The involvement of oxygen radicals during the autoxidation of adrenalin. 1. In unbuffered alkaline solutions, autoxidizing adrenalin generates superoxide anions: both the scavenging by adrenalin itself, leading to adrenochrome, and the formation of nitrite from hydroxylamine are inhibited by superoxide dismutase. No hydroxyl radical could be detected. 2. The yield of hydrogen peroxide increases with pH in a way similar to that of adrenochrome and nitrite. The dissociated form of adrenalin (pK = 8.5) is proposed as the source of superoxide anions. 3. Superoxide dismutase delays rather than inhibits the reaction. In addition to the diminished formation of adrenochrome due to the scavenging of superoxide anions and re-reduction of the semiquinone by hydrogen peroxide, respectively, adrenochrome is further removed by hydrogen peroxide, with final products absorbing at 310 nm. 4. The diminished inhibitory effect of superoxide dismutase above pH 10 is due to superoxide-independent reactions. This effect is masked by the alkaline conversion of adrenochrome to indole compounds. 5. It is concluded that monitoring the absorption of adrenochrome in alkaline solutions does not produce reliable evidence for superoxide anions.


A protease-like permeability factor in the guinea pig skin. 1. Partial purification and characterization. A permeability factor was extracted in a latent form from guinea pig skin and separated by ammonium sulfate fractionation into the pseudoglobulin fraction (30--50% saturation). The activation of the latent form of the permeability factor seemed to be caused in the desalting step by gel filtration with Sephadex G-50. The factor was partially purified by streptomycin treatment and column chromatography using hydroxyapatite, diethylaminoethyl cellulose and Sephadex G-75, in this order. Gel filtration showed that its molecular weight was approx. 35000. Its permeability activity was heat stable at 61 degrees C for 60 min at neutral pH, resistant at pH 5--10 and at ionic strengths from deionized water to 1 M NaCl at 4 degrees C. Its activity was transient and suppressed by guinea pig serum, but insensitive to an anti-histamic agent (triprolidine). Furthermore, its permeability activity was inhibited by diisopropylfluorophosphate, soybean trypsin inhibitor and leupeptin, and completely adsorbed by soybean trypsin inhibitor affinity column. These findings suggested that the permeability factor was a serine-type protease.


The mobility of methylmercury in biological systems. Toxicology studies indicate that methylmercury in humans and other species is bonded to sulfhydryl ligands and that the methylmercury in such complexes is labile even though their thermodynamic stability is large. It is shown in this paper that bimolecular nucleophilic displacement of complexed ligand by sulfhydryl-deprotonated ligand is the major pathway for ligand exchange at physiological pH, while at the pH of the stomach the proton-assisted dissociation of the complex is the predominant means by which exchange occurs. The dynamic and equilibrium aspects of the distribution of methylmercury between chloride and sulfhydryl ligands under the solution conditions of the stomach are also considered with respect to a possible role for lipid-soluble CH3HgCl in the absorption of methylmercury from the stomach.


A light-scattering study of the effect of calcium chloride on the molecular weight of Busycon hemocyanin. Solutions of Busycon canaliculatum have been studied by light scattering. In 0.05 M Trizma buffer +0.1 M NaCl at pH 7.0 at 14 degrees, the weight-average molecular weight is 8.9 X 10(6). In the presence of added CaCl2 (0.02 M), the molecular weight of the protein increases to 10.7 X 10(6), and the second virial coefficient is reduced. At pH 9.95, the molecular weights with and without 0.02 M CaCl2, are 3.7 X 10(6) and 1.3 X 10(6), respectively; and the effect of Ca++ in reducing the second virial coefficient is much greater than at pH 7.0. These results can be understood on the basis that at pH 7.0, ca++ increases the association of hemocyanin, by binding and intermolecular linkage through the carboxyl groups of protein side chains. At pH 9.95, amino groups are deprotonated and therefore also become available for Ca++ binding. The relative effect of Ca++ in enhancing the association of hemocyanin therefore becomes greater at the higher pH.


Effects of ammonium and bicarbonate-CO2 on intracellular chloride levels in Aplysia neurons. The level of intracellular free chloride in Aplysia giant neurons can be made to decline by pretreatment with 50 mM NH4+ solution followed by washing with 10 mM HCO3-/0.4% CO2-containing fluids. This effect can be completely blocked by the anion flux inhibitor, 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonic acid (SITS). The net change of free chloride in the cell cannot be explained by changes in the electrochemical gradient of chloride. These results support the hypothesis that at least one mechanism for intracellular pH regulation involves a Cl-/HCO-3 exchange process.


Effects of a new selective beta1-adrenoceptor agonist on amylase secretion from the rat parotid gland. The effects of a new selective beta1-adrenoceptor agonist, (--)-1-(4-hydroxyphenoxy)-3-isopropyl-amino-propanol-2-hydrochloride (H 133/22), on amylase secretion from the rat parotid gland were investigated in an in vitro system. The results were compared to the secretory responses obtained with noradrenaline, adrenaline, methoxyamine and terbutaline. H 133/22 was found to be a potent enzyme secretagogue and appeared even more effective than noradrenaline and adrenaline, particularly at low concentrations. The beta1-adrenoceptor agonist, terbutaline, also stimulated amylase discharge from the parotid gland but was much less potent than H 133/22. Methoxyamine had no effect on enzyme secretion. We suggest that the adrenergic stimulation of amylase secretion from the rat parotid gland is mainly mediated by beta1-receptors.


The effect of lysergic and diethylamide (LSD) and 2-bromolysergic acid diethylamide (BOL) on the striatal DOPA accumulation: influence of central 5-hydroxytryptaminergic pathways. Selective chronic lesions of the dorsal raphe nucleus or combined lesions of the dorsal and median raphe nuclei did not significantly change the in vivo tyrosine hydroxylation in the striatum as measured by the DOPA accumulation after decarboxylase inhibition. Neither did acute combined lesions of the raphe nuclei, nor did electrical stimulation of the dorsal raphe nucleus have any significant effect. p-Chloroamphetamine (PCA, 20 mg/kg, i.p.) and p-chlorophenylalanine (PCPA, 400 mg/kg, i.p.), known inhibitors of the 5-hydroxytryptamine (5-HT) synthesis, significantly decreased the DOPA accumulation. The increase in DOPA accumulation observed after LSD (0.5 mg/kg, i.p.) or BOL (0.5 mg/kg, i.p.) was seemingly unaffected by pretreatment with PCA or PCPA and also after lesion of the dorsal raphe nucleus. The results suggest that the effect of LSD or BOL on the DOPA accumulation in the striatum is not mediated via a 5-hydroxytryptaminergic control mechanism originating in the dorsal raphe nucleus. A control mediated via the median raphe nucleus cannot be excluded, since LSD did not increase the DOPA accumulation after combined chronic raphe lesions. Such a control would also be in agreement with our previous results suggesting that hte DOPA generation after LSD is controlled by 5-HT receptors.


Monoamines, estrogen and female sexual behavior in the golden hamster. In the golden hamster drugs which inhibit monoaminergic function (including p-chlorophenylalanine (PCPA), methysergide and a-methyl-p-tyrosine (a-MPT) facilitated lordosis in ovariectomized female hamsters as a function of the duration of estradiol benzoate (EB) priming. a-MPT (200 mg/kg), methysergide (6 mg/animal) or PCPA (150 or 360 mg/kg) potentiated lordosis if 6 days of EB priming preceded drug treatment. However, if female hamsters were primed with EB for only 2 days, a-MPT and methysergide were ineffective. PCPA (360 mg/kg) was less effective after 2 days of EB than after 6 days of EB priming. alpha-MPT produced a three-fold elevation in progesterone levels in ovariectomized females but methysergide and PCPA did not influence serum progesterone. PCPA (360 mg/kg) facilitated lordosis in adrenalectomized, ovariectomized females, eliminating the possibility that adrenal progesterone is essential for the behavioral effects of the drug. Luteinizing hormone-releasing hormone levels in the preoptic/anterior hypothalamic area and the medial basal hypothalamus were also not significantly altered at 1 h after PCPA injection. Pimozide (1.5 mg/kg) and pimozide (1.5 mg/kg) and amphetamine (2.5 mg/kg) did not potentiate lordosis in ovariectomized hamsters after either 2 or 6 days of EB priming. Pargyline, a monoamine oxidase inhibitor, inhibited female sexual behavior in females in estrogen alone-induced estrus. Lordosis in the female rat is more readily elicited both by drugs and estrogen. It is proposed with regard to female sexual behavior that species differences in estrogen sensitivity may underlie apparent differences in drug sensitivity.


Synaptic connexions and related postsynaptic pharmacology studied in the cerebral ganglion of Aplysia. Interneuronal connexions have been studied within the cerebral ganglion of Aplysia californica. Excitatory monosynaptic connexions between two groups of cells located on the dorsal surface of the ganglion near the cerebropleural connectives were analyzed in detail. The monosynapticity of these connexions was established not only by the strict one-to-one correlation between presynaptic action potential and excitatory postsynaptic response (EPSP) and the constant latency for any given cell pair, but also by the following criteria: (a) gradual change in the EPSP following tetraethylammonium injection into the presynaptic neurone, (b) sustained EPSP in the presence of a high external calcium ion concentration, (c) sensitivity of the EPSP amplitude to presynaptic polarization. Iontophoretic application of acetylcholine, 5-hydroxytryptamine and glutamate on the postsynaptic cells elicited excitatory responses in many cases. Inhibitory responses were obtained by local iontophoresis of dopamine, gamma-aminobutyric acid and occasionally also by acetylcholine. The only agent found to block the EPSP was bufotenine, which also readily blocked the 5-hydroxytryptamine response. Bufotenine was completely ineffective on the acetylcholine or glutamate excitatory responses. Of the various cholinolytics tested, none had an effect on the EPSP. Our data all point to 5-hydroxytryptamine as a transmitter in the studied synaptic connexions. However, it must be emphasized that in the absence of biochemical and histological evidence the role of the 5-hydroxytryptamine cannot be regarded as conclusive.


Effects of pH and flow rate on the release of 'bound' red cells from the splenic pulp. On perfusion of isolated, denervated spleens with Ringer solution, immature and abnormal red cells are released into the venous outflow much more slowly than normal mature cells, being delayed through adherence to fine structures of the red pulp (Am. J. Physiol. 231, 1665-1671 (1976)). Evidence suggested that the rate at which such cells are released from the 'bound' state might depend on local pH and fluid shear rate within the pulp. Therefore, the rate of washout for this slow component of red cells, from cat spleens, was measured as a function of pH and flow rate of the perfusate. The volume of solution (V 1/2) for 50% washout of 'bound' cells decreased as pH was lowered from 7.8 to 6.6, especially (from 97 to 18 ml/g) between 7.4 and 6.6. The percentage total red cell outflow thus represented rose from 0.06 to 0.5 as pH fell from 7.8 to 6.6. At a high perfusion rate (14-16 ml/min) the V 1/2 value was only one-half that prevailing at a lower rate (4-6 ml/min), and the percentage flow of 'bound' red cells was more than three times greater. Both acidic pH and augmented blood flow thus assist release of adherent red cells from the splenic pulp.


An alkaline phosphatase mutant of Pseudomonas aeruginosa. 1. Effects of regulatory, structural, and environmental shifts on enzyme function. An alkaline phosphatase mutant of Pseudomonas aeruginosa exhibiting both regulatory and catalytic changes was isolated. Under repression conditions (i.e. high inorganic phosphate (Pi)) the mutant culture produced an alkaline phosphatase (APase) displaying significant activity against both beta-glycerol phosphate (betaGP) and p-nitrophenyl phosphate (pNPP), while the wild type displayed no activity directed towards these substrates under the same conditions. In vivo, the mutant enzyme's ratio of specific activities was 45:1 in favour of betaGP versus pNPP, whereas this ratio was reversed to 1:9 betaGP versus pNPP for the same enzyme isolated from mutant cells. In addition, the kinetic parameters and stability requirements for the mutant-derived enzyme was altered in comparison with those of the wild type. A study of lipopolysaccharide (LPS) preparations from both the mutant and wild type indicated the mutant to be deficient in the core region of its LPS. The authors propose that the modifications in the catalytic activity of the mutant enzyme, demonstrated in vivo, are due to a change in the enzyme's microenvironment.


Proteolytic activity of Trichoderma viride in mixed culture with Sclerotium rolfsii in soil. Cultures of Sclerotium rolfsii and Trichoderma viride together in autoclaved soil were assayed at intervals during 8 days of incubation for proteolytic activity (PA) of T. viride. Significant proteolytic activity was detected only in soil containing T. viride (i.e., T. viride alone or S. rolfsii + T. viride); greatest activity occurred between 3 and 4 days after infestation and declined rapidly thereafter. Maximal PA in the mixed-culture soil was accompanied by an increase in soil pH. optimal pH values for PA was 5.5-6.5 with a maximum at 6.0.


Proliferation and viability in cellular spheroids of human origin. The capacity of cells to form growing tumor-like colonies in culture was tested by a new and unlaborious suspension technique. Six of nine tested cell lines formed spheroids, five of which started to grow. The growing spheroids reached a maximal size determined by balanced cell gain in a proliferative, superficial layer and cell death, particularly at larger depth. Sections of spheroids of different origin showed large variations in the thickness of the viable cell layers and in the shape of the proliferative gradients. The data, taken together with earlier published information, indicate that a difference between rodent and human cells generally exists, the former showing thinner viable and proliferative layers.


Enzymatic determination of blood ethanol, with amperometric measurement of rate of oxygen depletion. A rapid electrochemical measurement of blood ethanol is proposed. Alcohol is oxidized by NAD+ in the presence of alcohol dehydrogenase; and the NADH produced is aerobically oxidized by horseradish peroxidase. The rate of depletion of buffer-carried oxygen, which is directly proportional to the alcohol concentration in the sample, is amperometrically monitored with a membrane oxygen-sensing electrode. Only a 5-microliter sample of whole blood is required, with no deproteinization, incubation, extraction, or dilution. Results, obtained in less than 1 min, correlate well with those obtained by gas-chromatographic and spectrophotometric methods.


Simultaneous liquid-chromatographic determination of 12 common sedatives and hypnotics in serum. We present a method for simultaneously determining 12 hypnotics and sedatives (primidone, methyprylon, phenobarbital, butabarbital, butalbital, ethchlorvynol, pentobarbital, amobarbital, phenytoin, glutethimide, secobarbital and methaqualone) in 200 microliter of serum. Serum proteins are precipitated with an acetonitrile solution containing 5-(4-methylphenyl)-5-phenylhydantoin, the internal standard. The drugs are eluted from a reversed-phase column with a mobile phase consisting of an acetonitrile/phosphate buffer, at a flow rate of 3.0 ml/min. The eluted drugs are detected by their absorption at 195 nm; their quantities are estimated from their peak heights. Each analysis requires no longer than 30 min at the optimum column temperature of 50 degrees C. The lower limit of detection for all of these drugs is less than 10 ng/sample for drug standard. A sensitivity of 1.0 mg/liter of serum is attained routinely for each of the drugs. Analytical recoveries for the 12 drugs varied from 94 to 112%, with good day-to-day precision (CV between 3.8 and 10.4%). Of more than 35 drugs tested for possible interference, only ethotoin interferes with the analysis of phenobarbital.


The use of gamma-glutamyl-p-aminobenzoic acid as the substrate for determination of gamma-glutamyltranspeptidase activity in blood serum. By the phthaloyl method less toxic and readily soluble gamma-L-glutamyl-p-aminobenzoic acid was synthesized. This substance was used as a substrate for gamma-glutamyltranspeptidase activity assay in blood serum and urine. Close correlation was shown between the results obtained with the new method and with the old one which used gamma-L-glutamyl-p-nitroanilide.


The effect of urinary pH and flow rate on monoamine output. Three-hour urinary output values of dopamine, noradrenaline, adrenaline, 2-phenylethylamine and p-tyramine were measured in normal volunteers who had been induced, by pretreatment with ammonium chloride or sodium bicarbonate, to excrete maximally acid or alkaline urine. There were significant effects on the excretion of dopamine, adrenaline and 2-phenylethylamine, inversely proportional to urinary pH value. Adrenaline output increased with increasing urinary flow rate. There was a significant correlation between urinary concentrations of 2-phenylethylamine and p-tyramine. These findings may have important clinical implications.


Acid esterase activity in cultured skin fibroblasts and amniotic fluid cells using 4-methylumbelliferyl palmitate. The properties and levels of acid esterase in cultured skin fibroblasts and amniotic fluid cells were investigated using 4-methylumbelliferyl palmitate as substrate. Determinations of acid esterase activity could be made using as little as 1 microgram cell protein. Cardiolipin increased the activity 2--3 fold at the pH optimum 4.0. The apparent KM for both cell types studied was 196 micrometer without and 96 micrometer with cardiolipin. Acid esterase activity was inhibited by cyanide and thiomersal, but not by iodoacetate and N-ethylmaleimide. However activation by cardiolipin was prevented by iodoacetate, N-ethylmaleimide and also sodium chloride. Skin fibroblasts and primary amniotic fluid cells had similar levels with or without cardiolipin. A cyclic activity was found with subculture but no consistent pattern with passage. The acid esterase deficiency in Wolman's and cholesterol ester storage diseases was demonstrated with this substrate.


Cardiac and bronchial beta-adrenoceptor antagonistic potencies of atenolol, metoprolol, acebutolol, practolol, propranolol and pindolol in the anaesthetized dog. 1. The beta-adrenoceptor antagonists atenolol, metoprolol, acebutolol, practolol, propranolol and pindolol have been tested for their ability to reduce isoprenaline-induced bronchodilation and tachycardia in the anaesthetized dog. 2. Atenolol, metoprolol, acebutolol and practolol all possessed a similar degree of cardioselectivity in this animal model.


The influence of serotonin on the mitotic rate in the colonic crypt epithelium and in colonic adenocarcinoma in rats. 1. The mitotic rate in the crypts of Lieberkühn of the descending colon and in dimethylhydrazine-induced adenocarcinomata of the descending colon of rat was measured using a stathmokinetic technique. 2. Intraperitoneal injection of a small dose (10 microgram/kg) of serotonin resulted in an increase in the tumour cell mitotic rate. 3. Blockade of serotonin receptors by 2-bromolysergic acid diethylamide and depletion of tissue serotonin levels following injection of DL-6-fluorotryptophan both result in a decrease in the tumour cell mitotic rate. 4. Treatment with serotonin, 2-bromolysergic acid diethylamide and DL-6-fluorotryptophan were all without effect on the colonic crypt cell mitotic rate.


Kinetics, salivary excretion of amphetamine isomers, and effect of urinary pH. Amphetamine was administered to healthy subjects as the racemic mixture and as (+)- and (-)-isomers under conditions of urine acidification and alkalinization. Plasma and saliva concentration of each isomer was measured and the kinetics of the individual isomers were determined. (+)-amphetamine was eliminated more rapidly than the (-)-isomer. The difference in half-life between isomers was maximal under basic urinary pH conditions. Saliva amphetamine levels were higher than plasma levels and in the postabsorptive phase were predictably proportional to plasma drug levels.


Plasma level monitoring of antipsychotic drugs. Psychotic patients treated with identical doses of antipsychotic drugs have been shown to have great interindividual differences in their steady state plasma concentration. Therefore, monitoring treatment by dosage adjustment alone is of little value. If antipsychotic blood levels can be related to clinical response then their routine measurement may well result in well defined guidelines to individualised optimal dosage. Despite the considerable effort expended in this field and the many interesting testable hypotheses generated, little substantive evidence for an acceptable plasma level monitoring guide has been reported to date. Work on metabolite level profiles, intra- and extracellular drug concentration differences, more detailed clinical rating scales, and improved experimental design, all show great promise for the future. Investigation of the pharmacokinetics and the elucidation of the often complex metabolic pathways of individual antipsychotic drugs are generating the data base required for the rational pharmacotherapy of these most severely ill patients. Until more data are available, routine monitoring of antipsychotic drug plasma levels remains of research interest.


Diseases and drug protein binding.

Diseases and drug protein binding. In a number of pathological states a decrease in the plasma protein binding of drugs is observed. This may be due to many factors related either to the protein, or the ligand (drug), or to the binding conditions. The most important of these disease states quantitatively are probably hypoalbuminaemia, conditions resulting in modification of the albumin compartment volume and the presence on albumin binding sites of pathological inhibitors of drug binding. A decrease in the extent of drug plasma protein binding does not necessarily lead to enhanced drug effects and therefore raises two important therapeutic questions. Firstly, does reduced protein binding have a clinically significant influence on the pharmacological effects of the drug? Secondly, if it does, is it preferable to modify the dosage regimen of the drug or to correct the plasma protein concentration prior to the administration of the drug? At present, only tentative answers can be given.


Acid-base balance in small premature infants (less than 1500 grams) with type II RDS. Twenty-one small premature infants with clinical features and radiological findings of type II RDS were studied. Correction of blood pH, corresponding with the decrease of blood PaCO2, was usually seen in the first eight hours of age. Clinical inprovement, as judged by the RDS score, was noted only after 12 hours of age. Complete recovery was the rule although signs of respiratory distress in a few infants lasted for 48-72 hours. The calculated AaDO2 and total Qs/Qt weer slightly higher (27.8%) than reported in normal infants and remained unchanged until after 48 hours of age when it decreased to 24.5%.


Loxapine: a review of its pharmacological properties and therapeutic efficacy as an antipsychotic agent. Loxapine is a dibenzoxazepine, tricyclic compound recommended for the treatment of acute and chronic schizophrenia. In its therapeutic effectiveness and profile and incidence of side-effects, loxapine closely resembles the traditional antipsychotic agents. Although loxapine has tended to be less effective than some standard antipsychotic drugs in a few short-term (3 to 4 weeks) studies, it has been superior to a placebo and about as effective as chlorpromazine, haloperidol, trifluoperazine or thiothixene when evaluated after 4 to 12 weeks. Like the phenothiazine (e.g. chlorpromazine) and butyrophenone (e.g. haloperidol) antipsychotic agents, loxapine causes a high incidence of extrapyramidal reactions. Sedation occurs frequently, especially during early stages of treatment. Other, less common side-effects such as anticholinergic effects (dry mouth, blurred vision, etc.), hypotension, tachycardia and precipitation of epileptic seizures, which occur with the older antipsychotic drugs, have also been reported with loxapine.


[Hypotensive and antihypertensive activity of a newly synthesized derivative of 2-aminotetralin]. The report describes the hypotensive and antihypertensive activity of the compound N-(trans-3-hydroxy-1,2,3,4-tetrahydro-2-naphtyl)-N'-(3-oxo-3-phenylpropyl) piperazine dihydrochloride (P11). The work is made on normotensive cats and rats as well as on spontaneous and experimental (metacorticoid) hypertension. The studies were carried out under the conditions of acute and chronic experiment as the arteria pressure was recorded by blood and bloodless method. The compound P11 show a manifested hypotensive activity with favourable duration and therapeutic width. Its antihypertensive activity is revealed in rats with spontaneous and metacorticoid hypertension both after single and chronic applciation of the compound. The influence on the blood pressure weakens after reserpinization of the animals. The compound possesses slight ganglion-blocking action and manifested alpha and beta adreno-blocking activity: diminishes noradrenaline, adrenaline and isoprenaline responses on the arterial pressure and heart activity in anesthetised cats and rats, shifts to the right the cumulative dose-response curve of noradrenaline on vas deferens of a rat, ect. The hypotensive and antihypertensive effect of the compound P11 is discussed on the basis of its alpha and beta adrenoblocking action and its specific sedative effect.


Effects of local delayed hypersensitivity on the small intestine. There are many T and B cells in the small intestinal mucosa and local T cell immunity could have a role both in protective immunity and as a cause of disease (i.e. hypersensitivity). This latter aspect has been investigated by using several animal models to assess the effects of local delayed hypersensitivity on the structure and function of the small intestine. Heterotopically transplanted grafts of fetal small intestine in mice (isografts and allografts) have been examined by conventional histology, scanning and transmission electron microscopy, by making direct measurements of villi, crypts, and lymphoid cell infiltrate, and by counting the number of mitoses per crypt. This cell-mediated immune reaction causes lymphocyte infiltration which is most marked in the lamina propria, hyperplasia of the crypts of Lieberkühn, increased cell loss with villous atrophy and a flat surface, but the individual enterocytes appear fairly normal. Graft-versus-host disease cause exactly the same changes in structure and in cell kinetics as does rejection. However, crypt hyperplasia has been found to precede villous atrophy by several days. Preliminary experiments on local contact hypersensitivity suggest that intraluminal injection of oxazolone in the gut of sensitized mice also produces villous atrophy and crypt hyperplasia. It is postulated that these effects are likely to be produced via lymphokines: by an 'enteropathic' factor which damages the lamina propria and basement membrane, and a factor which is mitogenic for crypt stem cells. In mice infected with Giardia lamblia, crypt hyperplasia and lymphocyte infiltration of the epithelium are present and there is accelerated epithelial cell turnover. In rats infected with Nippostrongylus brasiliensis, the flat mucosa has been shown to be due to the thymus-dependent immune response and not directly to the damage produced by the parasite itself. A common factor in the variety of conditions associated with villous atrophy and crypt hyperplasia may well be a local cell-mediated immune reaction to food, microbial, parasite or other antigens which causes changes in enterocyte turnover rate and malabsorption.


[Study of a beta-adrenergic stimulant and its antidepressant activity in man]. In animals, the psychopharmacological profile of beta-adrenergic stimulants is very similar to that of tricyclic antidepressants. In patients, more particularly in endogenous depressive patients, the antidepressant effect of salbutamol was very clear. A definite improvement was observed in all of the 22 studied patients after 1 to 3 days of intermittent venous infusion. The place of salbutamol in the therapeutic armamentarium of depressive states has still to be defined exactly. It is speculated that the antidepressant effect of imipramine-like derivatives is related to the stimulation of central beta 2-adrenergic receptors.


Apparent effects of neurotransmitters on sexual differentiation of the brain without mediation of sex hormones. Male sexual behaviour was found to be permanently decreased in neonatally reserpinized or paragylinized male rats. On the other hand, hypoplasia of sex organs was only observed in reserpinized, but not in pargylinized newborn males. Furthermore, male sexual behavior was found to be permanently increased in neonatally pyridostigminized males which showed even a slight hypoplasia of seminal vesicles in neonatal life. These findings suggest that changes of neurotransmitter concentrations and/or turnover rates apparently induced by psychotrophic drugs can affect sex-specific brain differentiation by direct action without mediation of sex hormones. Hence, neurotransmitters may be regarded as organizers of the brain.


Effects of total fasting in obese women. II. Acid-base balance. Acetoacetate, beta-hydroxybutyrate, pyruvate and lactate in whole blood, the acid-base balance according to Astrup, SGOT and SGPT, and the serum and urinary sodium and potassium were estimated in 10 fasting obese women. Fasting led metabolic acidosis due to a tenfold increase of acetoacetate and beta-hydroxybutyrate, while pyruvate and lactate had a tendency to decrease. Serum and urinary sodium and potassium decreased; A tendency to increased SGOT and SGPT was observed. Sodium bicarbonate administered in general since the 8th or 9th day of fasting led in 4 to 5 days to an earlier correction of blood pH, while pCO2, actual HCO3, standard HCO3, base excess, buffer base, pO2, Hb saturation and total CO2 reached the prefasting values only several days after blood pH normalisation. The administration of NaHCO3 corrected the serum level of sodium while its excretion remained low. Bicarbonate had no influence on the decreasing trend of serum potassium, but the urinary output of potassium was stabilized. It is suggested that in fasting subjects 1. the correction of the metabolic acidosis by NaHCO3 does not reduce the rate of lipid catabolism and does not lead to fluid retention and 2. liver damage in fasting may be prevented by blood pH normalisation.


Effect of substrate composition and concentration on aortic cholesterol ester hydrolase activity. Cholesterol ester hydrolase activity of pig aorta has been examined under optimum experimental conditions for hydrolysis of different cholesterol esters. The enzyme specific activity values were in the numerical order of substrates hydrolyzed: cholesteryl linoleate larger than or equal to linolenate greater than palmitate larger than or equal to stearate greater than oleate. The results are discussed in relation to the arterial accumulation of cholesterol esters.


Butadiene diepoxide activation of guanylate cyclase. A variety of nitroso chemical carcinogens increase the activity of guanylate cyclase (EC 4.6.1.2), the enzyme catalyzing the production of guanosine 3',5'-monophosphate. In the present report, the first non-nitroso chemical carcinogen, butadiene diepoxide, was shown to activate guanylate in a variety of tissues over the concentration range 1-100 mmol/l. At 20 mmol/l concentration, increases were 2- to 17-fold above control. These observations have potential importance since guanosine 3',5'-monophosphate may be involved in cell growth and malignant transformation.


Purification and characterization of the two molecular forms of membrane acid protease from Aspergillus oryzae. Two forms (M1 and M2) of the membrane-bound acid protease of Aspergillus oryzae have been purified by extraction with Triton X-100, washing with cold acetone, and repeated gel filtration on Bio-Gel A-15 m in the presence and absence of Triton X-100. The purified membrane enzymes, M1 and M2, moved as a single band in acrylamide gel electrophoresis and had apparent molecular weights of 150 000 and 60 000, respectively, as estimated by sodium dodecyl sulfate/acrylamide gel electrophoresis. These two membrane enzymes activated bovine pancreatic trypsinogen and had the same pH optima in the acid pH range. They immunologically cross-reacted with each other and with an extracellular acid protease from A. oryzae, and contained carbohydrate, ranging from 52.5 to 80.5% and comprising three hexoses, glucose, galactose, and mannose. While these catalytic, chemical and immunological properties are similar to those of the extracellular acid protease from A. oryzae, both membrane enzyme differed in their hydrophobic properties from external enzymes. Thus they are activated by the detergent Triton X-100 and some polar lipids.


Spectrofluorometric measurement of the binding of ethidium to superhelical DNA from cell nuclei. Structures retaining many of the morphological features of nuclei may be released by lysing HeLa cells in solutions containing non-ionic detergents and high concentrations of salt. These nucleoids contain few chromatin proteins. We have shown that the DNA of nucleoids is quasicircular and supercoiled by measure spectrofluorometrically the amount of the intercalating dye, ethidium, bound to unirradiated and gamma-irradiated nucleoids. Ethidium binds to nucleoids in the manner characteristic of the binding to superhelical DNA: at low concentrations more ethidium binds to unirradiated nucleoids than to their gamma-irradiated counterparts with broken DNA, and at higher concentrations less ethidium binds to the unirradiated nucleoids. The quasi-circles in nucleoids are 22 times less sensitive to gamma-irradiation than are circles of pure PM2 DNA: they must contain about 2.2 X 10(5) base pairs. The constraints that maintain the quasi-circularity of nucleoid DNA are very resistant to extremes of temperature and alkali; some remain under conditions in which the duplex is denatured. The constraints are destabilised by ethidium suggesting that they are stabilised by free energy of supercoiling. Proteolytic enzymes, but not ribonucleases, remove the constraints. Possible structures for the constraining mechanism are discussed.


Characterization of beta-glucosidase isoenzymes possibly involved in lignification from chick pea (Cicer arietinum L.) cell suspension cultures. Crude cell wall preparations from Cicer arietinum L. cell suspension cultures show high activity for the hydrolysis of coniferyl alcohol beta-D-glucoside (coniferin). Various beta-glucosidase activities could be solubilized from these preparations by 0.5 M NaCl treatment and one of these could be shown to possess a high activity for the hydrolysis of coniferin. The enzyme activities were purified to near homogeneity by Sephadex G-200 and CM-Sephadex chromatography. Isoelectric focussing indicated the occurrence of beta-glucosidase isoenzymes with identical catalytic activity (pI 8.5-10). Molecular weights were determined as 110 000, with two subunits of 63 000 and 43 000. Maximum hydrolytic activity is at pH 5. The beta-glucosidase isoenzymes catalyze the hydrolysis of various beta-glucosides with aromatic aglycone structure and different sugar moieties. However, coniferin has been found to be one of the best substrates (km = 0.8 mM; V = 6 mumol.min-1.mg protein-1) for these beta-glucosidase isoenzymes. The data suggest that beta-glucosidase-catalyzed reaction might be involved in lignification of these plant cell cultures.


Intrinsic sympathomimetic activity of beta-adrenoceptor blocking agents. The pharmacological methods used to assess the intrinsic sympathomimetic activity (ISA) of beta-blockers are discussed. The clinical relevance of ISA to respiratory function, peripheral resistance and cardiac function is reviewed. It appears doubtful whether ISA is always of predominant clinical significance and an alternative explanation is offered for many clinical effects observed with certain beta-blockers, e.g. pindolol, oxprenolol, tolamolol, metoprolol, etc. Some effects of these beta-blockers resemble those of labetalol, a new drug with both alpha and beta-blocking activity. Some clinical effects of certain beta-blockers are more likely to be due to alpha-blocking activity than to their ISA.


Effect of diazepam on the frog neuromuscular junction. Diazepam (10(-5) M) had no effect on the compound action potential of frog sciatic nerve and its refractory period. At the neuromuscular junction, no changes were found in the amplitude and frequency of miniature endplate potentials in the presence of the drug. End-plate potential (e.p.p.) amplitude and early facilitation of the e.p.p. were unchanged. Diazepam had no effect on the amplitude of potentials evoked by ACh applied iontophoretically (0.1 Hz) at low frequencies in the chemosensitive region of the postjunctional membrane. Diazepam (10(-6) M) enhanced the progressive desensitization to ACh applied at a frequency of 1 Hz but did not affect recovery of sensitivity. A possible mechanism of action of diazepam at cholinergic synapses is discussed.


Kinetics of blockade of different receptors by chlorpromazine in rabbit stomach strips. The kinetics of receptor blockade by chlorpromazine (5 X 10(-8) M) were studied on fundic strips from the rabbit stomach. Onset of blockade by chlorpromazine proceeds at nearly identical rates when carbamylcholine and histamine are the agonists (t1/2 = approximately 5 min) while occurring more slowly against phenylephrine (t1/2 = 41 min). Similarly, offset of blockade by chlorpromazine is faster against carbamycholine and histamine (t1/2 = 83 and 34 min, respectively) than against phenylephrine (t1/2 = 385 min). The pA2 values of chlorpromazine on the alpha-adrenergic, muscarinic and histaminic (H1) receptors are 8.96, 7.17, and 7.90, respectively. No correlation  exists between the kinetics of blockade and the affinity of chlorpromazine for the receptors. It is suggested, therefore, that alpha-adrenoreceptors in the rabbit stomach are less accessible to chlorpromazine, or distributed differently within the tissue, than either the muscarinic or histaminic receptors.


Benzodiazepine receptors: labeling in intact animals with [3H] flunitrazepam. [3H]Flumitrazepam appears to label specific benzodiazepine receptors in vitro after i.v. injection in mice. Benzodiazepine potencies in reducing [3H]flunitrazepam binding in vivo correspond to pharmacological potencies and parallel relative affinites for [3H]flunitrazepam binding sites in isolated brain membranes. However, 50% occupation of [3H]-flunitrazepam sites by benzodiazepines in vivo requires brain concentrations of the drugs about 1000 times higher than their Ki values for the binding sites in vitro. In pharmacologically active doses sodium pentobarbital, strychnine, picrotoxin and bicuculline fail to influence [3H] flunitrazepam binding in vivo.


Amnesic effects of intravenous diazepam and lorazepam. The amnesic effects of 2 benzodiazepine drugs, diazepam (Valium) and lorazepam (Ativan), have been investigated. Some of the effects were similar to those of certain clinical amnesic syndromes. The effects were more extensive than previous work has indicated.


Neutral proteases in the guinea-pig lymphocytes. Partial purification of neutral proteolytic enzymes in guinea-pig lymphocytes yielded 2 enzymes. Both enzymes were heat-labile and inhibited by thiol reagents. The molecular weights were more than 200,000 and 150,000-200,000, and optimal pH around 9 and 8, respectively.


Effect of age on T cell differentiation. A brief overview of the area of T cell aging is presented by first discussing the age-related changes in T cell activities, and then by focusing attention on the possible mechanisms that may be responsible for the decline. Present evidence indicates that thymic involution precedes and therefore may be responsible for the age-dependent decline in the ability of the immune system to generate functional T cells. At this time, it appears that the primary effect of thymic involution is on a T cell differentiation pathway affecting the more mature T cells first with time, and then the less mature T cells. Thus, the thymus may be the aging clock for the immune system. Further studies should be centered around processes regulating growth and atrophy of the thymus.


Nasal absorption of insulin in dogs.

Nasal absorption of insulin in dogs. The intranasal application of an insulin solution in dogs resulted in the rise of plasma immunoreactive insulin and in dose-dependent hypoglycemia. The absorption of insulin from this site was found to be enhanced when insulin was dissolved in an acid medium. In addition, when an insulin preparation with some surfactant was used, the effectiveness of nasally administered insulin was 25 to 30 per cent of that achieved with intravenously administered insulin.


Trial of an alpha-adrenolytic drug (indoramin) for nocturnal enuresis. The investigation of methods of treatment known to be effective in controlling nocturnal enuresis may yield information on the pathophysiology of the condition. An attempt has been made to mimic the known alpha-adrenolytic action of imipramine by treating a group of 14 enuretic school-children with a competitive alpha-adrenoceptor blocking substance, indoramin. Treatment in the doses used had no significant effect on night-wetting frequency.


Intestinal mucins in health and disease. Intestinal mucins are complex glycoproteins which are secreted from goblet cells, and form a gel-like covering over the mucosal surface. They are assumed to provide lubrication and protection of the underlying epithelium against potentially injurious chemicals, enzymes, bacteria and dietary constituents. Recent advances in our understanding of mucin structure, secretion and functional properties are reviewed in this paper. Implications for diseases such as cystic fibrosis, peptic ulcer, malignancy and inflammatory bowel disease are briefly discussed.


Prognosis in children with Crohn's disease. The course of 86 children with Crohn's disease was examined during a 10-year period between 1966 and 1976. Patients were classified according to the initial site of disease. Ileocolitis was the most (52%) and colitis the least (9%) common form of disease with diffuse small bowel or ileal disease each comprising nearly 20% of the study group. These figures show a reversal from those of a previous decade when 42% of the patients had only terminal ileal disease and 17% had ileocolitis. Children with ileocolitis had the highest number of extracolonic manifestations and operations and required steroid therapy the longest. Those with only small bowel disease (with the exception of duodenal involvement) had fewer extraintestinal symptoms and operations and showed a consistently good response to medical treatment.


Inhibition of pancreatic secretion by enkephalin and morphine in dogs. The nature and extent of enkephalin- and morphine-induced inhibition of pancreatic bicarbonate and protein secretion were studied in dogs with chronic pancreatic fistulae after administering exogenous secretin or octapeptide of cholecystokinin and stimulants for the endogenous release of these hormones. Enkephalin and morphine competitively inhibited the pancreatic bicarbonate secretion induced either by exogenous secretin or duodenal acidification. This inhibition was partially reversed by naloxone, an opiate antagonist. Opiate substance also profoundly inhibited pancreatic protein response to octapeptide of cholecystokinin and to various stimulants of endogenous cholecystokinin release. We conclude that enkephalin and morphine strongly inhibit the pancreatic responses to exogenous and endogenous stimulants by a mechanism involving separate opiate receptors.


Some further characteristics of the growth of Naegleria fowleri and N. gruberi in axenic culture. The effects of pH, various viscosity of the medium, changed ratio between the concentrations of dissolved and corpuscular components in the medium, and dissolved inorganic salts on the growth of axenic cultures of Naegleria fowleri and N. gruberi have been studied. The cultures were grown in liquid CALYG and BCS media. The pH optimum was 6.5 for N. fowleri and 6.0--6.5 for N. gruberi. No negative influence on the growth of N. fowleri was observed even at 0.5% concentration of highly viscous methylcellulose, whereas the growth of N. gruberi was distinctly inhibited by more than 0.2% of methycellulose. N. fowleri preferred the osmotorphic and N. gruberi phagotrophic nutrition in the given system of cultivation. The growth of both Naegleria species was inhibited by 0.1 N concentration of sodium chloride and potassium chloride without any significant difference in the tolerance. The inhibitory effect of these salts correlated primarily with the concentration of chloride anion. The ability to grow in a medium with increased viscosity and the preference for osmotrophic nutrtion are, besides the higher temperature optimum determined earlier, further characteristics of the pathogenic species N. fowleri.


Virological examination of mosquito larvae from southern Moravia. 254 pools of 4,115 mosquito larvae belonging to nine species were examined by isolation experiments. The larvae were collected in breeding places in an inundated forest--a natural focus of Tahyna virus, in April, June and July 1974 and 1975. Tahyna virus was isolated from one pool of 10 Culiseta annulata larvae collected in July 1974. Ecological questions concerning this finding are discussed.


[Electroencephalographic effects of flurazepam in rabbits (author's transl)]. Electroencephalographic (EEG) effects of flurazepam were investigated in unanesthetized, unrestrained rabbits with chronic electrode implants and compared with those of diazepam. Flurazepam, at doses of 0.5 approximately 5 mg/kg i.v., induced a drowsy EEG pattern, i.e. high voltage slow waves in the cortex and amygdaloid complex and desynchronization of the hippocampal theta waves. In addition, low voltage fast waves were superimposed, especially on the cortical EEG. Flurazepam suppressed the EEG arousal responses induced not only by auditory stimulation but also by electrical stimulation of the mesencephalic reticular formation, posterior hypothalamus and centromedian thalamus. The EEG arousal response induced by i.v. injection of physostigmine was suppressed by flurazepam. Flurazepam depressed the photic driving response and the augmenting response. The recruiting response was slightly enhanced by flurazepam. The limbic afterdischarges elicited by either hippocampal or amygdaloid stimulation were suppressed by flurazepam. Flurazepam caused reductions of pressor responses to stimulation of the posterior hypothalamus and the mesencephalic reticular formation in anaesthetized rabbits. There was little or no effect on pressor responses to the injection of noradrenaline, carotid artery occulusion and asphyxia with flurazepam. In general, these effects of flurazepam were similar to those of diazepam, but the drug induced actions which differed from those of diazepam.


[Comparative studies of antihypertensive effects of several beta-blocking agents in conscious rats (author's transl)]. The present study was undertaken to compare the antihypertensive effects of beta-blocking agents and to clarify the relations between antihypertensive effects and beta-blocking actions. In this study, blood pressure was measured by a direct cannulation of the abdominal aorta. Subcutaneous administration of carteolol and pindolol caused a significant fall in mean blood pressure in both normotensive and spontaneously hypertensive rats, whereas propranolol produced a rise in blood pressure. Maximum fall in blood pressure was observed 3 approximately 7 hr after the administration of carteolol and pindolol. In order to determine the beta-blocking action, changes in heart rate and blood pressure in response to isoproterenol (3 microgram/kg i.v.) were observed during the experiment. beta-Blocking action was found as early as 1 hr after subcutaneous administration. Carteolol showed the most effective blocking action throughout the experiment. Although beta-blocking agents lowered the blood pressure in this experiment, there was no apparent parallel between antihypertensive effects and beta-blocking action on the cardiac function.


[Pharmacological studies of loperamide, an anti-diarrheal agent. I. Effects on diarrhea induced by castor oil and prostaglandin E. (author's transl)]. Effects of loperamide on diarrhea induced by castor oil and prostaglandin E1 were investigated in rats and mice and compared with those of narcotic analgesics, atropine, mecamylamine and local anesthetics. The following results were obtained. Loperamide markedly suppressed the appearance of diarrhea induced by oral administration of castor oil in rats and the ED50 values for 1 and 2 hr protection was 0.082 and 0.42 mg/kg p.o., respectively. Loperamide markedly suppressed the appearance of diarrhea induced by i.v. administration of prostaglandin E1 and the ED50 value for 2 hr protection was 0.24 mg/kg p.o. in rats. The ID120 min value of loperamide which was calculated on the basis of the dose producing a 20% or more inhibition of the charcoal transport in the small intestine for 120 min was 0.8 mg/kg p.o. in mice and this activity was 9.2 times more potent than that of morphine. The analgesic ED50 value (Haffner's method) and LD50 value of loperamide was 149 and 249 mg/kg p.o., respectively. These results suggest that loperamide has a potent anti-diarrheal activity and specificity to the gastrointestinal tract and inhibits the effect of prostaglandin E1 and ricinoleic acid on the intestinal tract in rats.


[Mechanism of methamphetamine toxicity in grouped mice and the effects of centrally acting drugs on its toxicity (author's transl)]. The mortality of ddK mice treated with 40 mg/kg i.p. of methamphetamine (MA) was 85% in grouped conditions (10 mice in a cage) and 3% in individually isolated conditions. This mortality was not altered by the social environments even when other mice in the cage were not treated with MA. The mortality of mice individually isolated in cages with transparent walls was significantly higher than that of completely isolated mice. Almost all neuroleptics dose-dependently antagonized the MA toxicity in grouped mice, in small doses. The antagonizing activity of clozapine was somewhat weak, and sulpiride potentiated MA toxicity. Phentolamine and propranolol antagonized the MA toxicity at higher doses than neuroleptics. Reserpine and tetrabenazine previously given to mice remarkably antagonized the MA toxicity. H44/68 (a tyrosine hydroxylase inhibitor) had a considerable effect in antagonizing the MA toxicity, but diethyldithiocarbamate, U-14, 624 and FLA 63 (dopamine-beta-hydroxylase inhibitors) prevented the MA toxicity to a lesser extent than did H44/68. Apomorphine had no effect on the MA toxicity. The present data show that the MA toxicity in grouped mice (the increase in mortality) was enhanced by the presence of other mice, and suggest that the norepinephrine neurons play an important role in promoting the MA toxicity. Neuroleptics antagonize MA toxicity probably by blocking alpha-receptors in the central nervous system.


[Digestant effects of a new digestive enzyme capsule, Excelase, on jejunectomized and pancreato-jejunectomized Beagle dogs (author's transl)]. The digestant effects of a new digestive capsule, excelase containing sanactase, proctase, meicelase, olipase-2S and pancreatic digestive enzyme TA, were investigated in vivo. Jejunectomy and pancreato-jejunectomy were performed to cause an artificial disturbance of gastro-intestinal digestion and absorption in Beagle dogs. Absorption of protein and fat was measured using chromic oxide as an indicator. Excelase (3 capsules/dog/day) was given orally to Beagle dogs 1 week after each operation for 7 weeks. Changes in body weight and absorption of protein and fat were observed during the administration. The decrease in body weight of dogs treated with excelase fully recovered, however, that of controls remained even 8 weeks after the surgery. Absorption of protein and fat in the groups of dogs treated with excelase was greatly improved as compared with controls. The digestant effects of excelase on percent absorption of protein and fat were more manifest in pancreato-jejunectomized dogs than in jejunectomized dogs. These results indicate that excelase is effective for gastro-intestinal disturbances of digestion and absorption. The digestant effects of excelase on starch, protein and cellulose were also investigated in vitro using a gastro-intestinal model.


[Relation between the drug-metabolizing activities of liver microsomes and multiplicity of cytochrome P-450 (author's transl)]. Wada et al. found a hyperbolic curve in the Lineweaver-Burk plots of the drug-metabolizing activity and competitive inhibition of steroid hormones to drugs, and suggested that several enzymes acting on different sites of various steroid hormone molecules might metabolize drugs without having specificities for hydroxylation sites. Recently, several cytochrome P-450s were separated and purified from liver microsomes. We studied the relation between the drug-metabolizing activities and multiplicity of cytochrome P-450 with the pH curve and the Lineweaver-Burk plots. Phenobarbital- and methylcholanthrene-treatment had remarkable effects on the pH curve of aniline hydroxylation by mouse liver microsomes. Addition of cortisol inhibited aniline hydroxylatin by liver microsomes from phenobarbital-treated mice more effectively at higher pH range and the pH curve was similar to that seen for normal mice. Haugen and Coon separated preparations [A] [B] and [C] with hydroxylapatite chromatography and we obtained [A'] (eluted by 100 mM K-phosphate buffer, pH 7.4) between [A] (50 mM) and [B] (300 mM). The preparation [A'] had the same absorption maximum (451 nm) of reduced CO complex as [A], but Km and pH curve for [A'] were similar to those for [B]. Each separated preparation of cytochrome P-450 showed a hyperbolic curve in Lineweaver-Burk plots and a different pH curve from each other.


[Comparative studies on the protective effect of etilefrin and dihydroergotamine in circulatory instability after blood donation]. In 20 female and 40 male subjects with circulatory instability, the response of pulse rate and blood pressure to blood loss and orthostasis was studied under the condition of protective pretreatment with drugs known to improve venous return by increasing the tone of capacitance vessels. Before blood loss (420 ml), in a cross-over double-blind procedure, each subject obtained 10 mg etilefrin or 2 mg dihydroergotamine, both administered orally. The procedure was repeated after at least 8 weeks. At this time, the subject obtained the drug which was not given in the first examination. By comparison of pulse rate and blood pressure taken after pretreatment, the statistical evaluation did not reveal any differences in the effectiveness of one or the other drug on the response to hypovolemic and orthostatic conditions. Patients preferred etilefrin.


Macrophage colony development: properties of colony stimulating factors from murine embryo and pregnant uterus. Extracts from embryonic and uterine tissue of mice, operationally defined as colony stimulating factor (CSF), promoted the growth of macrophage-granulocyte colonies in vitro. Uterine CSF focusses from pH 5.15 to 6.00 and embryonic CSF from pH 3.60 to 5.20, although both forms have similar biological activity. CSF is relatively resistant to denaturation but it is inactivated by periodate and dithiothreitol. Gel filtration indicates a molecular weight of 45,000 which is unchanged following treatment with insolubilized trypsin, a procedure which affords a useful purification (240-fold). Trypsin-sensitive material in CSF preparations modifies colonial form under certain conditions of culture, probably by increasing the motility of macrophages. Diaminoethane derivatives of CSF were prepared and retained biological activity at isoelectric points above pH 9.0. These derivatives may be covalently linked to Sepharose providing an insolubilized form of CSF to study interactions of CSF with the cell surface.


Effect of complement fixation on the release of lysosomal enzymes from rabbit alveolar macrophages. Macrophage lysosomal enzymes that have specificities for substrates found in mammalian tissue may contribute to the tissue damage observed in chronic inflammatory diseases. Although a variety of agents that stimulate the release of hydrolases from peritoneal macrophages have been identified, little work has been done to establish the conditions and specific stimuli responsible for enzyme release by alveolar macrophages (AM). To assess the effect of phagocytosis by AM on the release phenomenon, hydrolytic enzymes normally sequestered in AM lysosomes were quantified in the supernatant fluids of phagocytosing and non-phagocytosing AM monolayers in both the presence and absence of serum. Maximum release occurred under conditions known to favor complement fixation by the classical or alternative pathways. Thermal destruction or immune fixation of serum complement before use in culture reduced the magnitude of release to that observed in cultures incubated in the complete absence of serum. Phagocytosis was not essential for release, since cells exposed to particles but treated with a known inhibitor of phagocytosis (cytochalasin B) still showed maximal release in the presence of fresh serum. These data are interpreted to indicate that a heat-labile component of serum, probably a by-product of complement fixation, was primarily responsible for the hydrolase release by AM. On the basis of these findings, individuals suffering from chronic pulmonary disease may suffer acute episodes of lung destruction from endogenous AM enzymes upon the inhalation of materials capable of fixing complement.


Evidence for quantitative variability of bacterial opsonic requirements. We studied human serum opsonins by using combinations of heat inactivation and chelation to inhibit complement, adsorption to remove antibody, and trypan blue to inactivate the C3 receptor of human polymorphonuclear leukocytes. Streptococcus pneumoniae, serotype 25, required both complement and immunoglobulin for opsonization, even though that strain activated the alternative complement pathway. Both strains of Escherichia coli required antibody and complement, but varied in the degree of dependence on the C3 opsonin, since trypan blue moderately inhibited the killing of E. coli-1 and markedly inhibited the killing of E. coli-2. Serratia marcescens was opsonized in heat-inactivated serum (limited complement) or serum absorbed at 0 degrees C with S. marcescens (limited antibody), but depended on the C3 receptor in absorbed serum. S. marcescens activated the alternative pathway. Thus, opsonic requirements varied with the availability of opsonins. Requirements for bacterial opsonization vary with species and strains within species, perhaps reflecting quantitative relationships among alternative and classical pathway activation of C3, efficiency of adsorption of C3 or immunoglobulin G to bacterial surfaces, and efficiency of attachment of these ligands to polymorphonuclear leukocyte receptors. Furthermore, although not always sufficient for opsonization, the C3 opsonin (activated through either the classical or alternative pathway) appears necessary for effective phagocytosis and killing of all strains studied.


Interactions between effector cell activity and lymphokines: implications for recovery from herpesvirus infections. The destruction of herpesvirus-infected target cells by antibody-dependent and direct cell cytotoxicity was enhanced by the presence of bovine lymphokine-containing preparations. To relate these effects to possible in vivo mechanisms of recovery, several in vitro approaches were used to measure the effects of lymphokine-containing preparations on controlling viral spread. In the first approach it was shown that in the presence of lymphokines, virus-infected cells could be killed earlier in the replication cycle by the mechanism of antibody-dependent cell cytotoxicity, thus possibly limiting spread of virus. That this was indeed the case was demonstrated by a decrease in the area of viral-induced cytopathology as well as in the total number of infected cells present. Secondly, the amount of infectious virus released was also markedly reduced in cultures incubated with lymphokines and immune peripheral blood lymphocytes as compared to cultures treated with either component alone. Finally, lymphokines caused the activation of macrophages. These results are discussed in terms of how various immune parameters may interact in a positive way so as to aid in the recovery from virus infection.


Synthesis and biological activity of ovine beta-lipotropin-(41--91)-henkaipentekontapeptide. The 51-residue peptide ovine beta-lipotropin-(41--91) has been synthesized by the solid-phase method in about 5% overall yield. The synthetic product was characterized by partition chromatography on agarose gel, thin-layer chromatography in two solvent systems, paper electrophoresis at two pH values, polyacrylamide gel electrophoresis, amino acid analyses of acid and enzymic hydrolysates, and bioassay for lipolytic and melanotropic activities. The synthetic peptide is about 5.4 times as active on a weight basis as ovine beta-lipotropin in the lipolytic assay. In the melanotropic assay, it was about 2.4 times more active than the beta-lipotropin but only 5% as active as bovine beta-melanotropin. It had negligible opiate activity in the guinea pig ileum assay.


Glucose oxidation in the chick cornea: effect of diamide on the pentose shunt. Chick embryo corneas (stages 38 and 45) have been used to study variations in pentose shunt activity following the use of a glutathione-specific oxidizing agent, diamide, and a sulfydryl blocking agent, N-ethylmaleimide (NEM). Shunt activity was measured by the ratio of radiolabeled carbon 1 (14C-1) of glucose to radiolabeled carbon 6 (14C-6) of glucose derived as expired 14CO2. Diamide and NEM were both found to increase pentose shunt activity relative to glycolysis, although by different means. Diamide appeared to exert its effect by oxidizing glutathione and creating a demand for higher shunt activity to facilitate glutathione reduction by NADPH. Both C-1 and C-6 oxidation were increased, but C-1 oxidation was increased to a much greater extent. In contrast, NEM decreased both C-1 and C-6 oxidation, with C-6 preferentially affected. Thus NEM appears to preferentially inhibit the enzymatic machinery of the glycolytic-tricarboxylic acid cycle pathway and acts as an effective metabolic stress on the cornea. Our data suggest that the pentose shunt in the cornea may serve as an important alternative pathway under conditions of metabolic stress for glucose utilization and the production of energy (ATP) in the corneal cells.


Blood gas changes during panting in a small East African antelope, the dik-dik. Five adult male dik-dik (Madoqua kirkii) were exposed in a climatic chamber to an air temperature of 45 degrees C. Measurements were made of rectal temperature (Tre) and respiratory frequency (f) and arterial blood samples taken before and during heat exposure were analyzed for pH, PCO2 and PO2. During exposure, Tre and f increased in all animals. In the first 80 min dik-dik displayed thermal tachypnea and minor changes in blood gases. Continued exposure lead to hyperpnea accompanied by a fall in PaCO2 and a rise in pH. PaCO2 at first fell and then increased toward or above control levels. The dik-dik did not display second phase breathing. This observation confirms that second phase breathing is not essential to the development of respiratory alkalosis. The main conclusion of the study is that the dik-dik, unlike another heat-adapted antelope, the wildebeest (Taylor, Robertshaw, and Hoffmann. Am. J. Physiol. 217:907-910, 1969), is unable to resist alkalosis during heat stress.


Alpha-l-arabinofuranosidase from Rhodotorula flava. An alpha-L-arabinofuranosidase (EC 3.2.1.55) from the culture fluid of Rhodotorula flava IFO 0407 grown on beet arabinan as a carbon source has been highly purified. The purified enzyme has a pH optimum of 2.0. The enzyme is unusually acid stable, retaining 82% of its activity after being maintained for 24 h at pH 1.5 and at 30 degrees C. The apparent Km and Vmax values of the enzyme for phenyl alpha-L-arabinofuranoside were determined to be 9.1 mM and 72.5 mumol per min per mg of protein, respectively.


Protonmotive force and motility of Bacillus subtilis. Motility of Bacillus subtilis was inhibited within a few minutes by a combination of valinomycin and a high concentration of potassium ions in the medium at neutral pH. Motility was restored by lowering the concentration of valinomycin or potassium ions. The valinomycin concentration necessary for motility inhibition was determined at various concentrations of potassium ions and various pH's. At pH 7.5, valinomycin of any concentration did not inhibit the motility, when the potassium ion concentration was lower than 9 mM. In the presence of 230 mM potassium ion, the motility inhibition by valinomycin was not detected at pH lower than 6.1. These results are easily explained by the idea that the motility of B. subtilis is supported by the electrochemical potential difference of the proton across the membrane, or the protonmotive force. The electrochemical potential difference necessary for motility was estimated to be about -90 mV.


Fate of heterospecific transforming DNA bound to Streptococcus sanguis. The fate of 3H-labeled str-r fus-s DNA from Streptococcus pneumoniae, bound after a 1-min uptake to 14C-labeled str-s fus-r S. sanguis recipients, was followed by techniques previously developed for analyzing the fate of homospecific DNA. Heterospecific S. pneumoniae DNA was bound and formed complexes with recipient protein in a manner similar to that of homospecific DNA but transformed relatively poorly. The rate at which complexed heterospecific DNA becomes physically associated with recipient DNA, and at which donor markers are integrated into the chromosome, was slower than in the case of homospecific DNA. In addition, about half of the heterospecific donor counts initially bound in trichloracetic acid-insoluble form were gradually solubilized and released from the cell. The association of heterospecific DNA with the recipient chromosome was more unstable than that involving homospecific DNA, since only associations of the former type were largely dissociated by isolation and resedimentation. The donor DNA-containing material so dissociated had the same sedimentation properties as complexed heterospecific DNA before association, indicating that the complex of single-stranded donor DNA and recipient protein formed on uptake moves as a whole from its site of formation to synapse with the chromosome.


Cell-free biosynthesis of the O-acetylated N-acetylneuraminic acid capsular polysaccharide of group C meningococci. A cell-free system was established to study the biosynthesis of group C meningococcal capsular polysaccharide, an alpha-2 leads to 9-linked N-acetylneuraminic acid (NeuAc) homopolymer containing O-acetyl groups at either C7 or C8. Sialyltransferase activity, isolated from group C meningococcus strain C-11, catalyzed incorporation of [14C]NeuAc from CMP (CMP--[14C]NeuAc) into polymeric form. This sialyltransferase was stimulated by addition of meningococcus group C and Escherichia coli K92 capsular polysaccharides, the latter being an alpha-2 leads to 8- and alpha-2 leads to 9-linked NeuAc heteropolymer. Group C meningococcal sialyltransferase did not require divalent ions but was stimulated by Mn2+. Attempts to demonstrate a lipid-soluble intermediate in the biosynthesis of this NeuAc polymer were unsuccessful. Meningococcal group C sialyltransferase incorporated NeuAc into a membrane-associated product. The polysaccharide can be extracted from the membrane-bound fraction with Triton X-100. The newly synthesized polysaccharide coprecipitates with authentic group C antigen in meningococcal group C antiserum and is degraded by sodium metaperiodate, indicating that the NeuAc polymer synthesized by the cell-free system consists of alpha-2 leads to 9 linkage. Meningococcal group C spheroplast membranes contain an O-acetylase that can catalyze the transfer of acetyl groups from acetyl coenzyme A to the in vitro-synthesized polysaccharide.


Involvement of the product of the glnF gene in the autogenous regulation of glutamine synthetase formation in Klebsiella aerogenes. Mutations in a site, glnF, linked by P1-mediated transduction of argG on the chromosome of Klebsiella aerogenes, result in a requirement for glutamine. Mutants in this gene have in all media a level of glutamine synthetase (GS) corresponding to the level found in the wild-type strain grown in the medium producing the strongest repression of GS. The adenylylation and deadenylylation of GS in glnF mutants is normal. The glutamine requirement of glnF mutants could be suppressed by mutations in the structural gene for GS, glnA. These mutations result in altered regulation of GS synthesis, regardless of the presence or absence of the glnF mutation (GlnR phenotype). In GlnR mutants the GS level is higher than in the wild-type strain when the cells are cultured in strongly repressing medium, but lower than in the wild-type strain when cells are cultured in a derepressing medium. Heterozygous merodiploids carrying a normal glnA gene as well as a glnA gene responsible for the GlnR phenotype behave in every respect like merodiploids carrying two normal glnA genes. These results confirm autogenous regulation of GS synthesis and indicate that GS is both a repressor and an activator of GS synthesis. The mutation in glnA responsible for the GLnR phenotype has apparently resulted in the formation of a GS that is incompetent both as repressor and as activator of GS synthesis. According to this hypothesis, the product of the glnF gene is necessary for activation of the glnA gene by GS.


Glycerolipid biosynthesis in Saccharomyces cerevisiae: sn-glycerol-3-phosphate and dihydroxyacetone phosphate acyltransferase activities. Yeast acyl-coenzyme A:dihydroxyacetone-phosphate O-acyltransferase (DHAP acyltransferase; EC 2.3.1.42) was investigated to (i) determine whether its activity and that of acyl-coenzyme A:sn-glycerol-3-phosphate O-acyltransferase (glycerol-P acyltransferase; EC 2.3.1.15) represent dual catalytic functions of a single membranous enzyme, (ii) estimate the relative contributions of the glycerol-P and DHAP pathways for yeast glycerolipid synthesis, and (iii) evaluate the suitability of yeast for future genetic investigations of the eucaryotic glycerol-P and DHAP acyltransferase activities. The membranous DHAP acyltransferase activity showed an apparent Km of 0.79 mM for DHAP, with a Vmax of 5.3 nmol/min per mg, whereas the glycerol-P acyltransferase activity showed an apparent Km of 0.05 mM for glycerol-P, with a Vmax of 3.4 nmol/min per mg. Glycerol-P was a competitive inhibitor (Ki, 0.07 mM) of the DHAP acyltransferase activity, and DHAP was a competitive inhibitor (Ki, 0.91 mM) of the glycerol-P acyltransferase activity. The two acyltransferase activities exhibited marked similarities in their pH dependence, acyl-coenzyme A chain length preference and substrate concentration dependencies, thermolability, and patterns of inactivation by N-ethylmaleimide, trypsin, and detergents. Thus, the data strongly suggest that yeast glycerol-P and DHAP acyltransferase activities represent dual catalytic functions of a single membrane-bound enzyme. Furthermore, since no acyl-DHAP oxidoreductase activity could be detected in yeast membranes, the DHAP pathway for glycerolipid synthesis may not operate in yeast.


Intracellular serine protease of Bacillus subtilis: sequence homology with extracellular subtilisins. Intracellular serine protease was isolated from stationary-grown Bacillus subtilis A-50 cells and purified to homogeneity. The molecular weight of the enzyme is 31,000 +/- 1,000, with an isoelectric point of 4.3. Its amino acid composition is characteristically enriched in glutamic acid content, differing from that of extra-cellular subtilisins. The enzyme is completely inhibited with phenylmethylsulfonyl fluoride and ethylenediaminetetraacetic acid. Intracellular protease possesses negligible activity towards bovine serum albumin and hemoglobin, but has 5- to 20-fold higher specific activity against p-nitroanilides of benzyloxycarbonyl tripeptides than subtilisin BPN'. Esterolytic activity of the enzyme is also higher than that of subtilisin BPN'. The enzyme is sequence homologous with secretory subtilisins throughout 50 determined NH2-terminal residues, indicating the presence of duplicated structural genes for serine proteases in the B. subtilis genome. The occurrence of two homologous genes in the cell might accelerate the evolution of serine protease not only by the loosening of selective constrainst, but also by creation of sequence variants by means of intragenic recombination. Three molecular forms of intracellular protease were found, two of them with NH2-terminal glutamic acid and one minor form, three residues longer, with asparagine as NH2 terminus. These data indicate the possible presence of an enzyme precursor proteolytically modified during cell growth.


Acid proteases. II. Fluorescence study of the interaction of Cladosporium acid protease with glycyl-DL-norleucine methyl ester in the presence of cupric ions. Glycyl-DL-norleucine methyl ester (GN), a diazoacetyl-DL-norleucine methyl ester (DAN) analog, in the presence of cupric ions was found to partially quench the protein fluorescence of acid protease from Cladosporium sp. No. 45-2, and cupric ions were also found to quench the fluorescence. These quenchings were pH-dependent. GN alone did not quench the fluorescence of the enzyme. The interaction between the enzyme and GN in the presence of cupric ions was studied statically at pH 5.4 in terms of fluorescence change. The dissociation constant, Kd, of the enzyme-GN complex in the presence of a 20-fold molar excess of cupric ions (0.08 mM) determined by fluorescence titration at 30 degrees C (Kd = 1.86 mM) was in good agreement with that obtained for GN from kinetics of inhibition of DAN-induced inactivation in the presence of a 20-fold molar excess of cupric ions at 30 degrees C (KA = 1.94 mM) (Kanazawa, H. (1977) J. Biochem. 81, 1739-1744). At various concentrations of cupric ions, no change of Kd was found. These results suggest that cupric ions are attracted to a negatively charged carboxyl group responsible for the formation of the enzyme-GN complex.


Purification and some properties of a neutral muscle pyrophosphatase. In the water-soluble fraction of rabbit skeletal muscle, at least two types of inorganic pyro phosphatase (PPase) are distinguishable on ion exchange column chromatography. One of them, pyrophosphatase-A (PPase-A), was isolated in an electrophoretically homogeneous form. This enzyme catalyzed the hydrolysis of PPi but not that of other phosphate esters. Only Mg2+ was required for activity and stability. Other cations such as Ca2+, Co2+, Mn2+, and Zn2+ had no activating effect. The activity of this PPase was optimum at pH 7.4. ATP, ADP, sodium imidodiphosphate (PNP), p-chloromercuribenzoate, and Ca2+ inhibited its enzymic activity. The enzyme was protected by dithiothreitol (DTT) against heat denaturation. The molecular weight was estimated to be 67,000 by gel filtration and the molecular size of the subunit was found to be 35,000 by gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). The enzyme probably consists of two identical subunits of 35,000 daltons.


Bacteriolytic enzyme induced from pyocinogenic Pseudomonas aeruginosa. Purification and characterization of PR1-lysozyme. A bacteriolytic enzyme, PR1-lysozyme, has been purified from the lysate of mitomycin C-induced pyocinogenic Pseudomonas aeruginosa, by acrinol treatment, Amberlite CG-50 chromatography, ammonium sulfate fractionation, Sephadex G-100 gel filtration and two cycles of SP-Sephadex C-50 chromatography. Homogeneity of the preparation was demonstrated by three electrophoretic techniques. PR1-lysozyme is a basic protein (pI, 9.4) and consists of a single polypeptide chain having a molecular weight of 24,000. The amino acid composition of the protein was analyzed, and no cystein residue was found among more than 210 amino acid residues. The optimum pH for enzymatic activity was 6.4 and the enzyme exhibited about 50 to 70 times greater specific activity than hen egg-white lysozyme when assayed with chloroform-killed P. aeruginosa as a substrate. By analyzing the products of enzymatic action on purified peptidoglycan of P. aeruginosa, the enzyme was identified as an N-acetylmuramidase, i.e., the same classification as hen-egg-white lysozyme. PR1-lysozyme did not show any activity towards intact cells of gram-positive and gram-negative bacteria tested. However, the enzyme was able to lyse chloroform-killed gram-negative and gram-positive bacteria.


Studies on soybean trypsin inhibitors. XIII. Preparation and characterization of active fragments from Bowman-Birk proteinase inhibitor. Soybean Bowman-Birk inhibitor, a double-headed inhibitor of trypsin and alpha-chymotrypsin, was treated with cyanogen bromide and then pepsin to yield two inhibitory active fragments. Structural investigation showed that one of the fragments was derived from the trypsin inhibitory domain and the other from the chymotrypsin inhibitory domain of the inhibitor. In contrast to the unusual stability of the native inhibitor, the separated domains were less stable and could be inactivated with excess proteinases. These results suggest that the legume double-headed inhibitors acquired their unusual stability by duplicating an ancestral single-headed structure.


A new affinity adsorbent for guanyloribonuclease. Guanylyl-(2'-5')-guanosine coupled to aminohexyl-Sepharose. Guanylyl-(2'-5')-guanosine binds to RNase T1 in 1:1 stoichiometry with a dissociation constant of 0.22 mM at pH 5.0 and 25 degrees C. This nucleotide, coupled to aminohexyl-Sepharose 4B, is able to serve as an affinity adsorbent for guanyloribonuclease [EC 3.1.4.8]. The strength of interaction between the adsorbent and various guanyloribonucleases at pH 5.0 was found to decrease in the following order: RNase N1 greater than RNase F1 greater than RNase T1 greater than RNase St. The bound enzymes can be released from the adsorbent either by increase of ionic strength or by increasing the pH from 5.0 to 7.5. The interaction between RNase T1 and the adsorbent is weakened by the presence of a low concentration of 2', 3'-, or 5'-GMP, which are competitive inhibitors of the enzyme. RNase F1 was purified to homogeneity by use of this affinity adsorbent.


Purification and characterization of lysozyme produced by Streptomyces erythraeus. A species of lysozyme (SE lysozyme) was purified from culture filtrate of Streptomyces erythraeus. The enzyme has a molecular weight of 18,500 as determined by ultracentrifugation. Its isoelectric point is 9.5, and it shows optimal activity at pH 4.0 with an optimal ionic strength of 0.1. Investigation of the substrate specificity showed SE lysozyme to be an N-acetyl-muramidase. The simplest product in the digest of cell walls of Micrococcus lysodeikticus was identified as a disaccharide, [GlcNAcbeta(1 leads to 4) MurNAc]. While S. aureus as well as M. lysodeikticus was lysed by this lysozyme, chitin and its derivatives were not.


Induction of hepatic tyrosine aminotransferase mRNA by protein synthesis inhibitors. Several protein synthesis inhibitors were as effective as the inducers hydrocortisone or cyclic AMP in elevating rat liver tyrosine aminotransferase mRNA levels when assayed in the wheat germ cell-free translational system. Cycloheximide, emetine, or puromycin increased this mRNA activity 6- to 7-fold within 4 h after in vivo administration. No increase in total hepatic mRNA levels or tryptophan oxygenase mRNA was found after treatment with these protein synthesis inhibitors. Furthermesults suggest that a short lived protein may specifically regulate the level of functional hepatic tyrosine aminotransferase mRNA or that ongoing translation of this mRNA is required for its degradation.


The role of Ca2+ on pH-induced hydrodynamic changes of bovine pancreatic deoxyribonuclease A. DNase A studied by gel filtration on Sephadex G-100 at pH 7.4 in 40 mM Tris-HCl buffer, behaves hydrodynamically as a spherical monomeric macromolecule of around 31,000 molecular weight, with a Stokes radius = 24.7 A, f/fo = 1.19, and D20,W = 8.69. Similar results were obtained by analytical dialysis using zinc chloride-modified cellophane membranes. The elution volume of DNase A decreases as the pH increases between pH 4.7 and pH 9.5. This effect has been attributed to a change in the tridimensional structure of the protein and interpreted as a modification in the axial ratio due to unfolding of the polypeptide chain with increase in the apparent Stokes radius. The addition of Ca2+ produce reversion of the pH-induced changes at pH 9.5. The transition occurs when Ca2+ binds to at least two binding sites (n = 1.66 in a Hill plot) with a Kd = 8.9 X 10(-5) M and the effect appears to be cooperative. These findings support the hypothesis that Ca2+-binding to DNase A causes a conformational change that maintains a more active structure of the enzyme, especially when the pH-induced unfolding reduces its activity.


Energy transduction in the photosynthetic membranes of the cyanobacterium (blue-green alga) P-lectonema boryanum. Membrane preparations isolated from the photosynthetic lamellae of the cyanobacterium Plectonema boryanum generate upon illumination  a transmembrane pH gradient of approximately 2 to 3 pH units (acid inside), as determined from the distribution of either fluorescent or radioactive amines (9 aminoacridine and [14C]methylamine, respectively). Using the distribution of permeant ions to measure the electrical potential across the membrane, it was found that the latter is practically nil under conditions in which the deltapH is formed and photophosphorylation takes place. In agreement with the above findings cyclic photophosphorylation in this membrane preparation is inhibited by agents shown to collapse the deltapH but not by agents which should collapse the electrical potential. It is deduced that the pattern of proton movement in the photosynthetic lamellae of intact Plectonema spheroplasts corresponds to that of the cell-free membrane system, as both preparations show similar light dependent accumulation of fluorescent amine. It is concluded that the pattern of energy transduction in Plectonema photosynthetic lamellae is similar to that of chloroplast thylakoid membranes and not to that of bacterial cytoplasmic membranes. The evolutionary implications of the findings are discussed and a model for the directionality of H+ movements in the whole cell is presented.


The transport of L-alanine by the hamster kidney cell line BHK-21-C13. The uptake of L-alanine into BHK21-C13 cells in culture has been studied. This amino acid appears to be transported essentially via a relatively low affinity, high capacity, sodium ion dependent transport system. Inhibition studies using other amino acids or their analogues provided information about the specificity of this system. This alanine transport system was shown to exhibit a broad substrate specificity and appeared to be capable of transporting most naturally occurring neutral alpha-amino acids. Kinetic studies of the inhibition of L-alanine uptake also indicated the presence of a second neutral amino acid transport system capable of transporting this amino acid. However, it is unlikely that this second uptake system contributes greatly to L-alanine uptake. Inhibition of the uptake of L-leucine indicated that this transport system has a similar specificity to the "L"-system initially described for Ehrlich ascites carcinoma cells.


Coordinacy of lysosomal enzyme excretion in human urine. Assay conditions have been developed for the determination of urinary beta-glucuronidase, beta-galactosidase, alpha-galactosidase, and beta-hexosaminidase using fluorometric substrates. The assay conditions for beta-glucuronidase overcome interference by both low and high molecular weight inhibitors, a problem that has confused earlier studies of enzyme excretion. The four lysosomal enzymes are excreted corrdinately: although their absolute levels (in units per milligram of creatinine) vary during the day and from one day to the next, the ratio of one enzyme to another remains relatively constant. The lack of correlation betweem plasma and urine enzyme levels, together with the high molecular weights of these enzymes, suggests that the urinary enzymes are not derived by glomerular filtration. The lack of coordinacy with lactate dehydrogenase suggests they are not derived from exfoliated cells. by analogy with experimental animals, they may be derived from lysosomes extruded into the lumen of the proximal tubule by epithelial cells. There is considerable variation among a population of 125 healthy adult subjects for total enzyme excretion. Both total enzyme excretion and coordinacy ratios are log-normally distributed, suggesting that they are the resultants of many factors, each of which has a relative, or proportional, effect on enzyme excretion. About one-half the population variation resides in a process common to the excretion of all four enzymes (possibly the lysosome extrusion pathway), and about one-half resides in factors affecting each enzyme independently.


Diminished spectrin extraction from ATP-depleted human erythrocytes. Evidence relating spectrin to changes in erythrocyte shape and deformability. We measured spectrin "extractability" in erythrocytes which were metabolically depleted by incubation at 37 degrees C in plasma or glucose-free buffers. Membranes were extracted with 1 mM EDTA (pH 8, 40 h, 4 degrees C) and analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. This procedure solubilized 85--90% of the spectrin, actin, and residual hemoglobin from ghosts of fresh erythrocytes. In incubated erythrocytes, inextractable spectrin rapidly accumulated when ATP concentrations fell below 0--15% of normal. In severely depleted cells, 60--90% of the total ghost spectrin became inextractable. Inextractability was not abolished by physically disrupting the ghost before extraction, but was reversed when erythrocyte ATP was replenished with adenosine. The accumulation of inextractable spectrin correlated temporally with the increase in apparent membrane deformability and the increases in erythrocyte vicosity, calcium content, sodium gain, and potassium loss characteristic of ATP-depleted erythrocytes. No change in integral membrane protein topography (assessed by the distribution of intramembranous particles and concanavalin A surface-binding sites) was detected in depleted cells. Analogous changes were observed in erythrocytes exposed to extremes of pH and temperature. When the pH in the erythrocyte interior fell below 5.5, a pH where spectrin was aggregated and isoelectrically precipitated, erythrocyte and ghost viscosity increased coincident with a marked decrease in spectrin extractability. Similarly above 49 degrees C, a temperature where spectrin was denatured and precipitated, erythrocyte viscosity rose as inextractable spectrin accumulated. These observations provide direct evidence of a change in the physical state of spectrin associated with a change in erythrocyte shape and deformability. They support the concept that erythrocyte shape and deformability are largely determined by the shape and deformability of the spectrin-actin protein meshwork which laminates the inner membrane surface.


Alpah-adrenergic receptor modulation of beta-adrenergic, adenosine and prostaglandin E1 increased adenosine 3':5'-cyclic monophosphate levels in primary cultures of glia. Beta-adrenergic agonists, adenosine and prostaglandin E1 increased the level of adenosine 3':5'-monophosphate (cAMP) in glial cultures prepared from rat cerebral cortical tissue. In addition to these physiological effectors, cholera toxin also increased cAMP levels in these cultures. The accumulation of cAMP in response to each of these agen-s, including cholera toxin, was partially blocked (50--80%) by simultaneous alpha-adrenergic receptor stimulation. Basal levels of cAMP were not affected by alpha-adrenergic agonists. These results indicate that in glia, alpha-adrenergic receptors may serve to modulate the level of cAMP which normally accumulates in response to a number of neurohumoral substances. The modulatory effect of alpha-adrenergic agents does not appear to reduce cAMP accumulation by activating phosphodiesterase since the effect was not blocked by a potent inhibitor of this enzymemthe results suggest that the modulatory effect of alpha-adrenergic receptor activation results from an interaction which takes place at some point in between adenylate cyclase-associated-membrane receptors and the enzymatic degradation of cAMP.


Diazepam and lorazepam for intravenous surgical premedication. Diazepam, 10 and 20 mg, and 2 and 4 mg lorazepam were studied as intravenous surgical premedicants in 120 patients. Relief of anxiety, sedation, patient acceptance, lack of recall, and side effects were the variables evaluated. Both diazepam and lorazepam proved to be excellent surgical premedicants. The basic difference between the two drugs is temporal. Both medications produce similar relief of anxiety, sedation, patient acceptance, and lack of recall. The clinical effects of intravenous diazepam peaks in 2 to 3 minutes and diminishes thereafter. Intravenous lorazepam has a latent period of 8 to 15 minutes, with increasing effects at 15 to 30 minutes.


Loss of carbofuran from rice paddy water: chemical and physical factors. The loss of carbofuran was studied from rice paddy water treated with a granular formulation of the insecticide, and from ponds filled with drainage from the paddy. The average half-life (t 1/2) for carbofuran loss was 57 hr. Controlled experiments indicated that pH was the predominating factor governing carbofuran loss from water in the environment studied. The loss due to hydrolysis was over 700 times more rapid at pH (t 1/2 = 1.2 hr.) than at pH (t 1/2 = 864 hr.) in buffered deionized water. The average pH of the rice paddy was 8, but diurnal fluctuations of 7 to 9.5 are common in similar environments. Impurities in the water, sunlight, and temperature influence the rate of carbofuran loss but not nearly so much as pH. There was no evidence for significant loss due to evaporation or oxidation. The results have important implications for the duration of the insecticide's activity and the effect on fish within or downstream from treated paddies.


Preservation of organophosphorous pesticides in water samples. Four techniques were studied for capacity to preserve sixteen organophosphorous pesticides in distilled water and in creek water. A technique using chloroform effectively preserved all sixteen pesticides for the three weeks of the study and refrigeration was effective for fourteen of the pesticides, but buffers of pH 4 and pH 7 appeared undependable as preservatives.


Long-term hypnotic efficacy and safety of triazolam and flurazepam. Both triazolam and flurazepam are effective hypnotics when administered nightly for 12 consecutive weeks. However, at the dosages tested, 0.6 mg triazolam had a significantly faster onset of activity than 30 mg flurazepam. Long-term administration of either treatment did not influence the patient's capability to recognize the difference between active drug and placebo. This supports the conclusion that there was no tolerance development on either treatment. There were no deleterious effects attributable to either treatment as measured by the 35-Item Hopkins Symptom Checklist or by physical examinations, laboratory tests, ECGs, and ophthalmologic examinations. Side effects occurred more often on flurazepam than on triazolam, and the number of patients experiencing side effects was significantly higher in the flurazepam group. Drowsiness and grogginess were reported most frequently on both treatments, and the number of patients reporting drowsiness or grogginess was also significantly higher in the flurazepam group.


Biological characteristics of peptidoglycans of group A streptococcus and some other bacterial species. I. Tolerance and effect of antibody in fever response, and heart damaging effect in rabbits. Induced tolerance to the pyrogenic action of group A streptococcus peptidoglycan decreased after one week and was no longer detectable after the second week. However, one or two further doses of peptidoglycan rapidly restored the tolerance. The passive transfer of plasma from rabbits tolerant to streptococcus peptidoglycan to nontolerant animals failed to transfer tolerance. Antiserum to streptococcus peptidoglycan neutralized the pyrogenic effect of not only streptococcus but also staphylococcus and pneumococcus peptidoglycan; it did not influence the febrile response to endotoxin. Histopathologic changes in the rabbit heart produced by the intravenous injection of staphylococcus or pneumococcus peptidoglycans were similar and were characterized by various stages of degeneration and necrosis. The changes were less pronounced than after streptococcus peptidoglycan. Antiserum to streptococcus peptidoglycan had modest or no counteracting effect on the development of heart alterations after staphylococcus or pneumococcus peptidoglycan.


Studies on the mitogen responses of germfree allogeneic chimeras. II. Maturation of two cell types and partial restoration of responsiveness of the short-term chimeras. Germfree allogeneic bone marrow chimeras (ABMC) were produced by the i.v. injection of approximately 10(7) bone marrow cells from germfree DBA/2 mice into lethally irradiated germfree C3H mice. In the germfree state, the short-term ABMC showed no histologic signs of graft-vs-host reactions (GVHR), yet splenic lymphocytes were unable to respond to PHA, Con A, or SRBC. Attempts to remove responsiveness by the implantation of a DBA/2 thymus under the host kidney capsule also resulted in failure. However, when the donor thymus was enclosed in a cell-impermeable chamber to eliminate a GVH reaction, responsiveness to Con A was restored. The PHA and SRBC responses were unaffected by this treatment. Daily injections of thymosin caused both an increased Con A response and increased numbers of PFC, although the PHA response was again unaffected. Thus, soluble substances from thymic tissue can be used to overcome partially the histocompatibility barrier present in the ABMC that affects at least two different functional cell populations.


Tyrosine aminotransferase in AKR/J albino and C57BL/6J black mouse skin. L-Tyrosine aminotransferase is present in a high speed supernatant fraction of skin homogenate of AKR/J albino and C57BL/6J black mice. The conversion of tyrosine to p-hydroxyphenylpyruvate was shown to be catalyzed by an aminotransferase by the following observations: the reaction was partially dependent on the presence of low concentrations of alpha-ketoglutarate; catalase was ineffective in increasing the yield of p-hydroxyphenylpyruvate; there was potent inhibition by typical inhibitors of pyridoxal phosphate enzymes and of rat liver tyrosine aminotransferase; there was no inhibition by inhibitors of L-amino acid oxidase; and there was no oxidation of L-leucine, the best substrate for rat kidney L-amino acid oxidase. The aminotransferase was stimulated by mercaptoethanol and was inhibited by high concentrations of alpha-ketoglutarate. The apparent Km for tyrosine was 5 X 10(-3) M and the molecular weight, determined by sucrose density gradient centrifugation, was 150-200,000. Dopa was also transaminated by the crude enzyme. No tyrosine aminotransferase could be detected in extracts of hamster melanoma.


The relationships among arterial oxygen flow rate, oxygen binding by hemoglobin, and oxygen utilization in chronic cardiac decompensation. We have examined the interrelationships among CaO2, blood flow, oxygen binding by hemoglobin, and VO2 in cardiac patients with and without chronic cardiac decompensation. We have quantified the role that decreased oxygen-binding to hemoglobin may play in maintaining VO2 in the presence of low systemic blood flow rates. The volume rate of oxygen delivery to tissues was expressed as the OFIa, the product of CO2 and blood flow. OFIa varied from 738 to 262 ml/min/m2, whereas VO2 varied from 170 to 117 ml/min/m2. Thus, in the patients with lowest OFIa (63% below the highest OFIa), VO2 was only down 19%. VO2 was maintained because the extraction of oxygen rose from about 20% to 50% in close association with the decrease in OFIa. Oxygen binding to hemoglobin was lower in patients with the lowest OFIa--and therefore, at in vivo conditions of pH, PCO2, and temperature, P50 in vivo was higher. The resulting facilitation of oxygen release at the PO2 of tissue capillaries could explain about one third of the observed increment in oxygen extraction in patients with low OFIa. An alternative interpretation is that a high P50 in vivo minimizes the reduction in PVO2 needed to maintain VO2 when increased proportional extraction of O2 compensates for decreased OFIa.