Reduced nicotinamide adenine dinucleotide phosphate-dependent formation of 2,3-dihydro-2,3-dihydroxyaflatoxin B1 from aflatoxin B1 by hepatic microsomes. 2,3-Dihydro-2,3-dihydroxyaflatoxin B1 (dihydrodiol) was formed as a major metabolite in the incubation of aflatoxin B1 with rat and hamster liver microsomes. The yield of the dihydrodiol was maximal at pH 6.5, was reduced nicotinmide adenine dinucleotide phosphate- and cytochrome P-450-dependent, and was increased 2- to 4-fold by pretreatment of the animals with phenobarbital; pretreatment with 3-methylcholanthrene did not alter the activity of rat hepatic microsomes. Inhibitors of epoxide hydrase did not lower the yield of the dihydrodiol in these systems. Negligible yields of the dihydrodiol were formed from aflatoxin B1 and rat liver microsomes in the presence of DNA. Little or no formation of the dihydrodiol was noted with microsomes from rat intestinal mucosa, kidney, or lung. These results further support the formation of aflatoxin B1 2,3-oxide as a major electrophilic metabolite of aflatoxin B1 in rat and hamster liver microsomal systems, since this highly reactive epoxide would be expected to hydrolyze readily to form the dihydrodiol.


Quantitative characteristics of the Feyrter cells and neuroepithelial bodies of the fetal rabbit lung in normoxia and short term chronic hypoxia. We report here quantitative data on the Feyrter (single) cells (APUD cells) and neuroepithelial bodies (grouped Feyrter cells), in the lungs of rabbit fetuses at 26, 27.5 and 29 days gestational age, during normoxia and short term chronic hypoxia. The apparent number of these cells declines during this period; we suggest that this might be due to increased hypoxemia. Moreover, the number of cells in the lungs of fetuses from short term chronically hypoxic mothers is lower than in the normoxic animals. These findings are in agreement with our previous studies in short term chronically hypoxic neonatal rabbits, and suggest that the increased hypoxemia in the fetus, caused by the induction of hypoxia in the mother, constitutes a stimulus for secretory activity of the Feyrter cells and neuroepithelial bodies (NEBs). This in turn could be part of the mechanism responsible for maintaining the pulmonary vasoconstriction due to hypoxemia. Our results from fetuses of normoxic does provide base line data on the chronological importance of the Feyrter cells and NEBs.


pH-dependent behaviour of erythrocyte membrane elevations. Freeze-etch electron microscopy of human erythrocytes from heparinized normal blood reveals membrane elevations. These elevations disappear at low pH values and reappear under normal pH conditions in vitro, both in the absence and the presence of plasma. This pH-dependent behaviour is discussed as an intrinsic membrane event.


Kinetic studies on the interaction of gold (III) with nucleic acids. IV. RNA-Au (III) system. The kinetics of the interaction of Au(III) with whole yeast RNA has been studied using UV-spectrophotometry. The reaction is second order with respect to the nucleotide unit of RNA and first order with respect to Au(III) in the respective stoichiometry of 2 : 1. The effects of initial composition, temperature, ionic strength, pH and chloride ion on the kinetics have been studied. Activation energy is found to be 11.5 kcal/mol. Effect of ionic strength indicates that both the positively charged and neutral species of Au(III) take part in the rate limiting step, the former being dominant at low ionic strength. A plausible mechanism has been proposed which involves the interaction of two nucleotide units of RNA with one species of Au(III) in the rate limiting step.


[Culicidae of the French Territory of Afars and Issas. The genera Aedes, Culiseta, Uranotaenia and Mimomyia]. During an entomological survey conducted in French Territory of Afars and Issas from november 1973 to june 1975, the following species were collected: Aedes caspius, A. vittatus, Culiseta longeareolata, Uranotaenia balfouri, Mimomyia mimomyiaformis, M. mediolineata. To these mosquitoes, we must add the record of A. aegypti larvae on a dhow berthing in Djibuti while this mosquito does not exist actually in the Territory. The epidemiological importance of these culicids is discussed.


[Pharmacological study of para-aminoclonidine, an alpha sympathomimetic]. Para-aminoclonidine is a more potent alpha-adrenergic agent than clonidine; in vitro on Rat aorta it is 150 times more active than NE, in vivo on pithed Rat it is still twice as active. The hypotensive action of paraaminoclondine is only manifested when given intraventricularly. In the conscious Rat, it has a diuretic action when given per os. Following IV injection in the anaesthetized Dog, the para-aminoclonidine induces an essentially hydric diuresis which is independet of its hypertensive action.


[Significance of the various toxins present on dead coral substrata in the origin of ciguatera]. The authors look directly on the coral reefs the toxins carried by poisonous fish. Pointing out two major toxic substances, they discuss their respective role in ciguatera physiopathology and suggest a new hypothesis for the origin of the phenomenon.


[Respiratory function of blood and relative weight of certain organs in two species of shrew: Crocidura russula and Suncus etruscus (Soricidae mammals)]. A high oxygen capacity combined with a low oxygen affinity and a large Bohr effect achieve a high oxygen transport capacity in small shrews. The high metabolic rate (Suncus estruscus: 100 to 350 ml O2/kg. min) can be explained by the combination of the favorable blood parameters with a high relative heart weight (1,2% of body weight) and heart frequencies of 1,000 to 1,350 per minute.


[Examination of the enzymatic functions of the normal liver in black Africans (Apropos of 50 Senegalese cases)]. In order to establish the hepatic enzymogram in healthy African black people, four enzymes have been studied in 50 apparently healthy male Africans: transaminases (GOT, GPT), alcaline phosphatases, ornithine carbamoyltransferase (OCT) and gamma-glutamyl transpeptidase (GGT). The findings do not show any difference with the usually admitted levels in European countries, except for alcaline phosphatases which are situated at the upper limit of the normal.


[Effect of total and prolonged starvation on the acid soluble nucleotides in carp liver]. The composition of the acid soluble nucleotide pool of Carp liver was determined by column chromatography, and its changes were followed during prolonged total fast. Total prolonged starvation caused substantial changes in the distribution of hepatocyte free nucleotides. Eleven months of starvation caused a reduction in the concentration of di- and triphosphate nucleosides as well as a diminution of the oxido-reduction of the hepatocyte. Only pyrimidine nucleoside monophosphates remained at concentration approaching those found in regularly fed Carp.


[Modification of the effect of cardio-accelerator nerve stimulation in dogs by clonidine and several alpha-adrenolytics]. In dogs anaesthetized with pentobarbital (30 mg. kg -1 i.v.), clonidine (0,01 mg.kg-1 i.v.) reduced the tachycardia induced at low frequencies by stimulation of the cardiac nerve. The effects of some alpha-adrenoceptor blocking agents on this effect have been studied. Small doses of yohimbine (0.3 mg. kg-1 i.v.) or piperoxan (0.3 mg. kg-1 i.v.) increased the effects of the stimulation and in addition antagonized the inhibitory effects of clonidine and reversed the pressor response to adrenaline. Thymoxamine (1 mg.kg-1 i.v.) and prazosin (1 mg.kg-1 i.v.) did not increase the effect of the stimulation of the cardiac nerve, but reduced the effect of clonidine. ARC239 (0.05 mg.kg-1) reversed the pressor response to adrenaline but even at high doses did not increase the effects of the stimulation of the cardiac nerve or the effects of clonidine. These observations afford further evidence for a dissimilarity between pre and post-synaptic alpha-adrenoceptors.


[Changes in the distribution of gamma-glutamyl transferase in the organs of the mouse as a function of development]. In the Mouse, gamma-glutamyl transferase distribution changes with development in the kidney and the liver; on the contrary, its activity remains almost equal and low in other organs. In the liver, a low activity is observed in the fetus and the new-born up to the 3rd day of life; then it is no more measurable. In the kidney, the low enzyme activity of the fetus is multiplied by 10 in the 10 first days of life and by 50 in the 6 first weeks.


[Comparative action of toxins isolated from the venom of the scorpion (Androctonus australis) and the tentacles of the sea anemone (Anemonia Sulcata) on an isolated frog neuromuscular preparation]. We have shown that, if Scorpion venom is acting a skeletal muscle indirectly by releasing Acetycholine and directly by inducing an increase in intracellular free calcium, the main action of toxin II isolated from Anemonia Sulcata tentacles is presynaptic.


[Action of toxin II isolated from the tentacles of sea anemones on the neuromuscular transmission on normal and botulin toxin inhibited frogs]. In our experimental conditions, toxin II from Anemonia sulcata restored Frog neuromuscular transmission blocked by botulinum toxin, type A.


Observations on serum and urine alkaline ribonuclease activity and urate after burn injury in man. In an investigation into the disturbances of body function associated with burn injury we have measured the activity of alkaline ribonuclease (EC 3.1.4.22) and the level of urate in the serum and urine of patients sustaining burn injury. Ribonuclease activity was elevated in all patients. The degree of elevation can be related to the percentage of the body surface area burned and to a predictive index of burn mortality. Increased serum ribonuclease activity was accompanied by increased urine ribonuclease output. The relationship between serum urea and ribonuclease activity has been investigated. A significant correlation between these two parameters was observed during the first week post burn. We suggest that this correlation shows as a result of increased protein catabolism and renal dysfunction. After the first week a significant correlation between serum urea and ribonuclease activity was not observed. It is possible that, at this stage, increased ribonuclease activity is perhaps a result of tissue repair. Serum urate was found to be decreased in all patients after burn injury. Serum urate decrease expressed as a percentage of initial value, correlated very strongly with the predictive index of burn mortality. In severely burned patients the decrease in serum urate was accompanied by increased urine urate output and may indicate a change in renal handling of urate after burn injury.


Some pitfalls in the quality control of alkaline phosphatase activity. A study of the pH optimum of alkaline phosphatase activity in calibration and control serum preparations reveals a wide variation in pH optima. It is shown that as small an error of +/- 0.05 pH units in preparation of the buffered substrate results in an error in the order of +/- 5% in the response between the calibrating serum and patient specimens. It is recommended that a calibration serum should closely parallel the pH optimum of alkaline phosphatase in human sera and that a quality control serum containing alkaline phosphatase of alternative source to that present in the calibration serum be included with patient samples as a sensitive means of monitoring pH changes in the buffered substrate.


Characterization of beta-adrenergic receptor linked to adenylate cyclase in a human cancer cell line (COLO 16). 1. A human cancer cell line (COLO 16) derived originally from an epidermal squamous cell carcinoma was found to possess adenylate cyclase responsiveness to beta-adrenergic agonists. 2. The adenylate cyclase response was characterized with respect to activation constants (KA) for various beta-adrenergic agonists and inhibition constants (Ki) for antagonists. 3. Intact cells responded with dose-dependent increases in production of cyclic adenosine 3':5'-monophosphate. 4. Properties of the beta-adrenergic receptor were evaluated by using the specific binding of [3H]propranolol to cell membranes. Specific binding was saturable, with KD 5.79 nmol/l and binding sites 0.68 pmol/mg of protein. 5. Competition for binding to cell membranes was shown by beta-adrenergic agonists and antagonists and was stereospecific. There was close agreement between the affinity of these various agents on adenylate cyclase and receptor binding. 6. It is likely that the beta-adrenergic receptor-linked adenylate cyclase in COLO 16 cells represents persistence in a cancer cell line of a receptor present normally in epidermal cells.


The nature of inactive renin in human plasma and amniotic fluid. 1. The properties of inactive and active renin in human plasma and amniotic fluid were studied chromatographically. Activation was achieved at pH 3.3 with and without added pepsin. 2. Acid activation of renin was time- and temperature-dependent but was inhibited by dilution of the sample. The dilution effect was corrected by adding pepsin. Such characteristics indicate that activation at low pH is catalysed by intrinsic enzymes. 3. Separation and/or dilution of the activating enzyme during ion-exchange chromatography concealed the eluted position of inactive renin and reduced the amount recovered. Only after full activation of the eluted renin was achieved with added pepsin was a distinct peak of inactive renin exposed. 4. At pH 7.5 inactive renin carried a lower negative charge than the active enzyme. This charge difference was lost after activation. 5. No molecular-weight differences between active, inactive renin or the International Renin Standard were detected by gel filtration. No renin of larger molecular weight was present. 6. These findings will be helpful in purification studies of human inactive renin.


Immunologically mediated drug-induced acute renal failure. (1) AIN is the most frequent pattern of drug-induced immunologically mediated renal injury. A number of drugs may be responsible for AIN, namely methicillin and other penicillin derivatives, rifampicin, phenindione and sulfonamides. Particular clinical and pathological features often suggest an immune pathogenetic mechanism. IgG anti-TBM and IgE antibodies have been found in only a few cases and it is likely that antibody-mediated and cell-mediated injury may operate in the same patient. (2) Only few examples of drug-induced vasculitis and glomerulonephritis are known, and the pathophysiology of this kind of renal damage is poorly understood.


Coupling of cerebral metabolism and blood flow in epileptic seizures, hypoxia and hypoglycaemia. This study examines the possibility that changes of cerebral extracellular pH (PH e) or adenosine concentration may provide coupling mechanisms of a general nautre, adjusting cerebral blood flow (CBF) to metabolic demands. Although there is considerable indirect evidence that CBF varies inversely with pHe, results obtained during the last few years indicate that large increases in flow may occur in the absence of a fall in pHe. Thus, induction of hypoxia or epileptic seizures leads to maximal increase in CBF before pHe falls or even when there is initial alkalosis due to concomitant hypocapnia. Furthermore, CBF increases in hypoglycaemia and after administration of amphetamine, two conditions unassociated with tissue acidosis. The possibility that adenosine may be a coupling factor was examined in hypoxia and during epileptic seizures in rats. In both conditions a four- to fivefold increase in CBF occurs in spite of the fact that tissue adenosine concentrations remain at or below 1 mumolkg-u. It is concluded that adenosine accumulates first when there is a perturbation of cerebral energy state with a rise in AMP concentration. It seems unlikely that adenosine, formed by breakdown of AMP, acts as a general coupling factor.


Control of cerebral vascular smooth muscle during general anaesthesia. The effects of some general anaesthetics, for example thiopentone, Althesin (alphaxalone + alphadolone) and ketamine, on cerebral vascular smooth muscle are those which would be expected from their metabolic actions. With other anaesthetics, mainly those administered by inhalation, and especially the volatile agents, cerebral blood flow increases in excess of the metabolic activity, which is usually depressed to varying degrees. During general anaesthesia with any of these agents, responses to changes in arterial Pco2 or blood pressure are maintained. Furthermore, when seizure activity occurs during enflurane administration, there is a flow response to the associated metabolic stimulation. The time course of the flow response to the metabolically depressant drug Althesin has been measured in baboons and shown to be very rapid. Wtih this drug cerebrovascular resistance begins to increase within 2 s of its arrival in the brain. This rapid flow change occurs also after sympathetic denervation. Extracellular fluid pH of the cortex does not alter until after the initiation of the vascular smooth muscle response.


Production, metabolism and possible functions of adenosine in brain tissue in situ. Adenosine and H+ may act synergistically to regulate cerebral blood flow because adenosine production is enhanced under various experimental conditions associated with an imbalance between oxygen supply and oxygen need. Direct application of adenosine dilates the pial vessels, but changes in cerebral vascular resistance are not observed when adenosine is infused intraarterially. This is because adenosine does not readily cross the blood-brain barrier. The studies reported here show that in dogs the adenosine released into the interstitium is partly reincorporated into adenine nucleotides via an adenosine kinase (EC 2.7.1.20) reaction (salvage pathway) and partly degraded to inosine and hypoxanthine. However, in contrast to other tissues, the accumulation of iosine and hypoxanthine in brain tissue proceeds at a rate slower than that of adenosine because one of the degradative enzymes, namely purine-nucleoside phosphorylase (EC 2.4.2.1) is located only in the vessel wall, which is not readily permeable to adenosine. Thus, the slow access of adenosine to its degradative enzymes delays the appearance of its products, inosine and hypoxanthine.


Cellular microenvironment in relation to local blood flow. The investigation was designed to identify microenvironmental factors that might be important in the regulation of local blood flow. Cellular microenvironment in terms of Po2, pH, [K+], [Cl-], [Ca2+] and lactate was measured in rat brain by means of specific microelectrodes. Vascular endothelium was stained in vivo with Thioflavine S. Local blood flow was measured with micro hydrogen electrodes. Some intracellular measurements of pH were made in conjunction with extracellular measurements. The main findings were that local autoregulation responses and blood flow changes in response to imposed hypoxic changes were very rapid (1-1.5 s). Microflow responses to changes in local cell activity were limited to a region not more than 250 micron in diameter. Increased blood flow in acute hypoxia occurred within 1-2 s of the fall in tissue Po2 and was much more rapid than changes in either pH or potassium. Intracellular pH changed within 10 s of the onset of severe hypoxia but in all cases the blood flow followed the Po2 much more closely than any other parameter. It is suggested that changes in capillary endothelium and local membrane transmission may play a part in autoregulatory mechanisms.


[Diethylpentenamide, a substitute for carbromal? (author's transl)]. Bromcarbamide-containing sleeping pills are frequently used in suicide attempts and cause severe, often fatal, intoxication. Since 1975, the chemically related drug diethylpentenamide had been available (in the German Federal Republic) without prescription. The authors report four cases of attempted suicide with the drug. The signs were similar to those after carbromal intoxication. There was severe respiratory depression, successfully treated by extracorporeal detoxication with combined haemoperfusion and haemodialysis. One patient developed acute pancreatitis as a complication.


[Salazosulfapyridine-induced lupus erythematosus syndrome in ulcerative colitis (author's transl)]. In a 25-year old woman with ulcerative colitis a lupus erythematosus syndrome occurred during treatment with salazosulfapyridine. The connection with medication was ascertained by reexposition. Some of the systemic complications of ulcerative colitis may possibly be due to the salazosulfapyridine medication.


Pharmacological study of cholinergic mechanisms of compensatory ovarian hypertrophy in rats. Treatment of hemicastrated adult female rats with nicotine increased the compensatory ovarian hypertrophy (COH) while central n-cholinolytic IEM-506 decreased COH and prevented the estrogen-induced suppression of COH. Administration of L-DOPA abolished the effect of IEM-506, and disulfiram blocked gonadotropic action of nicotine. Elevation of the dose of arecoline decreased COH and the sensitivity of hypothalamo-gonadotropic complex to estrogen suppression. Treatment of rats with L-DOPA and disulfiram abolished the late effect of arecoline. Central m-cholinolytic metamizyl decreased COH, potentiated the effect of estrogen and prevented the gonadotropic effect of L-DOPA. The regulatory role of m-cholinergic systems in noradrenaline mediation of gonadotropic function and n-cholinergic system in dopamine ones are suggested.


The stimulus-secretion coupling of glucose-induced insulin release. Metabolic effects of menadione in isolated islets. Pancreatic islets contain an enzyme system which catalyzes the donation of hydrogen from NAD(P)H to menadione (2-methyl-1,4-naphthoquinone). In high concentrations (20 to 50 micrometer), menadione, in addition to lowering the concentration of reduced pyridine nucleotides in the islets, also impairs glycolysis and glucose oxidation, decreases ATP concentration, and inhibits proinsulin biosynthesis. However, at a 10 micrometer concentration, menadione fails to affect the concentration of adenine nucleotides, the utilization of glucose, the production of lactate and pyruvate, the oxidation of [6-14C]glucose and the synthesis of proinsulin; whereas the metabolism of glucose through the pentose shunt is markedly increased. The sole inhibitory effect of menadione 10 micrometer upon metabolic parameters is to reduce the concentration of both NADH and NADPH, such an effect being noticed in islets exposed to glucose 11.1 mM but not in those incubated at a higher glucose level (27.8 mM). Since, in the presence of glucose 11.1 mM, menadione 10 micrometer also severely decreases glucose-stimulated45 calcium net uptake and subsequent insulin release, it is concluded that the availability of reduced pyridine nucleotides may play an essential role in the secretory sequence by coupling metabolic to cationic events. Thus, when insulinotropic nutrients are oxidized in the B-cell, the increased availability of reduced pyridine nucleotides could modify the affinity for cations of native ionophoretic systems, eventually leading to the accumulation of calcium up to a level sufficient to trigger insulin release.


Characterization of deoxycytidylate methyltransferase in Xanthomonas oryzae infected with bacteriophage Xp12. Three methods, chromatographic, spectrophotometric and tritium-release assay, were used and compared for the assay of deoxycytidylate methyltransferase. All three methods can be used for assay of this enzyme but the tritium-release assay appears to be the most simple and convenient. With the help of this assay the deoxycytidylate methyltransferase has been isolated and purified from sonically disrupted cells of Xp12-infected Xanthomonas oryzae. Using a procedure that involves fractionation with streptomycin sulfate and ammonium sulfate, filtration through Sephadex G-100 and chromatography on DEAE-cellulose, a 214-fold increase in specific activity was obtained. The enzyme displays a narrow pH optimum at 6.0 Among the buffers tested, 6-morpholinoethane sulfonate with the addition of Mg2 is the best. The enzyme can utilize dCMP as a substrate. The enzyme can also convert tetrahydrofolic acid into dihydrofolic acid. The Km value for dCMP is 31.3 micrometer and the Km value for tetrahydrofolic acid is 71.4 micrometer. There is no absolute requirement of ions for the activity of the enzyme; however, the presence of ions causes stimulating or inhibiting effects on enzyme activity that are dependent on the variety and concentration of ions used.


Dependence upon pH of steady-state parameters for the beta-galactosidase-catalysed hydrolyses of beta-D-galactopyranosyl derivatives of different chemical types. The effect of pH upon the beta-galactosidase-catalyzed hydrolyses of aryl galactosides is essentially similar for each of the three steps of their hydrolysis. It differs markedly from that on the hydrolysis of galactosyl pyridinium salts; these proceed through a 'non-bottleneck' pathway. While pH increase abolishes the rate of every step of the reaction for aryl galactosides, it favors the first step of hydrolysis of the galactosyl pyridinium salts, which supports the hypothesis that catalysis of these compounds originates largely in non-covalent interactions.


Effect of pH on the process of ternary-complex interconversion in the liver-alcohol-dehydrogenase reaction. 1. Kinetic relationships referring to multiple-turnover conditions have been derived for the slowest exponential transient appearing in two-substrate enzyme reactions proceeding by an ordered ternary-complex mechanism. The validity of these and previously derived theoretical relationships for this mechanism has been tested by application to the liver alcohol dehydrogenase reaction. 2. All essential features of the transient-state kinetics of alcohol oxidation by NAD+ in the liver alcohol dehydrogenase system can be qualitatively and quantitatively explained in view of the compulsory-order mechanism in the proposed scheme. There is no kinetic evidence for any half-of-the-sites reactivity of the enzyme. A consistent set of rate constants is reported for the enzymic oxidation of benzyl alcohol at pH 8.75. 3. Transient-state rate parameters for benzyl alcohol/benzaldehyde catalysis by liver alcohol dehydrogenase have been determined at different pH. The interpretation of such rate parameters is critically discussed with reference to their informative value for the purpose of determination of rate constants (k and k') for the process of ternary-complex interconversion in the proposed scheme. It is concluded that the apparent rate constant (k') for hydride transfer from benzyl alcohol to NAD+ is dependent on a proton dissociation step with a pKa of 6.4, whereas the rate constant (k) for hydride transfer from NADH to benzaldehyde exhibits no corresponding dependence on proton association. 4. The asymmetric pH dependence of the forward and reverse rate of ternary-complex interconversion during liver alcohol dehydrogenase catalysis appears to reflect an obligatory step of alcohol/alcoholate ion equilibration occurring at the ternary-complex level. It is suggested that the observed pKa 6.4 dependence of the transient rate of alcohol oxidation can be attributed to a coupled acid-base system involving minimally the enzyme-bound alcohol and the protein residues Ser-48 and His-51.


Isolation of a multiprotein complex containing cytochrome b and c1 from Neurospora crassa mitochondria by affinity chromatography on immobilized cytochrome c. Difference in the binding between ferricytochrome c and ferrocytochrome c to the multiprotein complex. A multiprotein complex which contains in equimolar amounts two cytochromes b (Mr each about 27,000), one cytochrome c1 (Mr 31,000) and six subunits without known prosthetic groups (Mr 8000, 12,000, 14,000, 45,000, 45,000, and 50,000) has been isolated from the mitochondrial membranes of Neurospora crassa by affinity chromatography on immobilized cytochrome c. The chromatographic separation was based upon the specific binding of the complex to ferricytochrome c coupled to Sepharose and its specific release upon conversion of the coupled ferricytochrome c into ferrocytochrome c using ascorbate as a reductant. The chromatography was performed in the presence of the nonionic detergent Triton X-100 at low ionic strengths. A monodisperse preparation of the multiprotein complex was obtained which was used for binding studies with cytochrome c from Neurospora crassa, horse heart and Saccaromyces cerevisiae. At low ionic strength (20 mM Trisacetate) and slightly alkaline pH (pH 7 to 8), more than one molecule of ferricytochrome c were bound to the isolated multiprotein complex with dissociation constants below 1 x 10(-7) M. One of these bindings appeared different from the others, since its high affinity was preserved at an ionic strength at which the affinities of the other bindings decreased. Furthermore, the affinity of only this binding decreased upon reduction of cytochrome c. It is suggested that this binding is at or near the functionally active site(s) of the mulipprotein complex.


Reduction of methemoglobin by cobaltocytochrome c catalyzed by mediators. The reduction of methemoglobin by cobaltocytochrome c (Cocyt c) has been measured using nine mediators of different half-reduction potentials, Em, 7. The rate increases with the increase of Em, 7 for the mediator but dropped precipitously when it becomes more positive than the Em, 7 for the methemoglobin/hemoglobin couple. The reaction is most efficient with phenzaine methosulfate, therefore it was studied in detail. The reaction is first order in the concentrations of Cocyt c and phenazine methosulfate. The average second-order rate constant for Cocyt c + phenazine methosulfate (M) k1 leads to Cocyt c+ M-. is 2.9 x 10(4) M-1 s-1 at 25 degrees C, 0.1 M phosphate pH 7.0. There is a slight negative temperature dependence of k1 at low temperature; at higher temperatures the process has deltaH not equal to approximately 27 kJ mol-1 and deltaS not equal to approxmately - 75 J mol-1 K-1. The effect of anions reflects the dependence of Em, 7 for the methemoglobin/hemoglobin couple with various anions. There is no significant effect on k1 by the addition of inositol hexakisphosphate. The variation of k1 with pH is complicated. The experimental rate constants are compared with values calculated with the theory of nonadiabatic multiphonon process of electron tunneling.


Nuclear-magnetic-resonance studies of 5'-ribonucleotide and 5'-deoxyribonucleotide conformations in solution using the lanthanide probe method. The conformations of the metal-bound 5'-ribonucleotides and 5'-deoxyribonucleotides in aqueous solution at different pH values have been studied using the lanthanide probe method. The conformational analysis, based on mixing different conformations in fast exchange within the nuclear magnetic resonance time scale, agrees well with the results from coupling constants, nuclear Over-hauser effects and spin-lattice relaxation times, obtained for the metal-fixed systems. The equilibrium between the two basic conformational combinations for the 5'-nucleotides, anti-(N in equalibrium S)-gg-g'g' and syn-(N in equalibrium S)-gt-g'g' depends on the nature of the furanose ring, the base and also on the state of base protonation and phosphate ionization. The effect of base protonation is particularly strong for the guanine nucleotides.


Ecdysterone biosynthesis: a microsomal cytochrome-P-450-linked ecdysone 20-monooxygenase from tissues of the African migratory locust. Ecdysone 20-monooxygenase, an enzyme which converts ecdysone to ecdysterone (the major moulting hormone of insects) has been characterized in cell-free preparations of tissues from African migratory locust. The product of the reaction has been identified as ecdysterone on the basis of several microchemical derivatization and chromatographic methods. Ecdysone 20-monooxygenase activity is located primarily in the microsomal fraction which also carries NADPH cytochrome c reductase and cytochrome P-450, as shown by sucrose density gradient centrifugation. Optimal conditions for the ecdysone 20-monooxygenase assay have been determined. The enzyme has a Km for ecdysone of 2.7 x 10(-7) M and is competitvely inhibited by ecdysterone (Ki = 7.5 x 10(-7) M). Ecdysone 20-monooxygenase is a typical cytochrome P-450 linked monooxygenase: the reaction requires O2 and is inhibited by CO, an effect partially reversed by white light. The enzyme is effectively inhibited by several specific monooxygenase inhibitors and by sulfhydryl reagents, but not by cyanide ions. Ecdysone elicits a type I difference spectrum when added to oxidized microsomes. NADPH acts as preferential electron donor. The transfer of reducing equivalents proceeds through NADPH cytochrome c (P-450) reductase: ecdysone 20-monooxygenase is inhibited by cytochrome c. Both NADPH cytochrome c reductase and ecdysone 20-monooxygenase are inhibited by NADP+ and show a similar Km for NADPH. The Malpighian tubules have the highest specific activity of ecdysone 20-monooxygenase, while fat body contain most of the cytochrome P-450 and NADPH cytochrome c reductase.


Ultramicro-electrophoresis of protein in sweat from patients with cystic fibrosis of the pancreas and controls. 46 sweat-samples from 32 children with cystic fibrosis of the pancreas (C.F.) and 35 samples from 23 control-children were collected with glass micro-capillaries. Protein was determined by ultramicro-electrophoresis. 1. Protein was detected in 48% of the samples (C.F. and controls). 2. At pH 2.3, C.F.-sweat showed at least one more band than control-sweat. 3. At pH 8.9, C.F.-sweat occasionally showed one more band than the control-group. 4. At pH 2.3, more protein was found at the electrophoresis start point using C.F.-sweat, whereas at pH 8.9 the opposite was found. 5. At pH 2.3, protein was found in fewer samples than at pH 8.9. The "C.F.-factor" is postulated to represent a basic polyelectrolyte which induces the following pathogenic mechanisms: a) Aggregation of proteins giving rise to a high viscosity of secretions, such as saliva. b) Binding to the cell-membrane of the glandular epithelium, thus inducing a disturbance of active NaCl-reabsorption (e.g. by reduction of luminal passive Na+-influx).


Erythrocyte organic phosphates in the anemia of renal failure in childhood. Erythrocyte 2,3-diphosphoglycerate (2,3-DPG ) and adenosinetriphosphate (ATP) levels were determined in 43 children with chronic renal failure on conservative treatment (CT), and 12 children on regular hemodialysis (HD) immediately before and after a HD session. The results were compared to non-anemic and anemic controls. In spite of anemia, erythrocyte 2,3-DPG in renal failure was similar to non-anemic controls at normal blood pH, but rose during dialysis as a result of alkalosis. In contrast, ATP levels were high already at a normal blood pH. 2,3-DPG correlated with packed cell volume (PCV) in children with renal failure but at lower concentrations compared to controls. Both organic phosphates in the erythrocytes showed a significant correlation with blood pH. The poor increase of 2,3-DPG, in combination with elevated ATP levels, suggests uremia-induced inhibition of 2,3-DPG synthesis.


Methylmalonic acidemia.

Methylmalonic acidemia. A patient presenting with severe metabolic acidosis accompanied by hyperglycinemia, hyperuricemia, hypoglycemia and hypertammonemia is described. Metabolic acidosis was found to be due to accumulated methylmalonic acid and did not respond to vitamin B12 administration. The patient showed favorable growth and development when kept on a low isoleucine, methionine, threonine and valine diet. In vitro studies using a lymphoid cell line derived from the patient showed a deficiency of methylmalonyl-CoA carbonyl-mutase.


Controlled double-blind trial of camazepam, a new benzodiazepine derivative, versus chlordiazepoxide. 31 in-patients with anxiety entered at random in a double-blind clinical trial of chlordiazepoxide and camazepam, a benzodiazepine derivative. They were assessed by three different rating scales. Statistical evaluation of the results showed similar psychopharmacological activity of both drugs. It is concluded that camazepam is suitable for treatment of mild and severe anxiety states.


Phenprocoumon requirement, whole blood coagulation time, bleeding time and plasma gamma-GT in patients receiving mianserin. A possible interaction between the tetracyclic antidepressant mianserin and a coumarin derivative has been investigated. Sixty-three subjects, 61 of whom required anticoagulant therapy for a variety of medical conditions, were treated for 5 consecutive weeks with phenprocoumon, in a dose adjusted to reduce the prothrombin time to 15%-25%. After an initial control period of one week, subjects were randomly treated under double-blind conditions with mianserin 3 X 10 mg daily or 3 X 20 mg daily, or with a matching placebo. The dose of mianserin was gradually increased to reach the maximum by the 6th day. Three subjects dropped out and 60 completed the trial. The dose of phenprocoumon and the prothrombin, bleeding, and coagulation times were not significantly affected by administration of mianserin. It can be concluded that there is no clinically important interaction between phenprocoumon and doses of mianserin effective in depression. Sedation was more frequent in patients taking mianserin than in those given placebo.


Long-term blood pressure control with pindolol given once daily. The antihypertensive effect of beta-adrenoceptor blocking drugs has been documented in a number of papers [10--13] and over the last 10 yr beta-receptor blockade has become a more and more frequent primary therapy for hypertension. During this time, a number of different beta-blockers with various degrees of selectivity, potency and intrinsic sympathomimetic effect have come into use. There have also been attempts to reduce the number of daily doses in beta-adrenoceptor blocking therapy while maintaining the antihypertensive effect [5]. Pindolol (Viskén) is a beta-adrenoceptor blocking agent with intrinsic sympathomimetic effect and is characterized by high potency and long duration.


Participation of the sympathetic and the renin--angiotensin systems in blood pressure control during hypercapnia in the anaesthetized dog. The effects of hypercapnia on plasma renin concentration and blood pressure were studied in anaesthetized dogs, untreated and after pretreatment with guanethidine, propranolol or prazosin. An increase in plasma renin concentration which accompanied hypercapnia in untreated dogs was completely suppressed by pretreatment with guanethidine or propranolol. Prazosin significantly reduced but did not abolish renin release during hypercapnia. The pressor response normally occurring during hypercapnia was abolished by propranolol and reversed by guanethidine and prazosin.


Isoelectric focusing study of radiation damage to dry conalbumin. The effect of gamma-rays on iron-free conalbumin was studied by isoelectric focusing technique. The results obtained can be interpreted on the basis of radiation-induced conformational changes of the macromolecules through a cleavage of disulphide bridges. Treatments with reagents acting on disulphide bridges lead to isoelectric focusing patterns that confirm this hypothesis.


[Influence of isolanide and trasicor on the content of nicotinamide coenzymes in the myocardium of growing rats in hemic hypoxia]. Hemic hypoxia was produced in 7,30-day and 3--5-month old (adult) rats through introduction of sodium nitrite (100 mg/kg) with subsequent determination of the nicotinamide co-enzymes content in the myocardium. The effect of combined and separate application of the cardiac glycoside isolanide and beta-adrenoblocking agent trasicor under hypoxia at early stages of post-natal ontogenesis is shown.


The cation composition of the seminal plasma and prostatic fluid and its correlation to semen quality. The concentrations of calcium, magnesium, and zinc were measured by atomic absorption spectrometry. The concentrations of all three cations were found to be strongly correlated with one another and with acidity (pH). Analysis of the relative concentrations in prostatic fluid, split ejaculates, and whole seminal plasma confirmed an almost exclusively prostatic origin of these cations. Semen quality, as judged by motility, vitality, concentration, and morphology of spermatozoa, was inversely related to cation concentrations. Therefore, we recommend adoption of the measurement of seminal divalent cations as part of the routine andrologic examination.


Treatment of cryptorchidism with pernasal gonadotropin-releasing hormone therapy. Twenty-five boys between 1 and 10 years of age with unilateral or bilateral cryptorchidism were treated with 200 microgram of gonadotropin-releasing hormone (GnRH) pernasally six times daily until descensus was completed, or for 10 weeks at most. Complete descent of the tests occurred in 16 patients, usually after 2 to 5 weeks of treatment. No adverse side effects have been observed. Radioimmunologic measurements of serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone, and prolactin, carried out before treatment, at the end of treatment, and 6 months after treatment, showed a transitory increase of the LH responsiveness to GnRH in only four of the patients and in the group as a whole a slight but significant decrease of FSH responsiveness. There were no signs of precocious puberty. GnRH antibodies were not found.


Treatment of cryptorchidism with a potent analog of gonadotropin-releasing hormone. Pernasal therapy of cryptorchidism with D-Leu6-des-Gly10-gonadotropin-releasing hormone ethylamide (D-Leu6-des-Gly10-GnRH-EA), a potent, long-acting GnRH analog, was attempted. Eleven prepubertal cryptorchid boys received between 25 microgram once daily and 25 to 50 microgram twice daily for 5 to 12 weeks. Complete testicular descent was achieved in 4 of the 11 boys. GnRH tests (1.5 microgram/kg intravenously), conducted in six boys before treatment, after 4 weeks of treatment, and in 2 boys 3 months after treatment, did not reveal changes in gonadotropin secretion indicative of precocious puberty or of decreased hypophyseal sensitivity to GnRH. Antibodies to the GnRH analog or to GnRH could not be detected.


Histochemical demonstration of glucose-6-phosphate dehydrogenase, D-sorbitol dehydrogenase, and alkaline phosphatase in human ampulla ductus deferentis. A cytochemical study on the localization of the enzymes glucose-6-phosphate dehydrogenase, sorbitol dehydrogenase, and alkaline phosphatase was carried out in human ampullary glands. Although the former two enzymes showed a marked reactivity, alkaline phosphatase was absent from epithelial cells. On the basis of these results it is concluded that, as in the human seminal vesicle, in the ampullary gland fructose is probably secreted via the oxidative mechanism proposed by Hers (Le Metabolisme du Fructose. Bruxelles, Arscia, 1957).


Cytological analysis of bone marrow present in the bone nodules induced by human FL cells in mice. Bone marrow cells recovered from bone nodules induced in mice by intramuscular transplantation of human FL cells were compared with the host femoral bone marrow. The precursors and mature forms of all types of blood cells were found in the induced bone marrow. The concentration of lymphocytes was significantly higher, and that of erythropoietic cells significantly lower in the induced bone marrow as compared with heterotopic femoral bone marrow.


Evaluation of the local antihistaminic activity of dimetindene maleate by a quantitative method. The topical application of a 0.1% dimetindene maleate gel on the forearms of 11 subjects reduces the areas of both erythema and wheal produced by the intradermal injection of 0.025 mg of histamine hydrochloride to a statistically greater degree than 1% d-chlorpheniramine maleate cream (p less than 0.005 and p less than 0.0005, respectively). The quantitative method employed in this study has proved to be an excellent means for the evaluation of the antihistaminic activity of topical preparations.


[Sodium dichloracetate: its application to the therapeutics of experimental hyperiactatemia (author's transl)]. Different types of experimental hyperlactatemia and hyperpyruvicemia with or without lowering of blood pH were induced in anesthetized dogs. The initially studied experimental model was the hyperlactatemia and hyperpyruvicemia with lowering of blood pH induced by the intraduodenal administration of high doses of phenformin. Intravenous perfusion of sodium dichloroacetate (150 mg/kg infused during 20 minutes), which acts on the enzymatic complex of pyruvate dehydrogenase, reduced the hyperiactatemia and hyperpyruvicemia with or without acidosis provoked by phenformin injected intraduodenally (30 mg/kg), by intense muscular work, by hypoxia or by continous perfusion or adrenaline. Exogenous or endogenous insulin combined with sodium dichloracetate reduced the hyperlactatemia and hyperpyruvicemia as well as the changes in blood pH provoked by phenformin, more strongly than did sodium dichloroacetate alone. These findings have been confirmed in the conscious dog.


Increased trypsin sensitivity of cell surface macromolecules after malignant transformation. The effect of treatment with 0.04% (w/v) trypsin (EC 3.4.4.4) for 3 h on the electrophoretic mobility (EPM) of polyoma-virus malignantly transformed BHK21 cells (Py6) and their normal counterparts has been investigated. These particular conditions were chosen because an earlier study had shown that such treatment released material from the Py6 cells which was not obtained from the BHK21 cells. The negative EPM of the Py6 cells at pH 7.5 was greatly increased by this treatment; whereas the EPM of the BHK21 cells remained unchanged. Active enzyme was required to produce the change. No evidence was obtained for cytolysis, cytotoxicity or uptake of the enzyme by the treated Py6 cells. Measurement of the EPM of the Py6 cells at different pH levels before and after trypsin treatment suggested that the enzyme was removing cationic groupings from the cell surface.


Relationship between site of peptic ulceration and gastric acid-peptic activity: new evaluation of gastric analysis in patients with acute gastric bleeding and chronic peptic ulcer. In an attempt to elucidate the etiology of acute gastric bleeding and/or erosion and chronic peptic ulcer, a measurement of gastric juice and mucosal pepsin was carried out in surgically-treated patients. Patients with massive gastric mucosal bleeding in the fundic gland area showed high levels of fundic mucosal pepsin without acid-pepsin appearance in the gastric contents. In these patients, a significantly high value of the peptic activity ratio of gastric mucosa to gastric juice (MJPR, 36.4 +/- 6.7) was observed. It can be suggested that transient blockage of pepsin output from peptic cells with occur in the course of the acute mucosal bleeding, while acid-peptic digestion could be carried out within the fundic gland mucosa. On the other hand, a close correlation between relatively high acid-and-pepsin concentration of the gastric contents and a low level of MJPR (5.6 +/- 1.2) was observed in patients with chronic gastric ulcer. Patients who had a gastric ulcer within the pyloric gland mucosa had a highest acid-peptic activity among three groups with ulcers in fundic gland area, border zone and pyloric gland area. There is a rule that acid-peptic activity becomes low when the site of gastric ulcer moves from pylorus to fundus. A marked increase in acid-and-pepsin secretion into the gastric cavity was observed in patients suffering from chronic duodenal ulcer showing the lowest level of MJPR (3.40 +/- 0.50).


In vitro studies on mechanism for concentrating function of gallbladder. The mechanism for concentrating function of gallbladder was studied in vitro, turning and attention to changes in the gallbladder bile composition, especially a marked increase of Na+ and a marked decrease of Cl- in the concentrating process of hepatic bile to gallbladder one. The following results were obtained by the present investigations: 1) bile acids present in bile regulate the rate of Na+ and Cl- transport: 2) the epithelial membrane of the gallbladder can be regarded as being a simple semi-permeable membrane.


Food sensitivity in reflux esophagitis.

Food sensitivity in reflux esophagitis. We examined 66 patients with pain of possible esophageal origin for sensitivity to intraesophageal infusions of coffee, orange juice, spicy tomato drink, or HCl of varying concentrations as an addendum to their acid infusion (Bernstein) tests. Compared to Berstein-negative subjects, acid-sensitive patients were sensitive to infusion of coffee (P less than 0.01), orange juice (P less than 0.001), and tomato drink (P less than 0.001). Patients were largely insensitive to HCl solutions with a titratable acidity of 1 mEq per liter or less, less than the least acidic food solution tested. However, Berstein-positive patients were still highly sensitive to infusions of coffee, orange juice, and tomato drink adjusted to pH 7 (P less than 0.001). Patients were unable to differentiate symptoms caused by acid or food infusions, and solutions did not differ in the duration of infusion needed either to cause symptoms or to relieve them by saline. We conclude that the pain of esophagitis is nonspecific and can be precipitated by variety of seemingly unrelated substances.


Intraluminal pH esophageal motility.

Intraluminal pH esophageal motility. The aim of the present study was to investigate the effect of intraluminal pH on motor activity of the lower esophageal body. Liquid boluses of different pH values (7 to 2) were infused in the esophagus of 14 normal subjects during manometric recording of motor activity. Each test infusion elicited an esophageal motor response, either secondary peristalsis or simultaneous contractions. Secondary peristalsis was elicited by similar volumes of perfusates at pH 7, 6, 5, but significantly less volumes were needed with perfusates pH 4, 3, and 2. Simultaneous motor activity was not affected by intraesophageal pH value in the range investigated.


[Riboflavin biosynthesis operon of Bacillus subtilis. XIII. Genetic and biochemical study of mutants with regard to intermediate stages of biosynthesis]. New riboflavin dependent mutants of Bacillus subtilis accumulating different pteridines were studied. The data obtained show that the formation of ribityl side chain proceeds in a few steps at least on a part of riboflavin precursors. The oxidation of connected ribosyl into ribulose with subsequent restoration of it into ribityl proceeds at first. The corresponding genes are located on terminal part of riboflavin operon, as show the results of two-factor transformational crosses with different donors and recipients.


Experimental and clinical investigations on stem cell take and colony formation. First, lymphocyte transplantation into total body-irradiated rats is discussed. The effect on spleen colony formation caused by the transplantation of untreated lymphocytes, as well as of lymphocytes previously incubated with PHA, or with PHA plus L-asparaginase, or with lymphokines, was studied. Then the effect of the urinary colony-stimulating factor in vitro, and the in vitro feeder-layer activity of leucocytes on colony formation of human and mice bone marrow cells in haematological diseases is dealth with. The injection of rat lymphocytes previously incubated for 24 hours with PHA resulted in a higher number and a larger size of colonies in the spleen of the recipient rats. Lymphocytes preincubated with lymphokines gave rise to the formation of spleen colonies which were larger than those developing after the injection of untreated lymphocytes. When the lymphocytes were previously incubated with PHA plus L-asparaginase, PHA failed to stimulate colony formation in the spleen. The phenomenon is explained by assuming that PHA, as an aspecific stimulator of cell division, initiated the division of CFUs, thus the CFUs content of the preincubated samples increased, resulting in an increase in the number of colonies formed after the transplantation of lymphocytes pretreated with PHA. Another possible explanation is that CFUs division, or their spleen take is enhanced by the immunocompetent lymphocytes activated by PHA, either directly or via soluble mediators produced or released by immunocompetent lymphocytes such as lymphokines. The study of colony-forming cells and colony-stimulating activity in primary myelofibrosis (PM) showed an increase in the number of circulating CFUc in this condition, and an abnormal density of these cells reaching a peak below 1.062. The lowering of CSA in the first two peripheral blood gradient fractions agrees with the observation in the same fractions of a high percentage of CFUc at the expense of the CSC population. Thus, double cell population seems to exist in PM. One is greatly abnormal with a low specific density and high plating efficiency, whereas the other population is almost normal, showing a higher specific density and a lower plating efficiency.


Effect of lanthanum ion on platelet electrophoretic mobility. The effect of trivalent La ions on the elctrophoretic mobility of washed human platelets has been studied. The mobility of the human platelet in 0.145 M sodium chloride at pH 7.2 was -0.84 +/- 0.06 micron per sec per volt per cm with an isoelectric point approximately at pH 3.4 La3+ at a concentration corresponding to the physiological level of Ca2+ changed the sign of the electrophoretic mobility of the platelets at pH 5.5 and pH 4.0, but had only a faint or practically no effect at pH 3.0 and pH 2.0. It is assumed that La 3+ exerts its effect when bound to the phosphate groups on the surface.


Antihaemophilic factor (factor VIII) from human granulocytes. Factor VIII has been isolated and purified from human granulocytes using chromatography on Sephadex G-200 AND DEAE-Sephadex A-50. The final preparation was purified 60-fold. Some properties of this partly purified factor VIII were compared with the human plasma AHF preparation. Optimum pH activity and the highest activity after incubation at different pH values was found in buffer at pH 7.0. The preparation was thermolabile and had a molecular weight of about 214,000. Absorption curves indicated that it is a protein exhibiting four fractions in polyacrylamide gel disk electrophoresis. AHF preparations from plasma and granulocytes were almost identical in the characteristics tested.


Some properties of an endo-1,4-beta-D-xylanase from the ligniperdous fungus Trametes hirsuta. Some properties of purified endo-1,4-beta-D-xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) from the ligniperdous fungus Trametes hirsuta were investigated. The enzyme was stable between pH 4.0 and 8.0 were optimum activity at pH 5.0--5.5. The temperature optimum was 50 degrees C and the enzyme was stable for up to 30 min at 45 degrees C; however, it was denatured at higher temperatures. The Km for 4-O-methylglucurono-D-xylan was 6.36.10(-3) equivalents of D-xylose per litre, the activation energy was 28 kJ mol-1. The molecular weight determined by means of gel chromatography was 22000--24000. The enzyme was activated by Ca2+ and inhibited by Ag+ and Hg2+. On the basis of the effect of 2-hydroxy-5-nitrobenzyl bromide. N-bromosuccinimide and N-acetylimidazole it may be assumed that trytophan and possibly tyrosine residues influence the enzyme catalysis.


[Psychopharmacologic drugs in the rehabilitation after myocardial infarct]. The specific use of psychotropic agents in myocardial infarction has become possible due to interdisciplinary cooperation. Especially the differentiated therapeutic application of benzodiazepine derivatives will take into better account the different psychopathologic states in the various phases. The medicamentous facilities are extended by using the central and peripheral effects of the beta-blockers. After a myocardial infarction there are various somatic and mental symptoms. This requires precise medical and psychological management with reference to a diagnostic and combined-therapeutic schedule of treatment. An intensive co-operation with all medical departments is a conditio sine qua non.


[Insect bite allergy. Acute therapy and hyposensitization by means of insect venom]. Systemic reactions after an insect bite necessitate an allergologic investigation. In addition to taking the patient's history a skin test and an estimation of allergenspecific IgE should be performed. If insect bite allergy is confirmed immediate pharmacological treatment is necessary and hyposensitization therapy is recommended. Purified venom extracts are available.


[Acid-base equilibrium in the cerebrospinal fluid in diseases of the central nervous parenchyma]. A number of case studies reveal and dynamic changes of the acid-base balance in the blood and cerebrospinal fluid, such as the increasing decompensation of the metabolic lactate acidosis in the cerebrospinal fluid in lethal cases or the gradual regression in patients who were cured. Individual metabolic cerebrospinal parameters are correlated with the disturbance of consciousness. The cerebrospinal fluid parameters of patients with clouded consiciousness constitute prognostically critical values: lactate 3.6 mmol/l, pyruvate 0.19 mmol/l, pH 7.249 and bicarbonate 18.1 mmol/l. Affections of the central nervous parenchyma are better reflected in the cerebrospinal fluid than in the blood. The determination of the CSF/blood relationship of individual parameters shows a significant increase of the lactate quotient as well as a decrease of the pH quotient and of bicarbonate quotient. Respiratory parameters do not show any statistically significant changes. Statistically significant correlations show that, in the case of primary central nervous diseases, the increased hydrogen ion supply in the cerebrospinal fluid is quickly intercepted by the bicarbonate buffer and that, with a corresponding pH decrease, there is a rapid decompensation of the acid-base balance.


Unilateral cryptorchidism with compensatory hypertrophy of descended testicle in prepubertal boys. 5 prepubertal boys with unilateral cryptorchidism and compensatory hypertrophy of the descended testicle, 22 prepubertal boys with unilateral cryptorchidism and without CTH, and 14 prepubertal normal boys were submitted to LH-RH and to HCG tests in order to study the hormonal behaviour in CTH phenomenon before puberty. High but normal peaks of plasma LH and FSH were observed after LH-RH in CTH boys who showed a significant increase of testosterone after HCG stimulation. On the contrary the LH response to LH-RH and the testosterone response to HCG of the boys with unilateral cryptorchidism and without CTH were, as expected, significantly lower than in the control ones.


Carcinoma in situ of the testis: frequency and relationship to invasive germ cell tumours in infertile men. A light microscopical study on a total of 812 consecutive testicular biopsies from 555 infertile men revealed intratubular changes in germ cells compatible with a carcinoma in situ pattern in six oligospermic patients (I.I%); the changes were found in both testes in two of these men. Four of the six patients developed an invasive germ cell tumour within follow-up period of 1.3 to 4.5 years. The results confirm the malignant nature of these intratubular atypical germ cells. It is concluded that testicular biopsy may be useful for early detection and cure of germ cell carcinoma in patients at risk, i.e. patients with cryptorchidism, infertile men or patients with previous cancer of one testis.


G6PD Vientiane: a new glucose-6-phosphate dehydrogenase variant with increased stability. A new G6PD variant, called G6PD Vientiane, has been discovered in a patient from Laos. The characteristics of this variant are: mild enzyme deficiency (about 50% of the normal activity) in the granulocytes and the red cells, with normal G6PD-related antigen concentration; increased stability; normal Km glucose 6-phosphate and NADP+; increased inhibition constant by NADPH; decreased inhibition by ATP; slightly increased utilization of the substrate analogue; abnormal pH curve, with maximum activity at pH 9.5; slightly reduced starch gel electrophoretic migration. The implications of the molecular stability of a deficient mutant variant are discussed.


Growth and embryo formation in wild-carrot suspension cultures with ammonium ion as a sole nitrogen source. Wild-carrot (Daucus carota L.) suspension cultures grew and produced embryos on ammonium ion as a sole source of nitrogen in the absence of any exogenous Kreb's cycle acid when the pH of the medium was controlled by continuous titration with with KOH or KHCO3.


Initiation and characterization of a diploid cell line from larval tissues of Aedes dorsalis (Meigen). Mosquito cell cultures were initiated from the minced tissues of newly hatched Aedes dorsalis (Meigen) larvae. Continuous cell division occurred only after an adaptive period of approximately 6 months. Optimal growth of the cells required a relatively low pH of 6.5. Karyological studies showed that the cells have remained diploid (2n = 6) for 60 serial passages and that the cultures are free of contaminating cells. The cultures also were shown to be free of bacteria (including Mycoplasma), fungi and virions. Subpopulations (strains) of the original parental cultures have been selected and characterized on the basis of morphology, karyology, growth rate and monolayer formation.


gamma-Glutamyltransferase in human diploid fibroblasts and other mammalian cells. gamma-Glutamyltransferase was determined in WI-38 human diploid fibroblasts and compared to enzyme levels determined in several other mammalian cell lines including: fibroblast-like cells from human skin, tibia and foreskin; epithelial-like cells from human, bovine and monkey kidney; and transformed cells (Chinese hamster ovary, HeLa S3 and SV-40 transformed WI-38). Transformed cells had the lowest activity found followed in increasing order by fibroblasts, human and bovine epithelial cells and monkey kidney epithelial cells. The enzyme isolated from the plasma membrane of WI-38 cells, like the enzyme from kidney and brain, was found to be irreversibly inhibited by iodoacetamide, reversibly by serine-borate, and had a strong specificity for certain amino acids. The possibility exists that gamma-glutamyltransferase could be involved in transport of amino acids into cells in culture; and glutamine, used in media, is an excellent substrate for the enzyme.


Filamentous capsulated streptococci from the human respiratory tract: chemical and immunochemical characterization of a glycoprotein capsular antigen of provisional binary capsular type 87. A filamentous alpha-hemolytic streptococcus of provisional capsular type 87 isolated from the human respiratory tract has been shown to be binary capsulated. One of the capsular antigens appears to be a glycoprotein; the other appears to be a polysaccharide. Transformation reactions with deoxyribonucleic acid from streptococcus type 87 and a number of noncapsulated pneumococci yielded transformed pneumococci with either a glycoprotein capsule or a polysaccharide capsule, but not with both. Capsular precipitin (quellung) reactions were observed when streptococcus type 87 was treated with homologous antiserum or with antisera to either of the two distinct capsular transformants. Each of the transformed pneumococci gave a quellung reaction with its homologous antiserum or with antiserum to streptococcus type 87, but neither reacted with antiserum to the heterologous transformant. Chemical analysis showed the glycoprotein antigen of streptococcus type 87 to contain, in addition to amino acids, glucose, galactose, glucosamine, and phosphate. The amino acid composition of the glycoprotein capsular antigens from streptococcus type 87 and of those from transformed pneumococci were similar, showing only minor differences. The glycoprotein capsular antigen from streptococcus type 87 gave two closely associated precipitin bands with homologous antiserum or antisera to transformed pneumococci with the glycoprotein capsule. That the two precipitin bands represent two unrelated proteins is precluded largely on the basis of the unlikely probability of 100% cotransformation of the genes coding for both proteins in the pneumococcal transformants that were isolated. Chemical analyses of the various fractions of the glycoprotein indicate that the two precipitin bands may represent a glycoprotein and its corresponding apoprotein.


Filamentous capsulated streptococci from the human respiratory tract: chemical and immunochemical characterization of the polysaccharide capsular antigen of provisional binary capsular type 87. The polysaccharide capsular antigen of the filamentous binary capsulated streptococcus of provisional type 87 and the polysaccharide capsular antigens of two pneumoccal strains transformed with deoxyribonucleic acid of streptococus type 87 have been purified and analyzed with regard to their component monosaccharides. The purified polysaccharides from the three strains were immunochemically identical. Each was found to contain rhamnose, glucose, galactose, galactosamine, and phosphate. Rhamnose was the immunodominant sugar.


Heat-stable enterotoxin from Escherichia coli: factors involved in growth and toxin production. Six enterotoxigenic strains of Escherichia coli produced variable levels of heat-stable enterotoxin (ST) when grown under pH control at 8.5 in a simple synthetic medium containing neither amino acids nor vitamins. Bacterial growth and ST production were at levels as high as or higher than those observed in complex media. ST elaboration was detectable in the early logarithmic phase of growth and appeared to be related to disappearance of glucose in the growth medium. The results of this study did not suggest pH-dependent release of ST. Imposition of pH control in complex media resulted in increased growth rates, earlier detectable ST synthesis, and elevated levels of ST. In synthetic medium, attainment of the stationary growth phase was followed by a significant decrease in culture density and a concomitant increase in ST. Cellular autolysis experiments revealed that as much as 20% of the total ST activity was present in a cell-associated form.


Allosteric transformation of reduced nicotinamide adenine dinucleotide (phosphate) oxidase induced by phagocytosis in human polymorphonuclear leukocytes. We used sensitive isotopic and fluorometric assay procedures to investigate reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]oxidation in a particulate fraction derived from normal and chronic granulomatous disease leukocytes. Granules isolated from normal resting cells showed allosteric kinetics with regard to oxidation of either NADH or NADPH, so that no enzyme activity was observed at physiological concentrations of substrate. If the granules were isolated from cells that had previously phagocytized zymosan, normal hyperbolic kinetics were obtained, so that activity could now be observed at low levels of substrate. The activity towards NADPH was always substantially greater than that towards NADH at any given concentration of substrate. This alteration in kinetics with phagocytosis was not observed with the other granule enzymes, acid phosphatase or beta-glucuronidase, and thus appeared to be specific for the reduced pyridine nucleotide oxidase(s). In contrast, granules isolated from cells of patients with chronic granulomatous disease showed allosteric kinetics regardless of whether they were obtained from resting or phagocytizing cells, so that NADPH oxidation was not measurable at physiological concentrations of substrate. This defect in the oxidation of NADPH by granules isolated from phagocytizing chronic granulomatous disease cells was observed over the pH range of 4.0 to 7.0. These data suggest that initiation of the respiratory burst by pahgocytosis normally requires an allosteric transformation in a reduced pyridine nucleotide oxidase, which in turn allows expression of enzymatic activity at physiological concentrations of substrate. The defect in chronic granulomatous disease appears to lie in an inability to achieve this transformation, and the enzyme remains in the inactive, allosteric form.


Cell envelope of Neisseria gonorrhoeae: phospholipase activity and its relationship to autolysis. The relationship between conditions which permit or inhibit cell lysis and those which promote phospholipid hydrolysis in Neisseria gonorrhoeae was investigated. Suspension of exponential-phase gonococci in buffer in the absence of divalent cations resulted in autolysis but not in phosphlipid hydrolysis. The addition of Ca2+ or Mg2+ to the buffer inhibited autolysis and markedly stimulated the hydrolysis of phosphatidylethanolamine. Incubation of cells in buffer at pH 6 inhibited both autolysis and phospholipid hydrolysis.


Immunochemical studies on a Mycoplasma pneumoniae polysaccharide fraction: cross-reactions with type 23 and 32 antipneumococcal rabbit sera. Lipid-free polysaccharide fraction 2 extracted from Mycoplasma pneumoniae strain FH by Prescott et al. (J. Bacteriol. 91:2117-2115, 1966) was examined for its ability to cross-precipitate antibody from type-specific rabbit antipneumococcal sera types 1 to 34 inclusive. Cross-precipitation in type-specific pneumococcal anti-type 23 and anti-type 32 sera was examined in detail and could be attributed to a rhamnose-galactose-rich component of crude M. pneumoniae polysaccharide fraction 2 recovered from immunoprecipitates formed with anti-type 23 serum. Immunochemically isolated mycoplasma polysaccharide was found to contain glucose, galactose, rhamnose, and mannose in 1:14:5:4 molar proportions. Comparison of the ability of 6-O-alpha-L-rhamnosyl-D-glucose and free L-rhamnose to inhibit precepitation by homologous pneumococcal and heterologous mycoplasma polysaccharide antigens indicates a combining site specificity for anti-type 23 and anti-type 32 antibodies directed largely against the alpha-linked L-rhamnosyl determinants and the occurrence of alpha-L-rhamnosyl units in type 32 and M. pneumoniae polysaccharides. Hapten inhibition of the cross-precipitation of pneumococcal type 23 capsular polysaccharide in anti-type 32 serum helps to establish that cross-reactivity can be attributed to interaction of recurrent, alpha-L-rhamnosyl units of type 23 with anit-alpha-L-rhamnoside combining sites of anti-type 32 antibodies.


Activation of alveolar macrophages exposed to lavage-procured immunoglobulin G obtained from normal rabbit lungs. Pulmonary washings from rabbits were freed of cells and added to the monolayers of homologous alveolar macrophages (AM). At 1 h after incubation with the pulmonary washings, many more cells adhered to glass, spread out, and showed enhanced Nitro Blue Tetrazolium reduction. The maximal effect of the pulmonary washings on AM activation was obtained 12 h after incubation. The AM activated by the pulmonary washings showed a higher capacity to inhibit the growth of intracellular BCG, and that capacity was correlated with the intensity of Nitro Blue Tetrazolium reduction by the AM. Gel filtration of the pulmonary washings through Sepharose 4B yielded five fractions. The factor that activated the AM functions was in fraction 4. When the immunoglobulin G in the fraction was removed by an immunoadsorbent column, AM activity was abolished. The effect of the immunoglobulin G was dose dependent, and minimal responses to 10(6) cells per ml were obtained at a protein concentration of 20 mug/ml. Lymphokines had no effect on AM activation with respect to the morphological alterations and Nitro Blue Tetrazolium reduction during the 24-h observation time. In summary, AM from normal rabbits were soon activated markedly by lavage-procured immunoglobulin G, but not by lymphokines.


Immuno-stimulation by a ribosomal vaccine associated with a bacterial cell wall adjuvant in humans. We have studied a new vaccine of ribosomal nature associated with glycoprotein cell walls from Klebsiella pneumoniae which served as an immunoadjuvant. Thus vaccine was administered by the aerosol route to working men free of any important disease, especially of respiratory disease. A total of 104 men working for the Commissariat à l'Energie Atomique, all volunteers, were randomly placed into two groups. During the first period, 51 patients (group I) were vaccinated three times a week during 5 weeks, and the second group was used as control. During the second period, which started on day 225, the control group received the vaccine, and the first group was revaccinated. Results of this experience show a significant difference in the immunity of the two groups. The specific antibodies increased with vaccination as illustrated by chi-square test (Yates correction), which corresponds to an independent probability equal to 0 (P = 0.5 X 10-4).


Comparative investigation of the respiratory and cardiovascular effect of mepindolol, propranolol and pindolol in asthmatic patients. In a controlled double-blind crossover trial of 5 mg mepindolol versus 80 mg propranolol, 15 mg pindolol or placebo given as single oral doses in 16 asthmatic patients, respiratory function and cardiovascular parameters were measured simultaneously. The doses chosen of the 3 active drugs were almost equipotent as shown by the similarity in the percentage fall in pulse rates at 2 hr after ingestion, although it was observed that propranolol had a different time course of effect from the 2 other beta blockers. The effect on respiratory function was marked for propranolol, with statistically significant falls in FEV1 and FEV3 at all times; mild for pindolol with lesser falls which were statistically significant only at nearly 3 hr post-ingestion; and intermediate for mepindolol. Mepindolol gave rise to a rapid and statistically significant fall in lying diastolic blood pressure compared to control values; the reductions in diastolic B. P. for propranolol or pindolol were not statistically significant compared to pretreatment control values.


Assessment of beta-blocking activity of trimepranol in man. The beta-blocking potency and the duration of action of trimepranol were measured in healthy volunteers using isoprenaline antagonism and reduction in exercise tachycardia. Based on isoprenaline antagonism, trimepranol was four times as potent as propranolol on a weight basis. The degree of beta blockade increased linearly with dose from 5 mg to 20 mg, excluding a dose-dependent first-pass metabolism in this dose range. There was a significnat correlation between plasma concentration and the effect of 14C-trimepranol on isoprenaline and exercise tests. The elimination half-life of trimepranol, calculated both on the basis of its effects and plasma concentrations, was approximately three to four hours. The beta blockade due to 10 or 20 mg of trimepranol was extended at least up to 12 hours following p.o. administration, based both on isoprenaline and exercise tests and on the effect of resting heart rate. Twice-a-day administration thus seems sufficient to provide a continuous beta blockade in the clinical use of trimepranol.


Diazepam and halazepam in anxiety: some prognostic indicators. A multiple step-search regression procedure was applied to data obtained with 37 diazepam and 42 halazepam treated anxious outpatients. Good treatment outcome was predicted for those patients who reported a more adequate family adjustment, the presence of precipitating stress, and who either had no prior psychotropic drug treatment, or if they had received such treatment, had experienced a good response. Probably of greatest interest to the practicing clinician was the observation that patients high in initial anxiety but low in initial interpersonal problems improved the most with both medications. Differential drug effects indicated halazepam to do particularly poorly in less anxious patients and in those patients given a good prognosis by the doctor. Diazepam response was much less affected by these variables. It is speculated that the excessive sedating effect of the daily halazepam dosage (160 mg/d) used in this study may explain these differential drug effects. In the dosages employed, namely, diazepam 20 mg/d and halazepam 160 mg/d, diazepam produced the more consistent anti-anxiety effects. The indication that halazepam 160 mg/d was more effective than diazepam 20 mg/d in the initially sicker patients, while of interest, is probably simply a dose-related phenomenon, indicating that diazepam 20 mg/d was too low a daily dosage for severely anxious patients, a fact well known by most clinicians.


Drug interactions in psychiatry.

Drug interactions in psychiatry. Since maintenance therapy with neuroleptics, lithium and tricyclic antidepressants may have to continue over extended periods of time, drug interactions between these psychotherapeutic agents and non-psychotropic treatments have become of practical clinical significance. In this paper some of the more common or important drug interactions between psychotherapeutic and non-psychotherapeutic agents are discussed.


Localization of beta receptors in the anterior segment of the rat eye by a fluorescent analogue of propranolol. A fluorescent analogue of propranolol, 9-AAP, was injected intravenously in order to detect beta-adrenergic receptors in the anterior segment of the albino rat eye. Specific 9-AAP fluorescence was noted along cell membranes of the ciliary epithelium and to lesser extent in the walls of blood vessels in the ciliary processes and episclera at the limbus. The iris showed maximum 9-AAP binding in the region of the sphincter muscle. These data suggest that 9-AAP may label beta receptors in the anterior segment of the rat eye.


Light-evoked release of endogenous glycine into the perfused vitreous of the intact rat eye. Glycine, a putative neurotransmitter, is released into the perfused vitreous of anesthetized pigmented rats when the eye is stimulated by intermittent flashes of bright light. Other amino acids do not show stimulated release. This result provides further evidence for the role of glycine as a neural transmitter in the retina.


Proximal renal tubular acidosis in metachromatic leukodystrophy. A 2-year-old girl affected with the late infantile form of metachromatic leukodystrophy had a persistent and moderate metabolic acidosis. Renal functional studies demonstrated the presence of decreased tubular reabsorption of sodium, bicarbonate and some amino acids. Other tubular functions, including distal urinary acidification and concentrating mechanism were normal. Glomerular filtration rate was moderately decreased. Metachromatic inclusions were demonstrated along the nephron by histochemistry and electron microscopy. Tubular dysfunction in metachromatic leukodystrophy could have been overlooked until now given the severity of the neurological picture.


In vitro evaluations of monopolar intravascular oxygen sensors. Intravascular PO2 electrodes of a commercially available design were tested in vitro to establish their characteristics and to further elucidate on their clinical usability. In addition, four different cathode-anode combinations, utilizing the same geometrical design, were evaluated to establich the best combination. Au-Ag/AgCl exhibited the best characteristics as verified by the evaluation of stabilization time, sensitivity and reproducibility of the response, response time, linearity, drift, effects of temperature, flow, and pH, and the useful life of the sensors. Even though the Au-Ag/AgCl combination exhibited the best characteristics, it fell far below clinically recommended criteria for continuous monitoring of less than 10 Torr/24 h.


Regulation of intracellular pH in lungs and other tissues during hypercapnia. Using a 14C-labeled DMO, 36Cl and 3H method, we have determined the in vivo buffering capacity of lung, kidney, heart, skeletal muscle, and extracellular fluid (ECF) of guinea pigs during hypercapnia (FICO2 = 0.15). After 1 days' exposureto 15% CO2, both the relative CO2 buffer values (delta HCO3/deltapH) and the "%pH regulation" were lung greater than kidney greater than heart greater than ECF greater than skeletal muscle. For lung tissue the intracellular pH was significantly decreased only during acute (8 h) hypercapnia and had completely returned to control values after 7 days with arterial PCO2 congruent to 122 Torr. Kidney and cardiac muscle also showed ca. 100% regulation of pH at 7 days, whereas skeletal muscle and ECF showed only 80 and 70% pH regulation, respectively. The results are discussed with respect to the important (and pH-dependent) metabolic functions of the lung and kidney.


Red cell oxygen affinity in fetal sheep: role of 2,3-DPG and adult hemoglobin. Studies were carried out during fetal life in sheep to determine the relationship of 2,3-diphosphoglycerate (DPG), the intracellular red cell and extracellular pH, and the switchover to adult hemoglobin synthesis in regulating the position of the fetal red cell oxygen-affinity curve in utero. Adult hemoglobin first appeared near 120 days of gestation. The mean oxygen tension at which hemoglobin is half saturated (P50) prior to 120 days of gestation remained constant at 13.9 +/- 0.3 (SD) Torr and then increased gradually as gestation continued, reaching 19 Torr at term. During the interval of fetal life studied, the level of DPG was 4.43 +/- 1.63 (SD) micromol/g Hb and the deltapH between plasma and red blood cells was 0.227 +/- 0.038 (SD); neither was affected by gestational age. The decrease in the red cell oxygen affinity after 120 days of gestation ocrrelated with the amount of adult hemoglobin present in the fetus (r = 0.78; P less than 0.001). This decrease can be attributed only to the amount of the adult-type hemoglobin present, and not to DPG, or to changes in the deltapH between plasma and red blood cells, because both remained stable during the last trimester.


Management an experimental approach.

Management an experimental approach. An experimental teaching procedure was devised and tested for integrating the nursing process, nursing care plans, the research process and management principals. The integration defined as the problem oriented process (POP) was tested with 16 students over two semesters. The results indicated that the POP was a successful integration that aided the student in "getting it all together." As one of the students put it: This experience will give future students, as it did me, a better understanding of how research is best utilized. The students can use almost all their past learning experiences, bringing it all together in one situation. He (the student) can use his communication skills to detect a problem, his research skills to design  planned intervention and evaluation and his interpersonal skills to measure his effectiveness as a change agent. Finally, he can use his nursing skills and knowledge of pathophysiology in a setting most like what he will find upon graduation.


On "problems" with integrated curricula in nursing. Curriculum development is many things; it is the study of impact on students, a process through which faculty are moving. It usually does not represent a final decision- like all issues in higher education it is altered as different people and issues arise. The integrated curriculum movement in nursing, while a seminal change, has in a way been like a shadow- incompletely described with lack of clarity about base for comparison. It seems to have become the mode for many schools to develop their own conceptual frameworks and curricular plans. While adaptation to the local setting is expected, one must question how much of the total development effort is necessary and due in part to lack of fully developed curriculum models and dissemination and not building systematically toward improvement in the field. In such a situation we all may be making the same mistakes. If these identified "problems" are indeed just that, they all are amenable to correction. Each solution will require hard conceptual work.


Seduction and the hospitalized person.

Seduction and the hospitalized person. Seductive behavior seen in the convalescent individual is generally a signal that the patient is in the process of assessment. It is not a social invitation to sexual activity. The patient is merely trying to reassess his situation after having experienced an illness, injury or surgery. Nurses have an obligation to be informed regarding the effects of illness on sexuality, and an obligation to recognize the patient's seductive signals. Nurses need to recognize the mechanics of seductive behavior and be able to qualify the behavior in order to help the patient express his/her concerns during convalescence. Finally, nurses have an obligation to discover or create referral sources in their local area where patients may seek help with their altered sexual function, should that be warranted.


1-N HAPA gentamicin B, a new aminoglycoside active against gentamicin resistant isolates--activity compared to other aminoglycosides. 1-N HAPA gentamicin B is a new aminoglycoside active against most Enterobacteriaceae, Pseudomonas aeruginosa and Staphylococcus aureus. Among 504 clinical isolates at a concentration of 12.5 microgram/ml all Staph. aureus, Escherichia coli, Klebsiella, Enterobacter, Proteus rettgeri, Providencia and 78% of Pseudomonas, 86% of Proteus morganii were inhibited. Like other aminoglycosides, the activity was greatest at an alkaline ph and reduced by high cations concentrations. 1-N HAPA gentamicin B was equal in activity to amikacin against both gentamicin-sensitive and resistant isolates. It inhibited bacteria containing many of the aminoglycoside inactivating enzymes. When combined with carbenicillin it inhibited in a synergistic manner many Gram-negative bacteria, particularly Pseudomonas and Serratia.


Macrolide antibiotics M-4365 produced by Micromonospora. III. In vitro antimicrobial activity of antibiotic M-4365G2 (de-epoxy rosamicin). Antibiotic M-4365G2 (de-epoxy rosamicin) produced by Micromonospora capillata MCRL 0940 is a new basic 16-membered macrolide antibiotic with activity equal to or superior to erythromycin and josamycin against Gram-positive bacteria. Of interest are the high degree of activity against Gram-negative bacilli and mycoplasmas, and striking inhibitory effects against indole-producing Proteus spp. Bactericidal activity of M-4365G2 is also to be noticed.


Control of ammonium assimilation in Rhizobium 32H1. The symbiotic, nitrogen-fixing bacterium Rhizobium sp. 32H1 is a specialized ammonium producer during symbiosis. However, during free-living growth, Rhizobium 32H1 assimilates ammonium very poorly. Two pathways of ammonium assimilation exist in enteric bacteria. One is mediated by glutamate dehydrogenase, and the other is mediated by glutamine synthetase-glutamate synthase. The former pathway is altogether inoperative in Rhizobium 32H1; the latter pathway operates at a slow rate and is under strict negative control by ammonium itself. Rhizobium 32H1 glutamine synthetase activity is modulated by both repression-derepression and reversible adenylylation. For a biochemical process lacking an alternative pathway, such a regulatory pattern exacerbates the very process. This suggests that Rhizobium 32H1 restricts its own ammonium assimilation to maximize the contribution of fixed nitrogen to the host plant during symbiosis.


Purification and properties of a second enzyme catalyzing the splitting of carbon-mercury linkages from mercury-resistant Pseudomonas K-62. An enzyme (splitting enzyme 2) which catalyzes the splitting of carbon-mercury linkage of arylmercury compounds was found in extracts of mercury-resistant Pseudomonas K-62. This enzyme was purified about 725-fold by treatment with streptomycin, precipitation with ammonium sulfate, and successive chromatography on Sephadex G-75 and diethylaminoethyl-cellulose. A purified preparation of the enzyme showed a single band in electrophoresis either on polyacrylamide or sodium dodecyl sulfate-containing polyacrylamide gels. The molecular weight of the enzyme was estimated to be 20,000 (determined by Sephadex G-75 gel filtration) 17,000 (determined by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis). The enzyme showed a Km of 180 micron and a Vmax of 3.1 mumol/min per mg for p-chloromercuribenzoic acid and a Km of 250 micron and a Vmax of 20 mumol/min per mg for phenylmercuric acetate. The optimum temperature and pH for the reaction were 40 degrees C and 5.0, respectively.


Alcohol dehydrogenases from a facultative methylotrophic bacterium. Alcohol-oxidizing enzymes of the facultative methylotroph PAR were investigated after growth of the bacteria on methanol and ethanol. During methanol growth only a phenazine methosulfate-linked alcohol dehydrogenase was detected. This enzyme had broad specificity for primary alcohols and was also capable of oxidation of secondary alcohols. It had a molecular weight of 112,000, was composed of two subunits of equal molecular weight, and showed an absolute requirement for ammonium ion for activation. During ethanol growth this enzyme was absent and was replaced by a typical nicotinamide adenine dinucleotide-linked alcohol dehydrogenase of molecular weight 150,000. The latter enzyme also had broad specificity but could not oxidize methanol. This enzyme was not found during methanol growth. These data show that the organism has two distinctly separate mechanisms for oxidation of alcohols.


Glycolipids stimulate DNA polymerase activity in a DNA-membrane fraction and in a partially purified polymerase system extracted from pneumococci. We have assayed the ability of various lipids to affect DNA polymerases activity in a DNA-membrane complex extracted from Streptococcus pneumoniae by the Sarkosyl-M-band technique. In addition, to determine which DNA polymerases were affected by the lipids, we partially purified three DNA polymerase activities from cell lysates, the first such demonstration outside of Escherichia coli and Bacillus subtilis. Glycolipids are unique among polar lipids in stimulating the rate and extent of DNA polymerase activity in M-bands and in Sarkosyl lysates from which the M-band is derived. It appears that they exert this stimulatory effect, in part, by removing (neutralizing) detergent molecules which act as inhibitors, as well as by substituting for the detergent, thereby creating a favorable environment for the polymerases involved in DNA synthesis. That the stimulatory effect is not simply a detoxification of the detergent was shown by two observations. One, phospholipids, although interacting with Sarkosyl and therefore "potentially" capable of detoxifying the system, did not stimulate DNA polymerase activity in vitro. Two, glycolipids were capable of stimulating the activity of at least two DNA polymerases partially purified from cell lysates in the absence of any Sarkosyl. The stimulatory effect was greater for a polymerase that had four characteristics similar to those observed with polymerase III in other organisms.


EPR investigation of the Mn(II) binding sites in glutamine synthetase (Escherichia coli W). II. Intermediate-affinity binding sites. The nature of the intermediate-affinity (n2) Mn(II) binding sites in glutamine synthetase [EC 6.3.1.2] has been studied as a function of adenylylation in a variety of enzyme-metal complexes by EPR. In the absence of nucleotide the n2 Mn(II) environment is nearly isotropic, the Mn(II) bonds are highly ionic, and the interaction distance R greater than or equal to 12-14 A. Nucleotide binding at the n2 Mn(II) site renders the n2 Mn(II) signal unobservable and causes a reduction in signal amplitude (approximately 30%) and line broadening (approximately 6 G) at the high-affinity (n1) Mn(II) site. This behavior indicates that nucleotide binding induces a conformational change in the enzyme which brings the previously distant n1 and n2 sites into closer proximity (R less than or equal to 8-11 A), possibly for the purpose of activating the nucleotide for direct phosphoryl transfer to L-glutamate. In line with this suggestion, the broad, unresolved resonances in complexes containing both L-methionine SR-sulfoximine (MSOX) and nucleotide may result from the phosphorylation of MSOX. The n2 Mn(II) site is not affected by adenylylation in all the enzyme-metal complexes studied, which suggests that the regulatory effects of adenylylation may only act at the n1 Mn(II) sites.


Use of fluorescamine-labeled casein as a substrate for assay of proteinases. Conditions have been investigated for the use of fluorescamine-labeled casein as a substrate for fluorometric assay of proteinases. Fluorescamine-labeled casein can be prepared simply by mixing solutions of casein and fluorescamine at pH 8.0 and used without removal of the excess reagent or its hydrolysis product. The fluorescence of the labeled casein and its enzymatic digest is moderately stable in the range of pH 7.0 to 10.0. Activities can be determined by measuring the fluorescence of the hydrolysis products soluble in 0.1 M trichloro acetic acid solution at pH 4.0 after adjusting the pH of the acid-soluble fraction to 7.7. This method is suited for assay of proteinases active at neutral to slightly alkaline pH values, and is capable of quantitating about 0.05 microgram of trypsin or 0.5 microgram of alpha-chymotrypsin or papain. The assay can be done in the presence of large amounts of contaminating amino acid, protein and/or exopeptidases which may interfere with the ordinary assay of proteinases.


Heat activation of rat liver acetyl-CoA carboxylase in vitro. Acetyl-CoA carboxylase in rat liver homogenates was activated in vitro in a time- and temperature-dependent manner. The activity of acetyl-CoA carboxylase in rat liver preparations was determined in a 1-min assay to preclude the possibility of citrate activation of the enzyme during the assay period. Activation of the enzyme occurred more rapidly in liver preparations continuously maintained at ambient or greater temperatures than in homogenates of liver which had been chilled. High speed supernatant (105,000 X g, 60 min) did not heat-activate, and reconstitution of the heat-activatable 27,000 X g, 20-min, fraction by recombining the high speed pellet with the high speed supernatant only partially restored the heat activatability. Elution of the 105,000 X g supernatant from Sephadex G-25 resulted in an enzyme preparation which was heat-activatable. Addition of boiled 105,000 X g supernatant to the Sephadex G-25-treated enzyme again prevented heat activation. Dilution of the enzyme 5-fold did not prevent heat activation.


Transglutaminase-catalyzed cross-linking through diamines and polyamines. Transglutaminases were found to catalyze the formation of cross-links between peptide chains by means of a transfer reaction between the carboxamide group of a glutamine residue in each chain and both primary amino groups of a diamine or a polyamine. Production of this heretofore undescribed linkage by guinea pig liver transglutaminase was demonstrated by the use of high performance liquid chromatography in a model system using glutamine peptide derivatives and a variety of diamines and polyamines. Evidence for intermolecular cross-linking through polyamines with both the liver enzyme and thrombin-activated human plasma blood coagulation factor XIII was obtained by the use of a guanidinated derivative of beta-casein.


Direct inhibition of tyrosine hydroxylase activity by catechol estrogens. Catechol estrogens, the 2-hydroxylated metabolites of estrogens, recently shown to be formed in brain, inhibit tyrosine hydroxylase, the enzyme that catalyzes the pivotal step in the biosynthesis of the neurotransmitters dopamine and norepinephrine. The nature of the inhibition is by competition with the pterin cofactor and thus resembles feedback inhibition of the enzyme by catecholamines.


Determination of glucose oxidase oxidation-reduction potentials and the oxygen reactivity of fully reduced and semiquinoid forms. The oxidation-reduction potential values for the two electron transfers to glucose oxidase were obtained at pH 5.3, where the neutral radical is the stable form, and at pH 9.3, where the anion radical is the stable form. The midpoint potentials at 25 degrees were: pH 5.3 EFl1ox + e- H+ equilibrium EFlH. Em1 = -0.063 +/- 0.011 V EFlH. + e- + H+ equilibrium EFlredH2 Em2 = -0.065 +/- 0.007 V pH 9.3 EFlox + e- EFi- Em1 = -0.200 +/- 0.010 V EFi- + e- + H+ equilibrium EFlredH- Em2 = -0.240 +/- 0.005 V All potentials were measured versus the standard hydrogen electrode (SHE). The potentials indicated that glucose oxidase radicals are stabilized by kinetic factors and not by thermodynamic energy barriers. The pK for the glucose oxidase radical was 7.28 from dead time stopped flow measurements and the extinction coefficient of the neutral semiquinone was 4140 M-1 cm-1 at 570 nm. Both radical forms reacted with oxygen in a second order fashion. The rate at 25 degrees for the neutral semiquinone was 1.4 X 10(4) M-1 s-1; that for the anion radical was 3.5 X 10(4) M-1 s-1. The rate of oxidation of the neutral radical changed by a factor of 9 for a temperature difference of 22 degrees. For the anion radical, the oxidation rate changed by a factor of 6 for a 22 degrees change in temperature. We studied the oxygen reactivity of the 2-electron reduced form of the enzyme over a wide wavelength range and failed to detect either oxygenated flavin derivatives or semiquinoid forms as intermediates. The rate of reoxidation of fully reduced glucose oxidase at pH 9.3 was dependent on ionic strength.


Dependence of the catalytic activity of papain on the ionization of two acidic groups. The pH dependence of kcat/Km for the papain-catalyzed hydrolysis of ethyl hippurate, N-alpha-benzoyl-L-citrulline methyl ester, and the p-nitroanilide, amide, and ethyl ester derivatives of N-alpha-benzoyl-L-arginine was determined below pH 6.4. The value of kcat/Km was observed to be modulated by two acid ionizations rather than a single ionization as previously believed. For the five substrates studied, the average pK values for the two ionizations are 3.78 +/- 0.2 and 3.95 +/- 0.1 at T/2 0.3, 25 degrees C. The observation that similar pK values were obtained with different substrates was taken as evidence that the kinetically determined pK values are close in value to true macroscopic ionization constants for ionization of groups on the free enzyme.