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Specificity of serotoninergic inhibition in Limulus lateral eye.

The receptor specificity for synaptically mediated lateral inhibition in Limulus lateral eye retina was studied by structure-activity correlations of the action of the putative indoleaminergic neurotransmitter, serotonin (5-HT), and its isomers and structural analogs, tryptamine (TRYP), 6-hydroxytryptamine (6HT), 5,6-dihydroxytryptamine (5,6-DHT), 5-hydroxydimethyltryptamine (5-HDMT), and 5-hydroxytryptophan (5-HTP). The 5-HT blockers, lysergic acid diethylamide (LSD), bromo-LSD (BOL), and cinanserin, were also tested. The inhibitory action of the indoleaminergic agonists is highly structure-specific. An hydroxyl group in the 5 position of the indole nucleus, sterically unencumbered by hydroxyls in neighboing positions, is essential. In order of decreasing potency, 5-HT, 5-HDMT, and 5-HTP are active agonists; TRYP, 6-HT, and 5,6-DHT are inactive. Configuration and mobility of the side chains of the active agonists also affect the interaction, and these side-chain characteristics correlate with agonist potency. The receptors for inhibitory action and for transmembranal transport in reuptake are different. Both active agonists and inactive analogs appear to be taken up (Adolph and Ehinger, 1975. Cell Tissue Res. 163:1-14). LSD and BOL have bimodal actions: direct inhibition and agonist blockade. These actions may be mediated via low-specificity presynaptic uptake receptor sites rather than highly specific, postsynaptic, agonist receptor sites.

The longevity and hatchability of Philophthalmus megalurus and P. gralli miracidia in different environmental conditions.

The effects of salinity, pH, and temperature on the longevity and hatchability of miracidia of Philophthalmus megalurus and P. gralli were determined. Miracidia of both species are able to hatch and survive at saline concentrations much above physiological levels, although these processes are reduced in 2.0--2.4% saline and completely inhibited at 2.6%. The greatest hatching rates for both species were found near neutrality (pH 6--8) but some miracidia hatched at the extreme pH levels of 3 and 12. Philophthalmus megalurus miracidia exhibited longer half-lives under acid conditions (pH 2--6) than P. gralli miracidia; conversely, P. gralli miracidia showed longer half-lives in alkaline conditions (pH 8--11). Hatching and longevity were much greater below room temperature (5--20 C) than above (30--50 C) for miracidia of both species. Temperatures above 50 C proved lethal for eyefluke eggs. Except in acid pH, P. gralli miracidia showed longer half-lives than miracidia of P. megalurus. Comparison to studies on schistosomes revealed that the inhibitory effects of physiological saline and host body temperature on the hatching process of schistosome eggs does not occur in these 2 species of eyeflukes.

Inhibitory effect of 5-hydroxytryptamine on lipogenesis in rat adipose tissues.

5-Hydroxytryptamine (5-HT) inhibited the incorporation of 14C from 14C-labelled glucose, pyruvate, citrate and acetate into fatty acids but it did not inhibit the conversion of 14C from citrate and acetate into CO2, and the citrate conversion into glyceride-glycerol in epididymal and mesenteric adipose tissue from 24h-fasted rats. 5-HT stimulated the formation of lactate from glucose and pyruvate, and increased the ratio of lactate produced/pyruvate taken up. This ratio was similar to the NADH:NAD ratio. These results indicate that 5-HT inhibits fatty acid synthesis in rat white adipose tissue by mechanisms similar to those of the catecholamines.

"Fingerprinting" beta-haemolytic streptococci by their production of and sensitivity to bacteriocine-like inhibitors.

A scheme for the "fingerprinting" of streptococci according to their production of (P typing) and sensitivity to (S typing) bacteriocine-like inhibitory substances has been developed. P typing of 450 beta-haemolytic streptococci by their action on a set of nine standard indicator strains revealed that 80% of strains produced one or more detectable inhibitors, and that 17 different P types could be recognised. Production of some inhibitors seemed to be a property of strains of a particular serological group or type. Bacteriocine-like substances were produced by streptococci of serological groups, A, B, C, D, E, F and G. Nine strains were selected as standard producers for S typing. These strains differed in their spectra of inhibition, but all seemed to be active only against gram-positive bacteria. One producer, a group-F streptococcus, specifically inhibited group-A streptococci. The conditions of incubation were critical for demonstration of inhibitor production. A requirement for blood and for incubation at 32 degrees C were important factors. None of the inhibitors was induced by ultraviolet irradiation. The observed inhibitory effects were not attributable to either hydrogen peroxide or low pH, but to the production of a variety of substances having diverse physicochemical properties and production requirements. Most of the inhibitors do not seem to be produced in liquid media. The "fingerprinting" procedure is simple and inexpensive, and provides a reliable means of subdividing streptococcal strains that may find application as a supplement to the existing serological typing schemes.

Quantitative structural analysis and the secretory behaviour of the rat parotid gland after long and short term isoprenaline treatment.

Isoprenaline (IPR) was administered as daily subcutaneous injections into newborn rats for a period of 9 weeks (long-term treatment) and into 8 week-old rats for 10 days (short-term treatment). Both the parotid and the submandibular glands increased five- to six-fold in weight in the two groups due to hypoertrophy as well as to hyperplasia. The parotid glands were subjected to electron microscopic stereological analyses and to in vitro secretory studies. The results were compared with non-treated controls. Whereas mean total cell volume in the latter was 807 microns 3 it amounted to 5804 microns 3 in glands from long-term IPR-treated rats. A striking increase in size and number of cytoplasmic granules was noted after IPR treatment; there was also a marked decrease in granule electron density as compared with control cells. Granule volume density was 31.2 +/- 1.8% in controls and 58.0 +/- 1.5% in long-term IPR-treated rats. The increase in volume density, however, was accompanied by a relative decrease in amylase and cyclic AMP contents. On a percentage basis, the basal secretion of amylase from incubated IPR-treated parotid glands was markedly higher (roughly twice) than that of control glands; the absolute release of amylase into the medium, however, was only slightly increased. Basal secretion was not energy requiring, which would suggest a passive diffusion. Stimulation by beta-adrenoceptor agonists, including beta 1 and beta 2 selective agents, had no or only a small stimulatory effect in vitro on IPR-treated glands. Dibutyryl cyclic AMP was effective in controls but not in treated glands. Cholinergic stimulation caused a considerable amylase release from glands of IPR-treated rats; this release was comparable to that obtained in controls. The results suggest that superstimulation of the beta-adrenoceptor leads to a decreased sensitivity for adrenergic agonists. This may be due to membrane changes (e.g. modified receptor sites) and/or to an altered intracellular metabolism (e.g. a modified turnover of cyclic nucleotides).

[TEA-resistant outward current in the somatic membrane of perfused nerve cells].

The outward currents remaining after addition of 20-50 mM tetraethylammonium (TEA) to the extracellular solution were studied on perfused isolated neurons from Helix pomatia. A potassium-carried noninactivating outward current with potential-dependence and kinetics different from those of TEA-sensitive potassium currents was found. This TEA-resistant current includes a component depending on the presence of the inward calcium current. It could be abolished by replacing extracellular calcium by magnesium ions, by blocking the calcium channels with extracellular cadmium ions and their distruction by intracellular fluorid ions. An increase in the level of intracellular free carcium (by perfusing the cell with solutions containing Ca-EGTA buffer) potentiated the TEA-resistant component of the outward current and the removal of free calcium by EGTA decreased it. A conclusion is made that the somatic membrane contains outward current channels which can be activated only when calcium ions are bound to its inner surface.

The evolution of ageing and longevity.

Ageing is not adaptive since it reduces reproductive potential, and the argument that it evolved to provide offspring with living space is hard to sustain for most species. An alternative theory is based on the recognition that the force of natural selection declines with age, since in most environments individuals die from predation, disease or starvation. Ageing could therefore be the combined result of late-expressed deleterious genes which are beyond the reach of effective negative selection. However, this argument is circular, since the concept of 'late expression' itself implies the prior existence of adult age-related physiological processes. Organisms that do not age are essentially in a steady state in which chronologically young and old individuals are physiologically the same. In this situation the synthesis of macromolecules must be sufficiently accurate to prevent error feedback and the development of lethal 'error catastrophes'. This involves the expenditure of energy, which is required for both kinetic proof-reading and other accuracy promoting devices. It may be selectively advantageous for higher organisms to adopt an energy saving strategy of reduced accuracy in somatic cells to accelerate development and reproduction, but the consequence will be eventual deterioration and death. This 'disposable soma' theory of the evolution of ageing also proposes that a high level of accuracy is maintained in immortal germ line cells, or alternatively, that any defective germ cells are eliminated. The evolution of an increase in longevity in mammals may be due to a concomitant reduction in the rates of growth and reproduction and an increase in the accuracy of synthesis of macromolecules. The theory can be tested by measuring accuracy in germ line and somatic cells and also by comparing somatic cells from mammals with different longevities.

Distribution and environmental synchronization of the marine insect, Halobates robustus, in the Galapagos islands.

The following three aspects of the biology of the marine insect Halobates robustus were studied, during a two week observation period, at several sites in the Galapagos Islands: distribution, aggregation behaviour and rhythmicity of locomotory activity. H. robustus occurred in highest numbers on the water surface at shores fringed with mangroves. The aggregations of H. robustus varied according to their location and density. Copulating adults formed dense, floating aggregations, which tended to be close to rocks or mangroves. Late instar nymphs were less aggregated and, in lagoons (where there was some shelter from direct tidal forces), were furthest from the shore. In two types of habitat (mangrove-fringed, sandy shores and rocky shores) the aggregations of H. robustus showed a pronounced ability to maintain a floating station in relation to the surrounding environment, irrespective of tidal movements (in one case at 34 m from the nearest fixed objects). Evidence of the ability of the aggregations to maintain station on the water surface was also obtained by comparing the movements of H. robustus with those of floating polystyrene particles, which move passively with wind and tide. Laboratory observations and experiments indicated no clear periodicity in locomotory movements throughout a 24 h period. However, the frequency of encounters between individuals showed two daily peaks, post-dawn and pre-dusk, with fewer encounters during the day and only occasional encounters during the night. By shifting the light-dark cycle it was demonstrated that the daily bimodal rhythm of encounters is triggered by dawn and, since it is not maintained in constant light or dark, an 'hour-glass' mechanism is suggested. The contributions of single adults, of copulating pairs and of immature stages to the overall pattern of activity were also determined. Immature stages did not affect the overall rate of encounters significantly and the interactions between single and copulating pairs of adults appear to have been responsible for the bimodal pattern.

Covert transport dysfunction in the choroid plexus as a possible cause of schizophrenia.

Schizophrenia and certain forms of idiopathic mental retardation may result from covert immune complex disease of the basal lamina of the choroid plexus, a process already known to cause covert transport dysfunction in similar structures of, for example, skin, bowel, kidney, and endocrines. Plexial attack could lead to cerebrospinal fluid contamination and then, via an "open" ependyma, to neurotransmitter dysfunction in the periventricular limbic brain. The immune complex mechanism implies polygenic induction, direct or autoimmune, of immune sensitivity to exogenous agents and is thus compatible with the genetic picture in schizophrenia. Candidate agents include viral coat peptides and cereal grain glutens. The glutens are known to cause immune complex skin and bowell disease variants, and some empirical evidence links them to schizophrenia. Only newer immunofluorescence methods can detect the pathology, which is otherwise silent. Systemic lupus erythematosus provides a model since it is a genetic immune complex disease strongly associated with schizophreniform psychoses, exhibits choroid plexial immunofluorescence but no central nervous system pathology by ordinary methods, and may be triggered by viruses.

Epithelial cell kinetics in the descending colon of the rat. II. The effect of experimental bypass.

The influence of experimental bypass on the epithelial cell kinetics in the rat descending colon was studied. It was found that the number of cells per crypt was markedly reduced at 6 weeks after bypass. The percentage of labelled crypt cells, 1 h after 3HTdR, and the distribution of labelled cells in the crypt was normal. Also the life span of the epithelial cells was the same in control and bypassed colon. The response of crypt cell proliferation to ischaemia-induced cell loss in the bypassed descending colon was similar to the one previously described for normal descending colon. This indicates that the absence of the normal luminal contents does not result in a different response of colonic crypts to induced cell loss. Furthermore, it was found that the number of cells per crypt and the proliferative activity did not change in the transverse colon after temporary ischaemia of the bypassed descending colon. This indicates that the increase in crypt cell proliferation after ischaemia-induced cell loss is a local response.

Analysis of mammary tumors for cytochemical evidence of endogenous mammary peroxidase.

The purpose of this study was to determine whether or not endogenous mammary peroxidase can serve as a cytochemical marker to distinguish ovarian hormone-dependent from ovarian hormone independent mammary tumors. Spontaneous mammary tumors arising in virgin C3H and GR mice (hormone independent tumors) and hormone-dependent mammary tumors arising during pregnancy in GR mice were examined. None of these tumors contained mammary peroxidase. Mammary tumors induced in Sprague-Dawley rats with methylnitrousourea (MNU) and dimethylbenzanthracene (DMBA) were also examined. These tumors included hormone-dependent and hormone independent ones. Several of the DMBA-induced hormone-dependent tumors contained a few peroxidase-positive cells, but the hormone independent tumors were negative. All of the MNU-induced tumors examined were negative for mammary peroxidase. Twenty human breast tumors (malignant and non-malignant) removed from women at surgery, were also negative for mammary peroxidase. Our results indicate that endogenous mammary peroxidase cannot be used to distinguish hormone-dependent from hormone independent mammary tumors.

[Comparison of the activity and properties of 5'-nucleotidase in the homogenates and plasma membranes of the normal liver, hepatomas and of the liver in mice with inoculated hepatomas of varying degrees of malignancy].

Activity of 5'-nucleotidase was significantly lower in plasmatic membranes of highly malignant hepatoma 22 as compared with the activity found in normal liver tissue. The optimal activity of the enzyme from hepatoma 22 was found at pH 8.5 with AMP as a substrate. Decrease of pH value from 8.5 to 7.4 did not affect the enzymatic activity in homogenates and plasmatic membranes in the normal liver tissue. In all the experiments activity of 5'-nucleotidase was lower towards CMP as compared with AMP. The additive effect of the both substrates was observed only in experiments with hepatoma 22.

NADPH reduction of cytochrome P-450 at different integrational levels of the enzyme system.

The complex monooxygenatic enzyme exhibits different functional behaviour at different integrational levels, thus indicating distinct organizational states. The aerobic NADPH reduction of microsomes, solubilized and reconstituted systems follows a biphasic kinetics, the two phases are attributed to associated state (cluster) and random cytochrome P-450 reduction. States of different cytochrome P-450/reductase ratio (associates) could not be differentiated in rate. Detergents (Triton N-101, cholate) are capable of disintegrating the system, at last only monophasic slow reduction is observed. The hydroxylation activity follows the respective reduction behaviour. Sedimentation analysis proves the distinct structural states. Reconstitution of the system can be achieved by means of detergent dilution as well as by combining the constituents. The activity of the reconstituted system depends on the composition of the phospholipids as well as on its organizational state. The reassociation of the solubilized enzyme system at nearly microsomal components stoichiometry (Triton N-101 dilution) proves to be thermodynamically governed leading to self-organization of the system without matrix prerequisite. Individual step rate constants of the reduction reaction and other system parameters are accessible by means of a model treatment of the disintegrated system. Further application to mixed kinetics systems is in progress.

[Non arrhythmogenic sudden death as complication of coronary heart disease].

In a cohort of 417 patients admitted consecutively to the Coronary Care Unit for acute myocardial ischemia (unstable angina pectoris in 121, acute myocardial infarction in 296 patients) 21 cases of non arrhythmogenic sudden death occurred within 24 hours after admission. 16 of these patients suffered from acute myocardial infarction and 5 from unstable angina pectoris. Cause of death was cardiac rupture in 12 and pump failure in 4 patients with acute myocardial infarction, whereas all patients with unstable angina pectoris died from pump failure. Patients with cardiac rupture within 24 hours after admission, had significantly higher systolic and diastolic blood pressure in comparison with the other groups and with patients dying from cardiac rupture on the third day, or later. All patients dying from pump failure with unstable angina pectoris and one of the patients dying from pump failure with acute myocardial infarction had beta blocker therapy. Beta blockers were given to 68 of the patients with unstable angina pectoris. Acute pump failure occurred in this group only. The risk of pump failure with beta receptor blocking drugs is indicated by angina decubitus, marked dyspnea during anginal attacks (even in patients free of signs of cardial insufficiency outside their attacks) and a lack of responsiveness to beta blocking therapy. In these patients rapid coronary angiography and bypass surgery seems to be the prefered method of management. Beta blockers should not be given to these patients or discontinued in cases which lack responsiveness.

Development of tumours in mice chronically infected with herpes simplex virus.

Tumours developed in chronic infection lasting for 150--180 days in 39 (60%) of 65 mice infected with strain L2 of herpes simplex virus (HSV) type 1 and in 12 (20%) of 60 mice infected with strain 333 of HSV type 2. Similar results were obtained in 150 immunosuppressed mice chronically infected with HSV types 1 and 2. Pathomorphologically, the neoplasias in the first group (strain L2) were similar to adenocarcinoma and malignant lymphoma and in the second (strain 333) to lymphoma and angio- or fibrosarcoma. The respective HSV strains were isolated by cocultivation of blood leukocytes from chronically infected animals and cultures of the tumour cells with human embryo fibroblasts (HEF). HSV and Gross murine leukaemia virus antigens were detected in tumour cells by immunofluorescence and radioimmunoassay, respectively, and HSV antigen by immunofluorescence also in cultures of tumour cells and in cells of the brains, spinal cords, livers and spleens of the animals. HSV antibody was demonstrable in the blood serum from chronically infected tumour-bearing mice in a titre of 32.

Influence of mucosal and serosal pH on antidiuretic action in frog urinary bladder.

Mucosal acidification to pH 6.5 reduced by 88% the oxytocin- (2.2 x 10(-8) M) elicited increase of water permeability in frog urinary bladder. Mucosal alkalinization (pH 10.5) increased by as much as 200% the response to the same concentration of oxytocin. These effects were not observed when supramaximal concentrations of oxytocin were imployed. Similar changes were found when the serosal pH was modified. The hydrosmotic responses elicited by serosal hypertonicity or cyclic AMP plus theophylline were also affected by mucosal or serosal changes of the hydrogen in concentration, suggesting an effect at a post-cyclic AMP level. Important interactions were found between luminal pH and serosal hypertonicity when experimental conditions were employed similar to those observed in the collecting duct of mammalian nephron. Freeze-fracture studies showed that the number of intramembranous aggregates of particles induced by ADH in the luminal membrane was reduced by mucosal acidification and augmented by an increase in medium pH.

Clinical pharmacology of mephenytoin and ethotoin.

Effective prescribing of anticonvulsants requires foreknowledge of baseline pharmacokinetic data. Little such information is available about the hydantoins other than phenytoin, although one of them, mephenytoin, is widely used. Useful pharmacokinetic data should be derived from patients already exposed to anticonvulsants to reflect the induction of hepatic oxidative enzymes. Single-dose studies of mephenytoin (Mesantoin) and ethotoin (Peganone) were performed in adult inpatients on stable regimens of other anticonvulsants. Five patients received mephenytoin, 7 mg per kilogram of body weight. Serial blood sampling was performed rigorously. The time to peak concentration (Tmax) for mephenytoin was 1 hour, with a half-life (T 1/2) of 7 hours; the T 1/2 of its metabolite, 5-ethyl-5-phenylhydantion, was 96 hours. Ethotoin administration was 25 mg per kilogram in 5 patients. Ethotoin Tmax was 2 hours, with a T 1/2 of 5 hours. Saliva accurately represented the unbound fraction for all three agents. Mean salivary levels (as percentage of total levels) were 61% for mephenytoin, 73% for its metabolite, and 54% for ethotoin. The implications for therapy are that following mephenytoin administration, the metabolite 5-ethyl-5-phenylhydantoin will provide anticonvulsant effectiveness, with its long half-life producing stable blood levels on simple dose schedules. Ethotoin, in contrast, has a short half-life and would require divided daily doses to achieve a steady state. This, rather than pharmacological ineffectiveness, limits its usefulness.

pH modification of the effects of detergents on the stability of enteric viruses.

The effect of detergents on the stability of enteric viruses was found to be highly dependent on pH. This was demonstrated primarily with two ionic detergents, sodium dodecyl sulfate (an anionic detergent) and dodecyltrimethylammonium chloride (a cationic detergent). Both detergents were shown to be potent virucidal agents for reovirus, but the effects of sodium dodecyl sulfate were minimal near neutrality and much more pronounced at low than at high pH values. Dodecyltrimethylammonium chloride was extremely virucidal at high pH's but had little observable effect on reovirus stability at low pH values. In contrast, both detergents protected enteroviruses against heat at neutral and alkaline pH's. However, as was found with reovirus, sodium dodecyl sulfate was extremely virucidal at pH values below 5, even when the virus samples were incubated in ice. At different pH's the effects of detergents on the stabilities of coliphages T4, f1, and Q beta were qualitatively similar to those found with reovirus. Differences in viral stability in these experiments appeared to be due to the effects of pH on the ionic states of the viral capsid proteins.

Reinvestigation of the reaction of chymotrypsin with N-furylacryloyltryptophan derivatives at acidic pH.

The reaction of alpha-chymotrypsin with N alpha-3-(2-furyl)acryloyl-L-tryptophan methyl ester (FA-Trp-OMe) and amide has been investigated in aqueous and dimethylsulphoxide cryosolvent solutions from pH2 to 7 and over a wide temperature range. Previous reports have suggested that an intermediate preceding the acyl-enzyme can be detected spectrophotometrically in the reaction with methyl esters of FA-Trp and FA-Tyr at low pH [Yu & Viswanatha (1969) Eur. J. Biochem. 11, 347--352), and that this intermediate is an oxazolinone [Coletti-Previero et al. (1970) FEBS Lett. 11, 213--217]. We show that the previous interpretations of the time-dependent spectral changes were incorrect, and that the only detected intermediate is the acyl-enzyme. This may be isolated by gel filtration at pH less than 2.5, 1 degree C, owing to its relative stability. The pH-dependence of the rates of acylation and deacylation from pH 8.5 to 2.0 are consistent with a single ionization of pK congruent to 7.0 in both aqueous and cryosolvent solutions.

Pharmacokinetics and metabolism of 2-chloro-11-(2-dimethylaminoethoxy)-dibenzo[b,f]thiepine (zotepine) in rat, mouse, dog and man.

The absorption, distribution, metabolism and excretion of 2-chloro-11-(2-dimethylaminoethoxy)dibenzo [b,f]thiepine (zotepine), a new neuroleptic agent, were investigated in rat, mouse, dog and man. Zotepine was absorbed rapidly and almost completely from the gastrointestinal tract in all species after oral dosing. The serum level of the unchanged drug in man was comparatively higher than in animals. The serum half-lives of zotepine after i.v. dosing were approximately 3.2 h in rats, 1.5 h in mice and 3.0 h in dogs. The drug and radioactive metabolites were rapidly distributed to the tissues of rats and mice, and the brain levels of the unchanged drug were about 20 to 30 times higher than the serum levels. Only small amounts of the unchanged drug were excreted in the urine in all species; faecal excretion through the bile was the main route of elimination of the drug and metabolites. Extensive biliary excretion and enterohepatic circulation of the radioactive compound were observed in rats. Zotepine was well metabolized in the body. Besides N-demethylation and oxygenation of the N and/or S atoms, hydroxylation of the aromatic ring and consecutive conjugation were important metabolic pathways of zotepine.

Activation of human brain galactosylceramidase by phosphatidylserine.

Assays of sphingolipid hydrolases in vitro generally require bile salts or other detergents. A few 'activator proteins' have been reported that can partially replace the detergents in the assay mixture. We report here that phosphatidylserine from bovine brain is a relatively specific activator of human brain galactosylceramidase in the absence of sodium taurocholate (phosphatidylserine system). Activity similar to that obtained with the conventional assay system containing taurocholate and oleic acid (taurocholate system) could be obtained. Other lipids tested generally gave less than 10% of the taurocholate system activity, but sulfatide could activate human brain galactosylceramidase to 20--30% of the taurocholate system. The properties of the reaction in the phosphatidylserine system were examined with human brain whole homogenate, crude soluble post-concanavalin A preparations, and partially purified preparations as the enzyme source and compared with those obtained with the taurocholate system. The pH optimum shifted from 4.2 in the taurocholate system to 4.7 in the phosphatidylserine system. The phosphatidylserine system was superior in the linearity of the reaction with respect to the enzyme protein. Reasonably linear Lineweaver-Burk plots could be obtained. The Km values for the phosphatidylserine system were greater than those for the taurocholate system. The effect of phosphatidylserine was not additive to that of taurocholate. Additional phosphatidylserine to the taurocholate system was either without effect at lower concentrations or inhibitory at higher concentrations. The assays of galactosylceramidase with phosphatidylserine and without taurocholate do not necessarily provide pragmatic advantages but offer a potentially useful system with which to study the mechanism of in vivo degradation of the membrane-bound glycosphingolipid.

Modification of hemoglobin A with dimethyl adipimidate. Contribution of individual reacted subunits to changes in oxygen affinity.

The effect of dimethyl adipimidate, a bifunctional imidoester, on the oxygen affinity of hemoglobin A has been studied. Treatment of human oxyhemoglobin with 5 mM dimethyl adipimidate at pH 8.5, room temperature is accompanied by an increase in oxygen affinity in the presence and absence of 2,3-diphosphoglyceric acid. Circular dichroism measurements in the ultraviolet region indicate that dimethyl adipimidate-treated hemoglobin exhibits a reduced conformational change upon deoxygenation. In order to study the contribution of reacted individual subunits, alpha and beta subunits of dimethyl adipimidate-treated and untreated hemoglobin have been separated and reconstituted to form hybrid tetramers containing either the alpha-treated (alpha t beta c) or the beta-treated subunits (alpha c beta t). Electrophoresis on sodium dodecyl sulfate polyacrylamide gels of isolated alpha and beta globin subunits as well as hybrid tetramers from dimethyl adipimidate-treated hemoglobin reveals that 20% of the globin subunits are cross-linked. In the absence of 2,3-diphosphoglyceric acid, modification of alpha subunits increases the oxygen affinity and reduces the conformational change of the tetramer upon deoxygenation whereas modification of beta subunits has no effect. However, treatment of beta subunits decreases the effect of 2,3-diphosphoglyceric acid on the oxygen affinity of the hybrids and reduces the 2,3-diphosphoglyceric acid-induced spectral changes in oxyhemoglobin. Therefore the interaction of dimethyl adipimidate with both the alpha and beta subunits contributes to regulating the oxygen affinity of human hemoglobin.

Hydrolysis of urea by gelatin-immobilized urease: separation of kinetic and diffusion phenomena in a model immobilized-enzyme reactor system.

Experiments and appropriate mathematical models are presented in an attempt to elucidate and separate the effects of mass transfer and immobilization on the apparent kinetics of hydrolysis of urea by urease immobilized within a crosslinked gelatin film. Diffusion of urea through the gelatin matrix appears to exert the major influence on the observed kinetics. Diffusion coefficients are measured, and a model for the "effectiveness factor" is presented, accounting for this aspect of mass transfer control. A secondary, but significant, influence on apparent kinetics arises because the reaction products lead to an increased pH level which, because of diffusion resistance, remains high within the gelatin matrix. For pH levels in the 6.7 to 9.0 range the activity of urease is a strongly decreasing function of pH. An approximate model accounting for ionic equilibrium allows this pH-diffusion effect to be introduced in such a way as to lead to predictions of the apparent kinetics that are compared with experimental observations. Examination of these results indicates that the immobilization procedure leads to some loss of activity due to an interaction of the gelatin crosslinking reaction with the enzyme itself.

Use of drugs with dependence liability.

The term addictive as used by the popular press frequently confuses the more precise concepts of acute and chronic tolerance, physical dependence and withdrawal, and psychologic dependence. Serious physical dependence on psychoactive drugs is rare and is easily managed. In contrast, psychologic dependence, the most important reason for persistent drug use, is much more common and is difficult to treat. Some tactics are available - for example, confrontation and discussion with the patient about how a drug is not going to be effective over long periods. Treating the symptom of a complex problem should, of course, not be expected to solve the problem. The most important tactic is to prescribe dependence-associated drugs only when clearly indicated, when the problem is responsive to drug therapy and for the shortest period necessary, without the option for renewing the prescription. Many problems related to drug use long after the period of expected benefit is past can be avoided by far more restrictive drug prescribing. Barbiturates and nonbarbiturate sedative hypnotics (e.g., ethchlorvynol, glutethimide, meprobamate, methaqualone and methyprylon) should not be prescribed for insomnia, acute reactive anxiety, chronic anxiety neurosis or depressive illnesses, since the safer and equally effective benzodiazepines, which are less associated with dependence, are available.

Sleep-wake patterns and integrated values of luteinizing hormone, follicle stimulating hormone, prolactin, growth hormone and thyroid stimulating hormone in normal and cryptorchid pubertal patients.

The sleep-wake behaviour of LH, FSH, PRI, GH and TSH was studied in seven cryptorchid patients (four unilateral and three bilateral cryptorchids) average age 12 years and in nine normal pubertal boys of 13 years (mean age). Blood samples were collected by a continuous withdrawal pump, every hour, for 24 h. The hormonal concentration for every fraction of time was measured and related to the sleep (Sc-), wake (Wc-) and total 24 h period (Dc-). The integrated concentrations of the corresponding periods (IS, IW, ID) were calculated as well as their ratios (IS/IW; IS/ID%). For GH and TSH, the data obtained demonstrated no differences between cryptorchid and pubertal subjects. The PRL secretion in cryptorchid patients was moderately increased during the hours of nocturnal sleep. A normal pubertal sleep-wake rhythm was found for gonadotrophins in both groups of subjects. More marked levels of LH secretion were observed in cryptorchid boys compared to normal pubertals. The presence of a sleep-wake rhythm was also found in the cryptorchid patients and normal pubertal subjects in the P 1 stage. These data suggest that the CNS "programme" which controls the onset of puberty may be normal in cryptorchid patients.

Changes of serum testosterone and of LH-RH test after treatment of cryptorchidism by intranasal LH-RH.

The aim of the present investigation was to study, in a collaborative double-blind study, the treatment with intranasal LH-RH application of boys aged 1 1/2 to 12 years, who suffered from unilateral or bilateral cryptorchidism. A total of 88 subjects were randomly and blindly allocated to LH-RH and placebo therapy. Before and after 4 weeks of treatment basal testosterone serum levels were estimated and LH-RH tests were performed. Intranasal treatment with LH-RH resulted in partial or complete descents of testes in 23 out of 88 patients, whereas the position of testes remained unchanged in 17 subjects. 42 boys did not respond to placebo therapy, but complete descents were observed in 6 boys of the placebo group. Hormone analysis data of 4 different laboratories were recorded and statistically evaluated. No changes of LH, FSH and testosterone were found in the placebo groups. Only patients of the Frankfurt group responded to LH-RH test with augmented LH release after therapy (P less than 0.05). All patients treated with intranasal LH-RH showed a significant decrease of FSH release after therapy (P less than 0.05 to P less than 0.01). Basal testosterone serum levels were found to be increased after therapy only in the patients treated at Zürich (P less than 0.05). Data of the present study combine to suggest that chronic application of LH-RH may result in an overstimulation phenomenon, and that LH-RH test as well as basal testosterone levels cannot be used as prognostic index of therapy efficacy.

Crystallization and properties of cathepsin B from rat liver.

Cathepsin B from rat liver was purified to apparent homogeneity by cell-fractionation, freezing and thawing, acetone treatment, gel filtration, DEAE-Sephadex and CM-Sephadex column chromatography, and was crystallized. The purified enzyme formed spindle-shaped crystals and its homogeneity was proved by disc gel electrophoresis in the presence of sodium dodecyl sulfate and by ultracentrifugal analysis. Its s20,w value was 2.8 S and its relative molecular mass was calculated to be 22,500 (+/- 900) by sedimentation equilibrium analysis. Crystalline cathepsin B was shown to consist of four isozymes with isoelectric points between pH 4.9 and 5.3, the main isozyme having an isoelectric point of pH 5.0. The enzyme was irreversibly inactivated by exposure to weak alkali. The pH optimum was 6.0 with alpha-N-benzoyl-DL-arginine-4-nitroanilide as substrate. Amino acid analysis showed that the enzyme contained hexosamine, glucosamine and galactosamine. Cathepsin B inactivated aldolase, glucokinase, apo-ornithine aminotransferase, and apo-cystathionase, but the rates of inactivation of glucokinase, apo-ornithine aminotransferase, and apocystathionase were lower than that of aldolase. Studies by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate showed that cathepsin B degraded apo-ornithine aminotransferase to two polypeptide chains differing in relative molecular mass and electrophoretic mobility.

Tubal occlusion via laparoscopy in Latin America: an evaluation of 8186 cases.

Ease of performance, safety and effectiveness of laparoscopic sterilization techniques are evaluated for 8186 procedures (6995 interval, 680 postabortion and 511 postpartum) performed in Latin America from June 1972 through February 1978. The tubes were occluded via electrocoagulation or application of the spring-loaded clip or the tubal ring. Less than 1% of the procedures were declared technical failures (ie, those which were not completed as planned). For all the tubal occlusion techniques, interval patients had the lowest rate of surgical complications. Pregnancy rates were low for the electrocoagulation and for the tubal ring techniques; patients sterilized by the spring-loaded clip, however, had a significantly higher pregnancy rate.

[Clinical application of a supplement to the extracorporal circulation to produce a pulsatile flow (author's transl)].

Two groups of patients with atherosclerotic coronary artery disease, who underwent aortocoronary bypass operation, were perfused with nonpulsatile flow during extracorporeal circulation (ECC) using membrane oxygenators. One group (MO) was used as a control, while for the other group (PAD) a Pulsatile Assist Device in the arterial line was employed. This apparatus consists of a balloon of 80 ml placed inside a rigid housing. The balloon is compressed by pressurized air or expanded by vacuum supplied by a driving console. The apparatus produced pulse amplitudes between 30 and 50 mm of mercury. Other than a very short-lasting fall in mean arterial pressure, thus showing diminished peripheral resistance, no perceptable advantages were found. Base excess and pH-changes showed no differences, also the given amount of sodium bicarbonate in both groups was the same. On the other hand significantly higher hemolyses took place, increasing with the duration of pulsation. The application of the apparatus as an arterial counterpulsator was possible with limitation in only 5 of 15 patients. In all other patients after a short time massive blood foaming developed in the PAD and the attempts had to be stopped because of the risk of gas embolism. In our opinion this apparatus is an unnecessary supplement to the ECC and as an arterial counterpulsator it seems too dangerous.

Studies on trypsin inhibitors. Part IX. Synthesis and trypsin inhibitory activity of the duopentacontapeptide corresponding to the amino acid sequence of porcine pancreatic secretory trypsin inhibitor II (Kazal).

The synthesis of the protected duopentacontapeptide corresponding to the entire amino acid sequence I-52 of porcine pancreatic secretory trypsin inhibitor II (Kazal type) is described. The benzyloxycarbonyltetradecapeptide tert-butyloxycarbonylhydrazide (sequence 1-14) was selectively deblocked with trifluoroacetic acid and used to acylate, by the azide procedure, the peptide free base corresponding to the sequence 15-52. The isolated material was purified by ion exchange chromatography and the protecting groups were removed by successive treatments with anhydrous hydrogen fluoride, 1 M piperidine and mercuric acetate. F02M phosphate buffer, pH8. Determination of the inhibitory capacity indicated that the synthetic material is about 50% effective, at 30:1 inhibitor:trypsin molar ratio in inhibiting the tryptic hydrolysis of Nalpha-benzoyl-DL-arginine-4-nitroanilide. Full inhibition was achieved at a higher inhibitor:trypsin molar ratio. The stability constants and the standard free energy of binding of the complex between trypsin and the synthetic inhibitor have been determined.

Allantoate transport in Saccharomyces cerevisiae.

Allantoate uptake appears to be mediated by an energy-dependent active transport system with an apparent Michaelis constant of about 50 microM. Cells were able to accumulate allantoate to greater than 3,000 times the extracellular concentration. The rate of accumulation was maximum at pH 5.7 to 5.8. The energy source for allantoate uptake is probably different from that for uptake of the other allantoin pathway intermediates. The latter systems are inhibited by arsenate, fluoride, dinitrophenol, and carboxyl cyanide-m-chlorophenyl hydrazone, whereas allantoate accumulation was sensitive to only dinitrophenol and carboxyl cyanide-m-chlorophenyl hydrazone. Efflux of preloaded allanotate did not occur at detectable levels. However, exchange of intra- and extracellular allantoate was found to occur very slowly. The latter two characteristics are shared with the allantoin uptake system and may result from the sequestering of intracellular allantoate within the cell vacuole. During the course of these studies, we found that, contrary to earlier reports, the reaction catalyzed by allantoinase is freely reversible.

Preparation and characterization of an active lysozyme derivative: Kyn 62-lysozyme.

A novel method for the preparation of Kyn 62-lysozyme, in which tryptophan 62 is replaced by kynurenine, is reported. Hen egg-white lysozyme was ozonized in aqueous solution to yield one N'-formylkynurenine residue and deformylated with hydrochloric acid in frozen solution at -10 degrees C. Crude Kyn 62-lysozyme was purified by affinity and Bio Rex 70 chromatography successively. Kyn 62-lysozyme retains affinity for chitin and is essentially an active enzyme with a slightly weakened but distinct catalytic activity. After this modification, the enzyme activity was changed differently depending on the kind of substrate. At the individual optimum pH's, lytic activity was largely retained (80% active), but the catalytic efficiency for hydrolyzing glycol chitin was relatively low (30% active). Lysis of M. lysodeikticus cell suspensions was optimally catalyzed by Kyn 62-lysozyme at pH 6.2 and at 0.088 ionic strength. These values are lower by 1.3 pH unit and 0.04 ionic strength, respectively, than those of intact lysozyme. The optimum pH and ionic strength for the hydrolysis of neutral substrates were scarcely affected. These results suggest the significance of electrostatic interaction in the lysis of lysozyme. Relatively limited loss of activity induced by modification of the 62nd residue, which is thought to participate directly in the binding of the substrate at subsite C, is discussed on the basis of the similarity of side chain structure in tryptophan and kynurenine.

The contractile basis of ameboid movement. VI. The solation-contraction coupling hypothesis.

The contracted pellets derived from a high-speed supernate of Dictyostelium discoideum (S3) were investigated to determine the functional activity associated with this specific subset of the cellular motile apparatus. A partially purified model system of gelation and contraction (S6) was prepared from the contracted pellets, and the presence of calcium- and pH-sensitive gelation and contraction in this model demonstrated that a functional cytoskeletal-contratile complex remained at least partially associated with the actin and myosin during contraction. Semi-quantitative assays of gelation and solation in the myosin-free preparation S6 included measurements of turbidity, relative viscosity, and strain birefringence. The extent of gelation was optimal at pH 6.8 and a free calcium ion concentration of approximately 3.0 x 10(-8) M. Solation was favored when the free calcium ion concentration was greater than 7.6 x 10(-7) M or when the pH was increased or decreased from pH 6.8. Gelation was reversibly inhibited by increasing the free calcium ion concentration to approxomately 4.6 x 10(-6) M at pH 6.8. The solation-gelation process of this model has been interpreted to involve the reversible cross-linking of actin filaments. The addition of purified D. discoideum myosin to S6 served to reconstitute calcium- and pH-regulated contraction. The results from this study indicate that contraction is coupled functionally to the local breakdown (solation) of the gel. Therefore, solation has been identified as a structural requirement for extensive shortening during contraction. We have called this concept the solation-contraction coupling hypothesis. Fractionation of a preparation derived from the contracted pellets yielded a fraction consisting of actin and a 95,000-dalton polypeptide that exhibited calcium-sensitive gelation at 28 degrees C and a fraction composed of actin and 30,000- and 18,000-dalton polypeptides that demonstrated calcium-sensitive genlation at 0 degrees C.

An experimental investigation into the possible origin of pancreatic islet cells from rhombencephalic neurectoderm.

To determine whether or not any pancreatic islet cell type arises from rhombencephalic levels of neurectoderm, lengths of presumptive rhombencephalon (containing potential neural crest) of Black Australorp chick embryos at 6- to 9-somite stages were replaced isotopically and isochronically by neural tube of Japanese quail embryos. Some transplants included mesencephalic regions. In some cases various levels of the rhombencephalon were deleted and not replaced. The quail nuclear marker was detected in cranial ganglia in operated embryos sacrificed at 3 3/4 days of incubation and in enteric ganglia and cells accompanying some pancreatic nerves, in embryos killed at 7 days of incubation. This provided evidence of normal migration of crest cells from the grafts. Dopa was administered to the younger embryos, which were submitted to the formaldehyde-induced fluorescence procedure to demonstrate APUD (Amine Precursor Uptake and Decarboxylation) cells. No pancreatic APUD cells exhibited the quail nuclear marker. In 9- to 11-day embryos, A and B cells were identified by specific light and electron microscopic features. None showed the quail marker. The marker was also absent from those D cells seen and from cells of an as yet unidentified type, but not enough of these were found to warrant a conclusion. All islet cell types were found in embryos from which various levels of the rhombencephalon had been deleted. It is concluded that at least A and B islet cells are not derived from the rhombencephalic neurectoderm and probably not from mesencephalic levels. Their most likely origin remains the endoderm, which was the accepted source until recently.

Regulation of chloride in quiescent sheep-heart Purkinje fibres studied using intracellular chloride and pH-sensitive micro-electrodes.

1. The intracellular Cl activity, alpha iCl was measured inside quiescent sheep cardiac Purkinje fibres, bathed in normal Tyrode at pH 7.40, buffered with approximately 22 mM-bicarbonate/approximately 5% CO2 + 95% O2. The measurements were made using liquid ion-exchanger Cl-sensitive micro-electrodes. 2. After internal Cl levels had been depleted by prolonged exposure to Cl-free media (glururonate-substituted) when external Cl was restored, there was a rapid re-accumulation of Cl inside the fibres to levels that were much higher than those expected for a passive Cl distribution. Such a process can be conveniently defined as an active inward Cl pump. 3. The inward-pumping was noticeably temperature-sensitive (Q10 approximately 2.6), its rate was reduced about eighteenfold in the nominal absence of external bicarbonate/CO2 and it was substantially inhibited by the drug SITS (4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulphonic acid). 4. The fall of alpha iCl in Cl-free solution was slow and was also equally temperature-sensitive and substantially inhibited by SITS, but was only slightly impaired in the nominal absence of external bicarbonate/CO2. 5. pHi was measured using recessed-tip pH-sensitive micro-electrodes, and in some experiments both pHi and alpha iCl were monitored simultaneously. When alpha iCl slowly declined in Cl-free solution then pHi slowly became alkaline. Upon restoring external Cl, then there was, as usual, a rapid recovery of a high alpha iCl and this was accompanied by a rapid re-acidification of pHi. Both the recovery of alpha iCl and pHi were exponential with virtually the same time constant. 6. Both the slow alkalinization of pHi in Cl-free solution and the rapid re-acidification upon restoring external Cl were substantially inhibited by the drug SITS. 7. When [k]O was raised to 45 mM or more (by removing equivalent amounts of [Na]O), there was a large depolarization of Em and a slow rise of alpha iCl, which was not accompanied by a large change of pHi. The rise of alpha iCl appeared to be unaffected by SITS. 8. It is suggested that a Cl/CHO-3 exchange mechanism can operate reversibly across the membrane of quiescent Purkinje fibres, and that it can account, at least in part, for the high levels of alpha iCl measured in the resting state. It is also concluded that Cl can cross the membrane in other ways, especially in high-K solution possibly by moving passively through conductance channels that are open under these conditions.

Inhibin activity in ram rete testis fluid: depression of plasma FSH and LH in the castrated and cryptorchid ram.

Ram "rete testis" fluid (RTF) routinely collected throughout the year has been used as a source of inhibin. The mean flow rate and mean concentration of spermatozoa in the fluid remained constant during the first 12 days of cannulation. More than 50 castrated or cryptorchid rams have been treated with low doses of steroid-free RTF over a 25-h blood sampling period. Human serum albumin was injected as a control. RTF depressed both FSH and LH plasma levels although the pattern was different for each hormone. There was no change in prolactin secretion. LH secretion was affected first while FSH remained unchanged in castrated and in cryptorchid rams. Thereafter, the maximum depression of FSH plasma levels occurred at a time when LH started to return or had returned to preinjection levels in the cryptorchid and castrated animals respectively. In the cryptorchid rams, RTF suppressed pulsatile LH secretion which was present before treatment but in the castrated animals, RTF lowered LH plasma levels which were constant and showed no pulsatile changes before treatment. Both FSH and LH inhibitory activities have been found in all active fractions obtained by purification of RTF. These activities are papain-sensitive and active fractions have a high apparent molecular weight (greater than or equal to 100 000) as shown by gel filtration and ultrafiltration. These and other results in the literature have lead to a re-definition of inhibin as a protein factor of gonadal origin able to depress plasma levels of FSH and LH, even at low doses.

Lactation, fertility and the working women. Working Conference organized by International Planned Parenthood Federation and the International Union of Nutritional Sciences.

20 participants from 15 countries discussed lactation, fertility and the working woman at a conference held in Bellagio, Italy, July 1977. The purpose of the conference was to suggest ways that mothers can combine their 2 roles: salaried worker and mother, with necessary legislation and social measures. Breastfeeding, because of the demands of the work place, has been in decline in recent years. Lack of support by health services, inadequate education of health workers, and the production and promotion of milk substitutes by the food industry have played a part in the decline of breastfeeding. Bottle-feeding has contributed to serious health problems in children, e.g., marasmus and diarrhoea, as well as smaller birth intervals. Some of the measures suggested to help working mothers included a minimum period of 3 months maternity leave, the development of social support services suited to mother and child in different societies, and the provision of suitable facilities where a woman can breastfeed her child. The cost of such services should be paid by the society or community as a whole, not by the employer or employee. More research is needed regarding approprite family planning methods for working women in developing countries; the effects of hormonal contraception on breastfeeding and the health of the infant; and, the effects on working women and their babies of breastfeeding only during their time away from work.

Distribution and metabolic fate of adenosine nucleotides in the membrane of storage vesicles from bovine adrenal medulla.

The reactions of adenosine 14C-and gamma 32P-labelled ATP with isolated membranes from catecholamine storage vesicles of the bovine adrenal medulla were studied. In presence of Mg2+ about twice as much of 32P-radioactivity combined with the membrane as 14C-adenosine compounds at 31 degrees C and also at 0 degrees C, while in the absence of Mg2+ the amounts of 14C and 32P incorporated were similar for both substances. Autoradiography of the SDS-polyacrylamide gel after electrophoresis of the 32P-ATP-treated membrane protein showed two distinct zones corresponding to protein bands. Sonication released twice as much 32P-ATP as 14C-ATP from the space within the membrane particles indicating that at least half of the ATP present in space did not contain its original terminal phosphate group. About 40--45% of the 32P-radioactivity was incorporated in the membrane lipids, whereas only small amounts of 14C-radioactivity were extracted with lipids. About 1/3 of the incorporated 14C-radioactivity was not extractable with acids. The same amount remained in the 32P-ATP treated preparation acid-stably bound after extraction of the lipids and hus must be firmly bound ATP. When the reaction of the membrane preparation with labelled ATP was performed at 0 degrees C the fractions of the acid-stably bound 32P- and 14C-radioactivity increased. About 1 nmole/mg of protein (10--15%) of the bound 32P-radioactivity was exchangeable against unlabelled ATP, while only a very small fraction (less than 0.5 nmol/mg protein) of the 14C-radioactivity was exchanged against unlabelled ATP. Preincubation of the membrane particles with ATP-Mg2+ at 0 degrees C induced 30% inhibition of the ATPase activity and abolition of the net uptake of catecholamines. Different Km values obtained from initial velocity studies of ATPase activity and the overall-incorporation of 32P-radioactivity indicated that a direct correlation between these processes did not exist. Different strong inhibitory effects exerted by ADP on the ATPase activity and net uptake of catecholamine at the one hand and the overall 32P-and 14C-incorporation at the other hand supported that view. It is concluded that small fractions of the observed 32P-and 14C-incorporation can be involved in the ATP hydrolyzing reaction.

Crop-sac response after systemic and intraventricular administration of neuroleptic drugs.

Present experiments were aimed at studying in pigeons the effects of some neuroleptic agents given systemically or into the 3rd cerebral ventricle on PRL secretion and following morphological changes of the crop-sac mucosa both by classical histological methods and scanning electron microscopy. In addition, such changes were also evaluated on the basis of a semiquantitative method using a 1-4 rating scale. 3- or 5-day systemic treatment with reserpine, haloperidol and (ł/-)-sulpiride produced an intense crop-sac response consisting of a marked epithelial hyperplasia and presence of milk-like material. Similarly, a much lower dose of haloperidol, clozapine, and the two enantiomers of sulpiride given into the 3rd cerebral ventricle for 3 consecutive days produced a marked crop-sac response. The l-sulpiride was more active in comparison to the d-enantiomer. In conclusion, present experiments show that, similarly to mammals, in pigeons neuroleptic drugs are able to stimulate prolactin secretion and suggest that these effects are mediated through an action at the hypothalamic and/or pituitary level by removing a tonic dopaminergic inhibition.

Perivascular pH and pial arterial diameter during bicuculline induced seizures in cats.

The aim of the present study was to correlate locally at the same pial artery the vascular reaction with the perivascular pH during the initial phase of functional hyperemia. As a model of functional hyperemia, bicuculline (3 mg/kg i.v.) induced seizure was taken. Normally, a strong increase of blood pressure occurs together with the start of seizure. Since a discrimination between metabolically induced and pressure dependent vascular reactions is not possible under such conditions, the cats (anesthetized with 40--50 mg/kg chloralose) received in addition 3 mg/kg phentolamine and 10 mg/kg pentobarbital. Under these conditions a significant increase of blood pressure started only 50 s after the onset of seizure. Perivascular pH was recorded using spear type pH microelectrodes in the subarachnoid space surrounding a pial artery. The diameter of the respective artery was measured continuously. After onset of seizure an immediate, increasing perivascular acidosis developed which was accompanied by an increase in pial arterial diameter. The maximal decrease of pH was 0.29 units and occurred 30 s after the start of seizure. These data show that a decrease in perivascular pH can be one factor mediating functional hyperemia in the brain.

Epidermal growth factor stimulation of DNA synthesis is potentiated by compounds that inhibit its clustering in coated pits.

We have used inhibitors of receptor-mediated endocytosis to investigate the mechanism and function of epidermal growth factor uptake by cultured cells. When rhodamine-labeled epidermal growth factor is bound to cell surface receptors on confluent monolayers of BALB/c 3T3 cells, it rapidly collects in cell surface clusters and is internalized. The clustering of occupied receptors requires Ca(2+) and is inhibited by primary alkylamines; both of these properties are shared by the enzyme transglutaminase (R-glutaminyl-peptide:amine gamma-glutamyl-yltransferase, EC 2.3.2.13). In Chinese hamster ovary cell extracts, methylamine inhibits 25-50% of the transglutaminase activity with a K(i) of 0.2 mM, and it inhibits the remaining transglutaminase activity with a K(i) of 20 mM. Clustering is almost completely inhibited by 10 mM methylamine. The polypeptide antibiotic bacitracin inhibits clustering of rhodamine-labeled epidermal growth factor or alpha(2)-macroglobulin at 0.7 mM, and it inhibits approximately 40% of the transglutaminase activity in Chinese hamster ovary cells with a K(i) of 0.03 mM. Fluorescent ligands bound to cell surface receptors in the presence of bacitracin form clusters within 30 min after bacitracin is removed from the culture medium. These results indicate that a transglutaminase-like enzyme may be required for the clustering and subsequent internalization of occupied receptors. The effects of 10 mM methylamine and 0.7 mM bacitracin on epidermal growth factor stimulation of DNA synthesis were examined. The stimulation of DNA synthesis by epidermal growth factor was increased 2- to 7-fold in the presence of methylamine or bacitracin. Alone, methylamine or bacitracin increased DNA synthesis 1.1- to 3-fold. The stimulation of DNA synthesis resulting from the simultaneous presence of the hormone and the clustering inhibitor was always greater than the sum of the stimulations produced by the hormone and the clustering inhibitors alone. The potentiation of epidermal growth factor activity by clustering inhibitors suggests that the hormone acts at the cell surface. We propose that rapid internalization of occupied receptors via coated pits may be a mechanism to limit the response to hormones.

Social gliding is correlated with the presence of pili in Myxococcus xanthus.

Myxococcus xanthus, an organism whose motility involves cell interactions, normally bears pili. Myxococcal pili are found only at cell poles, are less than 10 nm in diameter, and may be longer than a cell. Myxococcus has two basic patterns of cell movement, adventurous (A-motility) and social (S-motility). Pili are found to be completely correlated with the presence of S-motility. (The S-motility pattern has many groups of cells, almost no single cells, and is governed by a set of genes called system S.) On the other hand, A-motility is in dependent of piliation. (The A-motility pattern has many single, isolated cells and it is governed by a second set of genes called system A.) Electron microscopic examination of more than 40 genetically different strains shows that all A+S+ (wild-type) and A-S+ strains have pili, but A+S- and A-S- strains lack them. Mutations in four different loci belonging to system S were tested and were found to stop productions of pili: the loci sg1A, sg1B, sg1G, and tg1. When brought into contact with tg1+ cells, cells of a tg1- strain, which lack pili, become phenotypically S+, produce pili, and become S-motile. Both motility and the production of pili are transient when initiated in this way. Thus it appears that pili permit cells that are close to one another to move.

Amplification and characterization of the proline transport carrier of Escherichia coli K-12 by using proT+ hybrid plasmids.

A previous report [Motojima, K., Yamato, I. & Anraku, Y. (1978) J. Bacteriol. 136, 5-9) described the characteristics of mutants (proT-) of Escherichia coli K-12 that are defective in proline transport carrier activity. Two hybrid plasmids from the Clarke and Carbon colony bank were found to complement the mutation by conjugation and transformation. Analysis with restriction endonucleases showed that both plasmid DNAs carried the same fragment of the E. coli chromosome. A polypeptide with a molecular weight of 24,000, specifically coded for by the proT+ plasmid, was identified in the cytoplasmic membrane by using a double-isotope labeling method in a minicell system. The strain harboring the proT+ plasmid has 6 times as much proline transport carrier as the wild strain. This amplification of the carrier makes it possible to measure proline binding to the carrier by microequilibrium dialysis. Detailed analysis of binding indicated that the maximal amount of proline bound to the carrier is 0.70 nmol/mg of protein of the cytoplasmic membrane of the amplified strain. From this value and the assumption that the carrier has one binding site per minimal molecular weight of 24,000, the content of the proline transport carrier in the cytoplasmic membrane was estimated to be 1.7%.

Alpha- and beta-adrenergic stimulation of arachidonic acid metabolism in cells in culture.

Madin-Darby canine kidney cells (MDCK) synthesize prostaglandin (PG) F(2alpha), PGI(2) (measured as 6-keto-PGE(1alpha)), PGE(2), PGD(2), and thromboxane A(2) (measured as thromboxane B(2)). When incubated in the presence of norepinephrine (6 muM), the syntheses of these arachidonic acid metabolites are stimulated 3-fold. Norepinephrine's effect can be antagonized by the addition of alpha-adrenergic receptor blocking agents (phenoxybenzamine>phentolamine>yohimbine>dibenamine>tolazoline) but not by the beta-adrenergic blocking drug propranolol. Norepinephrine's stimulation is also inhibited by low concentrations of dihydroergotamine, bromocryptine, ergocryptine, and ergotamine. The stimulation of PG synthesis by norepinephrine is reversible, continues during the 24 hr of incubation, and requires the presence of norepinephrine at the receptor site but it is not blocked by the addition of colchicine, cytochalasin B, or cycloheximide. Neither phenoxybenzamine nor ergotamine at concentrations that block norepinephrine's stimulation of PG biosynthesis suppresses the increase in PG synthesis induced by exogenous arachidonic acid, suggesting that the alpha-adrenergic regulation is not occurring primarily at the cyclooxygenase step in the metabolism of arachidonic acid. In mouse lymphoma cells (WEHI-5), low concentrations of isoproterenol or norepinephrine stimulate the synthesis of thromboxane, an effect that can be blocked by the addition of propranolol but not by relatively high concentrations of phenoxybenzamine or ergotamine. Taken together, these results suggest that alpha-adrenergic receptor stimulation promotes the deacylation of phospholipids by MDCK cells whereas beta-adrenergic mechanisms lead to activation of similar pathways in WEHI-5 cells.

Oesophageal sensors and their modulatory influence on oesophageal peristalsis in the lobster, Homarus gammarus.

The musculature and innervation of the oesophagus of Homarus gammarus are described as a prerequisite to studies on the mechanisms and control of food ingestion. Of particular interest are two paired sensors (the anterior and posterior oesophageal sensors) which are bilaterally situated at the oesophageal-cardiac sac valve. These are similar to contact chemoreceptors previously described in insects and are classified as such on morphological grounds and with indirect electrophysiological evidence. Oesophageal peristalsis is effected by the coordinated contraction of the Oesophageal musculature. This is controlled by rhythmical bursting neuronal activity, which can be recorded from the nerve trunks in the area. A characteristic burst recorded from the superior oesophageal nerve is used as an indication of oesophageal dilatation during peristalsis for studies on the feedback effects of the oesophageal sensors. Electrical and chemical stimulation of the posterior oesophageal sensors can initiate and increase the frequency of oesophageal peristalsis, while stimulation of the anterior oesophageal sensors can slow and terminate oesophageal peristalsis. The results are discussed and a model presented of the role of the oesophageal sensors in feeding.

Effect of hypocapnia on intracellular pH during metabolic acidosis.

Separate and combined effects of acute metabolic acidosis and hypocapnia were determined in skeletal and cardiac muscles of intact rats. Normocapnic metabolic acidosis, imposed by intraperitoneal injection of hydrochloric acid (6 mEq/kg), did not change skeletal muscle intracellular acid--base parameters. Hypocapnia, induced by mechanical hyperventilation, resulted in intracellular alkalosis within skeletal muscle during both respiratory alkalosis and compensated metabolic acidosis; changes of skeletal muscle intracellular bicarbonate concentration per unit change in carbon dioxide tension were identical during these two experimental procedures. These data suggest that processes other than physicochemical buffering neutralize protons taken into skeletal muscle cells during acute metabolic acidosis. The acid--base state of the heart was quite stable during these experimental manipulations; thus, it appears that cardiac muscle has an extraordinary buffering ability. Moreover, our data suggest that processes other than physicochemical buffering maintain cardiac intracellular pH normal during hypocapnia.

Drugs and breast feeding.

This paper reviews: 1) the basic mechanisms of drug passage into milk; 2) clinical considerations in the use of drugs in lactating women; and 3) use of specific classes of drugs during lactation. Milk can be considered as an oil in H20 emulsion which is separated from the blood plasma by a semipermeable lipoid membrane; which consists of small pores which allow H20 and small H20-soluble molecules and electrolytes to pass from plasma to milk. Larger molecules, including most drugs, are diffused though the lipid portion of the membrane by diffusion. Physiochemical and pharmacokinetic factors affect the passage of drugs into milk. When drugs are prescribed to a breast-feeding mother (e.g., treatment of urinary tract and vaginal infections), the benefits of using drugs should be weighed against the potential hazards of the drug in the infant. Consideration should also be given to the fact that infants, especially the neonates and preterms, have immature excretory mechanisms which allow excessive accumulation of certain drugs. Effects of drugs in the milk could be beneficial, detrimental, and nonexistent on the breast-fed intake. So far, the only known beneficial effect of a drug in breast milk is that of pyrimethamine. Potentially hazardous drugs to lactation include: corticosteroids; sex hormones; major tranquilizers; tricyclic antidepressants; antihypertensives; and antituberculars. A series of guidelines for prescribing drugs from various classes of agents is also provided.

Transport of radiolabelled glycoprotein to cell surface and lysosome-like bodies of absorptive cells in clutured small-intestinal tissue from normal subjects and patients with a lysosomal storage disease.

The transport of 3H-fucose- and 3H-glucosamine-labelled glycoproteins in the absorptive cells of cultured human small-intestinal tissue was investigated with light- and electron-microscopical autoradiography. The findings showed that these glycoproteins were completed in the Golgi apparatus and transported in small vesicular structures to the apical cytoplasm of these cells. Since this material arrived in the cell coat on the microvilli and in the lysosome-like bodies simultaneously, a crinophagic function of these organelles in the regulation of the transport or secretion of cell-coat material was supported. In the absorptive cells of patients with fucosidosis or Hunter's type of lysosomal storage disease, a smiliar transport of cell-coat material to the lysosome-like bodies and a congenital defect of a lysosomal hydrolase normally involved in the degradation of cell-coat material, can explain the accumulation of this material in the dense bodies.

Stereological study of B-cell mitochondria in alloxan-treated mice.

Using stereological techniques, including semi-automatic image analysis, the B-cell mitochondria were studied in the pancreatic islets from one group of control mice and two groups of mice killed 10 min and 60 min, respectively, after alloxan administration. Ten min following alloxan the mitochondrial volume and envelope surface densities, the mean mitochondrial volume and surface area, and the area of mitochondrial profiles were significantly increased, whereas the mitochondrial numerical density was not significantly altered. At the 60 min observation time the mitochondrial volume density, the mean mitochondrial volume and surface areas, and the area of mitochondrial profiles were significantly decreased, whereas the mitochondrial envelope surface was not significantly altered. The findings indicate a rapid swelling, followed by disintegration of the mitochondria in the B-cells of alloxan-treated mice, thereby supporting our view that mitochondrial lesions play a primary role in the development of alloxan diabetes. These lesions are believed to be due to ionic alterations in the B-cells ("Pi-pH hypothesis").

Effects on fetal breathing movements of maternal challenges. Cross-over study on dynamic work, static work, passive movements, hyperventilation and hyperoxygenation.

Ten women in the last trimester of a normal pregnancy were subjected to five different loads in a cross-over study. Fetal breathing movements (FBM), fetal heart rate (FHR), maternal heart rate (MHR), and mean arterial pressure (MAP), maternal transcutaneously measured pO2 (Tc-pO2), and the energy supply to the Tc-pO2 electrode were recorded continuously before, during, and after the load. Maternal capillary pH and pCO2 were measured at three representative time points. The immediate responses of the incidence of FBM to the different challenges were: increase after dynamic work (bicycle test); no change after static work (isometric muscle contraction) and passive movements; decrease after hyperventilation and hyperoxygenation. FHR was unaffected by all challenges. The FBM incidence varied in parallel with pCO2 after dynamic work and hyperventilation and inversely with the Tc-pO2 rise caused by hyperoxygenation. Maternal pH was increased after passive movements (no change in FBM) and after hyperventilation (decreased incidence of FBM), FBM seem to be more sensitive to environmental changes than is the FHR. Mechanical stimuli to the uterus were not responsible for the augmentation of FMB seen after the bicycle test. The present observations reveal the multifactorial nature of the regulation of FBM, and support the role of CO2 as a major stimulator of breathing movements also in prenatal life.

[Seroepidemiologic studies on reovirus infections of man, domestic and wild animals in Tanzania (author's transl)].

2238 sera of bovines, 95 of goats, 251 of antelopes (various species), 143 of zebras and 11 of warthogs collected in Tanzania as well as 811 sera of men and females of the city and the region of Dar es Salaam were tested for haemagglutination inhibiting antibodies (Ab) to reovirus serotypes (St) 1, 2 and 3. Ab to St 1 resp. 2 could be detected in 24--39% of bovines, goats, antelopes and zebras, and in warthogs to 64%. In human beings the positive percentage was 60%. Ab to St 3 were most prevalent: Sera of domestic animals were positive in 70--84%, of wild animals in 77--100% and of the human population in 91%. These figures include the occurrence of Ab to one St alone as well as to more than one St. A further analysis showed, that Ab to St 3 are definitely dominant especially in sera of animals, whereas the simultaneous occurrence of Ab to all 3 St was more frequently observed in sera of human beings. Other Ab-type combinations were apparently of far less importance. It seems to be significant that the highest infection rate was found in humans (except zebras and warthogs) and that their contact to animals did not result in a higher Ab conversion rate as without such a possibility. The results were discussed under seroepidemiologic aspects and it is concluded that reovirus infections are facultative viral zoonoses.

Determination of the stimulus-response relation for three alpha-adrenergic agonists on rabbit aorta.

A standard rabbit thoracic-aorta strip preparation was used to determine the effect of varying preload on KA(the dissociation constant), A50(the concentration that produces half maximal response) and the ratio KA/A50. Additionally, the stimulus-response relationships were obtained for the drug and preload conditions tested. Values of KA and A50 were calculated from dose-response curves obtained for three alpha-adrenergic agonists (phenylephrine, methoxamine and norepinephrine) in the presence and absence of partial irreversible blockade by phenoxybenzamine. For all three drugs, values of KA and A50 were found to be significantly greater at 1/4 gram preload than at 10 gram preload. However, the ratio KA/A50 was found to be independent of preload conditions. The stimulus-response relationships were all found to be non-linear, in contrast to the predictions of classical receptor theory. The significance of the constancy of KA/A50, rather than either KA or A50, and the non-linear relation between drug stimulus and response is discussed in terms of Stephenson's theory of drug action.

Wheat-germ aspartate transcarbamoylase. Purification and cold-lability.

1. Aspartate transcarbamoylase was purified approx. 3000-fold from wheat (Triticum vulgare) germ in 15-20% yield. The product has a specific activity of 14 mumol/min per mg of protein and is approx. 90% pure. The purification scheme includes the use of biospecific "imphilyte" chromatography as described by Yon [Biochem.J.(1977) 161, 233-237]. The enzyme was passed successively through columns of CPAD [N-(3-carboxypropionyl)aminodecyl]-Sepharose in the absence and presence respectively of the ligands UMP and L-aspartate. In the second passage the enzyme was specifically displaced away from impurities with which it co-migrated in the first passage. These two steps contributed a factor of 80 to the overall purification. 2. The enzyme is slowly inactivated on dilution at 0 degrees C and pH 7.0, the inactivation being partially reversible. A detailed investigation of the temperature- and pH-dependence of the cold-inactivation suggested that it was initiated by the perturbation of the pKa values of groups with a moderately high and positive heat of ionization, which were tentatively identified as histidine residues. These findings support a new concept of cold-lability proposed by Bock, Gilbert & Frieden [Biochem. Biophys. Res. Commun. (1975) 66, 564-569].

Regulation of microsomal stearoyl-coenzyme A desaturase. Purification of a non-substrate-binding protein that stimulates activity.

Crude cytosolic fraction from rat liver was examined for proteins that may be involved in regulation of microsomal stearoyl-CoA desaturase activity. Gel filtration revealed the presence of several components that either stimulate or inhibit this enzyme. In addition, other components bind the acyl-CoA substrate, thus affecting observed activities in vitro. A protein that stimulates stearoyl-CoA desaturase but does not bind substrate was purified approx. 1100-fold. The purified protein had no visible absorption spectrum and an approximate mol.wt. of 26500. Maximal stimulation of desaturase activity occurred with less than 2 micrometer purified protein. The protein was heat-labile and exhibited neither catalase nor glutathione peroxidase activity. Addition of the cytosolic protein produced no effect on the desaturase reaction stoicheiometry; the proportions O2 consumed/NADH oxidized/stearoyl-CoA desaturated remained 1:1:1. Because the Km' for stearoyl-CoA was also unchanged by addition of the cytosolic protein, no change in substrate affinity was suggested. Furthermore addition of the cytosolic protein also produced no effect on desaturase inhibition by oleoyl-CoA, which suggested that the protein does not simply relieve apparent product inhibition. These results indicate that, in analogy to other cytosolic proteins that stimulate microsomal oxidase activities, the protein may have a regulatory function, perhaps related to activity modulation via organization of the multienzymic desaturase in the membrane.

The metabolism of 4-methyl-2-oxopentanoate in rat pancreatic islets.

1. Radioactively labelled 4-methyl-2-oxopentanoate was taken up by isolated pancreatic islets in a concentration- and pH-dependent manner and led to the intracellular accumulation of labelled amino acid and to a decrease in the intracellular pH. Uptake of 4-methyl-2-oxopentanoate did not appear to be either electrogenic or Na+-dependent. The islet content of 2-oxo acid radioactivity was not affected by either 2-cyano-3-hydroxy-cinnamate (10mM) or pyruvate (10mM), although both these substances inhibited the oxidation of [U-14C]4-methyl-2-oxopentanoate by islet tissue. 2. 4-Methyl-2-oxopentanoate markedly stimulated islet-cell respiration, ketone-body formation and biosynthetic activity. The metabolism of endogenous nutrients by islets appeared to be little affected by the compound. 3. Studies with the 3H- and 14C-labelled substrate revealed that 4-methyl-2-oxopentanoate was incorporated by islets into CO2, water, acetoacetate, L-leucine and to a lesser extent into islet protein and lipid. Carbon atoms C-2, C-3 and C-4 of the acetoacetate produced were derived from the carbon skeleton of the 4-methyl-2-oxopentanoate, but the acetoacetate carboxy group was derived from the incorporation of CO2. These results, and consideration of the relative rates of 14CO2 and acetoacetate formation from 1-14C-labelled as opposed to U-14C-labelled 4-methyl-2-oxopentanoate, led to the conclusion that the pathway of catabolism of this 2-oxo acid in pancreatic islets is identical with that described in other tissues. The amination of 4-methyl-2-oxopentanoate by islets was attributed to the presence of a branched-chain amino acid aminotransferase (EC 2.6.1.42) activity in the tissue. Although glutamate dehydrogenase activity was demonstrated in islet tissue, the reductive amination of 2-oxoacids did not seem to be of importance in the formation of leucine from 4-methyl-2-oxopentanoate. 4. The results of experiments with respiratory inhibitors and uncouplers, and the finding that 14CO2 production and islet respiration were linked in a 1:1 stoicheiometry suggested that 4-methyl-2-oxopentanoate catabolism was coupled to mitochondrial oxidative phosphorylation. The catabolism of 4-methyl-2-oxopentanoate in islet tissue appeared to be regulated at the level of the initial 2-oxo acid dehydrogenase (EC 1.2.1.25) reaction.

Cardiovascular dynamics after acute and long-term alpha- and beta-adrenoceptor blockade at rest, supine and standing, and during exercise.

1 After acute intravenous administration labetalol reduced mean values for BP, total peripheral resistance, heart rate and cardiac output. All changes were more pronounced during bicycle exercise. 2 After a mean duration of 20 months' treatment with oral labetalol the haemodynamic findings were broadly similar except for a more marked reduction in the total peripheral resistance and cardiac output had returned to pretreatment level due to an increased stroke volume which had counter balanced the reduction in heart rate. These changes occurred at rest, in the erect position and during exercise but the reductions in BP and peripheral resistance were most marked during exercise. 3 Left ventricular filling pressures and stroke volume/filling pressure ratios were not significantly altered after intravenous labetalol compared with pretreatment values. 4 Systolic BP x heart rate product was lowered particularly during exercise after both intravenous and oral labetalol. 5 After long-term oral labetalol, the most striking haemodynamic change was in the elevated resting stroke volume supine and standing.

Lysosomal acid hydrolases in lymphocytes of I-cell disease.

Several lysosomal acid hydrolases were assayed in peripheral blood leukocytes from a patient with I-cell disease by the method of Hindman, J. and Cotlier, E. ((1972) Clin. Chem. 18, 971--975). The activities of lysosomal hydrolases in polymorphonuclear cells showed no significant differences between the patient, the parents, and normal controls, while lymphocytes from the patient exhibited the reduced activities of alpha- and beta-galactosidases, beta-glucuronidase, and N-acetyl-beta-glucosaminidase. After phytohemagglutinin-stimulated culture, lymphocytes from the patient showed a more definite reduction in the activities of these lysosomal enzymes; the activities of beta-glucuronidase, alpha-mannosidase, and N-acetyl-beta-glucosaminidase were reduced to about 20--30% of the activity in the phytohemagglutinin-stimulated control cultures, and the activities of alpha- and beta-galactosidases to 10% or less. Lymphocytes from the parents showed no significant difference in the activities of these enzymes from the controls, whether stimulated or not.

Studies on the persistence of basic amines in the rabbit lung.

We have investigated the time-course of the pulmonary deposition of imipramine (IMIP) following a single iv injection into rabbits. A pool of IMIP and its demethylated metabolites, which exhibited considerable persistence (half-life of decay = 4 hr), was formed in the lung. This pool, now called the slowly effluxable pool (SEP), appears to be synonymous with the noneffluxable pool (NEP), which we have previously shown to be formed with IMIP in the isolated perfused lung (PL). Furthermore, this pool appears to be responsible for the pulmonary persistence of IMIP, inasmuch as 24 hr after an iv injection it contributes greater than 90% of the total lung concentration. Chlorphentermine and methadone formed SEP's in the IPL of comparable size to that formed with IMIP, whereas phentermine formed a considerably smaller SEP. These results suggest that the degree of hydrophobicity of the amine is an important determinant of the size of the SEP formed. This further supported by the lack of an SEP with the relatively polar amines, 5-hydroxytryptamine and amphetamine. The 10-fold difference in the size of the SEP for compounds known to induce pulmonary phospholipidosis (chlorphentermine and IMIP) and known not to induce lipidosis (phentermine and amphetamine) may suggest a possible involvement of the SEP in the onset of phospholipidosis; this possibility is discussed.

Mechanism-controlled stereospecificity. Acylation of subtilisin with enantiomeric alkyl and nitrophenyl ester substrates.

The activation parameters of acylation of subtilisin with alkyl and p-nitrophenyl esters of N-acylamino acid enantiomers were determined. It was found that (1) the activation entropy is much higher with the nitrophenyl esters than with the corresponding methyl esters, (2) the difference in rate constants between enantiomers is 10(4)--10(5) with methyl esters whereas it is only of the order of 10 with nitrophenyl esters. The results indicate that the catalytic mechanism is simpler for nitrophenyl esters than for alkyl esters. The simple mechanism requires only general base catalysis, and thus permits more freedom of motion in the transition state, whereas the complex mechanism involves both general base and general acid catalysis. Furthermore, the strikingly low enantiomeric specificity with nitrophenyl esters indicates that not only binding but also the catalytic mechanism is an important factor in determining the stereospecificity of an enzyme. The activation parameters for enantiomeric nitrophenyl ester reactions suggest that structurally related substrates can be transformed by the enzyme in different conformations which may be energetically similar or not. The energetically different conformations may account for the activation enthalpy-entropy compensation.

Nonlinear dependence of biological activity on hydrophobic character: the bilinear model.

In homologous series of compounds biological activity is linearly dependent on hydrophobic character until a cut-off point is reached where this linear relationship changes to a nonlinear relationship: biological activity increases with increase of hydrophobic character, reaches a maximum and then decreases with further increase of hydrophobic character. Drug transport in biological systems is determined by the rate constants of transfer of the drug through aqueous and organic compartments. In simple in vitro systems the rate constant k1 of transport of a drug from an aqueous phase into an organic phase and the rate constant k2 of the reverse process can be described as functions of the partition coefficient P: log k1 = log P - log (beta P + 1) + c and log k2 = - log (beta P + 1) + c. Observed and calculated k1 and k2 values are used to simulate drug transport in different multicompartment systems. Based on the McFarland probability model a new model for the quantitative description of the dependence of biological activity on hydrophobic character, called bilinear model, log 1/C = a log P - b log (beta P + 1) + C, has been derived recently: unsymmetrical curves with linear ascending and descending sides and a parabolic part within the range of optimal lipophilicity result from this model. The bilinear model is applied to experimental data of drug absorption, drug distribution and drug activity in biological systems. A comparison of the parabolic model and the bilinear model shows that in nearly all cases a better fit of the data results from the bilinear model.

Testicular biopsy in the study of male infertility: its current usefulness, histologic techniques, and prospects for the future.

Testicular biopsy has been widely used for the diagnosis of male infertility for more than three decades. During that time, with advances in cytogenetics, radioimmunoassay, and endocrinology, the role of testicular biopsy has changed. Testicular biopsy is still useful in the diagnosis of azoospermic and oligospermic males without stigmata of gonadotropic insufficiency or Klinefelter's syndrome. A classification and description is presented of pathologic changes in the testis as seen on testicular biopsy by light microscopy. The present rationale for testicular biopsy in infertility, the processing and staining of histologic material, and the role of testicular biopsy in infertility and infertility related situations, including cryptorchidism, malignant disease, and chemotherapy related changes, are discussed. Further understanding of testicular function and disease will depend upon the correlation of histologic and ultramicroscopic changes, immunohistologic localization of hormones, and epidemiologic and endocrinologic data.

Effect of carbohydrate source and growth conditions on the production of lipoteichoic acid by Streptococcus mutans Ingbritt.

Streptococcus mutans Ingbritt was grown in a chemostat at defined dilution rates and pH values and under carbohydrate limitation. At a constant dilution rate of D = 0.1 h-1 and with either 0.5% glucose or 0.5% sucrose, the amounts of both cellular and extracellular lipoteichoic acid increased as the culture pH increased from 5.0 to 7.5. At a constant pH of 6.0, the amount of cellular lipoteichoic acid formed by cultures growing in 0.2% or 0.5% glucose was relatively constant over a range of dilution rates, although the amount of extracellular lipoteichoic acid formed in 0.2% glucose at intermediate dilution rates was less than that formed in 0.5% glucose. Organisms grown in 0.5% sucrose at pH 6.0 contained increasing amounts of cellular lipoteichoic acid as the dilution rate was increased. A comparison of the amounts of cellular lipoteichoic acid formed by organisms growing at D = 0.5 h-1 and pH 6.0 in glucose, sucrose, fructose, or mixtures of glucose and fructose in limiting amounts suggested that the enhanced production of lipoteichoic acic by sucrose-grown organisms was due to the fructose component. The culture fluids from both glucose- and sucrose-grown organisms contained detectable amounts of serotype c antigen, whereas glucose-grown cultures also contained significant amounts of an extracellular hexose-containing polymer.

Immunocytochemical localization of tyrosine hydroxylase, dopamine-beta-hydroxylase and phenylethanolamine-N-methyltransferase in the adrenal glands of the frog and rat by a peroxidase-antiperoxidase method.

The subcellular localizations of tryrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT) in the adrenal glands of the frog and rat have been examined by a peroxidase-antiperoxidase (PAP) method. TH was localized in the ground substance of the adrenaline-containing cells and noradrenaline-containing cells, but not in the nucleus or in the mitochondria. TH was also located on the outside of the membrane of the chromaffin granules. DBH was observed only inside the granules. PNMT was found not only in the ground substance but also on the membrane of some adrenaline-containing granules. Cortical lipid cells of the frog adrenals did not show TH-, DBH-, and PNMT-reactions. The negative reactions to TH-, DBH-, and PNMT-antiserum exhibited by the summer cells of the frog adrenals prove that they belong to the cortical cells.

Colorimetric and spectrophotometric determination of total and non-phenolic alkaloids in ipeca and its formulations.

A simple method, requiring no chromatographic separation, is presented for the determination of the total and non-phenolic alkaloids in ipeca and its preparations. The complex formed between the alkaloid and methyl orange at pH 5.0 is extracted with chloroform and treated with 0.1N NaOH. The liberated dye, determined at 460 nm, is a measure of the total alkaloids. The chloroform phase remaining is treated with 0.1N H2SO4, and the acid extract is measured at 283 nm for the non-phenolic alkaloids, calculated as emetine. The proposed method was successfully applied to samples of ipeca powder, ipeca tincture, and 3 British Pharmaceutical Codex mixtures containing ipeca tincture, namely, ipecacuanha mixture, pediatric; ipecacuanha and ammonia mixture, pediatric; and belladonna and ipecacuanha mixture, pediatric. The proposed method compares favorably with the Egyptian Pharmacopoeia, British Pharmacopoeia, and USP methods and has a relative standard deviation of 1.54%. The present procedure is less time-consuming and requires about 45 and 90 min for the assay of ipeca tincture and powder, respectively. Only a small sample (0.2 mL tincture of 1.0 g powder) is required.

Comparative gastrointestinal and biliary tract effects of morphine and butorphanol (Stadol).

Butorphanol, levo-N-cyclobutylmethyl-3,14 beta-dihydroxy-morphinan, is a new agonist-antagonist type analgetic agent which is 4 to 8 times more potent than morphine in experimental animals and man. Butorphanol and morphine were evaluated in mice and dogs for their gastrointestinal and biliary tract smooth muscle effects. Morphine decreased the propulsion of a charcoal meal through the gastrointestinal tract of the mouse in a dose related manner with the maximal inhibition obtainable being 90%. Butorphanol produced a maximal inhibition of motility of only 40% with very high doses producing less of an inhibitory effect than lower doses. In dogs, morphine caused a dose-dependent increase in duodenal smooth muscle activity and a dose-dependent decrease in bile duct flow. A clinically effective i.v. dose of morphine (0.1 mg/kg) produced a statistically significant spasmogenic effect on dog biliary tract and gastrointestinal smooth muscle, while a clinically effective equianalgetic i.v. dose of butorphanol (0.025 mg/kg i.v.) had little or not effect on the biliary or gastrointestinal systems. These findings indicate that at equianalgetic doses, butorphanol should produce less constipation and less biliary tract and gastrointestinal smooth muscle spasm than morphine.

The effects of bromocriptine on pre-synaptic and post-synaptic alpha-adrenoceptors in the mouse vas deferens.

Noradrenaline (NA) and dopamine (DA) contracted the mouse vas deferens and reduced the responses to low frequency nerve stimulation (0.1 Hz). The relative potencies of antagonists suggested that these effects were due to stimulation of post-synaptic and pre-synaptic alpha-adrenoceptors respectively. Bromocriptine produced a non-competitive antagonism of contractile responses to NA (pD2' = 7.6) and DA (pD2' = 8.0) but had no effect on responses to carbachol. Bromocriptine also reduced single twitch responses of the vas to low frequency field stimulation (0.1 Hz), but did not affect stimulation at higher frequencies (1--20 Hz). Yohimbine selectively and rapidly reversed the inhibiting effects of bromocriptine on single twitches, although they could not easily be reversed by washing. Bromocriptine produced a yohimbine-reversible reduction in the stimulated overflow of tritium from vasa previously loaded with 3H--NA. Thus the mouse vas deferens does not appear to contain specific DA receptors and the results suggest that bromocriptine acts as a pre-synaptic alpha-adrenoceptor agonist and post-synaptic alpha-adrenoceptor antagonist in this tissue.

Measurement of the aquatic toxicity of volatile nitrosamines.

The acute toxicity of N-nitrosodimethylamine (DMN) and N-nitrosodiethylamine (DEN) was determined for three groups of aquatic organisms: algae, invertebrates, and fish. Toxicity of DMN and DEN to algae was assessed as a repression in the growth rate of either Selenastrum capricornutum or Anabaena flos-aquae in static bioassay tests. DMN and DEN concentrations of 1-10 ppm depressed algal growth in all cases. Invertebrate toxicity was determined in 96-h static bioassay tests with Dugesia dorotocephala and Gammarus limnaeus. The data indicated that these organisms are not highly susceptible to nitrosamine toxicity. The 96-h LC50s for D. dorotocephala were 1365 and 1490 ppm for DMN and DEN, respectively. Similar studies with G. limnaeus indicated LC50s of 330 and 500 ppm for DMN and DEN, respectively. Fish toxicity was also determined in 96-h statis bioassays with the fathead minnow (Pimephales promelas). Acute toxicities were calculated as LC50s of 940 and 775 ppm for DMN and DEN, respectively. Algae were calculated as LC50s of 940 and 775 ppm for DMN and DEN, respectively. Algae were quite sensitive to relative low levels of volatile nitrosamines, but higher organisms (invertebrates and fish) were relatively insensitive.

Phospholipid metabolism in Neisseria gonorrhoeae: phospholipid hydrolysis in nongrowing cells.

Hydrolysis of cell envelope phospholipids was demonstrated in cells of both autolytic and nonautolytic strains of Neisseria gonorrhoeae that were labeled during growth in the presence of [3H] acetate. The label incorporated into the cellular phospholipids was located exclusively in the fatty acid acyl side chains. Labeled cells were incubated for 2 hr in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer, pH 8.5, containing various additions, and then examined for distribution of 3H in lipids. Ca++ selectively stimulated the deacylation of phosphatidylethanolamine (PE), whereas Mn++ stimulated the deacylation of phosphatidylglycerol (PG). Hydrolysis of phosphatidylethanolamine by phospholipase A was accompanied by the accumulation of lysophosphatidylethanolamine (LPE) and free fatty acids in the cells. Free fatty acids accumulated to a greater extent than lysophosphatidylethanolamine, suggesting that the latter was further hydrolyzed to glycerophosphorylethanolamine (GPE) and free fatty acids by a lysophospholipase. Methanol, ethanol, propanol, and isopropanol, added at concentrations which inhibited growth by 50%, stimulated phospholipase A, but not lysophospholipase activity. Differences in heat inactivation, metal ion requirements, and pH optima suggested that phospholipase A activities with phosphatidylethanolamine or phosphatidylglycerol as substrate and lysophospholipase may be separate enzymes.

Specific binding of 3H-adenosine to rat brain membranes.

The binding of 3H-adenosine to rat brain membranes was studied by a microcentrifugation technique. Specific binding of 3H-adenosine was rapid, reversible, saturable and dependent on pH and temperature. Scatchard plots of equilibrium binding data were nonlinear suggesting the existence of two different binding sites for adenosine. The dissociation constants (Kd) were 1.7 muM and 13.6 muM and the maximal number of binding sites (Bmax) 31 and 165 pmol adenosine bound per mg of membrane protein. Ten adenosine derivatives were studied for their ability to compete with 3H-adenosine binding. The phosphorylated adenosine compounds 5'-AMP, cyclic AMP and ATP were most potent in displacing 3H-adenosine from its binding sites and the IC50-values ranged from 11--25 muM. N6-Phenylisopropyladenosine produced only partial inhibition (30%) of 3H-adenosine binding and no stereospecific difference between the (-)- and (+)isomer was observed. Several methylxanthines known as adenosine antagonists competed for the 3H-adenosine binding sites parallel with their pharmacological potency. The results offer a first approach for the study of adenosine binding sites in brain membranes.

Pesticides for locust control.

The factors influencing the choice of insecticides for locust control are effectiveness, safety in use, relative cost, and the formulations available. The relative importance of these factors varies with the scale of control. In small scale control by farmers safety and simplicity are paramount and a BHC dust is commonly used, but in large scale control operations by specialized organizations more toxic formulations ultra-low-volume (u.l.v.) concentrates and methods of application requiring considerable skills can be used. The remarkable effectiveness of dieldrin as a stomach poison appears to be due to its conversion to photodieldrin after application. Cases of poisoning following large scale control operations are rare, and fatalities in man unknown. Alternatives to organochlorine insecticides include fenitrothion, already recommended and used on a large scale against adults. Against nymphs the correct dosage of fenitrothion would cost nearly 11 times as much as that of dieldren. Recent experimental work with new insecticides has included safety tests with domestic animals and measurements of persistence. 1 microgram deposits of cyanofenphos, decamethrin and mecarphon, as well as of dieldrin and photodieldrin, on wheat seedlings left in the open under tropical conditions for two days, killed 80% or more first instar nymphs of the Desert Locust which fed on them.

Geochemistry and health in the United Kingdom.

Before the 1960s, comparisons between the distribution of trace elements in the environment and health in the United Kingdom were primarily confined to ad hoc studies in areas associated with particular agricultural disorders or with unusual human mortality or morbidity records. More recently, increasing interest in the importance of trace elements in crop and animal production and in the hazards of environmental pollution have created a need for more systematic geochemical data. Geochemical reconnaissance maps for England, Wales, Northern Ireland and parts of Scotland have demonstrated the extent of many known clinical trace element problems in agriculture and have also been valuable in delineating areas within which subclinical disorders may occur. Their application to studies on the composition of soils, food crops and surface waters in relation to public health has proved encouraging. Current knowledge and present investigations into environmental geochemistry and human health in the U.K. are reviewed, together with future research requirements.

Geochemistry and cardiovascular diseases.

Deficiencies or excesses in the content or availability of trace elements in rocks and soils, or in water flowing through them, is hypothesized as a possible cause of certain chronic diseases, including cardiovascular diseases. Geographic distribution of cardiovascular diseases is often associated with geochemical differences. This trend is particularly evident in the United States and in Europe, with higher rates for cardiovascular mortalities being present in areas uunderlain by soils that are poor in most essential trace elements. Confirmation of this trend is found in connection with the degree of mineralization of local water supplies. Areas that are served by soft waters usually show higher rates of cardiovascular mortality and other forms of cardiovascular pathology, compared with the areas that are served by hard waters. Such a negative association between water hardness and cardiovascular pathology is evident in many countries, both industrialized and developing.

Trace elements in animals.

Trace element deficiency and toxicity in animals induces a wide variety of clinical effects although few are sufficiently specific to permit diagnosis without supporting investigation of changes in tissue trace element content or of the activity of metabolic processes influenced by trace element supply. Study of such trace element dependent processes has shown that extensive changes often arise before overt signs of disease appear. Some of these subclinical effects have pathological consequences and thus cannot be ignored when seeking correlations between geochemical anomalies and disease incidence. Many past estimates of the quantitative requirements of animals for the essential trace elements are imprecise. Although recent work is providing clearer definition of requirements, many common dietary components have a marked influence upon the efficiency with which such elements can be utilized from the diet. Recent evidence indicates that such antagonists influence both the absorption and the subsequent fate of essential and toxic elements in body tissues and these processes have to be taken into account when investigating the aetiology of disorders believed to be attributable to anomalies in trace element supply. Their existence is not always detectable if attention is confined to the trace element analysis of body tissues or to the nature of clinical lesions. Provided the complexity of soil-plant-animal relations with respect to trace element supply is fully recognized in the interpretation of data, the geochemical approach to the initial recognition of areas associated with a high risk of anomalies in trace element supply to animals and man has considerable potential value. This is already apparent from investigations upon the incidence of trace element problems in animals. As yet, its validity for similar purposes in man is less fully established.

Cat liver cystathionase.

Cat liver cystathionase was about 300-fold purified in comparison with the supernatant of the homogenate, and the characteristics were compared with those of rat. Optimum pHs for several substrates were found to be somewhat lower, and isoelectric point remarkably lower, in cat than in rat. Molecular weight of the cat liver cystathionase was about 158,000.

Prostaglandins have limited actions on abnormalities of beating induced in cultured heart cells.

Prostaglandins are antiarrhythmic in a variety of situations including ischaemic arrhythmias, but the mechanisms involved are not known. In view of this, the protective actions of prostaglandins A2, E2, F1 alpha, F2 beta, and I2 against abnormalities of beating induced in cultured heart cells were investigated. Abnormalities of beating were induced in single cells by variety of agents including ouabain Ca++, K+, dinitrophenol (DNP), and toxic material from the jellyfish Cyanea. Abnormalities were assessed in terms of rate, rate range, subjective arrhythmic behaviour and percent cells beating. The prostaglandins (at 10(-7)-10(-5) M) were added with the arrhythmogenic agent to test for their ability to modify agent-induced beating abnormalities and were compared with lidocaine and quinidine. Prostaglandins alone had minimal direct effects on the cells and only minimally reduced responses to arrhythmogenic agents. The most protective prostaglandins, PGE2 and PGF1 alpha, tended to normalise beating behaviour most noticeably in DNP-treated cells, unlike lidocaine and quinidine which were effective against Ca++-induced changes while worsening those of K+. Thus, a general ability to protect disturbed cardiac cells is not seen with high concentrations of prostaglandins.

Serum group I pepsinogens during prolonged infusion of pentagastrin and secretin in man.

Six 20- to 25-year-old healthy men were studied with an intravenous pentagastrin infusion in a dose of 6 micrograms/kg-h for 4.5 h. Four of these were also studied on separate days with an intravenous secretin infusion in a dose of 2 CU/kg-h for 4.5 h. Gastric juice was collected continuously for one 30-min period before and in 30-min periods throughout the infusion periods, and the gastric H+ and pepsin outputs were determined during the pentagastrin infusion only. Blood was drawn before, every 30 min throughout the infusion, and the next morning for determination of serum group I pepsinogens (PG I), serum gastrin, and plasma secretin. Pentagastrin evoked a sustained rise in gastric H+ and pepsin secretions, a more delayed and sustained increase in serum PG I in the four subjects with a normal pentagastrin-stimulated maximal gastric secretion, and a fall in serum PG I in the remaining two subjects with a low gastric secretion. Secretin also elicited a sustained elevation in serum PG I in all four examined, including one who showed a fall in serum PG I during pentagastrin infusion. It is proposed that pentagastrin may exert its stimulatory effect of pepsinogen synthesis subsequent to degranulation of the chief cells, whereas secretin may stimulate the pepsinogen synthesis more directly. Thus, the fall in serum PG I during pentagastrin infusion in the two subjects with low gastric secretion may possibly be due to a defective cellular storage of PG I in atrophic gastritis. Plasma secretin was not affected by gastric suction or by prolonged infusion of pentagastrin, whereas serum gastrin fell during secretion infusion accompanied by gastric suction.

Biogenic amines in neuroendocrine systems: multiple sources, messages, targets and controls.

Biogenic amines are small chemical mediator molecules synthesized from amino acids by the body. Our research has centered on functions and controls of three categories of biogenic amines in neuroendocrine and neural systems of laboratory species of mammals as models of similar processes in the human body. Experimental analyses have been made of possible contributions of particular of these compounds, or their sources, to regulation of reproductive and 24-hour rhythms, and to responses and adaptations to stress. Results from this and related work, by many other investigators as well, have general implications for biomedical research, including: (A) Biogenic amines have multiple sources, messages, targets and possible controls, depending on species (genetics), developmental stage and age, and in relation to tissue regions and responses during both adaptations and disease states. (B) Functions or actions of biogenic amines in either the nervous or the neuroendocrine system go beyond what can be accommodated by the classical paradigms or terms of neurophysiology. Labeling these compounds in their various tissue sites as "neurotransmitters", or "neuromodulators", or "neurohormones" adds little to our understanding of their actions and roles. (C) Future understanding of how these chemical mediators function and are controlled in health and in disease should be enhanced by investigation of the spectrum of adaptive tissue changes in which these compounds and their control mechanisms participate. This suggested approach contrasts with the usual and classic paradigm in which primary, if not unitary, sources, actions and controls are emphasized to the exclusion and ignoring of others that may have vital significance at particular times or in particular circumstances.

Continuous positive airway pressure during mechanical and spontaneous ventilation. Effects on central haemodynamics and oxygen transport.

The effect of continuous positive airway pressure during continuous mechanical (CMV + PEEP) and spontaneous (CPAP) ventilation on central haemodynamics and systemic oxygen transport was studied in 10 male patients who had undergone aortocoronary bypass graft operation 18 h earlier. With the change from CMV + PEEP 5 cmH2O to CPAP 5 cmH2O, cardiac index was found to increase from 2.58 +/- 0.44 (s.e. mean) to 2.88 +/- 0.19 l/min/m2 (P less than 0.005), and systemic oxygen transport improved from 8.5 +/- 0.6 to 9.5 +/- 1.0 ml/min/kg (P less than 0.05). Arterial oxygen tension and content did not change, but mixed venous blood oxygen tension increased from 3.5 +/- 0.2 to 4.2 +/- 0.2 kPa (P less than 0.005), reflecting the increase in cardiac output. Arteriovenous oxygen content difference decreased from 4.6 +/- 0.5 (CMV + PEEP) to 3.6 +/- 0.2 (CPAP) ml/100 ml (P less than 0.05), while total oxygen consumption remained unchanged. Mean systemic arterial pressure was found to increase from 10.8 +/- 0.4 to 11.6 +/- 0.4 kPa (P less than 0.05) and mean pulmonary arterial pressure changed from 2.2 +/- 0.1 to 2.4 +/- 0.1 kPa (P less than 0.05). Right atrial and pulmonary capillary wedge pressures did not change. Our observations suggest that, in terms of central haemodynamics and tissue oxygen supply, CPAP offers a noteworthy alternative weaning method and an alternative to CMV + PEEP in cases where therapy is prolonged and the patient is able to breathe spontaneously.

Rosaramicin versus penicillin G in experimental Pneumococcal meningitis.

Rosaramicin, a new macrolide antibiotic, was compared with penicillin G in the treatment of pneumococcal meningitis in rabbits. Animals were infected intracisternally with 10(4) colony-forming units of Streptococcus pneumoniae type III (rosaramicin minimal inhibitory/bactericidal concentrations, 0.25/0.5 mug/ml; penicillin G minimal inhibitory/bactericidal concentrations, 0.03/0.06 mug/ml). Treatment was instituted 96 h later. Infusion of rosaramicin at 25 mg/kg per h intravenously for 8 h produced a peak cerebrospinal fluid (CSF) drug concentration of 1.54 mug/ml (range, 0.87-3.6 mug/ml). During this infusion the numbers of pneumococci in CSF decreased from 6.2 +/- 0.5 to 3.36 +/- 1.12 log(10) colony-forming units per ml. Penicillin G, infused at 30 mg/kg per h for 8 h, reached a similar concentration in CSF but caused a greater reduction (P < 0.01) in CSF bacteria, from 6.4 +/- 0.36 to 1.3 +/- 0.67 log(10) colony-forming units per ml. Penicillin G, at 100 mg/kg per day intramuscularly for 5 days, cured 7 of 10 rabbits with pneumococcal meningitis. A higher dose, 300 mg/kg per day for 5 days, was no more efficacious: 11 of 14 rabbits were cured. Rosaramicin at 100 mg/kg per day intramuscularly for 5 days cured only 5 of 15 rabbits with meningitis, but a higher dosage regimen of that drug (250 mg/kg per day intramuscularly) produced acute, fulminant enterocecitis and death within 48 h in seven of eight rabbits. No cytotoxin was detected in the feces of one rabbit with acute enterocecitis. Thus the efficacy of rosaramicin in experimental pneumococcal meningitis, measured by bacterial clearance from CSF and by treatment outcome, was less than that of penicillin G. In addition, high-dose parenteral rosaramicin caused acute, fulminant enterocecitis in a high proportion of treated rabbits.

A model enzymic extracorporeal detoxification system.

Preliminary studies at the University of Oklahoma have incorporated the use of a continuous, seal-less blood centrifuge as an extracorporeal detoxification unit to aid in the removal of foreign chemicals from the blood. Detoxification is performed by immobilized enzymes in conjunction with a cofactor (NADPH) bound to a water-soluble macromolecule. A drug enters the device with the plasma and then passes across a semipermeable membrane which serves to retain the cofactor. At this point, a combination of the drug, the cofactor and the enzyme react to form the drug-oxide. The oxide then passes back through the membrane into the blood and back into the body. Concurrently, the macro-NADP+ is reduced by G-6-P and G-6-PD in the cofactor regeneration portion of the device. To facilitate detoxification, the centrifuge is employed to provide plasma rich in toxins, but void of potentially interfering blood components such as platelets and whole blood cells. These components tend to dilute the toxins or adhere to the interfacing membrane, decreasing the permeability of these toxins into the detoxification unit. It is felt that the centrifuge-detoxification combination will provide a potentially efficient hepatic assist device.

Biochemical studies on pili isolated from Pseudomonas aeruginosa strain PAO.

Pseudomonas aeruginosa strains PAO and PAK bear polar pili which are flexible filaments having a diameter of 6 nm and an average length of 2500 nm. Both types of pili are retractile and promote infection by a number of bacteriophages. The present communication describes the partial biochemical characterization of PAO pili isolated from a multipiliated nonretractile mutant of PAO. The observed properties are compared to those of PAK pili which were characterized previously. PAO pili were found to contain a single polypeptide subunit of 18 700 daltons. This is similar to PAK pili which contain a single polypeptide of 18 100 daltons. The amino acid composition of PAO pilin was also similar to that of PAK pilin. Neither protein contained phosphate or carbohydrate residues and both were found to contain N-methylphenylalanine at the amino terminus. Sequencing of 20 amino acid residues at the amino terminal end of PAO pilin revealed the sequence to be identical with that of PAK pilin, while tryptic peptide analyses of PAO and PAK pilin indicated that the two proteins probably contain a number of homologous regions within the polypeptide. It was concluded that PAO and PAK pili were closely related structures.

Interaction of gamma-glutamyltransferase from human tissues with insolubilized lectins.

We have characterized the binding of gamma-glutamyltransferase to three insolubilized lectins. Optimal binding was achieved in 2 hours at 25 degrees C for concanavalin A and at 4 derees C for ricinus communis agglutinin 120 and wheat germ agglutinin,and was also a function of the ratio of lectin protein to gamma-glutamyltransferase protein. The interaction of gamma-glutamyltransferase with these three lectins is specific, and release of bound enzyme by carbohydrates follows the same general order of specificity previously observed for the competition between mono or polysaccharides for the lectin carbohydrate binding sites. The binding of trypsin-solubilized liver gamma-glutamyltransferase to the three insolubilized lectins was virtually identical to that of detergent solubilized enzyme. We propose, therefore, that the release by proteolytic enzymes, of gamma-glutamyltransferase from plasma membrane matrix does not significantly alter its carbohydrate structure. We obtained great differences in binding to the three lectins between the liver, kidney, pancreatic and duodenal isoenzymes of gamma-glutamyltransferase. From this data we conclude that carbohydrate content and topography are important distinguishing features of gamma-glutamyltransferase isoenzymes.

[Pharmacological properties of procaterol, a newly synthesized, specific beta 2-adrenoceptor stimulant. Part II. Effects on the peripheral organs (author's transl)].

Pharmacological properties of procaterol (PRO) in the peripheral organs were examined in comparison with those of sulbutamol (SAL) and isoproterenol (ISO). PRO slightly enhanced twitch tension of the tibialis anterior muscle but affected little the mono- and poly-synaptic spinal reflexes and ganglionic transmission. PRO depressed spontaneous contractions of the isolated ileum, non-pregnant and pregnant uterus and also the gastrointestinal and uterine movements in vivo. PRO prolonged the time of peroral charcoal transport in the intestine. Potencies of PRO in producing these effects were between those of ISO and SAL except those on the uterus in which PRO was more potent than ISO and SAL. Pro depressed gastric and bile secretion but had no effect on pancreatic secretion. ISO, PRO and SAL reduced resistance of the common carotid, femoral and renal arteries and the relative potencies of PRO and SAL to ISO were significantly less in the renal artery than in the other arteries. In accelerating heart rate in conscious rats and dogs, PRO (p.o. or s.c.) was almost equipotent to SAL. Urine flow, GFR, RPF, free water and osmolar clearance and also excretion of electrolytes were reduced by PRO with the concomitant fall of systemic blood pressure. PRO has no effect on blood coagulation and hemolysis but inhibited carrageenin edema and an increase in permeability of blood vessels induced by acetic acid. PRO had no alpha-adrenolytic, cholinolytic and anti-histaminic effects.

Formation of aminosuccinyl peptides during acidolytic deprotection followed by their transformation to piperazine-2,5-dione derivatives in neutral media.

Prolonged acidic treatment of Boc-Leu-Asp(OBut)-Phe-NH2 with 4N HCl in acetic acid resulted in H-Leu-Asc-Phe-NH2 . HCl(Asc, aminosuccinyl), which transformed partially to cyclo[Leu-Asp(Phe-NH2)] during its purification by column chromatography on silica gel with a mixture of ethyl acetate/pyridine/acetic acid/water = 60:20:6:11, i.e. in neutral medium. Examination of the imide formation was extended to different reaction conditions (no imide derivative was detected in trifluoroacetic acid), to several protected derivatives of L-aspartyl-L-phenylalaninamide and to tripeptides containing an aspartyl residue in the middle position. It was clearly demonstrated that in strongly acidic media the imide derivatives are directly formed from the aspartyl peptides containing a free beta-carboxyl group. The influence of the C-terminal residue was greater than the N-terminal on both the rate of formation of the imide and its further transformation to piperazine-2,5-dione derivative. In aqueous ethanol the X-Asc-Y-NH2 (X, Pro, Leu; Y, Gly, Ala, Val, Phg, Phe) containing N-terminal proline are more readily transformed to piperazine-2,5-dione derivatives, but compared to simple proline dipeptides the rate of this transformation is relatively slow because of the crowdedness of the tricyclic transitional state.

Dopaminergic control of oxytocin release in lactating rats.

During suckling, anaesthetized lactating rats release regular (about every 7 min) but brief pulses of oxytocin (0.5--1.0 mu.) which produce single transient increases in intramammary pressure. Drugs which selectively impair synaptic transmission were used to determine the role of dopamine and noradrenaline in regulating this natural reflex. Diethyldithiocarbamate (100--200 mg/kg, i.v.) and alpha-methylparatyrosine (100--400 mg/kg, i.v.) which inhibit the synthesis of catecholamines both blocked the suckling-induced release of oxytocin. The milk-ejection reflex was also inhibited in a dose-dependent manner by the intravenous administration of the dopamine antagonists, fluphenazine (0.7 mg/kg), pimozide (1.4 mg/kg), cis-dupenthixol (4.5 mg/kg) and metoclopramide (6.0 mg/kg), and caused a significant inhibition P less than 0.01) of the reflex in 50% of the rats tested. The alpha-adrenoceptor antagonist phenoxybenzamine (1.4 mg/kg) was similarly effective. Dopamine (40 micrograms), bromocriptine (10 micrograms), apomorphine (100 micrograms), noradrenaline (10 micrograms) and phenylephrine (2 micrograms) injected into the cerebral ventricles evoked a sustained release of oxytocin which produced multiple increases in intramammary pressure; isoprenaline (4 micrograms) was ineffective. The release of oxytocin evoked by dopamine and noradrenaline was prevented by cis-flupenthixol and phenoxygenzamine respectively. None of the drugs used affected the mammary sensitivity to exogenous oxytocin nor were their actions modified by pretreatment with propranolol (1 mg/kg). The results suggest that the neural pathway for the reflex release of oxytocin during suckling in the rat contains both dopaminergic and noradrenergic synapses, the latter acting through alpha-adrenoceptors and being distal in the pathway to the dopaminergic component.

Light induced changes of internal pH in a barnacle photoreceptor and the effect of internal pH on the receptor potential.

1. Intracellular pH (pH1) was measured in Balanus photoreceptors using pH-sensitive glass micro-electrodes. The average pH1 of twelve photoreceptors which had been dark adapted for at least 30 min was 7.3 +/- 0.07 (S.D.). 2. Illumination reduced the recorded pH1 by as much as 0.2 pH unit. The change in pH1 was graded with light intensity. 3. When the cells were exposed to CO2 in the dark, pH1 declined monophasically. Saline equilibrated with 2% CO2; 98% O2 produced a steady reduction in pH1 of about 0.25 unit in 2--3 min. The buffering capacity of the receptor cell cytoplasm calculated from such experiments is approximately 15 slykes. 4. In the presence of HCO3-1, CO2 saline produced smaller, biphasic changes in pH1. 5. The membrane depolarization produced by a bright flash (depolarizing receptor potential) was reversibly reduced in the presence of external CO2 or by injection of H+. Iontophoretic injection of HCO2- increased the amplitude of the receptor potential. 6. In individual cells there was a close correlation between the amplitude of the receptor potential and pH1. 7. Saline equilibrated with CO2 reduced the light induced current (recorded under voltage-clamp) by 40--50% without affecting its reversal potential. 8. Exposure of the receptor to 95% CO2 saline for several minutes (pH0 5.5) not only abolished the receptor potential but also reversibly decreased the K conductance of the membrane in the dark. These effects were not reproduced by pH0 5.5 buffered saline or by a 5 min exposure to saline equilibrated with N2. 9. It is suggested that changes in pH1 induced by light modulate the sensitivity of the receptor under physiological conditions.

Studies on lithium transport across the red cell membrane. V. On the nature of the Na+-dependent Li+ countertransport system of mammalian erythrocytes.

Ouabain-resistant Na+-Li+ countertransport was studied on erythrocytes of man, sheep, rabbit, and beef. A transport system, exchanging Li+ for Na+ in a ratio of 1:1, was present in all four species. Li+ uptake by the exchange system increased 30-fold in the order man less than HK-sheep less than LK-sheep less than rabbit less than LK-beef. This order is identical to that of ouabain-resistant Na+-Na+ exchange in these species, but bears no relation to the Na+-K+ pump activity. The activity of the Na+-Li+ exchange system varied up to 7 and 16-fold among individual red cell specimens from man and beef, the variability being much smaller in sheep and rabbit erythrocytes. The affinities of the system for Li+ and Na+ were similar among the species and individuals (half saturation of the external site at about 1 mM Li+ and 50 mM Na+, respectively). 50-60% of Na+-Li+ exchange was blocked by N-ethylmaleimide in all species. p-Chloromercuribenzene sulfonate inhibited the exchange only in beef and sheep erythrocytes (60-80%). The two SH-reagents act by decreasing the maximum activity of the system, whilst leaving its affinity for Li+ unaltered. Phloretin was a potent inhibitor in all species. 1 mM each of furosemide, ethacrynic acid, and quinidine induced only a slight inhibition. The Na+-Li+ exchange of human and beef erythrocytes increased 3.5-fold upon elevation of the extracellular pH from 6 to 8.5, the pH-dependence arising from a change in affinity of the system for the cations and being similar to that reported for ouabain-resistant Na+-Na+ exchange in beef erythrocytes. It is concluded that a transport system exists in the red cell membranes of the four species which can mediate ouabain-resistant exchange of either Na+ for Na+, Na+ for Li+, or Li+ for Li+. The exchange system exhibits essentially identical transport characteristics in the four species, but shows a marked inter- and intra-species variability in maximum transport capacity and some differences in susceptibility towards inhibitors. A similar transport system is probably present also in other tissues. The exchange system seems to be distinct from the conventional Na+-K+ pump and shows no clear relation to one of the furosemide-sensitive, ouabain-resistant Na+ transport systems described in the literature.

Antiallergic action of 6-ethyl-3-(1h-tetrazol-5-YL) chromone (AA-344) on immediate hypersensitivity reaction in rats.

A newly synthesized compound, 6-ethyl-3-(1H-tetrazol-5-yl)chromone (AA-344) given intravenously or orally inhibited considerably the 72-hr passive cutaneous anaphylaxis (72-hr PCA) induced by IgE in rats. The antiallergic action of AA-344 was neither due to the antihistamine or antiserotonin effect nor was it mediated via adrenergic mechanisms. The results obtained in a double sensitization with two IgE antibodies suggest that AA-344 may not impair antigen-antibody combination but probably prevents the release of chemical mediators including histamine. This assumption was supported by observation that AA-344 inhibited a reduction in the skin histamine content caused by the 72-hr PCA, without effect on the compound 48/80-induced histamine reduction. AA-344 also partially inhibited the IgGa-mediated 3-hr PCA in rats. These results indicate that the inhibitory action of AA-344 on the immediate hypersensitivity reactions is due to prevention of the release of chemical mediators from the mast cells, by acting on some process in sequential events leading to the mediator release following antigen-antibody combination.

In vivo direct effects of cholinergic agents on the inferior mesenteric and cardiac ganglia with relation to their receptors in the dog.

The relative contribution of nicotinic and muscarinic receptors to the cholinergic transmission of the inferior mesenteric ganglion was studied in spinal dogs by recording changes in perfusion pressure of the inferior mesenteric artery as an indicator of ganglionic function. Preganglionic stimulation elicited a frequency (2.5--320 Hz)-dependent rise in the perfusion pressure, which was inhibited by i.v. hexamethonium (C6) (10 mg/kg) or atropine (0.1 mg/kg) administered after C6. Acetylcholine (ACh) (0.1--1000 microgram) administered into the inferior mesenteric artery to reach the mesenteric ganglion induced a dose-dependent rise in perfusion pressure and this dose-response curve was shifted to the right by C6 or atropine. Bethanechol (1--1000 microgram) i.a. produced a dose-dependent rise in the pressure, which was abolished after i.v. atropine. Tetramethylammonium (1--300 microgram) i.a. elicited an increase in the pressure thought the effects were decreased at larger doses, and these effects were strongly inhibited by i.v. C6. ACh (5--100 microgram) administered into the right subclavian artery to reach the cardiac sympathetic ganglia caused a dose-dependent positive chronotropic effect, which was inhibited by i.a. C6 or atropine. The results suggest that the inferior mesenteric ganglion seems to differ from the cardiac ganglia in relative contribution of nicotinic and muscarinic receptors to the cholinergic transmission.

An influence of diet on transplantation immunity.

It was discovered by chance that mice raised under otherwise entirely conventional conditions of husbandry but fed upon autoclaved diet (diet A) had stronger cell-mediated immune reactions than those of mice raised under the same conditions but with an unmodified diet (diet B) : skin allografts were rejected more quickly, transplantation tolerance was more difficult to procure and fibrosarcomas induced by the injection of methylcholanthrene (MCA) arose more slowly and less often. Analysis showed that these findings could be explained at least in part by the discovery of Mertin & Hunt (1976, p. 928) that a partial deprivation of polyinsaturated fatty acids led to an intensification of cell-mediated immunity; on the other hand, experiments with dietary mixtures made it seem unlikely that this was the whole explanation and pointed towards some positive immunopotentiation by an ingredient of autoclaved diet. This, it was proposed, might be a compound of unknown composition resulting from the interaction of vitamin A with other dietary constituents. This interpretation was not supported by direct evidence but by confirming that retinol derivatives, especially retinyl acetate, could exercise an immunopotentiation of the kind and degree under investigation: retinyl acetate could counteract the immunosuppressive action of linoleic acid, though retinyl methyl ether was ineffective. Although retinyl derivatives may protect against MCA tumours by impeding its metabolic conversion to an oncogenic form, the effects of an autoclaved diet upon skin allograft survival, the induction of tolerance and the formation of tumours is probably mediated through an immunological mechanism.

A simple analytical technique for the determination of hexavalent chromium in welding fumes and other complex matrices.

A systematic study of analytical methods for the determination of hexavalent chromium showed that currently existing techniques are unsatisfactory when used with welding fumes, the s-diphenylcarbazide method proposed by the U.S. National Institute for Occupational Safety and Health permitting less than 1% recovery of hexavalent chromium from synthetic welding fumes of known composition. A new (carbonate leaching) technique is proposed which permits better than 80% recovery of both soluble and insoluble chromium (VI). This technique is then used as part of a general method for the determination of total chemical composition as distributed among sample fractions of different solubility. The method is specifically designed for use in the analysis of small samples and is especially suitable for the routine evaluation of health risks as found in the work environment.

Congenital persistent proximal type renal tubular acidosis in two brothers.

Two brothers showed severe and persistent hyperchloraemic metabolic acidosis (capillary blood pH 7.07--7.15) due to a low renal bicarbonate threshold at 11 mmol/l. The maximal tubular capacity for bicarbonate reabsorption was reduced to about half the normal. A high dose of acetazolamide (25 mg/kg) lowered the tubular bicarbonate reabsorption substantially, indicating the presence of carbonic anhydrase. Both the glomerular filtration rate, the renal blood flow and the renal concentrating capacity were slightly reduced. The clinical characteristics were: growth retardation, mental retardation, nystagmus, corneal opacities, cataract, glaucoma and enamel defects of the permanent teeth. Serum thyroxine was pathological low without clinical signs of hypothyreosis. The erythrocytes showed an increased osmotic resistance. Autopsy of the younger brother, who died 4 1/2 years old, revealed thyroid and thymus weights of 25% of the normal. The kidney tubular cells were swollen with vacuoles. The glomeruli had a normal appearance.

Ixodes ricinus: vector of a hitherto undescribed spotted fever group agent in Switzerland.

A tick/rickettsial survey in various parts of Switzerland revealed the presence of a new, hitherto undescribed spotted fever group rickettsia ("Swiss agent") in up to 11.7% of I. ricinus collected off vegetation. Infection in ticks was found to be generalized with rickettsiae developing intracellularly and occasionally also intranuclearly. As a result of massive growth in ovarial tissues, including the germinative cells, the rate of transovarial and filial infection was 100%. The "Swiss agent" appears to be nonpathogenic for guinea pigs, domestic rabbits, and Swiss mice, but in male meadow voles (Microtus pennsylvanicus) it produces a microscopically detectable infection in the tunica vaginalis. The rickettsia grows well in tissue culture systems including chick embryo fibroblast, Vero, and vole tissue cells, when inoculated via yolk sac into 5-day-old hens' eggs, it kills 100% of the embryos after 5 to 7 days. Antigenic relatedness of the "Swiss agent" to rickettsiae of the spotted fever group was indicated by indirect and direct fluorescent antibody staining. Preliminary serologic typing by microimmunofluorescence and by microagglutination indicated that the "Swiss agent" differs from all prototype strains of spotted fever group rickettsiae studied so far.

Phenotypic changes in the chemistry of Aspergillus nidulans: influence of culture conditions on mycelial composition.

A quantitative study was made of macromolecular (nucleic acids, protein), carbohydrate and mineral (magnesium, potassium and phosphorus) components of Aspergillus nidulans in glucose limited chemostat cultures, under varying conditions of dilution rate, temperature, pH and NaCl concentration. The overall mineral content showed greatest variation in response to changes in culture salinity, which also affected the mycelial carbohydrate content. Concomitant and opposite changes in the content of cations and carbohydrates under conditions of increasing salinity may be interpreted in terms of mycelial osmoregulation. Slight variations in DNA content but gross fluctuations in the level of RNA were noted under the different cultural conditions examined. Co-ordinate changes in RNA and Mg2+ contents were evident only under certain conditions: dilution rate from 0.05--0.07 h-1 or temperature from 22--30 degrees C. The constant molar stoichiometry between RNA and Mg2+ characteristic of unicellular microorganisms was not a feature of fungal growth. The protein content was most affected by shifts of temperature and reached minimal values at 25 and 50 degrees C. The growth environment had a marked influence on the protein synthesising activity of RNA, which increased eightfold as the dilution rate was increased from 0.02--0.175 h-1, doubled within the temperature range 20--30 degrees C and fell by 50% between 40 and 50 degrees C. These observations are discussed in the context of the constant ribosomal efficiency in protein synthesis hypothesis.

Studies of acidosis in the ischaemic heart by phosphorus nuclear magnetic resonance.

1. Phosphorus-nuclear-magnetic-resonance measurements were made on perfused rat hearts at 37 degrees C. 2. With the improved sensitivity obtained by using a wide-bore 4.3 T superconducting magnet, spectra could be recorded in 1 min. 3. The concentrations of ATP, phosphocreatine and Pi and, from the position of the Pi resonance, the intracellular pH (pHi) were measured under a variety of conditions. 4. In a normal perfused heart pHi = 7.05 +/- 0.02 (mean +/- S.E.M. for seven hearts). 5. During global ischaemia pHi drops to 6.2 +/- 0.06 (mean +/- S.E.M.) in 13 min in a pseudoexponential decay with a rate constant of 0.25 min-1. 6. The relation between glycogen content and acidosis in ischaemia is studied in glycogen-depleted hearts. 7. Perfusion of hearts with a buffer containing 100 mM-Hepes before ischaemia gives a significant protective effect on the ischaemic myocardium. Intracellular pH and ATP and phosphocreatine concentrations decline more slowly under these conditions and metabolic recovery is observed on reperfusion after 30min of ischaemia at 37 degrees C. 8. The relation between acidosis and the export of protons is discussed and the significance of glycogenolysis in ischaemic acid production is evaluated.

The twenty aminoacyl-tRNA synthetases from Escherichia coli. General separation procedure, and comparison of the influence of pH and divalent cations on their catalytic activities.

A general separation procedure of the twenty E. coli aminoacyl-tRNA synthetases including either a 105 000 g centrifugation or a poly-ethyleneglycol-dextran two-phases partition fractionation, and chromatographies on DEAE-cellulose, phosphocellulose and hydroxyapatite is described. The specific activities of the synthetases have been determined after each chromatographic step and compared to their respective activities in the 105 000 g supernatant. Some aminoacyl-tRNA synthetases were obtained at 80 per cent purity. The presence of phenylmethylsulfonyl fluoride does not significantly modify either the elution patterns of the synthetases during the various chromatographic steps or their specific activities. Thus, contrarily to enzymes from various eukaryotic organisms no significant inactivation of the E. coli aminoacyl-tRNA synthetases occurs via proteolytic processes during the purification procedure. The effects of various factors: pH, magnesium, and other bivalent cations including spermidine, were tested on the aminoacylation and the [32P] PPi-ATP isotope-exchange reactions, and the optimal aminoacylation and isotope-exchange conditions determined for 18 of the 20 E. coli aminoacyl-tRNA synthetases.

Oxidation of reduced nicotinamide adenine dinucleotide phosphate by plant mitochondria.

Mitochondria isolated from various plant tissues (leaves, etiolated shoots and hypocotyls, and stem tubers) oxidize exogenous NADPH with respiratory control values and ADP:O ratios similar to those obtained with exogenous NADH as substrate. In all the mitochondria investigated, the electron-transfer inhibitors rotenone and amytal each had the same effect on the oxidation of NADPH as they had on the oxidation of NADH. The oxidation of exogenous NADPH by white potato tuber mitochondria was much more sensitive to inhibition by citrate or ethylene glycol bis-(beta-aminoethyl ether)-N,N-tetraacetic acid than was the oxidation of NADH. Mitochondria isolated from aged beetroot slices showed an increased capacity for the oxidation of exogenous NADH (compared with mitochondria from fresh tissue) but no such increase in the capacity to oxidize exogenous NADPH. These results suggest that exogenous NADPH and NADH are oxidized via different flavoproteins in plant mitochondria.

[Forced diuresis].

Forced diuresis (FD) is a frequently used method for eliminating toxins. Its therapeutic effect has yet to be evaluated by a controlled clinical trial. In the absence of such a trial its usefulness can be judged only indirectly from urinary excretion rates. In former times the usual clinical laboratory procedures could not differentiate between the unchanged toxin and its metabolites. Using more selective methods, the discussions of the effect of FD in eliminating various drugs were renewed. The problem for the indication of FD is not only a missing knowledge about the kind and amount of the ingested drug, but also when knowing it, the missing evaluation of the effect of FD on its excretion. In such cases the pharmacokinetic behaviour of the drug can be helpful. If paying enough attention to the contraindications, to the principles of electrolyte and water balance, the complication rate is low. Many infusion regimes are proposed for this treatment, but only a standardized procedure can increase the safety and efficiency of this method. More complicated and more expensive methods should be used, when there is an intoxication with a substance of high mortality or when there is no effect of FD on eliminating the toxin.

Elucidation of the inhibitory factors of yogurt against Salmonella typhimurium.

The inhibitory nature of yogurt against contaminating microorganisms has been studied extensively. Nevertheless, the factors responsible for the death of Salmonella typhimurium in yogurt have not been elucidated. An understanding of these factors is important for the determination of yogurt's safety to consumer health. Yogurt fermented for 18 h at 42 C had a stable environment with the following conditions: pH 3.85, oxidation-reduction potential -80 mV, lactic acid concentration 158 mM, and acetic acid concentration 3.7 mM. Under these conditions, lactic acid was responsible for virtually all of yogurt's bactericidal activity against S. typhimurium at 37 C. Die-off rates were observed when these conditions were reproduced artificially in milk (artificial milk yogurt) and when lactic acid was added back to 18-h yogurt from which acids were removed by passage of the whey through a Dowex 1 (Cl-) anion exchange column (cationic yogurt). Factors that augmented lactic acid inhibition of S. typhimurium were low pH and low oxidation-reduction potential. The die-off rate of S. typhimurium was more rapid in yogurt whey (yogurt minus the casein fraction) than in whole yogurt, indicating that the casein fraction was partially able to protect Salmonella.

Cell wall-associated 1,4-beta-D-xylanase in Cryptococcus albidus var. aerius: in situ characterization of the activity.

1,4-beta-D-Xylanase (1,4-beta-D-xylan xylanohydrolase; EC 3.2.1.8) has been detected in both cell-free extracts and culture fluids of the yeast Cryptococcus albidus var. aerius grown on glucose as the only carbon source. Mild acid treatment of whole cells proved that the enzyme was extracellularly located. The activity remained almost completely linked to the wall after cell breakage, only being liberated in the presence of salt at high concentration. After release, the enzyme became very unstable and so has been characterized in situ in 'permeabilized' cells. The maximum production took place at the beginning of the exponential growth phase. The optimum pH and temperature for activity were 5.0 and 40 degrees C, respectively. The enzyme degraded xylan and xylo-oligosides by an endo-splitting mechanism giving xylobiose, xylotriose and xylose as the main end-products. Activation energy and kinetic constants for xylan degradation were determined. Several metal ions such as Ag+ and Hg2+ inhibited the enzyme. The possible function of this endo-xylanase in Cr. albidus var. aerius is discussed.