Physiological disposition and metabolic fate of a new antiarrhythmic agent, alpha, alpha-dimethyl-4-(alpha, alpha, beta, beta-tetrafluorophenethyl) benzylamine in the rat, dog, monkey, baboon, and man. The physiological disposition of a new orally active antiarrhythmic drug, alpha, alpha-dimethyl-4-(alpha, alpha, beta, beta-tetrafluorophenethyl)benzylamine (MK-251) was investigated in the rat, dog, rhesus monkey, baboon, and man. MK-251 was extensively absorbed after oral administration in all species. Fecal excretion was the major route of tracer elimination in the rat (70%) and dog (80%), whereas the monkey, baboon, and man excreted the majority of the dose via the urine (40-80%). MK-251 and/or its metabolites were widely distributed in rat tissues and exhibited tissue/plasma ratios greater than one in most instances. The lung, liver, and kidney possessed a high tissue affinity for drug and metabolites. The plasma and urinary profile of radioactivity indicated extensive metabolism of MK-251 in all species. Less than 5% of the plasma and urinary radioactivity was identified as unchanged drug. In spite of extensive metabolic transformations, a remarkable feature of this drug is its persistence in the plasma for long periods of time. This is thought to be due to tissue affinity. The metabolic pattern for MK-251 was essentially the same in all species. The major metabolites present in the plasma and the urine were identified as the carbinol analog of MK-251, 2-[4-(alpha, alpha, beta, beta-tetrafluorophenethyl)phenyl]-2-propanol (I), and its glucuronide conjugate. Other metabolites characterized in the urine and plasma were: the N-glucuronide of MK-251, 2-[4-(alpha, alpha, beta, beta-tetrafluorophenethyl)phenyl]propene (II), 2-nitro-2-[4-(alpha, alpha, beta, beta-tetrafluorophenethyl)phenyl]propane (III), alpha, alpha-dimethyl-4-(alpha, alpha, beta, beta-tetrafluorophenethyl)benzyl methyl ether (IV-1) and 4-(alpha, alpha, beta, beta-tetrafluorophenethyl), acetophenone (IV-2). Two minor urinary metabolites were tentatively identified as the N-hydroxy analog of MK-251 and the glycol analog of carbinol I. The in vivo formation of the methyl ether represents the first report of alkylation of a tertiary alcohol.


[Treatment of hypertrophic obstructive cardiomyopathy with verapamil, a calcium antagonist (author's transl)]. Cardiac catheterisation with pressure measurements, left-ventricular cine-angiography and selective coronary angiography confirmed the diagnosis of hypertrophic obstructive cardiomyopathy in 20 patients. After a mean observation period of 20 months during which most of them were treated with beta-blockers, verapamil, 480 mg by mouth, was given for an average of 12 months. There was an impressive improvement in symptoms, compared with the state under beta-blocker treatment. There was a significant reduction in the ECG signs of left-ventricular hypertrophy and of the radiologically measured heart volume. Treatment of this condition with verapamil appeared to be superior to that with beta-blockers.


Effect of 3-methylcholanthrene pretreatment on the bioavailability of phenacetin in the rat. A thin-layer chromatographic procedure is described for the quantitative determination of phenacetin and acetaminophen in rat plasma. The method was used to determine the effect of 3-methylcholanthrene (3-MC) on the disposition and bioavailability of phenacetin following its oral and iv administration to rats. Pretreatment with 3-MC decreased the plasma half-life of phenacetin, after iv administration, from 28 min to 4.5 min and reduced the systemic bioavailability of phenacetin, after oral administration, from 45% in control rats to 6% in 3-MC-treated rats. By comparing the plasma levels of phenacetin in the portal circulation with those in the peripheral circulation, following the oral administration of phenacetin, it was concluded that the 7-fold reduction in the bioavailability of phenacetin observed in 3-MC treated rats was caused by a marked increase in the metabolism of phenacetin during its first pass through the liver.


Effect of methadone dose on the biliary excretion of methadone metabolites in the rat. The effect of three different doses of 14C-methadone (0.08, 1.0 and 2.5 mg/kg) on the biliary excretion of methadone metabolites was studied in the rat. After administration of the 0.08- and 1.0-mg/kg doses of 14C-methadone there was no difference in the percentage of administered 14C excreted into bile with time. However, after the 2.5-mg/kg dose a significant increase was observed in the percentage of administered 14C excreted into bile. Analysis of bile samples showed that this increase was due to increases in the biliary excretion of two of the major metabolites of methadone. Several mechanisms could be responsible for this disproportionate increase in biliary excretion of methadone metabolites after high doses of methadone. It was found that the effect of high methadone dose on the biliary excretion of its metabolites was nearly eliminated when studied in phenobarbital (PB)-pretreated rats. Pretreatment of rats with PB increases the biliary excretion of methadone metabolites, primarily by increasing rates of methadone metabolism. The lack of additivity of the effect of the high dose of methadone and PB pretreatment on the biliary excretion of methadone metabolites suggests that a high dose of methadone also stimulates methadone metabolism, which results in the observed increased percentage of the administered dose excreted into bile.


[Studies on optimising rubella virus haemagglutination-inhibitor tests]. Immuno-electrophoretic and serological studies were undertaken to characterise the non-specific serum inhibitor of rubella haemagglutination. The majority of such inhibitor effects come from the beta(1)-lipoproteins. In addition, small inhibitory effects which were due to alpha(1)-lipoproteins were noted in some sera. The result of the tests is influenced less by removing the inhibitor than by conditions of incubation of the serum-antigen mixture, especially pH. The test is more sensitive if it is not performed at optimal pH for haemagglutination.


Inactivation of porcine calcitonin by rat kidney microsome. Biological activity of porcine calcitonin was most actively inactivated by the rat kidney homogenate than by other tissue homogenates. Among the various subcellular fractions of the rat kidney homogenate examined, microsome fraction was most active in the in vitro inactivation of porcine calcitonin. Inactivation of porcine calcitonin by the rat kidney microsome was dependent on pH and temperature. Inactivating activity of the rat kidney microsome was inhibited by 1 X 10(-3) M monoiodoacetate and 1 X10(-5) M p-chloromercuribenzoate. These results suggest that porcine calcitonin is probably inactivated by a SH-enzyme in the rat kidney microsomes. However, the participation of other enzymes cannot be ruled out, since the inactivating activity of the rat kidney microsome fraction is also inhibited by 1 X 10(-4) M diisopropylfuorophosphate.


Estimation of testosterone binding capacity in the serum with hydrophobie resin. A method for the estimation of the serum testosterone binding capacity (TeBC) using hydrophobic resin, Amberlite XAD-2, was developed. The serum was incubated with a saturating amount of testosterone-1,2(-3)H at 15 degrees C, unbound testosterone was adsorbed to the resin and the 3H-radoiactivity remaining in the supernatant fluid was counted. Under these conditions, the normal levels and their standard deviations were 15.08+/-3.39 ng/ml (n=7) for male and 35.06+/-3.56 ng/ml (n=6) for female respectively. Precision of the method was 6.29%. TeBC in the third month of pregnancy was approximately 1.5 times more than that of non-pregnant women, and approximately 3 to 5 times in the tenth month.


Dynamic asymmetries in liver tyrosine aminotransferase induction. Changes in liver tyrosine aminotransferase activity were measured in mice and rats following a whole body X-irradiation in order to ascertain the dose-effect relationship. The response was either (1) linear in case of low pulses (25 rad/min); (2) parabolic, indicating the onset of a saturation process in case of medium pulses (60 rad/min), or (3) biphasic, suggesting the cooperation of at least two regulatory mechanisms following longtime exposure to strong stimuli (100 rad/min).


Amino acid metabolizing enzymes in rat submaxillary gland, normal or neoplastic, and in pancreas. The activities of 12 enzymes, many related to ornithine metabolism, were measured in rat submaxillary gland, submaxillary gland tumors and pancreas. In submaxillary gland, the activities of arginase, ornithine aminotransferase, pyrroline-5-carboxylate reductase and glutamine synthetase were high, but no ornithine transcarbamylase or proline oxidase could be detected. In the fetal submaxillary gland, arginase was at almost adult levels while ornithine aminotransferase reached 50% of its adult value postnatally. Submaxillary tumors deviated from their cognate tissue by lower levels of amino acid metabolizing enzymes and by high concentrations of thymidine kinase. In pancreas, none of the pyrroline-5-carboxylate metabolizing enzymes were as high as in either liver or submaxillary gland. The outstanding activities were those of gamma-glutamyl transpeptidase and glutamate dehydrogenase. Although arginase activities in submaxillary gland and pancreas were quantitatively similar, they differed qualitatively: submaxillary gland contained the same variant as liver while the pancreatic isozymes resembled those of other nonhepatic tissues.


Relationships of femoral venous [K+], PO2, osmolality, and [orthophosphate) with heart rate, ventilation, and leg blood flow during bicycle exercise in athletes and non-athletes. The relationship of femoral venous [K+], [H+], osmolality (OSM), PO2, and [inorganic phosphate] ([Pi]) with heart rate (HR), ventilation (VE), and calculated leg blood flow (Q) were investigated during bicycle exercise in endurance trained (TR) and untrained (UT) test subjects. At a given VO2 the increases of [K+], OSM, [Pi] and the decrease of PO2 were significantly lower in TR than in UT. In the same proportion the increases of HR, VE, and Q were diminished. Thus in TR and UT identical and highly significantly correlated regression lines of [K+], [H+], OSM, [Pi] and PO2 with HR, VE, and Q were obtained. These constituents changed in the same proportion as the relative VO2 in TR and UT. No relationships with [Na+], [Ca++], and [ Mg++] were found. By means of a multiple regression analysis the partial influence of K+, H+, OSM, PO2, and Pi upon the total change of HR, VE and Q was estimated to compare with data from infusion experiments. The findings were discussed in view of the hypothesis that these candidates may provide linkage between metabolic events, circulatory, and ventilatory adjustments during work.


On the transport of tripeptide antibiotics in bacteria. The two tripeptide antibiotics L-2-amino-4-methylphosphinobutyryl-alanyl-alanyl-alanine (L-phosphinothricyl-alanyl-alanine) and L-(N5-phosphono)methionine-S-sulfoximinyl-alanyl-alanine, both inhibitors of the glutamine synthetase, are transported into the cell of Escherichia coli K 12 via the oligopeptide transport system. The uptake by this system is proved first of all by cross-resistance with tri-L-ornithine using oligopeptide-transport-deficient mutants, and secondly by antagonism tests demonstrating competitive reversal of the action of the antibiotic by several peptides which have been shown to be transported via the oligopeptide transport system, e.g. tri-L-alanine, tetra-L-alanine, tri-L-lysine, tri-L-serine, tri-glycine, glycyl-glycyl-L-alanine and the synthetic tripeptide L-azadenyl-aminohexanoyl-alanyl-alanine. On the other hand, there is no effect on the action of the antibiotic in antagonism tests with compounds which use different transport systems, such as L-alanyl-alanine, L-lysyl-lysine, glutathione and the synthetic amino acid azaadenylaminohexanoic acid, i.e. 2-amino-6-(7-amino-3H-v-triazolo-[4,5-d]-pyrimidin-3-yl)hexanoic acid. Another inhibitor of the glutamine synthetase, L-methionine-S-dioxide (methioninesulfone) could be converted into a tripeptide form by linkage to L-alanyl-alanine analogously to the tripeptide antibiotics described above. Whereas the free L-methionine-S-dioxide seems to be transported via the methionine transport system, the tripeptide form is transported via the oligopeptide transport system. Thus, this glutamine synthetase inhibitor can be taken up by the cell via two different transport mechanisms. Our results indicate that this could provide a synergistic effect. The syntheses of the new tripeptides L-azaadenylaminohexanoyl-alanyl-alanine and L-methionine-S-dioxidyl-alanyl-alanine were performed by dicyclohexylcarbodiimide couplings of the unusual N-protected L-alpha-amino acids azaadenylaminohexanoic acid and L-methionine-S-dioxide to L-alanyl-alanine-tert-butyl ester followed by common deprotection steps. Tri-L-ornithine was synthesized without carboxyl protection via two successive couplings of hydroxybenzotriazol esters of Nalpha-butoxycarbonyl-Ndelta-benzyloxycarbonyl-L-ornithine.


Yeast hexokinase: substrate-induced association--dissociation reactions in the binding of glucose to hexokinase P-II. A method is described for the purification of native hexokinases P-I and P-II from yeast using preparative isoelectric focussing to separate the isozymes. The binding of glucose to hexokinase P-II, and the effect of this on the monomer--dimer association--dissociation reaction have been investigated quantitatively by a combination of titrations of intrinsic protein fluorescence and equilibrium ultracentrifugation. Association constants for the monomer-dimer reaction decreased with increasing pH, ionic strength and concentration of glucose. Saturating concentrations of glucose did not bring about complete dissociation of the enzyme showing that both sites were occupired in the dimer. At pH 8.0 and high ionic strength, where the enzyme existed as monomer, the dissociation constant of the enzyme-glucose complex was 3 X 10(-4) mol 1(-1) and was independent of the concentration of enzyme. Binding to the dimeric form at low pH and ionic strength (I=0.02 mol 1(-1), pH less than 7.5) was also independent of enzyme concentration (in the range 10-1000 mug ml-1) but was much weaker. The process could be described by a single dissociation constant, showing that the two available sites on the dimer were equivalent and non-cooperative; values of the intrinsic dissociation constant varied from 2.5 X 10(-3) mol 1(-1) at pH 7.0 to 6 X 10(-3) at pH 6.5. Under intermediate conditions (pH 7.0, ionic strength=0.15 mol 1(-1)), where monomer and dimer coexisted, the binding of glucose showed weak positive cooperatively (Hill coefficient 1.2); in addition, the binding was dependent upon the concentration of enzyme in the direction of stronger binding at lower concentrations. The results show that the phenomenon of half-sites reactivity observed in the binding of glucose to crystalline hexokinase P-II does not occur in solution; the simplest explanation of our finding the two sites to be equivalent is that the dimer results from the homologous association of two identical subunits.


Affinity chromatography and binding studies on immobilized 5'-monophosphate and adenosine 2',5'-bisphosphate of nicotinamide nucleotide transhydrogenase from Pseudomonas aeruginosa. 1. Nicotinamide nucleotide transhydrogenase from Pseudomonas aeruginosa was purified to apparent homogeneity with an improved method employing affinity chromatography on N6-(6aminohexyl)-adenosine 2', 5'-bisphosphate-Sepharose 4B. 2. Polyacrylamide gel electrophoresis of the purified transhydrogenase carried out in the presence of sodium dodecyl sulphate, indicated a minimal molecular weight of 55000 +/- 2000. 3. The kinetic and regulatory properties of the purified transhydrogenase resembled those of the crude enzyme, i.e., NADPH, adenosine 2'-monophosphate and Ca2+ were activators whereas NADP+ was inhibitory. 4. Nicotinamide nucleotide-specific release of binding of the transhydrogenase to N6-(6-aminohexyl)-adenosine-2',5'-bisphosphate-Sepharose and N6-(-aminohexyl)-adenosine-5'-monophosphate-Sepharose suggests the presence of at least two separate binding sites for nicotinamide nucleotides, one that is specific for NADP(H) and one that binds both NAD(H) and NADP(H). 5. Binding of transhydrogenase to N6-)6-aminohexyl)-adenosine-2',5'-bisphosphate-Sepharose and activation of the enzyme by adenosine-2',5'-bisphophate showed a marked pH dependence. In contrast, inhibition of the Ca2+-activated enzyme by adenosine 2',5'-bisphosphate was virtually constant at various pH values. This descrepancy was interpreted to indicate the existence of separate nucleotide-binding effector and active sites.


Somatic antigens of shigella. Structural investigation on the O-specific polysaccharide chain of Shigella dysenteriae type 1 lipopolysaccharide. The O-specific polysaccharide obtained from the lipopolysaccharide of Shigella dysenteriae type 1 (Shigella shiga) by mild acid hydrolysis followed by fractionation on Sephadex G-50 was found to be identical to that desribed by Morgan's group and was composed of L-rhamnose, D-galactose and N-acetyl-D-glycosamine in a ratio 2:1:1. On the basis of methylation analysis data the polysaccharide was proved to be a linear chain of monosaccharide residues in pyranose forms substituted at position 3, except for that of galactose substituted at position 2. Selective cleavage, based on the N-deacetylation reaction of the polymer, together with determination of linkage configurations by chromic anhydride oxidation showed that the O-specific polysaccharide is built up of repeating tetrasaccharide units whose proposed structure is given below -3)-alpha-L-Rhap (1-3)-alpha-L-Rhap(1-2)-alpha-D-Galp(1-3)-alphapD-GlcNAcp(1- where RHAP = rhamnopyranose, Galp = galactopyranose, and GlcNAcp = N-acetyl-glucosamine. The present findings confirmed the considerations of Heidelberger on the substitution patterns of L-rhamnose and D-galactose residues from the results of serological studies.


Purification and properties of 3-hexulosephosphate synthase from Methylomonas M 15. 3-Hexulosephosphate synthase, the first enzyme of the ribulose monophosphate cycle, was purified 15-fold from methanol-grown Methylomonas M 15. The purification procedure involved chromatography on DEAE-cellulose, Sephadex G-75, and DEAE-Sephadex A-50. The purified enzyme was more than 95% pure as judged by analytical polyacrylamide gel electrophoresis. The molecular weight was calculated to be 43000 from sedimentation equilibrium experiments. Electrophoresis in sodium dodecylsulfate gels gave a single band corresponding to a molecular weight of 22000. The enzyme catalyzes specifically the condensation formaldehyde with ribulose 5-phosphate to yield D-arabino-3-hexulose 6-phosphate. The Km values were found to be 1.1 mM for formaldehyde and 1.6 mM for ribulose 5-phosphate. A bivalent cation is essential for activity and stability of the enzyme, Mg2+ and Mn2+ serve best for this purpose. The optimum of pH for enzyme activity is 7.5--8.0.


Interest of isoproterenol in the treatment of hemorrhagic shock in dogs. Four groups of experiments were focused on two problems: first, the replacement of the substracted amount of blood, and second, the correction of the peripheral reactions to substantial blood loss, namely vasoconstriction and acidosis. We stuck to a simple plan in our experiments: lost blood was entirely collected and retransfused. In 26 cases out of 37, we used isoproterenol hydrochloride (Isuprel - WINTHROP) to open the peripheral vascular bed. In six different groups, we followed the response to variations of retranfusion and administration of isoproterenol. Several parameters were studied: arterial blood pressure, central venous pressure, rectal temperature and pH. The association of blood transfusion with injection of isoproterenol, in adequate amounts to correct hypovolemia and to prevent vasoconstriction, is undoubtedly the best treatment of hemorrhagic shock.


The noradrenergic cyclic AMP generating system in the limbic forebrain: pharmacological characterization in vitro and possible role of limbic noradrenergic mechanisms in the mode of action of antipsychotics. The cyclic AMP generating system in slices of the rat limbic forebrain was investigated. In consists of: (u) A noradrenergic system which responds to norepinephrine (NE) and isoproterenol. Though the rise of the nucleotide elicited by isoproterenol is more rapid than that caused by NE, the maximal effect is less than half of that induced by NE; (2) an adenosine-dependent system. The noradrenergic cyclic AMP generating system in the limbic forebrain displays a number of properties of a central NE receptor: it develops supersensitivity to NE and isoproterenol following prolonged deprivation of NE at postsynaptic sites (chronic treatment with reserpine or chemosympathectomy with 6-hydroxydopamine). When noradrenergic terminals are protected from 6-hydroxydopamine by desmethylimipramine, the responses to NE are not enhanced. Responses to NE are blocked by both propranolol and phentolamine, while responses to isoproterenol are blocked by propranolol but not by phentolamine. The adenosine-dependent system does not develop supersensitivity after central chemosympathectomy and is not blocked by either alpha- or beta-antagonists. While not altering the basal level of the nucleotide, clinically effective antipsychotic drugs caused a dose-dependent inhibition of the limbic noradrenergic cyclic AMP response with clozapine and pimozide being particularly potent (IC50 0.06 and 0.08 muM, respectively). Antipsychotic drugs do, however, not affect cyclic AMP responses elicited by adenosine. The results are compatible with the view that the central NE receptor is closely related to or may be an integral part of an adenylate cyclase system and that its blockade in the limbic forebrain by antipsychotic drugs may contribute to their therapeutic action.


Effect of propranolol of vascular responses to sympathetic nerve stimulation and plasma renin activity in mongrel dogs. I.v administration of propranolol (0.2 mg/kg and 1.0 mg/kg) to pentobarbital-anesthetized dogs produced blockade of cardiac beta-receptors and a significant decrease in heart rate. However, only the higher dose of propranolol demonstrated a significant hypotensive effect. Furthermore, this hypotensive action of propranolol was not associated with either adrenergic neruron blockade or changes in plasma renin activity. These results indicate that the initial hypotensive action of propranolol in mongrel dogs is not due to the blockade of beta-receptors, alterations in peripheral sympathetic nervous transmission or plasma renin activity. On the other hand, the action that propranolol is reported to have within the central system may play an important role in accounting for the acute blood pressure lowering action of the compound in mongrel dogs.


Hypotensive responses following oral adminstration of beta-adrenoceptor blocking drugs to the conscious cat. On oral administration, the non-selective beta-adrenoceptor blocking drugs (+/-)-bufuralol, (-)-bufuralol, propanolol, oxprenolol, pindolol and alprenolol produced hypotensive responses in the conscious cat; (+)-bufuralol was without effect. The selective beta-adrenoceptor blocking drugs practolol and atenolol had no effect on blood pressure but tolamolol elicited a hypotensive response. All the drugs tested reduced the tachycardia due to intravenous isoprenaline in the conscious cat; however, not all doses of these drugs reduced blood pressure. (+)-Bufuralol was devoid to beta-adrenoceptive blocking activity. Only tolamolol reduced the pressor response to i.v. phenylephrine in the conscious cat, indicating that alpha-adrenoceptive blocking activity may contribute to its hypotensive action. The results suggest that beta-adrenoceptive blocking activity is necessary for the hypotensive responses of these drugs. However, for the different drugs, there was no correlation between peripheral beta-adrenoceptive blocking activity and hypotensive response.


A kinetic analysis of a catechol-specific binding site in the microsomal fraction from the rabbit aorta. (-)-3/-Norepinephrine (3H-NE) binding to the microsomal fraction of the rabbit aorta has been studied. Binding appears to increase linearly with time up to at least 30 min, shows no evidence of stereoselectivity and may be inhibited only by compounds possessing the catechol or 3-methoxy-4hydroxyphenyl moieties, with the latter being 100-fold less effective. 3H-NE binding is saturable with a Km of 8.5 X 10(-8) M and V max of 28 pmoles/mg protein. A Hill plot indicates that binding is noncooperative whereas a Scatchard plot suggests that two sites may be present. Binding does not appear to require physiological concentrations of Ca2+ or Mg2+ and is inhibited significantly by EDTA and sodium metabisulfite. In addition, binding is markedly enhanced by low and high pH values. This binding is also inhibited by sodium metabisulfite which suggests that an oxidized form of the catecholamine is the active binding species. Experiments with several group specific reagents indicate that binding may require a free sulfhydryl group but not a carboxyl function. The binding process requires an energy of activation of 14.8 kcal/mole whose magnitude may be partly explained, with the aid of optical rotatory dispersion spectra, by a non-stereoslective conformational change in protein structure induced by the amine. The characteristics of the 3H-NE binding sites observed in the microsomal fractional of the rabbit aorta appear to be different from those expected if binding were to the adrenoreceptors. A possible mechanism for catecholamine binding to free sulfhydryl groups on protein is presented.


Alpha-adrenergic activity of N,N-dimethyldopamine (DMDA). The autonomic and cardiovascular activity of N,N-dimethyldopamine (DMDA) was studied in the dog and rabbit. DMDA appears to be a postganglionic sympathetic alpha-adrenoceptor agonist since it consistently caused vasconstriction in several isolated vascular beds, even in the presence of ganglionic blockade. The pressor activity of DMDA was attenuated by the alpha-adrenergic antagonist phentolamine. Calculated pA2 values for phentolamine against both DMDA and norepinephrine in isolated rabbit arteries were in close agreement. DMDA inhibits cardioaccelerator nerve stimulation induced chronotropic responses by a mechanism which appears to be alpha-adrenergic in the dog.


Glycoprotein biosynthesis in splenic cells. Purification of a microsomal galactosyl-transferase. One part of the microsomal galactosyl-transferase activity of splenic cells of rats can by solubilized by the action of Triton X-100 and Tween 20. Its purification on a Sephadex G-200 column and by electrophoresis on a polyacrylamide gel leads to a solution of high specific enzymic activity.


Beta-adrenergic blocking agents as potent antagonists of mescaline-induced contractions in the rat uterus. In the isolated rat uterus, mescaline induces contractions that are notably antagonized by catecholamines, by beta-adrenergic stimulants and certain beta-adrenergic blocking agents as well as by chlorpromazine, amitriptyline and methysergide.


Influence of castration and testosterone replacement on hypothalamic tyrosine hydroxylase activity in the rat. Hypothalamic tyrosine hydroxylase (TH) activity of castrate rats is modulated by testosterone propionate (TP) in vivo. Kinetic studies revealed that both Vmax and Km were virtually unaltered for substrate tyrosine in the presence of an excess of DMPH4 cofactor. TP replacement to castrate rats increased the Km for added DMPH4 cofactor, while Vmax decreased. These results suggest that TP decreases TH activity of castrate rats by inhibiting the enzyme-reduced pteridine cofactor complex.


Effects of autonomic drugs on epididymal contractions. The spontaneous contractility of rat epididymis was recorded in vivo and the effects of various autonomic drugs were studied. Norepinephrine, epinephrine, and orciprenaline produced a sudden increase in tonus and in the size and frequency of epididymal contractions. Phentolamine (an alpha-blocker agent) inhibited the effects of norepinephrine. On the other hand, alprenolol (a beta-blocker agent) inhibited the effects of orciprenaline but did not block the effects of norepinephrine. In addition, phentolamine and alprenolol decreased the spontaneous activity of the epididymis. Acetylcholine produced effects similar to those of norepinephrine. These effects were blocked by atropine. The results described would indicate the presence of the two receptors, alpha and beta, and that both are mediators of stimulatory effects.


[Studies of various factors in thyrotropoin releasing hromone TRH) radioimmunoassay for serum (author's transl)]. To study the significance of TRH in the hypothalamo-pituitary-thyroid axis measurement of TRH in body fluid are needed. We previously reported TRH radioimmunoassay for urine. TRH radioimmunoassay for serum has not established yet, because TRH immunoreactivity is inactivated with serum. We investigated effects of various factors on this inactivation and method for prevention of this inactivation. Synthetic TRH was added to normal human serum at 4 degrees C and incubated at 60 degrees C, 37 degrees C, 20 degrees C, 4 degrees C or -20 degrees C for various intervals. After incubation, recovery of TRH was measured. After one hour incubation, recovery of TRH was 9.2% at 37 degrees C, 34.5% at 20 degrees C, 100% at 4 degrees C or -20 degrees C. Incubation of TRH serum mixtures at 65 degrees C after incubation at 37 degrees C resulted in some recovery of TRH. After one hour incubation at 37 degrees C, recovery of TRH was 9.2% at serum pH 7.0, 100% at serum pH 3.0 to 5.0 or 11.0. Recovery of TRH was increased in accordance with stepwisely increase of serum dilution. Concentrations of serum thyroid hormone did not affect recovery of TRH. Smaller quantities of TRH were more rapidly inactivated. Inactivation of TRH immunoreactivity could be prevented addition of BAL (over 0.25 mg/ml) or mixture of 8-Hydroxyquinoline (HQ) and Tween 20(T) (over 0.1 mg/ml of HQ and 1 lmg/ml of T). Duration of effectiveness of BAL was short. Effectiveness of HQT continued for 12 weeks, if HQT treated serum was stored at -20 degrees C. From above data it was suggested that TRH immunoreactivity might be inactivated with enzyme system and other factors and TRH levels in the serum might be able to measure with addition of HQT to serum.


[Cutaneous side effects of systemic drugs. Part 4 of a synopsis. 6/7. Drugs affecting the central nervous system. C. Drug-induced photosensitivity. D. Drug-induced changes of skin color]. This, the fourth part of a synopisis of cutaneous side effects of drugs, covers the drugs affecting the central nervous system: antiepileptics, hypnotics, narcotics and psychopharmaceutics; the myorelaxants and antiallergics follow, and lastly there is a section on drug addiction and placebo. The various cutaneous side effects are listed in chart form referring to more than 500 sources. A drug index is attached for handy reference. The reviews of certain drug induced skin disorders are continued with tables covering photosensitivity and changes in skin colour. Phototoxicity, photoallergy and light sensitivity by porphyria are differentiated. The various pigmentation disorders, colour changes due to metal deposits as well as different localisations are included.


Gastric and extragastric gastrin release in normal subjects in duodenal ulcer patients, and in patients with partial gastrectomy (Billroth I). In 10 normal subjects, in 32 patients with duodenal ulcer (DU), and in 11 patients with partial gastrectomy (Billroth I), serum gastrin rose significantly after an oral and intraduodenal test meal. The highest increases were observed in DU patients after the oral as well as after the intraduodenal test meal. After the intraduodenal test meal in 4 normal subjects and in 17 DU patients an increase of gastric acid secretion and serum gastrin was measured. In basal state, after an intraduodenal or an oral test meal, DU patients with normal gastric acid secretory capacity had higher serum gastrin concentrations than DU patients with gastric hypersecretion. There was a good correlation between peak serum gastrin levels after the oral and after the intraduodenal test meal. From these data it is concluded: (1) Intraduodenal application of a test meal results in release of gastrin from extragastric sites. (2) Extragastric gastrin is biologically active. (3) DU patients are able to release more antral and more extragastric gastrin in response to a test meal. Further studies, however, are necessary to show the significance of these findings in the pathogenesis of peptic ulcer disease.


Effects of carbenoxolone on gastric mucosal permeability and blood flow in the dog. The effects of topical application of carbenoxolone at neutral and acidic pH were compared in exteriorized, chambered segments of canine gastric corpus. When dissolved in saline at pH 7.5 to 8.0, 0.25% carbenoxolone caused a rapid drop in gastric potential difference of 56 +/- 2 mv and greatly increased permeability to H+ ions. Blood flow, as measured by radioactive microspheres, was not changed by carbenoxolone treatment, but subsequent exposure to isotonic HC1 caused an abrupt rise in flow. Application of 0.25% carbenoxolone suspension in isotonic HC1 caused no change in potential difference, permeability, or blood flow. Neither carbenoxolone preparation had a significant effect on aspirin-induced H+ back-diffusion or injury.


Recombinant clone heterogeneity in Escherichia coli conjunction: effect of pH and partially replicated recipient deoxyribonucleic acid. At pH 6.8, a substantial fraction of recombinant colonies obtained from conjugation with an HfrH donor contained multiple recombinant classes in a single colony (polygenotype colony). In contrast, when the conjugation was performed at pH 7.6, the number of polygenotypic colonies was drastically reduced, and the recombinant colonies were predominantly monogenotypic or digenotypic. Genetic analysis revealed that the digenotypic recombinants differ in those donor markers near the origin of DNA replication but share those donor markers near the terminus. This integration pattern suggests that the formation of digenotypic recombinants involves recombination of a single copy of the exogenome with a partially replicated recipient DNA molecule. This suggestion was supported by examination of the genotype of recombinant colonies recovered from crosses with an HfrKL96 donor which was derived from HfrH but transfers its chromosome in the reverse direction.


[Galenic studies of a new gastrotherapeutic drug]. This paper presents the methods for galenical investigations of a new gastrotherapeutic preparation. The results and their possible relations to therapy are discussed. The preparation is presented a press-coated tablet. Its antacid characteristics concerning the rate of effectiveness, potency and duration are compared with leading brands on the German market. Pepsin inactivation together with the rapid liberation of the spasmolytic agent propantheline and the psychically stabilising agent peranzine are demonstrated. The results of the stability tests for the active ingredients and the in vitro availability are described. The new product is a gastrotherapeutic preparation, which fulfils all requirements for rapid action and reliable therapy.


[Field study on the decrease of lipids using etofibrate]. A field-trial on Etofibrat was performed on 4405 patients suffering from primary and secondary hyperlipoproteinemia. The results were proved statistically. After a 3 to 4 weeks treatment the concentration of cholesterol as well as tryglicerides in the serum decreased significantly. After 6 to 8 weeks treatment the lipid-lowering effect was even stronger. Nearly 90% of the patients investigated gave a positive response to the treatment. Part of the population had undergone a treatment with other lipid-lowering agents before-most likely without sufficient success-in these cases a further lipid-lowering effect due to Etofibrat could be shown. Under-dosage in premedication cannot be excluded. The stratification of the patients in different groups of diagnosis showed a nearly similarity of both blood lipids independent of the diagnosis. This could also be confirmed for the group of patients suffering from diabetes. To prove the lipid-lowering efficacy of Etofibrat a population was withdrawn from treatment. Cholesterol as well as tryglicerides increased significantly during the interval without treatment. During a long-term study both lipid fractions could be kept down without increasing of the daily Etofibrat dose. The tolerance of Etofibrat was stated to be good up to very good. Objectively the measured enzymes SGOT, SGPT and gamma-GT showed a decrease of the means. Subjectively the occurrence of an often intermediate heat sensation and/or rubedo was of relevance. The low daily dose compared with other lipid-lowering agents gives indication for a better pharmacocinetic behaviour of the drug;


Plasma insulin concentration during physiological variations in immunoreactive plasma secretin. The effect of endogenous and exogenous secretin on fasting plasma insulin and glucose concentrations in peripheral venous blood was studied. In 10 non-diabetic subjects intragastric instillation of 300 ml 0.1 mol/l hydrochloric acid increased the plasma secretin concentration significantly. This increment did not influence insulin or glucose concentration. Control experiments with intragastric instillation of 300 ml of isotonic saline did not influence the plasma concentration of secretin, insulin or glucose. In four other non-diabetic persons no significant changes were found in plasma insulin or glucose concentration during an i.v. infusion of pure natural porcine secretin in doses of 0.1, 0.3, 1.0 and 3.0 clinical units/kg/h. The results suggest that secretin is without effect on insulin secretion in the fasting normal subject.


Stability of the insoluble form of uridine kinase coupled to zn2+ or pb2+ ions. Partially purified calf brain uridine kinase precipitated by bivalent metal cations has been compared with the soluble enzyme fraction regarding its stability in the presence of inactivating factors. The freeze-dried preparations of uridine kinase precipitaated by Pb2+ or Zn2+ ions, althouth enzymatically highly active, are insoluble in aqueous solutions. The activity of metal-insolubilized enzymes disappears during their preincubation in acidic media or in the presence of silver ions. Also trypsin, chymotrypsin and cathepsin B1 caused decreases in enzyme activity. However, fractions which have been precipitated by metal ions and freeze-dried are stable at high temperatures, whereas the activity of soluble uridine kinase is completely lost. Both unheated metal-ion precipitated uridine kinase preparations and those heated at 100 degrees C are equally sensitive to the feedback inhibition by CTP.


[Effect of 2-hydroxyestradiol-17beta on NADPH-dependent electron transfer in rat liver microsomes in vitro (author's transl)]. If rat liver microsomes are incubated with NADPH and 2-hydroxyestradiol-17beta in vitro, the following is observed: 1. Inhibition of lipid peroxidation, 2.inhibition of cytochrome P-450 reduction, and 3.inhibition of cytochrome b5 reduction. Beyond this the catechole inhibits lipid peroxidation of liposomes in vitro. These phenomena can be explained by interaction of different states of oxidation of the estrogen with the NADPH-cytochrome reductase and with 0-2 radicals, which leads to terminal "uncoupling" of microsomal electron transport.


Purification and characterisation of four polypeptides with neurotoxic activity from Condylactis aurantiaca. Four toxic polypeptides, Toxins I, II, III and IV were isolated in pure form from the sea anemone Condylactis aurantiaca (Actinaria). Toxin isolation was achieved by alcoholic extraction of the homogenised sea anemones, batchwise adsorption onto cation exchangers, gel filtration on Sephadex G-50 and G-25 and ion-exchange chromatography on SP-Sephadex and QAE-Sephadex. Toxins from Condylactis aurantiaca all contain between 49 and 51 amino acids. Their amino acid compositions were compared to those of the Anemonia sulcata toxins. The toxins were tested on the shore crab Carcinus maenas by intramusclar injection. Crabs react highly sensitively to sea anemone toxins with muscle cramps and paralysis. For Condylactis toxins LD100 ranges from 2 to 6.6 mug/kg Carcinus maenas.


Hydrolysis-resynthesis equilibrium of the lysine-15--alanine-16 peptide bond in bovine trypsin inhibitor (Kunitz). Catalytic amounts of bovine beta-trypsin, bovine alpha-chymotrypsin and porcine plasmin establish a true thermodynamic equilibrium between virgin (I) (reactive site Lys15-Ala16 peptide bond intact) and modified (I) (this bond hydrolyzed) bovine trypsin/kallikrein inhibitor (Kunitz). The very slow reaction rates for attaining equilibrium are pH-dependent and differ for different enzymes. Optimal rates are for beta-trypsin at pH 3.75, for alpha-chymotrypsin at pH 5.5, and for plasmin at pH 5.0. Under conditions of optimum pH the equilibrium is reached with the highest rate by plasmin. In 10(-5)M inhibitor solutions the equilibrium concentrations of virgin and modified inhibitor are established by plasmin after almost 300 days starting from either pure virgin or pure modified inhibitor. Thus, the hydrolysis constant KHyd = [I]/[I] is determined to be 0.33 at pH 5.0. In spite of many unsuccessful attempts, this demonstrates that the reactive site peptide bond Lys15-Ala16 in the bovine trypsin inhibitor (Kunitz) can be hydrolyzed by catalytic amounts of endopeptidase. It further confirms that the hydrolyzed Lys15-Ala16 peptide bond in modified inhibitor is subject to thermodynamic control resynthesis.


Biological effects of Escherichia coli lipopolysaccharide (LPS) in vivo. I. Selection in the mouse thymus of killer and helper cells. In the present study we have investigated the biological effects on thymus lymphocytes resulting from Escherichia coli lipopolysaccharide (LPS) treatment in young adult mice. It has been established that LPS induces the following effects: (a) a dose-dependent reduction of thymus weight contemporaneous with a rise in the anti-LPS antibody response; (b) an increase of killer activity of thymus cells; (c) an enhancement of thymocytes helper activity; (d) a reduction of theta-positive cells in the thymus; (e) a cellular depletion in the thymus cortex. These data, indicating that LPS selects in the thymus a population of cells more efficient in expressing both killer and helper functions, are interpreted as caused by an increased rate of cortisol secretion induced by the LPS treatment.


Fertility control with sub-dermal silastic capsules containing a new progestin (ST-1435). The contraceptive action of silastic implants containing a new progestion ST-1435 was investigated in 285 women of reproductive age. A group of 52 women (group A) received three capsules containing each 35 mg of the steroid. A second group of 48 women (group B) received four capsules and a third and larger group of 185 women (group C) received five capsules. No pregnancies occurred during 530 months of use in group A. In 502 months of use recorded for group B, one pregnancy occurred. In group C, 2.142 months were recorded. One pregnancy was reported at the end of 11 months of use and another at the end of 19 months of use. Alterations in the pattern of menstrual bleeding was the most common side effect.


Human semen ribonuclease. Location, properties and inhibition by sodium dodecyl sulfate, zinc sulfate and EDTA. Optimal conditions were established for RNase activity measurement. The enzyme was measured in human seminal plasma as well as in spermatozoa. Results suggest that sperm enzyme may come from seminal plasma contamination and that RNase may be used as a marker enzyme for seminal plasma contamination. Sodium dodecylsulfate, a reagent utilized to produce the solubilization of the spermatozoa, produced a very strong inhibition of the RNase at low concentrations (530 muM). Zinc sulfate and EDTA also produced inhibition of the RNase activity. Such inhibition may be very useful in future studies of RNA metabolism in human spermatozoa.


Lack of effects of testosterone on norepinephrine turnover and tyrosine hydroxylase activity of the rat vas deferens and epididymis. To test the effects of testosterone on some aspects of the metabolism of norepinephrine (NE) in short adrenergic neurons of the male genital tract, orchiectomized rats received 500 mug of testosterone propionate twice daily for up to 18 days. Androgen treatment did not affect the levels, uptake or efflux of NE nor modified the activity of tyrosine hydroxylase in the vas deferens and epididymis. Likewise, formaline-induced fluorescence and ultrastructure of nerve endings remained unchanged after testosterone administration. These data suggest that short adrenergic neurons of male genital tract are relatively insensitive to androgens.


Fifteen years experience with artificial insemination. Artificial insemination is today an accepted procedure in circumventing sterility. In recent years this procedure gained much popularity as the number of infants available for adoption steadily decreased due to the liberal abortion laws. A review of 15 years of practiced experience (senior author) is presented. In all described cases (168 women) artificial insemination was performed personally by the author. The high rate of success (80% of the presented cases) is attributed to a very meticulous and highly individualized approach to each case. Review of literature is presented.


Reversible anti-fertility effect by non-occluding oviductal copper coils in the rabbit. Placement of two 1 cm copper (Cu) coils around the mid-region of each oviduct in a group of five female rabbits with proven fertility, 34 days before mating, virtually prevented fetal implantation in these animals. The oviducts remained patent during a 47 day implantation period and following removal of the Cu coils, does that were remated became pregnant. In two of these does an implantation rate (living fetuses/corpora lutea) of 33% (7/21) was observed and one post-Cu implanted doe had a litter of two. Among four does bearing a single Cu coil on their left oviduct for 28 days prior to mating there was 19% (4/21) implantation of the left side and 56.3% (9/16) on the untreated right side, suggesting a local action by the metal. The results indicate that Cu is an effective anti-fertility agent when implanted around the oviducts and, as a corollary, it is apparent that the metal is not restricted to placement in the uterus in order to prevent pregnancy in rabbits. Non-occluding Cu containing devices may offer a new approach to reversible fertility control in the female that merits further investigation.


The effect of exogenous gondadotropins on the pre-implantation period in the rat. It is well established that the egg implantation in the rat is dependent on a surge of gonadotropin secretion on day 4 of pregnancy. The present experiments were designed in order to demonstrate the possible effect of exogenous administration of gonadotropins during pre-implantation period in pseudopregnant rats, made pregnant after egg transfer. Groups of pseudopregnant rats by sterile mating were injected with different doses of LH and FSH, between 9:00-12:00 A.M. on day 4 of pseudopregnancy; blastocysts were transferred to them in the morning of day 6, instead of day 5. The implantation rate and the number of young born at term were the criteria of the successful extension of the sensitive period of the endometrium by the exogenous gonadotropins. From the results obtained it can be concluded that LH alone acts on the uterine sensitivity by extension of the implantation period.


Case report: sterility due to sperm autoagglutination. Autoagglutination in seminal fluid of husbands in three unexplained sterile couples was found in routine semen-analysis. Further exploration showed that sera and seminal fluid of these husbands agglutinate donors' sperm, while their wives' sera did not. Post-coital tests were not significant. After failure of conservative treatment with vitamin C, two couples agreed to AID followed promptly by pregnancies and deliveries. The possible connection between sterility and sperm-auto-agglutination is discussed, stressing the justification for looking at these phenomena more carefully and with more attention.


Distribution and removal of the acrosin of bull spermatozoa. The effects of Hyamine 2389, Triton X-100, Nadeoxycholate, acetic acid and hypertonic KCl and MgCl2 as well as freezing and thawing and sonication were studied on the solubilization of acrosin from washed bull spermatozoa, from Hyamine-pretreated spermatozoa (devoid of cell and outer acrosome membrane and of acrosomal material) and from isolated acrosomal caps and vesicles. Concurrent ultrastructural changes were observed. Hyamine, Triton, KCl, and acetic acid effectively solubilized acrosin from whole spermatozoa but MgCl2 had a poor effect. The outer acrosome membrane and acrosomal material extracted by Hyamine contained about 35-40% of the total acrosin activity, and three quarters of it was soluble. The rest of acrosin situated in the innter acrosome membrane or equatorial segment was best extracted by hypertonic KCl and MgCl2, but the detergents were ineffective in this case indicating that acrosin is bound differently to the outer and inner parts of acrosome. The opposite effect of MgCl2 on the acrosin activities extracted from these two parts could even be a suggestion of multiple forms of acrosin. The increase of the total acrosin activity during the Hyamine treatment indicates that acrosin is partly in an inactive form. due either to steric hindrance, inhibitor complex of existence of a proacrosin.


The effect of prostaglandins F1 alpha and F2 alpha on spermatogenesis. The effect of the prostaglandins F1 alpha and F2 alpha on spermatogenesis in the mature male mouse has been studied. Administration of prostaglandins F1 alpha and F2 alpha subcutaneously at a dose of 3 mg/kg once a day for 15 days produced a pronounced decrease in spermatogenesis primarily during the meiotic phase as reflected by a significant decrease in stage 7 spermatids as compared to controls. PGF2 alpha produced a stronger suppression than did PGF1 alpha. Accessory reproductive gland weights also appeared to be reduced by the prostaglandins, although not consistently. Histopathological examination revealed increased numbers of exfoliated immature germinal cells in the seminiferous tubules and epididymi of prostaglandin treated animals. When the effect of PGF1 alpha and PGF2 alpha were compared to those of PGE1 and PGE2 it was found that PGE2 produced the strongest suppression, with PGF2 alpha, PGE1 and PGF1 following in decreasing order respectively.


Immunological factors and post coital test in unexplained infertility. Twenty-four couples facing longstanding primary or secondary infertility underwent semen analysis, PCT, functional evaluation of menstrual cycle, hysterosalpingography and laparscopy. All clinical findings were normal with the exception of PCT which was positive in 13 and negative in 11. All the women underwent a multiple approach investigation of local and circulating antibodies production as well as cell-mediated immunity against live spermatozoa. Sperm Immobilization Test (SIT) and Spermatotoxicity Tests (STT) were performed in a single experimental design on cervical mucus and blood serum. Leucocyte Migration Inhibition Test in presence of sperms (LMIT) was done on peripheral leucocytes. Local antispermatic activity in the cervical mucus was negative in all PCT positive cases, and positive in six out of 11 PCT negative cases. SIT and STT were both positive in cervical mucus and in blood in one case only. No correlation could be found between PCT and serum SIT, STT or LMIT. The fertility pattern expressed by the number of pregnancies per years of exposure, already low in the whole group, was even lower in the sub-groups with positive immunological factors. Positive immunological factors do not exclude the possibility of conception but appear to be associated with a reduced rate of conception.


Pituitary and testicular response to hypothalamic LH-releasing hormone (LH-RH) in normal and oligospermic men. A comparative study was done of the basal and post-LH-RH stimulation levels of LH, FSH and Testosterone (T) in 10 healthy, male volunteers, aged 24-35 years, and in 25 normogonadotropic oligospermic men aged 23-43 years in whom other endocrine, urologic and/or vascular diseases had been rules out. Seventeen of the latter group underwent bilateral testicular biopsy. Each subject received rapid i.v. injection of 50 mug synthetic LH-RH at 8:00 A.M. Serum levels of LH, FSH and T were determined by radioimmunoassay on samples obtained immediately before and 30 and 45 minutes after injection. As compared to normals, oligospermic men had a lower average LH response, although the basal values did not differ significantly from normal. FSH levels did not differ significantly between groups, although some individual patients showed higher and some lower than normal values. The possibility of stimulating T secretion with LH-RH was confirmed. Average serum T levels rose more slowly and reached lower levels in the oligospermic men than in normal men. There was no correlation between LH and FSH rises. It is postulated that some patients with normogonadotropic oligospermia may have latent insufficiency of pituitary gonadotropic functional reserve for one or both hormones and/or of the Leydig cell function. The need for individual analysis of results and their correlation with testicular biopsy findings is commented.


Alkaline phosphatase, arylsulfatase and beta-glucuronidase in saliva of cyclic women. Levels of the enzymes alkaline phosphatase, arylsulfatase, and beta-glucuronidase have been measured in whole unstimulated saliva samples collected daily during normal menstrual cycles. The cellular content of the saliva has been shown to be a major source of the three enzymes. The enzymes were found to have peak maximum levels of activity during the periovulatory -hase of the cycle. This observation can be attributed to the presence of increased numbers of exfoliated cells from the oral mucosa resulting from the pre-ovulatory estrogen stimulus to cell proliferation.


A new model for antisperm autoimmunity in guine pigs. Adult outbread male guinea pigs were autoimmunized without adjuvant. Homogenates were prepared with one of their own testes previously submitted "in vivo" and "in vitro" to thermal injury. Animals received a single or daily repeated intradermal injection without added adjuvant, in one or different skin sites. Guinea pigs daily sensitized in the same site during 30 days showed the presence of: a) dermal granuloma at the site of injection; b) several foci of typical allergic orchitis; c) delayed hypersensitivity detected by inhibition of macrophage migration; d) moderate titres of spermagglutinins and negligible levels of hemagglutinating antibodies. Guinea pigs receiving a single dose in one site only developed delayed hypersensitivity. Animals daily sensitized with the same dose of altered antigen in different sites, or with normal testis antigen either in one or different sites, showed negative results. The correlation among testicular lesion, dermal granuloma and cellular immunity is discussed. It is concluded that testis autosensitization is obtained in the absence of added adjuvant provided that a thermally injured gonad used as antigen is repeatedly injected in the same site.


Quantitative study of spermatogenesis in vasectomized mice. Adult mice were vasectomized under semi-sterile conditions and examined five weeks later. The weight of the epididymis was increased but none of the animals had developed spermatic granulomas. The concentration of testosterone in the plasma, the body weight and the weight of the testes were not affected by vasectomy. Cross sections of the testes were prepared for histological examination and the nuclei of the various types of germinal cells were enumerated at stage VII of spermatogenesis. Vasectomy caused a small, but statistically significant, decrease in the number of preleptotene spermatocytes, pachytene spermatocytes and step 7 spermatids.


Fertility patterns in female mice following treatment with arginine vastocin or melatonin. Daily injections of either 2 mug arginine vasotocin (AVT) or 100 mug melatonin significantly prolonged the estrous cycles of mature female mice. If administered daily throughout pregnancy, none of the AVT-treated mice delivered viable pups.


The pH dependence of binding of alpha,alpha'-dibromo-p-xylenesulfonic acid to lysozyme. The chemical modification of lysozyme (I) has been accomplished with alpha, alpha'-dibromo-p-xylenesulfonic acid (DBX) at five different pH values. I was alkylated by DBX at room temperature (28 degrees C) with decrease in enzyme activity. The rate of inactivation depended upon the pH at which alkylation was carried out. The highest rate was seen at alkaline pH values; the lowest at more acidic pH values. Amino acid analyses showed that-two lysines and two tryptophan residues had been modified at pH 9; two lysines, one tryptophan and one methionine had reacted at pH 8. A histidine residue was bound at pH 6.5 together with a tryptophan residue. At the lower pH values (2.7, 4.5, 6.5), alkylation occurred with a single tryptophan residue each. Fluorescence and CD data both ruled out the participation of tryptophans 62 or 108. Labeling experiments showed that two residues  of DBX-35S were bound per molecule of I at both pH9 and pH8; one residue of DBX was bound per molecule of I at the other pH values. Sedimentation coefficients were characteristic of native lysozyme. The stoichiometry of binding and residue modification indicated that intra-molecular cross links were established. The pH dependence of the cross-linking provides means to measure several allowed intra-molecular distances. The results presented here are consistent with the existence of side chain motion in lysozyme.


Complete enzymic digestion of acidic proteins. Acidic proteins are usually resistant to complete enzymic hydrolysis. The increasing number of "unusual" amino acids, which are unstable to acid hydrolysis, makes it necessary to have a method of enzymic hydrolysis applicable to all proteins. The complete hydrolysis of four acidic proteins by subtilisin plus leucine amino-peptidase plus prolidase followed by carboxypeptidase C, is described. Recoveries of amino acids were in excellent agreement with the expected content from the known sequences.


Interpretation and applications of thermal difference spectra of proteins. The technique of thermal perturbation difference spectroscopy for examining chromophores in proteins has been set on a more acceptable theoretical and experimental foundation. (i) The origins of the thermal effect have been analysed to explain changes in spectral band width and wavelength for simple chromophores in various solvents. Comparison with theoretical curves shows that the effect of heating chromophore solutions is mainly spectral broadening coupled with an almost negligible blue shift. The apparently anomalous behavior of tryptophan and tyrosine in aqueous solvents, where the main effect is a red shift on heating, is traced to hydrogen bonding with water. A model in which tyrosine acts simultaneously as H-donor (Type I) and H-acceptor (Type II) and the later is the more temperature sensitive, is successful in explaining all available data for a variety of solute derivatives and solvents. (ii) The contribution of chromophores in the protein interior has been assessed using polyvinyl alcohol films of differing water content. There models simulate the rigidity and low thermal expansivity of the protein interior and confirm that buried chromophores contribute negligibly if at all to thermal difference spectra. (iii) Curve-fitting procedures have been used for matching protein difference spectra over a wavelength range, to those for mixtures of models, rather than relying as hitherto on data measured at extrema.


Studies on the acute toxicity of ionic and non-ionic contrast media following rapid intravenous injection. An experimental study in mice. The influence of the injection rate on the acute intravenous toxicity (LD50) on mice of the ionic high osmotic contrast medium Isopaque Coronar (370 mg I/ml) and the non-ionic low osmotic Amipaque (370 mg I/ml) was investigated. At slow injection (0.1 ml/sec). Amipaque had a significantly higher LD50 than Isopaque Coronar (17.3 respectively 9.8 gm I/kg). Also at rapid injection (2 ml/10 sec). Amipaque was significantly less toxic than Isopaque Coronar (13.4 respectively 6.1 g I/kg). Thus Isopaque Coronar increased approximately 60% in toxicity while Amipaque increased 28% when the rate of injection was increased. This difference was statistically significant (p less than 0.001). Several possible mechanisms that might explain this difference, with special interest in the disturbances on the pulmonary circulation following contrast media injection, are discussed.


Partial purification of pig aorta amine oxidases. Two amine oxidases have been partially purified from pig aorta and their porperties investigated: one is similar to pig plasma oxidase (benzylamine oxidase), the other to lysyloxidase. Both are inhibited by carbonyl reagents and cupric copper chelating agents, but they strongly differ in the kinetic properties. The benzylamine oxidase has a maximal reaction rate at acid pH values, the lysyloxidase has an optimum around pH 6.8.


Carbamylphosphate hydrolysis in donkey liver. Mitochondria, microsomes, and cytosol all hydrolyze carbamylphosphate. Microsomes show the greatest specific activity. The mitochondrial enzyme is localized in the inner membranes, has optimum pH 5.5 and is heat-stable; the cytosol enzyme has optimum pH 5.0 and is heat-labile. Km for carbamylphosphate is 1.1 to 1.2 X 10(-2 M for both the cytosol and the mitochondrial enzyme. Both enzymes are inhibited by high substrate concentrations.


[Ultrastructural localization of acid phosphatase and thiamine pyrophosphatase activities and of phosphotungstic staining at low pH in the placental labyrinth of the cat]. The localizations of acid phosphatase (ACPase) and thiamine pyrophosphatase (TPPase) activities were studied in the placental labyrinth of the cat during the last days of gestation. ACPase activities were observed essentially in the syncytiotrophoblast and in the "interstitial inert substance" (S.I.I.) that separates maternal from foetal tissues. Reaction product was localized in lysosomes, multivesicular bodies, and in the network of smooth-membraned tubules which open on the cell surface of the syncytiotrophoblast facing the S.I.I. The S.I.I. exhibit a strong activity, probably of syncytiotrophoblastic origin. TPPase activities were found in the syncytiotrophoblast, rarely in the Golgi apparatus but always in the lumen of the network of smooth-membrande tubules. The S.I.I. shows a moderate activity. These TPPase positive tubules being frequently observed very close to the Golgi cisternae, it is proposed that they arise from the Golgi complex and convey phosphatases to the S.I.I. After phosphotungstic acid staining at low pH, luminal surface coat of maternal endothelium is always strongly and continuously visualized, while the plasma membrane of the syncytiotrophoblast facing the S.I.I. is never stained. Staining is intense in the lumen of tubules, and continuous with the stain of the S.I.I. ACPase activity of the S.I.I. could be implicated in enzymatic degradation of maternal molecules during their transfer from maternal to foetal blood. The S.I.I. may correspond to the immunological buffer zone at the interface between maternal and foetal tissues.


A preliminary microspectrofluorometric study of NAD(P) reduction in dibenzo(a, e) fluoranthene-treated single living cells. The fluorescence increase, due to NAD(P) reduction, following microelectrophoretic injection of glucose 6-P (G6P) into EL2 and NCTC 8739 single living cells treated with diBenzo(ae) Fluoranthene (diB(ae)F) and non-treated, has been studied with a rapid microspectrofluorometer. This study shows the enhanced capacity of treated cells to utilize larger doses (6-10 times more) of G6P than control cells. The time course of the return to the initial fluorescence level is essentially related to the magnitude of the injection dose. There are alterations (e.g. red & blue shifts) in the fluorescence spectrum of diB(ae)F-treated cells before injection and in the increase spectrum after injection of G6P, as compared to the same spectra in the diB(ae)F-untreated cells. This is discussed in reference to the metabolization of diB(ae)F as an alternative pathway for the reoxidation of NAD(P)H.


Quantitative cytochemical measurement of glyceraldehyde 3-phosphate dehydrogenase activity. A system has been developed for the quantitative measurment of glyceraldehyde 3-phosphate dehydrogenase activity in tissue sections. An obstacle to the histochemical study of this enzyme has been the fact that the substrate, gylceraldehyde 3-phosphate, is very unstable. In the present system a stable compound, fructose 1, 6-diphosphate, is used as the primary substrate and the demonsatration of the glyceraldehyde 3-phosphate dehydrogenase activity depends on the conversion of this compound into the specific substrate by the aldolase present in the tissue. The characteristics of the dehydrogenase activity resulting from the addition of fructose 1, 6-diphosphate, resemble closely the known properties of purified glyceraldehyde 3-phosphate dehydrogenase. Use of polyvinyl alcohol in the reaction medium prevents release of enzymes from the sections, as occurs in aqueous media. Although in this study intrinsic aldolase activity was found to be adequate for the rapid conversion of fructose  1, 6-diphosphate into the specific substrate for the dehydrogenase, the use of exogenous aldolase may be of particular advantage in assessing the intergrity of the Embden-Meyerhof pathway.


Protocol management of male genitourinary infections. As part of a demonstration study, 567 male patients presenting to a "walk-in" clinic with common genitourinary complaints were interviewed by health assistants guided by a protocol. Independent examination of 19 patients by a health assistant and a physician formally demonstrated that the health assistants could collect the clinical data accurately. Forty-four patients were then randomly chosen to be examined, diagnosed and treated either by a health assistant guided by the protocol and supported by an available physician, or only by a physician. Using medical records and a follow-up interview, we assessed the thoroughness of the medical record, adequacy of diagnosis and treatment, symptom relief, patient satisfaction and patient education: the health assistant-protocol system proved as safe and effective as the MD-only system, and the health assistants were able to manage 68% of patients without involving the physician. The study suggests that briefly-trained health assistants may help save physician and nurse time, and that the development of protocols can help set standards for the medical management of defined problems while providing a mechanism for rapidly creating a complete medical record which can be easily audited for conformance with standards.


Gardimycin, a new antibiotic from Actinoplanes. III. Biological properties. The new antibiotic gardimycin has an interesting in vitro antibacterial activity against Gram-positive bacteria and Neisseria gonorrhoeae. Parenteral administration gives a high degree of protection against experimental infections in mice. It also shows some chemotherapeutic activity when given rectally.


Inhibitory activity of pyridindolol on beta-galactosidase. The activity of pyridindolol in inhibiting beta-galactosidases obtained from various sources has been studied. Whereas acid bovine liver beta-galactosidase (optimal pH 4.0) was not affected by this compound, neutral bovine liver beta-galactosidase (pH-optimum = 7.0) was inhibited by pyridindolol in reaction mixtures of pH 4.0 approximately 5.0. There was no inhibition at pH 7.0. The type of inhibition is non-competitive by formation of a pyridindolol-enzyme complex. Since beta-galactosidases from other sources are not affected by pyridindolol, the inhibitory action of this compound seems to be rather specific for neutral bovine liver beta-galactosidase.


Selective N-actylation of gentamicin antibiotics-synthesis of 1-N-acyl derivatives. Treatment of acid addition salts of aminoglycoside-aminocyclitol antibiotics of the gentamicin-sisomicin class, with one equivalent of triethylamine and an acylating agent results in selective formation of 1-N-acyl derivatives. This is in contrast to acylation of the antibiotic free bases which results in preferential acylation of other basic centres in the molecule. Origins of the observed selectivity are discussed. In vitro antibacterial activities of several new 1-N-acy1 derivatives of gentamicin, sisomicin and verdamicin are reported.


Propagated spikes and secretion in a coelenterate glandular epithelium. The rete mirabile of Hippopodius (Cl. Hydrozoa, O. Siphonophora) is a sheet of giant endoderm cells penetrated by branches of the ventral radial canal. The cells appear to be highly polyploid. The rough ER is very richly developed and expanded ER cisternae containing amorphous material (presumably synthesized protein) are observed near the outer cell surface. The cells are electrically coupled, and are connected by gap junctions. The rete is electrically excitable and cell to cell conduction of action potentials at 10 cm/s is observed. The action potentials are all-or-none, positive-going events, showing amplitudes of about 70 mV and arising from a 44 mV resting potential. Slowly developing and decaying secondary depolarizations, capable of summing to the 20 mV level, are also observed. After passage of a train of impulses, the rete cells swell and secretion drops appear at the surface, these changes becoming apparent within a few seconds. In 15 mM Mn2+ the response fails to occur, and secondary depolarizations ("secretion potentials") are not seen. Spike propagation is not affected. In Na+-free solutions the spikes are reduced and propagation eventually fails. It is suggested that the spikes are sodium-dependent events which trigger a calcium-dependent secretory process. The composition and biological activity of the secretion are uncertain, but indirect evidence suggests a possible defensive or repellant role for the response.


Respiratory adaptations in burrowing pocket gophers from sea level and high altitude. To examine the adaptations to low O2 and high CO2 among fossorial and nonfossorial rodents, hematological parameters were determined for laboratory rats, the valley pocket gopher (Thomomys bottae) from 250 m, and the mountain pocket gopher (T. umbrinus melanotis) from 3150 m. Hematocrit, hemoglobin concentration, and O2 capacity were higher in pocket gophers than in rats. Blood PO2 at 50% saturation and pH 7.4 was 33 mmHg for both gophers and 39 mmHg for rats. Bohr factors for all three rodents were similar (-0.55 to -0.61) but buffer value, delta log PCO2/delta pH, was -2.54 for T. umbrinus, -1.97 for T. bottae, and -0.98 for Rattus. Concentrations of total acid-soluble phosphates were 50-75% higher in gophers than in rats, while bicarbonate values were within the normal mammalian range. All three rodents had similar myoglobin concentrations in cardiac muscle. Myoglobin concentrations were significantly higher in skeletal muscles (diaphragm, gastrocnemius) of T. umbrinus when compared to T. bottae, and significantly higher in both gophers when compared to rats. These differences may constitute important adaptations to the hypoxia and hypercapnia in burrows; certain of these factors in pocket gophers respond to the additional stress of high altitude hypoxia.


Control of breathing in uremia: ventilatory response to CO2 after hemodialysis. The mechanisms responsible for the transient respiratory alkalosis which follows clinical hemodialysis were evaluated by studying the ventilatory response to carbon dioxide in chronic uremic patients, and in unanesthetized normal and chronic uremic goats. A significant increase in sensitivity to CO2 was found in acidotic uremic patients immediately (within 30 min) following hemodialysis (P less than 0.01). Sensitivity to CO2 returned to the predialysis value within 24 h. Lung volume and maximal breathing capacity were unchanged. A similar increase in sensitivity to CO2 was seen in nonacidotic uremic goats following hemodialysis. In the goats, these changes in sensitivity could not be explained by changes in cerebrospinal fluid acid-base status. Adding sufficient urea to the dialysate to prevent a fall in plasma urea concentration, eliminated this increase in sensitivity to CO2 in both uremic patients and goats. These results suggests that the transient respiratory alkalosis following hemodialysis is due to an increase in the sensitivity of the ventilatory response to carbon dioxide and is a consequence of dialysis-induced osmotic disequilibrium.


Characteristics and energy requirements of an alpha-aminoisobutyric acid transport system in Streptococcus lactis. Galactose-grown cells of Streptococcus lactis ML3 acculated alpha-aminoisobutyric acid (AIB) by using energy derived from glycolysis and arginine catabolism. The transport system displayed low-affinity Michaelis-Menten saturation kinetics. Using galactose or arginine as energy sources, similar V max and K m values for AIB entry were obtained, but on prolonged incubation the intracellular steady-state concentration of AIB in cells metabolizing arginine was only 65 to 70% that attained by glycolyzing cells. Efflux of AIB FROM PRELOADED CElls was temperature dependent and exhibited the characteristics of a first-order reaction. The rate of AIB exit was accelerated two- to threefold in the presence of metabolizable energy sources. Metabolic inhibitors including p-chloromercuribenzoate, dinitrophenol, azide, arsentate, and N, N'-dicyclohexylcarbodiimide either prevented or greatly reduced AIB uptake. Fluoride, iodoacetate and N-ethylmaleimide abolished galactose-dependent, but not arginine-energized, AIB uptake. K+ and Rb+ reduced the steady-state intracellular AIB concentration by approximately 40%, and these cations also induced rapid efflux of solute from actively transporting cells. Equivalent concentrations (10 mM) of Na+, Li+, or NH4+ were much less inhibitory. The proton-conducting ionophores tetrachlorosalicylanilide and carbonylcyanide m-chlorophenlyhydrazone abolished uptake and induced AIB efflux even though glycolysis and arginine catabolism continued at 60 and 140%, respectively, of control rates. A proton motive force is most likely involved in the active transport of AIB, whereas data from efflux studies suggest that energy is coupled to AIB exit in cells of S. lactis ML3.


Deoxyribonucleic acid-binding studies on the hut repressor and mutant forms of the hut repressor of Salmonella typhimurium. In Salmonella typhimurium the genes coding for the enzymes of histidine utilization (hut) are clustered in two adjacent operons, hutMIGC and hut(P,R,Q)UH. A single repressor, the product of the C gene, regulates both operons by binding at two operator sites, one near M and one in (P,R,Q). The deoxyribonucleic acid (DNA)-binding activity of the repressor was measured using DNA's containing separate operators. The repressor had greater activity when assayed using DNA containing the operator of the (P,R,Q)UH operon than when assayed using DNA containing the operator of the MIGC operon. The binding to either operator was absent in the presence of the inducer, urocanate. The DNA-binding activities were also determined for two super-repressors. The super-repressors had altered DNA-binding properties, although the self-regulated nature of the repressors complicated the analysis of the results. A purfication procedure for the wild-type repressor is presented. The purified repressor was somewhat unstable, and additional experiments using it were not performed.


Purification of properties of dihydroorotase, a zinc-containing metalloenzyme in Clostridium oroticum. Dihydroorotase +4,5-L-dihydro-orotate amidohydrolase [EC 3.5.2.3]), which catalyzes the reversible cyclization of N-carbamyl-L-aspartate to L-dihydroorotate, has been purified from orotate-grown Clostridium oroticum. The enzyme is homogeneous when subjected to polyacrylamide gel electrophoresis and is stable at pH 7.6 in 0.3 M NaCl containing 10 muM ZnSO4. The enzyme has a molecular weight of approximately 110,000. Sodium dodecyl sulfate gel electrophoresis, using three different buffer systems, indicated the enzyme is composed of two subunits, each having a molecular weight of 55,000. Dihydroorotase is shown by atomic absorption spectroscopy to be a zinc-containing metalloenzyme with 4 g-atoms of zinc per 110,000 g of protein. The pH optima for the conversion of N-carbamyl-L-aspartate to L-dihydroorotate and for L-dihydroorotate to N-carbamyl-L-aspartate are pH 6.0 and 8.2, respectively. The Km values for N-carbamyl-L-aspartate and for L-dihydroorotate are 0.13 and 0.07 mM, respectively. Inhibitor studies indicate that zinc may be involved in the catalytic activity of the enzyme.


Influence of amino acids on the growth of Bacteroides melaninogenicus. Addition of individual amino acids to a Trypticase-yeast extract-hemin medium affected growth rates and final yields of an asaccharolytic strain and a saccharolytic strain of Bacteroides melaninogenicus. L-Aspartate or L-asparagine produced maximal growth enhancement for both strains. L-[14C]aspartate was fermented by resting cells of the asaccharolytic strain. L-Cysteine or L-serine also enhanced growth for the saccharolytic strain. However, growth of the saccharolytic strain was inhibited by L-lysine, L-glutamate, L-glutamine, L-isoleucine, L-leucine, and L-proline; growth of the asaccharolytic strain was inhibited by DL-valine and L-serine. Both strains were inhibited by L-histidine, DL-methionine, L-tryptophan, L-arginine, and glycine.


Constitutive and repressivle enzymes of the common pathway of aromatic biosynthesis in Escherichia coli K-12: regulation of enzyme synthesis at different growth rates. Synthesis of five of the enzymes of the common pathway of aromatic biosynthesis has been shown to be unaffected by either the aromatic amino acids--the product of the first reaction (3-deoxy-D-arabinoheptulosonic acid-7-phosphate) or the product of the last reaction (chorismate)--or by the state of regulator gene loci tyrR. On the other hand, the rate of synthesis of these enzymes, and of several other enzymes for which repression control was inactive because of mutations in relevant regulator genes, was found to change with growth rate. These changes were found to correlate at faster growth rates than those observed in glucose minimal medium with the alterations in the relative frequencies of the corresponding structural genes which occur at these growth rates. It was found that when wild-type cells were grown at these faster growth rates in medium lacking the aromatic amino acids, complete derepression of the tyrosine-inhibitable 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthetase occurred, in strong contrast to the situation when wild-type cells are grown in glucose minimal medium.


Characterization of L-asparagine transport systems in Stemphylium botryosum. L-Asparagine uptake by Stemphylium botryosum is mediated by two distinct energy- and temperature-dependent transport systems. One permease is relatively specific for L-asparagine and L-glutamine and is present in nutrient-sufficient mycelium. The specific permease shows an optimum pH at 5.2, saturation kinetics (Km = 4.4 x 10(-4) M, Vmax = 1.1 mumol/g per min), competitive gradient of L-asparagine, and higher affinity towards the L-isomer of asparagine. Amide derivatives of L-asparagine (5-diazo-4-oxo-L-norvaline or L-aspartyl hydroxamate) are the most effective competitors, alpha-amino derivative (N-acetyl asparagine) is a moderate competitor, and alpha-carboxyl derivative (L-asparagine-t-butylester) shows only slight inhibition of the specific permease. Derivatives of L-glutamine are significantly less effective competitors than those of L-asparatine. The level of the specific permease is affected by nitrogen sources and increases approximately threefold upon starvation. The nonspecific permease possesses an optimum pH at 6.8, saturation kinetics (Km = 7 x 10(-5) M, Vmax = 5 mumol/g per min, Kt = 7.4 x 10(-5) M for L-leucine), and high affinity towards various types of amino acids.


Synthesis of omega-alicyclic fatty acids from cyclic precursors in Bacillus subtilis. A mutant of Bacillus subtilis synthesizes a variety of omega-alicyclic fatty acids when fed with the respective alicyclic carboxylic acids. These fatty acids are: omega-cyclopropane, omega-cyclobutane, omega-cyclopentane, omega-cyclohexane, and omega-cyclohexene fatty acids. These unusual fatty acids did not lead to an inhibition of growth at 37 degrees C and pH 7. The selective advantage of these fatty acids under extrene conditions was studied in comparison with the acidophilic, thermophilic bacterium B. acidocaldarius, which normally contains a high proportion of omega-cyclohexane fatty acids.


Isolation and characterization of dihydropteridine reductase from Pseudomonas species. Dihydropteridine reductase isolated from the bacterium Pseudomonas species (ATCC 11299a) has been purified approximately 450-fold byammonium sulfate precipitation and diethylaminoethyl-cellulose chromatographic procedures. The preparation is at least 80% pure as judged by polyacrylamide gels. Its molecular weight was determined to be about 44,000. Both dihydropteridine reductase and phenylalanine hydroxylase activities were found to be higher in cells adapted to a medium containing L-phenylalanine or L-tyrosine as the sole carbon source than in those grown in L-asparagine. The substrate of the reductase is quinonoid dihydropteridine, and the product is tentatively identified as a tetrahydropteridine through its ability to serve as a cofactor for phenylalanine hydroxylase. The enzyme shows no marked specificity for the pteridine cofactor that occurs naturally in this organism, L-threo-neopterin. The pH optimum for the reductase is 7.2, and nicotinamide adenine dinucleotide, reduced form, is the preferred cosubstrate. Inhibition of the reduced and untreated enzyme by several sulfhydryl reagents was observed. A metal requirement for the reductase could not be demonstrated. Dihydropteridine reductase was found to be inhibited by aminopterin in a competitive manner with respect to the quinonoid dihydro form of 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine.


Factors affecting growth and nitrogen fixation of Spirillum lipoferum. Spirillum lipoferum grows vigorously on malate, succinate, lactate, or pyruvate, moderately on galactose or acetate, and poorly on glucose or citrate. It reduces 15N2. Acetylene reduction rates decrease rapidly when the pH of the culture rises above 7.8. The organism is highly aerobic and had doubling times as low as 2 h when grown on NH4+. However, S. lipoferum reduces N2 well only under microaerophilic conditions. The optimal pO2 for acetylene reduction by stagnant cultures was 0.006 to 0.02 atm depending upon the cell density; aerated cultures grew well at dissolved O2 concentration corresponding to a pO2 of about 0.008 atm. Shaking S. lipoferum with air temporarily inactivates its nitrogenase; reactivation is inhibited by chloramphenicol. The organism assimilated 20 to 24 mg of N/g of organic acid oxidized during growth. The strains studied can be placed in two groups based upon their morphology and physiological characteristics.


Ornithine delta-transaminase activity in Escherichia coli: its identity with acetylornithine delta-transaminase. Procedures that have been developed for the purification of acetylornithine delta-transaminase from Escherichia coli W also lead to the simultaneous purification of ornithine delta-transaminase. These two enzymatic activities have the same electrophoretic mobility and are identical immunochemically. Studies of inhibition kinetics demonstrate that the two substrates, acetylornithine and ornithine, compete for the same active site of acetylornithine delta-transaminase; thus, the ornithine delta-transaminase activity in E coli is due to acetylornithine delta-transaminase and not to a separate specific ornithine delta-transaminase.


Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus. A stable deoxyribonucleic acid (DNA) polymerase (EC 2.7.7.7) with a temperature optimum of 80 degrees C has been purified from the extreme thermophile Thermus aquaticus. The enzyme is free from phosphomonoesterase, phosphodiesterase and single-stranded exonuclease activities. Maximal activity of the enzyme requires all four deoxyribonucleotides and activated calf thymus DNA. An absolute requirement for divalent cation cofactor was satisfied by Mg2+ or to a lesser extent by Mn2+. Monovalent cations at concentrations as high as 0.1 M did not show a significant inhibitory effect. The pH optimum was 8.0 in tris(hydroxymethyl)aminomethane-hydrochloride buffer. The molecular weight of the enzyme was estimated by sucrose gradient centrifugation and gel filtrations on Sephadex G-100 to be approximately 63,000 to 68,000. The elevated temperature requirement, small size, and lack of nuclease activity distinguish this polymerase from the DNA polymerase of Escherichia coli.


Studies on peroxisomes. VI. Relationship between the peroxisomal core and urate oxidase. The peroxisomal core from the liver of rats was purified 450-fold as a marker of urate oxidase [EC 1.7.3.3.] activity. This preparation has a high specific activity of urate oxidase but not of other peroxisomal enzymes: D-amino acid oxidase [EC 1.4.3.3.], L-alpha-hydroxy acid oxidase [EC 1.1.3.15], or catalase [EC 1.11.1.6]. No activity of marker enzymes for other subcellular particles; cytochrome c oxidase [EC1.9.3.1] (mitochondria), acid phosphatase [EC 3.1.3.2] (lysosomes), or glucose-6-phosphatase [EC 3.1.3.9] (microsomes), was detected in this preparation. The core obtained showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the position of the band was found to correspond to a molecular weight 35,000. When the peroxisomal core was subjected to treatment at various pH's with 0.1 M carbonate buffer, urate oxidase was almost completely solubulized at pH 11.0, although approximately 35% of the core protein still remained in the pellet After solubilization of the core at pH 11.0, the specific activity of urate oxidase in the supernatant increased about 1.6 times; the density of the insoluble protein remaining in the pellet was identical with the that of the original core on sucrose density gradient centrifugation.


Depolarization-induced calcium release from sarcoplasmic reticulum fragments. I. Release of calcium taken up upon using ATP. Ca2& taken up by sarcoplasmic reticulum membrane fragments (SRF) upon using ATP could be released rapidly by changing the anion outside the vesicles from methanesulfonate to chloride. It is considered that this anion exchange caused depolarization of the sarcoplasmic reticulum membrane. Similar rapid release of Ca2& taken up by SRF was also caused by a change from high to low osmotic pressure, probably due to bursting of the membrane. On the basis of experiments in which these two types of Ca2& release were discriminated, it was concluded that Ca2& bound inside the membrane was released directly by anion exchange (depolarization). However, Ca2& release was not caused by cation exchange. Sucrose inhibited these two types of Ca2& release. Cia2& taken up in the presence of oxalate could not be released by any treatment used. Liver microsome fraction also has Ca2& uptake activity. However, Ca2& was not released upon anion exchange, but was released upon oxmotic change. These results show that Ca2& release from SRF upon anion exchange is specific to the sarcoplasmic reticulum membrane. In conclusion, SRF membrane retains the ability to respond to the depolarization caused by ion exchange and can release the accumulated Ca2&.


Depolarization-induced calcium release from sarcoplasmic reticulum fragments. II. Release of calcium incorporated with ATP. Ca2& incorporated in vesicles of sarcoplasmic reticulum fragments (SRF) by diffusion could be released rapidly by changing the ionic environment, by dilution from methanesulfonate (MS) to chloride. This ion exchange is considered to make the membrane potential of SRF inside-negative. Much faster release of Ca2t was also observed upon osmotic change from high to low. These responses were very similar to the Ca2& release from SRF after take up using ATP, but the release rate was slow in the case of anion exchnage. The behavior of K&, Na&, sucrose, and inulin incorporated in SRF was followed upon similar treatment. These ions and nolecules were not released upon ion exchange, but were immediately released by osomtic treatment. Therefore, the Ca& release upon anion exchange was not due to the bursting of SRF, but to a direct effect such as a membrane potential change of the SRF. The behavior of anion such as C1- and propionate could not followed by the same method because of the large permability of these anions. It was also shown that Ca& release upon ion exchange was not a direct effect of pH change. Liver microsomes did not show Ca& release upon the same treatment as SRF.


Partial proteolysis of some cellulase components from Trichoderma viride and the substrate specificity of the modified products. An endo-cellulase component [EC 3.2.1.4] or random type, F II, was obtained from "Cellulase Onozuka," a commercial product from Trichoderma viride, and was subjected to partial proteolysiats with a protease preparation of the same fungal origin. The resulting modified cellulase was fractioned by two steps of column chromatography, and the resulting patterns, together with the substrate specificity expressed in terms of the randomness of CMC hydrolysis and the immunological properties against anti-F II-rabbit se-um, were examined. The chromatographic patterns were very similar to those of cellulase subfractions without proteolytic treatment. Moreover, the immunological response of the modified cellulases from F II was mostly positive and their randomness of CMC hydrolysis was generally lower, compared with subfractions of F II which were not subjected to proteolysis. The subfractions of Peak III, which were obtained from F II by proteolysis, showed mostly negative immunological response and higher randomness of CMC hydrolysis compared with subfractions of Peak III which were not subjected to proteolysis. Thus, some limited proteolysis of cellulase components may, at least in part, be responsible for its multiplicity in vivo.


Purification and properties of an endo-cellulase of avicelase type from Irpex lacteus (Polyporus tulipiferae). A culture filtrate of Irpex lacteus (Polyporus tulipiferae) was fractionated initially by salting out with ammonium sulfate, and a cellulase [EC 3.2.1.4.] fraction with high Avicel-hydrolyzing activity (formerly called Avicelase) was extensively purified by a series of column chromatography procedures. This purified endo-cellulase showed a less random hydrolytic mechanism, and was obtained in a yield of 0.04% with respect to the starting material. Its specific activity was enhanced approximately 30 times over that of the starting material. The cellulase component showed a single peak on both ultracentrifugal and acrylamide disc electrophoretic analyses. Its molecular weight was estimated to be 56,000. It contained 12.2% carbohydrate; the major sugar constituents were glucose and mannose. Regarding the amino acid composition, the contents of aspartic acid and glycine were highest, followed by those of glutamic acid, serine, and theonine. The cellulase component was not markedly inhibited by most metal ions tested excepted for Hg2+. This purified endo-cellulase attacked a series of cellooligosaccharides, beta-cellobioside, CM-cellulose, and insoluble, cellulosic substrates. In the digests from insoluble substrates, glucose, cellobiose, cellotriose, and cellotetraose were detectable, but the amount of cellobiose was the largest by far. In constrast, cellobiose and glucose were produced in almost equal amounts from beta-cellobioside.


Xylanase activity of an endo-cellulase of carboxymethyl-cellulase type from Irpex lacteus (Polyporus tulipiferae). An endo-cellulase [EC 3.2.1.4.] of carboxymethyl-cellulase type (F-1) which was fractionated from culture filtrate of Irpex lacetus and purified to electrophoretic and ultracentrifugal homogeneity, was found to show xylanase [EC 3.2.1.8.] activity. The activity was not removed from any of the intermediate fractions during the purification of the initial F-I peak, and the radio of xylanase to cellulase activity remained almost unchanged through the purification processes. The xylanase activity of F-I showed not only the same optiomal pH, heat stability, and pH stability as its cellulase activity, but also the same mobility as the cellulase activity upon cellulose acetate film and starch zone electrophoreses. The overall rates of hydrolysis of mixtures of variouis concentrations of CM-cellulose and xylan by F-1 coincided well with those calculated from the Michaelis-Menten treatment of two substances competing for the same active site of the enzyme. These results indicate that the xylanase activity of F-1 is intrinsic to the cellulase itself.


Active transport of alanine by thermostable membrane vesicles isolated from a thermophilic bacterium. 1. Thermostable membrane vesicles which were capable of active transport of alanine dependent on either respiration or an artificial membrane potential were isolated from the thermophilic aerobic bacterium PS3. 2. Uptake of alanine was dependent on the oxidation of ascorbate-phenazine methosulfate or on generated or exogenous NADH, but succinate and malate failed to drive the uptake. The optimum temperature for respiration-driven uptake of alanine was 45 to 60 degrees. 3. Potassium ion-loaded vesicles were prepared by incubating vesicles at 55 degrees in 0.5 M potassium phosphate. The addition of valinomycin elicited rapid and transient uptake of alanine under the test conditions. Uptake of alanine in response to valinomycin was progressively enhanced by the addition of dicylohexylcarbodiimide, but was completely abolished in the presence of a proton conductor or synthetic permeable cation. The effect of dicyclohexylcarbodiimide was dependent on its concentration and was maximal at a concentration of 0.4 mM. 4. The proton permeability of membrane vesicles was reduced by the addition of dicyclohexylcarbodiimide. A small but significant difference was found in the initial rates of proton uptake in the presence of dicyclohexylcarbodiimide with and without alanine. The results suggest that protons alanine are transported simultaneously in a stoichiometric ratio of 1 : 1. 5. The uptake of alanine was also driven by a pH gradient induced by an instantaneous pH drop in a suspension of alkali-loaded vesicles. Thus, alanine accumulation was driven not only by an electrical potential but also by a pH gradient. 6. Addition of ATP resulted in the inhibition of alanine uptake dependent on artificial membrane potential. ATP hydrolysis by membrane ATPase created a membrane potential which was inside-positive, and this might decrease the effective membrane potential (generated by K+ efflux mediated by valinomycin) available to drive alanine uptake.


Purification and characterization of methioninase from Pseudomonas putida. Methioninase of Pseudomonas putida was purified to homogeneity, as judged by polyacrylamide gel electrophoresis, with a specific activity 270-fold higher than that of the crude extract. 1. The purified enzyme had an S20,w of 8.37, a molecular weight of 160,000, and an isoelectric point of 5.6. 2. A break in the Arrhenius plot was observed at 40 degrees and the activation energies below and above this temperature were 15.5 and 2.97 kcal per mole, respectively. 3. In addition to L-methionine, various S-substituted derivatives of homocysteine and cysteine could serve as substrates. D-Methionine, 2-oxo-4-methylthiobutanoate, and related non sulfur-containing amino acids were inert. Equimolar formation of alpha-ketobutyrate and CH3SH was observed with methionine as a substrate. 4. In addition to the protein peak at 278 nm, two absorption maxima were observed at 345 and 430 nm at pH 7.5. Hydroxylamine removed the enzyme-bound pyridoxal phosphate, resulting in almost complete resolution with the concomitant disappearance of both peaks. Reconstruction of the treated enzyme could be achieved by addition of the cofactor; the Km value was calculated to be 0.37 muM. 5. The reported purified enzyme should be designated as L-methionine methanethiollyase (deaminating).