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Effect of clofibrate treatment on acylcarnitine oxidation in isolated rat liver mitochondria.

The oxidation of palmitoyl- and octanoylcarnitine in liver mitochondria from normal and clofibrate-treated male rats was studied by measuring the ADP-stimulated oxygen consumption and acetyl group production (the sum of formed ketone bodies, acetylcarnitine and citrate). In the absence of malate the treatment approximately doubled the rate of acylcarnitine oxidation. In normal mitochondria the acetyl groups consisted almost totally of ketone bodies. The clofibrate-induced increase in acetyl group production was attributable to enhanced rates of ketone body and acetylcarnitine formation. The observed increase in acylcarnitine oxidation was associated with an elevated beta-hydroxybutyrate: acetoacetate ratio, reflecting an increased mitochondrial NADH:NAD+ ratio. In normal mitochondria the addition of malate in the presence of fluorocitrate doubled the rate of beta oxidation by forming citrate. The beta oxidation in mitochondria from clofibrate-treated rats was virtually unresponsive to added malate. The clofibrate-induced increase in ketogenesis was confirmed in disintegrated mitochondria. The treatment approximately doubled the rate of ketone body production from acetyl-CoA in disrupted organelles. The enhanced capacity of ketogenesis was accompanied by increased activity of the specific acetoacetyl-CoA thiolase (EC 2.3.1.8), which is the first step enzyme of the pathway. Clofibrate administration also increased the activities of general oxoacyl-CoA thiolase (EC 2.3.1.16), palmitoyl-CoA dehydrogenase (EC 1.3.99.3), and butyryl-CoA dehydrogenase (EC 1.3.99.2), which all take part in the beta oxidation of fatty acids.

Myosin polymorphism in human skeletal muscles.

Myosins isolated from individual human muscles (primarily normal muscles) were investigated with respect to their structural and catalytic properties. The results indicate unexpected elements of uniformity shared by the several myosins, such as a three-banded, electrophoretic pattern of light chains in sodium dodecylsulfate (SDS) gels and a low degree of alkaline lability. The pH activity profile and the effect of KCl on myosin ATPase activities were also found to be the same for the myosins from predominantly fast (e.g., vastus lateralis and rectus abdominis) and slow (e.g,, soleus and pectoralis minor) muscles. Coelectrophoretic experiments lend further credence to the interrelationship between human myosin light chains and the light chains of rabbit fast-muscle myosin. However, several kinds of circumstantial evidence, such as that derived from the study of myosin in nemaline myopathy, suggest that one shoould exercise caution in interpreting these results. On the other hand, human muscle myosins, like those of other mammalian species, can be divided into two main categories according to the peptide composition of tryptic heavy meromyosin (HMM) and the banding pattern of light meromyosin (LMM) paracrystals. These results, which are indicative of differences in the primary structure of the heavy chains, allow us to identify these heavy chains as the main site of heterogeneity among myosins in human mucles.

The effect of antihistamine drugs on the neuroleptic-induced catalepsy.

The effect of atropine on the spiperone- or reserpine-induced catalepsy was compared with the effect pure antihistamines (chlorcyclizine, diphenhydramine, mepyramine) and antiserotonin -- antihistamine drugs (cyproheptadine, danitracen). All the drugs were used in equipotent doses in respect of their central cholinolytic action, assassed previously on the basis of the tremorine test. The potency of the antiserotonin action of chlorcyclizine, diphenhydramine and mepyramine was estimated by assessing the ID50 values of these compounds in the test based on antagonism to L-5-hydroxytryptophan action in the mouse. The spiperone-induced catelepsy, was most effectively inhibited by classical histaminolytics and less by drugs of a combined antiserotonin and antihistamine action. For the reserpine-induced catalepsy, differences in action of the two groups of drugs were less distinct. In both cases atropine produced the weakest anticataleptic effect. Amodiaquine, an inhibitor of histamine degradation, enhanced the catalepsy induced by either neuroleptic (the reserpine-induced catalepsy in a statistically significant menner). A possibility that the anticataleptic action of chlorcyclizine, cyproheptadine, diphenhydramine, mepyrymine and danitracen depends on the blockade of the histamine receptors in the brain is discussed.

Abomasal function following injections of elfazepam and 9-aza-cannabinol.

The feed intake stimulants elfazepam (E), a benzodiazepine, and 9-aza-cannabinol (9-AC) decrease rumen contractions and abomasal acid content in sheep and E increases rumen fluid volume, digestibility and overall nutrient availability. E has been hypothesized to decrease the propulsive activity of the entire GI tract. To further examine the effects of E and 9-AC on gastric function, 4 ewes were prepared with abomasal cannulas and 3 silver/silver chloride monopolar electrodes alternated with 2 strain gauges on the distal one-third of the abomasal serosa. Electromyographical (slow waves and action potentials) and contractile (rates and forces) activities and abomasal pH were measured. Treatment of 8 and 16 mg E had no effect on slow wave frequency, action potential rate, contraction rate, or contraction force. Abomasal content pH was decreased with 8 mg E. Treatments of 125 and 250 microgram 9-AC depressed action potential and contraction rates and contraction force but had no effect on slow wave frequency or pH.

Chlordiazepoxide loses its anxiolytic action with long-term treatment.

The biochemical and behavioural effects of acute, 5, 15 and 25 days treatment with chlordiazepoxide (CDP, 5 mg/kg) were examined in the rat. After 5 days of drug treatment, 5-hydroxytryptamine (5HT) levels in the midbrain, hypothalamus and cortex were significantly higher than those of the corresponding controls, and the level of 5-hydroxyindoleacetic acid (5HIAA) was significantly lower, indicating reduced turnover. After 15 days of drug treatment, 5HIAA levels were significantly elevated, compared with the controls, possibly indicating that CDP was blocking the transport of 5HIAA from the brain. This effect appears to be independent of the reduced turnover. After 25 days of drug treatment there were no significant differences compared with the controls. There were no marked changes in noradrenaline and dopamine in any of the areas investigated. It appears that the reduction in 5HT turnover is linked to the anxiolytic effects of CDP; the latter were found after 5 days of drug treatment, but not after 15 or 25 days, using the social interaction test of anxiety.

Intercellular communication in pancreatic islet monolayer cultures: a microfluorometric study.

Single islet cells in monolayer cultures of neonatal rat pancreas were microinjected with fluorescein and scanned topographically by microfluorometry. Fluorescein spread from an injected islet cell directly into neighboring islet cells, and, in the presence of 16.7 millimolar glucose, significantly more islet cells communicated with the injected cell than in glucose-free medium. Islet cells were also microinjected with glycolytic substrates and activators that produced transient changes in cellular levels of reduced pyridine nucleotides-nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate [NAD(P)H]. Changes in NAD(P)H fluorescence were observed in islet cells incubated first for 18 hours in very low glucose concentrations and then in a glucose-free medium and injected with glycolytic substrates and activators; however, little change of fluorescence occurred in adjacent islet cells. In contrast, after adding 16.7 millimolar glucose to the medium, injection of glycolytic substrates and activators produced transient changes in NAD(P)H fluorescence in the injected cell and in neighboring cells.

[Decontamination of hot beef carcases with organic acids (author's transl)].

The decontamination effect of spraying hot beef carcases with solutions of lactic acid and solutions of the commercial preparation of pH liquid was studied. Spraying a 0.5 per cent lactic acid solution only resulted in a significant reduction of aerobic, gram-negative and gram-positive bacterial counts on severely contaminated areas. Treatment with a 0.75 per cent solution of lactic acid caused a significant decrease of almost all bacterial counts, whereas a 1 per cent lactic solution produced a significant reduction of all bacterial counts at the sites sampled. The maximum reduction was 2 log units per sq.cm. and varied with the level of contamination. A 5 per cent pH Liquid solution was ineffective and a 20 per cent pH Liquid solution had an effect similar to that of a 0.5 per cent lactic acid solution. The reduced bacterial counts persisted during three days of storage in the chilling room. When the above concentrations are applied, this will not cause any permanent changes of colour of the treated carcases. These changes do occur when a 10 per cent solution of lactic acid is used. This treatment can only be used to spray severely contaminated superficial layers which are to be removed. Recommendations are made for the possible use of organic acids in the slaughter line. The legal aspects are discussed.

Effect of non-carbonic acidosis on total splanchnic perfusion and cardiac output during anaesthesia with O2-N2O-barbiturate-relaxant.

Seven dogs were anaesthetized using mebumal natrium-O2-N2O-gallamonijdidum. The PaCO2 was kept at a constant level by means of mechanical ventilation, and non-carbonic acidosis was induced with HCl infusion (0.3 normal). The arterial pH varied from 7.45 to 6.88. During this acidosis, a rising arterio-venous oxygen difference was observed, with an unchanged total oxygen consumption. The pulse fell, but the mean pressures in the right atrium and aorta were unchanged. The peripheral resistance rose by 50%, whereas the fall in cardiac output of 20% was non-significant (0.10 greater than P greater than 0.05). The total splanchnic perfusion fell by 28%, and the change in flow was correlated to the change in pH. Total splanchnic perfusion (ml min-1) = -4078+655x pH (N = 42, r = 0.67, P less than 0.001). Total splanchnic perfusion as a fraction of the cardiac output remained unchanged. The resistance in the splanchnic area rose by 50%. The oxygen saturation in the portal vein and mixed venous blood changed in parallel. It is concluded that contraction of the blood vessels is the most important effect on the circulation resulting from non-carbonic acidosis during the anaesthesia employed here.

The effect of propranolol on cerebral oxygen consumption and blood flow in the rat: measurements during normocapnia and hypercapnia.

The cerebral blood flow (CBF) and cerebral oxygen consumption (CMRO2) in the rat during normocapnia and hypercapnia were investigated by means of the intraarterial 133Xenon injection technique; measurements were performed during normocapnia and hypercapnia and the effect of propranolol upon CBF and CMRO2 was studied. The CBF technique applied to rat yield reliable results even in high flow situations. A steady state period of only 10--15 s is all that is necessary to obtain the initial slope of the 133Xenon clearance curve from which CBF is calculated and measurements may be repeated within minutes. Hypercapnia caused an increase in CMRO2 of 35% which confirms the findings of other investigators. The beta-adrenergic receptor blocker propranolol (2 mg per kg i.v.) prevented this increase and could eliminate an increase in CMRO2 already induced; this indicates that CO2 affects adrenergic mechanisms. Although propranolol eliminated the CMRO2 response to hypercapnia, it only reduced the CBF response; this dissociation of CBF and CMRO2 response occurred probably because the beta-receptor blockage only eliminated a CBF increase mediated through an increased CMRO2 (cellular response) whereas a direct CO2 effect upon the arterioles (vascular response) persisted.

A review of helisoma duryi in biological control.

Biological control of schistosomiasis by means of introduction of the north American planorbid snail, Helisoma duryi, as a competitor of the intermediate host snails has been proposed. The systematics of the genus Helisoma and the geographic distribution of the different species is described. Papers dealing with laboratory experiments or field observations on the competition between H. duryi and different intermediate host snails have been reviewed. The status of H. duryi as intermediate host of trematodes has been evaluated by searching the literature for all the trematode species that are recorded from the genus Helisoma. The list does not include trematodes of medical or veterinary importance and despite many attempts it has not been possible to infect H. duryi with Schistosoma mansoni and S. haematobium. Finally this paper makes a few comments on the experiments that should be performed in the laboratory, under semifield conditions and and field conditions before H. duryi should be actively dispersed in Africa. The aspects to be considered include the nature of the competitive interactions, the relation between H. duryi and different medical and veterinary important trematodes and the effect of H. duryi on the biotope.

Antigenic glycopolypeptides HA1 and HA2 of influenza virus haemagglutinin. IV. Immunogenic properties of separated haemagglutinin glycopolypeptides.

Highly purified haemagglutinin glycopolypeptides HA1 and HA2 were effective in eliciting an antibody response. HA2 had a markedly greater immunogenic potential than HA1. In gel double immunodiffusion, sera from rabbits immunized with HA2 produced more distinct precipitin lines than sera obtained by immunization with HA1. Both kinds of rabbit sera gave precipitation with homologous antigen and with bromelain-released and purified haemagglutinin (B-HA). In radioimmunoassay, sera from rabbits immunized with HA2 revealed considerable titres for 125I-labelled HA2 binding and reacted preferentially with 125I-labelled HA2. In general, sera from rabbits immunized with HA1 exhibited low titres for 125I-labelled HA1 binding: usually they reacted also with 125I-labelled B-HA and 125I-labelled HA2. Only rabbits injected with a few doses of HA1 at short intervals revealed preferential binding for 125I-labelled HA1. Glycopolypeptides HA1 and HA2 failed to induce haemagglutination-inhibiting and virus neutralizing antibodies in rabbits.

Cow red blood cells. II. Stimulation of bovine red cell glycolysis by plasma.

Cow red cell glycolysis, which can be stimulated by a variety of purines and pyrimidines, was also found to be elevated by its own plasma. Dialyzed or charcoal-treated plasma could no longer stimulate glycolysis, suggesting that the stimulating factors may be purines or pyrimidines. Determination of purines or pyrimidines in plasma revealed the presence of xanthine (0.31 muM), hypoxanthine (0.60 muM), and adenosine (0.05 muM), as well as unknown compounds. A physiologic level of hypoxanthine, with or without xanthine and adenosine approximating their concentrations in plasma, resulted in the stimulation of cow red cell glycolytic rate by 16% (P less than 0.01). These findings suggest that plasma-borne purines may act on cow red cells in concert with as yet unidentified factors. Moreover, exchanging calf and cow plasmas produced no stimulatory effect on either calf or cow red cell glycolysis, suggesting that a) calf red cells lack some of the cellular components that respond to this stimulator and, b) only cow plasma contains this specific stimulator. In other species, including dog, cat, rabbit, rat, guinea pig, and human, stimulation of glycolysis by plasma was not observed.

Humoral regulation of vascular resistance after 30 days of pulmonary artery constriction.

In an earlier study of guinea pigs with constriction of the pulmonary artery (PA) for 30 days, hindquarters' vascular resistance was maintained primarily by humoral mechanisms. In the present study, we investigated the contribution of circulating catecholamines, angiotensin II, and other constrictor stimuli to hindquarters' vascular resistance by observing vasodilator responses to specific competitive antagonists. Pressure-flow curves indicated vascular resistances in isolated, perfused, sympathectomized hindquarters of anesthetized guinea pigs. Phentolamine produced significantly greater (P less than 0.05) vasodilatation in animals with constriction of pulmonary artery than in sham animals [Sar1-Ala8]angiotensin II produced no vasodilation in either group. After alpha-adrenergic blockade, papaverine produced similar vasodilatation and similar final perfusion pressures in both groups. It appears that circulating catecholamines and augmented vasoconstrictor responsiveness to norepinephrine are totally responsible for the increased humoral regulation of vascular resistance in this experimental model of right ventricular hypertrophy.

No barbiturate protection in a dog model of complete cerebral ischemia.

Complete global ischemia was produced in 39 dogs by temporary ligation of the aorta. Prior to the ischemic episode, pentobarbital (30 to 45 mg per kilogram of body weight) was administered to 19 of these dogs. The neurological effects of cerebral ischemia episodes lasting 8, 9, or 10 minutes were compared in dogs treated with pentobarbital and those not treated. At 48 hours following the ischemic episode most of the dogs made ischemic for 8 minutes were normal, whereas most animals made ischemic for 10 minutes were dead or comatose. The 9-minute ischemic period resulted in a relatively even distribution of normal and damaged dogs. There were no differences between treated and untreated dogs. Cerebral blood flow, cerebral metabolic rate for oxygen, and various cerebral metabolites were measured in dogs surviving 48 hours. Again, there were no differences between treated and undertreated dogs. We conclude that barbiturates provide no protection in this model of complete global ischemia. This conclusion supports the hypothesis that the likely mechanism of barbiturate protection in models of incomplete ischemia or hypoxia is based on cerebral metabolic depression; such a mechanism would not be expected to be effective in complete global ischemia.

Relationship of muscle surface pH to noninvasive hemodynamic studies in arterial occlusive diseases.

To examine the possible relationship between Doppler pressures (DP) and pulse volume amplitude (PVR) with muscle surface pH (pHm), we studied 20 patients before, during, and after arterial reconstruction. The mean pHm for the claudication, rest pain, and ischemic gangrene groups differed from a control group and from each other. The pHm varied directly with DP and PVR for the 20 patients as a whole. After reconstructive surgery, improvement in pHm seemed to precede changes in DP and PVR in six patients with combined segment disease. Although pHm correlates generally with DP and PVR, it is invasive. Therefore, pHm should not be used as a routine screening test. Whereas DP and PVR may reflect the anaerobic activity of peripheral tissue, they may be less prompt than pHm in responding to acute changes in blood flow.

Effects of altitude and two decongestant-antihistamine preparations on physiological functions and performance.

Fourteen men were studied to determine the combined effects of two altitudes--388 and 3,810 m or 1,274 and 12,500 ft--and three preparations--lactose placebo, Compound A (Actified, and Compound B (Dristan). Subjects reported least attentiveness with A and greatest with placebo. Fatigue increased significantly with time while energy, interest, and attentiveness decreased. The Multiple Task Performance Battery (MTPB) showed no effects of altitude, drugs, or time on overall performance; however, performance declined with time in several tasks, while problem solving improved. Subjects enjoyed the problem-solving tasks and may have given them preference as levels of interest declined. Though the MTPB overall composite scores did not change significantly, physiological parameters and subjective evaluations indicate that type of compound and time after ingestion are important. Declines in energy and attentiveness 2.5 h after ingestion could result in neglect of important--although routine--tasks. Hypoxia might enhance this effect and consequences might be worse in subjects whose medical conditions require these drugs.

The effects of calcium ions and pH on bovine prothrombin fragment 1. Intrinsic fluroescence studies.

The effects of pH and Ca2+ on the intrinsic fluorescence of bovine prothrombin fragment 1 were investigated to deduce the nature of protein functional groups involved in Ca2+ binding to fragment 1. From pH values of 9 to 3, increasing the H3O+ concentration results in quenching of the fluorescence of fragment 1. Reversible pH-titration curves are obtained which appear to consist of two regions. From pH 4 to pH6.5 a broad titration curve is obtained, whereas from pH6.5 to 9 a more pronounced titration behaviour is evidenced by a group or groups on fragment 1 with an apparent pKa of approx. 7.5. In contrast, the apparent association constant for Ca2+ and fragment 1 shows a sharp pH-dependence in the region between pH7 and 8 with tighter Ca2+ binding at higher pH values. A PKa of approx. 7.5 can be estimated for the group or groups on fragment 1 linked to the tight binding of Ca2+. Both H3O+ and Ca2+ result in blue-shifts in the wave-lengths of fragment-1 emission. These results are interpreted in terms of H+ - and Ca2+ - induced changes in the conformation of fragment 1 as a result of surface-charge neutralization.

[In vitro method for estimation of enteral absorption of clanobutin and other drugs].

Two models have been developed for the determination of in vitro absorption of 4-[4-chloro-N-(4-methoxyphenyl)-benzamidol]-butyric acid (clanobutin) and other agents. Permeability coefficients PM are obtained on artificial phospholipid collodion membranes which permit calculation of absorption coefficients k1 for anticipated human enteral absorption with consideration of physiological parameters. At a pH of 7--8 (small intestine) in particular, k1 shows good agreement with in vitro absorption determined in swine intestine and absorption constant ka derived from pharmacological studies in man. The kinetic absorption model simulates gastro-intestinal transport of the substance as well as local changes in pH. It allows calculation of anticipated human gastric (pH 3) and intestinal (pH 7.5) absorption rates with k1 for different gastric filling volumes. The calculated invasion curves are held against invasion curves calculated from pharmacokinetic studies in man. The comparison shows that all invasion curves calculated according to the kinetic absorption model for gastric filling volumes of 125--1000 ml are within the 95% confidence range; very good agreement exists above all with filling volumes of 125--250 ml. The prognostic value of permeation studies on artificial phospholipid collodion membranes and of the use of the kinetic absorption model in drug design is demonstrated by corresponding studies with chenodesoxycholic acid, indometacin, and clofibrinic acid.

Response of the rat to saccharin with particular reference to the urinary bladder.

Male and female Wistar rats were administered sodium saccharin for life (2 yr) either in the drinking water or diet. The maximum palatable dose of saccharin in the drinking water was found to be 2 g/kg/day and, even then, there was some voluntary restriction of fluid intake in the males. By contrast, double this dose--namely 4 g/kg/day, was palatable in the diet. A control group of rats of both sexes received saccharin-free diet and drinking water. Mild urothelial hyperplasias developed from 85 weeks in rats of both sexes receiving saccharin either in the drinking water or diet; the incidence was statistically significant in both the bladders and kidneys of rats receiving the higher dose of saccharin in the diet, but in the kidneys only of rats receiving the lower dose of saccharin in the drinking water. Telangiectasia of the vasa recta was significant in saccharin-treated rats of both sexes at both doses. A very low incidence of bladder tumours, exclusively in males receiving the higher saccharin dose in the diet was seen from 95 weeks. No consistent relationship between bladder epithelial hyperplasias and crystalluria could be demonstrated, although all 3 bladder tumours were associated with some form of mineralisation. Results suggest a particular susceptibility of males to saccharin treatment. The possibility that saccharin may promote, or enhance, the development of latent tumour cells already present in the experimental population, rather than initiate carcinogenesis per se is considered.

Kinetics of Mg2+ flux into rat liver mitochondria.

Unidirectional fluxes of Mg2+ across the limiting membranes of rat liver mitochondria have been measured in the presence of the respiratory substrate succinate by means of the radioisotope 28Mg. Rates of both influx and efflux of Mg2+ are decreased when respiration is inhibited. A linear dependence of the reciprocal of the Mg2+ influx rate on the reciprocal of the Mg2+ concentration is observed. The apparent Km for Mg2+ averages about 0.7 mM. N-Ethyl-maleimide, an inhibitor of transmembrane phosphate-hydroxyl exchanges, enhances the observed pH dependence of Mg2+, influx. In the presence of MalNEt, the apparent Vmax of Mg2+ influx is greater at pH 8 than at pH 7, and there is a linear dependence of the Mg2+ influx rate on the external OH- concentration. The K+ analogue Tl+ inhibits Mg2+ influx, while La3+, an inhibitor of mitochondrial Ca2+ transport, has no effect on Mg2+ influx. Mg2+ competitively inhibits the flux of K+ into rat liver mitochondria. The mechanism(s) mediating mitochondrial Mg2+ and K+ fluxes appear to be similar in their energy dependence, pH dependence, sensitivity to Tl+, and insensitivity to La3+.

The isolation of stable cattle rod outer segments with an intact plasma membrane.

A procedure is described to purify and stabilize cattle rod outer segments with an intact plasma membrane. Three criteria are applied to assess the integrity of the latter. Upon photolysis in these rod outer segments: (1) exogenous ATP cannot phosphorylate rhodopsin located in the disk membrane. (2) Endogenous cofactors (NADPH, NADPH-regenerating system) are still available in the rod cytosol and consequently retinol is the final photoproduct of photolysis of rhodopsin. (3) The rod cytosol can maintain a pH different from that of the medium, since the later stages of rhodopsin photolysis are independent of the medium pH. The stability and homogeneity of the preparation appear to be much better than those of freshly isolated frog rod outer segments, which have been used most frequently so far for experiments on the physiology of rod outer segments. In addition, these cattle rod outer segments remain intact during various manipulations and therefore considerably extend the experimental possibilities when intact rod outer segments are required.

The role of cysteine residues in the catalytic activity of glycerol-3-phosphate dehydrogenase.

Glycerol-3-phosphate dehydrogenase (sn-glycerol-3-phosphate:NAD+ 2-oxido-reductase, EC 1.1.1.8) has been shown to be sensitive to inhibition by iodoacetate. The reaction of the enzyme with iodoacetate, which appears to be a simple bimolecular process, is accompanied by a corresponding loss of enzyme activity. In addition to changes in activity, the alkylation reaction was monitored by the incorporation of radioactivity from iodo[2-14C]acetate, by changes in amino acid composition, and by changes in the content of free sulfhydryl groups. It is concluded that there are two cysteine residues in the native dimeric enzyme which are essential for enzymic activity. The rate of inactivation was relatively insensitive to the presence of various compounds with the exception of NADH which markedly inhibited the reaction. Kinetic and binding studies showed that the binding of NADH prevents alkylation and, conversely, alkylation prevents NADH binding. From the pH dependence of the alkylation reaction, the pKa of the essential sulfhydryl groups was calculated to be 8.5 and it is suggested that the binding of coenzyme is independent of the state of ionization of these groups.

Solubilization and molecular weight determination of the (Na+ + K+)-ATPase from rectal glands of Squalus acanthias.

The membrane-bound (Na+ + K+)-activated ATPase (ATP phosphohydrolase, EC 3.6.1.3) system was treated with the nonionic detergent octaethylene-glycoldodecyl ether, yielding a transparent supernatant after centrifugation. The supernatant was highly active with both ATPase and p-nitrophenylphosphatase, with initial specific activities of 2300 mumol Pi released . mg-1 protein. h-1 and 350 mumol p-nitrophenol released.mg-1 protein.h-1, respectively. The supernatant was purified to 95--100%, with respect to the 96 000 dalton and the 56 000 dalton peptides. The solubilized enzyme was gel filtered in Sepharose 4B-Cl and displayed 2 peaks, both with catalytic activity. The low molecular weight particles eluted at Kav = 0.54, corresponding to a molecular weight of approximately 500 000 daltons and the particles had a specific activity of 2100 mumol Pi.mg-1 protein.h-1. Both peaks contained phospholipid with 60 mol phospholipid bound per 300 000 g protein. The low molecular weight particles had a molecular weight of 276 000 as determined by sedimentation equilibrium analysis.

Factors affecting the hydrolysis of ceramide-3 by alpha-galactosidase A from human liver.

1. The effect of detergents on the catalytic properties of alpha-galactosidase from human liver was studied using p-nitrophenyl-alpha-galactoside and galactosyl-alpha(1 leads to 4)-galactosyl-beta(1 leads to 4)-glucosylceramide (ceramide-3) as substrates. 2. The hydrolysis of p-nitrophenyl-alpha-galactoside by alpha-galactosidase was inhibited by commercial preparations of sodium taurocholate and by taurocholate purified from these preparations by thin-layer chromatography. The extent of inhibition was dependent on the concentration of the detergent and on the amount of protein present. The impurities present in the preparation also inhibited the hydrolysis. 3. The inhibition of taurocholate preparations of p-nitrophenyl-alpha-galactoside hydrolysis was pH-dependent. 4. The inhibition by taurocholate of p-nitrophenyl-alpha-galactoside hydrolysis can be partly overcome by adding glycosphingolipids. 5. No significant hydrolysis of ceramide-3 occurs in the absence of detergent. Upon adding increasing concentrations of taurocholate, the rate of hydrolysis increases to a maximum value. At still higher taurocholate concentrations the activity decreases. 6. The concentrations of taurocholate giving a maximal rate of hydrolysis of ceramide-3 is dependent on the amount of protein present and independent of the ceramide-3 concentration. 7. When the pH dependence of the rate of hydrolysis of ceramide-3 was measured in the presence of a commercially available preparation of pure taurocholate or of crude taurocholate, curves with different shapes were obtained.

Glycoprotein biosynthesis in calf kidney. Glycoprotein sialyltransferase activities towards serum glycoproteins and calf Tamm-Horsfall glycoprotein.

CMP-AcNeu:glycoprotein sialyltransltransltransltransltransferase of calf kidney cortex was characterized using serum glycoproteins and Tamm-Horsfall glycoprotein, obtained from calf urine, as acceptors. Native calf Tamm-Horsfall glycoprotein showed the best acceptor properties, followed by desialylated calf fetuin and desialylated human alpha 1-acid glycoprotein exhibiting V values of, respectively, 114, 63 and 41 nmol/h per g wet wt. of kidney cortex and Km values of 0.12, 0.16 and 0.26 mM glycoprotein acceptor. Desialylated ovine submaxillary mucine appeared to be a very poor acceptor. Tamm-Horsfall glycoprotein sialyltransferase could be distinguished from serum glycoprotein sialyltransferase by competition studies. In addition the two glycoprotein sialyltransferase activities showed different distributions over the three regions of the calf kidney: the ratios of the Tamm-Horsfall to serum glycoprotein sialyltransferase activities decreased from 3.3 in the cortex to 0.8 and 0.4 in the medulla and the papilla, respectively. It was concluded that in calf kidney at least two different sialyltransferases exist. The high cortical Tamm-Horsfall glycoprotein sialyltransferases activity corresponds markedly to the origin of the urinary Tamm-Horsfall glycoprotein, namely the distal part of the kidney tubule. Inactivation of glycoprotein sialyltransferase activity by preincubation at various temperatures and during storage at 0 degree C, could be reduced by the addition of CMP-AcNeu. The possible relevance towards the in vivo sialylation of this finding is discussed.

Dissociation between the presynaptic dopamine-sensitive adenylate cyclase and [3H]spiperone binding sites in rat substantia nigra.

[3H]Spiperone binding sites and the dopamine-sensitive adenylate cyclase were measured in rat substantia nigra (s. nigra) 7 or 14 days after various lesions. Hemisections, which resulted in a 66% decline in tyrosine hydroxylase and cyclic nucleotide phosphodiesterase and a 73% decrease in glutamate decarboxylase, led to a 50% decrease in [3H]spiperone binding and to the almost complete disappearance of the dopamine-sensitive adenylate cyclase from the s. nigra on the lesioned side. 6-Hydroxydopamine injection into the s. nigra, which depleted tyrosine hydroxylase activity within the s. nigra by 85%, while leaving phosphodiesterase unaffected, resulted in a 40% decrease in [3H]spiperone binding but no change in the dopamine-sensitive adenylate cyclase. Intrastriatal injections of kainic acid did not alter tyrosine hydroxylase activity in the s. nigra, but decreased both glutamate decarboxylase (54%) and phosphodiesterase (68%); [3H]spiperone binding was unaffected by this lesion while the dopamine-sensitive adenylate cyclase was greatly reduced (50-75%). These results suggest that within the s. nigra the dopamine receptor binding sites as defined using [3H]spiperone are located on dopamine neurones while the dopamine-sensitive adenylate cyclase is located presynaptically on striatonigral nerve terminals.

Serum enzymes in colorectal cancer.

A study of the value of serum enzymes in 184 patients with colorectal cancer has been performed. The enzymes studied were gamma glutamyltransferase (gammaGT), alkaline phosphatase (AP), lactate dehydrogenase (LDH), 5'-nucleotidase (5'-NT), glutathione reductase (GR), alanine and aspartate transaminases. In patients without liver metastases, elevated enzyme levels were found in 11-55% preoperatively. 5'-NT showed the least number of elevated activities, while gammaGT activities were increased in 29% and LDH in 55%. The percentage of elevated enzyme levels rose significantly in the early postoperative period. Patients with liver metastases showed increased enzyme activities in 40-60% preoperatively: gammaGT was the most sensitive indicator. Increased enzyme activity was related to the degree of liver involvement with secondary tumor. With extensive liver metastases, gammaGT levels were increased in 82%. It is concluded that serum enzymes are of limited value in the preoperative detection of liver metastases, and particularly when tumor involvement of the liver is small.

Lorazepam kinetics in the elderly.

Lorazepam is a 3-hydroxy-1,4-benzodiazepine derivative biotransformed by glucuronide conjugation, followed by urinary excretion of the glucuronide metabolite. The kinetic properties of single 1.5- to 3.0-mg doses of intravenous lorazepam were assessed in 15 healthy elderly subjects, 60 to 84 yr of age, and in 15 healthy young subjects, 19 to 38 yr of age. Volumes of distribution for lorazepam in the elderly group (mean, 0.99 1/kg), were slightly but significantly smaller than in the young group (1.11 1/kg), suggesting less extensive drug distribution in the elderly. Values of elimination half-life (t1/2beta) in the elderly (15.9 hr) did not differ significantly from those in the young group (14.1 hr), but total clearance in the elderly (0.77 ml/min/kg) was 22% less (p less than 0.05) than in the young subjects (0.99 ml/min/kg). Age differences in lorazepam clearance were partly explained by more frequent cigarette smoking in the young subjects. Gender had no apparent relationship to kinetics. The rate and completeness of absorption of intramuscular (IM) and oral loraxepam was assessed in 10 of the elderly subjects. Deltoid IM injection and oral administration of tablets in the fasting state led to rapid absorption of lorazepam into the systemic circulation. Peak plasma lorazepam concentrations were always reached within 2.5 hr, and values of absorption half-life (t1/2a) did not exceed 45 min. Absorption of IM and oral lorazepam was 80% to 100% complete. Thus, the aging process is associated with small changes in the kinetics of lorazepam. IM and oral administration of lorazepam in elderly persons, as in the case of young individuals, leads to rapid and nearly complete absorption into the systemic circulation.

Biochemical and physiological studies of long-term synaptic plasticity.

High frequency stimulation of fiber systems in the mammalian hippocampus produces a semipermanent increase in synaptic efficacy. This effect, long-term potentiation (LTP), has been of considerable interest as a potential substrate of memory due to its rapid onset and extreme persistence. Experiments are described that indicate that the locus of LTP is confined to the synaptic complex of the fibers stimulated; further, Ca2+ is shown to be essential for the initiation of LTP and may play a role in triggering this increase in synaptic efficiency. Data from biochemical analyses of LTP indicate that a 40,000 dalton synaptic membrane protein shows a highly reliable change in its endogenous phosphorylation following high frequency hippocampal stimulation. Phosphorylase kinase, a Ca2+ sensitive enzyme, is shown to specifically catalyse the phosphorylation of this 40,000 dalton protein. The data are discussed in terms of a working model in which the Ca2+ dependent phosphorylation of the 40,000 dalton protein produced by high frequency stimulation is a biochemical intermediate in the production of LTP.

Are epinephrine and gastrin accelerative factors of acute cinchophen ulcer?: Studies on gastric mucosal microcirculation and gastric secretion.

Measurements of serum epinephrine and gastrin, simultaneously, gastric mucosal blood flow and gastric secretion were carried out in cinchophen treated dogs. No significant changes in either serum concentrations of epinephrine and gastrin or fundic mucosal microcirculation after a single 100 mg/kg cinchophen administration were found while gastric secretion increased markedly after the medication. On the other hand, a significant increase in serum epinephrine and gastrin levels was observed while gastric secretion decreased significantly after large doses of cinchophen (300 mg/kg) were injected intravenously. Here gastric mucosal microcirculation is decreased. Repeated administration of 100 mg/kg cinchophen for 3 to 7 days brought about an increase in epinephrine and gastrin levels and caused an occurrence of fundic mucosal hemorrhage. Sympathetic discharge and gastrin release were not seen after a 3-week period of cinchophen administration. Cinchophen ulcers were produced, even when contact between the bile and the stomach mucosa was avoided. Vagotomy had no connection with ulceration and gastric secretion.

Effects of drugs on brain neurotransmitter and pituitary-testicular function in male rats.

The effects of different drugs influencing brain neurotransmitter contents have been tested on the pituitary-testicular function in male rats. L-dopa (200 mg/kg body weight, i.p.) increased the dopamine and noradrenaline contents of the hypothalamus, amygdala, striatum and mesencephalon, but it was ineffective as regards the 5-hydroxytryptamine contents of the same brain areas, and increased the plasma testosterone level. alpha-Methyl-p-tyrosine (250 mg/kg b.w., i.p.) decreased the dopamine and noradrenaline contents of these brain areas, but it was ineffective to 5-hydroxytryptamine, and decreased the plasma testosterone level. Diethyldithiocarbamate (400 mg/kg b.w., i.p. twice a day) increased the dopamine levels in the hypothalamus, amygdala, striatum and mesencephalon, decreased the noradrenaline contents in the same brain regions but had no effect on the 5-hydroxytryptamine contents of these brain areas or on the testosterone level in the peripheral blood. p-Chlorophenylalanine (300 mg/kg b.w., i.p.) decreased the 5-hydroxytryptamine contents of the different brain areas, while it had no effect on the dopamine and noradrenaline levels or on the plasma testosterone level. 5-Hydroxytryptophan (200 mg/kg b.w., i.p.) increased the 5-hydroxytryptamine contents of all brain areas studied, but was without effect on the dopamine and noradrenaline contents or the plasma testosterone level. The data suggest that both dopamine and noradrenaline may be involved in the regulation of the pituitary-testicular function, and the ratio of the two transmitters might be more important that their actual levels in definite brain areas.

Purification and some properties of cholesterol oxidase from Schizophyllum commune with covalently bound flavin.

Cholesterol oxidase [EC 1.1.3.6] from Schizophyllum commune was purified by an affinity chromatography using 3-O-succinylcholesterol-ethylenediamine (3-cholesteryl-3-[2-aminoethylamido]propionate) Sepharose gels. The resulting preparation was homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 53,000 by SDS-gel electrophoresis and 46,000 by sedimentation equilibrium. The enzyme contained 483 amino acid residues as calculated on the basis of the molecular weight of 53,000. The enzyme consumed 60 mumol of O2/min per mg of protein with 1.3 mM cholesterol at 37 degrees C. The enzyme showed the highest activity with cholesterol; 3 beta-hydroxysteroids, such as dehydroepiandrosterone, pregnenolone, and lanosterol, were also oxidized at slower rates. Ergosterol was not oxidized by the enzyme. The Km for cholesterol was 0.33 mM and the optimal pH was 5.0. The enzyme is a flavoprotein which shows a visible absorption spectrum having peaks at 353 nm and 455 nm in 0.1 M acetate buffer, pH 4.0. The spectrum was characterized by the hypsochromic shift of the second absorption peak of the bound flavin. The bound flavin was reduced on anaerobic addition of a model substrate, dehydroepiandrosterone. Neither acid not heat treatment released the flavin coenzyme from the enzyme protein. The flavin of the enzyme could be easily released from the enzyme protein in acid-soluble form as flavin peptides when the enzyme protein was digested with trypsin plus chymotrypsin. The mobilities of the aminoacyl flavin after hydrolysis of the flavin peptides on thin layer chromatography and high voltage electrophoresis differed from those of free FAD, FMN, and riboflavin. A pKa value of 5.1 was obtained from pH-dependent fluorescence quenching process of the aminoacyl flavin. AMP was detected by hydrolysis of the flavin peptides with nucleotide pyrophosphatase. The results indicate strongly that cholesterol oxidase from Schizophyllum commune contains FAD as the prothetic group, which is covalently linked to the enzyme protein. The properties of the bound FAD were comparable to those of N (1)-histidyl FAD.

Metal-free southern bean mosaic virus crystals.

Native southern bean mosaic virus contains a significant number of Mg2+ and Ca2+ ions. These can be removed by treatment with EDTA causing the virus to swell by 7% in radius at alkaline pH values. The swollen virions are susceptible to protease and nuclease digestion. They are likely to be an intermediate during assembly and disassembly. Crystals of the metal-free virus have been grown and were found to be approximately isomorphous with the orthorhombic type III southern bean mosaic virus crystals (Akimoto, T., Wagner, M.A., Johnson, J.E., and Rossmann, M.G. (1975) J. Ultrastruct. Res. 53, 306-318), although the cell dimensions are longer by 2%. Native rhombohedral type II crystals disintegrate on changing the pH or increasing the ionic strength of the mother liquor. Damage can be prevented by addition of ethylene glycol. At alkaline pH values, these crystals also show a 2% increase in their cell dimensions as well as a significant alteration in their diffraction patterns. In the type II and III crystals, the viruses pack with only their 5-fold axes in contact. Thus, the difference of the apparent swelling in solution and in the crystals may be one of differential swelling over the virus surface.

Regulated release of Ca2+ from respiring mitochondria by Ca2+/2H+ antiport.

Simultaneous measurements of oxygen consumption and transmembrane transport of Ca2+, H+, and phosphate show that the efflux of Ca2+ from respiring tightly coupled rat liver mitochondria takes place by an electroneutral Ca2+/2H+ antiport process that is ruthenium red-insensitive and that is regulated by the oxidation-reduction state of the mitochondrial pyridine nucleotides. When mitochondrial pyridine nucleotides are kept in a reduced steady state, the efflux of Ca2+ is inhibited; when they are in an oxidized state, Ca2+ efflux is activated. These processes were demonstrated by allowing phosphate-depleted mitochondria respiring on succinate in the presence of rotenone to take up Ca2+ from the medium. Upon subsequent addition of ruthenium red to block Ca2+ transport via the electrophoretic influx pathway, and acetoacetate, to bring mitochondrial pyridine nucleotides into the oxidized state, Ca2+ efflux and H+ influx ensued. The observed H+ influx/Ca2+ efflux ratio was close to the value 2.0 predicted for the operation of an electrically neutral Ca2+/2H+ antiport process.

Chick embryo liver beta-glucuronidase. Comparison of activity on natural and artificial substrates.

The beta-glucuronidase in homogenates of 12-day chick embryo livers catalyzed the release of glucuronic acid from 4-methylumbelliferyl-beta-D-glucuronide and from the nonreducing terminals of the hexasaccharides of chondroitin-6-SO4 and chondroitin-4-SO4 at rates of 143, 114, and 108 nmol of glucuronic acid/h/mg of protein, respectively, when assayed at pH 3.5 in 0.05 M sodium acetate buffer. During a 60-fold purification of the enzyme, the ratios of the activities on these substrates did not change. When 4-methylumbelliferyl-beta-D-glucuronide was used as substrate the enzyme was active at pH values from 3.0 to 5.5, with maximal activity between pH values 4.0 and 4.5. Concentrations of NaCl from 0.15 to 0.3 M inhibited the activity at low pH values but activated the enzyme between pH 4.0 and 5.5. The enzyme was active on the chondroitin-6-SO4 hexasaccharide from pH 3.0 to 5.5, with a broad optimum between 3.0 and 4.5. NaCl inhibited the activity on the oligosaccharide substrate at all pH values. Eadie-Scatchard plots of rates of 4-methylumbelliferyl-beta-D-glucuronide hydrolysis at substrate concentrations ranging from 2 to 1000 microM showed multiple kinetic forms of the enzyme, a form with a Km of approximately 11 microM, and a second form with a Km of approximately 225 microM. The pH optimum of the low Km form was 3.5 to 4.0; that of the high Km form was pH 4.5. NaCl inhibited the activity of the low Km form, but activated the high Km form of the enzyme. Chondroitin SO4 oligosaccharides competed with 4-methylumbelliferyl-beta-D-glucuronide for the low Km form of the enzyme but had little effect on the hydrolysis of 4-methylumbelliferyl-beta-D-glucuronide by the high Km form of the enzyme. The activities of the beta-glucuronidase on tetra-, hexa-, octa-, and decasaccharides of chondroitin-6-SO4 and chondroitin-4-SO4, measured using a new assay procedure which can detect the formation of 1 nmol of product, were similar, although rates were somewhat lower for the higher oligosaccharides. With the exception of the chondroitin-4-SO4 tetrasaccharide, all of the oligosaccharide substrates saturated the enzyme at concentrations of 20 to 30 microM, indicating Km values of less than 10 to 15 microM for the oligosaccharides. Highly purified beta-glcuronidases from human placenta and from rat preputial gland also showed multiple kinetic forms when assayed using 4-methylumbelliferyl-beta-D-glucuronide as substrate.

An extrarenal role for parathyroid hormone in the disposal of acute acid loads in rats and dogs.

Acid infusion studies were performed in nephrectomized rats and dogs with either intact parathyroid glands (intact) or after thyroparathyroidectomy (thyroparathyroidectomized [TPTX]) to determine the role of parathyroid hormone (PTH) in extrarenal disposal and buffering of acutely administered acid. 29 intact rats given 5 mM/kg HCl and 6 intact dogs given 7 mM/kg HCl developed severe metabolic acidosis but all survived. However, each of 12 TPTX rats and 4 TPTX dogs given the same acid loads died. Intact rats and dogs buffered 39 and 50% of administered acid extracellularly, respectively, whereas extracellular buffering of administered acid was 97 and 78% in TPTX rats and dogs, respectively. 17 TPTX rats and 6 TPTX dogs given synthetic PTH 2 h before acid infusion survived. The blood bicarbonate and extracellular buffering in these animals, measured 2 h after acid infusion, was similar to intact animals. Changes in liver, heart, and skeletal muscle pH determined from [(14)C]5,5-dimethyl-2,4 oxazolidinedione distribution seemed insufficient to account for the increased cell buffering of PTH-replaced animals. Indeed, muscle pH in TPTX dogs given PTH and acid was only 0.06 pH units lower than in control dogs given no acid, suggesting that another tissue, presumably bone, was the target for PTH-mediated increased cell buffering. This conclusion was supported by the observation that PTH did not alter the pH of intact rat diaphragms in vitro. These results indicate that PTH is necessary for the optimal buffering of large, acute acid loads presumably by increasing bone buffering.

Circulating DNA:anti-DNA complexes in systemic lupus erythematosus. Detection and characterization by ultracentrifugation.

Although it is generally accepted that DNA:anti-DNA immune complexes play a significant role in the pathogenesis of tissue injury in systemic lupus erythematosus, their presence in the circulation is still a matter of controversy. In this study, we detected DNA:anti-DNA compexes by identification of both the antigen and(or) the antibody, the necessary requisites for immune complex definition, in 14 of 24 plasmas (7 of 11 patients). These antibodies were specific for native DNA and could be adsorbed by anti-immunoglobulin (Ig)G antisera. The DNA recovered was, at least in part, of low molecular weight. The presence of DNA:anti-DNA compexes was not related to high molecular weight IgG, cryoprecipitins, positive polyethylene glycol precipitation, or low plasma C3 levels. It was related significantly to low plasma C4 levels and to the presence of diffuse proliferative nephritis. The lack of correlation with other methods of detection of immune complexes and with the presence of heavy IgG (above 13 S) is in favor of the existence of other antigen-antibody systems (or aggregated immunoglobulins) in systemic lupus erythematosus plasmas. From the results, it appears that methods directed towards the demonstration of specific immune complexes are more informative than those detecting heavy or altered immunoglobulins.

Effects of ascorbic acid and sodium ascorbate on cyclic nucleotide metabolism in human lymphocytes.

L-ascorbic acid (LAA) augmented cGMP many-fold in highly purified human peripheral blood lymphocytes. The cGMP response occurred within 10 sec and persisted for at least 60 min. D-ascorbic acid (DAA) and dehydroascorbic acid (DHAA) were also equally active in enhancing cGMP concentrations but metabolic precursors of ascorbic acid and other inorganic acids did not increase cGMP levels. Determination of the amount of DHAA contaminating the LAA precluded the possibility that it was solely responsible for the enhanced cGMP levels. The sodium or calcium salts of ascorbic acid did not increase cGMP concentrations. If these neutralized preparations were acidified, increased cGMP concentrations were then noted. In broken cell preparations, LAA, DAA, and DHAA and to a lesser extent sodium ascorbate (NaA) enhanced guanylate cyclase activity while neither inhibited cAMP or cGMP phosphodiesterase (PDE) activity. The possible role of H2O2, fatty acid liberation, prostaglandin production, oxidizing-reducing agents, and free radical formation in mediating the effects of ascorbic acid on cGMP levels were evaluated, but none of these potential mechanisms were definitively proven to be a required intermediary for the cGMP enhancing activity of ascorbic acid. LAA, DHAA or NaA did not induce lymphocyte transformation or modulate lectin-induced mitogenesis.

Fatigue effects on intelligence test performance in the elderly.

The purpose of the study was to investigate effects of fatigue on intelligence test performance in the elderly. Dependent variables were Verbal Comprehension, Numerical Facility, Perceptual Speed, and Word Fluency tests. Fatigue effects were investigated by varying the number of previous tests administered, by introducing breaks between tests in some conditions, and by using a pre-test fatigue-producing condition, a modified form of the Finding A's test. Subjects' ages were between 57 to 91-years. It was hypothesized that the Finding A's test would be more fatiguing than a long battery of tests and that introducing a break condition between the Finding A's test and the main battery would alleviate fatigue effects. Analyses of Variance resulted in a main effect due to a pre-test condition for the Perceptual Speed test only, and only when the main battery was preceded by the Finding A's task (p less than .001). It appears that the elderly are not as susceptible to test fatigue as previous results seemed to suggest.

7alpha-Dehydroxylation of cholic acid and chenodeoxycholic acid by Clostridium leptum.

The rate of 7alpha-dehydroxylation of primary bile acids was quantitatively measured radiochromatographically in anaerobically washed whole cell suspensions of Clostridium leptum. The pH optimum for the 7alpha-dehydroxylation of both cholic and chenodeoxycholic acid was 6.5-7.0. Substrate saturation curves were observed for the 7alpha-dehydroxylation of cholic and chenodeoxycholic acid. However, cholic acid whole cell K0.5 (0.37 micron) and V (0.20 mumol hr-1mg protein-1) values differed significantly from chenodeoxycholic acid whole cell K0.5 (0.18 micron) and V (0.50 mumol-1 hr-1 mg protein-1). 7alpha-Dehydroxylation activity was not detected using glycine and taurine-conjugated primary bile acids, ursodeoxycholic acid, cholic acid methyl ester, or hyocholic acid as substrates. Substrate competition experiments showed that cholic acid 7 alpha-dehydroxylation was reduced by increasing concentrations of chendeoxycholic acid; however, chenodeoxycholic acid 7alpha-dehydroxylation activity was unaffected by increasing concentrations of cholic acid. A 10-fold increase in cholic and 7alpha-dehydroxylation activity occurred during the transition from logarithmic to stationary phase growth whether cells were cultured in the presence or absence of sodium cholate. In the same culture, a similar increase in chenodeoxycholic acid 7alpha-dehydroxylation was detected only in cells cultured in the presence of sodium cholate. These results indicate the possible existence of two independent systems for 7alpha-dehydroxylation in C. Leptum.

A comparative study of Zn(II) and Co(II) binding to glycyl-L-tyrosine, a pseudosubstrate for carboxypeptidase A.

A comprehensive investigation of the interaction of Zn(II) and Co(II) with the dipeptide glycyl-L-tyrosine has been carried out. The carboxyl, amino, and tyrosyl pKa values, as well as the distribution of solution complexes, have been determined by analytical potentiometry. The amide pKa value was determined by relating the proton magnetic resonance (PMR) titration behavior of the tyrosyl alpha-hydrogen resonance to an H2-acidity function for concentrated solutions of aqueous base. Both metals behave in a qualitatively similar manner, yielding equivalent species as a function of pH. Both metals formed bis-peptide complexes, involving amino and peptide carbonyl coordination near pH = 8, with Zn(II) demonstrating a substantially higher affinity for the ligand. No evidence could be found for direct, metal-promoted phenolic dissociation, although the tyrosyl pKa value was sensitive to metal binding at other loci on the dipeptide molecule. At high pH, both systems ionized two additional protons. In the Co(II) system, these correspond to amide protons. However, it is not entirely clear whether the protons in the Zn(II) system originate from the peptide linkage or metal-bound water molecules.

Characterization of receptors on postganglionic cholinergic neurons in the guinea-pig isolated ileum.

Dopamine, apomorphine, noradrenaline and isoprenaline reduced the response of the isolated guinea-pig ileum to exogenous acetylcholine by a maximum of 40%. Propranolol reversed this inhibition whilst phentolamine and pimozide were ineffective, suggesting that the drugs were acting on a post-synaptic beta-adrenoceptor. The same agonists were more effective as inhibitors of the response to transmural electrical stimulation of the ileum, lower doses producing almost complete inhibition. This inhibition was partially antagonized by phentolamine, pimozide and propranolol. Clonidine proved to be the most potent inhibitor of the response to transmural electrical stimulation, whilst phenylephrine was ineffective. pA2 determinations showed that phentolamine was a potent antagonist of clonidine but a weak antagonist of apomorphine whilst for pimozide the opposite was true. The results suggest that there are two populations of prejunctional receptors on the cholinergic nerves innervating the smooth muscle of the guinea-pig ileum. One receptor is similar to a classical prejunctional alpha-adrenoceptor and the other resembles a central dopamine receptor.

Intracellular pH and plateau duration of internally perfused squid giant axons.

The effects of changing the intracellular pH on the action potential duration and other electrophysiological properties were studied in squid giant axons perfused intracellularly with TEA(tetraethylammonium)-containing solutions and under Ca-Na bi-ionic conditions. The duration of the action potential plateau, produced by TEA, was markedly decreased with acidic intracellular solutions and increased with alkaline intracellular solutions. The normalized duration, d, was calculated by dividing the plateau duration by that of the standard intracellular pH of 7.3, and plotted against the intracellular pH. The curve could be expressed by the formula log [d/(D-d)] = n(pH-pK') where D is the saturated value of d, n a constant and pK' the value of pH at which d becomes D/2. The values selected for D and n were 2.6 and 1.4, respectively. The pK' was found to be 7.5. Lowering of the extracellular pH to 6.2 only slightly changed the plateau duration. Voltage clamp analysis revealed that acidic intracellular solutions decreased the size of the inward current and the slope conductance which were measured at the late period of the depolarizing clamping pulse. Alkaline intracellular solutions increased the size of the inward current and the slope conductance measured at this late period. Intracellular perfusion with a low pH solution also shortened the duration of the Ca-Na bi-ionic action potential. It is argued that the remarkable generality of the pH effect on the plateau duration implies the existence of a common mechanism of formation of the plateau under widely differing experimental conditions.

Protein synthesis in neural cells in culture: role of cell density and neurohumors.

Protein sythesis was studied in C-6 glial cells and neuroblastoma (NB) cells as a function of cell density and after differentiation with dibutyryl cyclic AMP and treatment with either norepinephrine (NE), dopamine or L-dopa. In both C-6 glial cells and NB cells, unincorporated 3H-leucine decreased, whereas incorporation of 3H-leucine into protein increased with increasing cell density, particularly at high cell densities. Exposure of C-6 glial cells of NE at various dose for 60 minutes stimulated the efficiency of 3H-leucine corporation into protein. This effect was not seen with L-dopa or dopamine. In contrast to the glial cells, in neuroblastoma cells, all three neurohumors caused a decrease in the incorporation of 3H-leucine into protein. The increase in protein synthesis by NE was also seen in DBcAMP-differentiated glial cells. These findings suggest that cellular activity as reflected by protein synthesis is cell density dependent. In addition, neurohumor substances may play a regulatory role in the cellular activity of glial cells.

[Construction of partial denaturation charts for DNA with random base distribution].

By measuring DNA temperature transition profiles the evidence was obtained that DNA of phages Tg9 and OB infecting Bacillus thuringiensis and Brevibacterium flavum respectively have a random base distribution. After alkaline treatment in the presence of 10 per cent formaldehyde at room temperature partially denatured molecules of phage Tg9 and OB DNA's with different degrees of denaturation were obtained. Using electron microscopy the maps of distribution of melted regions along these DNA's were obtained. The location of the main peaks on histograms did not vary with the increase of the degree denaturation and changes of pH values. The map of Tg9 DNA is characterized by four main peaks located at the positions 0.031, 0.075, 0.145 and 0.885 but the map of OB DBA is characterized by seven main peaks located at positions: 0.09, 0.20, 0.34 0.42,0.49, 0.80 and 0.88 from the left end (in relative units). The increase of denaturation up to a certain value did not change the half-width of the main peaks at the maps of these DNA's. It may be due to the presence in the DNA's some "heat stolle" sequences limiting early melting DNA regions.

[Clinical trials in psychiatry. Recent evolution of the methodology in France (author's transl)].

The comparison, with the help of a check list of criteria, of psychotropic clinical trials published in France, with an interval of five years, covered 80 trials in 1970 and 75 trials in 1975. The differences found between these two years all indicate a stricter methodological approach in 1975. Certain gaps persist however, such as the extreme rarity of codified pretherapeutic evaluation, preliminary wash out periods, references to an official nosography and a sufficient homogeneity of treatment groups. On the other hand, in the majority of trials, the exclusion criteria are not given and a global clinical assessment is the only criterium of change used. Finally the conclusions proposed coincide with the data in only two thirds of the publications. The comparative revue of 120 controlled psychotropic trials so far published in French show above all the extreme variability of the type of control utilized. Only 58 trials out of 120 have been randomized and a minority of these were carried out according to a strict methodology, with quantitative evaluation of the results and an appropriate statistical analysis. The most frequent methodological insufficiences are the obvious heterogeneity of the treatment groups, the insufficient use of standardized evaluation instruments and the inadapted ness of certain experimental protocols to the goals being pursued. The points most frequently omitted in the presentation of the results are the drop-outs of treatment, information relating to associated and previous treatment, the dates of evaluation of the therapeutic effects as well as the approach used to established side effects. The recent application of the 1975 French Law has deeply changed the habits of French experimentators and it is necessary to correctly interpret the term of "control". The control of non-specific variables intervening in the therapeutical results is the only means of identifying the pharmacological effects of drugs. The best control of these variables is the randomized comparative trial of simultaneous treatment groups using double-blind prescriptions. In order to further comparisons of trials from different sources, it is absolutely necesary that evaluation studies in French be carried out and transmitted according to internationally recognized recommendations based on general rules of protocols and on standardized evaluation instruments.

H+/site, charge/site, and ATP/site ratios in mitochondrial electron transport.

H(+)/site, charge/site, and ATP/site ratios have been determined at coupling sites I, II, and III. Three e(-) donors have been used for coupling site III: ferrocyanide, ascorbate + tetramethyl-p-phenylenediamine (TMPD), and succinate + TMPD. The H(+)/site ratios are 4.0 with ferrocyanide and 6.0 with succinate + TMPD (at pH <7.0); the charge/site ratios are 6.0 with ferrocyanide and with succinate + TMPD (at pH <7.0) and 4.0 with ascorbate + TMPD; the ATP/site ratio is 1.34 with ascorbate + ferrocyanide. These ratios have been obtained in the presence of amounts of antimycin A that provide full inhibition of site II. For coupling sites I and II, ferricyanide has been used as e(-) acceptor and succinate or NAD-linked substrates as e(-) donors. The H(+)/site ratios are 4.0 at sites I and II; the charge/site ratios are 4.0 at site I and 2.0 at site II; the ATP/site ratios are 1.0 at site I and 0.5 at site II. Two major factors affect the stoichiometries: (i) dimension of [unk](H) and (ii) supply of H(+) from the matrix. There is a correlation between collapse of [unk](H) and increase of H(+)/site and charge/site ratios. This indicates that approximation of the phenomenologic stoichiometry of the H(+) pump is obtained when flow ratios are measured at level flow. That charge/site and ATP/site ratios increase when ferrocyanide is e(-) donor and decrease when ferricyanide is e(-) acceptor is attributed to the localization of the redox couple. This leads to separation of 1 charge/e(-) when ferrocyanide is e(-) donor and to consumption of 1 charge/e(-) when ferricyanide is e(-) acceptor. To account for an extrusion of H(+) in excess of that predicted by the loop model, it is proposed that each coupling site contains a channel acting as a H(+) pump.

From extracellular to intracellular: the establishment of a symbiosis.

The colonization of host cells by modern symbionts is surveyed. The morphological distinction between extracellular and intracellular symbionts is not sharp, and the various kinds of association can be arranged in a graded series of increasing morphological integration of the symbiont into the host cell. Apart from some aggressive parasitic infections, the great majority of symbionts are enclosed by a host membrane in a vacuole. Those not enclosed in a host vacuole usually cannot be cultivated outside the cell. It is therefore surmised that encirclement by a vacuolar membrane would only disappear, if at all, in the later stages of the evolution of intracellular symbiosis. Recognition mechanisms between host and symbiont occur, but have been little studied. In some associations, recognition at surface contact occurs, and there is evidence for the involvement of lectins in certain cases. In other associations, recognition may occur wholly or in part after the entry of symbiont into host cells. After entry, special mechanisms for the biotrophic transfer of nutrients from symbiont to host develop. Both the symbiont population size and its rate of increase are strictly regulated by the host cell; symbiont metabolism may be controlled likewise. Rates of evolution of intracellular symbionts are probably very rapid, owing in part to responses of the host cell to its symbiont.

The Rhizobium--legume symbiosis.

The rhizobia are soil microorganisms that can interact with leguminous plants to form root nodules within which conditions are favourable for bacterial nitrogen fixation. Legumes allow the development of very large rhizobial populations in the vicinity of their roots. Infections and nodule formation require the specific recognition of host and Rhizobium, probably mediated by plant lectins. Penetration of the host by a compatible Rhizobium species usually provokes host root cell division to form the nodule, and a process of differentiation by both partners then ensues. In most cases the rhizobia alter morphologically to form bacteroids, which are usually larger than the free-living bacteria and have altered cell walls. At all stages during infection, the bacteria are bounded by host cell plasmalemma. The enzyme nitrogenase is synthesized by the bacteria and, if leghaemoglobin is present, nitrogen fixation will occur. Leghaemoglobin is a product of the symbiotic interaction, since the globin is produced by the plant while the haem is synthesized by the bacteria. In the intracellular habitat the bacteria are dependent upon the plant for supplies of energy and the bacteroids, in particular, appear to differentiate so that they are no longer able to utilize the nitrogen that they fix. Regulation of the supply of carbohydrate and the use of the fixed nitrogen thus appear to be largely governed by the host.

Symbionticism revisited: a discussion of the evolutionary impact of intracellular symbioses.

Wallin (1927) first published the notion that the fusion of bacteria with host cells was the principal source of genetic novelty for speciation. He suggested that mitochondria are transitional elements in this process. While the significance that he attributed to symbiosis now seem excessive, he was one of the first authors to be aware of the evolutionary potential of symbiotic events and his view of mitochondria may not seem strange to many cell biologist today. The most significant evolutionary development which has been attributed to intracellular symbiosis is the origin of eukaryotic cellular organization. The current status of the 'serial endosymbiosis hypothesis' is briefly review. The case for the symbiotic origin of the chloroplast, based principally on 16 S RNA oligonucleotide cataloguing, is very strong. Mitochondrial origins are more obscure but also appear to be symbiotic due to recent 18 S cataloguing from wheat embryos. The probablility of the multiple origin of some eukaryotic organelles is also examined, the processes in question being the acquisition of distinct stocks of chloroplasts from disparate photosynthetic prokaryotes and the secondary donation of organelles from degenerate eukaryotic endosymbionts to their hosts, with specific reference to the dinoflagellates Peridinium balticum, Kryptoperidinium foliaceum and the ciliate Mesodinium rubrum. It is concluded that the evolutionary potential of intracellular symbiosis ('cytobiosis': a term introduced in this paper) is great, with the best established influence being on the origin of eukaryotic chloroplasts. Together with the potential effects of viral vectors, symbiosis serves as a supplementary speciation mechanism capable of producing directed evolutionary changes. It is likely that these processes will explain some of the apparent anomalies in evolutionary rates and direction which are not readily explicable by the conventional synthetic theory of evolution.

The in vitro binding of 2,2', 5,5'-tetrachlorobiphenyl metabolites to rat liver microsomal proteins.

The in vitro association of tritium labeled 2,2', 5,5'-tetrachlorobiphenyl (TCB) with the microsomal fraction isolated from rat livers has been investigated in a metabolizing system. It was found that the control microsomes, capable of only minimal TCB metabolism, had 92% of the total microsomal radioactivity associated with lipids, while only 61% was associated with the phenobarbital induced microsomal lipid. The radioactivity per mg microsomal protein was the same for both induced and noninduced microsomes; however, very important qualitative differences were found. Only the proteins of the induced system contained a protein (s) (MW = 45,000 g/mole) capable of specifically binding a TCB metabolite. This binding required metabolism and was TCB concentration dependent. The specificity of this association was confirmed by dialysis and this data could be analyzed by the Scatchard-Klotz equation. These calculations allowed the evaluation of the number of binding sites (38 micron moles/g of total microsomal protein) and the apparent binding constant (1.4 x 10(7) l/mole). These data are consistent with strong noncovalent interaction of a 2,2', 5,5'-TCB metabolites, but do not exclude the possibility of covalent binding of other non-dialyzable metabolites.

Gastroesophageal reflux and pulmonary aspiration: incidence, functional abnormality, and results of surgical therapy.

The incidence of aspiration, the causative esophageal pathophysiology, and the results of surgical therapy were evaluated in 100 patients with abnormal gastroesophageal reflux documented by 24-hour esophageal pH monitoring. Based on historical evidence, 48 patients were suspected to be aspirators. Eight patients had documented episodes of aspiration (drop on esophagela pH, followed by acid taste in mouth and onset of cough or wheezing spell) during the monitoring period. Nine patients were considered to be potential aspirators because they presented oral acid regurgitation without development of pulmonary symptoms. In five patients a primary respiratory disorder (PRD) induced gastroesophageal reflux. The remaining 78 patients had abnormal reflux without aspiartion or regurgitation. Aspirators had a 75% incidence of esophageal motor abnormality on manometry, and the clearance of refluxed acid was significantly delayed in the supine position. A history of heartburn and endoscopic evidence of esophagitis were present in only half of the patients who were documented aspirators. Potential aspirators were spared from aspiration by rapid esophageal clearance of refluxed acid unaffected by changes in body position. Patients with a PRD had higher distal esophageal segment (DES) pressure and normal esophageal motility with minimal esophagitis. Nonaspirators significantly improved their clearance while in the supine position, emphasizing the protective effect of esophageal peristalsis against aspiration. An antireflux procedure in five aspirators raised the DES pressure significantly and returned the reflux status to normal by 24-hour pH-monitoring standards. The incidence of aspiration appears to be less than that suspected by history and is due to a motor disorder that interferes with the ability of the esophagus to clear reflex acid. Abnormal pulmonary symptoms can induce or result from gastroesophageal reflux and, when the latter occurs, an antireflex procedure stops both reflux and aspiration.

Deranged tyrosine metabolism in cirrhosis.

In normal individuals, the main route for tyrosine degradation is the hepatic pathway tyrosine→4-hydroxyphenylpyruvic acid→homogentisic acid→CO(2). Quantitatively minor pathways, in large part extrahepatic, are: tyrosine→tyramine→octopamine and tyrosine→dopa→catecholamines.In cirrhosis, the main hepatic pathway is blocked to varying degrees at the first three stages. This appears to be due to lack of activity of the enzymes tyrosine transaminase, PHPA oxidase, and HGA oxidase, the first step being rate limiting. Hypertyrosinemia and tyrosine intolerance result.With the main hepatic pathway partially blocked, an abnormally large amount of tyrosine passes into the normally minor extrahepatic pathway leading to the false neurotransmitters tyramine and octopamine. Overproduction of these amines ensues and they accumulate in the body fluid.The false neurotransmitters can displace catecholamines from their storage sites in the peripheral and central nervous system, and thereby disrupt adrenergic processes in arterioles, kidneys, and brain. Their accumulation in cirrhotic patients may play a role in the pathogenesis of hepatic encephalopathy, hepatorenal syndrome, and hyperdynamic circulation.

HL-A antigens in Takayasu's disease.

Takayasu's disease is characterized by a "pulseless" condition which most often occurs in young females from Asian or South American areas. The cause of this disease remains obscure. Recently, we encountered monozygotic, Japanese, identical twin sisters, both of whom were diagnosed as having Takayasu's disease. The parents, two sisters, and one brother are healthy. HL-A typing analyses revealed that one haplotype found in the father had passed only to these twins. Such observations led us to search HL-A typing in Takayasu's disease to determine the possible participation of genetic factors in the pathogenesis of this morbid condition. Ten families, including that of our own patient, have been reported in the literature in Japan, as family cases of Takayasu's disease. HL-A typings in A and B locus analyzed in all family members of six families in attempts to find a common haplotype composed of A9, A10, B5, or BW40 in patients with Takayasu's disease, were confirmed statistically (chi 2 = 7.8, 0.01 less than p less than 0.05). In a population study, HL-A typing analyses of 65 patients with Takayasu's disease also revealed a high frequency of HL-A A10 and HL-A B5 with the level of 15.3 and 17.0 in the chi 2-test (p less than 10(-4)), as compared with the frequency in 128 healthy Japanese. These data strongly suggest that a genetic-related factor has to be given serious consideration.

Phenothiazine analgesia--fact or fantasy?

Double-blind clinical trials involving the use of phenothiazines as analgesics or potentiators of analgesics (aspirin, meperidine, morphine sulfate) and adverse effects of phenothiazines are reviewed and evaluated. Promethazine, promazine and propiomazine were not found to possess analgesic or potentiating properties. One chlorpromazine study contained important design and reporting deficiencies which precluded a recommendation for use of chlorpromazine in the treatment of pain. Methotrimeprazine was determined by numerous authors to have analgesic properties; however, most of the studies also were deficient in design or data presented, or both. Adverse reactions to phenothiazines, including hypotension, sedation, drowsiness, extrapyramidal symptoms, tardive dyskinesia, cardiac toxicity and agranulocytosis, are often more common and severe than those attributed to narcotic analgesics. Because of the lack of data supportive of analgesic activity and the adverse reactions associated with phenothiazines, use of these agents in the management of pain should be discouraged. The prophylactic use of phenothiazine for narcotic analgesic-induced emesis also is, in most cases, a questionable practice.

Beta-agonists and secretory cell number and intracellular glycoproteins in airway epithelium. The effect of isoproterenol and salbutamol.

This study describes the effect of systemic administration of the beta-adrenergic agonists isoproterenol and salbutamol on the secretory cell populations in seven regions of rat airway epithelium (three extrapulmonary and four intrapulmonary) and on the size of salivary glands and heart. Isoproterenol (a nonselective beta-adrenergic agonist) significantly increases secretory cell number in all airway regions except the midtrachea; salbutamol (a selective beta 2 agonist) increases secretory cell number only in proximal and peripheral regions. The absolute number of secretory cells is greatest in the most peripheral region after isoproterenol administration and in the most proximal region after salbutamol, although both drugs produce the greatest relative increase at the periphery. In proximal and, particularly, peripheral regions, the increase by isoproterenol (less than 3- and 14-fold, respectively) is greater than by salbutamol (less than 2- and less than 3-fold, respectively). In all airway regions, both drugs modify intracellular glycoprotein in the secretory cell population; within a given region, modification is much the same. In the most proximal region, the population of cells synthesizing only granules of neutral glycoprotein significantly increases while in other regions increase is in cells synthesizing only granules of acid. A significant shift in glycoprotein synthesis occurs whether or not the secretory cell population is increased, which suggests that existing as well as newly appearing cells modify their product. Isoproterenol significantly increases the size of the parotid and submaxillary glands; salbutamol increases the size of the parotid only. Isoproterenol significantly increases the weight of both ventricles of the heart; salbutamol has no such effect.

[Aminoglycoside-3'-phosphotransferase from Actinomyces fradiae. Its isolation, purification and properties].

Aminoglycoside phosphotransferase was isolated from the mycelium of Act. fradiae, the neomycin-producing organism, with paromomycin, neomycin and to a less extent ribostamycin being substrates of aminoglycoside-phosphotransferase. It was purified to homogenous state. The maximum activity of the enzyme preparations was observed at pH 7.7--7.8;KM for neomycin and paromomycin was about 20 micron and KM for ATP was 150 micron. Mg2+ ions were necessary for the enzyme activity. None of the divalent cations tested could replace the magnesium ions in the reaction of phosphorylation catalyzed by the enzyme. High sensitivity to the ionic strength of the buffer was characteristic of the enzyme. It lost about 80 per cent of the initial activity at a concentration of KC1 equal to 1.0 M. The molecular mass of the enzyme from the mycelium of Act. fradiae was determined by the method of gel-filtration through sefadex G-100. It was about 22,000. High stability was characteristic of the enzyme. The fingings indicate that aminoglycoside phosphotransferase from Act. fradiae differs from the described aminoglycoside-3'-phosphotransferases isolated from antibiotic resistant bacteria.

Development of a quantitative method for the detection of enteroviruses in soil.

A method is described for efficiently concentrating enteroviruses from soil. Viruses were eluted from soil by mechanical agitation in high pH glycine buffer containing ethylenediaminetetraacetic acid. The eluted viruses were concentrated on a floc that formed de novo upon adjustment of the soil eluate to 0.06 M aluminum chloride and pH 3.5. Viruses not pelleted with the floc were concentrated by adsorption to and elution from membrane filters. This method yielded an average efficiency of 66% recovery from loamy sand soil for four enteroviruses. Virus recovery from soil was consistently high, with samples ranging in size from 25 to 500 g. The method was used successfully to isolate naturally occurring viruses from soil beneath a wastewater land treatment site. Recovery of enteroviruses by this method form different types of soil was dependent on percentage of clay, surface area, and cation exchange capacity. Recovery was not dependent on soil saturation pH or on percentage of organic matter. This method should prove useful for studying enterovirus migration and survival during the land application of domestic sewage.

Graft patency in coronary bypass surgery.

A total of 514 vein bypass grafts and 49 internal mammary (IMA) grafts in 328 patients were studied after operation. Forty-two vein bypass grafts were performed without the use of a pump oxygenator, with a patency rate of 52%. When a pump oxygenator was used, the patency rate for vein bypass grafts was 78%. Patency rates for IMA grafts were 70% and 86%, respectively. In a small group of patients, endarterectomy with vein bypass grafts resulted in a patency rate of 59% in the right coronary artery, 88% in the left anterior descending coronary artery, and 74% in the circumflex artery. Except for the right coronary artery, these results compare favorably with those from vein bypass graft patency without endarterectomy. On the basis of these findings, insertion of bypass grafts into the coronary arteries without the use of a pump oxygenator cannot be recommended, unless the technique employed can be shown to produce graft patency rates comparable to those resulting from grafts done with the use of a pump oxygenator. Endarterectomy to the left anterior descending and circumflex arteries would not appear to affect vein bypass graft patency.

NIMH clinical research branch collaborative program on the psychobiology of depression.

This is a report on the history and implications of the collaborative effort that evolved from the 1969 National Institute of Mental Health conference on the psychobiology of depression. The major issues identified at that time were the need to (1) assess relative validities of current systems of nosology and (2) retest critical biological hypotheses concerning the etiology and nature of the depressive disorders. Research was required that would be multidisciplinary and involve clinical settings treating diverse types of depression. The objectives and the nature of the biological and clinical collaborative programs that were designed to address these problems are described. These unique programs, initiated in the early 1970s, currently span research on nosology, genetics, neurochemistry, neuroendocrinology, and psychosocial factors. Although these studies are still in the early stages, they have resulted in significant methodologic developments in diagnosis, descriptive psychopathology, and biological measurements.

Characterization of prolactin binding by membrane preparations from rat liver.

Binding sites for prolactin were identified in a plasma-membrane-enriched fraction isolated from livers of mature female rats. 125I-labelled sheep prolactin prepared by the lactoperoxidase procedure retained the same molecular integrity and binding affinity as the native hormone at physiological pH. The receptors bound prolactin from different species, whereas non-lactogenic hormones were not bound. The binding of 125I-labelled sheep prolactin was activated equally by bivalent and univalent cations, bivalent cations exerting their maximal effect at much lower concentrations. The association of 125I-labelled sheep prolactin with the receptor was a time- and temperature-dependent process. Partial dissociation was detected. The binding of 125I-labelled sheep prolactin was strongly influenced by pH, with an optimum observed at pH 6.5. Receptor activity was destroyed by Pronase and phospholipase C, whereas neuraminidase increased binding. Treatment of the membranes by ribonuclease and deoxyribonuclease did not affect the binding. Binding of 125I-labelled sheep prolactin was inhibited by p-chloromercuribenzoic acid, dithiothreitol and by brief exposure to high temperatures. Scatchard analysis of the binding of 125I-labelled sheep prolactin to receptors indicated that prolactin has a high affinity for its receptor. Binding of prolactin to liver membranes showed some properties different from those observed with mammary cells. Binding by these tissues differed in pH optimum, in effects of ions, and in response to neuraminidase.

Potassium-stimulated ATPase activity and hydrogen transport in gastric microsomal vesicles.

The Mg2+-dependent, K+-stimulated ATPase of microsomes from pig gastric mucosa has been studied in relation to observed active H+ transport into vesicular space. Uptake of fluorescent dyes (acridine orange and 9-aminoacridine) was used to monitor the generated pH gradient. Freeze-fracture electron microscopy showed that the vesicular gastric microsomes have an asymmetric distribution of intramembraneous particles (P-face was particulate; E-face was relatively smooth. Valinomycin stimulated both dye uptake and K+-ATPase (valinomycin-stimulated K+-ATPase); stimulation by valinomycin was due to increased K+ entry to some intravesicular activating site, which in turn depends upon the accompanying anion. Using the valinomycin-stimulated K+-ATPase and H+ accumulation as an index, the sequence for anion permeation was NO-3 greater than Br- greater than Cl- greater than I- greater than acetate approximately isethionate. When permeability to both K+ and H+ was increased (e.g using valinomycin plus a protonophore or nigericin), stimulation of K+-ATPase was much less dependent on the anion and the observed dissipation of the vesicular pH gradient was consistent with an 'uncoupling' of ATP hydrolysis from H+ accumulation. Thiocyanate interacts with valinomycin inhibiting the typical action of the K+ ionophore. But stimulation of ATPase activity was seen by adding 10 mM SCN- to membranes preincubated with valinomycin. From the relative activation of the valinomycin-stimulated K+-ATPase, it appears that SCN- is a very permeant anion which can be placed before NO-3 in the sequence of permeation. Valinomycin-stimulated ATPase and H+ uptake showed similar dependent correlations, including: dependence on [ATP] and [K+], pH optima, temperature activation, and selective inhibition by SH- or NH2-group reagents. These results are consistent with a pump-leak model for the gastric microsomal K+-ATPase which was simulated using Nernst-Planck conditions for passive pathways and simple kinetics for the pump. The pump is a K+/H+ exchange pump requiring K+ at an internal site. Rate of K+ entry would depend on permeability to K+ as well as the counterion, either (1) the anion to accompany K+ or (2) the H+ efflux path as an exchange ion. The former leads to net accumulation of H+ and anion, while the latter results in non-productive stimulation of ATP hydrolysis.

Enzymatic reduction and methylation of folate following pH-dependent, carrier-mediated transport in rat jejunum.

Intestinal transport of [3H] folate was studied using everted sacs of rat jejunum. The proximal small intestine transports folate against a concentration gradient by a system which is saturable, pH-dependent, energy-dependent, sodium-dependent, sensitive to temperature, and appears to be a common transport system for folate compounds. Chromatographic analysis of folate compounds in the serosal compartment after a 60 min incubation with folate in the mucosal medium in sodium phosohate buffer indicated that metabolism of folate to 5-methyltetrahydrofolate was extensive at pH 6.0 and negligible at pH 7.5. The percent conversion of folate to 5-methyltetrahydrofolate at pH 6.0 was reduced by increasing the concentration of folate in the mucosal medium, thus indicating saturation of the reduction and methylation process. These findings indicate that folate transport in rat jejunum occurs by an energy-dependent, carried-mediated system and that both folate transport and intestinal conversion of folate to 5-methyltetrahydrofolate are pH-dependent.

[Stability and specificity of extracellular protein inhibitor for trypsin from Actinomyces janthinus 118].

Some properties of protein inhibitor for trypsin (TI) from Act. janthinus 118 were studied. It was shown that TI has an antitrypsin activity within a wide pH range with a maximum at about 9,5. At 4 degrees and 20 degrees C TI is stable for 24 hours within the pH range of 6,0--11,0. At 100 degrees C TI is more stable in the slightly acid region of pH than at neutral or alkaline conditions. Trypsin and chymotrypsin inactivate the inhibitor for 8 hours. TI inhibits trypsin, fibrinolysin, subtilisin, pronase and terrilytin, but have no effect on chymotrypsin, thrombin, papain and pepsin. The dissociation constants for the trypsin-inhibitor complex were found to be 1,7.10-8 M, 4,1.10-9 M and 2,4.10-10 M, with casein, p-nitroanilide benzoylarginine and tosylarginine methyl ester used as substrates, respectively. The corresponding dissociation rate constants for the subtilisin-inhibitor complex were equal to 1.10-9 M and 4.10-10 M with casein and carbobenzoxy-L-alanyl-L-alanyl-L-leucin p-nitroanilide used as substrates, respectively.

The effects of nitrilotriacetic acid on solubilities of zinc, copper, manganese, and iron in the stomach of sheep.

Four sheep, each prepared with a rumen fistula and reetrant cannula in the proximal duodenum, were used to study the effects of ruminal administration of nitrilotriacetic acid on solubilities of zinc, copper, manganese, and iron in rumen and duodenal digesta. The sheep received a pelleted diet and were dosed with 0, 300, 600 and 1200 microgram of nitrilotriacetic acid per gram of diet via the rumen fistula. Higher concentration of soluble zinc, manganese, and iron but not copper, were found in the rumen of the sheep when they were dosed with nitrilotriacetic acid. The concentrations increased with increasing dose of the acid. However, only the solubilty of iron was increased in the duodenal digesta. Concentrations of soluble zinc and manganese in the rumen increased, whereas copper decreased, during the first 2 h after feeding. The pattern was reversed thereafter. Changes in the concentrations of soluble during 6 h afther feeding were comparatively small. It is concluded that the solubilty of iron in the stomach of sheep is increased by ruminal administration of nitrilotriacetic acid.

Subcellular localization of gamma-glutamyltransferase activity in guinea pig liver. Effect of phenobarbital on the enzyme activity levels.

The localization of gamma-glutamyltransferase activity in guinea pig liver was studied after subcellular fractionation. The enzyme activity was essentially connected with plasma membranes whereas only low activity was found in the endoplasmic reticulum. A similar activity distribution was demonstrated for 5'-nucleotidase. Highest specific activity of gamma-glutamyltransferase was found in plasma membranes enriched in bile canaliculi. In this fraction the specific activity was 35 times greater than the specific activity of the total homogenate, a value similar to the relative specific activity of (Na+,K+)-ATPase. More than 90% of the total gamma-glutamyltransferase activity in guinea pig liver was connected with parenchymal cells and the enzyme seemed to have an outside orientation. Animals treated with phenobarbital showed moderate increased in gamma-glutamyltransferase activity in serum and liver, whereas high activities were found in most bile samples. No particular liver subfraction showed substantial accumulation of gamma-glutamyltransferase activity. The present findings do not support the suggested use of serum gamma-glutamyltransferase measurements as a direct index of "microsomal enzyme induction".

Effects of azatadine maleate on subjective appraisal and psychomotor functions relevant to driving performance.

Studies were carried out in normal healthy male subjects to assess the effects on psychomotor functions and subjective ratings of performance after acute administration of azatadine maleate, a potent antihistamine with additional antiserotonin activity. In the first trial, 2 mg azatadine was compared with another new antihistamine Sch 12169 (2 mg) and placebo. In a second trial, higher doses of azatadine (4 mg and 8 mg) were compared with dexchlorpheniramine (4 mg) and placebo. Both trials were of a double-blind, randomized Latin square design and subjects were assessed using a battery of tests, after administration of each trial drug. The time and sequence of tests were standarized, with a 1-week interval between test sessions. The results showed that azatadine did not produce significant impairment of psychomotor function at either the standard 2 mg or the maximum recommended 4 mg per day dosage level. Permormance was only significantly impaired, compared with that after placebo, at the 8 mg dose level and was of a similar order to that observed after dexchlorpheniramine at the usual 4 mg dosage. It is suggested, therefore, that at the normal recommended dosage of 2 mg per day, azatadine is not likely to impair driving ability.

Physiological and morphological characteristics of progressive disruption of the canine gastric mucosal barrier.

The investigation had two major goals: to define the progression of physiological changes associated with disruption of the gastric mucosal barrier to sodium and hydrogen and to identify the morphological correlates of the physiological alterations. Fluxes of ions and water were determined before and after treatment of oxyntic mucosa with graded concentrations of butyric acid using dogs with gastric pouches. Three phases of barrier disruption were characterized: I, acceleration of normal Na+/H+ exchange; II, neutralization of H+; III, exudation of interstitial fluid. Parallel studies assessed morphological damage associated with these phases. In Phase I, cellular bulging into the lumen and dilation of intercellular spaces were evident. Some cellular erosion and extreme intercellular dilation were prominent in Phase II. Phase III was represented by necrotic changes and desquamation. It is concluded that disruption of transport mechanisms occurs sequentially and is closely correlated with morphological signs of progressive damage.

Cefuroxime: a review of its antibacterial activity, pharmacological properties and therapeutic use.

Cefuroxime is a new semisynthetic cephalosporin for parenteral administration. It is resistant to destruction by beta-lactamases produced by staphylococci and most Gram-negative aerobic bacteria and is active against many bacteria resistant to cephalothin. Cefuroxime is the most active of the cephalosporins against gonococci and Haemophilus influenzae particularly against beta-lactamase producing strains. Given by intramuscular or intravenous injection cefuroxime is effective against a wide variety of infections caused by Gram-positive or Gram-negative aerobes, but has no effect against infections caused by Pseudomonas aeruginosa or B. fragilis. Cefuroxime is of value in the treatment of respiratory infections due to Haemophilus influenzae and Streptocococcus pneumoniae and is useful against cephalosporin-resistant Klebsiella and Enterobacter infections. Cefuroxime is an alternative to spectinomycin for the treatment of beta-lactamase producing Neisseria gonorrhoeae infections. It is generally well tolerated and appears not to be nephrotoxic when given alone at usual dosages.

Kinetic studies of the interaction between MS2 phage and F pilus of Escherichia coli.

The kinetics of the binding reaction of MS2 phage to free F pili, which were highly purified from Escherichia coli, has been studied using a membrane filter assay. The rate of dissociation (kd) of the MS2-phage--F-pilus complex is very slow and follows first-order kinetics with a half-life of 4.2 h at 30 degrees C in the standard buffer. The dissociation rate is rather insensitive to temperature, but becomes more rapid at high ionic strength or at basic pH. In a 0.25 M ionic strength buffer, the half-life of the complex is about 1.0 min. The rate of association is very fast and follows second-order kinetics with the rate constant for association (ka) being 8 x 10(7) M-1 s-1 at 30 degrees C in the standard buffer. The rate of association is almost insensitive to ionic strength but slightly sensitive to pH or temperature. Monovalent cations can also promote the binding reaction as well as divalent cations but the complex formed with monovalent cation is unstable. A study of the kinetics of dissociation suggests that there are two types of interaction between MS2 phage and F pilus; one is a strong interaction formed with divalent cations and the other is a weak one formed with monovalent cations. The physical nature of the bonds involved in the former and the latter seems to be mainly electrostatic and non-electrostatic respectively. The mechanism of the binding reaction is discussed.

Pharmacokinetics of clorazepate in pregnant and non-pregnant women.

A single dose of clorazepate 20 mg was injected i.m. in 7 pregnant and 7 non-pregnant women. Blood samples were collected for one week, and urine was collected for 24 h after the dose. The concentrations of clorazepate and its metabolite nordiazepam were determined by electron capture gas liquid chromatography. There was no difference between the two groups on physical examinations. Clorazepate was rapidly absorbed and the peak concentration was reached within 2 h. Mean pharmacokinetic parameters for clorazepate were absorption half life 0.77 h in pregnant women and 0.56 h in non-pregnant women; elimination half life 1.3 h in pregnant women and 2.0 h in non-pregnant women; volume of distribution: 0.43 1 . kg-1 in the pregnant women and 0.33 1 . kg-1 in non-pregnant women. Nordiazepam reached its peak concentration within 12 h after dosing; its mean half life of elimination was 180 h in pregnant women and 60 h in non-pregnant women. Within 24 h, 1.3% of the clorazepate was recovered in urine from pregnant women and 7% in urine from the non-pregnant women.

A versatile new sustained-action neuroleptic: pipotiazine palmitate in psychiatric practice.

The long-term clinical effects of pipotiazine palmitate were tested in 206 men and women who were either not responding well to their previous neuroleptic therapy or who were negligent about pursuing protracted oral drug therapy. Of the 206 patients, 130 were suffering from some form of chronic schizophrenia; the remainder presented with depression, psychoneurotic or behavioural disorders. Pipotiazine palmitate, a long-acting depot neuroleptic, was given as a monthly intramuscular injection for up to 23 months. The average starting dose was 50 mg/injection and the average final dose was 65 mg/injection. These doses were somewhat lower than those usually reported in the literature, however all but a few patients received oral neuroleptics or antidepressants concomitantly. Psychiatric testing using the Brief Psychiatric Rating Scale revealed that significant improvement was achieved over time in all diagnostic groups represented. Individual as well as cumulative scores improved steadily for 6 momths at which time symptomatology was minimal in most patients. Pipotiazine palmitate was well tolerated, and only seven (3.4%) of the 206 patients had to interrupt therapy because of unwanted effects. The most frequent side-effects were extrapyramidal symptoms, particularly tremor and rigidity, yet these effects led to the discontinuation of therapy in only five patients.

[Therapy with antacids].

The usefulness of antacids in peptic ulcer disease is based on their ability to neutralize secreted gastric acid and to inhibit pepsin activity. The neutralizing capacity (expressed as mval of neutralized hydrochloric acid at a given pH) of different antacids depends upon their chemical composition. To achieve adequate neutralization of stimulated gastric acid secretion antacids have to be taken one and three hours after a meal in a dosage neutralizing 40-80 mval of acid. So far antacids have not been shown to be more effective in relieving ulcer symptoms than placebo. On the contrary an adequate antacid regimen will promote the healing of both gastric and duodenal ulcers in outpatients. Furthermore antacid prophylaxis will reduce the occurrence of acute gastrointestinal bleeding in critically ill patients.

Modification of antibody response to type III pneumopolysaccharide by route of injection of pertussis vaccine.

Pertussis vaccine (PV) or diphtheria toxoid-PV-tetanus toxoid (DPT) altered the antibody response of BALB/c female mice to type III pneumococcal polysaccharide antigen (S3). The key factor affecting the magnitude of the response to S3 was the route of injection of PV or DPT, whereas the route of injection of S3 was not crucial. Subcutaneous injections of DPT augmented the antibody response to low, optimal, and tolerogenic (high) doses of S3 injected either subcutaneously or intraperitoneally. This enhancement was persistent and was observed both when S3 and DPT were mixed and injected subcutaneously and when S3 and DPT were injected concurrently at separate subcutaneous sites. When either PV or DPT was injected intraperitoneally, the antibody response to subcutaneously or intraperitoneally injected S3 was significantly decreased. These experiments demonstrate a dichotomy of effect dependent on the route of administration of PV. Most studies in mice utilizing PV employ the intraperitoneal injection route, and it is important to consider whether PV treatment by this route may have unique effects. Our data suggest that intraperitoneal injection of PV suppresses B cells, possibly by influencing regulatory cell function.

Characterization of sperm agglutinins in sera from infertile women.

Sperm agglutinins detectable by a tray agglutination technique in sera from women from infertile couples were characterized. Preparative zone electrophoresis of seven head-to-head-agglutinating sera revealed two patterns. In two sera sperm agglutinins were obtained in the gamma-globulin fraction, whereas in five sera the agglutinating activity was detected in the beta-globulin fraction only. By immunoaffinity chromatography, IgM and IgG sperm antibodies were demonstrated in the former two sera and also in two tail-to-tail-agglutinating sera. The beta"sperm agglutinin was not found to share physicochemical or immunological characteristics with beta-lipoprotein in the present investigation by ultracentrifugation, by precipitation of beta-lipoprotein with heparin and MnCl2, by absorption of beta-lipoprotein with colloidal silic acid, or by affinity chromatography on an anti-beta-lipoprotein column. An apparent neutralizing effect of anti-beta-lipoprotein by simple addition of antiserum to sera containing the beta-sperm agglutinin was shown to be due to a reaction with sperm components released from the spermatozoa by which the antiserum had been absorbed before use. These sperm components were found to neutralize the activity of the beta-sperm agglutinin only, whereas no effect was observed in sera containing sperm antibodies. By Sephadex G-200 fractionation the beta-sperm agglutinin was shown to have a high molecular weight (MW greater than or equal to 600,000). Ultrafiltration and dialysis experiments gave no evidence of involvement of low-molecular compounds in head-to-head agglutination.

Evaluation of histochemical observations of activity of acid hydrolases obtained with semipermeable membrane techniques. 3. The substrate specificity of isoenzymes of acid phosphatase in m.gastrocnemius of rabbits.

Three distinct isoenzymes of acid phosphatase have been separated from extracts of m.gastrocnemius of normal and of vitamin E deficient rabbits by gel filtration and polyacrylamide gel electrophoresis. These isoenzymes, termed I, II and III, have molecular weights of: 110,000--130,000, 60,000--78,000 and 12,500--14,500. Isoenzymes I and II split the substrates 4-methylumbelliferyl phosphate and naphthol AS-BI phosphate and the activity is strongly increased in the muscles of vitamin E deficient rabbits. Isoenzyme III splits only 4-methylumbelliferyl phosphate and the activity is not increased in the muscles of vitamin E deficient rabbits. The pH-optimum for isoenzymes I and II is 4.8 and for isoenzyme III 5.5. It has been shown that the histochemical semipermeable membrane technique, using substrate naphthol AS-BI phosphate, is a very reliable technique for demonstrating activity of the isoenzymes I and II in tissue sections. On the other hand, activity of isoenzyme III cannot be demonstrated with this histochemical technique. In pathologically altered muscles, the activity of the isoenzymes I and II is greatly increased whilst the activity of isoenzyme III is not significantly altered.

Solubilization and properties of a particulate hydrogenase from Methanobacterium strain G2R.

Mechanical disruption of cells of Methanobacterium strain G2R resulted in a 78-fold increase in the specific activity of the hydrogenase as measured by the benzyl viologen reduction assay. Approximately 50% of the activity in disrupted cells was associated with the particulate fraction. Between 69 and 85% of the particulate hydrogenase was released by treatment with the detergents Triton X-100, deoxycholate, and octyl-beta-d-glucopyranoside. The relative electrophoretic mobilities of the soluble hydrogenases were identical, indicating that G2R possessed a single electrophoretically distinct hydrogenase. The particulate enzyme was inactivated by oxygen and could be reactivated with dithionite or glucose plus glucose oxidase. The enzyme had a pH optimum of 8.5 and resisted heating at 52 but not 77 degrees C. A number of nonspecific dyes, flavin adenine dinucleotide, and riboflavin 5'-phosphate were effective electron acceptors; oxidized nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, and factor 420 were apparently not reduced. Hydrogenase activity was inhibited by p-hydroxymercuribenzoate, cyanide, chloroform, and chloramphenicol. The molecular weight of the solubilized enzyme was 900,000, with subunits of molecular weights 38,500, 50,700, and approximately 80,000. It is suggested that, in intact cells of G2R, the large hydrogenase complex is loosely bound to the cell wall or membrane.

Thermosensitive, extracellular neutral proteases in Bacillus subtilis: isolation, characterization, and genetics.

Two mutants (NT02 and NT17), each producing a thermosensitive neutral protease, were isolated from Bacillus subtilis NP58, a transformant which acquired the property of hyperproduction of neutral protease from Bacillus natto IAM 1212. The neutral proteases produced by these two mutants were partially purified and enzymologically characterized. The two mutant neutral proteases displayed increased thermosensitivity as well as altered pH optima compared with those of the NP58 enzyme. In addition, the hydrolytic activity of the thermosensitive neutral proteases on synthetic peptide substrates was found to be extremely different. These results strongly suggest that the site of mutation in each of the temperature-sensitive strains is located within the structural gene for neutral protease (nprE). Previous studies indicated the existence of a specific regulator gene (nprR) in addition to the structural gene for neutral protease. Phage PBS1-mediated transduction and deoxyribonucleic acid-mediated transformation studies with the parental and mutant strains suggest that the chromosomal order of these genes is recA-pyrA-nprR-nprE-fruB-metC. Moreover, the results of these genetic analyses imply that the mutations to thermosensitivity are located proximate to each other within the nprE gene.

Role of renal prostaglandins in sympathetically mediated renin relase in the rat.

Renal prostaglandins (PG) appear to mediate renin release due to stimulation of the intrarenal baroreceptor, but not that due to activation of the macula densa. However, as the role of PG in sympathetically mediated renin release remains unclear, a possible interrelationship between these factors was examined in conscious rats. Hydralazine increased the serum renin levels from 3.1+/-0.8 to 16.7+/-3.0 ng/ml per h at a dose of 1 mg/kg. Indomethacin (5 mg/kg) suppressed urinary PGE(2) and PGF(2alpha) excretion by 89 and 74%, respectively, arachidonate hypotension by 82%, and inhibited the elevated renin levels from hydralazine by 100% without altering the hypotensive effect of the drug. Another PG synthetase inhibitor, meclofenamate, was also effective in attenuating hydralazine-induced renin release, urinary PGE(2) and PGF(2alpha) excretion, and arachidonate hypotension. Isoproterenol, a nonselective beta-adrenergic agonist, increased heart rate, lowered blood pressure, and also stimulated the release of renin when administered intraperitoneally. However, intrarenal infusion of the drug only resulted in increased renin release. Indomethacin inhibited isoproterenol-induced renin release by 66 and 67%, respectively, without altering the hemodynamic effects associated with the intraperitoneal administration of the drug. The selective beta(1) agonist, H133/22, increased the release of renin and heart rate in a dose-related manner without altering blood pressure. H133/22-induced renin release was inhibited by 80% by indomethacin pretreatment. Finally, intrarenal infusions of dibutyryl cyclic AMP (3 mg/kg per min) increased the serum activity from 4.1+/-0.2 to 20.4+/-3.9 ng/ml per h without altering mean arterial pressure. Indomethacin inhibited this renin response to dibutyryl cyclic AMP by 96%. Thus, renal PG appear to be important mediators of sympathetically stimulated renin release acting as a site distal to the beta-adrenergic receptor.

Divergent results in radial immunodiffusion. III. Isolation and specificity test of class-specific antibodies.

A method is described for the isolation of antibodies specific for immunoglobulin class determinants from antisera containing antibodies both for the immunoglobulin class and for its subclasses, using affinity chromatography. The model system used is bovine IgG with its subclasses IgG1 and IgG2, together with antisera raised in goats and sheep. The specificity of the isolated class-specific antibodies was demonstrated by double diffusion (DI) and radial immunodiffusion (RID). The levels of total IgG in normal bovine sera measured with the class-specific antibodies were the same as the sum of the IgG1 and IgG2 levels obtained with subclass-specific antisera. However, sera from cattle infected with Fasciola hepatica contain IgG1 fragments without class-specific determinants. In these sera, contrary to expectation, the class-specific antibodies gave higher total IgG levels than the subclass-specific antisera. The yield of class-specific antibodies varied with different batches of antisera. The minimum amount of each class-specific antibody preparation needed for a RID plate also varied. Class-specific antibodies isolated from antisera raised against IgG1 gave more intense precipitation reactions in RID against IgG1 than IgG2 and vice versa. Possible reasons for these differences are discussed.

Uptake of 5-hydroxytryptamine in blood platelets and its inhibition by drugs: role of plasma membrane and granular storage.

The initial uptake of 3H-5-hydroxytryptamine (3H-5-HT) showed linearity for short time intervals in normal and reserpinized blood platelets of guinea-pigs, but was lower in reserpinized platelets. Teh Km values for the 3H-5-HT uptake were virtually identical in normal and reserpinized platelets, whereas Vmax was lower in the latter. Imipramine and chlorpromazine caused the same percentage inhibition of 3H-5-HT uptake in normal and reserpinized platelets; the reserpine-like compound Ro 4-1284 inhibited the uptake of 3H-5-HT in the normal, but not markedly in the reserpinized platelets. Haloperidol, prenylamine and Ro 4-9040 were more potent inhibitors in normal than in reserpinized platelets. It is concluded that (a) the Km of the initial uptake of 5-HT by platelets is probably determined by the mechanism at the plasma membrane, whereas Vmax may be codetermined by the intracellular storage capacity, (b) platelets are models for differentiating the site of action (plasma membrane or storage organelles) of drugs interfering with 5-HT uptake, and (c) neuroleptics- and reserpine-like compounds may either act selectively on the plasma membrane or on the intracellular storage organelles, or affect both of these subcellular sites.

GI absorption of beta-lactam antibiotics. III: Kinetic evidence for in situ absorption of ionized species of monobasic penicillins and cefazolin from the rat small intestine and structure-absorption rate relationships.

Absorption rates of monobasic beta-lactam antibiotics were measured as a function of lumen solution pH between 4 and 9 by utilizing the rat intestinal recirculating method in situ. Between pH 6.5 and 9, the absorption rate constants of ionized antibiotics were almost identical; but, at pH 4, the unionized species were highly absorbed, depending on their lipophilicity through the GI membrane lipoidal barrier. The structure-absorption rate relationship was established with the unstirred layer model.

Coupled transport of protons and anions through lipid bilayer membranes containing a long-chain secondary amine.

Transport of protons and halide ions through planar lipid bilayers made from egg lecithin and a long-chain secondary amine (n-lauryl [trialkylmethyl] amine) in n-decane was studied. Net proton fluxes were measured with a pH electrode, and halide fluxes were measured with 82Br- and 36Cl-. In membranes containing the secondary amine, a large net proton flux was produced either by a Br- gradient with symmetrical pH or by a pH gradient with symmetrical Br-, but not by a pH gradient in Br--free solutions. This H+ flux was electrically silent (nonconductive), and the H+ permeability coefficient was greater than 10(-3) cm sec-1 in 0.1 M NaBr. In Br--free solutions, H+ selectivity was observed electrically by measuring conductances and zero-current potentials generated by H+ activity gradients. The permeability coefficient for this ionic (conductive) H+ flux was about 10(-5) cm sec-1, several orders of magnitude smaller than the H+ permeability of the electroneutral pathway. Large electroneutral Br- exchange fluxes occurred under symmetrical conditions, and the permeability coefficient for Br- exchange was about 10(-3) cm sec-1 at pH 5. The one-way Br- flux was inhibited by substituting SO4= for Br- on the "trans" side of the membrane. These results support a "titratable carrier" model in which the secondary amine exists in three forms (C, CH+ and CHBr). Protons can cross the membrane either as CHBr (nonconductive) or as CH+ (conductive), whereas Br- crosses the membrane primarily as CHBr (nonconductive). In addition to these three types of transport, there is also a pH-dependent conductive flux of Br- which has a permeability coefficient of about 10(-7) cm sec-1 at pH 5. Experiments with lipid monolayers suggest that the pH dependence of this conductive flux is caused by a change in surface potential of about +100 mV between pH 9.5 and 5.0.

The involvement of the sympathetic nervous system in tetanus.

Besides the characteristic disturbances of the motor nervous system symptoms indicating an overactivity of the sympathetic nervous system can complicate the course of severe cases of tetanus. These symptoms include fluctuating tachycardia and hypertension, electrocardiographic changes, sweating, constipation with development of paralytic ileus and metabolic disorders. These symptoms are comparable to these developing in patients with phaeochromocytoma. Elevated catecholamine levels in plasma and urine have been found in several patients with tetanus who developed these symptoms. The prolonged over-activity of the sympathetic nervous system is thought to contribute to the still considerably high mortality rate. Myocardial lesions observed at necropsy are comparable to those found in patients dying of phaeochromocytoma. These lesions are suggested to be associated with sudden death from arrhythmias or cardiac failure in patients with tetanus. For the protection of the organism against the overactivity of the sympathetic nervous system a treatment using the combination of beta-adrenergic receptor blocking agents and adrenergic neuron blocking agents has been introduced. A reduction of the mortality rate was achievable by this treatment. Experimental evidence is accumulating that the tetanus toxin affects not only the motor, but also the sympathetic and sensory neurons.

[Granulomatous and allergic angiitis (Churg and Strauss). A case report (author's transl)].

The autopsy findings in a case of granulomatous and allergic angiitis, or Chung and Strauss' disease, are reported. Certain uncommon peculiarities were seen during the course of the disease, such as the presence of multiple lymph nodes presumed from a clinical standpoint to be lymphomas. Its differential diagnosis from other forms of granulomatous vasculitis, especially from those which affect the lungs preferentially or exclusively, is discussed. Special consideration is given to the limited forms of Wegener's disease, which is surprisingly similar morphologically; its more significant differences lie in the clinical and topographic areas, rather than in morphology. The lymph nodes, presumed to be lymphomas from a clinical view-point, are a truly exceptional circumstances in this disease. In the author's opinion the histologic pattern clearly indicates that the etiopathogenesis lies in a congenital or acquired immune disturbance. The recent literature is review, while the present knowledge about the broad and confusing spectrum of these conditions --arteritis, and pulmonary granulomatosis-- is discussed.

[Cooperative interaction of serum albumin with quaternized poly-4-vinyl pyridine and structure of the complexes].

Interaction of bovine serum albumin (BSA) with quaternized poly-4-vinyl pyridine (PE) in aqueous solutions at pH 7 was studied. It was shown that in a wide range of the ratios of the components (nBSA/nPE) soluble stable cooperative complexes were formed. At the same time a certain critical content of the protein exists at which the system loses its homogeneity. Complex formation is not accompanied by protein denaturation. At smaller nBSA/nPE ratios non-homogeneous distribution of protein globulas among polyelectrolite macromolecules was found; this corresponded to the "all or none" principle. Using ultracentrifugation technique viscosimetric measurements and electron microscopy it was shown that the soluble complexes exist in the form of rode-like particles consisting of protein globules stabilized by polycation chains. Such particle can be considered as a model of nucleoprotein complex. At certain crytical nBSA/nPE rations the rod-like particles aggregate with additional number of BSA-molecules and form more complicate soluble and insoluble cooperative complexes. Possible structural models of the complexes described were suggested and the thermodinamic and kinetic cryteria of their self-assembly were discussed.

Monoamine metabolites in the CSF of epileptic patients.

To assess the possible role of amine neurotransmitters in human epilepsy, we measured metabolites of serotonin (5-hydroxyindoleacetic acid [5-HIAA]), dopamine (homovanillic acid [HVA]), and norepinephrine (3-methoxy-4-hydroxyphenylethylene glycol [MHPG]) in the lumbar cerebrospinal fluid (CSF) of patients with partial complex seizures and in neurologic controls. Untreated epileptic patients had lower concentrations of 5-HIAA and HVA in the lumbar CSF than the controls, but the differences were not statistically significant. Among epileptic patients receiving effective antiepileptic drug treatment, the HVA concentration was within the control range. Mean MHPG concentrations were similar in patients and controls. From the epileptic patients whose CSF was obtained at pneumoencephalography we obtained a second sample of CSF that was originally in the basal cisterns. No significant differences between treated and untreated patients were found for any of the three metabolites. The concentrations of HVA and 5-HIAA were higher in cisternal than in lumbar CSF, but there was no such gradient for MHPG.

Exocytotic shedding and glial uptake of photoreceptor membrane by a salticid spider.

Receptors in the anterior lateral eyes of salticid spiders possess paired rhabdomeres. The tips of the rhabdomeral microvilli lie adjacent to non-pigmented glial processes. Photoreceptor membrane is lost during turnover by a hitherto undescribed process: individual microvilli lengthen at their tips, taper, and are received by corresponding, coated endocytotic pits in the glial membrane. Pits detach as coated vesicles with coherent fragments of microvilli within them, lose their coats, and accumulate in the glial processes as disorderly membranous detritus. Some microvillus membrane disintegrates before local endocytosis, and appears to get into the glial arms distant from the parent rhabdomere by invaginations which are either endocytotic clefts or a tubulo-cisternal system, but whose precise nature is not yet clear. No photoreceptor membrane is lost by pinocytosis into the receptor cytoplasm. Analogies between the behaviour of this system and the phagocytosis of shed vertebrate photoreceptor membrane are briefly discussed.

Trophoblast transferrin and transferrin receptors in the host--parasite relationship of human pregnancy.

Transferrin and specific transferrin receptors are demonstrated on the microvillous surface of syncytiotrophoblast in human immature and term placentae by immuno histological techniques with the use of light and electron microscopy. That the distribution of transferrin is limited to the materno-foetal interface supports the hypothesis that binding of maternal transferrin to trophoblast receptors is involved in the process of iron transport to the foetus. Parallel studies with baboon placentae demonstrate the presence of trophoblast receptors which bind both baboon and human transferrin, thereby putting forward an experimental model which might be used to test the biological significance of placental transferrin receptors in primates. In addition, investigation of a large number of human cell lines shows that many transformed cells, but no normal cells (such as blood lymphocytes) or cells from primary culture (such as neonatal foreskin fibroblasts), possess the ability to bind transferrin to their membranes. These findings suggest that transferrin receptors may play important biological roles in addition to that of iron transport from mother to foetus. One such role could be the limitation of iron in intervillous spaces, thus depriving iron-requiring microorganisms of iron, hence serving as a non-specific factor of resistance for placentae. Another role for foetal transferrin receptors on trophoblasts could be to bind maternal transferrin at the materno-foetal interface, thus frustrating maternal immunosurveillance. This is similar to a mechahism used by schistosomes in the host-parasite relation where host proteins are bound by the parasite to escape immunological recognition. The presence of transferrin receptors on transformed cells suggests that this mechanism might also be employed by tumour cells. Finally, in view of previous studies which show that transferrin is required by stimulated lymphocytes to pass from the G1 to the S phase of cellular replication, it is proposed that trophoblast transferrin receptors could limit the amount of transferrin in intervillous spaces and thus impede the proliferation and possible cytotoxicity of maternal activated lymphocytes at the materno-foetal interface.

Differentiated regions of human placental cell surface associated with attachment of chorionic villi, phagocytosis of maternal erythrocytes and syncytiotrophoblast repair.

Scanning electron micrographs of human placental cell surface show: (1) Differentiated zones of trophoblast which may be covered by fewer 'microvilli' than the adjacent syncytial cell surface and which extend as a narrow, usually distal protrusion of the chorionic villus. This narrow outgrowth terminates as a fractured end. Presumably since preparations were obtained from therapeutic terminations of pregnancy or Caesarian deliveries these broken ends represent the yield point in the anchoring 'villi' ruptured as a result of surgery. Similar anchoring 'villi' with fractured ends were observed in unfixed material with the use of Nomarski interference contrast microscopy. (2) It appears that, during apparent phagocytic uptake of maternal erythrocytes by syncytiotrophoblast, cell surface lining the forming vacuole still retains an irregular microvillous surface. This observation indicates the potential location of phagocytosis receptors for red blood cells in the placental cell surface. (3) Areas of human placenta which appears to have been damaged and may be undergoing repair exhibit masses of cells with conspicuous microvillar cell surfaces. The origin of these cells is discussed in relation to the usual processes of syncytiotrophoblast formation.

Bedtime flurazepam and the human circadian rhythm of spontaneous motility.

Sixteen male students received bedtime placebo and flurazepam 30 mg at home in a counter-balanced double-blind, crossover design. For 24 h after each treatment the subjects' spontaneous motor activity was recorded each 15 min with an unobtrusive actometer, worn as the subjects attended classes. The circadian activity curves were submitted to cosinor analyses. The 24 h post-flurazepam activity was a mean of 15.1% lower than post-placebo activity (P less than 0.025). On the average, both the nocturnal through and the daytime peak of motility dropped; the latter change was greater, reducing by a mean of 19.4% the amplitude of the circadian rhythm of activity (P greater than 0.01). the timing, or phase, of the rhythm was not shifted. Although the drug did not consistently modify reports of subjective feelings on the Profile of Mood States (POMS), 13 subjects correctly discriminated drug from placebo sessions (P less than 0.05). A bedtime dose of 30 mg of flurazepam appears to significantly reduce spontaneous human motility that night and during the next day. Activity recording revealed an important residual, behavioral effect of the drug which was not reflected in POMS reports of subjective feelings, suggesting that activity recording may provide a more sensitive measure for psychotropic drug effects.

Biochemical evidence of cocarcinogenesis: tumor promoting agent enhances methylnitrosourea activation of rat guanylate cyclase activity.

The two-stage or cocarcinogenic hypothesis of carcinogenesis involves an initiator (carcinogen) and a promotor (cocarcinogen) being utilized in combination to produce more tumors than either would alone. This theory was tested at the cellular level utilizing Tumor Promoting Agent, 12-0-tetradecanoly-phorbol-13-acetate, (promotor) in combination with submaximal and maximal doses of methylnitrosourea (initiator). Tumor promoting agent, which can cause some tumors itself, was found to enhance the activity of guanylate cyclase (E.C.4.6.1.2.), an enzyme that has been associated with normal and abnormal growth. Tumor promoting agent when utilized in combination with submaximal stimulatory doses of methylnitrosourea had an additive effect on guanylate cyclase activity, but the agent had no further additive effect on guanylate cyclase activation when methylnitrosourea was utilized in maximal stimulatory doses. These results indicate a carcinogen acting alone without a promoter can maximally activate guanylate cyclase and would suggest that at the cellular level a promotor is not absolutely necessary for the changes observed morphologically in canerous cells. The promotor, however, did enhance the enzyme's activity when a submaximal dose of the carcinogen was used indicating that promoting agents, at least biochemically, appear capable of potentially contributing to the development of a cancerous cell.

A non-neuroleptic treatment for schizophrenia: analysis of the two-year postdischarge risk of relapse.

The efficacy of antipsychotic drug maintenance in reducing the risk of relapse among previously hospitalized schizophrenic patients has been well documented. However, data from an ongoing study comparing two cohorts of young first admission schizophrenics--one receiving neuroleptic-oriented treatment on the wards of a community mental health center (CMHC), the other an intensive interpersonal approach in a small homelike facility in the community (Soteria House)--raise questions about the routine use of neuroleptics with this population. Our questioning of this practice is based on data analyzed from these two cohorts by means of the life table, a statistical technique appropriate for longitudinal studies. Data are presented in two ways: (1) The overall effectiveness of the two independent treatment programs (Soteria, N = 32, vs. CMHC, N = 36) is compared in terms of the probabilities of not being readmitted over the 2-year postdischarge interval. (2) Analyses that look at the influence of the original treatment setting and postdischarge antipsychotic drug status on readmission rates are presented. Program comparisons reveal Soteria patients to have a consistently higher survival rate than CMHC patients throughout 2 years postdischarge. At 12 months postdischarge, the cumulative probability of remaining well (no readmissions) significantly favors the Soteria patients (p less than .05, Mantel chi2). The overall results of the Soteria program were achieved despite the fact that all CMHC patients received neuroleptics during their original inpatient stays and about 50 percent were maintained on neuroleptics up to the point of readmission or study termination, whereas only 10 percent of Soteria subjects were treated with or maintained on neuroleptics. The survival rates by postdischarge drug status and program affiliation show the Soteria no-drug group to have the highest proportion of survivors at almost every interval throughout 24 months, the CMHC drug-maintained group to have the lowest survival rate, and the CMHC unmaintained group to be surviving at a rate generally comparable to the Soteria no-drug group.

Spontaneous platelet aggregation in cerebrovascular disease II. Further characterisation of the platelet defect.

A group of 186 patients with Transient Ischaemic Attacks (TIA) or cerebral infarction (CI) was found to demonstrate in vitro Spontaneous Platelet Aggregation (SPA) in 39% of those studied. Of the 176 normal subjects studied the incidence on in vitro SPA was found to be 5%. Further investigation of the phenomenon of SPA revealed that: 1. it is associated with ADP-hyperaggregability, i. e. the threshold concentration to induce second wave aggregation is decreased; 2. it is dependant on the increase in pH which occurs in platelet-rich plasma stirring in an aggregometer while concurrent ADP-hyperaggregability is independant of this change in pH; 3. it is associated with malondialdehyde production and the release of endogenous 5-hydroxytryptamine; and that 4. in addition Km and Vmax values for [14c]-5HT incorporation are normal; and that 5. no gross abnormalities of the platelet membrane glycoproteins were apparent although occasionally glycoprotein III was found to be increased. This study demonstrates abnormal platelet behaviour in patients with TIA and CI where the enzyme system involved in thromboxane production is sufficiently stimulated, by stirring alone, to induce aggregation of platelets and the release reaction. Acetylsalicylic acid abolishes SPA and prolongs the bleeding time with similar characteristics as has been described for normal individuals. Plasma beta-thromboglobulin levels are significantly increased in the patients studied. However, no correlation was established with the incidence of in vitro SPA.

Suppression of graft-versus-host reactions in rats bearing implants in the anterior chamber of the eye.

The graft-versus-host (GVH) response of spleen cells from rats bearing either orthotopic skin grafts or allogeneic implants in the anterior chamber of the eye was evaluated using popliteal lymph node (PLN) assay. When a viable implant remained in the anterior chamber, the spleen cells of these rats produced a popliteal lymph node enlargement in F1 hybrids which was approximately 50% of that produced by a similar number of cells from a normal animal. Conversely, the GVH response of spleen cells from orthotopically skin-grafted rats was noted to be significantly increased over the response of spleen cells from normal animals. The decrease in the GVH response of implanted rat spleen cells was a specific reaction and not because of trauma or implantation, since spleen cells from rats bearing syngeneic implants had shown no reduction in their GVH-inducing ability. The PLN weights of rats receiving mixed population of normal and implanted rat spleen cells were always less than the weights observed with an equal number of normal spleen cells. These findings permit the assumption that implant-bearing rats may be lacking or low in cells that induce GVH reactions or that there is a delayed conversion of effector cells after early immune recognition.

Survival of rabbits after prolonged cerebral ischemia.

Cerebral ischemia was produced by a combination of vascular occlusion and mild systemic hypotension in 2 groups of rabbits. Arterial blood pressure, arterial pH, arterial blood gases, blood glucose and PCV were monitored and recorded before, during and for 3 hours after reperfusion. Return of EEG activity, vasomotor control, spontaneous ventilation and corneal reflex were also recorded. At 4, 8, 12, 24 and 48 hours after reperfusion, the rabbits' neurologic status was assessed according to an arbitrary scale based on motor function. The 2 groups differed in return of reflexes and motor function. Eighty percent of the rabbits ischemic for 20 minutes and 75% of the rabbits ischemic for 30 minutes survived. The graduated response of motor function to cerebral ischemia is attributed to the ventilatory and circulatory support given the rabbits for the first 3 hours after reperfusion. The graduate response of motor function to ischemia supports the suggestion that motor function can be used as an index of neurologic damage.

[Clinical chemical parameter changes in patients with liver metastases].

Certain biochemical serum parameters: GOT, GPT, LAP, HK, AP and Regan isoenzyme, GGTP, CE, ESR, Weltman test, thymol test, serum bilirubin and urine urobilogen were determined in 39 patients with different localization of malignant processes in the abdominal cavity (stomach, large intestines, pancreas, ovaries). The patients were subdivided into two groups, depending on the presence or absence of liver metastases, confirmed at laparatomy, laparascopy or necropsy material examination. The results revealed that in patients with liver metastases AP, CE, GOT, GGTP, ESR and Weltman test are most commonly and simultaneously abnormal. In patients without liver metastases, those indices are also changed but to a lesser degree, whereas LAP and Regan isoenyzme are with elevated activity in a higher per cent of these cases, than in the patients with liver metastases, being in unison with literature data. The determination of the above biochemical parameters could direct the clinicist to the presence of liver metastases but the more reliable diagnostic methods as laparascopy and laparatomy cannot be substituted for.

Irritant actions of unphysiological pH values. A controlled procedure to test for topical irritancy.

The abdominal skin of juvenile white mice was used. Topical application was accomplished by intracutaneous injection. The solutions tested were thoroughly standardized. By using a special buffer system, a mixture of histidine glutamate and lysine glutamate, each pH value could be tested with the same buffer capacity. The pH values varied from pH 3 to pH 11, the solutions being always iso-osmolalic with plasma. Two criteria were used to judge the irritancy: (a) the edematous reaction for the first 6 h and (b) the macroscopic aspect after 24 h. A very special procedure enabled us to obtain objective numerical values for criterion (a). The results disclose that irritation becomes manifest at pH 4 and pH 10, becomes clear-cut with pH 3 and even more so with pH 11, and depends strongly on the buffer capacity employed. With two substances-amidosulfuric acid and (tri) sodium phosphate--it was exemplified how the procedure could be used to routinely screen for topical irritancy in an informative, inexpensive and decent manner.

Depression, facilitation, and mobilization of transmitter at the rat diaphragm neuromuscular junction.

The characteristics of depression, facilitation, and mobilization of transmitter were examined at the rat diaphragm neuromuscular junction. Intracellular recording techniques were used to monitor end-plate potentials (EPPs), miniature end-plate potentials (MEPPs) and the muscle resting potentials. The cut-muscle technique was used to prevent muscle action potentials. Quantal release was determined by the direct method. The binomial statistical parameters, releasable store (n) and probability of release (p), were examined under various stimulating conditions to determine the basis for depression and facilitation. The present experiments demonstrate that p remains unchanged during repetitive nerve stimulation at low or moderately high frequencies. The experiments demonstrate that depression is due to a decrease in n and facilitation is due to an elevation in n. It is suggested that the increase in n during facilitation is due to a transient recruitment of inactive releasing sites. Substantial replenishment of n by mobilization occurs within a few ms after a stimulus but a slow residual rate of mobilization is needed to replenish n to resting levels.

Experimental evaluation of myocardial preservation techniques: IV. Potassium cardioplegia.

To investigate whether potassium per se plays a significant role in cold potassium cardioplegia, isolated blood-perfused rabbit papillary muscle preparations were used to determine the recovery of myocardial contractility after normothermic anoxia. Cardioplegia was induced by infusing an isotonic electrolyte solution containing either 5 or 40 mEq/liter of potassium chloride. Anoxic periods of 30 minutes (9 experiments each) 45 minutes (10 experiments each), and 60 minutes (1 experiment each) were compared. Hearts stopped contracting in 45 seconds with infusion of a 40mEq/liter of potassium chloride solution compared to 5 minutes with a 5 mEq/liter solution. After 30 minutes of anoxia, myocardial recovery was 65.39 +/- 24.48 per cent with 5 mEq/liter of potassium chloride and 90.05 +/- 6.40 per cent with 40mEq/liter of potassium chloride. The difference was highly significant (p less than 0.01). After 45 minutes of anoxia the same trend as just described was noted, but the difference was statistically insignificant. After 60 minutes of anoxia, recovery was extremely poor regardless of the potassium concentration of the cardioplegic solution. Our conclusion was that a high potassium solution will arrest the heart rapidly and provide protection against anoxic injury of the myocardium. Its protective effect becomes less significant, however, as the anoxic time is prolonged.