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Specific immune recognition by lymphocytes: an evolutionary perspective.

In this review, we analyze data pertinent to the origins of specific immune recognition by lymphocytes. The phenomena of immunity in invertebrates and the cells that might be involved in these processes are considered. We conclude that the existence of vertebrate-type immunocompetent lymphocytes in invertebrates is not yet proven. All vertebrates apparently possess immunologically competent lymphocytes, and the following conclusions may be drawn: (1) Specific antigen recognition by lymphocytes of all vertebrates appears to be mediated by membrane-bound immunoglobulins. These immunoglobulins are, in all probability, distinct from secreted, serum immunoglobulins (antibodies), although serum and surface immunoglobulins share combining sites for antigen which are formed of variable regions. (2) There is evidence for similar functional divisions among the immune systems of all vertebrates, as reflected in the results of anatomical, functional, and physicochemical investigations of lymphocytes from animals as diverse as fish and mammals.

Effect of pH on bile salt degradation by mixed fecal cultures.

Stoll specimens from 3 healthy volunteers were cultured under an-aerobic conditions in brain heart infusion broth with and without the addition of cholate, deoxycholate of chenodeoxycholate. The initial pH of the medium was adjusted to 5.5, 6.3, 7.3 (unadjusted), 8.0, and 9.0. Cell-free extracts prepared from the resulting bacterial growth contained increased levels of NAD- and NADP-dependent 3alpha-, 7alpha-, and 12alpha-hydroxysteroid oxidoreductases when the initial pH was 8.0 or 9.0 and depressed levels of these activities when the initial pH was 5.5 or 6.3 (as compared to control values obtained at 7.3). At pH 5,5 all activities except NAD-dependent 7alpha-hydroxysteroid oxidoreductase were absent. A powerful selective effect was imposed on NAD-dependent 7alpha-hydroxysteroid oxidoreductase when deoxycholate or chenodeoxycholate were incorporated into or chenodeoxycholate were incorporated into the medium. Thin-layer chromatography of either extracts of cholate-containing, acidified spent bacterial medium showed alkaline or neutral (optimal at pH 8). The precent hydroxyl group estimations at the 3alpha-, 7alpha-, and 12alpha-positions revealed an increase in disappearance of OH groups at all three positions with increasing initial pH value. The order of extent of bioconversion was 7alpha-OH greater than 3alpha-OH; at pH 8 AND 9, approximately 90% 7alpha-OH bioconversion was observed. Spent bacterial media and a number of commercial secondary bile salts were all negative in the Ames' assay for mutagenicity.

[Treatment of general psychovegetative disorders with a single night dose of dipotassium-clorazepate within a multicenter study (author's transl)].

The therapeutical effect of Tranxilium was studied in an open multicenter study by 66 general practitioners and 26 specialists. 1.027 patients were treated with Tranxilium 20, given in a single dose at night, for a period of three weeks. During this time, the patient's psychic status and functional organic disorders were assessed by means of rating scales. A complete medical assessment followed at the end of the study. States of anxiety, restlessness, depressive mood, irritation and insomnia as well as functional organic disorders improved significantly (p is less than 0.001) already after the first week of treatment. At the end of treatment, with the majority of patients the above symptoms had either completely disappeared or were found only to a small extent. In 85% of the patients, all investigators rated the stabilization and brightening effect on psychic conditions as well as the harmonizing effect on disturbed organic functions as "very good" to "good". In 89% of the patients, the overall therapeutic success was judged as "very good" to "good". The overall tolerance was excellent in 96% of the cases.

On the capacity of the beta-oxidation of palmitate and palmitoyl-esters in rat liver mitochondria.

The beta-oxidation of palmitate, palmitoyl-CoA and palmitoyl-L-carnitine proceeded at a high rate in isolated rat liver mitochondria. At high concentrations (100 nmol/mg protein) the oxidation of palmitate and palmitoyl-CoA was only partly carnitine dependent. All substrates were most rapidly oxidized in the presence of oxaloacetate and state 3 conditions. Succinate inhibited beta-oxidation especially in state 4 conditions. beta-Oxidation was faster in hypotonic than in isotonic medium both in state 3 and state 4 conditions. Hypertonicity inhibited beta-oxidation. The initial formation of palmitoyl-CoA from palmitate, CoA and ATP was faster than the oxidation of palmitate under identical conditions. The presence of bovine serum albumin inhibited the beta-oxidation, especially with palmitoyl-CoA or free palmitate as the substrates. Mitochondria contain a palmitoyl-CoA hydrolase which may influence the available intramitochondrial palmitoyl-CoA. The present results demonstrate no single rate limiting step in the beta-oxidation in vitro. Both the NADH/NAD ratio, competition for the respiratory chain, the level of ADP, binding of palmitoyl-CoA to extramitochondrial protein, and possibly intramitochondrial hydrolysis of palmitoyl-CoA all seem to influence the rate of beta-oxidation in vitro. It is suggested that in vivo the most important factor is the availability of acyl-CoA to the outer carnitine palmitoyl-transferase of the mitochondria.

Circulatory shock in pregnant sheep. IV. Fetal and neonatal circulatory responses to hypovolemia--influence of anesthesia.

Hemodynamic responses to hypovolemia were investigated in unanesthetized, unstressed fetal lambs and in acute fetal and neonatal preparations. The unstressed fetus tolerated twice the amount of blood loss of the acutely studied fetus or newborn lamb and with a lesser hypotension. Hemodynamic behavior of the newborn lamb and fetus anesthetized with pentobarbital during hypovolemia was markedly different from that of the fetus studied under spinal anesthesia or chronically. Besides tolerating greater blood loss, the unstressed fetus reversed the state of hypovolemic shock rapidly as contrasted to the stressed fetus which was unable to do so even with total blood reinfusion. All animals exhibited bradycardia in response to hypovolemia. The following conclusions were drawn: (a) cardiovascular response to hypovolemia in the perinatal period depends on the initial status of the animal, (b) the fetus tolerates a greater degree of blood loss than a newborn or adult animal, and (c) anesthesia and stress of surgery modify considerably circulatory behavior during blood loss.

[Streptokinase in the treatment of deep venous thrombosis and pulmonary embolism].

Fifty-two deep venous thromboses and 35 pulmonary emboli were treated by Streptokinase administered in accordance with a standard protocol. Radiological examinations revealed total lysis of clots in 22 cases, partial lysis in 42 and failure in 23. The latter more commonly involved venous clots than pulmonary emboli. Early treatment was more effective (21 total lyses out of 22) than late treatment. However, in venous thrombosis, late treatment may give partial lysis and free important venous junctions. With standard treatment, lysis was biologically correct in 70 p. 100 of cases. It was inadequate in 20 p 100 of cases and nil in 10 p. 100 of cases. The results could thus have been improved by treatment established and adjusted in the light of laboratory results. The extent of the thrombosis played an important role. Total lysis was obtained in 9 out of 10 cases of localised deep venous thrombosis. In pulmonary embolism there was an average gain of approximately 30 p. 100 in obstructed surface area. However, in these latter cases, it is important to take into account not only the pulmonary surface area obstructed but also the origin of the clots.

[Process of fumigatin and spinulosin formation by Aspergillus fumigatus Fres and polarographic assay of these toxins (author's transl)].

A strain of Aspergillus fumigatus (Fresenius) isolated from a milk food for calves was grown on a culture medium containing added saccharose. The purpose was to study the synthesis of two recently discovered mycotoxins, fumigatin and spinulosin. The work was performed under many different conditions of temperature, pH and inoculum. These mycotoxins were measured by analytical differential pulse polarography. Correlations were observed between the growth rate of A. fumigatus and variation in pH of the medium and the formation of fumigatin, which is only possible when pH falls to less than 4.0. Fumigatin appears promptly at the beginning of the growth phase of the fungus but quickly disappears. The production of metabolite depends on limited conditions of culture. Spinulosin, very similar to fumigatin, is substituted for fumigatin in slightly different conditions. During growth, the fungus degrades both metabolites. The nature of the substitution and the reason of these modifications have not been investigated. Fumigatin and spinulosin formation is observed in both toxigenic and non-toxigenic strains.

Uptake and metabolism of alpha-aminoadipic acid by Penicillium chrysogenum Wis 54-1255.

The uptake of 1-14C-DL-alpha-aminoadipate in resting mycelium of Penicillium chrysogenum Wis 54-1255 and its metabolism during benzylpenicillin formation were studied. The pH optimum for uptake at 25 degrees C was 6.4. Over a range of concentrations from 0.01--1.0 mM, approximately 45% of 1-14C-DL-alpha-aminoadipate was taken up by carbon-starved mycelium. 14CO2 was formed at a low rate, and the total formed amounted to only 1--3% of the 1-14C-DL-alpha-aminoadipate supplied. The intracellular pool of alpha-aminoadipate appears to be expandable, depending on the concentration of alpha-aminoadipate in the medium. The rate of penicillin synthesis depended on the intracellular concentration of alpha-aminoadipate. Penicillin biosynthesis achieved half of the maximum rate at an intracellular concentration of 0.06 nmol alpha-aminoadipate/mg dry cell weight. This low concentration, the result of adding 0.01 mM DL-alpha-aminoadipate to the medium, was sufficient to reverse the inhibition of penicillin biosynthesis caused by 10 mM extracellular L-lysine. Aminoadipate appears to be recycled during penicillin formation. Labeled alpha-ketoadipate was formed from alpha-aminoadipate to the extent of about 25%.

[On the toxicology of carbromal. IV. Binding of carbromal and its hypnotically active metabolites to human plasma proteins (author's transl)].

The binding of carbromal and its metabolites bromoethylbutyramide and ethylbutyrylurea to human plasma proteins was investigated in vitro by use of Sephadex-gelfiltration, equilibrium dialysis and ultrafiltration. No differences appeared in the binding characteristics of human plasma and of human albumine. In a concentration range between 3.10-8 and 1.5.10-6 moles/ml about 40% of the carbromal, and in a concentration range between 3.10-8 and 1.10-5 moles/ml about 30% of the bromoethylbutyramide are bound to plasma proteins. Proteinbinding of ethylbutyrylurea was found to be less than 5%. The binding constants, Ka, to human albumine and the binding energies deltaF 0 were found to be in the range of 0.5--1.2.10(3) L/Mol and 1.7--4.4 kcal/Mol, respectively. Protein binding of carbromal, bromoethylbutyramide, their chlorinated analgous compounds, chloroethylbutyrylurea and chloroethylbutyramide, and of ethylbutyrylurea is strongly correlated to the partition coefficients of these compounds between n-octanol and water, indicating that the intensity of proteinbinding depends on the hydrophobic character of the substances tested.

Hyperanodic forms of human glucose-6-phosphate dehydrogenase.

Pure glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) is transformed into 'hyperanodic forms' when incubated at acidic pH and in the presence of NADP+ with excess of glucose-6-phosphate or with some 'NADP+ modifying proteins' purified from the same cells. The enzyme hyperanodic forms exhibit low isoelectric point, altered kinetic properties and high lability to heat, urea, and proteolysis. Differences between hyperanodic and native forms of glucose-6-phosphate dehydrogenase are also noted by microcomplement fixation analysis, ultraviolet absorbance difference spectrum and fluorescence emission spectrum. Drastic denaturation of the enzyme by urea and acid treatment did not suppress the difference of isoelectric point between native and hyperanodic forms of glucose-6-phosphate dehydrogenase. From our data we suggest that the conversion into hyperanodic forms could be due to the covalent binding on the enzyme of a degradation product of the pyridine nucleotide coenzyme. This modification could constitute a physiological transient step toward the definitive degradation of the enzyme.

Reaction of alpha-mannosidase from Phaseolus vulgaris with group-specific reagents. Essential carboxyl groups.

When the pKm of alpha-mannosidase was determined at different pH values, the results indicated that ionizable groups with pK values of approx. 3.8 and 5.7 could be essential. Modification with carbodiimide or Woodward's Reagent K abolished the enzyme activity. The substrate analogue, alpha-methyl-D-mannoside, protected the enzyme against inactivation. Incorporation of a 14C-labeled nucleophile reagent in the presence or absence of the analogue suggested that 2--4 carboxyl groups were protected. Exchange studies indicated that the essential Zn2+ could be bound to such groups. There was no indication that hydroxyl groups, sulphydryl groups, guanidino groups or amino groups take part in the catalytic activity.

The relative electron affinities of the alpha and beta chains of oxyhaemoglobin as a function of pH and added inositol hexaphosphate. An electron spin resonance study.

Exposure of aqueous glasses of oxyhaemoglobin to 60Co gamma-rays at 77K results in electron addition to the FeO2 unit, the ESR spectrum for the alpha-chain electron adduct being well separated from that for the beta-chain. The relative yields of these two centres has been measured in the pH range 4.5 to 8.5, with or without added inositol hexaphosphate. We find that, in the absence of inositol hexaphosphate, the yield of beta-chain adduct is almost equal to that of the alpha-chains in the pH 4--5 region, but these rapidly diverge with increasing pH, the beta-yield increasing and the alpha-yield decreasing. After a plateau in the pH 6--8 region, the yield of beta-chain adduct decreases, but that of the alpha-chain adduct remains constant. In the presence of an excess of inositol hexaphosphate the pH change for the beta-adduct remains, but at low pH values the yield of the alpha-adduct is much greater than that of the beta-adduct. This constraint is removed with a pK of approx. 7.7 and at high pH values the yield of the beta-adduct is once again greater than that of the alpha-adduct. These results are significant in that they suggest that the electron affinities of the alpha and beta chains in oxyhaemoglobin are a function of pH, with that of the beta-chains being greater than that of the alpha-chains in the neutral region. Also inositol hexaphosphate clearly binds to one or both chains, and this has the effect of reversing the relative electron affinities of the two chains.

Pheochromocytoma in the modern context.

The hypertensive patient faces an uncertain future. Therefore a search for those cases in which the condition is potentially curable is eminently worth while and has become increasingly productive because of the specific diagnostic aids now available and because of the present safety of surgical intervention. Detection and localization of a pheochromocytoma allows a planned surgical approach. Correction of hypovolemia preoperatively ensures a safe course postoperatively. The use of blocking agents pre- and peroperatively prevents the hazardous hypertensive crises and arrhythmias that were a part of early surgical experience. Monitoring of central venous and arterial pressures as well as the electrocardiographic tracing during the operation permits prophylactic therapy when necessary. On the basis of a series of 31 patients the authors compare many aspects of the current management with earlier experience of pheochromocytoma in the same institution. The incidence of extra-adrenal lesions (3.8%), multicentric tumours (3.8%) and malignant change (11.5%) was lower in this group of patients than that usually reported. Abdominal exploration remains the approach of choice in most cases. Treatment of the solitary pheochromocytoma has become straightforward. However, management of the familial syndromes and the malignant from of the disease still requires careful scrutiny.

Atenolol and three nonselective beta-blockers in hypertension.

The object of this study was to establish whether cardioselectivity of atenolol confers any advantage over noncardioselective beta-blockade in the treatment of hypertension. A dose of atenolol was established on the basis of morning mean systolic blood pressure (mean of 5 readings) in 27 long-standing hypertensive patients previously controlled on one of three nonselective beta-blockers: propranolol, oxprenolol, or pindolol. Most patients were also taking a diuretic. A crossover trial was then conducted of atenolol and the previous nonselective beta-blocker, each drug being given for 8 wk in randomized order. Other drugs were kept constant. At the end of each 8-wk period a morning test of blood pressure and pulse rate was done, an 11:30 A.M. blood sample was taken for estimation of drug concentration, and spirometry was performed. During the eighth week a glucose tolerance test, fasting lipids, and other biochemical and hematologic estimations were done. On a separate occasion a late morning study was done on the response of blood pressure and pulse rate to three kinds of stress: bicycle ergometer, mental arithmetic, and handgrip. At dosage levels of atenolol giving a mean resting systolic blood pressure equal to that during nonselective beta-blockade, diastolic levels on atenolol tended to be lower at rest and during the mental and handgrip forms of stress. Serum creatinine levels on atenolol were lower than during nonselective beta-blockade. Anti-dioxyribonucleic acid (DNA) titers remained normal in all patients. There was no difference in lung function. There was little difference in glucose and insulin levels during glucose tolerance tests in these patients, half of whom were diabetic. There were no serious side effects but there were a few surprising ones such as vivid dreams in three and muscle cramps in one patient.

Cobalt hemoglobin: pH dependence of Adair constants and the Bohr effects.

Precise oxygen equilibrium curves have been obtained for cobalt hemoglobin at pH values from 5.5 to 8.2. The Hill plots are symmetric having asymptotes with slopes of unity. At pH 7.0, cobalt hemoglobin has p0.5 = 116 toor (15.45 kPa), pm = 117 torr (15.58 kPa) and a Hill coefficient of n = 1.72. The values of n decrease slightly with either decrease or increase of pH; the protein is almost non-cooperative at pH greater than 8.2. The Adair constants have been calculated with a non-linear least-squares program. From deltalnpm/deltapH a maximum of 2.5 Bohr protons was calculated at physiological pH values. The majority of alkaline Bohr protons are released after binding of the first and the third oxygen with maxima at pH 7.6 and 7.3, respectively. The acid Bohr effect was also observed with the majority of the protons taken up following the first and third oxygen bound. Smaller alkaline Bohr effects were obtained by differential titration and at higher pH than that calculated from oxygen equilibria. The discrepancy can be largely attributed to the binding of salt components to cobalt hemoglobin.

[Effects of a new beta-blocker (CI 775) on left ventricular hemodynamics at rest and during exercise (author's transl)].

The effects of a new beta-blocking agent (CI 778) were studied in 8 patients in whom non-invasive data suggested absence of significant organic heart disease. The left ventricular hemodynamics at rest and during bicycle exercise were measured before and after intravenous administration of 0,9 mg/kg body weight. With exercise there was a significantly smaller increment of heart rate (18%) after beta-blockade. Stroke volume index at rest was significantly lower (17%) after administration of CI 775; the difference disappeared with exercise. There was an 18% decrease of the resting cardiac output after CI 775 and a 23% decrease with exercise. Significant changes at rest and with exercise indicating a negative inotropic action of CI 775 were noted for max dP/dt and peak measured velocity of circumferential fiber shortening (Vpm). The left ventricular enddiastolic pressure with exercise increased with borderline significance by 41% after CI 775. Also left ventricular stroke work index at rest and with exercise decreased moderately (n.s.), the systemic arterial resistance changed to the same extent as cardiac output decreased. Also the arterial venous oxygen difference increased after CI 775 only according to the decrease of cardiac output. The data suggest the hemodynamic properties of CI 775 are located between propranolol and practolol within the spectrum of available beta-blockers.

Purification and properties of a new cathepsin from rat liver.

1) A lysosomal protease, a new cathepsin that inactivates glucose-6-phosphate dehydrogenase [EC 1.1.1.49] and some other enzymes and differs from cathepsin B [EC 3.4.22.1] was purified about 2,200-fold from crude extracts of rat liver by cell-fractionation, freezing and thawing, acetone treatment, gel filtration, and DEAE Sephadex and CM-Sephadex column chromatographies. 2) The new cathepsin was markedly activated by the thiol-reagent, 2-mercaptoethanol and inhibited by monoiodoacetate. 3) The molecular weight of the new cathepsin was found by Sephadex G-75 column chromatography to be 22,000, which is smaller than that of cathepsin B. 4) The optimum pH of the enzyme for inactivation of glucose-6-phosphate dehydrogenase was pH 5.0--5.5. The enzyme was unstable in alkali and on heat treatment. 5) The rates of inactivation of glucose-6-phosphate dehydrogenase, apo-ornithine aminotransferase [EC 2.6.1.13], apo-tyrosine aminotransferase [EC 2.6.1.5], apo-cystathionase [EC 4.4.1.1], glucokinase [EC 2.7.1.2], glyceraldehyde-3-phosphate dehydrogenase [EC 1.2.1.12], and malate dehydrogenase [EC 1.1.1.37] by the new cathepsin were higher than those by cathepsin B. However aldolase [EC 4.1.2.13] was inactivated more rapidly by cathepsin B than by the new cathepsin. Lactate dehydrogenase [EC 1.1.1.27], glutamate dehydrogenase [EC 1.4.1.2] and alcohol dehydrogenase [EC 1.1.1.1] were not inactivated by either cathepsin. Unlike cathepsin B, the new cathepsin scarcely hydrolyzes N-substituted derivatives of arginine.

Immobilization of rat lung soluble guanylate cyclase on alkyl-agarose gels.

The soluble guanylate cyclase from rat lung was immobilized by absorption rather than covalent attachment on hexyl-, octyl-, or decyl-agarose. The enzyme retained activity after being bound to these matrices and could be compared to the soluble, mobile form of the enzyme. Compared to the soluble enzyme, the immobilized guanylate cyclase had a lower apparent maximal velocity and a higher apparent Km for MeGTP in the presence of Mg2+, Ca2+, or Mn2+. The apparent maximum velocity was reduced to the same extent by hexyl-, octyl-, or decyl-agarose, but the reduction in activity was greater with Mg2+ than with Ca2+ or Mn2+. Both the soluble and immobilized guanylate cyclase displayed concave downward patterns on double reciprocal polots as a function of Mn2+, and Ca2+ caused apparent activation of either form of the enzyme. MnATP appeared to be a linear competitive inhibitor with respect to MnGTP for both forms of the enzymes but the ki was 3 micron for the soluble form and 30 micron for the immobilized form. These results demonstrate that the soluble form of guanylate cyclase from rat lung retains many of its basic properties after being immobilized on a hydrophobic matrix; however, rather pronounced decreases in the maximum velocity and increases in the apparent Michaelis constant for MeGTP, particularly for MgGTP, are observed upon immobilization.

Models for assessing the effect of toxicants on immunocompetence in mice. Part I: The effect of diphtheria, pertussis, and tetanus vaccine on antibody response to type III pneumococcal polysaccharide.

This study characterizes the response of BALB/c mice to Type III pneumococcal polysaccharides (S3) administered alone or with a vaccine consisting of diphtheria toxoid, pertussis vaccine, and tetanus toxoid (DPT). The usefulness of this vaccine in toxicological protocols for determining immunocompetence is discussed. There were significant differences in antibody titers depending upon the route of injection and whether the S3 was administered in saline or mixed with DPT. Subcutaneous injections induced higher antibody titers to S3 than did intraperitoneal injections. The effect of DPT varied depending upon the manner of administration. Subcutaneous injections of DPT mixed with S3 increased the S3 antibody titers to a dose of 0.1 microgram S3, but had little or no effect at 0.25, 0.5, and 0.75 microgram. Antibody titers were suppressed only at the highest dose administered (1.0 microgram). On the other hand, when administered intraperitoneally DPT has a consistently suppressive effect on 5 day peak S3 antibody titers. Intraperitoneal injections of DPT appeared to delay the rate of antibody induction, since antibody titers at 14 and 21 days were higher in the DPT treated mice than in those animals receiving S3 alone.

The effect of some formulation and process variables on the surface roughness of film-coated tablets.

The effect of some formulation and process variables on the surface appearance of film-coated tablets has been examined by measuring the arithmetic mean roughness, Ra, values across the faces of tablets before and after they were coated with hydroxypropyl methyl-cellulose. For all tablet cores except those that were very porous, film coating resulted in an increasing surface roughness; for very porous cores a decrease was found. Tablets with rough surfaces were produced by coating with low molecular weight grades of the polymer; increasing the polymer molecular weight resulted in a smoother finish with a minimum roughness at intermediate molecular weight grades. Increasing the polymer concentration above 2% w/v caused an increase in roughness as did increasing film thickness to 140 micrometer. There was a minimum in roughness at film thickness of 20 micrometer. The addition of pigment in low concentrations (0--25% v/v) caused a marginal increase in surface roughness but at concentrations above the critical pigment volume concentration, the surfaces were very rough. The results illustrate the potential of the method in the optimization of film formulations and process conditions during product development.

Loss of hydrophobic amine from solution by adsorption onto container surfaces.

The apparent loss of the hydrophobic amine drug alpha-[(dibutylamino)methyl]-6,8-dichloro-2-(3',4'-dichlorophenyl)-4-quinoline-methanol monohydrochloride from solution due to its adsorption onto the surface of its storage container was studied. The drug appeared to be adsorbed only as the free base. Therefore, any perturbations to the solution phase that will help solubilize the drug and thus lower its chemical potential will minimize adsorption. Multilayer drug adsorption to the container surfaces appeared to take place, with some evidence of a highly organized system in the adsorbed phase. Adsorption was minimized when the heterogenous polar functionalities on glass surfaces were covered by a layer of silicone or methacrylate polymer, which yielded less reactive, more hydrophobic surfaces. Loss was also minimized when the environment was kept acidic (pH less than or equal to 4,8), the drug was dissolved in a proton-donating solvent (e.g., chloroform), and an ion-pairing agent (e.g., trichloroacetate) was present to solubilize further the monocationic form of the drug in organic media.

Comparison of adenylate cyclase activity and in vitro secretion in the parotid and sublingual glands of the mouse.

1. Adenylate cyclase (EC 4.6.1.1) activity has been determined in the parotid and sublingual glands of the mouse. Optimal activity of the enzyme was obtained at a Mg2+-concentration of 8 mM at pH 8.2, using AMP-PNP as the substrate. 2. Cyclic AMP degradation during the adenylate cyclase assay was relatively high in both the homogenate and the 40,000 g pellet-fraction of the glands. Theophylline was effective in inhibiting this degradation only in the parotid hemogenate, whereas isobutylmethylxanthine inhibited the cyclic AMP degradation in both salivary glands. Using the latter phosphodiesterase inhibitor, we observed a higher adenylate cyclase activity in the sublingual glands than in the parotid glands. 3. Various receptor-selective sympathetic and parasympathetic agonists and antagonists have been tested for their capacity to influence the adenylate cyclase activity and the glycoprotein secretion in the parotid and sublingual glands of the mouse, in vitro. (a) The parotid glycoprotein secretion was increased by beta-adrenergic agonists, which stimulate adenylate cyclase, and by cholinergic muscarinic drugs, which do not activate this enzyme. The adrenergic alpha-agonist phenylephrine appeared to be involved neither in the glycoprotein secretion nor in the direct regulation of the adenylate cyclase activity. (b) The sublingual protein and mucin secretion was increased by cholinergic muscarinic agents. The over-all protein secretion was stimulated also by phenylephrine, but this effect could be blocked by propranolol. The adenylate cyclase activity in membrane preparations was not stimulated by these secretogogues.

Fatty acid biosynthesis in yeast.

Fatty acid synthetase and acetyl CoA carboxylase mutants have been used to study several aspects of fatty acid biosynthesis in yeast: the contribution of the various enzymes of fatty acid biosynthesis and modification to the overall cellular fatty acid composition, the mechanism of fatty acyl chain elongation in yeast, the molecular structure and the reaction mechanism of the fatty acid synthetase complex and the genetic control of the biosynthesis of this multi-enzyme system. Genetic and biochemical evidence suggest an alpha6beta6 molecular structure of this complex, where alpha and beta are multifunctional proteins comprising, respectively, 3 and 5 of the various fatty acid synthetase component functions. The two subunits alpha and beta are synthesized on two different, unliked genes, fas 2 and fas 1. The biosynthesis of both is coordinated. The various component enzyme activities reside in distinct domains on the multifunctional chains. While most domains appear to be functionally independent, the three acyl transferases exhibit extensive mutual interactions. It is suggested that the biosynthesis of a multifunctional protein is favoured on the grounds of kinetics and regulation as compared with the formation of a complex of the corresponding individual enzymes.

A comparison of the circular dichroism spectra of synthetic DNA sequences of the homopurine . homopyrimidine and mixed purine- pyrimidine types.

We have obtained the ultraviolet circular dichroism spectra of two repeating trinucleotide DNAs, poly [d(A-G-G).d(C-C-T)] and poly[d(A-A-G).d(C-T-T)], that have all purines on one strand and all pyrimidines on the other. These spectra, together with spectra of other synthetic polymers, can be combined to give 3 first-neighbor calculations of the spectrum of poly[d(A).d(T)] and 2 first-neighbor calculations of the spectrum of poly [d(G).d(C)]. The results show (1) that first-neighbor calculations utilizing only spectra of homopurine.homopyrimidine DNA sequences are no more accurate than are similar calculations that involve spectra of mixed purine-pyrimidine sequences, demonstrating that double-stranded homopurine.homopyrimidine sequences do not obviously belong to a special class of secondary conformations, and (2) that the wavelength region above 250 nm in the CD spectra of synthetic DNAs is least predictable from first-neighbor equations, probably because this region is especially sensitive to sequence-dependent conformational differences.

[Preparation of extracellular ribonuclease form Actinomyces rimosus 994].

By sequential acid treatment, gel filtration and KM-cellulose sorption a 18--20-fold purified preparation of ribonuclease with a yield of 50--60% was obtained from the culture liquid filtrate of Actinomyces rimosus 994. The preparation had a high specific activity of 450,000--600,000 units/mg protein, contained 85--98% protein, insignificant amounts of carbohydrates and hydroxytetracycline, and no quantities of DNase, phosphomonoesterases, phosphodiesterase or proteases. In RNA degradation (preparation of the total yeast RNA of the Sigma Co.) optimal results were obtained at 50 degrees C and pH 7.0--7.2 in phosphate buffer and 7.6--8.0 IN Tris-HCl buffer. The preparation was stable at high temperatures (80--100 degrees) in the wide pH range and during storage in the lyophilized form and in buffer solutions. RNase effect was inhibited by zinc, copper, iron and cobalt cations and activated by beta-mercaptoethanol, citrate and EDTA. Protamine sulphate and urea in low concentrations (0.01% and 1--4 M, respectively) accelerated and in high concentrations (1% and 8 M, respectively) terminated the enzyme reaction. With respect to many properties RNase from Act. rimosus 994 was similar to extracellular RNases, produced by other actinomycetes and fungi.

The effects of some stimulants and anti-psychotic drugs on urinary unconjugated tyramine levels in the rat.

The effects of intraperitoneal injections of amphetamine and methylphenidate (ritalin) at relatively low and high doses, the anti-psychotic drugs chlorpromazine and haloperidol and combinations of haloperidol with methylphenidate and amphetamine on the urinary excretion levels of unconjugated meta and para tyramine in the rat have been investigated. With the exception of a high dose of methylphenidate, none of the drug treatments changed significantly the urinary excretion level of para tyramine. meta Tyramine was significantly reduced by a low dose of amphetamine and a high dose of methylphenidate but significantly increased by a high dose of amphetamine. Such effects are different from those reported by Juorio (1977a, b) and Danielson, et al (1976) with respect to the rat striatum and mesolimbic systems where para tyramine was decreased and meta tyramine increased. The effect of adding anti-psychotic drugs in these latter studies was to potentiate their differential effects. Such differences indicate perhaps variations in the metabolism of the tyramines in peripheral tissues as compared with certain brain regions.

Myocardial protection during aortic valve replacement. Physiological and metabolic effects of selective coronary perfusion on the fibrillating heart.

The physiological effects and certain aspects of cardiac metabolism were studied in 14 patients undergoing primary aortic valve replacement. The operations were performed under moderate hypothermia (30 degrees +/- 2 degrees C) and blood for coronary perfusion was taken from a sidebranch of the arterial line. The majority of the hearts went spontaneously into ventricular fibrillation at some stage of the operation. In spite of the high resistance measured in the coronary perfusion cannulae, an intraluminar coronary blood flow of 380 ml/min was recorded. The myocardial oxygen uptake decreased to 6.0 ml/min at 29 degrees C compared with 20.0 ml/min at 36 degrees C. The elevated coronary sinus lactate throughout the period of coronary perfusion and the increasing level of ASAT-enzyme indicated that this technique could not fully protect the myocardium from ischaemic changes. One patient died of myocardial infarction and two others needed vasopressor support postoperatively, in spite of documented effective coronary perfusion throughout the procedure. Cannulation of the coronary sinus is a valuable adjunct for the study of cardiac metabolism during ECC and it was accomplished without complications.

Orchiopexy in prepubertal boys. Five-year survey.

A series of 141 prepubertal boys with undescended testes operated on in a provincial teaching hospital has been analyzed five years after operation. The main features noted at presentation were the mature age of the patients and the small number of boys referred by pediatricians. The incidence of unsatisfactory results was 36% in unilateral and 35% in bilateral operations. The majority of the patients (81%) were referred for surgery after the age of five years, commonly regarded as the most suitable time for surgical correction. Three patients required a primary orchiectomy for a small atrophic testis, while 2 patients had an orchiectomy done on a previously operated testis. The complication rate for the series was 4.5%. Testicular biopsy was not done at the time of operation, and no patients were referred for semen analysis. Eight patients underwent a second orchiopexy after the first operation failed. In 6 patients an atrophic testis developed after the second procedure. The need for more than one postoperative examination is stressed in view of the fact that an initially favorable result may not persist since the testes may be found, at a later date, to have retracted into an unsatisfactory position. The reasons for the poor results are discussed and compared briefly with previous reports.

Effects of acute hypercapnia and hypocapnia on plasma and red cell potassium, blood lactate and base excess in man during anesthesia.

In order to test the relationship between changes in plasma potassium concentration and pH changes of respiratory origin, we produced hypercapnia (mean PaCO2 71 mmHg = 9.5 kPa) in a group of 17 patients and hypocapnia (mean PaCO2 21 mmHg = 2.8 kPa) in another 20 patients during neurolept analgesia and intraabdominal operations. A control group of 19 patients was studied under normocapnia but otherwise identical conditions. During hypercapnia, serum potassium rose, deltaK/deltapH amounting to -0.82, -1.05 and -1.34 after 30, 60 and 90 min, respectively. During hypocapnia, serum potassium decreased, deltaK/deltapH being a little more negative than during hypercapnia (mean values -1.62, -2.44 and -1.60). Red cell potassium concentration decreased in all three groups to a similar extent. Blood lactate levels during hypercapnia decreased to 75% of control and during hypocapnia rose to a maximum of 186% of control. In order to obtain reasonable values for base excess in primarily respiratory acid-base disorders, it is necessary to use nomograms based on in vivo ECF-CO2-titration curves. With this premise, hypercapnia or hypocapnia in our patients was not associated with significant changes in base excess.

Effect of intraantral pH on basal gastrin release into the circulation and antral lumen in anesthetized cats.

In acute experiments on cats antral pouches were perfused with solutions of different pH (1-13). After antrum passage the gastrin levels in the perfusates were measured with radioimmunoassay and the amounts of gastrin released into the antral lumen per minute calculated. The venous gastrin levels were determined concomitantly. Small amounts of gastrin (1,000--1,500 pg/min) were released into the antrum during perfusion with 0.1 M HCl. Subsequent perfusion with 0.15 M NaCl (pH 6.8) did not significantly increase the release of gastrin. On the other hand, 0.1 M phosphate buffer (pH 7.4) caused a dramatic augmentation of the gastrin output into the antral lumen (approximately 17 fold). A concomitant increase of peripheral gastrin levels was observed. Also other alkaline solutions such as 0.15 M NaHCO3 (pH 8), 0.15 M Tris buffer (pH) or 0.01 and 0.1 M NaOH (pH 12 and 13) promoted the release of gastrin. It is discussed whether the gastrin release at alkaline pH is induced by the alkaline pH itself or by anions such as HPO-4, HCO-3 and OH-. The apparent effect of pH could then be due to the formation of these ions at higher pH.

Cefoxitin sodium compatibility with intravenous infusions and additives.

The compatibility and stability of cefoxitin sodium in solution with a series of frequently used intravenous infusion fluids and injectable additives were studied. Cefoxitin sodium's stability in various solutions was measured by ultraviolet spectrophotometry, iodometry, thin-layer chromatography, high-pressure liquid chromatography, ion-exchange chromatography and microbiological assay. Cefoxitin sodium was shown to maintain 90% of its initial concentration in aqueous solution for 40 hours at room temperature (25 C) and about 30 days at 5 C. The stability of cefoxitin sodium in common i.v. infusion fluids was independent of the concentrations (1 mg/ml to 400 mg/ml) and containers used, and was retained after 30 weeks storage at -20 C. Similar stability patterns were demonstrated for cefoxitin sodium in protein hydrolysate solutions and multivitamin formulations. Cefoxitin sodium was chemically and visually compatible with amikacin sulfate, gentamicin sulfate, kanamycin sulfate and tobramycin sulfate when admixed with normal saline or 5% dextrose in water injections. Cefoxitin sodium (397 mg/ml) in 0.5% lidocaine hydrochloride was stable after 26 weeks of storage at -20 C. Sodium cefoxitin is compatible with a wide variety of commonly used infusion solutions. Its stability is independent of concentration or pH within the ranges studied, and of types of common containers.

Subcellular distribution of nucleotide cyclases in rat intestinal epithelium.

The subcellular distributions of adenylate cyclase and guanylate cyclase were determined for the mature enterocyte from the rat duodenum. Brush-border and basolateral membranes were prepared from isolated cells by an analytical isolation procedure, and multiple linear regression analysis was used to obtain a quantitative estimate of the distribution of recovered cyclase activities between the brush borders and basolateral membranes. Adenylate cyclase was largely confined to the basolateral surface of the epithelium, whereas guanylate cyclase was found on the brush-border and basolateral membrane fractions in the ratio 2.4:1. There was no evidence for the presence of nucleotide cyclases in the cytosol. Guanylate cyclase in both the brush-border and basolateral membranes was stimulated by epinephrine, insulin, and Triton X-100, but not by carbachol. Adenylate cyclase was not influenced by epinephrine, but was markedly stimulated by NaF and vasoactive intestinal peptide. These results are discussed in relation to the effects of hormones on transport across the small intestine.

Protection of gastric mucosa against acute ulceration by intravenous infusion of sodium bicarbonate.

The influence of intravenous infusion of sodium bicarbonate on gastric mucosal injury induced by topical sodium taurocholate and hemorrhagic shock was assessed in a canine ex vivo model. As expected, exposure of the gastric mucosa to sodium taurocholate and acid resulted in excessive back diffusion of hydrogen ions (H+) and mild mucosal damage. This mucosal injury was enhanced by hemorrhagic shock in the control dogs. The degree of mucosal injury was significantly less in the test dogs that received intravenous infusions of sodium bicarbonate. The protection afforded by intravenous bicarbonate was not due to a reduction in the amount of H+ entering the tissue, since the net H+ loss from the lumen was not significantly different between the control and the test dogs. The protection effect of intravenous infusion of sodium bicarbonate is probably secondary to an enhancement of mucosal tolerance to H+. These results support the hypothesis that the enhancement of mucosal injury during hemorrhagic shock may be a result of a decrease in the ability of the gastric mucosa to buffer the influxing H+.

[Influence of age on biochemical parameters of the rat liver following partial hepatectomy (author's transl)].

In the present paper measurements of DNA and protein content and lysosomal enzyme (beta-glucuronidase, beta-acetylglucosaminidase, cathepsin D) acitivities were performed in the rat liver following partial hepatectomy. The rats were divided into three age groups: 6 weeks, 10 months and 18 months. The regenerating liver of the 6 week and 10 month old animals disclosed significant higher concentrations of DNA than the controls. The 18 month old rats revealed no differences of the DNA content. In all age groups the protein content of the regenerating liver was significant diminished. There were age dependent differences of the activities of the three lysosomal enzymes. In comparison to the controls the beta-glucuronidase activity of the regenerating liver was significantly decreased in the 6 week and 18 month old animals, but significantly increased in the 10 month old rats. Refering to the protein content there were no differences of the activities of beta-acetylglucosaminidase and cathepsin D between the regenerating and control livers. Refering to the liver fresh weight the beta-acetylglucosaminidase activity of the regenerating liver was significant diminished in the 10 and 18 month old rats.

[Problem in the management of arterial hypertension resistent to drug treatment. Study of 28 cases].

The antihypertensive effect of the following therapeutic regimens: diuretic alone (DA), diuretic plus sympathetic inhibitor (DSI), diuretic plus betablocker (DB) and diuretic plus, betablocker plus vasodilator (DBV) was studied for 34.1 +/- 5.4 months in 28 patients with resistant essential hypertension (REH). Depending of treatment tolerability and the optimal antihypertensive action of drugs 21, 24, 26 and 10 cases were treated continuously or alternately with DA (9.9), DSI (15.0), DB (4.8), and DBV (14.6), respectively (in paragraph average duration of treatment in months). On admission the 89.3% and 42.8% of population had electrocardiographic signs of left ventricular hypertrophy or past-history of cardio-vascular complications, respectively. Arithmethic average and standard deviation of individual changes of systolic and diastolic blood pressure obtained during DA, DSI, DB and DBV treatment were -32.4 +/- 31.8, -19.3 +/- 27.2, -18.9 +/- 15.9 and -18.2 +/- 21.3 for systolic and -35.8 +/- 20.2, -12.3 +/- 17.2, -15.1 +/- 16.9 and -15 +/- 13.1 (mm. de Hg.) for diastolic blood pressure respectively. Average blood pressure before treatment was 222.4 +/- 30.3/128.0 +/- 20.8 (mm of Hg) and under the most effective treatment was 175.5 +/- 21.8/106.5 +/- 12.1 with a p less than or equal to 0.001 for either sistolic and diastolic pressure. There were not significant regressive electrocardiographic changes during the therapeutic period, neither significant changes in urea and creatinine blood values. 46.4% and 25% of cases exhibited collateral drug symptoms and cardio-vascular no fatal complications, respectively. Three of the last group patients died outside of the Hospital (2 sudden deaths and 1 renal insufficiency death). RH still constitutes a challenge to medical therapy. Nevertheless individualized therapy may modify the natural history of this hypertensive variety.

Reconstitution of chlorophyllide formation by isolated etioplast membranes.

1. The reconstitution of chlorophyllide biosynthesis by barley etioplast membranes is described. 2. The process is dependent on the additon of NADPH and protochlorophyllide and on illumination, which can be either continuous or intermittent. 3. The reconstituted process involves spectroscopically similar intermediates to the native reaction in whole leaves. 4. Steps in the process are an initial enzymic formation in the dark of a photoactive complex, P638/652 (probably a ternary protochlorophyllide-NADPH-enzyme complex), followed by a very rapid light-dependent hydrogen transfer from the NADPH to the protochlorophyllide giving chlorophyllide giving chlorophyllide, finally releasing the enzyme for repeating the process. 5. A continuous assay for the system regenerating complex P638/652 was devised on the basis of monitoring chlorophyllide formation. 6. The pH optimum of the reaction is at 6.9 and Km values for protochlorophyllide and NADPH are 0.46 and 35 micron respectively. 7. The reaction is associated specifically with the etioplast membrane fraction. 8. Activities of the system assayed in vitro are more than adequate to account for rates of chlorophyll formation in vivo.

The role of peroxide in haem degradation. A study of the oxidation of ferrihaems by hydrogen peroxide.

The oxidation of ferrihaems by H2O2 was studied as a model for haem catabolism. Rates of ferrihaem oxidation were evaluated by using a new computer-based method that measures the loss in catalytic activity of the ferrihaem during oxidation. For protoferrihaem, deuteroferrihaem, coproferrihaem and mesoferrihaem, oxidation proceeded via the monomeric species and no dimer contribution was detectable. The pH-dependence of oxidation was studied in the range 6.5--11. Within experimental error, the data were compatible with an inverse linear dependence on [H+]. This was interpreted in terms of attack by HO2- on monomeric ferrihaem. The specific second-order rate constants for oxidation of monomeric species by HO2- were of the same order of magnitude for all the ferrihaems, and were in the sequence coproferrihaem greater than protoferrihaem greater than mesoferrihaem congruent to deuteroferrihaem. A model is suggested involving formation of a ferrihaem monomerperoxide complex, which may either dissociate with the formation of a peroxidatic intermediate or be involved in an intramolecular oxidation of the ferrihaem. Haem catabolism may occur via the same or a similar intermediate.

Subunit dissociation in the allosteric regulation of Glycerol kinase from Escherichia coli. 3. Role in desensitization.

The mechanism of desensitization of glycerol kinase to allosteric inhibition by fructose 1,6-bisphosphate caused by salt, urea, and high pH has been examined in the light of the model proposed in an earlier paper [de Riel, J. K., and Paulus H. (1978), Biochemistry 17] relating subunit dissociation and ligand binding. KCl (0.4 M) causes a tenfold decrease in the affinity of tetrameric glycerol kinase for fructose, 1,6-bisphosphate but has no significant effect on the dissociation process itself. Urea (2 M) causes a large increase in the equilibrium constant for the dissociation of the glycerol kinase tetramer to dimer but has no effect on the affinity of the tetramer for the allosteric inhibitor. High pH (9--10) has only a small effect on the subunit dissociation constant but greatly reduces the rates of subunit association and dissociation. Desensitization of glycerol kinase to allosteric inhibition can thus occur by three different mechanisms, two of which are directly related to the polysteric nature of the enzyme.

Proton stoichiometry of the cytochrome c peroxidase mechanism as a function of pH.

The proton stoichiometry for the oxidation of cytochrome c peroxidase (ferrocytochrome c: hydrogen-peroxide oxidoreductase, EC 1.11.1.5) to cytochrome c peroxidase Compound I by H2O2, for the reduction of cytochrome c peroxidase Compound I to cytochrome c peroxidase Compound II by ferrocyanide, and for the reduction of cytochrome c peroxidase Compound II to the native enzyme by ferrocyanide has been determined as a function of pH between pH 4 and 8. The basic stoichiometry for the reaction is that no protons are required for the oxidation of the native enzyme to Compound I, while one proton is required for the reduction of Compound I to Compound II, and one proton is required for the reduction of Compound II to the native enzyme. Superimposed upon the basic stoichiometry is a contribution due to the perturbation of two ionizable groups in the enzyme by the redox reactions. The pKa values for the two groups are 4.9 +/- 0.3 and 5.7 +/- 0.2 in the native enzyme, 4.1 +/- 0.4 and 7.8 +/- 0.2 in Compound I, and 4.3 +/- 0.4 and 6.7 +/- 0.2 in Compound II.

Effects of amino acids, adenine nucleotides and inorganic pyrophosphate on glutamine synthetase from Anabaena cylindrica.

Glutamine synthetase (L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2) from Anabaena cylindrica was inhibited by alanine, glycine, serine and aspartate. The effects of alanine and serine were uncompetitive with respect to glutamate, while those of glycine and asparatate were uncompetitive with respect to glutamate, while those of glycine and aspartate were non-competitive and mixed type respectively. Different pairs of amino acids and their various combinations caused a cumulative inhibition of the enzyme activity. Glutamine synthetase was also inhibited by ADP and AMP and both nucleotides affected the enzyme competitively with respect to ATP and non-competitively for glutamate. Inorganic pyrophosphate, between 2 and 3 mM, produced a very pronounced inhibiton of enzyme activity. The inhibition by PPi was uncompetitive for ATP. Various combinations of the adenine nucleotides, PPi and Pi exerted a cumulative inhibitory effect on the enzyme activity, as did the amino acids, in different combinations with either adenine nucleotides, PPi or Pi. The effects of the adenine nucleotides and the amino acids were more pronounced at higher concentrations of ammonia. Except for serine similar responses of these effectors were obtained with increasing concentrations of Mg2+. It is proposed that changes in the free concentrations of Mg2+ are important in energy-dependent regulation of the enzyme activity in this alga.

Tyrosine aminotransferase converting factor: kinetic properties, cellular localization, and tissue distribution.

Utilizing a more rapid procedure for determining tyrosine aminotransferase-converting factor activity, the kinetic properties of this factor were characterized further. Tyrosine aminotransferase-converting factor is a heat-labile substance present in the particulate fraction of rat liver that converts tyrosine aminotransferase form III to I at 4 degrees C. Analysis of the distribution of marker enzymes for mitochondria and lysosomes, and of converting factor, following differential and discontinuous sucrose gradient centrifugation indicated that this factor was associated with light lysosomes. The activity of converting factor was not altered following administration of cortisol. Converting factor activity, equivalent to that in liver, was also observed in particulate fractions from kidney and spleen, and to a lesser extent, in pancreas and salivary gland. No detectable activity was observed in brain, heart, small intestine, skeletal muscle, red blood cells, serum, or plasma. The presence of converting factor activity in kidney and spleen suggests that other proteins are substrates for this factor since tyrosine aminotransferase is virtually absent from these tissues. Alternatively, the absence of converting factor from other tissues need not indicate they are devoid of converting factor-like activity merely, that such activity in these tissues has different specificities and does not utilize tyrosine aminotransferase as a substrate.

Effect of pH on chloroplast photosynthesis. Inhibition of O2 evolution by inorganic phosphate and magnesium.

1. The pH optimum of CO2-dependent O2 evolution by barley (Hordeum vulgare L.) chloroplasts was found to be between 7.8 and 8.2. The addition of 1 mM MgCl2 in the dark inhibited O2 evolution over the entire pH range tested and resulted in a much sharper pH profile centered around pH 8.2. 2. The pH optimum for O2 evolution, in the presence and absence of 1 mM MgCl2, was acid-shifted 0.3--0.4 pH units by 2 mM NH4Cl. The pH optimum of O2 evolution, with and without 1 mM MgCl2, was base-shifted by 2 mM sodium acetate, approx. 0.5 pH units relative to the controls. 3. O2 evolution in the presence of bicarbonate plus 3-phosphoglycerate or ribose-5-phosphate was considerably less sensitive to pH than CO2-dependent O2 evolution in the absence of substrate. With these substrates, both in the presence and absence of 1 mM MgCl2, the pH optimum was broad and was centered around pH 7.8. 4. Inhibition of CO2-dependent O2 evolution by inorganic phosphate and magnesium increased as the pH of the reaction mixture was decreased below the optimum. Decreasing the pH from 8.2 to 7.6, reduced over 3-fold the concentration of inorganic phosphate required to inhibit O2 evolution completely. For magnesium, a similar change in pH reduced the concentration required to inhibit O2 evolution 50% approx. 5-fold. At pH 8.2, magnesium inhibition required inorganic phosphate. Magnesium was not required for inhibition of O2 evolution by inorganic phosphate, but incresaed the relative inhibition observed. 5. Illumination of intact barley chloroplasts increased the activity of NADP-glyceraldehyde-3-P dehydrogenase, phosphoribulokinase and fructose-1,6-diphosphatase. MgCl2 and inorganic phosphate prevented this increase in enzyme activity at concentrations that completely inhibited CO2-dependent O2 evolution. 6. The results obtained suggest that magnesium inhibition of O2 evolution may be caused by enhanced phosphate exchange across the chloroplast envelope.

Natural plant enzyme inhibitors. VI. Studies on trypsin inhibitors of Colocasia antiquorum tubers.

A trypsin inhibitor was purified from the tubers of Colocasia antiquorum. The inhibitor acted on bovine trypsin, human trypsin and weakly on bovine chymotrypsin. The inhibitor, which had a molecular weight of 40 000, contained trace amounts of carbohydrates. The purified inhibitor was stable over a pH range of 2.0--12.0 and was more thermostable than the crude preparations. Trinitrobenzene sulphonate treatment resulted in the inactivation of the inhibitor. Chymotrypsin, pepsin and pronase digested the inhibitor. Pretreatment with trypsin at neutral pH resulted in the partial loss of antitryptic activity, whereas treatment at pH 3.7 led to complete inactivation. Evidence for the formation of a trypsin-inhibitor complex at pH 7.6 is provided. During the plant growth, in the early phase (0--40 days) there was a gradual increase in protein content and in antitryptic activity. The middle phase (40--55 days) was characterized by a rapid fall and abolition of the antitryptic activity and a diminution in protein content in the tubers. The immature tubers had low antitryptic activity compared to the mature ones. Mild heat treatment caused a sharp rise in antitryptic activity in the extracts of immature tubers but not with the mature tuber preparations.

Nuclear phosphoprotein phosphatase from calf liver.

Calf liver nuclear phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) has been purified approx. 850-fold. The enzyme has a mol. wt. of 34 000 as determined by SDS-polyacrylamide gel electrophoresis. The purified enzyme has a pH optimum between 7.0 and 7.5 with phosphophosphorylase, phosphohistones f1 and f2b, and phosphoprotamine as substrates. The enzyme activity towards these substrates follows the order, phosphophosphorylase greater than phosphohistone f1 greater than phosphohistone f2b greater than phosphoprotamine. The Km values toward phosphophospharylase and phosphohistone f1 are 17 and 28 micron phosphate, respectively. Dephosphorylated histone f1 and orthophosphate are competitive inhibitors of the enzyme with respective Ki values of 11 micron and 4.1 mM. NaCl and divalent metal ions inhibit the enzyme but CaCl2 is slightly stimulatory. It appears that metal ion inhibition occurs at two sites, one on the enzyme and the other on the substrate. The enzyme is also inhibited by NaF and EDTA. Nucleotides bearing the pyrophosphate structure are potent inhibitors of the enzyme while mononucleotides are slightly inhibitory. DNA and other polyions also inhibit the enzyme. The enzyme appears to require free sulfhydryl groups for activity since it is inhibited by N-ethylmaleimide and p-hydroxymercuribenzoate; the latter inhibition can be reversed by mercaptoethanol and dithiothreitol.

Factors influencing the release of acetylcholine from the myenteric plexus of the ileum of the guinea-pig and rabbit.

1 The effects of electrical stimulation, changes in external ion concentrations and various drugs on acetylcholine release from the myenteric plexus were measured by bioassay in the presence of physostigmine and by recording the responses of the longitudinal muscle. In preparations from the guinea-pig, the acetylcholine output per pulse increased with decreasing frequency of stimulation and reached its maximum at a frequency of 0.017 Hz (1/min) and thus ensured that the output per unit of time was constant at frequencies below 0.5 Hz. Spontaneous release was suppressed during stimulation at 0.017 Hz. 2 In the rabbit, the fractional acetylcholine release was lower than in the guinea-pig. The output per pulse increased with decreasing frequency of stimulation but at a lesser rate, with the effect that the output per unit decreased between 0.5 and 0.017 Hz. 3 In the guinea-pig, reduction of the Ca2+ concentration, addition to the bath fluid of Mn2+, ganglion-blocking drugs, morphine and catecholamines reduced output more at low than at high frequencies of stimulation. In the rabbit, acetylcholine output was less sensitive to changes in Ca2+ concentration and insensitive to Mn2+ and morphine. 4 In the guinea-pig, morphine and catecholamines depressed both the contractile response and acetylcholine output whereas Mn2+ in concentrations up to 125 muM, bretylium and ganglion-blocking drugs depressed only acetylcholine output. 5 In preparations from the guinea-pig, drugs blocking noradrenergic neurons or alpha-adrenoceptors, e.g. bretylium, phenoxybenzamine, thymoxamine and phentolamine, increased acetylcholine output during stimulation at high (1.5 to 10 Hz) but not at low frequencies. 6 The implications of these findings for the release of acetylcholine from different pools in the heterogeneous myenteric plexus are considered. The possible errors, introduced by the effects of physostigmine, on the size of the acetylcholine pools and on the transmission of impulses within the myenteric plexus are discussed.

Purification and studies of some physicochemical properties of glutamine synthetase of Neurospora crassa.

Glutamine synthetase (EC 6.3.1.2) of Neurospora crassa was purified to near homogeneity by chromatography on a glutamate-Sepharose affinity column. Its properties, including molecular weight, subunit structure, amino acid composition, and approximate alpha-helix content, have been examined. In the native state, this enzyme has been demonstrated by gel filtration to be an octamer of molecular weight 360,000 and as having a sedimetation coefficient of 13.2 S by sedimentation velocity measurements. Circular dichroism spectra in the far ultraviolet range suggest an approximate alpha-helix content of 23-24%. The subunit generated by treatment with urea was found to be 45,000 daltons by gel filtration methods and a molecular weight of 46,000 was calculated for the monomer obtained by sodium dodecyl sulphate (SDS) treatment and electrophoresis in SDS-polyacrylamide gels. Interprotomeric cross-linking experiments, using diimidoesters, suggest the presence of two noncovalently linked tetramers comprising the native octameric structure. Amino acid analyses revealed the presence of six tryptophans, four half cystines, and nine methionine residues per monomer of 45,000 daltons.

Mobility of histones on the chromosome of simian virus 40.

Linear simian virus 40 (SV40) chromosomes were prepared by Eco R1 nuclease cleavage of the circular SV40 chromosomes released from virions with dithiothreitol at pH 9,8. Chromatin-DNA hybrids were constructed with segments of 3H-labeled, naked SV40 DNA covalently joined via the Eco R1-generated cohesive ends to segments of linear SV40 chromosome. Upon incubation of chromatin-DNA hybrids at 37 degrees C and moderate ionic strength, histones migrated onto the labeled DNA while retaining the nucleosome structure. This was shown first, by the pattern of micrococcal nuclease digestion of labeled DNA; second by nitrocellulose filter binding of labeled DNA after redigestion of the chromatin-DNA hybrids with Eco R1; and third, by examination of chromatin-DNA hybrids in the electron microscope. Migration was slow, being apparent after several hours. Parallel experiments in which naked DNA and chromosomes were mixed without joining showed no transfer of nucleosomal histones between DNA molecules. The kinetics of Eco R1 cleavage of the DNA in virion-derived SV40 chromosomes are also consistent with the notion that nucleosomal histones, in the absence of other proteins, can move on DNA.

The interaction between prazosin and clonidine.

1. The interaction between prazosin and clonidine was studied in anaesthetized rats, pithed rats and in anaesthetized cats. 2. Prazosin diminished the clonidine-induced hypotensive effect in anaesthetized rats, probably via an antagonism at the level of central alpha-adrenoreceptors. 3. In pithed rats, stimulation of the Nervi accelerantes caused tachycardia, which was diminished considerably by clonidine. The antagonism by clonidine was partly reversed by prazosin, suggesting that prazosin possesses a certain degree of presynaptic activity apart from its predominant effect at the postsynaptic alpha-receptor. Piperoxan was more active than prazosin. 4. The central hypotensive effect of clonidine, injected into the left vertebral artery of cats was significantly reduced by prazosin, administered before clonidine via the same route. Intravenously injected prazosin did not diminish the central hypotensive effect of clonidine. The antagonism is, therefore, caused by a central mechanism. 5. The combined application of clonidine and prazosin in antihypertensive treatment is probably not only irrational but ought to be discouraged in view of the interaction between the drugs, which leads to a reduced antihypertensive potency of clonidine.

[Studies on staphylococcal enterotoxin B. II. Production and regulation (author's transl)].

Enterotoxigenic strain of Staphylococcus aureus (ATCC 14458) was grown under various conditions with constant shaking to determine the requirements for maximum toxin production. It was evident that 3% tryptic soy broth, 3% NZ-Amine NAK + 3% casein hydrolysate, 3% NZ-Amine NAK + 1% yeast extract, and 3% NZ-Amine NAK + 1% yeast extract + 0.2% glucose are most available toxin production media. But concentration of glucose could strictly triggered the enterotoxin producing efficiency. When glucose concentration was less than 0.5%, although with higher yield, the toxin production was delayed for certain period of time. However, if glucose concentration was up to more than 0.5%, the enterotoxin production was almost inhibited. Some metabolites of glucose to elucidate the inhibitory effect have also investigated. Our results indicated that glycerol and citric acid inhibited the toxin production directly, while the inhibitory effect of lactic acid and acetic acid were due to those acidic metabolites, decreased the pH value of media, and adversely suppressed the bacterial growth.

Influence of detergents and organic solvent extraction on human gamma-glutamyltransferase activity.

Human serum and urinary gamma-glutamyltransferase (gamma-GT) have been found to react differently towards the detergents Triton X-100 and sodium lauryl sulphate (SDS). Serum gamma-GT was virtually unaffected by Triton X-100 at a concentration of 5% whereas urinary gamma-GT was 10-15% activated under similar conditions. There was a 100-fold difference in the response of serum and urinary gamma-GT to SDS. The enzyme activity in urine was completely destroyed by 0.02% SDS, whereas it required 2.0% to destroy the serum enzyme. These latter differences, however, were found to result from the environments of the serum and urinary enzyme rather than to intrinsic factors within the molecule. Extraction of serum gamma-GT from normal individuals with n-butanol resulted in little or no loss of activity from the aqueous phase, whereas more than 30% activity was lost from normal urines. Extraction using various mixtures of butanol and di-isopropyl ether (DIPE) produced largely similar results, except that 25% DIPE: 75% butanol mixture produced a marked loss of activity from serum. It is suggested that gamma-GT activity in human body fluids may be dependent on the presence of a lipid fraction.

Properties of human hemoglobin immobilized on Sepharose 4B.

This paper reports the properties of human hemoglobin covalently bound to Sepharose 4B both in 'high-affinity' and 'low-affinity' conformations. The results suggest that the coupling reaction is strongly affected by the conformational changes linked to oxygenation of the protein. The rate and the extent of the reaction are different for the oxy and deoxyderivatives, probably due to the change in reactivity of the amino groups in the liganded and unliganded tetramer. The data on the equilibrium which is established between matrix-bound and soluble subunits, measured by the 'subunit-exchange chromatography', indicate that the system displays a minimal heterogeneity when hemoglobin is coupled to the gel in the deoxy state at intermediate protein concentration and pH 8. Maxtrix-bound hemoglobin is characterized by a higher oxygen affinity and by decreased homotropic and heterotropic interactions with respect to hemoglobin in solution, but the changes depend strongly on the conditions used in the coupling procedure.

Comparative effects of clozapine and alpha-adrenoceptor blocking drugs on regional noradrenaline metabolism in rat brain.

Clozapine increased brain noradrenaline (NA) metabolism, as indicated by changes in 3-methoxy-4-hydroxyphenylglycol sulfate content, in brain regions corresponding to the predominance of alpha- over beta-receptors, i.e., hypothalamus, medulla, midbrain and cortex, but not corpus striatum or cerebellum. Phenoxybenzamine had a stronger effect in the hypothalamus than did clozapine, but did not change cortical NA metabolism within a 60 min treatment time; however, cortical NA metabolism was increased 150 min after phenoxybenzamine. The delayed effect of phenoxybenzamine may be due to either a poor affinity for some central receptors or a slow rate of entry into certain brain regions. Thioridazine and the benzodioxane, dibozane, had regional effects similar to clozapine. The similarity between clozapine and dibozane in ther effects on regional brain NA metabolism may reflect a preference for presynaptic alpha-receptors. It is unlikely that the antipsychotic activity of clozapine is related to a specific adrenolytic effect, but may reflect the combined activity of this drug on several transmitter systems.

Effects of the unilateral nigral application of dopaminergic drugs on the in vivo release of dopamine in the two caudate nuclei of the cat.

The effects of the unilateral application of d-amphetamine, benztropine, haloperidol and thioproperazine to one substantia nigra on the release of 3H-dopamine (3H-DA) in the two caudate nuclei were examined in halothane-anesthetized cats. For this purpose animals were implanted with push-pull cannulae and 3H-DA was estimated in superfusates during the continuous delivery of L-3,5-3H-tyrosine. The nigral application of d-amphetamine (10-6 M) or benztropine (10-6 M) reduced the release of 3-H-DA in in the ipsilateral caudate nucleus and induced an opposite effect in the contralateral side. In contrast, the nigral application of haloperidol (10-6 M) or thioproperazine (10-6 M) slightly increased the release of 3H-DA in the ipsilateral caudate nucleus and induced a reduction of 3H-transmitter release in the contralateral side. These results emphasize the role of the dendritic release of DA in the control of the activity of dopaminergic neurons and confirm our previous findings concerning the existence of a reciprocal control in the activity of the two dopaminergic pathways.

Systemic reactions with total dose infusion of iron dextran complex in obstetric patients.

Iron dextran complex (Imferon) total dose infusion (TDI) was used to treat iron deficiency anemia in 310 obstetric patients. Systemic reactions were reported for 13.5% of the group. Most (90.5%) of the severe reactions occurred during the test dose, while 66.6% recorded during or after TDI were mild. Antihistamine premedication included administration of promethazine hydrochloride to 104 patients and intramuscular and intravenous drip chlorpheniramine hydrogen maleate to 206 patients. Total systemic reactions were statistically greater with intramuscular promethazine hydrochloride than with intramuscular and intravenous chlorpheniramine maleate (p less than 0.001). On the other hand, the rates of severe reactions and reactions during the test dose were similar for both antihistamine schedules (p greater than 0.05). The incidence of all systemic reactions was the same for pregnant and postnatal women (p greater than 0.05), but severe reactions were more frequent among pregnant than postnatal patients (p less than 0.001). There were no fatalities.

Degradation of levan by Actinomyces viscosus.

Actinomyces viscosus ATCC 15987 was examined for its ability to hydrolyze its own levan. Washed whole cells and an ammonium sulfate fraction from cell-free culture fluids were shown to possess levan hydrolase activity. Analyses of reaction mixtures by gel filtration and thin-layer chromatography demonstrated that the product of levan hydrolysis was free fructose. The cell-associated and extracellular enzyme preparations also hydrolyzed inulin and the levans synthesized by Aerobacter levanicum and Bacillus subtilis. Growth of A. viscosus in media supplemented with 0.1% A. viscosus levan resulted in a 33-fold increase and a 7-fold increase in the specific activities of the respective extracellular and cell-associated enzymes when compared with those from 55 mM glucose cultures. Growth in the presence of 29.2 mM sucrose resulted in a 28-fold increase and a 5-fold increase in the specific activities of the respective enzymes when compared with those from the glucose cultures. The extracellular enzyme exhibited high activity over a wide pH range, with 87 and 89% of its pH 6.0 optimum activity at pH 5.0 and 7.0, respectively. The cell-associated enzyme also exhibited optimum activity at pH 6.0, but this was decreased to 10 and 20% at pH 5.0 and 7.0, respectively. Analysis for the presence of extracellular levan during growth of A. viscosus in sucrose broths demonstrated that peak levan concentrations occurred during the mid-exponential to late-exponential phase of growth followed by a rapid decline in extracellular levan as a result of levan hydrolase activity.

Dissociation between clinical and exercise responsiveness to beta-blockade in angina.

Twenty outpatients with mild angina were prescribed placebo tablets b.i.d. for 7 weeks followed by acebutolol, a cardioselective beta-blocker, 200 mg b.i.d. for 21 weeks under single-blind conditions. One graded multistage treadmill test was carried out after each treatment period and an angina diary was filled daily for the 6 months of the trial. Attack frequency declined by 71% from 2.59 per week on placebo to 0.76 per week on acebutolol (p less than 0.05). Exercise duration on the treadmill increased by 56%, from 5.95 minutes on placebo to 9.32 minutes on acebutolol (p less than 0.001). A satisfactory clinical response (50% or greater decline in attack frequency per week) occurred in 15 out of 19 patients (79%; a 100% or greater increase in exercise duration on the treadmill was observed in 10 out of 19 cases (53%). Exercise responsiveness was well predicted by exercise duration on placebo (r = 0.91, p less than 0.0005), patients with the least initial tolerance being the most improved. Clinical responsiveness was not well predicted by initial exercise tolerance (r = 0.38, N;S.) or by the improvement in exercise tolerance (r = 0.33, N.S.). It is concluded that acebutolol substantially reduces anginal attack frequency even in patients in whom exercise tolerance is not significantly improved, at the dose of 400 mg/day.

Slow postcapillary pH changes in blood in anesthetized animals.

To investigate the hypothesis that blood pH and PCO2 continue to change after the blood leaves an exchange capillary, we used a rapidly responding, pressure-insensitive, stopped-flow pH electrode apparatus. Arterial blood from an anesthetized dog or cat is drawn through the apparatus into a syringe. Syringe movement is then suddenly stopped. Temperature and pH of the blood in the electrode assembly are continuously monitored, both before and after blood withdrawal ceases. Hemolysis was reduced by coating all blood contact surfaces with silicone and fasting the animal overnight, anesthetizing it with crystalline pentobarbital sodium, and allowing it to ventilate spontaneously. After stopping withdrawal, pH of blood in the electrode chamber continued to change, rising 0.01 unit with t1/2 of 4.4 s. After lysed blood was returned to the animal to provide carbonic anhydrase to the plasma, no pH change was seen after stopping the flow. The small pH rise occurring in arterial blood in vivo is probably due in large part to disequilibrium of [H+] between red blood cells and plasma at the end of the pulmonary capillary, the equilibration process being rate-limited by the extracellular CO2 hydration-dehydration reaction.

Crystalline reduced nicotinamide adenine dinucleotide phosphate-adrenodoxin reductase from pig adrenocortical mitochondria. Essential histidyl and cysteinyl residues of the NADPH-binding site and environment of the adrenodoxin-binding site.

Pig NADPH-adrenodoxin reductase was crystallized from pig adrenocortical mitochondria and its physicochemical properties were investigated. Pig NADPH-adrenodoxin reductase is a typical flavoprotein. Its optical absorption spectrum showed peaks at 272, 377, and 450 nm in the oxidized form. The adrenodoxin reductase contained one FAD per mol. The molecular weight was 49,000. The isoelectric points of the adrenodoxin reductase and its complex with adrenodoxin were 5.3 and 4.6, respectively. Pig NADPH-adrenodoxin reductase, unlike bovine NADPH-adrenodoxin reductase, was found to be free of carbohydrate. The fluorescences of tryptophanyl residues and FAD of the adrenodoxin reductase were quenched by holo- and apo-adrenodoxins. The NADPH-binding site of the adrenodoxin reductase was examined by photooxidation and selective chemical modifications with diethyl pyrocarbonate and sulfhydryl reagents. The results indicate that a histidyl and a cysteinyl residue of the adrenodoxin reductase are essential for the NADPH-binding site. The circular dichroism spectrum of the adrenodoxin reductase showed negative ellipticity in the visible region. Spur formation was observed between pig and bovine NADPH-adrenodoxin reductases against the antibody to bovine NADPH-adrenodoxin reductase in Ouchterlony double-diffusion agar plates. The antibody did not interact with spinach ferredoxin-NADP+ reductase.

[The dental soldering by means of high frequency induction heating (author's transl)].

The authors examined the methods of high frequency induction soldering, especially Loop-method, other than the gas flame soldering which was known generally. The details which was done are: (1) about the form of induction coil, and the relative places of the coil and restrative appliance, and some supplementary appliances of them. (2) about strength of soldered joints of Co-Cr wire using silver solder and Pd-solder, and observation on the corrosion of soldered-joints by the scanning electron-microscope. (3) about the comparison of the characteristics of Co-Cr wire by means of high-frequency induction heating and gas-flame one. (4) about the examples of soldering of porcelain crown-bridge and clasp wire attached on the dental cast, and possibility of soldering techniques of them on the dental cast. The authors found that the high frequency induction method was preeminent from the other method on the point of (a) the characteristic of heated wire, (b) the strength of soldered joints, (c) the easiness of operating of them, and (d) the possibility of soldering using the high-melting point of Pd-solder, and the soldering of them on the dental cast. Therefore we can enough respect the application on the dental area of this apparatus and this techniques of the high-frequency induction soldering.

Polychlorinated biphenyls: in vivo and in vitro modifications of cholesterol and fatty acid biosynthesis.

Aroclor 1254 (0.1 percent w/w) administered in the diet caused moderate to severe vacuolar degeneration of periportal hepatocytes, heptocyte enlargement, lipid accumulation, and necrosis of the liver. The incorporation of [2-14C]mevalonate into nonsaponifiable lipids was inhibited 18 percent and 26 percent after 14 days and 30 days, respectively. Biosynthesis of cholesterol from [2-14C]acetate and [2-14C]mevalonate was decreased by 51 percent and 31 percent respectively after 30 days, but no significant inhibition was observed after 14 days of feeding Aroclor 1254. [2-14C]Acetate incorporation into non-saponifiable lipids was 1.66 times greater in homogenates from Aroclor-treated rats than in those from control rats. Similar results were obtained when 3H2O, Mevalonate-14C, and acetate-2-14C were incubated in vivo. The conversion of [2-14C-A1acetate to fatty acids was decreased 43 percent by Aroclor 1254 (0.1 percent w/w, dietary) and 73 percent by Aroclor 1254, 500 ppm, in vitro. The in vitro incorporation of each [2-14C]acetate, [2-14C]mevalonate and [1-14C]isopentenyl pyrophosphate into cholesterol was inhibited by Aroclor 1254. There was no inhibition of the conversion of [1-14C]mevalonate to CO2, indicating that there was no inhibition of mevalonate-5-pyrophosphate anhydrodecarboxylase. Fatty acid synthase was not inhibited by PCB. Citrate cleavage enzyme was inhibited by Aroclor 1254. When ATP and citrate concentrations were varied, the Ki's were 5.3 X 10(-5)M and 11.5 X 13(-5)M, respectively. Acetyl CoA carboxylase activity was not inhibited by 1000 ppm Aroclor 1254 in vitro. Inhibition of citrate cleavage enzyme is a possible explanation for the observed decrease in fatty acid synthesis. There was an apparent diversion of acetate from fatty acid synthesis into the formation of non-saponifiable lipids, accompanied by an inhibition of the biosynthesis of cholesterol per se.

Hypertension: its effect on the stimulated release of dopamine-beta-hydroxylase in the rat.

Plasma dopamine-beta-hydroxylase (DBH) concentrations have been postulated as providing an index of sympathetic nerve activity. Using a microspectrophotometric assay system, plasma DBH concentrations have been measured in emergent blood from autoperfused heart, spleen and mesentery of normotensive, deoxycorticosterone (doca)/NaCl-treated, Goldblatt (1 kidney) renal and spontaneously hypertensive rats following sympathetic nerve outflow stimulation. Changes in plasma DBH concentrations as a result of sympathetic nerve outflow stimulation rates of 1--25 Hz for the mesentery and spleen and 1--4 Hz for the heart, were found to be frequency-dependent in all groups. Significantly greater amounts of DBH were found in the perfusate from the spleen (1--25 Hz) and mesentery (3--25 Hz) but not the heart (0.5--4Hz) of renal hypertensive rats compared with normotensive controls. Significantly greater concentrations of DBH were released from the spleen but not the mesentery in all hypertensive groups following high stimulation frequencies of 12 and 25 Hz. It is concluded that there is a relation between plasma DBH concentrations and sympathetic nerve activity. Furthermore, greater amounts of the enzyme are released from the spleen and mesentery of chronic renal hypertensive rats following sympathetic nerve stimulation.

Regulation by histamine of cyclic nucleotide levels in sympathetic ganglia.

An investigation has been carried out of the role of histamine H1- and H2-receptors in the control of cyclic guanosine 3':5'-monophosphate (cGMP) and cyclic adenosine 3':5'-monophosphate (cAMP) levels in blocks of bovine superior cervical ganglion. The data suggest that activation of H1-receptors is associated with cGMP accumulation and that activation of H2-receptors is associated with cGMP accumulation. Histamine increased both cGMP and cGMP levels with similar time course and concentration-response relationships. Low concentrations of the H1-receptor agonist 2-(2-aminoethyl)thiazole increased cGMP but not cAMP levels. Conversely low concentrations of the H2-receptor agonist 4-methylhistamine increased cAMP but not cGMP levels. H1-receptor antagonists blocked the histamine-induced increase in cGMP at low concentrations but blocked the cAMP increase only at substantially higher concentrations. Conversely, H2-receptor antagonists blocked the histamine-induced increased in cAMP but not cGMP. The effects of histamine on cyclic nucleotide levels did not appear to be mediated via the release of an endogenous neurotransmitter. The histamine-induced increase in cGMP appeared to be mediated through calcium: the increase in cGMP required the presence of calcium in the extracellular medium, and the calcium ionophore A23187 caused a calcium-dependent increase in cGMP. When considered with previous electrophysiological and biochemical findings in sympathetic ganglia, a correspondence can be seen: both histamine (at H1-receptors) and acetylcholine (at muscarinic receptors) raise cGMP levels and are associated with excitatory actions; both histamine (at H2- receptors) and dopamine raise cAMP levels and are associated with inhibitory actions.

Purification of a nontoxic phospholipase A2 from the venom of Indian krait (Bungarus caeruleus).

A nontoxic phospholipase A2 was purified from the venom of Indian krait (Bungarus caeruleus) by a four-step procedure involving electrophoresis, gel filtration and ion-exchange chromatography. The recovery of the enzyme activity was 37% and the purified preparation was 38 times as active as the crude venom. The purified enzyme had a molecular weight of 12,500 and the optimum pH of 7.2. The enzyme showed higher specificity toward phosphatidylethanolamine than phosphatidylcholine. The preparation was not very labile to heat and its activity was dependent on the presence of divalent cations, calcium ions being the most effective activators. The enzyme was completely inhibited by iodoacetic acid but showed high stability against 8 M urea. Purified phospholipase A2 was nontoxic at an iv dose of 5 microgram/g mouse. The high specific activity, the high yield and the nontoxic nature of the enzyme indicate that the major form of phospholipase A2 in Bungarus caeruleus venom is not associated with any toxicity and has properties somewhat similar to that of phospholipase A2 from some other venoms.

Humoral and neurohormonal aspects of blood pressure regulation: focus on angiotensin.

Angiotensin circulates in the blood as a hormone. Its main target organs are vascular smooth muscle, adrenal gland and the kidney. Hormonal angiotensin increases blood pressure by its vasoconstrictor action, by stimulation of aldosterone secretion and subsequent sodium and water retention, and by the stimulation of catecholamine release. Circulating plasma angiotensin also effects brain mechanisms of blood pressure regulation. In addition to this hormonal function, angiotensin is present in the brain as part of an endogenous brain renin-angiotensin system. Brain angiotensin is not secreted into the blood and can be considered a neurohormone with local function. A role of brain angiotensin in the maintenance of high blood pressure of spontaneously hypertensive rats has been demonstrated. Circulating plasma angiotensin appears to influence brain renin levels and vice versa. Stimulation of specific areas in the brain known to be involved in the regulation of the cardiovascular system, stimulate renin secretion from the kidney. The renin-angiotensin system can therefore serve as an example for the intimate interrelationship between humoral and neurohumoral mechanisms of blood pressure regulation.

The masquerade of vasculitis: head and neck diagnosis and management.

Wegener's granulomatosis and forms of giant cell arteritis result from vasculitis and masquerade with symptoms of common head and neck disease entities. Recognition of the manifestations of vasculitis can be made early in the disease course and confirmed pathologically, allowing effective therapy. Current therapy of Wegener's granulomatosis with Cytoxin and Imuran and steroids for giant cell arteritis frequently results in reversal of head and neck involvement, prevention of systemic disease, and prolonged survival.

Impaired distal nephron acidification in chronically phosphate depleted rats.

Renal tubular bicarbonate reabsorption and acidification were evaluated in phosphate depleted rats (PD) and controls. After 33 days of phosphate depletion, urine pH of PD rats (N = 5, 5.36 +/- 0.15) was significantly higher than control (N = 5, 5.64 +/- 0.09, P less than 0.005) following an NH4Cl load. Urinary titratable acid of PD rats (9.6 +/- 1.8) was significantly reduced compared to control (117.2 +/- 19.7 muEq/3 h, P less than 0.001), whereas NH+4 excretion was not different. The plasma HCO-3 thresholds at which bicarbonaturia occurred (approximately 25 mEq/l) were identical in controls and phosphate depleted rats during isotonic bicarbonate infusion. The higher urine pH of phosphate depleted rats following NH4Cl administration was not due to low urinary phosphate as 3-day phosphate depleted rats could normally acidify urine after NH4Cl (pH = 5.86 +/- 0.09, N = 6 vs. control 5.87 +/- 0.08, N = 6, P = N.S.) despite urinary phosphate excretion as low as in 33-day PD rats. These data indicate the presence of impaired distal tubular acidification in chronically phosphate depleted rats.

Dendro-dendritic and reciprocal synapses in the primate motor cortex.

Dendro-dendritic synapses have been observed infrequently in the deep layers of the motor cortex. The presynaptic dendrites are of a varicose type and themselves receive a considerable density of synapses both of the asymmetric and symmetrical type. The ultrastructure of the dendro-dendritic synapse itself shows the typical arrangement of presynaptic and postsynaptic membrane densities, often with presynaptic dense projections, and the membrane specialization is of the symmetrical type. There is the usual cleft containing electron-dense material between the presynaptic and postsynaptic profiles. The synaptic vesicles occur in a small cluster confined to a region close to the presynaptic membrane specialization; some of the vesicles are flattened and were shown by tilt analysis to be of the discoid type. Two examples were found of reciprocal dendro-dendritic synapses, both components being of the symmetrical type. A single axon terminal may make a synapse on to both dendrites involved in a dendro-dendritic synapse.

Growth hormone and cortisol responses, tranquillizer usage, and their association with survival from myocardial infarction.

Sixty-six patients consecutively admitted to a Coronary Care Unit with a diagnosis of myocardial infarction were evaluated physically, psychologically, and neuroendocrinologically. Records were also kept of appetite and drug intake. Serum cortisol and growth hormone, physical, and mood variables were evaluated daily and the neuroendocrine measures were also taken serially at the time of removal from the cardiac monitor. At the end of the 2 years the patient's functional status was evaluated and the outcome correlated with the aforementioned indices. Three factors emerged as being significantly related to outcome. Survivors' appetites were significantly better than the appetities of nonsurvivors. Those who were alive at follow-up had taken significantly more minor tranquilizers than those who had died. Survivors had consistently lower levels of growth hormone than nonsurvivors and these differences were statistically significant for those samples taken at the time of removal from the monitor. The findings are incorporated in two new indices predictive of both survival and functional outcome.

Twice daily dosage of bacampicillin in chronic bronchitis. A double-blind study.

Three groups of patients (total 48) with acute exacerbations of chronic bronchitis were treated orally for 10 days, in a double-blind clinical trial, with bacampicillin (an ampicillin ester) 1600 mg twice daily, 800 mg three times daily and oral ampicillin 1000 mg three times daily. Most exacerbations were caused by Haemophilus influenzae or Streptococcus pneumoniae. Clinical and bacteriological results were significantly more favourable in the two bacampicillin-treated groups. Both drugs were generally well tolerated. Serum and sputum ampicillin assays after the first dose showed higher and earlier peak levels after bacampicillin. Only after bacampicillin did the sputum levels regularly exceed the ampicillin M.I.C. for the Haemophilus influenzae strains. After the 1600 mg and 800 mg dose these levels averaged 0.85 and 0.41 mg/l respectively. One third of the Haemophilus influenzae strains studied required more than 0.25 mg/l ampicillin for inhibition of growth. Bacampicillin 1600 mg twice daily appears to be an effective and safe treatment for most episodes of acute exacerbations of chronic bronchitis.

Regulative influence of o-aminobenzoic acid on the biosynthesis of nourseothricin in cultures of Streptomyces noursei JA 3890b. III. Change of redox state of nicotinamide-adenine-dinucleotides in the presence of aminobenzoic acids.

o-Aminobenzoic acid (OABA, anthranilic acid) and related compounds which are known to stimulate the biosynthesis of streptothricin-type antibiotic nourseothricin by Streptomyces noursei JA 3890b were found to increase strongly the NADH/NAD+ ratio in growing mycelium of this strain suggesting that these effectors are capable of interfering with the function of the respiratory chain. In parallel, a complex shift of metabolism was induced shown by simultaneous alteration of mycelial activities of alanine dehydrogenase, glutamine synthetase, and glutamate dehydrogenase. These changes may be responsible for the observed delay of amino acid catabolism and may improve the precursor supply of the secondary metabolism.

Influence of soil pH and calcium nutrition on resistance of alfalfa to bacterial and verticillium wilt.

Alfalfa plants of a resistant, a susceptible and a highly susceptible strains were grown in unlimed soil at pH 5.8 and in limed one at pH 6.9 and inoculated by the pathogens of vascular wilt, Corynebacterium insidiosum and Verticillium albo-atrum. Two types of liming were performed: 1) before inoculation and 2) after inoculation. Liming of the soil led to an increase in number of resistant plants. In susceptible plants the external symptoms of disease on the plant tops were delayed or alleviated. This phenomen was more conspicuous with Verticillium wilt than with bacterial wilt. The favourable effect of liming was less distinct in resistant strains than in susceptible ones. For an increase in resistance, post-infection liming of the soil was more effective in the case of bacterial wilt, while pre-infection liming provided the best results in the Verticillium wilt. The nitrogen content in the dry matter of roots from plants grown in limed soil was higher by more than a quarter as compared to roots from plants growing in unlimed soil.

Physico-chemical modification of lidocaine uptake in rat lung tissue.

The uptake of lidocaine, methyllidocaine, bupivacaine, etidocaine was studied in rat lung slices at different pH-values. The accumulation of the quaternary analogue, methyllidocaine, was not changed in the pH interval 7.0--8.0. The uptake of the three other substances was about 3--4 times lower at pH 7.0 than at pH 8.0. The rank order of distribution at a fixed cation/base ratio was bupivacaine greater than etidocaine greater than lidocaine. Interactions between lidocaine and other substances were studied in lung slices and in isolated perfused lungs. Bupivacaine and nortriptyline counteracted the accumulation of 14C-lidocaine in lung slices in a dose-dependent manner. Nortriptyline was more effective than bupivacaine. In isolated perfused lung, bolus injections of nortriptyline and lidocaine rapidly displaced 14C-lidocaine from the tissue. In this study we suggest that the base form of local anaesthetics accumulate in the lung tissue, while the cationic form binds to accessible binding sites in the cell membranes.

Cardiac and uterine hemodynamic responses to ritodrine hydrochloride administration in pregnant sheep.

The changes in the maternal circulation following administration of ritodrine hydrochloride were investigated in chronically prepared pregnant sheep. Low infusion rates of ritodrine (see text) elevated the maternal heart rate and cardiac output and decreased peripheral vascular resistance. Stroke work fell while minute work increased. The distribution of uterine blood flow did not change, as measured with microspheres. Simultaneously measured fetal cardiac output and umbilical blood flow were not altered. When ritodrine infusion rates (see text) were increased there was a slight but significant decrease in uterine perfusion pressure, and an increase in uterine vascular resistance with uterine blood flow decreasing. These changes were observed when the ewes were not in labor, and similar changes were again recovered with ewes in labor despite the simultaneous inhibition of uterine contractions. Selective beta blockade with practolol during ritodrine administration decreased the maternal tachycardia without affecting cardiac output, peripheral vascular resistance, or uterine vascular resistance.

Volume overload heart failure: length-tension curves, and response to beta-agonists, Ca2+, and glucagon.

Left ventricular force-generating capacity was determined in 19 anesthetized dogs with heart failure (HF) from aortocaval fistula. At the time of study all dogs had ascites, edema, and elevated pulmonary wedge pressure. Length-contractile force (CF) curves recorded from the left ventricle (LV) with a modified Walton-Brodie arch indicated that the LV was operating on the ascending limb of the length-CF curve at 62.4 +/- 0.1% Lmax in the normal group and in the HF group at 83.4 +/- 2.7% Lmax. In HF the length-CF curve was depressed when compared to normal and was further depressed when CF in grams was normalized for changes in LV wall thickness and expressed as g/cm2. Additionally, dose-response curves of CF in response to injected norepinephrine, isoproterenol, glucagon, and calcium were depressed when compared to the normal group while the response of heart rate and blood pressure was not different. These findings indicate that volume overload HF is associated with depressed ventricular muscle function and a depressed response to inotropic drugs.

Leydig cells within the lamina propria of seminiferous tubules in four patients with azoospermia.

Mature, polygonal Leydig cells within the thickened lamina propria of the seminiferous tubules are commonly found in three cases of cryptorchidism and one case of Sertoli-cell-only syndrome. By contrast, under normal conditions Leydig cells between the peripheral layers of the peritubular tissue were only occasionally met in a spindle-shaped variant. In both instances, Leydig cells were positively identified in the electron microscope by characteristic features such as anabundant smooth endoplasmic reticulum, tubular inclusions and crystals of Reinke. The development of Leydig cells from myoid contractile cells within the lamina propria is discussed.

Injury to Staphylococcus aureus during sausage fermentation.

Staphylococcus aureus 196E added to a beef sausage containing starter culture and 0.5 to 2.0% glucose and incubated at 35 degrees C was unable to grow when plated on tryptic soy agar (TSA) containing 7.5% NaCl. The injury, presumed to be due to the lactic acid produced during fermentation, was more pronounced at the lower concentrations of glucose (and lower acid levels). In the absence of glucose and/or starter culture, no injury was observed. When sausages containing S. aureus injured by fermentation at 35 degrees C were incubated at 5 degrees C, the counts on TSA (measures both injured and uninjured cells) and TSA containing 7.5% NaCl (measures uninjured cells only) remained constant; however, upon reincubation of the cold-stored sausage at 35 degrees C, the staphylococcus counts on TSA and TSA containing 7.5% NaCl and were similar to the counts of S. aureus present in fermenting sausages that had never been subjected to 5 degrees C. The demonstration of acid injury indicated that the injury phenomenon must be considered when determining numbers of viable S. aureus in fermented sausages.

A pharmacokinetic study of doxapram in patients and volunteers.

1. Following intravenous bolus injections or brief infusions in healthy volunteers, plasma concentrations of doxapram declined in a multi-exponential fashion. The mean half-life from 4-12 h was 3.4 h (range 2.4-4.1h), the mean apparent volume of distribution was 1.5 1 kg-1 and the whole body clearance was 370 ml min-1. 2. Enteric-coated capsules of doxapram base were absorbed rapidly after an initial delay, and the systemic availability was about 60%. 3. Doxapram is extensively metabolized and less than 5% of an i.v. dose was excreted unchanged in the urine in 24 h. A metabolite (AHR 5955) was present in plasma in amounts comparable to the parent compound and had a similar half-life. 4. The disposition of doxapram appears to be similar in healthy volunteers and patients with respiratory failure. 5. The previously held belief that plasma concentrations fall rapidly when an infusion is stopped is only true following short duration infusions. The pharmacokinetic properties of doxapram are such that steady-state plasma concentrations will not be achieved for many hours with the recommended constant rate infusion régime.

3-Hydroxy-3-methylglutaryl coenzyme A reductase from avian liver. Catalytic properties.

The catalytic properties of microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase from avian liver have been investigated. Solubilized and highly purified reductase preparations were not cold labile, and enzymic activity remained unchanged following preincubation at 37 degrees C. The pH optimum was 6.8--7.0 and maximal catalytic activity was achieved with 2 mM dithiothreitol and 0.75 M KCl. The heat stability of the enzyme was studied and the addition of 0.75 M KCl, 0.8 mg/ml bovine serum albumin and 5 mM NADPH reduced the inactivation of the purified reductase associated with heat treatment at 65 degrees C. At 37 degrees C, 0.8 mg/ml bovine serum albumin enhanced the purified reductase activity by 100 (+/- 20)%. An improved assay was developed for the avian hydroxymethylglutaryl-CoA reductase and the specific activity of the purified enzyme increased from 1550 to 3300 nmol . min-1 . mg-1. The Km values of solubilized and purified reductase for D-hydroxymethylglutaryl-CoA were 1.05 micrometer and 1.62 micrometer, and for NADPH, 1 mM and 263 micrometer, respectively. The activities of the reductase preparations were non-competitively inhibited by coenzyme A, acyl-CoA esters, and hydroxymethylglutarate. MgATP also reduced avian reductase activity. These modulators may play a role in the cellular regulation of the reductase activity.

[Chemical modification of lysine epsilon-NH2-groups in horseradish peroxidase. Its effect on enzyme stability. Temperature dependence of thermo-inactivation constants for native and modified peroxidase].

Thermostability of horseradish peroxidase modified by acetic, propionic, butyric, valeric and succinic anhydrides and trinitrobenzolsulfonic acid (TNBS) is studied within the temperature range of 56-80 degrees C. Acylation of 4 amino groups and arylation of 3 amino groups with TNBS are found to stabilize the enzyme, while modification of 6 groups decreases the enzyme stability. Chemical modification of peroxidase does not change its pH-dependence with respect to enzyme thermostability. Thermodynamic activation parameters of irreversible thermoinactivation are determined for native and modified peroxidase. Native peroxidase has deltaH not equal to = 30+/-1 kcal/mole and deltaS not equal to = 14 e. e.; modified by acid anhydrides peroxidase has deltaH not equal to within 64-87 kcal/mole and deltaS not equal to within 110-178 e. e. depending on the nature of a modifying agent. The effect of the structure of a radical introduced into the enzyme molecule, and of a number of modified epsilon-amino groups on thermoinactivation deltaH not equal to and deltaS not equal to values is discussed.

Differentiation of the roles of histamine H1- and H2-receptors in the mediation of the effects of histamine in the isolated working heart of the guinea-pig.

1 Differentiation of the roles of histamine H1- and H2-receptors in the mediation of the effects of histamine on the isolated working heart of the guinea-pig was achieved through the use of histamine and selective histamine receptor agonists and antagonists. 2 Histamine over the dose range 10(-9) mol to 10(-6) mol produced dose-related increases in sinus rate, left intraventricular pressure (LVP)max, LVdP/dtmax, coronary flow, aortic flow, total cardiac output and external pressure-volume work. 3 Dimaprit, a selective histamine H2-receptor agonist, produced very similar responses to histamine. 4 2-Pyridylethylamine, a selective histamine H1-receptor agonist, had little effect on cardiac function unless large doses were administered. Such doses produced increases in all measured parameters. 5 Cimetidine, a selective histamine H2-receptor antagonist, antagonized the effects of histamine and dimaprit and some but not all effects of 2-pyridylethylamine. In the presence of cimetidine a decrease in all parameters with the exception of sinus rate was observed with both histamine and 2-pyridylethylamine. 6 The selective histamine H1-receptor antagonist, mepyramine, had little effect on responses to all three agonists. However, the depressant effects observed with histamine and 2-pyridylethylamine in the presence of cimetidine were antagonized by mepyramine. 7 The results indicate the important role of the histamine H2-receptor in the mediation of the gross cardiac effects of histamine and also indicate that histamine H1-receptors can mediate cardiac depression.

Treatment of irritable bowel syndrome with lorazepam, hyoscine butylbromide, and ispaghula husk.

A double-blind controlled therapeutic trial of factorial design was used to study the therapeutic effects of lorazepam, hyoscine butylbromide, and ispaghula husk in 12 randomised blocks of eight patients with the irritable bowel syndrome (IBS). Each of the three agents caused a sustained symptomatic improvement in some of the patients, although only with ispaghula was the difference between the real and dummy preparation statistically significant. When the eight possible combinations of treatment were analysed none of the 12 patients who received only dummy preparations of the three agents had maintained any improvement over the three months of the trial. Seven patients improved among the 12 who received potent preparations of all three agents, and between four and six patients improved in the groups receiving one or two of the potent preparations. These therapeutic results, though far from perfect, show that the types of drug commonly used to treat IBS are of some value and may be additive in their effects. Similar combinations of other therapeutic agents may be more effective, but it will be possible to determine this only by carrying out factorial therapeutic trials.

gamma-Glutamyl transpeptidase and malignant transformation of cultured liver cells.

The relationship between gamma-glutamyl transpeptidase (GGT)and malignant cell transformation was analyzed in malignant and nonmalignant culture epithelial cell lines derived from rat livers and fibroblastic cell types derived from hamsters and mice. GGT activity was prominent (25 to 90% of cells) in 3 of 5 malignant epithelial liver cell lines. None of the 9 fibroblastic or 4 nonmalignant epithelial cell lines exhibited GGT activity. Our results suggest that by use of GGT activity we can detect in cultured liver cells a significant fraction of the spontaneously or chemically induced malignant cells. Thus, in conjunction with other markers, this marker may help in identifying tumorigenic cells in liver epithelial cultures.

A study of the relationship between cardiac beta-adrenoceptor blockade and intrinsic sympathomimetic activity in rats depleted of catecholamines.

1. The intrinsic sympathomimetic activity of a range of beta-adrenoceptor antagonists and its relationship to beta-adrenoceptor blockade was studied in pentobarbitone-anaesthetized, vagotomized rats which had been depleted of catecholamines by pretreatment with syrosingopine. Dichlorisoprenaline, practolol, oxprenolol, pindolol and acebutolol, produced dose-dependent positive chronotropic responses in this preparation. 2. The relationship between the dose requirements for this intrinsic sympathomimetic activity and beta-adrenoceptor-blocking activity was not the same for all drugs: (i) dichlorisoprenaline and practolol had intrinsic activity at all beta-adrenoceptor-blocking doses; and (ii) oxprenolol, pindolol and acebutolol had predominantly beta-adrenoceptor blockade at the lower dose levels and agonist activity only became significant at high doses relative to those producing beta-adrenoceptor blockade. 3. The positive chronotropic response to both practolol and pindolol was observed in rats which had been pithed and was antagonized by propranolol (0.1-3.0 mg/kg, i.v.), indicating that beta-adrenoceptors were involved. 4. It was concluded that the intrinsic sympathomimetic activity of beta-adrenoceptor antagonists was not a simple property as it was described by the relationship between the dose requirements for intrinsic sympathomimetic activity and for beta-adrenoceptor blockade as well as the degree of partial agonist activity.

Studies in the rat on the haemodynamic overshoot response to withdrawal of guanfacine or clonidine treatment.

1. Normal rats were injected intramuscularly twice daily for either 3 days or 3 weeks with guanfacine (0.1 or 1.0 mg/kg), clonidine (0.01 or 0.1 mg/kg) or 0.9% saline. All were anaesthetized at various times before or after the last injection, and their arterial pressures and heart rates were monitored via a carotid artery catheter. 2. Overshoots in systolic and diastolic pressure and heart rate, reaching peak values as soon as 16 h after the last injection, occurred in all rats withdrawn from guanfacine or clonidine treatment, but in no control rats. 3. Post-withdrawal blood pressures and heart rates of rats which had received the low dose of guanfacine or clonidine were as great as those of rats which had received the ten-fold greater dosage. Moreover, withdrawal responses were as great in rats which had been treated for only 3 days as in those treated for 3 weeks. 4. The dosages and duration of treatments used in these experiments thus did not influence the magnitude of the haemodynamic overshoots provoked by withdrawal of guanfacine or clonidine. However, all groups of rats from which guanfacine was withheld exhibited significantly greater peak overshoots in systolic and diastolic pressure than did those withdrawn from clonidine treatment.

A three hours study of the response of plasma corticosterone and of three liver enzymes in rats subjected to stress in the late afternoon or during the morning hours.

In adult male rats kept under controlled illumination (lights on from 6 a.m.--6 p.m.) the existence of higher afternoon values of plasma corticosterone ("B") is reconfirmed. Moreover, plasma "B" increases induced by 400 revolutions in Noble-Collip drums or immobilization for 2.5 h were found substantially less pronounced if stressor application took place at 4 p.m. rather than 8 a.m. so that essentially no difference between morning and afternoon poststress peaks was observed. Also the response to 0.05 mg ACTH i.p. was smaller in the afternoon experiments. Additionally three liver enzymes the activity of which is increased by corticosterone released under conditions of stress were also studied. Higher afternoon activity of liver tyrosine aminotransferase (TAT) and tryptophan pyrrolase (TP) was observed in spite of fasting from 8 a.m. imposed to the animals taking part in the afternoon experiments. The activity of phosphoenolpyruvate carboxykinase (PEPCK) was also increased in the afternoon probably because of the food deprivation. Immobilization-induced increase in the activity of all three enzymes showed a similar blunting as plasma "B" response, so that no differences between morning and afternoon were found in peak poststress values. In conclusion, the low plasma "B" response of rats at a time when the animals begin the active part of their day seems to have important metabolic implications.

The 1H nuclear-magnetic-resonance spectra of Neurotoxin I and cardiotoxin Vii4 from Naja mossambica mossambica.

Two toxins from the venom of Naja mossambica mossambica, neurotoxin I and cardiotoxin VII4, were investigated in aqueous solution by high-resolution 1H nuclear magnetic resonance (NMR) techniques at 360 MHz. The spectral characterization of the proteins included determination of the number of slowly exchanging amide protons which can be observed in 2H2O solution, measurement of the amide proton chemical shifts and exchange rates, characterization of the aromatic spin systems and the internal mobilities of aromatic rings, and studies of the pH dependence of the NMR spectra. For numerous resonances of labile and non-labile protons quite outstanding pH titration shifts were observed. It is suggested that these NMR parameters provide a useful basis for comparative structural studies of different proteins in the large group of homologous snake toxins. As a first application the NMR data presently available in the literature on neurotoxin II from Naja naja oxiana, toxin alpha from Naja nigricollis and erabutoxin a and b from Laticauda semifasciata have been used to compare these three proteins with neurotoxin I from Naja mossambica mossambica. This preliminary comparative study provides evidence that the same type of spatial structure prevails for these four homologous neurotoxins and that the folding of the backbone corresponds quite closely to that observed in the crystal structure of erabutoxin b. A second application is the comparison of cardiotoxin VII4 from Naja mossambica mossambica with the neurotoxins. The experimental data indicate that the folding of the polypeptide backbone is closely similar, but that the cardiotoxin molecule is markedly more flexible than the neurotoxins.

Collagenolytic activity and steroid levels after administration of dehydroepiandrosterone sulfate.

Seventy-five pregnant women in the 38th to 41st weeks of gestation were given a single intravenous injection of 200 mg of dehydroepiandrosterone sulfate (DHAS). The changes in estriol, 17beta-estradiol and progesterone levels in the serum, the uterine cervix and the myometrium of the placenta-implanting site were then determined. Estriol levels remained unchanged both in serum and tissue, but the level of 17beta-estradiol increased sharply both in serum and tissue after four hours. The increases of 17beta-estradiol in the serum and the portio vaginalis of the same patient were well correlated (r = 0.79898), but the percentage of increase was much higher in the portio vaginalis than in the serum (p less than 0.001). Serum progesterone levels did not change initially, but always decreased within four or eight hours in cases in which labor had started or delivery was accomplished within 24 hours (p less than 0.01 at four hours). The total amount of collagenase was determined in ten subjects before and after the administration of DHAS. The total collagenase activity was elevated by an average of 152.3%. The peak of activity was accelerated from the fourth to the second day (p less than 0.001) of the culture. A probable mechanism of DHAS action in accelerating cervical ripening is presented.

Oxygen availability from the blood and the effect of phosphate replacement on erythrocyte 2,3-diphosphoglycerate and haemoglobin-oxygen affinity in diabetic ketoacidosis.

Eleven patients with diabetic ketoacidosis were given intravenous phosphate in doses (mean 118 mmol; range 83--320 mmol) adequate to maintain normal plasma phosphate, in addition to a standard treatment regime. Prevention of hypophosphataemia stimulated recovery of the initially low red-cell 2,3-diphosphoglycerate concentrations (10.6 +/- 5.8 (SD) mumol/g Hb) after twenty-four hours. In ten control patients (initial concentration 8.1 +/- 4.4 mumol/g Hb) treated without phosphate replacement, significantly lower red-cell 2,3-diphosphoglycerate concentrations were found between 2 and 6 days after admission (forty-eight hour value for control patients 14.6 +/- 1.6 and for phosphate-treated patients 18.9 +/- 4.1 mumol/g Hb; p less than 0.01). However, no effect on in vivo p 50 or on the availability of oxygen from the blood resulted from the higher 2,3-diphosphoglycerate levels. Maintenance of normal plasma phosphate levels by intravenous phosphate is, therefore, not indicated to improve tissue oxygenation in diabetic ketoacidosis.

Study on malnutrition. III. Biochemical assessment of the dietary treatment and evolution of the illness.

During the course of a malnutrition study, the efficiency of two diets has been followed by clinical observations and biochemical tests. The diets were adapted to the physiological state of 25 patients (16 children, 6 young mothers, 1 adolescent and 2 men) undergoing treatment in a rural hospital at Yasa-Bonga, Zaïre. One group of patients was examined after two weeks of treatment (6 children, 2 mothers), the other group after two months (8 children, 5 adults). In order to study the evolution of the illness, 9 children and 3 young mothers were examined regularly after the return to their villages every 6 months for 2-4 years. The patients responded positively to both diets. After two months of treatment they had clinically recovered, except for the most serious cases. After two weeks of treatment a deficiency in electrolytes, low levels of prealbumin and a net increase in transferrin were noted. After 2 months of treatment the children had regained their normal growth (hydroxyproline index), and most of the biochemical parameters had reached their normal value despite a few deficiencies in electrolytes, phosphorus and magnesium. However, the prealbumin level remained low, particularly amongst children suffering from relapses. In a few cases the activities of cholinesterase (CHE) and gamma-glutamyltranspeptidase (gamma-GT) remained low, which was taken as a sign of poor response to treatment. Generally, the adults responded more slowly to treatment than the children. Regular examinations carried out on certain patients on their return to the villages permitted 2 groups to be distinguished: the first one was composed of patients recuperating well due to good social conditions; the second group of patients suffered from relapses due to lack of hygiene and of rudimentary knowledge of nutrition, and above all severe social problems. The condition of the liver of all these patients was very important; it could be estimated by the determination of the serum levels of prealbumin and the activities of CHE, gamma-GT and isoenzymes of gamma-GT.

Tumor inhibitory and non-tumor inhibitory L-asparaginases from Pseudomonas geniculata.

Two enzymes that catalyze the hydrolysis of l-asparagine have been isolated from extracts of Pseudomonas geniculata. After initial salt fractionation, the enzymes were separated by chromatography on diethylaminoethyl-Sephadex and purified to homogeneity by gel filtration, ion-exchange chromatography, and preparative polyacrylamide electrophoresis. The enzymes differ markedly in physicochemical properties. One enzyme, termed asparaginase A, has a molecular weight of approximately 96,000 whereas the other, termed asparaginase AG, has a molecular weight of approximately 135,000. Both enzymes are tetrameric. The asparaginase A shows activity only with l-asparagine as substrate, whereas the asparaginase AG hydrolyzes l-asparagine and l-glutamine at approximately equal rates and it is also active with d-asparagine and d-glutamine as substrates. The asparaginase A was found to be devoid of antitumor activity in mice, whereas the asparaginase AG was effective in increasing the mean survival times of both C3H mice carrying the asparagine-requiring Gardner 6C3HED tumor line and Swiss mice bearing the glutamine-requiring Ehrlich ascites tumor line. These differences in antitumor activity were related to differences in the K(m) values for l-asparagine for the two enzymes. The asparaginase A has a K(m) value of 1 x 10(-3) M for this substrate whereas the corresponding value for the AG enzyme is 1.5 x 10(-5) M. Thus the concentration of asparagine necessary for maximal activity of the asparaginase A is very high compared with that of the normal plasma level of asparagine, which is approximately 50 muM.

Bacteriophage-associated gene transfer in pneumococcus: transduction or pseudotransduction?

Lysates of pneumococcal phage PG24 transferred genes from one host to another in a process with many of the properties of generalized transduction, in that the host genes were packaged in DNase-resistant particles that closely resembled infectious phage in physical properties, adsorbed to the recipient cells like phage, and were inhibited by antisera to the phage and by trypsin. However, phage processes did not complete the transfer of host DNA as they did phage DNA. Instead, gene transfer required development of competence and entry of the host DNA by the endonuclease-dependent pathway used for transforming and transfecting DNA. This process often occurred on the assay plate hours after adsorption of the particles to the cells, and the transfer was DNase sensitive if challenged at this time. Phenotypic expression was therefore also delayed. The product of entry was like that in transformation, a single strand of DNA that integrates by formation of a hex-sensitive donor-recipient heteroduplex. Whether this gene transfer process is unique to this system or is only the first one described is not clear. The term "pseudotransduction" may be useful in calling attention to its unexpected features. The DNA of PG24 phage has anomalous physical properties reflecting unusual bases.

Mixed function oxidases in sterol metabolism. Separate routes for electron transfer from NADH and NADPH.

Oxidative deformylation of 4-hydroxy[14C]methylene-5alpha-cholest-7-en-3-one and oxidative demethylation of [30,31-14C]4,4-dimethyl-5alpha-cholest-7-en-3beta-ol by rat liver microsomes have been compared with regard to the manner in which electrons are introduced from both NADH and NADPH. Evidence suggests that NADH and NADPH support oxidation of both substrates via separate routes of electron transfer. Thus, 10 micron cytochrome c will inhibit NADPH-supported oxidation to 40 to 50% of control activity leaving NADH-supported oxidation unaffected. Also, treatment of microsomes with subtilisin diminishes NADPH-supported oxidation to 10 to 30% of control activity for either substrate to 70 to 90% of control activity while NADH-supported oxidative activity is virtually unaffected. Studies on the oxidase activities and NADPH-cytochrome c reductase as well as NADH-ferricyanide reductase have shown marked differences in activity in the presence of inhibitors. Thus, 9 mM 2'-AMP inhibits NADPH-cytochrome c reductase to 10 to 20% of control activity while NADPH-supported oxidative demethyl ation and deformylation are essentially unchanged. Mersalyl at 15 to 25 nmol/mg of microsomal protein inhibits both reductases to 20 to 40% of control activity; oxidative demethylation is unaffected and oxidative deformylation stimulated slightly when NADPH is used. Finally, antibody to NADPH-cytochrome c reductase inhibits oxidase activity for either substrate to 70 to 90% of control activity while reductase activity is inhibited to 10 to 30% of control activity.

Evidence for multiple electronic forms of two-electron-reduced lipoamide dehydrogenase from Escherichia coli.

Results are presented which demonstrate that the 2-electron-reduced lipoamide dehydrogenase (EC 1.6.4.3) from Escherichia coli is a mixture of species. In catalysis, this enzyme cycles between the oxidized and the 2-electron-reduced forms. Three spectrally distinct species are indicated in the pH range 5.8 to 8.0 from measurements of the fluorescence and visible spectra during dithionite titration. These have the following properties. 1) A fluorescent form where the FAD is oxidized and the active center disulfide is reduced. This species is unable to charge transfer and predominates at low pH. 2) A form in which there is a facile charge transfer between thiolate and FAD (epsilon530 - 3300 M-1 cm-1). This species, which predominates at high pH, is very similar to the 2-electron-reduced pig heart enzyme at high pH. 3) A form where the flavin is reduced and the disulfide is oxidized. The spectra of these three species have been determined. Anaerobic reduction of the enzyme with stoichiometric dihydrolipoamide leads to the formation of the charge transfer species in less than 1 s. Subsequently, in a process requiring about 12 s, the charge transfer complex relaxes to a mixture of species observed in dithionite titrations. The pH dependence of the oxidation-reduction potential, the fluorescence, the charge transfer absorbance (530 nm), and the 455 nm absorbance indicates the presence of a base which is able to stabilize the thiolate anion generated upon reduction of the active center disulfide. The pH dependence of the oxidation-reduction potential indicates that the reduction of the enzyme by dihydrolipoamide involves 2 protons as well as 2 electrons. These potentials are somewhat more positive than those determined for the pig heart enzyme and thus explain the ready further reduction of the E. coli enzyme to the 4-electron-reduced enzyme. The pH-independent formation constant (Kf) for the disproportionation of 2-electron-reduced enzyme (2EH2 in equilibrium E + EH4) is about 55 as calculated from dithionite titrations. Therefore at equilibrium there is about 80% 2-electron-reduced enzyme, 1-% oxidized enzyme, and 10% 4-electron-reduced enzyme. The spectrum of fully formed 2-electron-reduced enzyme has been calculated at several pH values from these data. The results confirm the previous conclusion that lipoamide dehydrogenase from E. coli is qualitatively similar to the pig heart enzyme, differing only in certain quantitative features such as the distribution between the various forms at the 2-electron-reduced level.

Regulation of mitochondrial pyruvate carboxylation and gluconeogenesis in rat hepatocytes via an alpha-adrenergic, adenosine 3':5'-monophosphate-independent mechanism.

Experiments were performed to determine if catecholamines can regulate control points in the gluconeogenic pathway, such as mitochondrial pyruvate carboxylation and pyruvate kinase activity, via an alpha-adrenergic, adenosine 3':5'-monophosphate-independent mechanism. Of a number of alpha agonists tested, only norepinephrine, epinephrine, and phenylephrine caused an increase in mitochondrial pyruvate metabolism. The effects of catecholamines on pyruvate carboxylation were not attenuated by 1-propranolol which abolishes changes in cyclic nucleotide levels but were blocked by alpha antagonists such as ergotamine, phenoxybenzamine, and phentolamine. Time course experiments demonstrated that the effects of catecholamines on the mitochondria and on carbohydrate metabolism correlated temporally with the concentration of epinephrine in the medium but not with the small changes in adenosine 3':5'-monophosphate. The effects of catecholamines appeared to require extracellular Ca2+ ion. The observation that catecholamines do not increase gluconeogenesis to the same extent as glucagon was not due to a differential effect on mitochondrial CO2 fixation. Rather, catecholamines caused a smaller inhibition of pyruvate kinase activity than did glucagon. The effects of catecholamines on pyruvate kinase also appeared to be mediated by an alpha-adrenergic, adenosine 3':5'-monophosphate-independent mechanism.

Quantitative analysis of methylguanidine and guanidine in physiologic fluids by high-performance liquid chromatography-fluorescence detection method.

A rapid and sensitive high-performance liquid chromatographic method has been developed for the quantitative analysis of methylguanidine and guanidine in physiological fluids. These quanidino compounds are separated on a 6 x 0.23 cm cation-exchange column with 0.5 M sodium hydroxide solution. The guanidino compounds are detected with a fluorometer, which monitors the fluorescent guanidine derivatives produced by the reaction of the eluted constituents with 9,10-phenanthrenequinone. Sensitivity to sub-nanomole levels of methylguanidine and guanidine is demonstrated. The method was successfully applied to physiological fluids such as serum and cerebrospinal fluid from uremic patients.

Biological apatite crystal disolution.

The dissolution curves of human acid-treated enamel powder are characterized by a rapid initial step followed, after 10 or 15 minutes, by a second stage, with release of very small amounts of calcium. Increase in pH and addition of fluoride ions tend to diminish, for short intervals, the amount of dissolved apatites. For higher pH values the decrease is noted over a longer time. Study of the relative ionic variation of human enamel powder from young and adult patients subjected to acid requires infra-red vibration band analyses. Special attention was given to the absorptions at 610 cm-1 (phosphate groups), 880 cm-1 (CO32- in OH- sites and HPO42-), 1410 cm-1 (CO32- in phosphate sites) and 1550 cm-1 (CO32- in OH- position). All results were related to vibration band at 610 cm-1. A preferential loss of carbonates in the two possible sites was always observed for 10 or 15 minutes, followed by a high release of phosphate. The increase of pH or small amounts of fluoride displaced the preferential carbonate loss to longer times. In the presence of higher fluoride levels a disappearance of the preferential loss of carbonates was noted. A continuous increase of HPO42- in the absence of fluoride occurred; however a straight line with a smaller shape was present, with fluoride addition.

[Horseradish peroxidase: a study of the kinetics and the determination of optimal reaction conditions, using hydrogen peroxide and 2,2'-azinobis 3-ethylbenzthiazoline-6-sulfonic acid (ABTS) as substrates (author's transl)].

The reaction of the two substrates hydrogen peroxide and ABTS with horseradish peroxidase was studied kinetically. Enzyme activity was determined as a function of substrate concentration and pH. Michaelis constants were determined for the two substrates at various pH values. It was found that the affinity of the enzyme for ABTS decreases with increasing pH, and that with higher ABTS concentrations the pH optima of the reaction are shifted towards neutrality. Maximal rate is reached at pH 4.2 with an ABTS concentration of 2 mmol/l. For hydrogen peroxide the data show that the dissociated O2H- is the proper substrate, its affinity for the enzyme being independent of pH. The two substrates show competitive binding to the peroxidase, and each therefore influences the binding constant of the other. A procedure is proposed which allows the determination of peroxidase down to a concentration of 10 ng/l or 2.5 x 10(-13) mol/l.

[The effect of chenodesoxycholic acid on cholesterol gallstones (author's transl)].

30 patients with radiolucent gallstones and 2 patients with radioopaque stones recieved chenodesoxycholic acid (CDC). In 12 patients stones were dissolved completely by CDC therapy, in 6 of the patients the size of the stones decreased by 40-70%. Only 1 patient developed severe diarrhea, so therapy had to be stopped. In 5 patients there was a slight and transient increase in SGOT and SGPT, in 2 other patient gammaGT increased; value normalized while treatment was continued. In 23 cases liver biopsies were done before and during treatment: no histological changes could be found in the specimens. 5 of the patients, whose gallstones had been dissolved, received 500 mg CDC/day thereafter, another 5 patients received 500 mg/d every other day. No recurrence of lithiasis was observed in either group. One patient on 500 mg CDC twice a week developed lithiasis after 6 months, another one stopped treatment completely and gallbladder stones reappeared after 8 months. The patients with radioopaque stones did not show any changes during treatment.

Neuropharmacology of sedatives and anxiolytics.

Sedative drugs are intended to cause various degrees of drowsiness. Animal experiments indicate that barbiturates induce these effects primarily by depression of the reticular activating system in the rostral brainstem. This in turn potentiates the thalamic recruiting system, thereby inducing 'barbiturate bursts' in the EEG. Anxiolytic drugs are intended to reduce anxiety or tension at doses which do not cause sedation or sleep. Propanediols may depress deactivating centers in the caudal brainstem, thereby releasing the activating centers in the rostral brainstem and depressing the thalamic recruiting response. These drugs may also act on the amygdala. Benzodiazepines have depressant effects on the amydala or hippocampus. These effects may release the reticular formation from inhibition. Enhanced activity of the activating and deactivating centers, to a different extent in different animals, would produce restlessness in some animals and sedation in others, accompanied by a mixture of fast and slow waves in the EEG. Sedative and anxiolytic agents also have central relaxant effects. The barbiturates act directly on the spinal cord, depressing both monosynaptic and polysynaptic reflexes. Propanediols and benzodiazepines act primarily on the descending facilitatory influence of the brainstem. Reduction of this influence depresses spinal polysynaptic but not monosynaptic reflexes. Biochemical studies suggest that barbiturates may act by antagonizing synaptic excitation induced by glutamate. Benzodiazepines may act by enhancing presynaptic inhibition mediated by GABA. The mechanism of action of propanediols is unknown.

Blood gas measurements.

pH and blood gas measurements are used to detect and monitor ventilation, oxygenation, and acid-base disturbances. The blood sample must be drawn anaerobically and transported in ice water to the laboratory. Partial pressure of carbon dioxide in arterial blood (PaCO2) reflects alveolar ventilation; partial pressure of oxygen in arterial blood (PaO2) reflects oxygen loading. The alveolar-arterial PO2 gradient (PA-aO2) distinguishes hypoxemia due to hypoventilation from that due to inefficent pulmonary gas exchange. The demand status of the cardiovascular system and the hemoglobin value reflect oxygen delivery to tissues. The relationship among pH, PaCO2, and bicarbonate concentration, when interpreted in the light of clinical findings, specifies the type and duration of acid-base disturbance.