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[Influence of narcotic analgesics, droperidol, diazepam, and flunitrazepam on the smooth muscles of small arteries (author's transl)].

In isolated segments of the rat tail artery, the influence of narcotic analgesics, their antagonists, droperidol, diazepam, and flunitrazepam on the vascular smooth muscle was investigated. The wall of this vessel consists of about 75 per cent smooth muscle fibres. For all agents studied, pressure-diameter relations were determined before and after a virtually maximum constriction which was induced with noradrenaline. The results were compared with those obtained under control conditions, i.e. prior to the addition of the drugs to the bath solution. Tested in pure Tyrode's solution, none of the agents produced a noticeable constriction or relaxation. Among the analgesics and their antagonists, morphine was the most potent in inhibiting the noradrenaline-induced constriction of the vessel (mean increase in diameter 15%). Pentazocine and levallorphan were less effective (mean increase 8%), and the administration of pethidine and naloxone was followed by even less relaxation, resulting in mean diameter increases of 4%. Piritramide and fentanyl did not show any effect. Depending on the pressure level, droperidol caused the diameter to increase 2 to 10%. Flunitrazepam brought about a marked relaxation (mean increase in diameter 15%), diazepam, however, did not influence the vessel segments. The results obtained show that the haemodynamic effects of the drugs used in this study, in particular those on the peripheral resistance, may, at least partially, be due to their direct influences on the vascular smooth muscle. The real pharmacologic mechanisms by which these effects are initiated, in most cases still remain to be investigated.

[Temperature effects at the NADP+ and NAD+ levels in the mycelium of P. nigricans Thom. strains grown on different carbon sources].

The effect of elevated temperature of the medium on the levels of NADP+ and NAD+ in the mycelium of highly productive strain 117 and low productive strain B of P. nigricans Thom was studied at various developmental periods on the mineral medium in the presence of glucose, succinate or acetate. It was found that in the presence of glucose the elevated temperature markedly stimulated the initial growth of the culture, decreased the level of NADP+ in strain 117 by the 48th hour and increased the level of NAD+ in both the strains. At later periods it decreased the rate of the dinucleotide accumulation during the culture development. With the use of succinate under these conditions the levels of NADP+ in both the strains during the growth phase (the 2nd and 5th days) and by the 9th day markedly increased, while the NAD+ concentration increased only by the 2nd day in both the strains and by the 9th day in strain B. When the strains were grown in the presence of acetate, the elevated temperature especially affected the level of NAD+.

[Overacidified tissue and microcirculation (author's transl)].

A discussion of physiological fundamentals with respect to the inhibition of blood microcirculation in (tumor) tissue at reduced pH values around 6.0 is followed by a report on principles, design and results obtained with a light probe array which permits to determine in vivo reference values of the relative intensity of microcirulation in both normal and tumor tissues under various conditions. An analysis of the discussed records has shown that--as compared to a value of 80-66% without glucose infusion--the relative mean intensity of microcirculation in tumor tissue drops to approximately 8-4% about 300 min after the onset of glucose infusion under CMT administration at 37 degrees C. By adding the CMT step of hyperthermy, the relative mean intensity of microcirculation--compared to normal tissue at 37 degrees C--will further drop below 1%. With such a decline of microcirculation--and an adequate duration of, say, 8 hours--local hyperthermy at 41-42 degrees C is likely to cause a very pronounced damaging action on tumor tissue because the then noticeably reduced substrate offer proves to be insufficient to ensure the structure-maintaining metabolic rate of cancer cells.

[Studies on the effect of oxprenolol on experimentally induced anxiety (author's transl)].

This experiment studied the question of whether the receptor blocking agent oxprenolol (20 mg) influences somatic and subjective responses to experimentally induced anxiety. 72 male students were Ss in a two-factorial design with type of drug (oxprenolol, placebo) and type of situation as the two factors. Anxiety was induced by the signaled application of shocks to one arm, under condition A with and under condition B without time control (clock available or not). Condition C was a control condition with neither shock nor time control. Heart rate, blood pressure and verbal reports of emotional experience were measured. An analysis of variance revealed the following: Oxprenolol showed physiological effects typical of a receptor blocking agent. The drug positively affected emotional experience not related to the experimentally induced anxiety but did not affect the emotional responses induced by the anxiety-provoking conditions. For explaining the results the possible meaning of the variable "internal vs external control of emotional responses" was considered.

Possible significance of the pharmacological differentiation of beta-blockers for therapy of hypertension.

1 Cardioselective and non-selective beta-blockers affect to a different degree several aspects of the circulatory homeostasis. The evidence available in this regard has been evaluated and the possible clinical importance of these differences has been discussed. 2 Venous return in partly regulated by beta-receptors (possibly of the beta 2 type) in the venous resistance vessels. Differences in blockade of venous return by the two classes of beta-blockers may, therefore, influence the degree of increase in left ventricular size, left ventricular end diastolic BPs and stroke volume during beta-blockade. 3 At the first part of the dose-reponse curve, non-selective beta-blockers seem to block more effectively renin release than cardioselective beta-blockers. 4 The direction and the extent to which beta-blockers 'directly' affect total peripheral resistance (TPR), is determined by the resultant of the degree of decrease in TPR by blockade of renin release and the extent of the increase in TPR by blockade of the beta 2-receptors in the arteriolar wall. 5 The clinical relevance of these differences could be that--especially in the low doses range--non-selective beta-blockers may be more 'safe' in patients with compromised cardiac function and may be more appropriate for the therapy of high renin hypertension than cardioselective blockers, whereas the latter may be more appropriate for the majority of hypertensive patients who have low to normal renin hypertension.

The digestion of pectin in the human gut and its effect on calcium absorption and large bowel function.

1. The effect of dietary fibre digestion in the human gut on its ability to alter bowel habit and impair mineral absorption has been investigated using the technique of metablic balance. 2. Five healthy male students were studied for 9 weeks under controlled dietary conditions and during the last 6 weeks they took 36 g pectin/d. Bowel habit, transit through the gut, faecal fibre excretion, calcium balance and faecal composition were measured. 3. During the control period only 15% of the dietary fibre ingested was excreted in the stools and when pectin was added to the diet there was no increase in fibre excretion. Stool frequency and mean transit time were unchanged by pectin but stool wet weight increased by 33% and faecal excretion increased (%) for fatty acids 80, nitrogen 47, total dry matter 28 and bile acids 35. Ca balance remained unchanged. 4. It may be concluded from these results that dietary fibre is largely metabolized in the human gut and dietary pectin completely so. This could explain its lack of effect on bowel habit and Ca balance. Other changes in the faeces may be related to an increase in bacterial mass.

Turnover and vectorial properties of cytochrome c oxidase in reconstituted vesicles.

1. Proteoliposomes containing cytochrome c oxidase and phospholipid have been made by sonication and by the cholate dialysis procedure. In both methods of preparation, only about 50% of the enzyme molecules are oriented in the membrane with their cytochrome c reaction sites exposed to the outside of the vesicle. 2. The activity of cytochrome c oxidase in the reconstituted vesicles in not increased by incubation in 1% Tween 80. Experiments on reconstituted vesicles containing internal (entrapped) cytochrome c indicate that turnover of enzyme oxidising entrapped cytochrome c in the presence of N,N,N',N'-tetramethyl-p-phenylenediamine or 2,3,5,6-tetramethyl-p-phenylendediamine is at a very much lower rate than enzyme oxidising external ferrocytochrome c. 3. Oxidation of ascorbate by externally added cytochrome c results in an electrogenic production of OH- inside the vesicles, which can be monitored using entrapped phenol red. Polylysine inhibits, but does not abolish, the internal alkalinity change in reconstituted vesicles oxidising internal (entrapped) cytochrome c using externably added ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine. When 2,3,5,6-tetramethyl-p-phenylenediamine is used as the permeable redox mediator, an increase in internal acidity can be monitored under the same conditions.

A high potential acceptor for photosystem II.

The effects of ferricyanide on Photosystem II reactions have been investigated by measurements of microsecond and millisecond prompt fluorescence and microsecond-delayed fluorescence in dark-adapted chloroplasts: (1) Titrations using ferri-ferrocyanide mixtures on: (a) the fast phase of the increase in fluorescence yield observed during a xenon flash, and (b) the normalised area above the millisecond fluorescence induction curve for chloroplasts inhibited by DCMU, showed a pH dependent mid point potential of 400 mV at pH 7.0 which varied by approx. -60 mV/pH unit between pH 6 and 8.5. (2) A saturating laser flash induced a fluorescence increase (as monitored by a weak measuring beam) of only 50% of that reached following a second flash in chloroplasts preincubated with ferricyanide and inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) prior to illumination. In the absence of ferricyanide, the fluorescence level reached after a single flash was initially close to that measured after a second flash (although the level subsequently declined). (3) The initial amplitude of the microsecond-delayed fluorescence excited by a single laser flash was diminished in chloroplasts dark-adapted with ferricyanide. In the presence of DCMU and ferricyanide, the amplitude was also diminished for the first flash of a series, but subsequently enhanced above the level obtained in chloroplasts in the presence of DCMU alone. (4) The above effects were not seen if DCMU was added to the chloroplasts before ferricyanide, or if the period of incubation with ferricyanide was much less than 4 min. (5) These results suggested the presence of a second acceptor Q2, with Em7 = 400 mV and n = 1, before the DCMU block in Photosystem II. There is 0.35--1 equivalent of the acceptor per reaction centre, and its reduction occurs within less than 5 mus. The role of the acceptor in double turnovers of the photochemistry during a single flash and its likely operating redox potential are discussed.

Fluorescein mercuric acetate as a probe of the dynamic structure of double-helical DNA.

Fluorescein mercuric acetate causes the unwinding of DNA and binds to the separated bases. The kinetics of this unwinding process were studied using both untreated DNA and sonicated DNA at various pH values (6.8--9.3) and Na+ concentrations (10--250 mM). The unwinding process is explained by assuming a nucleation in the middle of DNA (as a function of time) as well as at the helix ends (immediately after addition of this reagent) and the subsequent growth of the nuclei. The frequency of the nucleation in the middle of DNA appears to be markedly affected by pH and Na+ concentration. In contrast, the reaction rate of this reagent with heat-denatured DNA was almost insensitive to these environmental variables. The growth rate of the unwinding nuclei in double-stranded DNA also appears insensitive. The most important implication of this study is that in the low pH range (6.8--7.5) the reactivity of thermally-induced locally open regions in the middle of double-helical DNA toward this reagent appears much higher than that of heat-denatured DNA. Since this reagent is negatively charged, these findings are discussed in view of its electrostatic interaction with the locally open regions.

[Stability of glucose-6-phosphate dehydrogenase in human cells cultivated in vitro].

It was shown that the thermal stability of glucose-6-phosphate dehydrogenase in human diploid cells is much higher than in human heteroploid cell lines HeLa and T-9. The purified enzymes from human diploid cells and from HeLa and T-9 cells possess similar thermal stabilities. Mixing of T-9 extracts with the purified enzyme preparations revealed that the non-stability factors of the dehydrogenase are present in the T-9 extracts. An addition of NADP- and NADPH-containing buffers and crystalline NADP to the heteroploid cell extracts stabilizes the enzyme. The thermal stability of the enzyme from "in vitro" cultivated human cells depends on the concentration of the coenzyme. It was also demonstrated that glucose-6-phosphate dehydrogenase stability in HeLa and T-9 extracts is the same at low concentrations of the coenzyme and after addition of crystalline NADP. However, at NADP concentration of 10(-3) M the enzyme stability in HeLa and T-9 extracts is different. It is assumed that the destabilizing factors are the enzymes possessing the nucleotidases activity, which is different in various cell lines.

Nonplasminogen-dependent protease in human plasma.

Equal volumes of plasma and 0.3 M K2HPO4, pH 7.4, were mixed, diluted 20-fold, and adjusted to pH 5.2. After incubation at 37 degrees C for 30 min, the euglobulin percipitate, redissolved in 0.1 M K2HPO4, pH 7.4, developed caseinolytic activity (0.05 CTA U/ml). Na2HPO4 or NaCl of similar ionic strength could replace K2HPO4. The pH optimum of the protease was 6.5, activity falling off sharply below pH 6.0 and above 7.4. The proteolytic activity was inhibited by diisopropylphosphofluoridate and by pancreatic trypsin inhibitor, but was not inhibited by soybean trypsin inhibitor. The activity was not due to plasmin, contact activation, or coagulation factors, since it was fully generated in plasminogen-depleted, factors XII, XI, VII deficient, and prekallikrein-deficient plasmas. Purified Cl-esterase was not caseinolytic in our system. Redissolved euglobulin precipitate prepared from normal plasma without salt addition could serve as starting material for the generation of caseinolytic activity, as could serum, indicating that the Hageman factor cofactor and thrombin are not required. The protease had no detectable procoagulant or fibrinolytic activity.

The inhibitory effects of presynaptic alpha-adrenoceptor agonists on contractions of guinea-pig ileum and mouse vas deferens in the morphine-dependent and withdrawn states produced in vitro.

1 Isolated ilea from guinea-pigs implanted with morphine pellets were stimulated coaxially, either with or without morphine present in the bath fluid, and the longitudinal contractions recorded. 2 In the absence of morphine the inhibitory effects of the presynaptic alpha-adrenoceptor agonists, clonidine and oxymetazoline were much reduced and the dose-response curve was flat. This state of 'withdrawal' was readily reversed by morphine and levorphanol but not its inactive (+)-isomer, dextrophan. 3 The kappa-agonists, ketazocine and ethylketazocine, also restored the effects of clonidine as did the opioid peptides Tyr-D-Ala-Gly-Phe-D-Leu, acting preferentially on delta-receptors, and Tyr-D-Ala-Gly-MePhe-Met(O)-ol, acting mainly on micro-receptors. 4 The inhibitory effects of adrenaline and adenosine 3',5'-diphosphate were reduced at low but not at high concentrations. 5 In contrast, the inhibitory effect of clonidine on the electrically evoked contractions of vasa deferentia from mice implanted with morphine pellets was not abolished by the lack of morphine in the bath fluid or by addition of naloxone. 6 A possible explanation is suggested for the loss of the inhibitory effects of presynaptic alpha-adrenoceptor agonists in the withdrawn state of the dependent ileum.

Pharmacological characterization of perifornical hypothalamic dopamine receptors mediating feeding inhibition in the rat.

Mapping studies with central drug injections in the hungry rat have identified the perifornical lateral hypothalamus as being uniquely sensitive to the feeding suppressive effects of exogenous dopamine, as well as anorexic drugs which release endogenous catecholamines. In the present study, the hypothalamic dopamine-receptive sites mediating this phenomenon were pharmacologically characterized. These sites, studied via direct drug injection into the perifornical hypothalamus of freely moving, brain-cannulated rats, were found to be most responsive to dopamine, in a dose-dependent manner, but were also activated by other catecholamine receptor stimulants, with the order of potency being dopamine greater than apomorphine = epinine greater than norepinephrine. Clinically effective neuroleptic compounds antagonized these dopamine-sensitive sites, apparently in a competitive and stereochemically specific manner. The relative potency of the neuroleptics and structurally related compounds was calculated to be haloperidol greater than fluphenazine greater than chlorpromazine greater than pimozide greater than promazine. The ineffective neuroleptic promethazine, the tricyclic antidepressants imipramine and desipramine, and the antagonists of alpha-adrenergic, beta-adrenergic, cholinergic, and serotonergic receptors, did not manifest the ability to reverse dopamine's action. These results thus reveal properties of these hypothalamic dopamine-sensitive, feeding inhibitory sites which match to a large extent the characteristics recently identified for dopamine receptors in the periphery and extrahypothalamic brain areas.

Analogs of L-aspartic acid in chemotherapy for cancer.

The interaction of analogs of L-aspartic acid with adenylosuccinic acid synthetase, L-asparagine synthetase, and L-aspartic acid transcarbamylase is discussed. Each of these enzymes is of critical importance in the economy of certain types of tumor cells. L-Alanosine, a new antitumor antibiotic, is shown to be accepted as a substrate by the enzymes of de novo purine biosynthesis which ordinarily use L-aspartic acid as a substrate; as a consequence of this interaction, an anabolite is thought to be produced which impairs the formation of adenine nucleotides by inhibiting adenylosuccinate synthetase, leading to an interruption in DNA synthesis. Homoserine-beta-adenylate, guanidinosuccinic acid, and PA2LA [3-(phosphonacetylamido)-L-alanine] are shown to be inhibitors of L-asparagine synthetase from murine lymphoblasts; each of these analogs of L-aspartic acid exhibits novel structural properties which can be used by synthetic chemists in the design of molecules with an even greater ability to block the biosynthesis of L-asparagine. Certain aspects of the mechanism of action of PALA (N-phosphonacetyl-L-aspartic acid) were examined. This agent, which is a potent inhibitor of mammalian L-aspartic acid transcarbamylase, is capable of stimulating the homologous enzyme from Escherichia coli under certain circumstances. In vivo the duration of inhibition produced by this agent is shown to be unusually protracted; for example, L-aspartic acid transcarbamylase in mouse liver remains at 30% of treatment levels for greater than or equal to 20 days after a single therapeutic dose of PALA. This long-lasting effect reflects either sluggish synthesis of new enzyme molecules in this organ or shuttling of the inhibitor from old to new molecules. It is suggested that new and still more potent analogs of L-aspartic acid be sought, and that they be screened, inter alia, against these target enzymes.

Effect of ethanol on the renal excretion and metabolism of choline in the isolated perfused rat kidney.

The isolated perfused rat kidney was used to investigate the effect of ethanol on the renal excretion and metabolism of choline. Choline at an initial perfusate concentration of 2.8 mM, with tracer amounts of [methyl-14C]choline, was recirculated through kidneys and radioactivity measured in perfusate, urine, and kidney. 14C-Choline and its metabolites were identified by chromatographic and electrophoretic procedures. Tubular excretion of choline was demonstrated and a transport maximum (Tm) of 1.6 mumol/kidney/min was reached at a choline perfusate concentration of 1.2 mM. Addition of 50 mM ethanol resulted in a 56% increase in the choline Tm and 100 mM ethanol decreased the choline Tm by 25%. The rate of loss of 14C-choline from the perfusate was increased by the lower ethanol concentration and decreased by the higher ethanol concentration. Ethanol at both concentrations diminished the amount of 14C remaining in the kidney. 14C-Betaine was the major choline metabolite and the only 14C-metabolite present in perfusate or urine. Addition of either 50 or 100 mM ethanol increased both glomerular filtration rate and urine volume.

Metabolism and disposition by the rat of 35S-sulfadiazine alone and in the presence of trimethoprim.

The tissue distribution and metabolism of 35S-sulfadiazine (I, SDZ) alone and in the presence of trimethoprim (TMP) was studied in the male rat. In the 72-hr period following a single oral dose (30 mg/kg) of 35S-SDZ/TMP (5/1, w/w), 87% of the radioactivity was recovered in the urine and 15% of the radioactivity was recovered in the feces. The concentrations of drug-related material in the plasma or tissues after 72 hr were less than 0.1 ppm with the exception of the liver (0.13 ppm). Aside from intact drug, the two major urinary metabolites (greater than 5% of the radioactivity in urine) were N4-acetylsulfadiazine (II) and sulfadiazine N4-glucuronide (VI). Three minor urinary metabolites (less than 5%) were identified as N4-acetyl-2-sulfanilamido-4-hydroxypyrimidine (IV), 2-sulfanilamido-4-hydroxypyrimidine (III) and 2-sulfanilamido-5-hydroxypyrimidine (V). Metabolites IV and V are novel metabolites of SDZ and have not been reported previously for any species. The relative amounts of sulfadiazine and its metabolites excreted in the urine and feces as well as the distribution of intact drug and 35S in rat tissues were determined. The metabolites were screened for antibacterial activity; the N4-acetylated metabolites II and IV were inactive, whereas the hydroxypyrimidine metabolites III and V were active against a few organisms but in general much less active than I.

Enhancement of reductive metabolism of p-nitrobenzoate and nitrazepam in isolated perfused rat liver by ethanol.

Reductive metabolism of p-nitrobenzoate (2 mM) was studied in the isolated perfused rat liver, after acute ethanol dosing, with use of a hemoglobin-free perfusion medium. Formation of reduced metabolites under control conditions (0.3 mumol per g of liver per hr) was enhanced fivefold (1.4 mumol/g/hr) in the presence of ethanol (38 mM), thus reaching hepatic reductase activities occurring under anaerobic conditions (1.4 mumol/g/hr). Ethanol failed to increase hepatic nitro reduction when alcohol dehydrogenase was inhibited by pyrazole. Addition of acetaldehyde led to a marked stimulation of nitroreductase activity. Carbon monoxide did not influence the ethanol-mediated enhancement of nitroreductase activity but almost abolished the enhancement caused by anoxia. Reductive azo cleavage of salazosulfamide was not enhanced by ethanol. When nitrazepam was used as the substrate (1 mM) for the isolated perfused rat liver, addition of ethanol (38 mM) led to an enhanced content of 7-amino derivative in the liver and in the perfusate, whereas the formation of 7-acetylamino derivative remained unchanged. The distribution of nitrazepam in liver and perfusate was not altered by ethanol.

[Inhibition in neurophysiology].

In Neurophysiology, inhibition is the effect of one neuron upon another tending to prevent it from initiating impulses. At cellular level, there are 3 modes of inhibition: postsynaptic inhibition, presynaptic inhibition and electrical inhibition. The formers are mediated by chemical transmitter. Examples of functional inhibition are presented. They explain its role in the coordination of movement and posture, in the integration of the sensation and in the behaviour.

Interaction of ADP with skeletal and cardiac myosin and their active fragments observed by proton release.

The technique of proton release measurement has been used to explore the binding of ADP to skeletal and cardiac myosins and their active fragments in a variety of conditions. It has proved possible to obtain binding profiles on intact myosin in the filamentous, undissolved form in physiological solvent conditions. Binding constants are given. At higher ionic strength (0.5 M potassium chloride) the binding profile of magnesium-ADP. is compatible with the presence of two types of site, differing from one another both in respect of affinity and the number of protons released per site. Studies with cardiac myosin reveal no such indications of heterogeneity, and are consistent with the presence of a single population of thermodynamically indistinguishable sites. In the absence of divalent cations, in solutions containing potassium ions and EDTA, ADP binds with absorption rather than liberation of protons. The pH profile of proton absorption at saturation can be fitted in terms of an ionising group with an unperturbed pK of 9.4, and at least one of lower pK(5.9). The dissociation constant (pH8 at 5 degrees C) is about 8 microM, and the affinity for uncomplexed ADP is thus only slightly weaker than that for magnesium-ADP

Precordial mapping of ST-segment elevation and Q waves following anterior myocardial infarction. The effects of established beta-blocking drugs.

The electrocardiographic (ECG) signs of ST-segment elevation and the development of Q was using 72-lead precordial surface mapping, and the release of creatine kinase (CK) activity has been studied in 47 patients with uncomplicated anterior myocardial infarction. These findings were compared with a further nine patients who had acute myocardial infarction but were receiving long-term beta-blocking drugs. It was found that ST-segment elevation and Q waves had rapidly changing and different natural histories and that beta-blocking drugs altered the natural history of ST-segment changes but had no effect on the pattern and time course for the loss of electrically active myocardium. There was a close relationship between the precordial area of ST-segment elevation at 2--3 h and the final development of Q waves in the patients with uncomplicated anterior myocardial infarction. No similar relationship could be found in those on beta-blocking drugs. The pattern of changes in plasma CK and its MB isoenzymes activity were similar for both groups. The relationship between early ST-segment elevation and the final area of Q waves may prove useful in clinical practice. This may not apply where beta-blocking drugs are commenced before the initial recording of ST-segment elevation.

Treatment of severe diabetic ketoacidosis. A comparative study of two methods.

Patients with severe diabetic ketoacidosis (pH less than 7.10) were treated according to two protocols. Protocol I consisted of high-dose insulin therapy by intravenous and intramuscular injections and bicarbonate infusion and was used in the first 12 patients; they received an average of 260 U insulin and 167 mmol bicarbonate in the first 6 h of treatment. Protocol II consisted of low-dose continuous intravenous insulin therapy, 8 U/hour, without bicarbonate in a further 12 patients. Rehydration and potassium-supplementation were the same in both methods. Basal data of both groups were not significantly different. The fall of plasma glucose concentration, rise in arterial pH and decrease in 3-hydroxybutyrate were similar in the two groups. The mean time to achieve a pH equal to or greater than 7.30 was 6.8 hours in the high-dose group and 7.6 hours in the low-dose group (p greater than 0.10). Potassium supplementation and potassium concentration during both treatments were the same. During the low-dose treatment the mean (+/- SD) plasma insulin concentration was 121 +/- 46 microU/ml. The presence of insulin binding antibodies did not result in lower free insulin concentrations. Thus, in the treatment of severe ketoacidosis continuous intravenous therapy with low-dose insulin is as effective as high-dose therapy and bicarbonate-administration is probably unnecessary.

Radiographic findings of the National Cooperative Crohn's Disease Study.

On-study barium radiographs of 535 patients in the National Cooperative Crohn's Disease Study have been analyzed for the pattern of distribution of bowel disease and the individual features that characterized bowel involvement. On-study and off-study radiographs of 403 of these patients were compared under code to judge radiographic response to drug treatment and discover correlations of radiographic findings with clinical response. Patients with more clinically active disease had more colonic disease on x-ray. Duodenal abnormalities were recognized in 22% of the patients and radiographically typicaly Crohn's disease of the duodenum in 8%. Recurrent Crohn's disease and that characterized by small bowel obstruction each displayed a characteristic appearance. Overall there was little evidence of radiographic improvement during the study, and little correlation between clinical response and evidence of radiologic improvement. Only patients treated with prednisone for more than 6 mo showed statistically significant radiologic improvement. Patients with definite radiographic progression or regression were found in each treatment group. Both fistula and stricture with obstruction were associated with a poor clinical response to all therapies. In view of the evidence from this study that radiographic findings do not correlate with clinical symptoms or response, the ritual use of x-ray to follow patients with Crohn's disease is unnecessary.

Alpha- and beta-adrenoceptor blocking properties of labetalol in renin release.

The effect of labetalol, an alpha- and beta-adrenergic receptor blocking antihypertensive, on plasma renin activity (PRA) and the hemodynamics of healthy volunteers at rest and during an ergometric exercise test was studied. Oral doses of 200 and 400 mg labetalol were tested against a placebo in a crossover manner. The labetalol plasma concentrations were determined. Systolic and diastolic blood pressures in the supine position decreased after 400 mg labetalol as did the response of the heart rate to exercise. The lower dose decreased the resting heart rate, but had no effect on the heart rate during exercise. The ergometric exercise induced an increase in PRA which was partly inhibited after 200 mg labetalol in a manner similar to that induced by beta-blockers in our earlier studies. After 400 mg labetalol PRA was already increased at one hour at sitting rest and this higher basal level was maintained for four hours. After this higher dose of labetalol the reaction of PRA to exercise was not significantly inhibited. In renin release the vasodilating alpha-blockade thus dominated the beta-blocking property of labetalol at the dose which decreased the blood pressure.

Active groups of bicyclomycin and the reaction with thiols.

The binding of [14C]bicyclomycin to whole cells of E. coli and to the inner membrane proteins was inhibited by dithiothreitol and 2-mercaptoethanol. The reactivity of the drug with the sulfhydryl group was further studied, using methanethiol as a model compound. The kinetics revealed that the reaction was of pseudo-first-order in excess of thiolate anion. Analysis with gas chromatography-mass spectrometry showed that the main product was an adduct of thiol with bicyclomycin in an equal molar ratio. The structure of the adduct was determined by 1H-NMR spectrometry, showing that thiolate attacked the olefinic double bond of the antibiotic. 3'-Acyl derivatives of bicyclomycin did not significantly affect the binding of [14C] bicyclomycin to inner membrane proteins of E. coli. The results suggested that 4,5-double bond hydrocarbons and 3'-hydroxy group of bicyclomycin participate in the binding to E. coli inner membrane proteins, which are presumably the receptors of the antibiotic. The olefinic double bond seems to be the active center of bicyclomycin, reacting with the sulfhydryl group of the receptor protein, although the whole molecular is needed for the activity.

Electron spin resonance study of the role of NO . catalase in the activation of guanylate cyclase by NaN3 and NH2OH. Modulation of enzyme responses by heme proteins and their nitrosyl derivatives.

The role of NO . catalase in the activation of partially purified soluble guanylate cyclase of rat liver by NaN3 and NH2OH was examined by electron spin resonance (ESR) spectroscopy. Equilibration of bovine liver catalase with NO resulted in formation of a paramagnetic species exhibiting a three-line ESR spectrum similar to that of NO . catalase. This paramagnetic complex produced concentration-dependent stimulation of preparations of partially purified guanylate cyclase that were devoid of detectable endogenous heme content. The stimulation of partially purified guanylate cyclase by NO . catalase was similar to that obtained with NO . hemoglobin and with NO . cytochrome P-420 prepared by reaction of hepatic microsomes of phenobarbital-treated rats with NO. By contrast, these same enzyme preparations did not respond to NO or catalase alone. Addition of hematin or hemoglobin plus a reducing agent to purified guanylate cyclase restored enzyme responsiveness to NO and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), but not to NaN3 or NH2OH. Responses to the latter agents were restored by catalase and potentiated by a H2O2-generating system. Formation of the NO . catalase complex was evident by ESR spectroscopy in test solutions containing NaN3 or nh2oh, catalase, and a glucose-glucose oxidase, H2O2-generating system. The presence of NO . catalase correlated well with the ability of test solutions to activate purified guanylate cyclase. These results provide evidence for catalase-dependent NO generation from NaN3 and NH2OH under conditions leading to guanylate cyclase activation. Preformed NO . hemoglobin or NO . cytochrome P-420 also activated heme-deficient partially purified guanylate cyclase. The ability of several preformed NO . heme protein complexes, but not NO, to stimulate heme-deficient guanylate cyclase supports the concept that formation of the paramagnetic nitrosyl . heme complex, mediated by either enzymatic or nonenzymatic reactions, is a common and essential step in the process by which NO or NO-forming compounds activate guanylate cyclase. In the absence of the NO ligand, both hemoglobin and catalase suppress the stimulatory effects of the corresponding NO . heme proteins on guanylate cyclase. Release of each heme protein from the NO . heme protein complex occurs more rapidly under aerobic compared to anaerobic conditions. However, hemoglobin is approximately 2000 times more effective as an inhibitor of NO . hemoglobin stimulation of guanylate cyclase than is catalase as an inhibitor of NO . catalase action. This finding may explain the more pronounced decline in the rate of cGMP generation in air in the presence of NO . hemoglobin compared to NO . catalase. The results imply that guanylate cyclase responses to activators that can form NO are determined by both the stimulatory activity of the endogenous heme acceptors of NO and the relative inhibitory effects of the unliganded heme proteins present.

Specific fluorescent labeling of chicken myofibril Z-line proteins catalyzed by guinea pig liver transglutaminase.

Guinea pig liver transglutaminase has been found to catalyze the covalent incorporation of dansylcadaverine into chicken skeletal muscle myofibril proteins. Epifluorescence microscopy reveals that the incorporated dansylcadaverine is specifically localized at or near the myofibril Z line. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) indicates that actin constitutes a major fraction of the labeled material; the Z-line proteins alpha-actinin and desmin also show significant labeling, as well as tropomyosin, several additional unidentified proteins, and material with an extremely high molecular weight. The Z-line-specific fluorescence can be removed by brief trypsinization, which releases fluorescent alpha-actinin into the supernate. The majority of the fluorescent protein species are resistant to extraction by either 0.6 M KCl or KI. These results, in conjunction with the microscopic localization, suggest that the dansyl-labeled proteins are constituents of the myofibril Z line. A significant amount of fluorescently labeled transglutaminase is also present in labeled myofibrils, which is resistant to extraction with either 0.6 M KCl or KI. This result indicates a strong, noncovalent interaction between the transglutaminase molecule and the myofibril Z line.

Influence of diet and metabolism on urinary acid excretion in the rat and the rabbit.

Rabbits normally excrete an alkaline urine, whereas rat urine is usually acidic. This study was designed to determine whether this difference was attributable to the composition of the diet or to specific metabolism of the species. Balance studies were performed in rabbits and rats with comparable growth rates who were consuming standard laboratory feed. The diets for both species had an excess of fixed cations over anions. Rats fed Rat Chow excreted urine containing net acid, whereas rabbits fed the same diet excreted urine containing net base. Rabbits eating Rabbit Chow excreted a very alkaline urine, but rats eating the same diet excreted much less alkali when expressed per kilogram of body weight. Balance studies demonstrated that rabbits absorbed a much higher proportion of dietary organic anions and excreted a larger fraction of this as total CO2. Collecting-duct hydrogen ion secretion and ammonium excretion, which are normally low in the rabbit during metabolic acidosis, were not increased when Rat Chow was consumed by rabbits for 2 weeks. In summary, feeding identical diets to rabbits or rats led to vastly different values for NAE. There appeared to be a gastrointestinal difference, as judged by the quantity of absorbed organic anions, as well as a metabolic difference reflected in the very different ratios of urinary total CO2 to organic anion.

Analysis of the contractile effect of 5-hydroxytryptamine on the isolated posterior communicating artery of the cat.

5-Hydroxytryptamine (5-HT) induced dose-dependent increases in tension on the isolated posterior communicating artery (PCA) of the cat were significantly antagonized by lysergic acid diethylamide (LSD, 6 X 10(-9). In the presence of the phentolamine (10(-6) M) the contraction induced by the two lowest doses of 5-HT was significantly reduced. Pretreatment of the animals with reserpine (3 mg kg-1, i.p., total dose) did not modify the dose-response curve to 5-HT except for the lowest dose. Removal of both superior cervical sympathetic ganglia 15 days before the experiment brought about a significant increase in the vasoconstriction induced by 5-HT at all the doses compared with the control. Cocaine (10(-6) M) induced a significant shift to the left of the dose-response curve to 5-HT but the maximum response was the same as in the control. The augmented response to 5-HT after denervation was partially antagonized by LSD (6 X 10(-9) M) but not by phentolamine (10(-6) M). These results show that the vasoconstriction elicited by 5-HT in the PCA of the cat is mainly due to direct stimulation of tryptaminergic receptors. The participation of an indirect adrenergic component in the contractile effects of 5-HT seems to be negligible.

Hypothalamic receptors influencing the secretion of corticotrophin releasing hormone in the rat.

1. The production of corticotrophin releasing hormone (CRH) by the rat hypothalamus in vitro was studied in the presence and absence of various neurotransmitter substances and drugs which mimic or antagonize their actions.2. Acetylcholine, nicotine and bethanechol increased, in a dose-related manner, hypothalamic CRH release and content but the maximal responses to bethanechol or nicotine were less than those to acetylcholine.3. The actions of acetylcholine were antagonized by atropine, pempidine and hexamethonium but were completely inhibited only when atropine and pempidine were given together. The effects of nicotine were abolished by pempidine but not by atropine while those of bethanechol were abolished by atropine but not by pempidine.4. Acetylcholine-induced hypothalamic CRH activity was also antagonized by cyproheptadine but not by methysergide.5. 5-Hydroxytryptamine caused dose-related increases in hypothalamic CRH release and content. Its effects were antagonized by cyproheptadine and methysergide but not by atropine, pempidine or hexamethonium.6. Acetylcholine-induced increases in hypothalamic CRH production were reduced by GABA, noradrenaline, adrenaline, methoxamine and phenylephrine but not by isoprenaline. The actions of GABA were antagonized by bicuculline and those of noradrenaline by phentolamine but not by atenolol.7. The results suggest the presence of nicotinic and muscarinic cholinoceptors, 5-hydroxytryptamine receptors, alpha-adrenoceptors and GABA-receptors within the hypothalamus all of which may be important in the control of CRH secretion.

[Biochemical effects of psychotropic drugs in central nervous system (author's transl)].

Studies on the biochemical effects of clinically used psychotropic drugs in brain have shown that they all exert their action by a direct or indirect interference with synaptic transmission. Thus, animal studies in vivo and in vitro have shown that the clinical efficacy of antipsychotic drugs correlates with their inhibitory action on dopamine receptors. In vivo these compounds enhance dopamine turnover in the brain and in vitro they inhibit the dopamine sensitive adenylate cyclase and the binding of dopamine to its receptor at neuronal membranes. Tricyclic antidepressants are drugs which have effects on many transmitter systems. No specific biochemical action has been found which is closely correlated with their clinical potency. However, it appears that a stimulation of the function of the noradrenergic system might have some clinical relevance. Benzodiazepines exert their pharmacological activity in the CNS by interacting with a brain specific receptor. This receptor appears to be part of a larger complex including a GABA receptor and the chloride conductance mechanism associated with the GABA receptor. By binding to their receptor, benzodiazepines appear to enhance the sensitivity of the GABA receptor, thus indirectly potentiating GABA-ergic neurotransmission in the brain.

Fundamental difference between the molecular interactions of agonists and antagonists with the beta-adrenergic receptor.

Antagonist binding to the beta-adrenergic receptor is largely entropy driven, with only a small enthalpy component. The binding of agonists, on the other hand, is associated with a large decrease in enthalpy which permits a highly unfavourable decrease in entropy. The thermodynamic differences between the binding of agonists and antagonists may provide new insights into the molecular basis for hormone stimulation of adenylate cyclase activity.

Inhibition of nitroso chemical carcinogen activation of rat hepatic guanylate cyclase by anticancer agents.

Recent studies have demonstrated that nitroso chemical carcinogens activate guanylate cyclase (EC 4.6.1.2) which catalyzes the production of guanosine 3',5'-monophosphate. This nucleotide is thought to be involved in normal and abnormal cell growth. We examined the effect of 3 major classes of anticancer chemotherapeutic agents, the antimetabolites (methotrexate and 6-mercaptopurine), antitumor antibiotics (adriamycin and actinomycin D), and alkylating agents (cytoxan, uracil mustard, isophosphamide, chlornaphazine, and 1-propranol-3,3'-iminodimethane sulfonate) on the activation of guanylate cyclase by nitroso chemical carcinogens. The anticancer chemotherapeutic agents noncompetitively blocked the activation of rat hepatic guanylate cyclase by N'-nitro-N-nitroso-N-propylguanidine (NNPG) and hydrazine. Adriamycin, methotrexate, and uracil mustard were the most effective inhibitors completely abolishing the effect of 1 mM NNPG on guanylate cyclase activity. The remainder of the anticancer chemotherapeutic agents abolished the NNPG activation of guanylate cyclase 40--70%. Since a previously described guanylate cyclase inhibitor has been shown to terminate the growth of an undifferentiated prostatic cancer in tissue culture the present data may indicate that one of the mechanisms by which anticancer chemotherapeutic agents exert their effects is by inhibition of tumor guanylate cyclase activity.

Effect of cranberry juice on urinary pH.

Twenty-one female and 19 male subjects who had normal physical and laboratory examinations were randomly assigned into four groups of 10 subjects each. Each group was then randomly assigned a number (150, 180, 210, 240) which determined the amount of cranberry juice, in milliliters, members of that group would ingest with each meal during the experimental phase of the study. The study took place over a 12-day period. A one-group before-and-after design was used, with each subject serving as his or her own control. Diet was controlled; menus on days 1 through 6 were repeated on days 7 through 12 with the addition of cranberry juice at each meal. Subjects used nitrazine pH tape to measure the pH of midstream urine at each voiding. There were significant (.01 level) differences in mean urinary pH between each control group and its corresponding experimental group. Anticipated problems with increased number of bowel movements, weight gain, increased voiding frequency, and subject pH measurement inaccuracy did not occur.

The development of the binocular depth cells in the secondary visual cortex of the lamb.

In most respects, the response properties of cells in the secondary visual cortex of the newborn lamb were indistinguishable from those in the adult. The cells were sharply selective to orientation; the orientation preferences were the same in each eye, and they varied systematically as the electrode penetrated the cortex. The receptive-field organization did not differ noticeably from that in adults, and complex, hypercomplex, and a few simple cells were all observed. The ocular dominance distribution was similar to that in the adult. Most importantly, binocular cells were found with disparate receptive fields even in newborn, visually inexperienced animals. As in the adult, the disparities were largely horizontal, and they appeared to be arranged in columns. Many of the cells responded preferentially to a binocular stimulus at a particular disparity setting (often approximately zero), but unlike those in the adult almost all the binocular cells in the newborn lamb would also respond monocularly, and the enhancement at the optimal disparity was less than in the adult. The full development of binocular selectivity took several weeks, and was blocked by binocular deprivation. We conclude that the basic wiring of stereoscopic mechanisms is innate, but the development of mature binocular interaction may depend on an adaptive process which makes use of the visual information received during binocular stimulation.

Subcellular distribution and some properties of alanine aminotransferase in striated muscles of the crayfish, trout, carp, frog, pigeon and rabbit.

Subcellular distribution and some physicochemical properties of alanine aminotransferase in striated muscles of the crayfish, trout, carp, frog, pigeon and rabbit were studied. It was established that: (1) Alanine aminotransferase activity in all mentioned animals occurred almost entirely in the cytosolic fraction of the muscles. Total activity and activity per mg protein were highest in crayfish and pigeon muscles and lowest in carp and trout muscles. (2) The pH optimum for the muscles of homoiotherms and poikilotherms ranged from 7.5 to 8, Km values for L-alanine were of the order 10(-3)--10(-2) M and those for alpha-ketoglutarate 10(-4) M. (3) A 10 degree C temperature increase of the incubation medium was accompanied by a 70--90% increase in activity. (4) The higher the alanine aminotransferase activity of the muscles, the relatively higher their alanine production during electrical stimulation. (5) From the above results it is concluded that alanine aminotransferase in striated muscles regulates the rate of glycolysis and energy production under conditions of anaerobiosis through the formation of alanine.

Acid-base balance of pleural liquid in dogs.

Acid-base balance and electrolyte concentrations were measured in dogs on small artificial hydrothoraces and in vitro on bicarbonate buffered Ringer solution on serosal and interstitial side of specimens of parietal pleura. Under steady conditions, pleural liquid PCO 2 was similar to and pH higher (delta = 0.022 +/- 0.006 SE) than that in mixed venous blood. Computed pleural liquid [HCO-3] was similar to that in venous plasma and hence less than that set by the Donnan effect, with which Na+ and Cl- approximately complied. In vitro, pH, [Na+], [Cl-], and computed [HCO-3] were significantly lower (delta = 0.030 +/- 0.004; -2.6 +/- 0.5; -1.2 +/- 0.5 and -1.7 +/- 0.2 meq/L, respectively) on the serosal than on interstitial side of pleural specimens, PCO2 being 42 mm Hg on both sides . HCO-3 and Na+ were not distributed according to transpleural potential (-0.4 +/- 0.1 mV on serosal side), suggesting an active transport of Na+ and HCO-3 from pleural liquid to blood. This, however, does not seem to add to the absorption pressure of plasma proteins in setting pleural liquid pressure.

Hydrogen ion excretion and urine osmolality in patients with obstructive uropathy secondary to Schistosoma haematobium.

Sixty-one patients with urinary schistosomiasis were studied to determine the effects of obstruction and bacteriuria on renal function. 39 (12 with bacteriuria) had demonstrable obstruction and 22 (5 with bacteriuria) had no obstruction. Total hydrogen ion excretion (T.H.+) for obstructed patients with sterile urine did not differ from that in controls; patients with bacteriuria with and without obstruction had a significantly lower T.H.+ (all P values less than 0.05). Obstructed patients (with or without bacteriuria) had significant impairment of maximal (U max) and minimal (U min) urine osmolality. Patients with bilateral obstruction and bacteriuria had a significantly lower U max (P less than 0.01 than obstructed patients without bacteriuria. The T.H.+ is mainly associated with bacteriuria but that the effects of unilateral or bilateral obstruction and bacteriuria on urine osmolality are additive. These abnormalities are usually corrected after therapy.

Ultrastructural localization of apo-b and apo-c binding to very low density lipoproteins in rat liver.

Hepatic synthesis of apo-B and apo-C and their binding to nascent very low density lipoproteins (VLDL) have been studied in fat-fed rats. Apolipoproteins were located in hepatocyte organelles by light and electron microscopy after immunoenzymatic staining using peroxidase-conjugated antibodies. Our results indicate that apo-B and apo-C are synthesized by membrane-bound ribosomes. Both apoproteins seem to be adsorbed simultaneously to the lipid core of VLDL in the lumen of the endoplasmic reticulum channels, at the junction zone between rough and smooth endoplasmic reticulum. Some additional protein presumably binds nascent VLDL in the Golgi apparatus as judged by the strong positive reaction of lipoprotein particles with peroxidase-labeled antibodies. Finally our data show that significant amounts of apo-B and apo-C are bound to the sinusoidal plasma membrane in fed rat livers which probably represent remnants of lipoprotein of intestinal origin since membrane-bound apolipoproteins virtually disappeared 24 h after lymphatic duct cannulation. It is suggested that nascent VLDL (apo-C poor) could be enriched in apo-C from lipoprotein remnants at the space of Disse.

Histofluorescence study on monoamine entry into the brain before and after opening of the blood-brain barrier by various mechanisms.

The relationship between exogenous, circulating monoamines to the wall of cerebral microvessels, and the entrance of these amines into the cerebral parenchyma was studied by the formaldehyde histofluorescence technique in rats. No monoamine fluorescence could be detected in the wall tissue of the microvessels (pericytes and andothelial cells) unless either MAO or COMT were inhibited; these are integral to the blood-brain barrier mechanisms to monoamines. After transient opening of the morphologic blood-brain barrier by either a hypertonic of hypertensive insult, the amine fluorescence in the walls of the microvessels was intensified compared to that which was noted after monoamine oxidase inhibition by itself. Following opening of the structural blood-brain barrier, the circulating amines also passed through into the neuropil where they were concentrated within neurons, as demonstrated by prior depletion of endogenous monoamine transmitters by reserpine. Thus, both enzymatic and morphologic mechanisms in the blood-brain barrier ar involved in impeding the passage of monoamines into the cerebral parenchyma.

Specificity of cultured anterior pituitary cells in detecting corticotropin releasing factor(s): the effect of biologically active peptides and neurotransmitter substances on ACTH release in pituitary cell cultures.

Biologically active peptides and neurotransmitter substances were added to anterior pituitary cell cultures to examine the presence of corticotropin releasing factor (CRF)-like activity. Hypothalamic extract (HE) induced significant dose-related increase of ACTH, and the lowest effective dose was 0.01 HE/ml. Other tested substances including luteinizing hormone-releasing hormone, thyrotropin releasing hormone, melanocyte stimulating hormone release inhibiting factor, somatostatin, substance P, neurotensin, beta-endorphin. leu-enkephalin, met-enkephalin, bradykinin, norepinephrine, dopamine, serotonin, acetylcholine, histamine, gamma-amino butyric acid or gamma-hydroxy butyric acid showed no CRF-like activity. Relatively high doses of lysine vasopressin, arginine vasopressin and angiotensin II increased the release of ACTH in pituitary cell cultures, but the maximal ACTH response was markedly less than with HE. These results indicate that cultured anterior pituitary cells are sensitive and fairly specific in detecting CRF(s) comparing with other detecting procedures.

Mechanical properties and functions of the myoepithelium in the eccrine sweat gland.

Contractile properties of an isolated segment of the secretory coil of the monkey palm eccrine sweat gland were studied in vitro with a transducer. Contraction of up to 10 mgf was induced with acetylcholine but not with alpha or beta adrenergic agonists, caffeine, prostaglandin E1, or by a calcium ionophore A23187. Other features included K+ contracture, staircase effect, poor extensibility, length-tension relationship with a peak tensile response at 115--120% of the resting length, and requirement of Ca2+. The function of myoepithelium is unlikely to expulse the preformed sweat because the amount of preformed sweat is small, K+-contracture failed to expulse sweat, and because myoepithelial contraction was not induced by such stimulants of sweat secretion as A23187, phenylephrine, isoproterenol, and prostaglandin E1. The maximal transverse tension of 20 mgf during acetylcholine stimulation under resting tension was calculated to support the luminal hydrostatic pressure of approximately 500 mmHg. Thus the function of the myoepithelium may be to render structural support for the secretory epithelium.

[Resuscitation of the elderly brain].

The little poses the question of the usefulness of resuscitation of elderly individuals, victims of cerebral damage or aggression. The author reviews the difficulties inherent in the definition of the "elderly patient" and the "elderly brain". The results obtained in 195 patients aged 60 or over are reported. In a 12 month period, this accounted for 32.77 p. 100 of "resuscitated" patients in a department of neurosurgery. Resuscitation implied a state of coma with artificial ventilation for varying periods. It thus differed from simple surveillance, even specialised. Survival rates are analysed in terms of neurological state, aetiology, whether or not there was surgery under general anaesthesia, past history, complications and the gravity of the resuscitation methods employed. The overall rate of 32.31 p. 100 was already lower than that in the elderly in a general intensive care unit. The picture in geriatric neurological resuscitation worsens further if only good quality survivals are noted: approximately 21.03 p. 100. Nevertheless, all the indications are that these figures are likely to improve in this recent field of resuscitation.

Bactericidal and bacteriostatic action of chloramphenicol against memingeal pathogens.

The bacteriostatic and bactericidal effects of chloramphenicol, ampicillin, tetracycline, and sulfisoxazole were compared against several potential meningeal pathogens. Chloramphenicol is bactericidal at clinically achievable concentrations against Haemophilus influenzae, Streptococcus pneumoniae, and Neisseria meningitidis. It is bacteriostatic against gram-negative bacilli of the family Enterobacteriaceae and against Staphylococcus aureus. Chloramphenicol has proven highly efficacious in the treatment of bacterial meningitis caused by those organisms against which it is bactericidal at low concentrations. Because leukocytic phagocytosis in the subarachnoid space is inefficient, we propose that bactericidal activity in cerebrospinal fluid is important for optimal therapy of bacterial meningitis. Chloramphenicol does not provide such activity in meningitis caused by enteric gram-negative bacilli.

An improved fluorimetric determination of alpha-mannosidase activity in bovine plasma.

This paper describes a manual fluorimetric method for the assay of acidic alpha-mannosidase activity in bovine plasma. The optimum conditions for the assay of this enzyme were studied. The assay method devised includes the addition of zinc to the substrate, which stimulates activity by approximately twofold, and reduces the optimum substrate concentration. This latter feature affords considerable cost saving in each test. We have also shown that the alpha-mannosidase activity in lithium heparin plasma, EDTA plasma and blood serum is the same whether the plasma/serum is separated from the cells/clot at 6, 20 or 25 hours after sample collection. This has eliminated the previous necessity of having to deliver whole blood samples to the laboratory within 6 hours of collection. Furthermore samples for the supplementary neutrophil assay can now be taken at the same time as those for the plasma test, and both samples forwarded together. The plasma alpha-mannosidase assay is a rapid and reliable screening test for the mannosidosis genotype and for detecting carrier animals. Carrying out this plasma assay in conjunction with the more definitive neutrophil assay provides a reliable method of distinguishing homozygotes and heterozygotes from normal animals.

Enzyme-substrate and enzyme-inhibitor complexes of triose phosphate isomerase studied by 31P nuclear magnetic resonance.

The complex formed between the enzyme triose phosphate isomerase (EC 5.3.1.1.), from rabbit and chicken muscle, and its substrate dihydroxyacetone phosphate was studied by 31P n.m.r. Two other enzyme-ligant complexes examined were those formed by glycerol 3-phosphate (a substrate analogue) and by 2-phosphoglycollate (potential transition-state analogue). Separate resonances were observed in the 31P n.m.r. spectrum for free and bound 2-phosphoglycollate, and this sets an upper limit to the rate constant for dissociation of the enzyme-inhibitor complex; the linewidth of the resonance assigned to the bound inhibitor provided further kinetic information. The position of this resonance did not vary with pH but remained close to that of the fully ionized form of the free 2-phosphoglycollate. It is the fully ionized form of this ligand that binds to the enzyme. The proton uptake that accompanies binding shows protonation of a group on the enzyme. On the basis of chemical and crystallographic information [Hartman (1971) Biochemistry 10, 146--154; Miller & Waley (1971) Biochem. J. 123, 163--170; De la Mare, Coulson, Knowles, Priddle & Offord )1972) Biochem. J. 129, 321--331; Phillips, Rivers, Sternberg, Thornton & Wilson (1977) Biochem. Soc. Trans. 5, 642--647] this group is believed to be glutamate-165. On the other hand, the position of the resonance of D-glycerol 3 phosphate (sn-glycerol 1-phosphate) in the enzyme-ligand complex changes with pH, and both monoanion and dianon of the ligand bind, although dianion binds better. The substrate, dihydroxyacetone phosphate, behaves essentially like glycerol 3-phosphate. The experiments with dihydroxy-acetone phosphate and triose phosphate isomerase have to be carried out at 1 degree C because at 37 degrees C there is conversion into methyl glyoxal and orthophosphate. The mechanismof the enzymic reaction and the reasons for rate-enhancement are considered, and aspects of the pH-dependence are discussed in an Appendix.

Stereochemistry of viminol, a novel central analgesic.

The synthesis of the stereoisomers of the centrally acting analgesic 1-[1-(2-chlorobenzyl)-pyrrol-2-yl]-2-di-sec.-butylamino-ethanol (viminol) is described. Their absolute configuration has been shown by comparing the circular dichroism (CD) curves with those of some phenyl analogs: for one of the viminol stereoisomers the postulated configurational assignment has been recently confirmed by an X-ray analysis. The pharmacological properties shared by the viminol stereoisomers are also described. The R,R configuration of the sec.-butyl groups and the S configuration of the hydroxy group appear to be essential for the agonistic effects: analgesia, tolerance and physical dependence in rodents. The S,R,R configurated viminol does not substitute, however, for morphine in monkeys, using the single dose suppression test. S,S or R,S(S,R) configurations of the sec.-butyl groups are associated with antagonistic properties. The binding capacity of the viminol stereoisomers to the opiate receptors and their influence on the acetylcholine release from the intestinal cholinergic terminals electrically stimulated are also described.

Investigation of the interactions of oxytocin with neurophysins at low pH using carbon-13 nuclear magnetic resonance and carbon-13-labeled hormones.

The specifically 13C-labeled (90% 13C-enriched) peptide hormone derivatives [1-hem[2-13C]cystine]oxytocin, [1-hemi[1-13C]cystine]oxytocin, and [2-[-2-13C]tyrosine[-oxytocin and the analogue [3-[2-13C]leucine]oxytocin were prepared by total synthesis and used to study the interactions of the neurohypophyseal hormones with the bovine neurophysins as a function of pH and temperature. Under all conditions, whether high or low pH, the chemical shifts of the labeled carbon atoms of the bound hormones are the same, but they are shifted significantly from their positions in the free hormone. These results indicate that interactions of the side chain and disulfide moieties of the hormone with the neurophysins do not change as a function of pH. At neutral pH and 20--35 degrees C, the labeled atoms of the hormone are in slow exchange (1--5 s-1) with the neurophysins for the above hormone derivatives, but at low pH they are in intermediate or fast exchange depending upon the pH and temperature. At low pH, the dissociation rate constant (koff) is about 100-fold greater than the value at neutral pH, and this increase appears to be due exclusively to the breaking of the salt bridge involving the N-terminal amino group of oxytocin and a side-chain carboxyl group of neurophysin. Since the dissociation constant (Kd) also increases by about 100-fold in going from neutral to low pH, the association rate constant is deduced to be the same at neutral and low pH. In contrast to the low pH results, an increase in pH (from 6.6 to 10.5) leads to a continual decrease in the binding constant but to no apparent change in the dissociation rate constant. The bound hormone is always in slow exchange at high pH, even when the binding constant has been reduced by 2 or 3 orders of magnitude. At high pH, the decrease in binding affinity is due solely to the deprotonation of the alpha-amino group of the free hormone. Thus, at high pH the apparent association rate constant decreases, while the dissociation rate constant remains unchanged.

Membrane damage by a toxin from the sea anemone Stoichactis helianthus. I. Formation of transmembrane channels in lipid bilayers.

The addition of nanomolar amounts of a toxin preparation derived from the sea anemone Stoichactis helianthus to black lipid membranes increases their electrical conductance by one million-fold. In addition, the membranes become permeable predominantly to monovalent cations. The elevated bilayer conductance is voltage-dependent, and the current-voltage curves of these bilayers display rectification as well as a region of negative resistance. The membrane activity of the toxin is proportional to the third power of its concentration, and at very low concentrations the membrane conductance increases in discrete uniform steps. These observations indicate that the mechanism of toxin action involves the formation of transmembrane channels constructed by the aggregation of protein molecules which are inserted in the bilayer. The voltage-dependent membrane conductance arises from two distinct channel characteristics: (1) the unit conductance of individual channels is dependent on the polarity of applied voltage; (2) the number of ion-conducting channels is influenced by the polarity as well as the magnitude of applied potential. It is believed that these effects are due to the influence of an electric field on the insertion of toxin molecules into the bilayer or on their subsequent association with each other to produce channels. Partial chemical characterization of the toxin material has shown that the membrane active factor is a basic protein with a molecular weight of 17,500.

Characterization of a C21 neutral steroid hormone transforming enzyme, 21-dehydroxylase, in crude cell extracts of Eubacterium lentum.

A strain of the obligate anaerobe, Eubacterium lentum, isolated from human feces, catalyzes the 21-dehydroxylation of 11-deoxycorticosterone to progesterone. A quantitative radiochromatographic assay was developed to measure 21-dehydroxylase activity in cell extracts. Maximum enzyme activity in cell extracts required both a reduced pyridine nucleotide and an oxidized flavin coenzyme. However, photochemically reduced flavin (FMNH2) could replace the requirement for NAD(P)H plus oxidized flavin. NAD(P)H : flavin (either FMN or FAD) oxidoreductase activity was detected spectrophotometrically in cell extracts assayed under anaerobic conditions. 21-Dehydroxylase was active from pH 5.4 to 8.5 with an apparent optimum between 6.4 and 6.8 using mixtures of NADH plus FMN as coenzymes. The substrate concentration at half-maximal reaction velocity was 8.0 microM and a specific acitivity of 5.8 nmol [3H]progesterone formed . h-1 . mg-1 protein was determined using [3th]deoxycorticosterone as substrate. Atabrine, rotenone, acriflavin, and 2,4-dinitrophenol (all at 1 mM) inhibited 21-dehydroxylase activity in cell extracts by 25, 24, 35 and 84%, respectively. These results suggest that 21-dehydrogenase may be coupled to a NAD(P)H : flavin oxidoreductase system in E. lentum.

[Photo-induced H+ transport in Nitellopsis obtusa cells].

Light-induced changes of pH in the vacuole as well as changes of the electric potential difference across the plasmalemma and the tonoplast of Nitellopsis obtusa were measured simultaneously by means of conventional and H+-specific glass or antimony microelectrodes. Illumination is found to produce a decrease in pH of the vacuolar sap by 0.1-0.5 units concomitant with cell depolarization. Cells suspended in a medium with pH 9.0 exhibit great (up to 100 mV) light-induced potential changes but only small pH changes of the vacuolar sap. When pH of the external medium (pH0 in shifted from 9.0 to more acid values the amplitude of photoinduced changes of pH in the vacuole rises up to 0,3-0.5 pH units and the amplitude of the potential changes at the plasmalemma gets smaller. At pH0 = 9.0 a transient acidification of the medium is observed upon illumination whereas at lower pH light-induced alkalinization was only seen. Transition of the cells from pH0 9.0 to pH0 7,5 results in cell hyperpolarization by 60-80 mv and a decrease of vacuolar pH by 0.4-0.5 units under light conditions but has no significant effect on the potential and the vacuolar pH in the darkness. It is proposed that mechanisms of active H+ extrusion from the cytoplasm are located both at the plasmalemma and tonoplast. Light-induced depolarization seems to be determined by the increase of H+-conductance of the plasmalemma and by a correspondent decrease in the electrogenic components of the membrane potential. The ratio of light-induced H+-fluxes across the tonoplast and the plasmalemma depends crucially on the level of H+-conductance of the plasmalemma.

The reactions of Escherichia coli citrate synthase with the sulfhydryl reagents 5,5'-dithiobis-(2-nitrobenzoic acid) and 4,4'-dithiodipyridine.

Citrate synthase of Escherichia coli reacts rapidly with 1 equivalent of Ellman's reagent, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), per subunit, losing completely its sensitivity to the allosteric inhibitor, NADH. When the enzyme is treated instead with 4,4'-dithiodipyridine (4,4'-PDS), all activity is lost. Certain evidence in this paper is consistent with the belief that the sulfhydryl group modified by DTNB, and that whose modification by 4,4'-PDS inactivates the enzyme, are the same. (i) Both reagents abolish NADH fluorescence enhancement by the enzyme. (ii) Saturating levels of NADH and some other adenylic acid derivatives inhibit the reactions with both reagents. (iii) When the enzyme is modified with one equivalent of DTNB or 4,4'-PDS, subsequent reactivity toward the other reagent is greatly decreased. (iv) Following modifications, the DTNB and 4,4'-PDS derivatives spontaneously lose thionitrobenzoate (TNB) or pyridine-4-thione (PT), respectively, in reactions which are thought to involve displacement of TNB or PT by a second enzyme sulfhydryl group, so that an enzyme disulfide is introduced. The introduction of the disulfide bond, if this is what occurs, does not lead to cross-linking of citrate synthase polypeptide chains, as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis under nonreducing conditions. Certain evidence has also been found, however, that the sites of modification by DTNB and 4,4'-PDS are not the same. (i) DTNB modification desensitizes to NADH but does not inactivate, while 4,4'-PDS inactivates at least 99.9%. (ii) The presumed disulfide from elimination of TNB is also active, while that from PT modification is no more active than the original 4,4'-PDS modified product. (iii) Prior modification of the enzyme with DTNB affords no protection against later inactivation by 4,4'-PDS. The studies therefore indicate a close relationship between the DTNB desensitization and 4,4'-PDS inactivation, but they are unable to identify it exactly. Other properties of the DTNB reaction are also described, and a hypothesis is offered to explain quantitatively the finding that desensitization lags behind modification during the modification of citrate synthase by DTNB.

Immunosuppressive effects of sulfato-trans-(-)-1,2-diaminocyclohexaneplatinum(II).

Sulfato-trans-(-)-1,2-diaminocyclohexane platinum(II) is a new antitumor agent. Its effect on immune responses was investigated. This agent inhibited lymphocyte transformation in response to phytohemagglutinin, pokeweed mitogen, and concanavalin A. It inhibited antibody plaque-forming spleen cells in AKR/J mice at doses ranging from 2.5 to 7.5 mg/kg i.p. It also prolonged the survival of skin grafts against major histocompatibility barriers in C57BL/6J mice transplanted with tail skin from CBA/J mice. The effective dose ranged from 2.5 to 5 mg/kg. This compound inhibited graft-versus-host reaction in three-week-old AKR/J X DBA/2JF1 mice receiving spleen cells from AKR/J mice i.p. Significant inhibition of graft-versus-host reaction was seen with doses ranging from 1 to 7.5 mg/kg i.p. These results suggest that this drug is immunosuppressive.

Cardiac atrial myosin adenosine triphosphatase of animals and humans: distinctive enzymatic properties compared with cardiac ventricular myosin.

Cardiac myosin obtained from atria had a higher Ca2+-activated ATPase activity than did cardiac myosin from ventricles in various species of animals and in humans. The increased specific activity of Ca2+-activated adenosine triphosphatase (ATPase) of atrial myosin appeared to correlate with the level of the activity of ventricular myosin ATPase in the animal, since the same order in ATPase activity, as observed in ventricular myosins from various animals, was noted in atrial myosins. The enzymatic properties of atrial myosin also were characterized by no activation by N-ethylmaleimide, low activating energy, and a lower rate of inactivation at alkaline pH compared with the same properties of ventricular myosin. These findings suggest a difference in the myosin molecule at or near the active site, involving some sulfhydryl groups, between the two types of cardiac myosin. The Mg2+-activated ATPase activity, both in the presence and absence of actin (which is thought to be closely related to the basic contraction mechanism), also was enhanced in atrial myosin. Thus, the ATPase activities of atrial and ventricular myosins were different with special reference to the reaction pathway involving calcium and magnesium ions and appear to account for the difference in the velocity of contraction between the atria and the ventricles.

Ammoniagenesis by the isolated perfused rat kidney: the critical role of urinary acidification.

1. The effect of metabolic acidosis simulated in vitro on ammoniagenesis was investigated by using the isolated kidney of the rat perfused with an albumin Krebs-Henseleit medium containing glutamine and glucose. 2. Addition of HCl to a perfusate of normal bicarbonate concentration resulted in a prompt increase in urine flow rate, decrease in fractional sodium reabsorption and decrease in urine pH. 3. A minimum urine pH as low as 5.15 was achieved, with an average value of 5.92, indicating that this preparation has the capacity to acidify normally. 4. In contrast with studies in vitro with other preparations, with the functional perfused kidney a diminution in perfusate bicarbonate concentration resulted in a prompt increase in ammonia production, which was strikingly correlated with the decrease in urine pH. 5. The increase in ammonia production was diminished in studies carried out with a non-urinating kidney, in comparison with those that exhibited significant urine acidification. 6. These data suggest that a decrease in urine pH with trapping of ammonia in the urine may be a critical stimulus for increased ammonia production in acute metabolic acidosis.

Biochemical discrimination of Hurler and Scheie syndromes.

1. Homogenates of cultured skin fibroblasts derived from patients with alpha-L-iduronidase-deficiency disorders (Hurler and Scheie syndromes) were capable of hydrolysing iduronosyl anhydro-[1-3H]mannitol 6-sulphate although at considerably reduced rates compared with normal controls. 2. The Vmax. values of alpha-L-iduronidase from patients with Hurler or Scheie syndromes and from normal controls were 11, 12 and 833 pmol min-1 mg-1 of protein respectively; the corresponding apparent Km values were 656, 50 and 53 mumol/l respectively. The alpha-L-iduronidases from normal and Scheie fibroblast homogenates were shown to exhibit pH optima at 3.6 and 4.1 and were competitively inhibited by both chloride and sulphate ions: Hurler alpha-L-iduronidase activity exhibited the pH optimum at 3.8 and was also inhibited by chloride and to a lesser extent by sulphate ions. 3. The thermal stability of Hurler, Scheie and normal alpha-L-iduronidase activities at 55 degrees C gave half-lives of approximately 1.0, 2.5 and 1.0 h respectively. 4. These biochemical findings clearly demonstrate enzyme differences for these two clinically distinct phenotypes and provide biochemical evidence that the Hurler and Scheie syndromes result from different allelic mutations.

Bone marrow transplantation for severe combined immunodeficiency disease. Reported from 1968 to 1977.

Patients who received bone marrow transplantation (= BMT) for the treatment of severe combined immunodeficiency (= SCID), and who were reported in the medical literature from 1968 to 1977, were collected and analysed. Eighteen of these 80 children are still alive, 10 months to 9 years after transplantation. It is thus the first successful form of therapy for this otherwise invariably fatal disease. Fifteen of the 18 survivors received bone marrow cells from HLA and MLC compatible donors; the remaining 3 survivors received grafts from MLC-compatible but HLA-incompatible donors. Bone marrow transplantation is the treatment of choice for SCID when recipient and donor are HLA- and MLC-identical. All patients who received MLC-incompatible grafts died, and bone marrow transplantation for SCID from MLC-incompatible donors should be abandoned. Milt-to-severe graft-versus-host disease (= GVHD) occurred in spite of HLA- and/or MLC-compatibility, with some correlation to the number of cells transplanted. This should preferably be kept below 50 million cells per kilo body weight. Infection was the chief cause of death in all groups. Strict reverse isolation, bowel decontamination and routine pre- and post-transplant Pneumocystis carinii prophylactic treatment are recommended. The clinical picture and laboratory findings of these 80 children before BMT did not differ from non-transplanted SCID patients. Three of the 18 survivors are adenosinedeaminase deficient.

Myeloperoxidase-hydrogen peroxide-chloride antimicrobial system: effect of exogenous amines on antibacterial action against Escherichia coli.

Exogenous ammonium ions (NH(4) (+)) and amine compounds had a profound influence on the antibacterial activity of the myeloperoxidase-hydrogen peroxide-chloride system against Escherichia coli. The rate of killing increased in the presence of NH(4) (+) and certain guanidino compounds and decreased in the presence of alpha-amino acids, polylysine, taurine, or tris (hydroxymethyl) aminomethane. Myeloperoxidase catalyzed the oxidation of chloride to hypochlorous acid, which reacted either with bacterial amine or amide components or both or with the exogenous compounds to yield chloramine or chloramide derivatives or both. These nitrogen-chlorine derivatives could oxidize bacterial components. Killing was correlated with oxidation of bacterial components. The rate of oxidation of bacterial sulfhydryls increased in the presence of the compounds that increased the rate of killing and decreased in the presence of the other compounds. The reaction of HOCl with NH(4) (+) yielded monochloramine (NH(2)Cl), which could be extracted into organic solvents. The N-Cl derivatives of bacterial components or of polylysine, taurine, or tris(hydroxymethyl)aminomethane could not be extracted. The effect of NH(4) (+) on killing is attributed to the ability of NH(2)Cl to penetrate the hydrophobic cell membrane and thus to oxidize intracellular components. Polylysine, taurine, and tris(hydroxymethyl)aminomethane formed high-molecular-weight, charged, or polar N-Cl derivatives that would be unable to penetrate the cell membrane. These results suggest an important role for leukocyte amine components in myeloperoxidase-catalyzed antimicrobial activity in vivo.

Effect of growth conditions on the formation of extracellular lipoteichoic acid by Streptococcus mutans BHT.

Streptococcus mutans BHT was grown in a chemostat with glucose limitation and at defined dilution rates and pH values. Lipoteichoic acid was estimated by determining the ability of dilutions of culture fluid to sensitize erythrocytes. The greatest amounts of extracellular lipoteichoic acid were produced by organisms growing at a low dilution rate and at pH 6.0 or 6.5. To enable a more accurate estimation of the total amount of extracellular material, rocket immunoelectrophoresis was employed. These results confirmed that the greatest amounts of reactive material were produced by slow-growing organisms, although there were discrepancies between these results and those obtained by hemagglutination. The extracellular material was fractionated by column chromatography and membrane ultrafiltration to yield a lipoteichoic acid-containing fraction and a presumptive deacylated lipoteichoic acid fraction. The relative proportions detected by rocket immunoelectrophoresis differed with the growth conditions, particularly the dilution rate. Analysis of the phenol-extracted cellular material also indicated the presence of deacylated lipoteichoic acid, although less than in the culture fluid.

Induction of cellulolytic enzymes in Trichoderma reesei by sophorose.

Sophorose (2-O-beta-glucopyranosyl-D-glucose) induces carboxymethyl cellulase in Trichoderma reesei QM6a mycelium with 1.5 to 2 h. The induction response to sophorose concentration, although complicated by the metabolism of sophorose, shows saturation kinetics. Most of the cellulase appears after most of the sophorose has been taken up, but the presence of an inducer is required to maintain cellulase synthesis because enzyme production ceases after separation of the mycelium from the induction medium. Cellulase appears simultaneously in the medium and in the mycelium, and no appreciable levels accumulate in the mycelium. Response to pH suggest either that synthesis and secretion of the enzyme are closely associated or concurrent events affected by surface interactions with the medium. Effects of temperature and pH on cellulase induction by sophorose are similar to those reported for induction by cellulose. The kinetics of absorption by mycelium differs from that of other beta-linked saccharides and glucose, the uptake of sophorose being much slower. Under our cultural conditions, sophorose appears to induce an incomplete array of cellulase enzymes, as indicated by enzymatic and electrophoretic studies.

Conformational stability of ribonuclease T1. I. Thermal denaturation and effects of salts.

The thermal transition of RNase T1 was studied by two different methods; tryptophan residue fluorescence and circular dichroism. The fluorescence measurements provide information about the environment of the indole group and CD measurements on the gross conformation of the polypeptide chain. Both measurements at pH 5 gave the same transition temperature of 56 degrees C and the same thermodynamic quantities, delta Htr (= 120 kcal/mol) and delta Str (= 360 eu/mol), for the transition from the native state to the thermally denatured state, indicating simultaneous melting of the whole molecule including the hydrophobic region where the tryptophan residue is buried. Stabilization by salts was observed in the pH range from 2 to 10, since the presence of 0.5 m NaCL caused an increase of about 5 degrees C to 10 degrees C in the transition temperature, depending on the pH. The fluorescence measurements on the RNase T1 complexed with 2'-GMP showed a transition with delta Htr =167 kcal/mol and delta Str =497 eu/mol at a transition temperature about 6 degrees C higher than that for the free enzyme. The large value of delta Htr for RNase T1 indicates the highly cooperative nature of the thermal transition; this value is much higher than those of other globular proteins. Analysis of the CD spectrum of thermally denatured RNase T1 suggests that the denatured state is not completely random but retains some ordered structures.

Freeze-fracture studies of frog neuromuscular junctions during intense release of neurotransmitter. II. Effects of electrical stimulation and high potassium.

Frog cutaneous pectoris nerve muscle preparations were studied by the freeze-fracture technique under the following conditions: (a) during repetitive indirect stimulation for 20 min, 10/s; (b) during recovery from this stimulation; and (c) during treatment with 20 mM K+. Indirect stimulation causes numerous dimples or protuberances to appear on the presynaptic membrane of nerve terminal, and most are located near the active zones. Deep infoldings of the axolemma often develop between the active zones. Neither the number nor the distribution of dimples, protuberances, of infoldings changes markedly during the first minute of recovery. The number of dimples, protuberances, and infoldings is greatly reduced after 10 min of recovery. Since endocytosis proceeds vigorously during the recovery periods, we conclude that endocytosis occurs mostly at the active zones, close to the sites of exocytosis. 20 mM K+ also causes many dimples or protuberances to appear on the axolemma of the nerve terminal but they are distributed almost uniformly along the presynaptic membrane. Experiments with horseradish peroxidase (HRP) show that recycling of synaptic vesicles occurs in 20 mM K+. This recycling is not accompanied by changes in the number of coated vesicles. Since both exocytosis and endocytosis occur in 20 mM K+, it is difficult to account for this unique distribution. However, we suggest that K+ causes dimples or protuberances to appear between the active zones because it activates latent sites of exocytosis specified by small numbers of large intramembrane particles located between active zones. The activation of latent release sites may be related to the complex effects that K+ has on the quantal release of neurotransmitter.

Ultrastructural localization of D-amino acid oxidase in microperoxisomes of the rat nervous system.

A recently developed procedure for the localization of D-amino acid oxidase (D-AAO) has been used to investigate the distribution of this enzyme in rat nervous tissue. Initial studies were carried out on kidney to validate the methods. The cytochemically demonstrable enzyme in kidney is inhibited by kojic acid, a known competitive D-AAO inhibitor. Omission of the catalse inhibitor, aminotriazole, from the cytochemical medium produces a marked diminution of D-AAO reaction product in kidney peroxisomes. This would be expected if catalase and D-AAO are present in the same particles. In brain, kojic acid-inhibitable D-AAO is demonstrable in numerous bodies within astrocytes especially in the cerebellum, a brain region known from biochemistry to contain particularly high levels of the oxidase. In preparations incubated for catalase, far fewer positive bodies are seen in the cerebellum. Moreover, omission of aminotriazole has little evident effect on the D-AAO reaction. Thus, the oxidase-containing cerebellar bodies may be relatively poor in catalse. In contrast, several nervous system cell types that contain relatively numerous catalase-positive bodies, contain none with detectable D-AAO. Such heterogeneity of peroxisome enzyme content is in accord with reports from biochemical studies of brain.

Production of maximally acid urine by the isolated dog kidney.

The isolated kidney has not been reported to acidify urine maximally. To study this defect, kidneys from dogs fed NH4Cl were perfused with autologous blood. Perfusate pH was 7.20 +/- 0.03 [HCO3] was 14 +/- 1 mEq/L, and urine pH was abnormally high, 6.60 +/- 0.08. When corrected for difference in GFR, UNH4+V was similar to that seen in vivo, but UTAV and UNet H+V were low. FEHCO3- was 2.3% +/- 0.8% and HCO3- excretion persisted to a small degree at perfusate [HCO3-] of 8 to 9 mEq/L. In response to HCO3- infusions, large increases in excretion were not seen until perfusate values were over 24 to 26 mEq/L. HCO3- Tmax was 2.94 +/- 0.07 mEq/dl of glomerular filtrate. The isolated kidney failed to raise U-B PCO2 with HCO3- infusion secondary to low urine [HCO3-] and [Pi]. During perfusion in another group of kidneys from dogs fed NH4Cl and given DOC, perfusate pH and [HCO3-] were similar to those in the first group. Urine pH was also inappropriately high, 7.12 +/- 0.09, and there was no UNet H+V. In response to Na2SO4 infusion, urinary pH fell to 5.00 +/- 0.27. Log10UUAV was correlated to urine pH during the control perfusions in both groups and after Na2SO4 in the NH4Cl + DOC group. Thus production of a low urine pH in the isolated kidney may be mediated by changes in transtubular potential difference resulting from increased distal nephron delivery of Na+ and nonabsorbable anion. The defect in acidification is similar to that observed in incomplete forms of clinical type 1 (distal) renal tubular acidosis.

Comparison of ionic selectivity of batrachotoxin-activated channels with different tetrodotoxin dissociation constants.

The purpose of these experiments is to test whether the differences between normal and tetrodotoxin-resistant Na+ channels reside in the selectivity filter. To do this, we have compared the selectivity of batrachotoxin-activated channels for alkali cations, organic cations, and nonelectrolytes in two neuroblastoma clonal cell lines: N18, which has normal tetrodotoxin (TTX) sensitivity, and C9, which is relatively TTX-resistant. We have also studied the effect of H+ on Na+ permeability and on the interaction between TTX and its receptor site in both cell lines. There is no qualitative difference between the two cell lines in any of these properties. In both cell lines the batrachotoxin-activated Na+ channels have a selectivity sequence of Tl+ greater than Na+ greater than K+, guanidinium greater than Rb+ greater than Cs+, methylamine. Also, in both cell lines H+ blocks Na+ channels with a pKa of 5.5 and inhibits the action of TTX with the same pKa. These observations indicate that the selectivity filters of the Na+ channels in C9 and N18 do not differ significantly despite the 100-fold difference in TTX-affinity. Our selectivity studies of batrachotoxin-activated Na+ channels for both cell lines suggest that these toxin-activated Na+ channels have a limiting pore size of 3.8 x 6.0 A, as compared to a pore size of 3.0 x 5.0 A for potential-activated Na+ channels.

Rapid, large-scale isolation of Plasmodium berghei sporozoites from infected mosquitoes.

The discontinuous gradient technique for recovery of malarial sporozoites from mosquitoes (Beaudoin et al., 1977) has been modified to speed up recovery and prevent sensitization of mice by components of the gradient which contaminate the sporozoites used as antigen. Mouse serum was substituted for BSA in the gradient because the latter produced hypersensitivity. Best results were obtained with gradients consisting of Medium 199, Renografin and mouse serum. Heavy and light solution of gradient components are layered in a centrifuge tube. Centrifugation of comminuted, infected mosquitoes applied to the top of the discontinuous gradient concentrates sporozoites at the interface. Sporozoites recovered from the gradient were infective, immunogenic, and relatively free of mosquito tissue. This improved method enables recovery of 100,000 sporozoites from each Anopheles stephensi infected with the ANKA strain of Plasmodium berghei. As many as 2,800 mosquitoes have been processed in 2 hr without a significant decrease in yield.

The surface activity and self-association of some beta-adrenoceptor blocking agents in aqueous solution.

The surface activity at the air-solution interface of a series of beta-adrenoceptor blocking agents has been determined. The drugs investigated included, propranolol, sotalol, oxprenolol, labetolol, timolol, metoprolol and acebutolol. Correlation between surface activity and local anaesthetic potency of the drugs is examined. Light scattering measurements have indicated self-association of sotalol, oxprenolol, acebutolol and metoprolol in 0.5 mol kg-1 sodium chloride. Critical micelle concentrations and aggregation numbers are reported.

Pancreatic acinar cells: the effect of carbon dioxide, ammonium chloride and acetylcholine on intercellular communication.

1. Segments of mouse pancreatic or exorbital lacrimal gland were superfused with saline solutions. Under visual control two micro-electrodes were inserted into neighbouring cells within the same acinus or into neighbouring acini. Cell to cell electrical coupling was assessed by injecting rectangular current pulses through one electrode and measuring the electrotonic potential change in the same cell (V(1)) and in the neighbouring cell (V(2)). Acetylcholine (ACh) was added locally to impaled acini by micro-ionophoresis from an extracellular micropipette.2. Exposure of the tissues to a Krebs solution equilibrated with 100% CO(2) caused a rapid increase in the size of electrotonic potential changes in the current injection cell and disappearance of the electrotonic potential changes in a neighbouring acinus or cell. This electrical uncoupling of previously coupled cells was rapidly reversible upon return to a solution equilibrated with 95% O(2) and 5% CO(2).3. Reduction of electrical intercellular coupling was also obtained using smaller CO(2) concentrations (50, 20 or 10%). In these cases the effects developed more slowly and were less dramatic. Reducing the extracellular HCO(3) concentration enhanced the uncoupling effect of 10 or 20% CO(2). However, weak uncoupling effects were still observed using 10 or 20% CO(2) in combination with a high bicarbonate concentration maintaining a constant extracellular pH (7.4).4. Reductions in extracellular pH (down to 5.5) achieved by varying combinations of Tris base and Tris HCl had no effect on electrical coupling. Brief periods of anoxia (100% N(2)) also had no effect.5. Exposure to 20% CO(2) markedly enhanced the uncoupling effect of a brief ionophoretic pulse of ACh.6. Exposure of the tissue to 10 mM-NH(4)Cl, a procedure expected to increase the intracellular pH, counteracted the uncoupling effect of ACh. During sustained uncoupling caused by a sustained ACh stimulation a brief period of exposure to NH(4)Cl caused an immediate and fully reversible recoupling.7. It is concluded that variations in intracellular pH have marked effects on the electrical coupling between neighbouring cells in the pancreatic and lacrimal acinar tissue.

Partial purification and characterization of a bacteriolytic enzyme secreted by Tetrahymena.

Tetrahymena pyriformis strain HSM secretes large quantities of acid hydrolases into the culture medium. An enzyme secreted by the ciliate and capable of degrading walls of streptococci was identified and purified to a considerable degree. The pH optimum of this enzyme was 3--4, and it was eluted after cytochrome c from Sephadex G-75 columns. Unlike lysozyme, the enzyme was thermolabile at pH 2.9, but relatively thermostable at pH 8.1. It degraded 14C-labeled cell walls of streptococci releasing reducing groups. Cell walls prepared from different strains of streptococci differed in susceptibility to this enzyme, the most sensitive strain tested being of group A, type T12. It was shown in immunologic studies that this hydrolase released the group-specific carbohydrate from the walls. Secretions of Tetrahymena from early stationary-phase cultures had more bacteriolytic activity than those from cells from late stationary-phase cultures. Further, cells from cultures grown in glucose-supplemented medium secreted less of the enzyme than ciliates of comparable age grown in unsupplemented proteose-peptone. The newly isolated bacteriolytic enzyme, presumably of lysosomal origin, may be helpful in characterizing streptococcal cell walls.

pH-Dependence of water and solute transport in toad urinary bladder.

Stimulation of urea and water transport by vasopressin (ADH) appears to occur via independent pathways. We examined the effects of altering serosal or mucosal bath pH on transport of water, urea, and sodium. Compared to bladders with a serosal bath pH of 7.4 to 8.0, reducing the serosal bath pH to 6.8 led to a 60% fall in ADH-stimulated osmotic water flow, without decreasing the permeability of urea. Raising the serosal pH to 9.5 had the opposite effect: urea permeability was inhibited by 40% without altering water flow. Exogenous cyclic AMP-stimulated water and urea permeabilities were not dissociated, but were changed in the same direction by alterations in serosal pH: serosal acidification enhanced the effect of exogenous cyclic AMP on both urea and water, whereas the cyclic AMP effect on both was diminished by serosal alkalinization. This was especially marked for urea, suggesting that an alteration in the urea response to cyclic AMP may be particularly important in defining vasopressin-stimulated urea permeability as the serosal bath pH is altered. Mucosal acidification increased short circuit current but decreased both the urea and water response to ADH and 8-bromo-cyclic AMP. The response to cyclic AMP was less consistent. Mucosal alkalinization did not cause significant changes in either basal or stimulated transport. The data demonstrate distinct and separable effects of bath pH alterations on each of the transport systems examined.

Tricyclic antidepressants and histamine H1 receptors.

Tricyclic antidepressants and some structurally related compounds were tested for their ability to antagonize histamine H1 and muscarinic acetylcholine receptors of cultured mouse neuroblastoma cells. As a group, tertiary amine tricyclic antidepressants tended to be more potent than secondary amine drugs at both receptors. The most potent antihistamine, doxepin hydrocholoride, was about 4 times more potent than amitriptyline hydrochloride, about 800 times more potent than diphenhydramine hydrochloride, and about 8,000 times more potent than desipramine hydrochloride, the least potent tricyclic antidepressant at both the histamine H1 and the muscarinic acetylcholine receptors. All tricyclic drugs except desipramine hydrochloride were more potent as antihistamines than as anticholinergics. Doxepin hydrochloride and amitriptyline hydrochloride may be the most potent antihistamines known, and the antihistaminic potencies of these and the other tricyclic antidepressant drugs may relate directly to their ability to cause sedation and drowsiness in patients.

Clostridium botulinum can grow and form toxin at pH values lower than 4.6.

It is generally accepted that in Clostridium botulinum both growth and toxin formation are completely inhibited at pH values below 4.6. This critical pH value has been confirmed by many investigators using food as substrate or culture media. Occasionally growth of C. botulinum and toxin formation at pH values lower than 4.6 have been reported. In these cases the authors ascribed the unexpected outgrowth and toxin formation to local pH differences in inhomogeneous media and growth of C. botulinum before pH equilibration, or to the fact that fungi created microenvironments within or adjacent to the mycelial mat, where the pH was higher than 4.6 as was demonstrated by Odlaug and Pflug. We show here that the general assumption that C. botulinum does not grow below pH 4.6 is incorrect. We have observed that growth and toxin formation by C. botulinum can take place in homogeneous protein rich substrates (containing 3% or more soya or milk protein) at pH values lower than 4.6.

Excitation-contraction coupling in frog sartorius and the role of the surface charge due to the carboxyl group of sialic acid.

Frog sartorius muscle fibres were incubated with the enzyme neuraminidase which is known to remove surface-bound sialic acids. The sialic acid content of the incubation media was analysed, and the relationship between the threshold of contraction and the altered pH and divalent cation concentration was investigated. The threshold potential of fibres treated with 3.3, 5 or 6.7 units of neuraminidase (at pH 5.5 and 30 degrees C for 2 h) was more positive than that of the control muscle fibres incubated under the same conditions, but without the enzyme. The potential shift is positively correlated with the enzyme concentration and with the amount of sialic acid released. After incubation with 5 units of neuraminidase the potential shift rose to +8.5 mV, depending on [Ca2+]0, [Mg2+]0 and pH. The threshold shift is greatest at low divalent cation concentration (0.5 mM), and not significant at high concentrations of divalent cations (50 mM). In both neuraminidase-treated and control muscles, the effectiveness of Mg2+ is half of that of Ca2+. The dependence of the contraction threshold on pH in the range 5.5--10 is even more pronounced in enzyme-treated than in control muscle fibres. Resting potential, time-course and overshoot of action potential are not affected by treatment with neuraminidase. Threshold shifts are explained by shifts of an external surface potential upon variation of [Ca2+]0, [Mg2+]0 and pH. Treating the muscles with neuraminidase diminishes the net negative charge density, and hence shifts the surface potential to more positive values, by release of negatively charged sialic acid. The different effectiveness of Ca2+ and Mg2+ is ascribed to their different effectiveness of Ca2+ Mg2+ is ascribed to their different binding behaviour towards the negative surface charges.

Biochemical genetic analysis of pyrimidine biosynthesis in mammalian cells: III. Association of carbamyl phosphate synthetase, aspartate transcarbamylase, and dihydroorotase in mutants of cultured Chinese hamster cells.

Carbamyl phosphate synthetase (EC 2.7.2.9), aspartate transcarbamylase (EC 2.1.3.2), and dihydroorotase (EC 3.5.2.3), the first three enzymes in de novo pyrimidine synthesis in Chinese hamster ovary cell strain Kl (CHO-Kl), cose diment through a glycerol gradient. When an extract from Urd- A, a pyrimidine-requiring auxotroph reduced in all three activities, is run on a glycerol gradient, the enzyme activities appear in two peaks higher in the gradient, a peak of aspartate transcarbamylase separated from a peak of carbamyl phosphate synthetase and dihydroorotase. Revertants of Urd- A have increased activity of all three enzymes and give glycerol gradient patterns similar to either CHO-Kl or Urd- A. The gradient pattern for Urd- A and some of its revertants can be mimicked by treating the CHO-Kl cell extract with trypsin. Hybrids made between a CHO-Kl purine-requiring auxotroph (Ade- C) and a Urd- A revertant gave a glycerol gradient pattern which is a composite of the CHO-Kl and revertant patterns. A model is presented for the structure of this multifunctional protein.

Polypoid gastric heterotopy of the small intestine in a patient with primary hyperparathyroidism and alpha-1-antitrypsin deficiency belonging to a MEA-family. With particular reference to the ultrastructure of the epithelial cells.

A patient with a solitary polypoid gastric heterotopy in the jejunum and severe bleeding as a complication is described. Previous reports on this rare disorder and the conditions of its development are discussed. The patient belongs to a family with multiple endocrine adenomatosis (MEA), some members of which had an alpha-1-antitrypsin deficiency. The association between the familial disease and the gastric heterotopy in this case might be another argument for the assumed congenital nature of the latter. The polyp was studied by means of light and electron microscopy. It was mostly lined by fundic mucosa and only partially by antral mucosa. Focal foveolar hyperplasia, cysts and lymphoplasmacellular infiltration of the mucosa are regarded as secondary tissue alteration. Parietal cells, chief and endocrine cells were identified. The parietal cells were in the nonsecreting state and appeared in two distinct forms which are described in detail.

Contrasting effects of acute beta blockade with propranolol on plasma catecholamines and renin in essential hypertension: a possible basis for the delayed antihypertensive response.

Blood pressure, heart rate, plasma renin activity, plasma norepinephrine and plasma epinephrine were determined in 11 patients with essential hypertension at rest before and 15, 30, 45, and 60 minutes after an intravenous infusion of 0.12 mg./Kg. propranolol given over five minutes. After propranolol mean blood pressure was unchanged; heart rate decreased by 14 per cent within 15 minutes and showed no further changes. Plasma renin activity decreased progressively by 48 per cent 60 minutes after propranolol, whereas plasma norepinephrine and epinephrine were always higher after propranolol than control values. Increases in norepinephrine were statistically significant at 30, 45, and 60 minutes (respectively 49, 39, and 45 per cent, P less than 0.005 at least) and those of epinephrine even at 15 minutes (respectively 60, 82, 62, and 94 per cent P less than 0.01 for all). These results indicate that acute beta blockade with propranolol incudes increases in circulating plasma norepinephrine and epinephrine which might be consequent to rapidly induced hemodynamic changes. This augmented sympathetic activity might explain why propranolol, when acutely infused, does not decrease blood pressure despite effective cardiac and renin blockade.

Echocardiographic findings in patients with aortitis syndrome.

Echocardiography was performed in 18 patients with the aortitis syndrome and in 20 age-matched normal volunteers. The aortic root dimension, the aortic dimension at the level of the sinotubular ridge, the aortic arch dimension, the left ventricular internal dimension, the left atrial dimension, the interventricular septal thickness, and the left ventricular posterior wall thickness were measured. All measurements, except for the left atrial dimension, were significantly greater in patients with aortitis syndrome than in the control subjects. We concluded (1) that the patients with the aortitis syndrome may have an enlarged or narrowed aorta, a dilated left ventricle and left atrium, and a thickened interventricular septum and left ventricular posterior wall; (2) that the incidence and the degree of these abnormalities depend on the presence of complications such as aortic regurgitation and arterial hypertension; and (3) that M-mode as well a cross-sectional echocardiography plays an important role in the assessment of the aorta and heart in the aortitis syndrome.

Antimicrobial susceptibility testing of pneumococci: determination of Kirby-Bauer breakpoints for penicillin G, erythromycin, clindamycin, tetracycline, chloramphenicol, and rifampin.

Antimicrobial susceptibility testing of pneumococci is now essential to monitor for the presence of resistance to agents such as the penicillins, macrolides, lincomycins, chloramphenicol, and tetracycline. In this study, clinical isolates of a selection of resistant South African strains were tested for antimicrobial susceptibility by minimal inhibitory concentration (MIC) determination and by a modified Kirby-Bauer disk diffusion technique, using Mueller-Hinton medium supplemented with 5% horse blood. Disk diffusion breakpoints were determined for penicillin G, erythromycin, clindamycin, tetracycline, chloramphenicol, and rifampin. Reliable results were obtained on disk diffusion for all these agents except for penicillin G. With 6-mug penicillin G disks, zones of strains with intermediate penicillin susceptibility overlapped those of sensitive and resistant strains. With 5-mug methicillin disks, clearer separation of strains based on susceptibility to penicillin G occurred. Strains with zones of <35 mm around penicillin G disks and <25 mm around methicillin disks should have penicillin G MICs determined to confirm their resistance to penicillin G. In view of the potential for pneumococci to be resistant to the agents used in this study, antimicrobial susceptibility of all clinically significant isolates should be determined.

Microbial oxidation of gaseous hydrocarbons: epoxidation of C2 to C4 n-alkenes by methylotrophic bacteria.

Over 20 new cultures of methane-utilizing microbes, including obligate (types I and III) and facultative methylotrophic bacteria were isolated. In addition to their ability to oxidize methane to methanol, resting cell-suspensions of three distinct types of methane-grown bacteria (Methylosinus trichosporium OB3b [type II, obligate]; Methylococcus capsulatus CRL M1 NRRL B-11219 [type I, obligate]; and Methylobacterium organophilum CRL-26 NRRL B-11222 [facultative]) oxidize C2 to C4 n-alkenes to their corresponding 1,2-epoxides. The product 1,2-epoxides are not further metabolized and accumulate extracellularly. Methanol-grown cells do not have either the epoxidation or the hydroxylation activities. Among the substrate gaseous alkenes, propylene is oxidized at the highest rate. Methane inhibits the epoxidation of propylene. The stoichiometry of the consumption of propylene and oxygen and the production of propylene oxide is 1:1:1. The optimal conditions for in vivo epoxidation are described. Results from inhibition studies indicate that the same monooxygenase system catalyzes both the hydroxylation and the epoxidation reactions. Both the hydroxylation and epoxidation activities are located in the cell-free particulate fraction precipitated between 10,000 and 40,000 x g centrifugation.

Tissue levels of several radiolabelled beta-adrenoceptor antagonists after intravenous administration in rats.

The uptake of radioactively labelled propranolol, oxprenolol, metoprolol, acebutolol, practolol and atenolol into brain, liver and lung tissue was studied five min after intravenous administration (1.0 mg/kg) in normotensive Wistar rats anaesthetised with nitrous oxide and halothane. All of the beta-adrenoceptor antagonists including the least lipophilic compounds atenolol and practolol were detected in brain tissue five min after systemic administration. However, the level of propranolol (as measured by total radioactivity) in the bran was 40 and 67 times greater than the levels found for atenolol and practolol, respectively. Additionally, significantly more radioactivity was detected in lung tissue compared to that in liver tissue for the lipophilic, non-selective beta-adrenoceptor antagonists propranolol and oxprenolol. Levels of radioactivity in blood, brain, liver and lung were measured 5, 15, 30 and 60 min after administration of either propranolol or atenolol (1.0 mg/kg, i.v.) to both conscious and anaesthetised rats and a tendency towards high tissue levels of radioactivity was found in the animals which received the beta-adrenoceptor antagonists under anaesthesia. Pretreatment of groups of rats for 7, 14 and 21 days with unlabelled atenolol (1.0 mg/kg/day, i.p.) caused an increase in the subsequent central uptake of labelled atenolol whilst both the blood levels of radioactivity and the uptake into peripheral tissues were significantly lower in the 2 and 3 week pretreated rats compared to the control animals. These results are consistent with the hypothesis that a central action may contribute to the antihypertensive effect of beta-adrenoceptor antagonists.

The effect of ribonuclease on the replicative forms of Sindbis virus RNA.

Three species of double-stranded RNA, designated RF I, RF II, and RF III in order of decreasing size (25), are produced by ribonuclease treatment of extracts of chicken embryo cells infected for 6 hours with Sindbis virus. Only one class of replicative form RNA is present in extracts not treated with ribonuclease; this class contains some molecules which can be enzymatically cleaved to produce the other two replicative forms. At a low level of enzyme (0.001 microgram/ml) the major species obtained was RF I, the replicative form of the genome. When the enzyme concentration was increased 10-, 100-, and 1000-fold, there was a progressive increase in the proportions of RF's II and III and a concomitant decrease in the proportion of RF I. The generation of RF's II and III by nuclease resulted in the ratio expected for these two species if they are produced by cleavage of RF I-like molecules. In preparations of isolated double-stranded RNA, only RF I and replicative intermediate RNA were present. Mild nuclease treatment of these preparations converted the replicative intermediates primarily to RF I. Higher enzyme levels generated greater proportions of RF II and RF III, but RF I-like molecules were the major source for these increased proportions. Treatment of the isolated naturally occurring replicative form with 0.01 microgram of ribonuclease per ml cleaved some molecules migrating as RF I during gel electrophoresis into molecules which migrated as RF II and RF III.

Interactions of small molecules with phospholipid bilayers. Binding to egg phosphatidylcholine of some organic anions (bromosulphophthalein, oestrone sulphate, haem and bilirubin) that bind to ligandin and aminoazo-dye-binding protein A.

1. To assess the possible involvement of ligandin and aminoazo-dye-binding protein A in intracellular transport it is necessary to know how their ligands, most of which are molecules with hydrophobic moieties, interact with cellular membranes. To obtain such information we examined the interactions of bromosulphophthalein, oestrone sulphate, haem and bilirubin with aqueous dispersions of egg phosphatidylcholine and egg phosphatidylchone/cholesterol (1:1, molar ratio) by equilibrium dialysis and spectrophotometry. 2. In all four cases, saturation effects were observed. Values of Vmax (v = mol of compound bound/mol of lipid phosphorus) at 25 degrees C were: for bromosulphophthalein, approximately 0.1; for oestrone sulphate, approximately 0.25; for haem, approximately 0.25 (all at pH 7.4); and for bilirubin 0.1--0.2 (at pH 8.2). 3. Limiting values of v/c (c = unbound concentration) as v leads to 0 at 25 degrees C and pH 7.4 are: for bromosulphophthalein, 6.25 x 10(4) litre-mol-1; for oestrone sulphate, 7.8 x 10(2) litre-mol-1; for haem, 4.5 x 10(5) litre-mol-1; and for bilirubin, approximately 1.2 x 10(4) litre-mol-1. For haem the result depends on the assumption that only the monomeric form binds to the lipid. 4. The binding of each compound was decreased by cholesterol; bromosulphophthalein and oestrone sulphate were affected more than haem and bilirubin. 5. Bromosulphophthalein at saturating concentration decreased the limiting values of v/c of the other three compounds by approximately one order of magnitude. 6. By assuming that the interactions with egg phosphatidylcholine resemble those with the phospholipid components of mammalian intracellular membranes the binding data for phosphyatidylcholine, together with data for binding to the intracellular proteins ligandin and aminoazo-dye-binding protein A, enable the subcellular distributions of the four compounds to be estimated. For the rat hepatocyte up to 92, 51, 98 and 47% of the total bromosulphophthalein, oestrone sulphate, haem and bilirubin respectively may be membrane-bound.

The assay and partial characterization of macromolecular heparin depolymerase activity in rat small intestine.

Homogenates of rat small intestine can depolymerize macromolecular rat skin heparin (RS heparin) to products similar in size to commercial heparin [Horner (1972) Proc. Natl. Acad. Sci. U.S.A. 69, 3469--3473]. This activity is attributed to an enzyme provisionally named 'macromolecular heparin depolymerase'. An assay for macromolecular heparin depolymerase activity in rat small intestine has been developed, based on the action of the enzyme on 35S-labelled macromolecular RS heparin. The depolymerized products are separated into two peaks by gel chromatography through columns of Bio-Gel A-15m. The amount of label in the second peak, expressed as a percentage of the total radioactivity, is the index of enzyme activity. The pH optimum was found to be 6.0 and the temperature optimum 45 degrees C. The enzyme was shown to be most stable in 50mM-Tris/maleate buffer containing 1 mM-EDTA. Macromolecular heparin depolymerase activity measured as a function of time and substrate concentration produced curves typical of an enzymic reaction. Evidence was obtained demonstrating that the activity did not originate from bacteria in the intestine. Macromolecular heparin depolymerase activity was increased by dilution and storage at 7 degrees C for 24 h. This suggests that homogenates of rat small intestine contain an unstable inhibitor of the enzyme.

Effects of 6-(o-chlorophenyl)-8-ethyl-1-methyl-4H-s-triazolo [3,4-c]thieno[2,3-e][1,4]diazepine (Y-7131) on the metabolism of biogenic amines in brain.

A new derivative of thienodiazepine, 6-(o-chlorophenyl)-8-ethyl-1-methyl-4H-s-triazolo[3,4-c]thieno[2,3-e][1,4]diazepine (Y-7131) decreased the turnover of 5-hydroxytryptamine (5-HT) but not that of dopamine (DA) in the rat brain. The potency of Y-7131 in inhibiting the 5-HT turnover was stronger than that of diazepam. Imipramine decreased the turnover of 5-HT, whereas chlorpromazine increased the turnover of DA. Y-7131 decreased the turnover of norepinephrine (NE) in the mouse brain. With diazepam, such an action was not recognized. Imipramine did not affect the turnover of NE, whereas chlorpromazine increased it. Y-7131 inhibited the uptake of NE in the mouse brain, although it did not inhibit the uptake of 5-HT. Diazepam was devoid of such an action. Imipramine inhibited the uptake of both amines. The increase of the turnover of 5-HT, NE and DA due to foot-shock stress in the rat brain was suppressed by the administration of Y-7131. In this antagonistic effect, Y-7131 was more potent than diazepam. Neither imipramine nor chlorpromazine showed these antagonistic effects. The results obtained suggest that Y-7131 has biochemical profiles similar to diazepam but differs from it in exhibiting inhibitory effect on the NE turnover and its uptake.

A comparative study of the effects of prostaglandins and H2-receptor antagonists on gastric acid secretion, mucosal blood flow and ulcer formation.

The prostaglandins E2 and 16,16-dimethyl-PGE2 methyl ester were compared with the H2-receptor antagonists burimamide and metiamide for their effects on gastric acid secretion (GAS) and gastric mucosal blood flow (MBF) in rats and dogs, and on ulcer formation in rats. Orally, both 16,16-dimethyl-PGE2 methyl ester (20 microgram/kg) and metiamide (6 mg/kg) were markedly effective inhibitors of GAS stimulated by histamine acid phosphate or pentagastrin in Heidenhain pouch dogs, producing a reduction both in volume of gastric juice and in the concentration of titratable acid. In anaesthetised rats, however, the H2-receptor antagonists, when perfused into the gastric lumen, did not consistently inhibit the increased GAS caused by various secretagogues. In contrast, the prostaglandins, under the same conditions, caused marked inhibition of GAS provoked by all secretagogues. Intravenously, both burimamide and metiamide were effective in inhibiting GAS in rats but were less potent than the prostaglandins. The order of potency was: 16,16-dimethyl-PGE2 methyl ester greater than PGE2 greater than metiamide greater than burimamide. By the oral route, the H2-receptor antagonists were found to be very weak inhibitors of indometacin-induced gastric ulcer in rats, as compared to the prostaglandins. MBF studies in rats and in Heidenhain dogs showed that i.v. or p.o. administration inhibited both GAS and MBF in most cases. The ratio r = [MBF (ml/min)/GAS (mumol H+/min)] was generally increased by both types of compounds, suggesting a preferential effect on GAS.

Kinetics of carboxymethylation of histidine hydantoin.

The reaction of the imidazole group of histidine hydantoin with bromoacetate was studied as a model for carboxymethylation of histidine residues in proteins. pK values of 6.4 and 9.1 (25 degrees C) and apparent heats of ionization of 7.8 and 8.7 kcal/mol were determined for the imidazole and hydantoin rings, respectively. At pH values corresponding to the isoelectric points for histidine hydantoin, the rates of carboxymethylation at 12, 25, 37, and 50 degrees C were determined; the modified hydantoins were hydrolyzed to the corresponding histidine derivatives for quantitative amino acid analysis. At pH 7.72 and 25 degrees C, the imidazole tele-N was alkylated (k = 3.9 X 10(-5) M-1 s-1) twice as fast as the pros-N. The monocarboxymethyl derivatives were carboxymethylated at the same rate at the pros-N (k = 2.1 X 10(-5) M-1 s-1) but 3 times faster at the tele-N (k = 11 X 10(-5) M-1 s-1). The enthalpies of activation determined for carboxymethylation of the imidazole ring and its monocarboxymethyl derivatives were similar (15.9 +/- 0.7 kcal/mol). delta S for the four carboxymethylations was -25 +/- 2 eu. The electrostatic component of delta S (delta S es) was calculated from the influence of the dielectric constant on the reaction rate at 25 degrees C. delta S es was slightly negative (-4 +/- 1 eu) for mono- or dicarboxymethylations, indicating some charge separation in the transition state. The nonelectrostatic entropy of activation was -21 +/- 2 eu for all four carboxymethylations.

Equilibrium and kinetic measurements of the redox potentials of cytochromes c2 in vitro and in vivo.

The equilibrium oxidation-reduction mipoint potential (Em) of isolated Rhodopseudomonas sphaeroides cytochrome c2 exhibits a pH-dependent behavior which can be ascribed to a pK on the oxidized form at pH 8.0 (Pettigrew et al. (1975) Biochim. Biophys. Acta 430, 197-208). However, as with mammalian cytochrome c (Brandt, K.G. Parks, P.C., Czerlinski, G.H. and Hess, G.P. (1966) J. Biol. Chem. 241, 4180-4185) this pK can more properly be attributed to the combination of a pK beyond pH 11, and a slow conformational change of the ferricytochrome. This has been demonstrated by resolving the Em of cytochrome c2 before and after the conformational change. The Em of the unaltered form is essentially pH independent between pH 7 and 11.5, and the lower equilibrium Em is due solely to the conformational change. In vivo the conformational change is prevented by the binding of the cytochrome c2 to the photochemical reaction center, and the cytochrome exhibits an essentially pH-independent Em from pH 5 to 11. The alkaline transition thus has little physiological significance, and it is unlikely that the redox reactions of cytochrome c2 in vivo involve protons.

Purification and comparison of several catalytic parameters of the gamma-glutamyltranspeptidase of rat mammary adenocarcinoma (13762) and of normal rat mammary gland.

A method for the purification of a membrane-bound glycoprotein, gamma-glutamyltranspeptidase ((gamma-glutamyl)-peptide:amino-acid gamma-glutamyltransferase, EC 2.3.2.2), from a transplantable rat mammary tumor (13762 MT) is described. The properties of the tumor enzyme were compared with those of gamma-glutamyltranspeptidase similarly isolated from mammary tissue of nonpregnant multiparous rats. Evidence has been presented elsewhere that the mammary and tumor enzymes exist as groups of species differing in isoelectric point and that the tumor enzyme contains more of the those species with lower isoelectric points. In this study the normal and tumor enzyme preparations are found to be identical or very similar in regards to the effect of papain on molecular size, the ratios of the enzymatic activities as measured with various amino acids, the Km for gamma-glutamyl-p-nitroanilide, and the Ki for inhibition by glutathione. Neuraminidase treatment had no effect on these catalytic properties. The properties observed were generally similar to those previously reported for highly purified rat kidney preparations.

Immobilization of Escherichia coli cells containing aspartase activity with kappa-carrageenan. Enzymic properties and application for L-aspartic acid production.

Whole cells of Escherichia coli having high aspartase (L-asparate ammonialyase, EC 4.3.1.1) activity were immobilized by entrapping into a kappa-carrageenan gel. The obtained immobilized cells were treated with glutaraldehyde or with glutaraldehyde and hexamethylenediamine. The enzymic properties of three immobilized cell preparations were investigated, and compared with those of the soluble aspartate. The optimum pH of the aspartase reaction was 9.0 for the three immobilized cell preparations and 9.5 for the soluble enzyme. The optimum temperature for three immobilized cell preparations was 5--10 degrees C higher than that for the soluble enzyme. The apparent Km values of immobilized cell preparations were about five times higher than that of the soluble enzyme. The heat stability of intact cells was increased by immobilization. The operational stability of the immobilized cell columns was higher at pH 8.5 than at optimum pH of the aspartase reaction. From the column effluents, L-aspartic acid was obtained in a good yield.

Isolation and properties of a particulate fraction for the desaturation of palmitic acid from Alcaligenes faecalis.

A particulate fraction obtained from Alcaligenes faecalis could desaturate palmitic acid to palmitoleic acid. NADPH, ATP, CoA, Fe2+ and Mg2+ were essential cofactors for the reaction. The desaturation showed an absolute requirement for O2. Metal ions like Mn2+, Mo6+ and Cu2+ did not affect the desaturation, while Zn2+ was inhibitory. Sulfhydryl agents such as cysteine, glutathione and beta-mercaptoethanol had no effect, but SH-blocking agents like HgCl2 and p-hydroxymercuribenzoate inhibited the reaction. Azide and cyanide strongly inhibited the reaction while CO had no effect. The presence of a b-type cytochrome in the enzyme preparation was confirmed by the spectral studies on the reaction of enzyme with NADPH. Involvement of b-type cytochrome in the desaturation reaction was demonstrated by the reoxidation of b-type cytochrome initially reduced with NADPH, by the addition of palmitic acid and other cofactors. The pH optimum for the enzyme activity was 7.4. The optimum temperature for enzyme activity was 25 degrees C and maximum activity was obtained at the end of 45 min.

Purification and characterisation of alpha-L-fucosidase from human placenta. pH-dependent changes in molecular size.

alpha-L-Fucosidase has been purified 12 000 fold from human placenta. The enzyme is a glycoprotein containing, by weight: 0.9% galactose; 1.9% mannose, 1.9% N-acetylglucosamine and 1.9% N-acetylneuraminic acid. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate separated proteins with molecular weights ot 55 000, 51 400 and 25 000. Resolution of the two larger protein bands varied with the gel system and these proteins may differ only in carbohydrate content. Gel filtration of te purified enzyme failed to separate the three proteins. Treatments with the cross-linking reagent dimethyl suberimidate prior to electrophoresis, resulted in a diminution of the original protein bands and the formation of oligomers with molecular weights of 80 000, 100 000, 130 000, and 144 000. These results suggest that the heavy (55 000 and 51 400) and light (25 000) proteins are structurally associated. The molecular weight of the native enzyme, measured by gel filtration, was dependent on the pH of the eluting buffer. At pH 5.0 or 6.0 a catalytically active peak was observed, with a molecular weight of 305 000. At pH 7.5 this peak was completely absent and the enzyme eluted as an asymmetrical peak with an apparent molecular weight of about 60 000. The reduction in apparent molecular weight at pH 7.5 was reversible by dialysis of isolated fractions at pH 6.0. In agreement with these findings the sedimentation coefficient was 8.5 S at pH 5.0 but only 3.6 S at pH 7.5. The results can be accounted for by the existence of a pH-dependent equilibrium between aggregated and dissociated forms of the enzyme or by pH-depedent conformational changes.

Effects on induction of tyrosine aminotransferase in fetal mouse liver in vitro of prednisolone, insulin and thyroxine.

The hormonal requirements for formation of tyrosine aminotransferase (EC 2.6.1.5) in fetal mouse liver were investigated in organ culture using chemically defined medium. The hormones tested were insulin, thyroxine and prednisolone. Prednisolone alone resulted in a two-fold increase in tyrosine amino-transferase activity in explanted liver in hormone-free medium on day 6, and its effect was dose dependent, but neither insulin nor thyroxine alone induced the enzyme. Addition of prednisolone plus thyroxine and prednisolone plus insulin increased the enzyme activity 1.4- and 1.3-fold, respectively, over that of explants with prednisolone alone. These three hormones together had the greatest effect, causing induction of 1.5-fold more activity than that with prednisolone plus insulin or plus thyroxine. The three hormones were not all needed continuously during the culture period: prednisolone and insulin were required during the early part of cultivation and thyroxine during the later part. The effects of these hormones were blocked by actinomycin D or puromycin, suggesting that these hormones increase de novo synthesis of tyrosine aminotransferase. Phase-contrast microscopy showed that prednisolone stimulated liver epithelial cell outgrowth, probably acting with insulin.

Chromatographic evidence of the self-association of oxyhemoglobin in concentrated solutions: its biological implications.

Expressions that take into account the effects of thermodynamic non-ideality, described in terms of a high-order virial expansion, are derived for the concentration-dependence of the weight-average partition coefficient in exclusion chromatography of a single solute and of a solute undergoing reversible self-association. Comparison of the concentration-dependences predicted by those expressions with results obtained for bovine and human oxyhemoglobins on CPG-10-120 porous glass beads in 0.156 I phosphate-chloride buffer, pH 7.3, shows that neither oxyhemoglobin conforms with the concept of it being a single alpha 2 beta 2 entity with Stokes radius of 3.13 nm, the experimental value. Previously published osmotic pressure and sedimentation equilibrium results are also shown to be inconsistent with this concept. On the other hand, both sets of exclusion chromatography results are consistent with the joint operation of thermodynamic non-ideality and reversible association of the alpha 2 beta 2 species. From the magnitude of the equilibrium constant, derived for either of two possible modes of association, it is calculated that only half of the oxyhemoglobin would be in the alpha 2 beta 2 states under conditions of oxygen saturation and a concentration of 320 g/liter, that pertaining in the red blood cell. The consequences of this association phenomenon are discussed in relation to the oxygen binding curves obtained by others in the presence and absence of 2,3-diphosphoglycerate (DPG). An explanation is provided of the observed dependence on hemoglobin concentration of oxygen-binding in the presence of DPG, and of the absence of such an effect in DPG-free solutions. It is concluded that the control of oxygen binding to hemoglobin in the physiological situation involves the joint operation of self-association and allosteric effects.

Phosphoester specificity of purified human liver alkaline phosphatase.

Kinetic parameters for the hydrolysis of a number of physiologically important phosphoesters by purified human liver alkaline phosphatase have been determined. The enzyme was studied at pH values of 7.0 to 10.0. The affinity of the enzyme for the compounds was determined by competition experiments and by their direct employment as substrates. Phosphodiesters and phosphonates were not hydrolysed but the latter were inhibitors. Calcium and magnesium ions inhibited the hydrolysis of ATP and PP1 and evidence is presented to show that the metal complexes of these substrates are not hydrolysed by alkaline phosphatase. A calcium-stimulated ATPase activity could not be demonstrated for the purified enzyme or the enzyme in the presence of a calcium-dependent regulator protein. Nevertheless, the influence of magnesium and calcium ions on the ATPase activity of alkaline phosphatase means that precautions must be taken when assaying for Ca2+-ATPase in the presence of alkaline phosphatase. The low substrate Km values and the hydrolysis which occurs at pH 7.4 mean that the enzyme could have a significant phosphohydrolytic role. However, liver cell phosphate concentrations, if accessible to the enzyme, are sufficient to strongly inhibit this activity.

In vitro activation and behavior of the ameboid sperm of Ascaris suum (Nematoda).

A system is described for the study of activation and motility of Ascaris spermatozoa in vitro. Activation was accomplished by addition of the sperm-activating substances (SAS), extracted from the male accessory gland, to cells incubated in phosphate-buffered saline (pH 7.4) at 37-39 degrees C under anaerobic conditions (95% N2, 5% CO2). Activation is characterized by a change from spherical to ameboid shape with coalescence of the refringent granules. The normal ameboid spermatozoa bear several stubby and needle-like filopodia at the lamellipodial margin. Within the lamellipodium are bundles of microfilament-like structures extending toward the pseudopodial membrane and concentrating within the needle-like filopodia. These filopodia exhibit a pendulous, sweeping motion with subsequent retraction and disappearence within the main lamellipodium. Membranes of the ameboid cells interact at the pseudopodial regions with partial fusion, as suggested by apparent membrane breakdown between interdigitating portions of the pseudopodia. Activation is complete in 5-15 min, is totally inhibited at 4 degrees C and/or by an atmospheric environment, but can be reinitiated by transfer to anaerobic conditions at 22-39 degrees C. Activation also requires favorable pH (6.8-8.7) and continual exposure to sufficiently high sodium concentrations (134-154 mM), i.e., lowering of sodium concentration to 10 mM causes irreversible inactivation. Sodium may be replaced by potassium or lithium but not by Tris or sucrose. Proteinases (10 microgram/ml) can act as activators even though SAS lack detectable proteolytic activity against azoalbumin, azocasein, TAME and BTEE and SAS activation was not inhibited by TLCK or soybean trypsin inhibitor.

Suitability of commercial control sera for the quality control of activity determination of alkaline phosphatase.

The suitability of thirteen commercially available control sera for measuring alkaline phosphatase (EC 3.1.3.1; orthophosphoric acid monoester phosphohydrolase, ALP) activity in human serum was tested. Apart from differences in ALP activity observed in some reconstituted commercial sera, the behaviour of control materials towards experimental variables such as the nature and concentration of the substrate, pH and type of buffer (or PO4-acceptor) together with the composition of the isoenzymes present in human serum highlights the problems and difficulties if commercial materials are to be used as control sera. The half-saturation constants in control sera were in all cases smaller than those of ALP isoenzymes from bone and liver. The shape of substrate activity curves and the pH optimum in most of control sera differed from that of human serum. The discrepant kinetic data of control materials and human serum may mask or suggest changes relevant to commercial quality control serum but not to samples of human serum.

Interaction of alpha- and beta-adrenergic receptor blocking agents: circulatory effects in the conscious rat.

The alpha-receptor blockers phenoxybenzamine and phentolamine produced similar circulatory effects, e.g. hypotension and tachycardia in the conscious rat. The hypotension was more pronounced than that seen after an acute cervical transection of the spinal cord or after hexamethonium treatment. The tachycardia was blocked by drugs with beta 1-receptor blocking capacity while the hypotensive response was blocked by drugs with beta 2-receptor blocking capacity. The pronounced hypotension and tachycardia was absent after spinal transection, hexamethonium pretreatment or adrenal demedullation. In adrenal demedullated rats substitution with adrenaline after alpha-receptor blockade produced tachycardia and hypotension of the same degree as seen in intact rats after alpha-receptor blockade. There was no correlation between the degree of beta-blocker induced decrease in heart frequency and increase in blood pressure after alpha-receptor blockade, while a significant correlation was found between the alpha-blocker induced decrease in blood pressure and the subsequent beta 2-blocker induced increase in blood pressure. In spinal rats, pretreated with phentolamine, adrenaline caused a depressor response. This depressor response was converted into a pressor response by administration of beta-blockers at doses which seemed to correlate well with the doses of beta-blockers needed to effectively block the alpha-blocker induced hypotension in intact animals. It is concluded that acute administration of phentolamine or phenoxybenzamine, by blocking alpha-receptors causes a reflex increase in adrenaline output, which subsequently further decreases the blood pressure and increases the heart frequency by stimulation of beta-receptors.

In vitro metabolism of 1,2-dihaloethanes to ethylene.

1,2-Dichloroethane (DCE), a solvent and byproduct of the manufacture of polymers, and 1,2-dibromoethane, a soil fumigant, are known to be metabolized by conjugation with glutathione (GSH) to yield mercapturic acid derivatives. An alternate route of metabolism of the GSH conjugate involves beta-elimination of halide ion to form an olefin. In the present study, ethylene production from DCE was measured by gas chromatography in rat tissues as an index of this latter route of metabolism. The rate of enzymic ethylene production was linear over a 1-hr incubation time and from 1 to 8 mg of protein per ml reaction volume. The temperature optimum for ethylene formation from DCE was 55 degrees C and no distinct pH optimum was observed. DCE metabolism was highly dependent on the presence of reduced GSH. Metabolic activity was limited to hepatic and renal cytosolic fractions. The reaction was inhibited only by p-chloromercuribenzoic acid and by diethyl maleate and methyl iodide, which are substrates for GSH S-transferases. (S-(-2-Chloroethyl)-DL-cysteine . HCl, an analog of the conjugate formed from DCE and GSH, was nonenzymically converted to ethylene.

Models of neuroendocrine regulation: use of monosodium glutamate as an investigational tool.

The administration of monosodium-L-glutamate (MSG) during the neonatal period is known to result in central nervous system lesions in the arcuate nucleus of the hypothalamus and the retina. Rodents so treated exhibit behavioral deficts and endocrinopathies including obesity, hypogonadism, hypothyroidism, pituitary atrophy, tail automutilation and diminished locomotor activity. Assessment of endocrine status revealed normal serum levels of glucagon, thyroid-stimulating hormone and luteinizing hormone, and diminished levels of thyroid hormones and growth hormone in MSG-treated rats. Prolactin levels were elevated in the glutamate-treated male rats. Within the brain hypothalamic levels of thyrotropin-releasing hormone, luteinizing hormone-releasing hormone, and somatostatin were unchanged. Measurement of neurotransmitters and neurotransmitter-related enzymes in individual hypothalamic nuclei derived from MSG-treated rats revealed normal levels of norepinephrine, serotonin and glutamic acid decarboxylase, but reduced levels of choline acetyltransferase and dopamine in the arcuate nucleus and median eminence. Histochemical methods for visualization of dopamine and acetylcholinesterase in the mediobasal hypothalamus confirmed these findings. The MSG-treated animals exhibited a normal diurnal rhythm of pineal serotonin N-acetyltransferase activity. These data indicate that the MSG-induced endocrine deficiency syndrome results at least partly from destruction of cholinergic and dopamingeric tuberoinfundibular systems in the hypothalamus.

The aromatic residues of bovine pancreatic ribonuclease studied by 1H nuclear magnetic resonance.

1. The aromatic proton resonances in the 360-MHz 1H nuclear magnetic resonance (NMR) spectrum of bovine pancreatic ribonuclease were divided into histidine, tyrosine and phenylalanine resonances by means of pH titrations and double resonance experiments. 2. Photochemically induced dynamic nuclear polarization spectra showed that one histidine (His-119) and two tyrosines are accessibly to photo-excited flavin. This permitted the identification of the C-4 proton resonance of His-119. 3. The resonances of the ring protons of Tyr-25, Tyr-76 and Tyr-115 and the C-4 proton of His-12 were identified by comparison with subtilisin-modified and nitrated ribonucleases. Other resonances were assigned tentatively to Tyr-73, Tyr-92 and Phe-46. 4. On addition of active-site inhibitors, all phenylalanine resonances broadened or disappeared. The resonance that was most affected was assigned tentatively to Phe-120. 5. Four of the six tyrosines of bovine RNase, identified as Tyr-76, Tyr-115 and, tentatively, Tyr-73 and Tyr-92, are titratable above pH 9. The rings of Tyr-73 and Tyr-115 are rapidly rotating or flipping by 180 degrees about their C beta--C gamma bond and are accessible to flavin in photochemically induced dynamic nuclear polarization experiments. Tyr-25 is involved in a pH-dependent conformational transition, together with Asp-14 and His-48. A scheme for this transition is proposed. 6. Binding of active-site inhibitors to bovine RNase only influences the active site and its immediate surroundings. These conformational changes are probably not connected with the pH-dependent transition in the region of Asp-14, Tyr-25 and His-48. 7. In NMR spectra of RNase A at elevated temperatures, no local unfolding below the temperature of the thermal denaturation was observed. NMR spectra of thermally unfolded RNase A indicated that the deviations from a random coil are small and might be caused by interactions between neighbouring residues.

Mouse-liver glutathione reductase. Purification, kinetics, and regulation.

Glutathione reductase from the liver of DBA/2J mice was purified to homogeneity by means of ammonium sulfate fractionation and two subsequent affinity chromatography steps using 8-(6-aminohexyl)-amino-2'-phospho-adenosine diphosphoribose and N6-(6-aminohexyl)-adenosine 2',5'-biphosphate-Sephadex columns. A facile procedure for the synthesis of 8-(6-aminohexyl)-amino-2'-phospho-adenosine diphosphoribose is also presented. The purified enzyme exhibits a specific activity of 158 U/mg and an A280/A460 of 6.8. It was shown to be a dimer of Mr 105000 with a Stokes radius of 4.18 nm and an isoelectric point of 6.46. Amino acid composition revealed some similarity between the mouse and the human enzyme. Antibodies against mouse glutathione reductase were raised in rabbits and exhibited high specificity. The catalytic properties of mouse liver glutathione reductase have been studied under a variety of experimental conditions. As with the same enzyme from other sources, the kinetic data are consistent with a 'branched' mechanism. The enzyme was stabilized against thermal inactivation at 80 degrees C by GSSG and less markedly by NADP+ and GSH, but not by NADPH or FAD. Incubation of mouse glutathione reductase in the presence of NADPH or NADH, but not NADP+ or NAD+, produced an almost complete inactivation. The inactivation by NADPH was time, pH and concentration dependent. Oxidized glutathione protected the enzyme against inactivation, which could also be reversed by GSSG or other electron acceptors. The enzyme remained in the inactive state even after eliminating the excess NADPH. The inactive enzyme showed the same molecular weight as the active glutathione reductase. The spectral properties of the inactive enzyme have also been studied. It is proposed that auto-inactivation of glutathione reductase by NADPH and the protection as well as reactivation by GSSG play in vivo an important regulatory role.