturized derivatives. For example, increasing the amount of high protein cottonseed flour in wheat suspensions from 2% to 10% improved the capacity (54-97 ml of oil) and viscosity (5,000-100,000+ cps) of emulsions. The 10% suspension formed emulsions with increasing oil capacity (84-100 ml) and viscosity (28,000-100,000+ cps) as the pH was adjusted from 4.5 to 9.5. Consistencies of the products ranged from that of salad dressing (low percent suspensions, or acid pH) to that of mayonnaise (high percent, or basic pH). These data were utilized to derive a multiple regression model to predict optimum use of cottonseed proteins in emulsions of varying consistencies. A coprecipitated isolate containing greater than 94% protein was prepared from a blend of cottonseed and peanut flours. Amino acid content of the co-isolate reflected that of the protein in the two flours of the composite. The co-isolate has lower gossypol level and improved color and functional properties than a cottonseed protein isolate. Storage protein isolate of cottonseed suspended in aqueous solution and heated with constant stirring forms a texturized product; the quality of the product depends on heat, pH, salt, and the quantity of nonstorage proteins. Protein and amino acid content of meat products were improved by the addition of the texturized protein of cottonseed.

Effect of oxygen at high pressure on spontaneous transmitter release.

The effect of oxygen at high pressure (OHP) on resting membrane properties (effective membrane resistance (Reff) and membrane potential (Vm)) and the spontaneous release of excitatory transmitter were examined at the lobster neuromuscular junction. Pressurization with 100% oxygen to 150 pounds per square inch gauge pressure (psig) or with nitrogen to 150 psig (7,000 mmHg nitrogen and 135 mmHg oxygen) produced a decrease in Reff associated with a hyperpolarization of Vm. These changes, however, returned to control values within 20--30 min after completion of pressurization. Spontaneous release of excitatory transmitter was shown to increase dramatically in the presence of 100% oxygen at 150 psig. The increase in miniature end-plate potential (MEPP) frequency persisted beyond the transient changes seen with Reff and Vm. This effect was selective to oxygen, as pressurization with nitrogen did not produce an increase in MEPP frequency. No change in average MEPP amplitude was seen with either OHP or pressure alone. An OHP-induced increase in MEPP frequency was also seen at the frog neuromuscular junction. The results indicate that both glutamate-mediated and acetylcholine-mediated synaptic transmission are altered by OHP.

Pancreatic response to intestinal perfusion with lactic acid or acidified albumin.

To test the hypothesis that the permeability of weak acids across the intestinal mucosa affects their ability to stimulate pancreatic bicarbonate output, we compared pancreatic bicarbonate secretion in response to intestinal perfusion with an acid presumed to be permeable to cell membranes, lactic acid (90 daltons), and an acid presumed to be impermeable, acidified bovine serum albumin (about 70,000 daltons). These two substances have similar titration curves from pH 2.00 to pH 4.50. In four conscious dogs with pancreatic fistulas, solutions of these weak acids were perfused at 50 ml/15 min into the intestine at concentrations adjusted to deliver 1, 2, or 4 mmol/15 min of acid titratable to pH 4.50 (threshold pH for bicarbonate stimulation) from an initial pH of 2.00 or 3.50. At both pH 2.00 and 3.50 and at all titratable acid loads, bicarbonate secretory responses to lactic acid and acidified albumin were not significantly different. Equal titratable acid loads of HCl produced much larger secretory responses. The data do not support the hypothesis that permeability of weak acids is a factor, but confirm the observation that weak acids are less potent than strong acids in stimulating pancreatic bicarbonate secretion.

Clinical and in vitro analysis of determinants of gastroesophageal competence. A study of the principles of antireflux surgery.

The analysis of esophageal manometry and 24 hour esophageal pH monitoring in 266 consecutive patients indicates that the competency of the cardia depends upon the amplitude of the distal esophageal high pressure zone and the length of the abdominal esophagus. These two determinants of competency were examined using human esophagi in a unique in vitro model which allowed control of these parameters, as well as intraabdominal, intragastric, and intrathoracic pressures. The following principles of the function of the abdominal esophagus were graphically illustrated: (1) Competency of a segment of intraabdominal esophagus without intrinsic tone occurs only when intraabdominal pressure is equal to or greater than intragastric pressure. (2) Competency of a segment of intraabdominal esophagus without intrinsic tone is directly related to its length. (3) The length of intraabdominal esophagus necessary to maintain competency is indirectly related to variations in intraabdominal pressure. (4) Competency of a segment of intraabdominal esophagus is augmented by the presence of intrinsic tone, and the shorter the length, the greater the intrinsic tone needed. (5) Competency of a segment of intraabdominal esophagus is augmented by negative intrathoracic pressure. These findings beautifully illustrate the mechanical valvelike function of the abdominal esophagus and the objectives to be accomplished in the surgical treatment of gastroesophageal reflux.

Cardiovascular, acid-base, electrolyte, and plasma volume changes in ponies developing alimentary laminitis.

Twelve Shetland ponies were fed a high-starch ration. Seven ponies which had a transitory metabolic acidosis developed laminitis 56 hours (+/- 3.5, SEM) after overfeeding. These ponies also developed persistent hypokalemia, hyperthermia, and increased heart rate 24 hours before the onset of lameness. Serum sodium, serum chloride, hematocrit, plasma volume, and blood volume were unchanged. At the onset of clinical signs of laminitis, cardiac output and blood pressure increased, but total peripheral resistance was unchanged. None of the measured or calculated values predicted the onset of laminitis. Hypertension appeared to be a response to, rather than a cause of, lameness. Three of the remaining ponies apparently died of shock 29.3 +/- 2.7 hours after overfeeding. All 3 had severe metabolic acidosis; decreased cardiac output, systemic arterial pressure, and plasma volume; and increased hematocrit, total peripheral resistance, and pulmonary vascular resistance. The 11th pony was unaffected and the 12th pony was euthanatized.

The effects of agents modifying sympathetic nerve function on the response of the isolated rat tail artery to etilefrine and tyramine.

The effects of etilefrine on the ventral caudal artery of the rat have been examined in the presence of agents modifying sympathetic nerve function. Catecholamine levels were also measured in adjacent segments of artery to those studied pharmacologically and an attempt made to relate vascular response to etilefrine (and tyramine) with catecholamine content. Both guanethidine and reserpine produced significant attenuation of the vascular effects of etilefrine and tyramine. Pre-treatment with a monoamine oxidase inhibitor caused an increase in tissue catecholamine levels but, paradoxically, depressed the vascular response to etilefrine. The significance of some of the findings in terms of an indirect component to etilefrine's action are discussed.

Effects of glucagon and insulin on fatty acid synthesis and glycogen degradation in the perfused liver of normal and genetically obese (ob/ob) mice.

1. Rapid effects of hormones on glycogen metabolism and fatty acid synthesis in the perfused liver of the mouse were studied. 2. In perfusions lasting 2h, of livers from normal mice, glucagon in successive doses, each producing concentrations of 10(-10) or 10(-9)M, inhibited fatty acid and cholesterol synthesis. In perfusions lasting 40--50 min, in which medium was not recycled, inhibition of fatty acid synthesis was only observed with glucagon at concentrations greater than 10(-9)M. This concentration was about two orders of magnitude higher than that required for the stimulation of glycogen breakdown. Glucagon did not inhibit the activity of acetyl-CoA carboxylase, assayed 10 or 20 min after addition of glucagon (10(-9) or 10(-10)M). It is proposed that the action of glucagon on hepatic fatty acid biosynthesis could be secondary in time to depletion of glycogen. Insulin prevented the effect of glucagon (10(-10)M) on glycogenolysis, but not that of vasopressin. 3. Livers of genetically obese (ob/ob) mice did not show significant inhibition of lipid biosynthesis in response to glucagon, although there was normal acceleration of glycogen breakdown. This resistance to glucagon action was not reversed by food deprivation. Livers of obese mice exhibited resistance to the counteraction by insulin of glucagon-stimulated glycogenolysis, which was reversible by partial food deprivation.

Properties of pyruvate kinase and phosphoenolpyruvate carboxykinase in relation to the direction and regulation of phosphoenolpyruvate metabolism in muscles of the frog and marine invertebrates.

1. The properties of pyruvate kinase and, if present, phosphoenolpyruvate carboxykinase from the muscles of the sea anemone, scallop, oyster, crab, lobster and frog were investigated. 2. In general, the properties of pyruvate kinase from all muscles were similar, except for those of the enzyme from the oyster (adductor muscle); the pH optima were between 7.1 and 7.4, whereas that for oyster was 8.2; fructose bisphosphate lowered the optimum pH of the oyster enzyme from 8.2 to 7.1, but it had no effect on the enzymes from other muscles. Hill coefficients for the effect of the concentration of phosphoenolpyruvate were close to unity in the absence of added alanine for the enzymes from all muscles except oyster adductor muscle; it was 1.5 for this enzyme. Alanine inhibited the enzyme from all muscles except the frog; this inhibition was relieved by fructose bisphosphate. Low concentrations of alanine were very effective with the enzyme from the oyster (50% inhibition was observed at 0.4mm). Fructose bisphosphate activated the enzyme from all muscles, but extremely low concentrations were effective with the oyster enzyme (0.13mum produced 50% activation). 3. In general, the properties of phosphoenolpyruvate carboxykinase from the sea anemone and oyster muscles are similar: the K(m) values for phosphoenolpyruvate are low (0.10 and 0.13mm); the enzymes require Mn(2+) in addition to Mg(2+) for activity; and ITP inhibits the enzymes and the inhibition is relieved by alanine. These latter compounds had no effect on enzymes from other muscles. 4. It is suggested that changes in concentrations of fructose bisphosphate, alanine and ITP produce a coordinated mechanism of control of the activities of pyruvate kinase and phosphoenolpyruvate carboxykinase in the sea anemone and oyster muscles, which ensures that phosphoenolpyruvate is converted into oxaloacetate and then into succinate in these muscles under anaerobic conditions. 5. It is suggested that in the muscles of the crab, lobster and frog, phosphoenolpyruvate carboxykinase catalyses the conversion of oxaloacetate into phosphoenolpyruvate. This may be part of a pathway for the oxidation of some amino acids in these muscles.

Acyl carrier protein from Escherichia coli: characterization by proton and fluorine-19 nuclear magnetic resonance and evidence for restricted mobility of the fatty acid chain in tetradecanoyl-acyl-carrier protein.

The acyl-carrier protein (ACP) of Escherichia coli is a protein of molecular weight 8847 with a 4'-phosphopanthetheine prosthetic group. ACP functions (via the SH of the prosthetic group) as a coenzyme in the synthesis of fatty acids and complex lipids. We report proton nuclear magnetic resonance (NMR) studies of the structure of ACP under various experimental conditions. The motion of the fatty acyl chain of acyl-ACP has been investigated by 19FNMR studies of difluorotetradecanoyl-ACP. 31PNMR studies of the prosthetic group phosphorus of ACP and acyl-ACP are also reported. We make the following conclusions: (1) the structure of ACP is stabilized by surface charge, and (2) the fatty acid residue of acyl-ACP does not move freely and seems immobilized by an interaction with the protein moiety.

Acetyl-CoA acetyltransferase from bovine liver mitochondria. Molecular properties of multiple forms.

Bovine liver mitochondrial acetyl-CoA acetyltransferase (acetyl-CoA:acetyl-CoA C-acetyltransferase, EC 2.3.1.9) has been obtained in three forms designated transferase I, A and B on the basis of their elution positions from chromatography on phosphocellulose. All forms have been shown to have a molecular weight of about 152 000, each being composed of four similar subunits. Amino acid analysis of transferase A and B, the two major forms, revealed a close relationship between both forms with almost identical amino acid composition and arginine as N-terminal residue. The three transferases differ with respect to their redox state and their multiplicity of forms with isoelectric points of 6.9, 7.5 and 8.8, into which the transferases I and A were spontaneously transformed upon isoelectric focusing or rechromatography on phosphocellulose. Transferase B represents a stable enzyme form with an isoelectric point of 8.8. Although the redox state of transferase B can be adjusted to that of transferase A still a difference in charge and in the multiplicity of forms exists, thus indicating different protein states.

Acid Bohr effects in myoglobin characterized by proton NMR hyperfine shifts and oxygen binding studies.

Proton NMR studies of sperm whale and horse deoxymyoglobin have revealed that both proteins exhibit a single, well defined, pH-induced structural change. The changes in hyperfine shifts are clearly observed not only at the heme peripheral substituents, but also at the proximal histidyl imidazole, which suggest that heme-apoprotein contacts are looser in the acidic than alkaline conformations. The hyperfine shift changes are modulated by a single titratable group with a pK of approx. 5.7 in both proteins. Oxygen binding studies of sperm whale myoglobin over a range of temperature and pH showed that, while the oxygen affinity was independent of pH at 25 degrees C, it increased below pH 7 at 0 degrees C and decreased below pH 7 at 37 degrees C. Hence, sperm whale myoglobin exhibits a small acid Bohr effect which most likely arises from the characterized structural changes in the deoxy proteins. While horse myoglobin failed to exhibit a resolvable acid Bohr effect between 0 and 37 degrees C, it did show a weak alkaline Bohr effect at 25 degrees C which disappeared at lower temperatures. Since the oxygen affinity changed smoothly over several pH units, this alkaline Bohr effect can not be associated with any well defined conformational change detected by NMR.

Effects of hemolysate concentration, ionic strength and cytochrome b5 concentration on the rate of methemoglobin reduction in hemolysates of human erythrocytes.

An assay for determining the rate of methemoglobin reduction in hemolysates of human erythrocytes has been developed. The rates obtained by this assay, when corrected for dilution, are comparable to those obtained with intact cells. Increased ionic strength inhibits the reaction, whereas EDTA increases the rate of reduction. The rate with NADPH as electron donor is 65-70% of the rate with NADH. Added cytochrome b5 stimulates the reaction. The assay has been used to examine erythrocytes from two methemoglobinemic sisters and their asymptomatic mother. Hemolysates of the two patients have both decreased dichlorophenolindophenol reductase activity and decreased ability to reduce methemoglobin. Hemolysates from the heterozygous mother have intermediate dichlorophenolindophenol reductase activity and intermediate methemoglobin reduction ability. The data presented in this paper indicate that the concentrations of cytochrome b5 and cytochrome b5 reductase determine the rate of methemoglobin reduction in hemolysates.

Studies on the succinate dehydrogenating system. I. Kinetics of the succinate dehydrogenase interaction with a semiquindiimine radical of N,N,N',N'-tetramethyl-p-phenylenediamine.

1. The activities of the soluble reconstitutively active succinate dehydrogenase (EC 1.3.99.1) measured with three artificial electron acceptors, e.g. ferricyanide, phenazine methosulfate and free radical of N,N,N',N'-tetramethyl-p-phenylenediamine (WB), have been compared. The values estimated by extrapolation to infinite acceptor concentration using double reciprocal plots 1/v versus 1/[acceptor] are nearly the same for ferricyanide and phenazine methosulfate and about twice as high for the WB. 2. The double reciprocal plots 1/v versus 1/[succinate] in the presence of malonate at various concentrations of WB give a series of straight lines intercepting in the third quadrant. The data support the mechanism of the overall reaction, in which the reduced enzyme is oxidized by WB before dissociation of the enzyme-product complex. 3. The dependence of the rate of the overall reaction on WB concentration shows that only one kinetically significant redox site of the soluble succinate dehydrogenase is involved in the reduction of WB. 4. Studies of the change of V and Km values during aerobic inactivation of the soluble enzyme suggest that only 'the low Km ferricyanide reactive site' (Vinogradov, A.D., Gavrikova, E.V. and Goloveshkina, V.G. (1975) Biochem. Biophys, Res. Commun. 65, 1264--1269) is involved in reoxidation of the reduced enzyme by WB. 5. The pH dependence of V for the succinate-WB reductase reaction shows that the group of the enzyme with the pKa value of 6.7 at 22 degrees C is responsible for the reduction of dehydrogenase in the enzyme-substrate complex. 6. When WB interacts with the succinate-ubiquinone region of the respiratory chain, the double reciprocal plot 1/v versus 1/[WB] gives a straight line. The thenoyltrifluoroacetone inhibition of succinate-ubiquinone reductase or extraction of ubiquinone alter the 1/v versus 1/[WB] plots for the curves with a positive initial slope intercepting the ordinate at the same V as in the native particles. The data support the mechanism of succinate-ubiquinone reduction, in which no positive modulation of succinate dehydrogenase by ubiquinone exist in the membrane.

Particulate guanylate cyclase of skeletal muscle: effects of Ca2+ and other divalent cations on enzyme activity.

The properties of particulate guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) from purified rabbit skeletal muscle membrane fragments were studied. Four membrane fractions were prepared by sucrose gradient centrifugation and the fractions characterized by analysis of marker enzymes. Guanylate cyclase activity was highest in the fraction possessing enzymatic properties typical of sarcolemma, while fractions enriched with sarcoplasmic reticulum had lower activities. In the presence of suboptimal Mn2+ concentrations, Mg2+ stimulated particulate guanylate cyclase activity both before and after solubilization in 1% Triton X-100. Guanylate cyclase activity was biphasic in the presence of Ca2+. Increasing the Ca2+ concentration from 10(-8) to 10(-5) M decreased the specific activity. As the Ca2+ concentration was further increased to 5 . 10(-4) M enzyme activity again increased. After solubilization of the membranes in 1% Triton X-100, Ca2+ suppressed enzyme activity. Studies utilizing ionophore X537A indicated that the altered effect of Ca2+ upon the solubilized membranes was independent of asymmetric distribution of Ca2+ and Mg2+.

Response waveforms of vertebrate photoreceptors: what are the underlying mechanisms?

A class of models is investigated using computer simulation in which the inner and outer segments of the vertebrate photoreceptor are coupled through a pump. The outer segment membrane conductance is controlled by an internal transmitter, activated by photolysis of the photosensitive molecules in the cell. Several possibilities for the coupling dynamics are investigated. The analysis favors the conclusion that the hyperpolarizing transient at high intensity stimuli arises from the coupling dynamics, (unless there is an extracellular current shunt path). It predicts, moreover, that the transient should be observed intracellularly, but not extracellularly to the outer segment. This is, in fact, the case. It also predicts that the transient should become more marked, as the steady state ratio of inner to outer segment currents decreases. The computer simulations are concerned with the intracellularly recorded responses; the long term adaptation parallel to pigment bleaching and regeneration is not considered explicitly here. In conclusion, it is shown that the state conditions as well as the response waveforms can be related to physiologically significant variables.

4-Aminopyridine and evoked transmitter release from motor nerve endings.

1 In the presence of tetrodotoxin, electrotonic depolarization of frog motor nerve terminals causes the appearance of stimulus-graded endplate potentials. When 4-aminopyridine is added, the graded endplate potential is converted into a triggered all-or-none response resulting in giant endplate potentials of about 70 mV amplitude and 50 ms duration. The triggered endplate potentials are abolished in Ca(2+)-free saline and are blocked by Mn(2+) ions. Sr(2+) but not Ba(2+) can replace Ca(2+) in supporting transmitter release. Mg(2+) fails, even in concentrations as high as 32 mM, to affect the amplitude and the shape of the endplate potential but abolishes it when the Ca(2+) concentration is reduced to 0.2 mM.2 Despite the large amplitude of the triggered endplate potential in the presence of 4-aminopyridine and tetrodotoxin, repetitive stimulation up to 10 Hz causes only a small decline in amplitude of successive endplate potentials. However, in the presence of (+)-tubocurarine or gallamine, repetitive nerve stimulation produces a marked decline in successive endplate potential amplitude. The fall is counteracted when evoked transmitter release is reduced in the presence of 0.2 mM Ca(2+). The results suggest that in the presence of 4-aminopyridine such large amounts of transmitter are released that even during repetitive stimulation (5 to 10 Hz) endplate potentials are of maximal amplitude.3 4-Aminopyridine causes a prallel shift to the right of the dose-response curve to Mg(2+) for blockade of nerve impulse-evoked transmitter release (in the absence of tetrodotoxin). A similar parallel shift occurs in the presence of tetraethylammonium and guanidine.4 It is concluded that 4-aminopyridine increases transmitter release by enhancing the transport efficacy for Ca(2+) across the nerve terminal membrane during nerve terminal depolarization.

Fractionation of avian erythrocyte histones by hydrophobic interaction chromatography.

The interactions of H1 (H1A, H1B), H2A, H2B, H3, H4, and H5 with phenyl cross-linked agarose were studied. Procedures are described whereby all six histones can be bound, released, and fractionated by using appropriate salt concentrations or pH. The binding can be totally abolished by inclusion of hydrophobic disrupting agents. Control experiments with nonderivated cross-linked agarose ruled out a passive aggregation-disaggregation phenomenon governing the binding patterns. The absorption sequence based on the identification and quantitation of individual histones from either unfractionated (whole) histone or separate histone classes is as follows: H3 greater than or equal to H4 greater than H2B greater than or equal to H5 greater than or equal to H2A greater than H1A greater than or equal to H1B. The order differs only slightly from the reverse of the desorption sequence, H1B less than or equal to H1A less than or equal to H5 less than H2A less than or equal to H3. Preferential interaction of H2A-H2B, H3-H4, and H2A-H2B-H4 occur; these interactions can modify the original relative affinity of each individual component for the matrix. The variability in matrix affinity appears to involve simple stoichiometry of the histone components.

Prospects for the prevention of bacterial meningitis with polysaccharide vaccines.

MOST SUPPURATIVE INFECTIONS OF THE MENINGES ARE CAUSED BY FIVE BACTERIAL SPECIES: Escherichia coli, Haemophilus influenzae type b, Streptococcus pneumoniae, Neisseria meningitidis, and group B streptococcus. The immune response of adults to pneumococcal capsular polysaccharides has been studied in great detail and their responses to meningococcal and H. influenzae type b capsular polysaccharides are quite similar. Immune responses of adults to E. coli and group B streptococcal antigens are disappointing. The responses of children below the age of 7 years differ both quantitatively and in duration. Early experience shows that useful antibody titres can be achieved with certain antigens but further studies are required. In order to prevent bacterial meningitis by immunization, three vaccine formulations will need to be developed. When epidemic meningococcal disease occurs in a population, the vaccine containing only components of the meningococcus would be applied to a large segment of the population to terminate the epidemic. The second vaccine would contain components of H. influenzae type b, pneumococcus, and the meningococcus and would be administered in the first year of life, and repeated at suitable intervals to maintain life-long immunity. The third vaccine, designed to prevent neonatal meningitis caused by E. coli K1 and group B streptococci, would be administered to women preferably during the third trimester of pregnancy, so that their offspring would inherit sufficient antibodies to protect them during the first 3 months of life.The vaccine against the meningococcus is a reality and has been used extensively during major epidemics, with excellent results. The two vaccines for control of endemic bacterial meningitides do not exist as yet, but the prospects are good.

Labetalol in hypertensive patients with angina pectoris: beneficial effect of combined alpha- and beta-adrenoreceptor blockade.

1. Eight hypertensive patients with angina pectoris had placebo added to their existing medications for 8 weeks, then incremental doses of active labetalol with simultaneous stepwise reduction in other medicines until blood pressure was satisfactorily controlled; after that only labetalol and thiazide (8 weeks) and finally labetalol-placebo together with previous beta-adrenoreceptor antagonists and thiazide for 4 weeks were administered. 2. During the labetalol plus thiazide period resting blood pressures and measurements obtained during isotonic exercise, isometric exercise and the cold pressor test were significantly lower than during the initial placebo addition period. Angina scores were significantly reduced during this period. 3. During the final treatment with placebo, beta-adrenoreceptor antagonist and thiazide, blood pressures remained reduced, but angina was significantly worse. 4. Labetalol which antagonizes both alpha- and beta-adrenoreceptors produced better relief of angina pectoris than beta-adrenoreceptor antagonists during improvement in blood pressure in hypertensive patients.

The role of the septo-hippocampal system and its noradrenergic afferents in behavioural responses to none-reward.

Our experiments were designed with two purposes: (i) to examine the effects on one behaviour of differing interventions in the septo-hippocampal system; (ii) to compare these effects with those of minor tranquillizers. The behaviour studied (in rats) is extinction in the alley after continuous (CRF) or partial (PRF) reinforcement. Minor tranquillizers and large septal lesions produce three effects: (1) resistance to extinction is increased after CRF; (2) resistance to extinction is decreased after PRF; (3) the partial reinforcement extinction effect (PREE) is abolished. Small septal lesions fractionate this syndrome: either effect (1) or an actual increase in the size of the PREE is produced by medial septal lesions abolishing hippocampal theta; effects (2) and (3), but not (1), are produced by lateral septal lesions sparing theta. Dorso-medial fornix section, abolishing theta, reproduces the effects of medial septal lesions. Fimbrial section, sparing theta, reproduces some of the effects of lateral septal lesions. Minor tranquillizers produce a rise in the threshold for septal driving of hippocampal theta specifically at 7.7 Hz. This effect is reproduced by blockade of noradrenergic transmission or destruction of the dorsal noradrenergic bundle with 6-hydroxydopamine. This lesion reproduces all three behavioural changes listed above. These results suggest a model for the role of the septo-hippocampal system and its noradrenergic inputs in the PREE. This model is compared with other approaches to the septo-hippocampal system.

Agarose-bound horse-liver alcohol dehydrogenase. Dependence of molecular properties and activity on coupling conditions.

1. Spectroscopic methods for protein and active-site determination with the same sample of immobilised horse liver alcohol dehydrogenase have been developed. 2. The influence of pH, active-site protection of the soluble enzyme and protein concentration on coupling of alcohol dehydrogenase with cyanogen-bromide-activated Sepharose has been investigated. In phosphate buffer (pH 8.0) products with over 90% active-site retention have been synthesized. The binary complex alcohol-dehydrogenase . NADH gives a preparation with the same active-site content but a lower apparent specific activity compared to the unprotected enzyme. Increase in protein concentration yields products with the same active-site content relative to bound protein but the apparent specific activity is decreased. 3. The great similarity in spectroscopic properties of soluble and immobilised enzyme, as well as of their ternary complexes, shows that no significant conformational change has taken place during immobilisation. 4. Exchange of the non-catalytic Zn2+ against Co2+ yields a hybrid Sepharose--Co2Zn2-alcohol-dehydrogenase with over 90% active-site retention during metal exchange. The absorption spectra of the soluble and immobilised hybrid are identical.

Acute influence of different beta-blocking agents upon left heart hemodynamics at rest and during exercise in patients with coronary artery disease.

The study investigated the acute hemodynamic changes induced in patients with angiographically confirmed coronary artery disease by 3 beta-blockers: metoprolol, cardioselective without intrinsic sympathomimetic activity (ISA), group I, 11 patients; bunitrolol, noncardioselective with ISA, group II, 11 patients; oxprenolol, noncardioselective with ISA, group III, 11 patients. Hemodynamic variables were obtained at rest and during exercise, before and 45 min after 10 mg i.v. of the drug. Changes in LVEDP and cardiac indexes were such as LV function was improved in 1 patient of group I, 7 patients of group II and 5 patients of group III; impaired in 4 patients of group I and in 1 patient of group III; unchanged in the others. Contractility indexes were less influenced by bunitrolol and oxprenolol. During exercise there was a significant difference between groups for LVEDP which was lower in group II (P less than 0.01). The data seem to indicate that the choice of the beta-blocker may be of importance when it is desirable that an already compromised cardiac function be not further impaired by pharmacological intervention.

Irreversible beta-adrenoceptor blockade of atrial rate and tension responses.

The competitive reversible beta-adrenoceptor antagonist activity of Ro 03-5255 [1-(5-acetylaminobenzfuran-2-yl)-2-isopropylaminoethanol] upon isoprenaline-induced increases of the rate and tension of guinea-pig isolated atria is described. The chlorinated derivative [Ro 03-7894; 1-[5-chloracetylaminobenzfuran-2-yl)-2-isopropyl-aminoethanol] in contrast exhibited concentration-dependent non-competitive irreversible blocking activity as measured by depression of the maximum responses which were not restored by a washout period that successfully reversed Ro 03-5255. When orciprenaline was used as a weak agonist of low efficacy, the maximum responses were depressed to a greater extent. The blockade by Ro 03-7894 was relatively specific for beta-adrenoceptors since it did not antagonize histamine or calcium chloride. The depression of the maximum responses to orciprenaline was reduced by the presence of sodium thiosulphate. Sodium thiosulphate was ineffective in reversing an established blockade. The blockade by Ro 03-7894 was therefore assumed to involve irreversible binding to the beta-adrenoceptor after conversion to an appropriate electrophilic ligand. The significance of this is discussed.

[Hormone and metabolic profile in diabetic hyperosomolar coma. Plasma insulin response to intravenous tolbutamide (author's transl)].

Fifteen patients with non-ketotic hyperosmolar diabetic coma were investigated and compared with ketoacidotic patients. Basal plasma insulin levels were low in all patients (14.8 +/- 1.0 micronU/ml in hyperosmolar coma, 11.0 +/- 1.3 in keto-acidosis), but insulin level increased after intravenous tolbutamide (between 30 and 105 micronU/ml) in eight hyperosmolar comas. Insulin showed no increase in seven hyperosmolar comas and in none of the ketoacidotic patients. In hyperosmolar coma plasma free fatty acids (1710 +/- 197 micronEq/1), triglycerides (3,4 +/- 0,4 g/1) and cortisol levels (49,7 +/- 9,0 microgram/100 ml) were increased, must as in keto-acidosis. Growth hormone (1,7 +/- 0,1 ng/ml) was normal, unlike the case in keto-acidosis. Plasma lactate concentrations were elevated and account for the frequent mild acidosis found in hyperosmolar coma. In spite of the low peripheral "insulin/glycemia ratio", the positive response to tolbutamide in half of the hyperosmolar cases suggests a less complete pancreatic deficiency than in keto-acidosis. The plasma high free fatty acid and triglyceride levels suggest that the lack of ketosis is not due to inhibition of lipolysis but could be a consequence of inhibition of hepatic ketogenesis.

The prevalence and persistence of group B streptococcal colonization among hospital personnel.

A prospective study was performed to determine the prevalence and persistence of group B streptococcal colonization among obstetric (high-risk) and nonobstetric (low-risk) personnel. Seventy-four individuals participated in the study and the following sites were sampled: throat, rectum, vagina (females) and anterior urethra (males). The overall colonization rate was 32.4% and no statistical difference was found between high- and low-risk groups. The most frequently recovered serotypes were type III (37.5%) and type II/Ic (33.3%). Individuals older than 30 years were more likely to carry type II/Ic, whereas personnel in their twenties were most frequently colonized with type III. The rectum was the most frequently colonized site (83.3%). The vagina/urethra was colonized in 62.5% and the throat in 8.4% of carriers. Twenty-three culture-positive individuals were recultured from all sites three to six months later and persistent colonization was found in 56.5%. There was no statistical difference in persistence between the high- and low-risk groups. Type III carriers tended to become culture-negative, whereas type II/Ic carriers were significantly more likely to remain colonized with group B streptococci.

Binding of cholesterol by Neisseria gonorrhoeae.

The binding of [1,2-3H]cholesterol to Neisseria gonorrhoeae CS-7, Pseudomonas aeruginosa, and Salmonella typhimurium (smooth and rough strains) was investigated. The kinetics of cholesterol binding to N. gonorrhoeae CS-7 demonstrated that binding occurred slowly with maximum binding by 10 h. Under optimum conditions, a large percentage (65%) of the added cholesterol was associated with the cells. Chemical fractionation revealed that ca. 98% of the labeled cholesterol was associated with the cell membrane(s). The bound cholesterol was not esterified and was associated primarily with the cytoplasmic membrane. Intact gonococci bound 4 to 30 times more cholesterol than the deep rough mutant S. typhimurium TA1535, the wild-type S. typhimurium DB-21, and P. aeruginosa. In contrast, isolated cell membranes from all organisms rapidly bound cholesterol to the same extent. Therefore, the outer membrane can function as a permeability barrier to cholesterol. Cholesterol binding to both whole cells and isolated cell membranes was influenced by the incubation temperature. The rate of cholesterol binding by whole cells of N. gonorrhoeae decreased markedly at lower temperatures, with almost complete cessation of binding at 0 degrees C. A similar temperature effect on the binding of cholesterol to isolated membranes was not observed. Thus, the effect of temperature on the binding of cholesterol to whole cells was an effect not on the actual binding process but rather on the ability of the cholesterol molecule to penetrate the lipid domain of the gonococcal outer membrane.

Extramitochondrial protein synthesis in calf brain synaptosomes.

Isolated synaptosomes of calf brain cortex incorporated labelled amino acids into their mitochondrial, membranous and soluble proteins in an approximate ratio of 1:1:0.5 Synaptosomal protein synthesis was sensitive to ATP, noradrenaline, cycloheximide and puromycin, and together with mitochondrial protein synthesis, also to chloramphenicol, 2.4-dinitrophenol, KCN and hyperosmotic conditions. The absence of Na+ and K+ ions slightly inhibited both synaptosomal and mitochondrial protein synthesis. Using incorporated radioactivity as an indicator of synthesized proteins, the synaptosomal soluble proteins could be obtained in one large peak in gel and indicator of synthesized protein, the synaptosomal soluble proteins could be obtained in one large peak in gel filtration on Sephadex G-100 and G-25, and in two components in disc electrophoresis on a 7% polyacrylamide gel. An approximate molecular weight was calculated for the synthesized proteins using known proteins as standards, giving 15000-35000 in the gel filtration eluant, and 27000 and 36000 in the disc electrophoresis bands.

Sensitivity of the carotid body to within-breath changes in arterial PCO2.

Respiration, sinus nerve chemoreceptor discharge, and carotid arterial pH were monitored in cats. Chemoreceptor discharge frequency showed oscillations that had a respiratory period when averaged over many respiratory cycles. These oscillations disappeared when pH oscillations of respiratory period were eliminated from the carotid arterial blood. The maximum sinus nerve discharge was associated with the most acid point of the recorded pH oscillation. Briefly increasing PCO2 by giving CO2-rich saline into the aortic root resulted in brief reduction in carotid arterial pH, and when this reduction occurred during inspiration tidal volume increased, even with a pH change no larger than the pH oscillations. However, increased chemoreceptor discharge could only be demonstrated when each pH change had twice the amplitude of the pH oscillations. Injections of fixed acid mixed with free carbonic anhydrase transiently increased chemoreceptor frequency, whereas injections of fixed acid alone had no effect. The carotid body is therefore sensitive to small rapid changes in arterial PCO2, and the pH electrode record indicates the size of the stimulus except when fixed acid changes are produced too closely upstream.

Purification and some properties of three forms of glucoamylase from a Rhizopus species.

1. Three forms of glucoamylase [EC 3.2.1.3] were simultaneously purified from a Rhizopus species by (NH4)2SO4 fractionation and successive chromatographies on Sephadex G-75, DEAE-Sephadex, and CM-Sephadex, and were finally separated from each other by means of recycling chromatography on Bio-Gel P-150. The purification achieved was 3--4 fold from crude extract with respect to each glucoamylase; the yields of the three glucoamylases, designated as Gluc1, Gluc2, and Gluc3 in order of content, were 39, 7, and 0.4%, respectively. All the purified enzymes were homogeneous in polyacrylamide gel electrophoresis, isoelectric focusing, and ultracentrifugation. 2. The three glucoamylases were glycoproteins differing in both amino acid composition and carbohydrate content, but showed a common antigenicity in immunodiffusion. The molecular weights of Gluc1, Gluc2, and Gluc3 were estimated to be 74,000, 58,600, and 61,400, respectively, by sedimentation equilibrium and these values were verified by SDS-polyacrylamide gel electrophoresis. The specific activities of the three enzymes toward starch were in the opposite order to their molecular weights. 3. The three glucoamylases had the same broad pH optima in the range pH 4.5--5.0 and shared a common susceptibility to inactivation by heat, extreme pH, and such divalent cations as Hg2+, Pb2+, and Mn2+, indicating close similarity in enzymatic properties.

Net charge and oxygen affinity of human hemoglobin are independent of hemoglobin concentration.

The dependence of net charge and oxygen affinity of human hemoglobin upon hemoglobin concentration was reinvestigated. In contrast to earlier reports from various laboratories, both functional properties of hemoglobin were found to be independent of hemoglobin concentration. Two findings indicate a concentration-independent net charge of carbonmonoxy hemoglobin at pH 6.6: (A) The pH value of a given carbonmonoty hemoglobin solution remains constant at 6.6 when the hemoglobin concentration is raised from 10 to 40 g/dl, indicating that there is no change in protonation of titratable groups of hemoglobin: (b) the net charge of carbonmonoxy hemoglobin as estimated from the Donnan distribution of 22Na+ shows no dependence on hemoglobin concentration in this concentration range. The oxygen affinity of human hemoglobin was determined from measurements of oxygen concentrations in equilibrated samples using a Lex-O2-Con apparatus (Lexington Instruments, Waltham, Mass.). P50 averaged 11.4 mm Hg at 37 degrees C, pH = 7.2, and ionic strength approximately 0.15. Neither P50 nor Hill's n showed any variation with hemoglobin concentrations increasing from 10 to 40 g/dl.

Kleine-Levin syndrome with periodic apnea during hypersomnic stages--E.E.G. study.

A 33 year old male, suffering from Kleine-Levine syndrome associated with periods of apnea during the hypersomnic attacks, is reported. Ventilatory studies negate the Pickwickian syndrome. The E.E.G.'s recorded during the hypersomnic attacks and the apneic periods showed a direct correlation between high-voltage delta waves paroxysmal E.E.G. activity, and apneic period. Medications known to improve Kleine-Levin syndrome, in our case, had no effect upon the clinical hypersomnic and apnea periods, nor on the correlatives E.E.G.'s pattern and spirometric studies. Theoretical considerations let us assume that these paroxysmal E.E.G. patterns associated with apnea are NRem-sleep serotonin dependent, and have an inhibitory influence on the respiratory centers, by alternating the equilibrium between the catecholamines and acetylcholine activities.

The release of a model low-dose drug (riboflavine) from hard gelatin capsule formulations.

The in vitro release of a model low dose drug, riboflavine, from hard gelatin capsules, formulated with a range of diluents, in the absence and presence of magnesium stearate (0.5, 1.0 and 2.0% w/w), has been assessed by a dissolution technique. Comparison of the values of the time for 50% of the drug content of the capsule to appear in solution T50, by analysis of variance, indicated that the type of diluent significantly influenced the drug release. Irrespective of the magnesium stearate content, the diluents could be ranked in the following order of effectiveness: Primojel greater than sodium bicarbonate greater than Avicel congruent to Dri-flo starch congruent to lactose greater than Emcompress congruent to kaolin greater than starch. Correction of the T50 values for possible adsorption of riboflavine onto the water insoluble diluents, using experimentally determined adsorption isotherms, altered the relative order of effectiveness of the diluents to Primojel greater than sodium bicarbonate greater than kaolin congruent to lactose greater than Avicel congruent to Dri-flo starch greater than Emcompress greater than starch. Comparison of the urinary excretion of riboflavine, after administration of capsule formulations containing lactose, Emcompress or kaolin as the diluent, to volunteers, suggests that the dissolution results not corrected for adsorption provide a better indication of the in vivo performance of the formulations.

Effects of antipsychotic and antianxiety drugs on the morphine abstinence syndrome in rats.

The effects of representative antipsychotic and antianxiety drugs on the abstinence syndrome in morphine-dependent rats were compared. Groups of 10 to 22 male albino Sprague-Dawley rats (250-300 g) were individually implanted s.c. with either two 75-mg morphine base pellets or two placebo pellets. After 72 hr, chlorpromazine (CPZ 1,2 and 4 mg/kg), haloperidol (0.2, 0.4 and 0.8 mg/kg), thioridazine (10, 20 and 40 mg/kg), chlordiazepoxide (2,4 and 8 mg/kg), diazepam (DPM, 1, 2 and 4 mg/kg) or vehicle was injected s.c. 55 min before precipitation of abstinence with naloxone (1 mg/kg s.c.). Jumping was exacerbated by CPZ (4 mg/kg), chlordiazepoxide (4 and 8 mg/kg) and DPM (1, 2 and 4 mg/kg); haloperidol and thioridazine had no significant effect on this sign. Weight less over 1 hr was decreased by CPZ (4 mg/kg) and DPM (4 mg/kg). Wet-dog shakes were decreased by all doses of haloperidol but increased by chlordiazepoxide (8 mg/kg) and DPM (1, 2 and 4 mg/kg). CPZ (2 and 4 mg/kg) significantly increased the incidence of teeth chattering. Other abstinence signs were not affected in a dose-related manner. Although the antipsychotic agents each decrease dopamine availability at the postsynaptic receptor, this mechanism alone cannot explain their actions on individual signs of abstinence. Perhaps it is therefore time to question how modifying agents can be meaningfully compared in morphine-abstinent rats.

Antiarrhythmic effect of oxprenolol on halothane-epinephrine and coronary ligation induced ventricular arrhythmias in beagle dogs.

Antiarrhythmic effects of oxprenolol, a beta-blocker, were studied quantitatively on arhythmias produced by epinephrine during halothane anesthesia and by two-stage coronary ligation, and were compared to those of other beta-blockers, propranolol and Kö 1400, which have been already reported. Though oxprenolol has potent beta-blocking activity, the antiarrhythmic effect on halothane-epinephrine arrhythmia was significantly weaker than those of propranolol and Kö 1400. The effective dose of oxprenolol was 60 +/- 18 microgram/kg (mean +/- S.E., N = 6), which is in the range of the so-called beta-adrenergic blocking dose. The weaker antiarrhythmic effect of oxprenolol as compared to propranolol and Kö 1400 is probably due to the intrinsic positive chronotropic effect, which is most clearly observed in oxprenolol as compared to the other two drugs. As for two-stage coronary ligation arrhythmia, oxprenolol suppressed only that observed 48 hours after coronary ligation using higher doses (5 to 10 mg/kg). Other beta-blockers also showed similar effects. Because of the high doses necessary for the antiarrhythmic effects on the coronary ligation arrhythmia, the mechanism for suppressing the arrhythmia is probably due to the local anesthetic action of the beta-blockers.

Isolation and partial characterization of human erythrocyte membrane NADH: (acceptor) oxidoreductase.

The NADH: (acceptor) oxidoreductase (EC 1.6.99.3) was isolated from human erythrocyte ghosts by a procedure including Triton X-100 solubilization, affinity chromatography on an NAD+-Sepharose 4B column, ammonium sulfate precipitation, and isoelectric focusing. This enzyme preparation was characterized by a single band on the urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by a single precipitin line with its corresponding antiserum on double diffusion and immunoelectrophoresis. A 103-fold purification indicates that the oxidoreductase represents approximately 1% of the ghost protein mass. The specific activity of the purified enzyme was 112 units/mg protein. The pH optimum was 6.8 and the isoelectric point, pI, was 6.6 The oxidoreductase has a specificity for NADH as a cofactor. The NADPH was ineffective as a reducing agent. The enzyme activity was strongly temperature-dependent, displaying maximal activity between 35 and 40 degrees C. The energy of activation was 4.9 kcal. The enzyme activity was inhibited by sulfhydryl reagents, anionic detergents, and divalent ions. The amino acid composition of the purified enzyme is characterized by the presence of all common amino acids including half-cystine and tryptophan. The results of carbohydrate and lipid analyses indicated that the oxidoreductase is a glycolipoprotein with fucose, galactose, mannose, and glucosamine as the sugar components and cholesterol and sphingomyelin as the lipid constituents. The apparent subunit molecular weight estimated by urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence and presence of 2-mercaptoethanol was 40,000. The antiserum completely inhibited the enzymic activity at the equivalence point. We suggest that the membrane-bound NADH: (acceptor) oxidoreductase might be a transmembrane protein.

Studies on the glycosidases of semen: purification and properties of alpha-D-mannopyranosidase from goat seminal plasma.

Alpha D-mannosidase activity in goat semen was observed to be distributed in sperm and seminal plasma. In sperm the enzyme, present in soluble and bound forms, was located within the acrosome. The bound enzyme was associated with the denuded sperm. Seminal plasma alpha-mannosidase was purified 100-fold and the final preparation was shown to be homogeneous by polyacrylamide and SDS gel electrophoresis and on isoelectric focusing. The molecular weight of the enzyme, determined by gel filtration and disc electrophoresis in the presence of SDS, was 220,000. The isoelectric pH was 7.42 and the amino acid composition is reported. alpha-Mannosidase catalyzed the hydrolysis of both synthetic and natural substrates. The Km of p-nitrophenyl alpha-D-mannoside and alpha-methyl D-mannoside were 0.695 mM and 71.9 mM at pH 4.0, the optimum pH. The natural substrates were hydrolysed to varying degrees. Zn2+ was not essential though it activated the enzyme activity over longer incubations. The enzyme was observed to be more stable at wider pH range in the presence of Zn2+ than in its absence. EDTA which did not affect the enzyme activity has effect on enzyme stability similar to Zn.2+ Seminal alpha-mannosidase is not a zinc metalloenzyme but is activated by Zn2+.

Time course of changes of extracellular H+ and K+ activities during and after direct electrical stimulation of the brain cortex.

The kinetics of H+ and K+ activities were recorded during and after direct electrical activation of the brain cortex (cat). H+ activity was measured with H+-sensitive glass microelectrodes (tip diameters of 1--4 micron) and K+ activity was registered with double-barrelled ion-sensitive microelectrodes (tip diameters of 1--3 micron). It could be shown that extracellular H+ activity initially decreased for a few seconds and increased only after the 7.s. Maximum acidosis was always noticed after stimulation ended. Alkalotic as well as acidotic changes were the higher the stronger the stimulation parameters were. K+ activity increased very rapidly after stimulation began, reached its maximum when stimulation ended and then decreased to its initial value with an undershoot. It is concluded that the functional hyperemia of microflow could be triggered by the rapid increase in K+ activity, whereas the initial alkalotic change of extracellular pH means that H+ activity does not play a role in the first phase of this kind of hyperemia. The alkalotic shift is interpreted to be caused by the washout of C02 due to the rapid increase in microflow. In the further course, H+ activity obviously contributes to the maintenance of functional hyperemia. In this later period K+ activity is always below the control value.

Some critical qualitative details of lorica construction in the type species of Calliacantha Leadbeater (Choanoflagellata).

Qualitative structural details, amplifying or correcting previous accounts in the literature on this species have been compiled by means of light and/or electron microscopy of dry whole mounts, prepared in situ from freshly gathered wild material mainly from temperate sources in Denmark, Britain and south Alaska. The more important findings are summarized diagrammatically. These include elaborate and constant details of assembly at the anterior end of the lorica, combined with much greater variability at the hind end. The absence of continuity between the three anterior spines and any of the six longitudinal costae present at the front end of the lorica chamber is confirmed, but a range of conditions involving numerical reduction in costal numbers at the hind end is illustrated. The overall size range encountered in the three temperate localities listed is illustrated photographically, but the large cells characteristic of arctic sources are represented, for a qualitative purpose only, by means of a single photograph recording a specimen collected beneath sea ice in N. Alaska, a source which will be considered more fully on a later occasion.

Streptococcus mutans, an assessment of its physiological potential in relation to dental caries.

Streptococcus mutans converts low levels of sucrose to lactic acid, but at high levels favours synthesis of glucans for plaque accumulation. Thus, the continued exposure to sucrose fluxes would select microorganisms in the oral cavity (S. mutans being a prototype) with highly specialized adaptation and potential dental caries activity. The bacteria that have evolved physiological systems to function efficiently under these conditions are the lactic acid bacteria. These organisms survive in environments where carbohydrate availability is constantly changing. High tolerances to acidic environments may be an important determinant in establishing the ecology of the carious lesion. Also, the intercellular polysaccharide storgae (glycogenamylopectin) and extracellular polymer reserves (levan and soluble glucan) are important during carbohydrate depletion. Further, the formation of insoluble glucans is a prerequisite for the caries process on smooth surfaces of teeth through plaque development. These conditions could result in an increase in S. mutans and cariogenic microorganisms. As a result, this process may be best understood as a manifestation of an amphibiotic shift.

[Abnormal involuntary movements in the elderly and their treatment (author's transl)].

In the elderly, there are two main types of abnormal involuntary movements: tremors on one hand and dyskinesias on the other. Among tremors, senile and parkinsonian types have to be separated because they have different semeiologic signs and distinct therapeutics. Senile tremor is present during movements and tonically maintained attitude. It affects upper extremities (often asymmetrically) and the head; it is reduced by alcohol. When possible (in the absence of contrindications) its best treatment is by beta-blockers. Parkinsonian tremor is typically present at rest and is reduced by a voluntary movement. L-dopa is active but in the elderly the dosis has to be reduced. Dyskinesias are repetitive but not rythmic involuntary movements which are made at the speed of a normal movement. There are at least two types of dyskinesias: spontaneous and post neuroleptics (i.e. tradive). Spontaneous dyskinesias essentially involve the axial muscles and are chiefly bucco-linguo-facial. They are well controlled by various neuroleptics. If eventual side effects are taken into account, tiapride appears to represent the good choice. Tardive dyskinesias do not disappear when responsible neuroleptics are stopped and are usually permanent. Paradoxically, when necessary, their treatment consists in resuming a neuroleptic prescription.

Evidence for altered cyclic nucleotide metabolism during compensatory renal hypertrophy and neonatal kidney growth.

In adult male Sprague-Dawley rats contralateral nephrectomy was followed by an initial fall of the concentration of cGMP in renal cortical tissue followed by a rise to a peak level of 300 percent of the initial concentration within two hours. cGMP concentration in the remaining renal cortex remained at about 300 percent of the initial value during the subsequent 72 hours and slowly declined to 150-200 percent in the following two weeks. The changes in cGMP concentration were due to exactly parallel changes in the soluble fraction of renal cortical guanylate cyclase activity, while cGMP-phosphodiesterase activity remained unchanged. cAMP concentration after contralateral nephrectomy fell significantly by about 25 percent within two hours and remained below baseline level for up to eight hours. In the kidneys of newborn rats the concentration of cAMP was approximately one-half that found in adult kidneys: it slightly fell between the fourth and the seventh day after birth and subsequently continuously rose to reach adult values approximately two weeks after birth. The concentration of cGMP was significantly greater four days after birth than in adult rats, further rose between the fourth and the seventh day after birth and subsequently gradually declined to adult levels. The increased cGMP concentration appears to be due to an increase of guanylate cyclase activity in total kidney homogenates which, in turn, was mainly due to an increase of the particulate (membrane-bound) fraction of the enzyme. cGMP-phosphodiesterase activity, however, was also increased in respect to adult levels, one or three weeks after birth. Renal growth from the seventh day after birth to adulthood is accompanied by a continuous increase of the ratio cAMP/cGMP. Removal of one kidney four to seven days after birth resulted in a slower increase of this ratio. The data suggest that cGMP may trigger renal growth and that increases of cGMP concentration in the kidneys are the result of a primary increase in the activity of guanylate cyclase.

[Aqueous iodine solutions as disinfectants: composition, stability, comparison with chlorine and bromine solution (author's transl)].

The equilibrium concentrations of aqueous iodine solutions in dependence of the total concentration and the pH-value have been calculated with and without regard of the iodate formation. The values obtained by the latter methode enabled by application of the known rate law to calculate the initial rate of the iodate formation and to draw from this conclusions concerning the stability of iodine solutions. On the grounds of these calculations to aqueous iodine solutions in the concentration and pH-range which is relevant for disinfection (greater than 10(-5) M/l, pH 6--9) one can attribute a stability sufficient for the use in practice and - unlike chlorine and bromine solutions - a content of bactericidal "free halogene" which is higher and independent of the pH-value. The disinfecting action of the iodine cation (H2O+J) which is supposed to be very powerful can be neglected because of its low concentration (10(-3)--10(-6%) of the total concentration). Hypoiodic acid which has already been converted into iodate by disproportionation is as good as lost for the disinfection because of the extremely slow reverse reaction.

Studies on Trypanosoma (nannomonas) congolense. I. On the morphological appearance of the parasite in the mouse.

The pleomorphism of bloodstream Trypanosoma (Nannomonas) congolense was studied during the course of the first parasitaemic wave in mice using cloned and uncloned derivatives of three recent field isolates. The different morphological types were identified using the criteria described by Godfrey (1960). It was found that at any point of parasitaemia there were several morphological types of the parasite present, ranging from short to long forms. In the rising phase of parasitaemia, the short forms predominated, while at peak parasitaemia the parasites were highly pleomorphic, with significant proportions of short and "intermediate" forms although the long forms predominated. Pleomorphism was observed both in normal and in lethally irradiated (900 R) mice, even when the infection was initiated using a single organism. Such pleomorphism may result from physiological differences between the different forms of this parasite since these morphological types of T. congolense also differed in their ability to infect a new mammalian host.

Anesthesia for cesarean section: further studies.

This study was designed to re-evaluate neonatal condition at birth following elective cesarean section performed with epidural anesthesia and a modified technique of general anesthesia. Two groups of 20 patients were studied. Twenty received epidural anesthesia with 2 per cent lidocaine-carbon dioxide-epinephrine, and 20 patients were given general anesthesia. Modifications of our previous general anesthetic technique included the administration, to the mother, of high inspired concentrations of oxygen (66 per cent) prior to delivery, short induction-to-delivery intervals, and positioning of the mother in a 20 degrees left lateral tilt position. No significant differences in oxygen tension and acid-base balance in umbilical venous and arterial blood were demonstrated between the two sets of neonates. One-and five-minute Apgar scores and time to sustained respiration were similar in both groups. Our observations of the infants immediately after delivery led us to conclude that either anesthesia technique is acceptable for elective cesarean section.

Injuries in soccer.

The injury profile of a professional soccer team from the 1976 to 1977 season is compiled in this report. Injuries are spread across all of the playing positions, with midfielders and forwards being most prone to injury. During the entire season of this report, 60 injuries were encountered, 35 during game situations and 25 in practice. The most common injuries noted were foot and ankle sprains, but muscle strains were also frequent. Most injuries were of a minor variety. Seventeen injuries resulted in missed games while 46 injuries resulted in no games being missed. Only one player had an injury which did not allow him to return to play during the season. Although little is written in the American literature concerning soccer injuries, it does appear that the injury potential is much less when compared with that of football. The rapid increase in interest in this sport will require physicians and trainers to become familiar with its specific needs in order to render adequate care and participate in injury prevention programs.

[Plasma concentration of fentanyl during and after its administration at constant flow].

Using the technique of the radio-immunological estimation with fentanyl-H3, a study was made in sixteen adults anaesthetised by the administration at a constant rate of alfadione and fentanyl, of plasma concentrations of fentanyl during and after anaesthesia. Anaesthesia was induced by the administration of 4.2ml of alfadione and 0.084mg of fentanyl. The maintenance dose was 0.147 ml/kg/hour of alfadione and 2.95 microgram/hg/hour of fentanyl. Five minutes after induction, the concentration of fentanyl was 2.7 microgram/l. The level fell significantly to 2.56 microgram/l at the 45th minute. From this point onwards, it increased regularly up to the 120th minute, when it reached a level of 3.7 microgram/l. When the infusion was stopped, the level first decreased rapidly, the excretion curve then becoming flattened out. At the 120th minute, a level of 1 microgram/l persisted. This study indicates that the administration of fentanyl at a constant rate is not accompanied by a constant blood concentration up to the 120th minute, the point at which the study was terminated. The residual level found after administration and in the absence of any clinical effect implies the need for a reduction in dose at the time of any complementary administration of fentanyl during the postoperative period.

Effects of glucose, pH, and dissolved-oxygen tension on Bacillus cereus growth and permeability factor production in batch culture.

The production of a Bacillus cereus enterotoxin, measured as rabbit skin permeability factor (PF), in response to differences in glucose availability, pH, and dissolved oxygen tension was studied in a 1-liter batch fermentor system. Glucose had to be present for toxigenesis to occur. In uncontrolled fermentation an increasing inhibition of PF production and growth occurred as pH dropped occurred below 6.5. Optimum pH for toxigenesis was 7.0 to 7.5, and fermentations maintained at this level yielded 10- to 20-fold more PF than comparable uncontrolled fermentations. PF production was appreciably diminished at or below pH 6.0 and at or above pH 8.5. Peak PF titer was associated with a drop in acid output, and the titrant utilization profile could be used as an indication of this point. Productivity was greatest in the early exponential phase of growth and decreased to zero at the transition phase. Differences in dissolved oxygen tension affected both the maximum productivity early in the fermentation and the rate of its decrease as growth progressed. The optimum dissolved oxygen tension for toxigenesis was 0.002 atm, and the most rapid growth occurred at 0.10 atm. Productivity and growth were reduced under anerobic conditions, whereas a hyperoxic environment severely reduced productivity, but not growth. Postexponential-phase loss of toxic activity coincided with a rapid increase in cellular oxygen demand. Neither was inhibited by the presence of glucose. However, PF loss was completely prevented by stringent oxygen limitation. Extracellular proteolytic activity did not appear to be responsible for the loss of toxic activity.

The kinetics of the active and de-energized transport of O-methyl glucose in Ustilago maydis.

The kinetics of the uptake and efflux of 3-O-methyl-glucose in sporidia of Ustilago maydis were measured, both in active cells and in cells whose metabolic activity had been inhibited by azide and iodoacetate. The de-energized transport system proved to be carrier mediated with apparent affinity constants 13 +/- 2 mM outside (Ko) and 18 +/- 2 mM inside (K1). The apparent maximum rate constants for the same system were 0.66 +/- 0.05 mmol/1 cell water per min for uptake (V+) and 0.53 +/- 0.04 mmol/l cell water per min for efflux (V-). For the active system K0 = 0.08 +/- 0.01, K1 greater than 40, V+ = 9.7 +/- 0.5 and V- = 1.1 +/- 0.9 (in equivalent units). These results are discussed in the context of the carrier mechanism as proposed by Regen and Morgan (Regen, D.M. and Morgan, H.E. (1964) Biochim. Biophys. Acta 79, 151--166). The antifungal compound carboxin had no effect on de-energized transport but was shown to decrease both K0 And V+ in the active system. Phloretin and phlorizin were also found to be without effect on de-energized cells but the former enhanced while the latter inhibited active uptake.

The thermal unfolding of ribonuclease A. A 13C NMR study.

The 13C nuclear magnetic resonance (NMR) spectra of ribonuclease A over the pH range 1-7 and between 6 and 70 degrees C reveal many of the details of its reversible unfolding. Although the unfolding may loosely be described as 'two-state', evidence is presented for intermediate unfolding stages at least 10 degrees C on either side of the main unfolding transition, particularly at low pH. The first residues to unfold are 17-24, in agreement with other results. The C-terminal region shows a steeper temperature dependence of its unfolding than does the main transition, which itself is shown to lead at all pH values to a semi-structured but internally flexible state which is far from being truly random-coil. This is confirmed by measurements of T1 and of nuclear Overhauser enhancement. Indeed, even at pH 1.1 and 70 degrees C there is evidence for considerable motional restriction of cysteine and proline residues, amongst others. The native protein has more variability of structure at low pH than at neutral pH, and also interchanges more rapidly with the semi-structured, denatured state.

[Effect of glucose and its derivatives on systems of riboflavin uptake and excretion in the yeast Pichia guilliermondii].

Riboflavin uptake by washed cells of riboflavin deficient mutant MS1-3 of Pichia guilliermondii yeast was strongly depressed by D-glucose, L-sorbose, alpha-methyl-D-glucoside, sucrose, trehalose, maltose and salicin but not by D-mannose, D-galactose, D-fructose or ribitol. Glucose decreased also the initial uptake rate of riboflavin analogue, 8-piperidyl-10-(1'-D-galactityl) isoalloxazine; the inhibition having a competitive character (Ki==5,7 mM). Apparently riboflavin permease is able to accept not only riboflavin and its analogues but also glucose and some of glucose derivates. Cells preloaded with riboflavin and transferred into riboflavin-free medium excreted vitamin B2 into the medium. This excretion was strongly stimulated by D-glucose, D-fructose, D-mannose but not by citrate or succinate. In contrast to riboflavin, 8-piperidyl-10-(1'-D-galactityl) isoalloxazine was not excreted into the medium even in the presence of glucose. The rate of riboflavin excretion depended on temperature and pH of incubation medium (pH optimum approximately 7.0) and was decreased in the presence of different inhibitors of energy metabolism. It seems that the exit of riboflavin from the cells is accomplished by energy-dependent specific system of excretion (excretase) which in some properties is different from that of riboflavin permease.

The metabolism of 5-hydroxytryptamine and beta-phenylethylamine in perfused rat lung and in vitro.

1 Metabolism of 5-hydroxytryptamine (5-HT) and beta-phenylethylamine (PHE) by monoamine oxidase (MAO) was investigated in rat isolated lungs and in mitochondrial preparations from rat lung. 2. In perfused lungs 5-HT metabolism had an apparent Km of 2 microgram and PHE metaoblism a Km of 54 microgram, whereas in vitro the Km values were 330 microgram and 28 microgram respectively. 3 In vitro, MAO activity had substrate and inhibitor specificities compatible with the presence of A and B types of MAO. 4 In perfused lung, metabolism of 5-HT but not that of PHE was inhibited by desmethylimipramine. 5 These results show that PHE metabolism in perfused lung, unlike that of other metabolized amines, is not limited by transport and the transport process for PHE is unlike that of 5-HT or noradrenaline. 6 These results also show that the kinetic parameters obtained for MAO activity in vitro do not generally apply to the isolated lung where transport of substrate can be the deciding factor. This discrepancy emphasizes that the enzymic properties of the whole organ cannot relaibly be deduced from its enzymic content.

Neurotransmitter synthesis, storage and release by aggregating cell cultures of rat brain.

Rotation-mediated aggregating cell cultures of mechanically dissociated fetal (15-16 days gestation) rat brains between 25 and 35 days in vitro were examined for their ability to synthesize neurotransmitters and putative neurotransmitters from radioactively labeled precursors added to the culture medium. Cultures derived from whole brain synthesized [3H]acetylcholine from [3H]choline, [3H]gamma-aminobutyric acid from L-[3H]glutamic acid, [3H]dopamine from L-[3H]tyrosine, [3H]dopamine and [3H]norepinephrine from L-[3H]dihydroxyphenylalanine, and [3H]serotonin from L-[3H]tryptophan. Veratridine increased and tetrodotoxin decreased the rate of [3H]-dopamine synthesized by aggregates derived from midbrain plus hindbrain. In chase experiments in which aggregates were incubated for 4 h with radioactively labeled precursors and then for 4 h with non-radioactively labeled precursors, addition of veratridine (50 micronM) during the second 4 h incubation significantly decreased the amounts of radioactively labeled acetylcholine, L-glutamic acid, dopamine and serotonin recovered from aggregates. Tetrodotoxin (5 micronM) present during the chase significantly increased the amounts of [3H]acetylcholine and [3H]dopamine recovered from the aggregates. In addition, reserpine (4 micronM) markedly depleted [3H]dopamine from aggregates in these experiments. These results indicate that these cultured cells synthesized neurotransmitters and in addition suggest that some of these compounds are stored by and released from electrically active cells within the aggregates.

The dilating effect of histamine on pial arteries of cats and its mediation by H2 receptors.

We studied the effect of histamine and H1 or H2 blockers on the diameter of pial arteries (39-227 micron) using microapplication into the perivascular space. Concentration-response curves for histamine showed dilations which started at 10(-7) M and were maximal at 10(-5) and 10(-4) M. The H2 blocker, cimetidine, induced no vascular reaction over the whole concentration range tested (10(-7) to 10(-3) M). The H1 blocker, mepyramine, was not vasoactive in the concentration range from 10(-7) to 5 X 10(-5) M and evoked dilations at higher concentrations. The concentration-response curve for histamine was only slightly displaced by 10(-5) M mepyramine but was significantly shifted to the right by 10(-5) M cimetidine. The dilating effect of histamine could be reduced in a stepwise manner by increasing concentrations of cimetidine. These findings are in accordance with a selective antagonism between histamine and cimetidine at the H2 receptors of smooth muscle cells of pial arteries. The insignificant role of H1 receptors in histamine-induced dilations is supported by the finding that a combination of H1 and H2 blockers resulted in the same reduction of histamine-induced dilation as did the application of the H2 blocker.

The influence of the intrinsic sympathomimetic activity of beta-adrenoceptor antagonists on haemodynamic effects in anaesthetized dogs.

1. The effects of propranolol, atenolol (ICI 66,082), practolol and pindolol on heart rate and maximal left ventricular dp/dt, atrioventricular conduction time, mean aortic flow and diastolic blood pressure during cardiac pacing were investigated over a wide dose range (0.025-4.0 mg/kg, i.v.) in dogs anaesthetized with pentobarbitone.2. Propranolol and atenolol produced similar reductions in haemodynamic parameters. Propranolol had no further effect in dogs pretreated with atenolol. 3. Practolol tended to cause smaller reductions in the haemodynamic parameters than either propranolol or atenolol. Subsequent administration of propranolol still had some depressant activity. 4. Pindolol produced a biphasic response, with depression of cardiac function at the low doses (0.025 and 0.1 mg/kg), but a reversal of effect as the dose was increased. 5. It is therefore concluded that, in anaesthetized dogs, the intrinsic activity of practolol and pindolol limits the fall in heart rate, cardiac conduction, aortic flow and maximal dp/dt observed with beta-adrenoceptor blockade. With pindolol, however, the influence of intrinsic activity is observed only in high doses related to beta-adrenoceptor blockade.

The effect of established beta-adrenoreceptor-blocking therapy on the release of cytosolic and lysosomal enzymes after acute myocardial infarction in man.

1. Serial venous blood samples were obtained from 45 patients with acute myocardial infarction. Ten of these patients were receiving beta-adreno-receptor-blocking drugs at the time of onset of chest pain and continued on these drugs during their stay in the coronary care unit. The activities of creatine kinase and its MB-isoenzyme (CK-MB) were assayed in the plasma. A lysosomal enzyme, beta-N-acetylglucosaminidase, was also assayed. 2. In the 35 untreated patients it was found that creatine kinase activity was maximal at a mean time of 21.3 +/- 1.3 h after the onset of chest pain, whereas in the patients receiving beta-adrenoreceptor-blocking drugs peak activity of the enzyme occurred at 24.4 +/- 0.7 h. 3. Peak CK-MB acitivity was also delayed from 18.1 +/- 1.6 h in the control group to 22.4 +/- 1.2 h in the treated patients. 4. The lysosomal enzyme showed a similar pattern of changes to that of CK-MB. Maximum activity in plasma occurred at 18.0 +/- 1.0 h after the onset of chest pain in the control group of patients. In the treated patients peak lysosomal enzyme activity was not found until 24.2 +/- 1.2 h. 5. These alterations in the time-course of plasma enzyme changes after acute myocardial infarction are consistent with the suggestion that beta-receptor antagonists may delay tissue damage during myocardial ischaemia.

Trimethoprim-induced elevation of dihydrofolate reductase activity in human leukocytes.

The administration of trimethoprim (TMP)--a diamino benzylpyrimidine compound which binds very tightly the bacterial dihydrofolate reductase--was accompanied by the appearance of measurable levels of dihydrofolate reductase in peripheral leukocytes from patients with nonhematological diseases. In all instances, enzyme activity rose rapidly between the fourth and eighth day after TMP. The time course of the rise and fall of dihydrofolate activity approaches cellular life span and is similar to that obtained after methotrexate or triamterene administration. Dihydrofolate reductases, partially purified from leukocytes of patients treated with TMP, bone marrow and leukemic leukocytes, had simila molecular weights, pH optima, Ki of inhibitor (methotrexate); they were stimulated to the same degree by KCl and urea. Electrophoresis of the enzyme on cellulose acetate strip resulted in the separation of two enzymatically active protein components. No differences in the electrophoretic behavior of the three blood cell enzymes were noted. The findings noted above are consistent with the suggestion that the observed rise in dihydrofolate reductase activity is a quantitative one. Moreover, the effect of TMP in vivo is discussed in comparison with the currently held hypothesis for methotrexate action (stabilization by the drug of a previously synthetized enzyme).

Calcium-induced inactivation of microtubule formation in brain extracts. Presence of a calcium-dependent protease acting on polymerization-stimulating microtubule-associated proteins.

Incubation of brain extracts in the presence of 1 mM CaCl2 results in the permanent loss of tubulin polymerization, even after later addition of ethyleneglycol-bis(beta-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), when assembly conditions are chosen which rely on the presence of microtubule-associated proteins (such as MAP1 and MAP2). Purified microtubular protein, by contrast, recovers readily from calcium inhibition by the later addition of EGTA. Mixing experiments, using purified microtubular protein and brain extract, show that permanent loss of tubulin assembly is always accompanied by proteolysis of high-molecular-weight microtubular-associated proteins. Addition of purified protein MAP2 after chelation of calcium by EGTA, immediately restores microtubule assembly. Furthermore, substitution of guanosine 5'-[alpha, beta-methylene]triphosphate for GTP after EGTA treatment results in the typical tubulin polymerization process, which is independent of the presence of microtubule-associated proteins. Thus, the proteolytic action of a calcium-dependent protease is specific for high-molecular-weight microtubule-associated proteins and not tubulin itself. The protease is soluble and therefore removing during the purification of microtubular protein by cycles of temperature-dependent polymerization and depolymerization. We discuss the potential physiological importance of this calcium-dependent protease.

Treating dysmenorrhea with anti-inflammatory agents: a double-blind trial with naproxen sodium.

Thirty-two dysmenorrheic patients participated in a double-blind trial of naproxen sodium for three consecutive menstrual cycles. The women were divided into two groups: 15 women were given naproxen sodium (the sodium salt of d-2-(6-methoxy-2-naphthyl) propionic acid) and 17 women received placebo tablets. The women were prescribed two tablets (550 mg) at the first sign of menstrual pain and one tablet (275 mg) thereafter every six hours, as required. There were no significant differences between the two groups in physical characteristics, obstetric and gynecologic histories, including the character of dysmenorrhea and pretreatment pain intensity scores (p = 0.7). Following intake of the drug or placebo, the participants rated the relief provided by the medication with a six-point scoring system. When the scores for pain relief were tallied for the three treatment cycles, the naproxen sodium group averaged 13.7 +/- 0.65 standard error, while the placebo group averaged 8.8 +/- 0.95 standard error out of a possible maximum relief score of 18. The difference between the two groups was statistically significant at p = 0.0004. Few patients reported side effects.

The capacity of microsomally-activated cyclophosphamide to induce immunosuppression in vitro.

Cyclophosphamide (CY) was activated in vitro with washed rat liver microsomes and cofactors. Pretreatment of mouse spleen cells in vitro with the activated drug abolished their capacity to give a primary antibody response to SRBC and levan on transfer to irradiated syngeneic recipients. However, responsiveness returned if challenge was delayed for 7 or more days after transfer. Part of this was shown to be of donor origin by an allotype marker. The treatment of normal spleen cells with activated CY in vitro also prevented B cells from regenerating their immunoglobulin receptors after capping with anti-immunoglobulin serum. The induction of suppression required contact between lymphocytes and activated CY for at least 30 min at 37 degrees and did not appear following incubation for 1 h at 0 degrees. Since the antibody response of drug-treated spleen cells to SRBC could not be restored with purified normal B or T cells, it is probable that B and T lymphocytes are both susceptible to suppression by activated CY in vitro. Similar pretreatment abrogated the graft-versus-host (GVH) reactivity of spleen cells as measured by survival and in a popliteal lymph node assay. B cell chimerism in F1 recipients of drug-treated parental spleen cells was demonstrated by the presence of congenic allotype markers. This suggests a possible approach for the attenuation of GVH disease which is associated with bone marrow transplantation in man.

Further characterization of the reduced nicotinamide adenine dinucleotide phosphate: nitrate oxidoreductase in Aspergillus nidulans.

The reduced nicotinamide adenine dinucleotide phosphate (NADPH):nitrate oxidoreductase (EC 1.6.6.2) from Aspergillus nidulans wild-type bi-1 was purified by means of salt fractionation, gel filtration, affinity chromatography, and polyacrylamide gel electrophoresis. Enzyme which was adsorbed on Cibacron blue agarose could be eluted with 2 mM NADPH or 2 mM oxidized NADP (NADP(+)), the former being about three times more effective than the latter. About half the total NADPH:nitrate reductase activity adsorbed on agarose required elution with 1 M NaCl. This salt-elutable form remained active with NADPH and was not converted to the NADPH-elutable form after readsorption on Cibacron blue agarose. The NADPH-eluted enzyme exhibited a markedly different electrophoretic mobility than the enzyme eluted with NADP(+) or NaCl. After electrophoresis on polyacrylamide gels, the NADPH-eluted NADPH:nitrate reductase was separated into four proteins, two of which contained nonheme iron and exhibited reduced methyl viologen-nitrate reductase activity. None of these proteins, singly or in combination, reduced nitrate with NADPH as substrate. Difference spectra analyses and specific heme iron stains revealed the presence of cytochrome b(557) in the largest of the proteins. The molecular weights of the four proteins, which were determined from the relationship of their mobilities on varied concentrations of acrylamide gel, were 360,000, 300,000, 240,000, and 118,000. The subunit molecular weights of these, which are determined via sodium dodecyl sulfate slab gel electrophoresis, were 49,000, 50,000, and 75,000. The key role of NADPH in maintenance of the active form of the heteromultimer is further substantiated.

Transport of coenzyme M (2-mercaptoethanesulfonic acid) in Methanobacterium ruminantium.

A system for transport of coenzyme M, 2-mercaptoethanesulfonic acid (HS--CoM), in Methanobacterium ruminatium strain M1 required energy, showed saturation kinetics, and concentrated the coenzyme against a gradient. The process was sensitive to temperature and was maximally active at pH 7.1. Cells took up HS--CoM at a linear rate, with a Vmax of 312 pmol/min per mg (dry weight) and an apparent Km of 73 nM. An intracellular pool of up to 5 mM accumulated which was not exchangeable with the medium. Uptake required both hydrogen and carbon dioxide; it was inhibited by O2. Bromoethanesulfonic acid (BrCH2CH2SO3-), a potent inhibitor of methanogenesis in cell-free extracts, inhibited both uptake and methane production. Results of inhibitor studies with derivatives and analogs of the coenzyme showed that the specificity of the carrier is restricted to a limited range of thioether, thioester, and thiocarbonate derivatives. 2-(Methylthio)ethanesulfonic acid (CH3--S--CoM) showed an apparent Ki for HS--CoM uptake of 15 nM, being taken up itself with a Vmax of 320 pmol/min per mg (dry weight) and an apparent Km of 50 nM. An analysis of intracellular pools after HS--CoM uptake indicated that the predominant forms are a heterodisulfide of unknown composition and CH3--S--CoM.

Phospholipid-deacylating enzymes of rat small intestinal mucosa.

1. Two phospholipase activities, provisionally designated as phospholipase activity I and phospholipase activity II, were found to be present in the mucosal homogenates of rat small intestine. These phospholipase activities were present in the membraneous particle fraction and were characterized in this study without further purification, using phosphatidylcholine as a substrate. Phospholipase activity I was assayed at pH 5.9 in the absence of deoxycholate, whereas phospholipase activity II was assayed at pH 9.4 in the presence of deoxycholate. Phospholipase activity I was more easily inactivated by heat treatment and trypsin digestion than phospholipase activity II. Both phospholipase activities were inhibited by diisopropyl-fluorophosphate but not by SH-binding reagents. 2. Phospholipase activity I had a pH optimum at 5.9. A sigmoid curve was obtained when the amount of the enzyme preparation was plotted against the phospholipase activity I. The unusually low activity found at low enzyme concentrations was enhanced by addition of the heat-inactivated enzyme preparation to a level where a linear relationship was found between the amount of enzyme and the activity. The effector present in the enzyme preparation was tentatively identified as fatty acid(s). The addition of oleic acid or linoleic acid to the incubation mixture enhanced the phospholipase activity I. At 1 mM levels of these fatty acids the highest activity was obtained when 1.5 mM phosphatidylcholine was used as a substrate. 3. The phospholipase activity II increased on addition of deoxycholate. In the presence of 5 mM deoxycholate, a pH optimum was found at 9.6. It was found that the maximal extent of hydrolysis of phosphatidylcholine in the incubation mixture was dependent on the concentration of deoxycholate. This indicates that deoxycholate facilitates the action of phospholipase activity II, presumably by forming deoxycholate-phosphatidylcholine mixed micelles. Phospholipase activity II was found to deacylate specifically the 2-acyl moiety of phospholipids.

In vitro phosphorylation of bovine adrenal tyrosine hydroxylase by adenosine 3':5'-monophosphate-dependent protein kinase.

We have studied the effects of adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase on the phosphorylative and functional modification of bovine adrenal tyrosine hydroxylase. Incubation of partially purified tyrosine hydroxylase with cAMP-dependent protein kinase in the presence of [gamma32P]ATP and 5 micron cAMP led to a 3- to 5-fold activation of tyrosine hydroxylase and to incorporation of [32P]phosphate into protein. When tyrosine hydroxylase preparations activated by exposure to enzymatic phosphorylating conditions were analyzed by sucrose density gradient centrifugation, polyacrylamide gel electrophoresis, and gel electrofocusing, the radioactivity of 32P was coincident with the activity of tyrosine hydroxylase, suggesting incorporation of 32P from [gamma-32P]ATP into tyrosine hydroxylase. Polyacrylamide gel electrophoresis of the phosphorylated tyrosine hydroxylase preparation in the presence of 0.1% sodium dodecyl sulfate revealed that the 60,000-dalton polypeptide subunit of tyrosine hydroxylase served as the phosphate acceptor.

A comparison by 220-MHz NMR of histidine hydronium ion titrations in porcine pancreatic ribonuclease and an extensively deglycosylated derivative.

220-MHz NMR was used to observe the titration behavior of the 5 histidine residues in porcine pancreatic ribonuclease (ribonucleate pyrimidine-nucleotido-2'-transferase (cyclizing), EC 3.1.4.22) and a derivative prepared by removal of 80% of the attached carbohydrate from this glycoprotein. Resonances due to histidine C-2 protons were observed over the full pH range for 3 of the residues; such resonances for the remaining 2 histidine residues broadened out as the pH was increased. Resonances due to histidine C-4 protons were also observed for 2 of the residues. The titration curves for both proteins were identical within experimental error. Resonances were assigned by comparison with histidine NMR titrations in ribonucleases from other species. Histidine 105, immediately adjacent to the site of attachment of a heterosaccharide side chain, has a C-2 proton chemical shift and pK that are insensitive to the large alteration in the bulk of the carbohydrate side chain. The chemical shifts of the C-2 proton of histidine 48 and of the C-4 proton of histidine 80, histidine residues that are close to one another and to another heterosaccharide side chain, show a similar insensitivity. The observations are direct evidence in support of the thesis that the heterosaccharides in porcine ribonuclease project away from the surface of the protein into the solution environment.

Regulation of rat hepatic stearoyl coenzyme A desaturase. The roles of insulin and carbohydrate.

The rat hepatic stearoyl-CoA desaturation decreased by 3.7-fold in streptozotocin-induced diabetes. Insulin treatment of diabetic rats increased the enzyme activity by 7-fold. In marked contrast to glucose administration, fructose feeding in diabetic rats resulted in 20-fold stimulation of stearoyl-CoA desaturation, although both carbohydrates stimulated stearoyl-CoA desaturation in normal rats. Measurement of the microsomal electron transfer components showed no significant changes in the NADH-cytochrome b5 reductase activity or in the concentration of cytochrome b5. However, the activity of the terminal desaturase changed in a parallel fashion as the amount of terminal desaturase reflect changes in the overall desaturation. Supplementation of various microsomes with the saturating amount of purified terminal desaturase resulted in the formation of similar amounts of catalytically active complex and increased the stearoyl-CoA desaturation to the same level suggesting that the changes in the amount of terminal desaturase reflect changes in the overall desaturation. The results support the suggestion that both insulin and the intermediates of carbohydrate metabolism are involved in the regulation of terminal desaturase.

Gas-liquid chromatography of submicrogram amounts of drugs. V. Preparation of low-activity packed columns and their application to the toxicological analysis of underivatized polar drugs in the low nanogram range.

When diatomaceous earth and glass columns are acylated prior to coating with a silicone liquid phase, then subjected to heat treatment in an atmosphere of nitrogen, gas chromatographic columns can be prepared that show a marked reduction in adsorption. These columns can be used with a nitrogen-specific detector to chromatograph unmodified polar compounds such as morphine and cyclobarbital in nanogram amounts. Virtually no alteration of peak shape and no variation of retention time are observed over the range 10(-6)-10(-9) g of polar drugs. This represents, for these polar drugs, an "improvement" in chromatographic capability of the order of about 1000-fold in comparison with the best conventional commercial columns. Application to toxicological analysis of morphine in urine is described.

The role of H-2 and Ia antigens in graft-versus-host reactions (GVHR). Presence of host alloantigens on donor cells after GVHR and suppression of GVHR with an anti-Ia antiserum against hose Ia antigens.

By using an indirect immunofluorescence technique, the presence of host cell derived H-2K, H-2D, and Ia alloantigens on donor cells recovered from recipient spleens after a graft-versus-host response (GVHR) was demonstrated. Mapping studies indicated that only host K, D, and I-A region gene products could be identified on the donor cells. Host I-E/C- and I-J-subregion products were not absorbed by donor cells. Treatment of activated donor cells with anti-Ly sera plus C' revealed that donor cells carrying host Ia antigens have a Ly-1+,2-,3- phenotype, whereas donor cells carrying H-2K and H-2D host antigens have a Ly-1-,2+,3+ phenotype. A GVHR that resulted from only an I-region incompatibility was suppressed by the injection of recipient mice with an anti-Ia antiserum directed against self Ia antigens. The degree of suppression was proportional to the amount of anti-Ia antiserum administered.

Improved gas chromatographic method and micro-extraction technique for the measurement of nicotine in biological fluids.

A rapid and sensitive method for the measurement of nicotine in plasma, urine, saliva and breast milk is described. An internal standard (quinoline) is added to the samples and these are made alkaline and extracted with diethyl ether. The solvent is evaporated to small bulk and extracted with dilute acid which is then made alkaline. The nicotine is finally extracted into butyl acetate and an aliquot of this extract is injected onto a gas-chromatograph fitted with a nitrogen detector. Quantitation relies on comparison of peak areas and the calibration curve is linear over the concentration range 0.5 to 100 ng ml-1. Nicotine concentrations as low as 0.1 ng ml-1 can be measured. In addition, a micro-method is described which requires only 100 microliter of sample and yields an accurate result in 5 min.

Flow of reducing equivalents into isolated intestinal mitochondria.

A system of enzymes is required for the transport of reducing equivalents from reduced nicotinamide adenine dinucleotide (NADH) generated in the cytosol into the mitochondria by the substrate cycles. Also, the intestinal mitochondria must be capable of oxidizing the substrates of the cycles. Both substrate cycle enzymes and permeable mitochondria are necessary for the flow of pyruvate derived from glucose into the mitochondria for oxidative decarboxylation and for the efficient production of adenosine 5'-triphosphate (ATP) for the unique intestinal nutrient transport functions. Mitochondria from hamster intestinal mucosa were prepared exhibiting good respiratory control ratios. The isolated intestinal mitochondria would not oxidize NADH unless N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) was added as a carrier of reducing equivalents. The rates of oxidation of the substrates of the L-glycerol 3-phosphate and the L-malate/1-aspartate substrate cycles were measured with the mitochondria isolated from the small intestinal mucosa. The key enzymes measured in the cytosol and mitochondria from the mucosa were NAD-L-glycerol 3-phosphate dehydrogenase, Fp-L-glycerol 3-phosphate dehydrogenase, L-malate dehydrogenase and L-glutamate-oxaloacetate transaminase. In addition, the substrate cyclase were simulated in vitro by following NADH oxidation by isolated mitochondria in the presence of added cytosolic constituents.

Cataleptic and anticataleptic effects of muscimol and gabaculine injected into globus pallidus and substantia nigra, and interactions with haloperidol or benzodiazepines.

Intranigral injection of muscimol induced hyperactivity in rats and antagonized haloperidol-induced catalepsy. Intranigral injection of gabaculine, an inhibitor of GABA transaminase, induced similar effects 5h after injection, when the nigral GABA content was increased 7-fold. On the other hand, injections of muscimol (30 ng) into the globus pallidus potentiated the cataleptic effect of haloperidol, and muscimol alone in high doses (100 and 200 ng) induced catalepsy. Gabaculine also induced catalepsy of medium intensity and potentiated the effect of haloperidol 24h after injection, when GABA was increased in the globus pallidus as well as in the substantia nigra. Injections of muscimol into either the globus pallidus or substantia nigra increased striatal HVA and enhanced haloperidol-induced elevation of HVA. Three benzodiazepines, nitrazepam, diazepam and chlordiazepoxide administered orally, potentiated the effect of muscimol (30 ng) injected into the globus pallidus and induced catalepsy. A similar effect was not obtained with phenobarbital. It is suggested that stimulation of GABA receptor or increase of GABA content in the sustantia nigra antagonize haloperidol-induced catalepsy by activation of nigral dopaminergic system, and that enhancement of pallidal GABA function induces catalepsy by non-dopaminergic mechanisms. Potentiation of haloperidol-induced catalepsy by benzodiazepines may be due to enhancement of GABA-ergic transmission within the globus pallidus.

The influence of neuroleptics on the behavioural effect of 5-hydroxytryptophan.

The antagonism of neuroleptics of various groups (chlorpromazine, chlorprothixene, clopenthixol, clozapine, flupenthixol, fluphenazine, haloperidol, levomepromazine, mepazine, perazine, perphenazine, pimozide, prochlorperazine, promazine, spiperone, thiopromazine, thioridazine, trifluperazine, trifluperidol, triflupromazine) towards L-5-hydroxytryptophan (5-HTP) was assessed on the basis of inhibition of characteristic head-twitches. ED50 was assayed in mice and rats. The results indicate that all investigated neuroleptics inhibit the action of 5-HTP and their ED50 values are, as a rule, lower than the values of ED50 for catalepsy. That action is for the majority of neuroleptics more pronounced in mice than in rats. The present paper disucsses possible serotonergic, dopaminergic and also noradrenergic mechanism of action of neuroleptics in the 5-HTP test.