The effects of alpha- and beta-adrenergic receptor blockers on the pressure responses to isometric exercise in hypertensive patients. 1. The cardiovascular responses to handgrip exercise have been studied in ten patients with uncomplicated essential hypertension in a randomized crossover study of propranolol and prazosin. 2. Isometric handgrip exercise was performed with a calibrated strain gauge dynamometer at 30% of maximum voluntary contraction for 3 min. 3. Blood pressure and heart rate were measured in the supine position at rest and in the last 10 s of the exercise period. 4. These exercise studies were undertaken at the end of a run-in period and at the end of 1 month's optimal therapy with the two drugs. 5. The active treatment periods were separated by a 2 weeks placebo washout period. 6. Both drugs lowered the supine and standing systolic and diastolic pressures and there was no difference between these drugs in their effect on these variables. 7. Propranolol lowered the resting heart rate and neither drug suppressed the pressor response to isometric exercise. 8. The degree of pressure rise was similar with both drugs but propranolol suppressed isometric exercise-induced tachycardia.


Influence of biogenic amines on the growth of xenografted human colorectal carcinomas. The influence of some biogenic amines and amine-receptor-blocking drugs in the growth rate of human colorectal carcinomas propagated as s.c. xenografts in immune-deprived mice was studied. In mice treated with adrenaline, a beta-adrenergic agonist, the growth of xenografts was suppressed for 2 days, after which growth was resumed at a rate similar to that in control animals. Treatment with the phosphodiesterase inhibitor theophylline prolonged the adrenaline-induced inhibition of growth. Treatment with the beta-adrenergic antagonist sotalol or practolol increased the rate of tumour growth. Treatment with either serotonin or the histamine H2-receptor agonist Dimiprit had no effect on tumour growth rate. However, the antiserotoninergic drug BW 501C and the histamine H2-receptor antagonist cimetidine each caused short-term suppression of tumour growth.


Sequential analysis of hepatic carcinogenesis: the comparative architecture of preneoplastic, malignant, prenatal, postnatal and regenerating liver. The organizational pattern of hepatocytes in hyperplastic nodules, probable precursors of hepatocellular carcinoma, was examined sequentially at different stages in the carcinogenic process, and compared with the patterns in hepatocellular carcinomas, in developing liver and in regnerating liver. Scanning as well as transmission electron microscopy, and histochemistry with light microscopy were used. The hepatocytes in the hyperplastic lesions were arranged in plates 2 or more cells thick and glands, in contrast to the one-cell-thick plates of hepatocytes in normal mature liver, and showed unusualy separation from eachother, with irregularly dilated bile canaliculi. The organizational pattern found in the hyperplastic lesions shared properties with developing liver in the perinatal period, regenerating liver following the peak of cell division, and some hepatocellular carcinomas. Unlike the normal, in which there is a highly predictable time scale for change, an apparent delay or interruption of maturation may be of importance in lesions that persist and ultimately evolve into hepatocullular carcinoma.


Favism: erythrocyte metabolism during haemolysis and reticulocytosis. The reduced activity of glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate; NADP+ 1-oxidoreductase; G6PF) in Mediterranean erythrocytes explains the precarious equilibrium of the hexose monophosphate pathway (HMP) and the susceptibility of these cells to haemolytic agents. G6PD-deficient erythrocytes, in steady-state conditions, have a low NADPH/NADP+ ratio, thus allowing the HMP to operate at its maximal intracellular rate and to compensate the intrinsic erythrocyte enzyme deficiency. Studies started soon after accidental intake of fava beans by sensitive G6PD-deficient subjects demonstrate a decrease of both NADPH/NADP+ ratio and reduced glutathione. The metabolic effects induced by fava beans may be attributed to oxidative stress probably associated with an inhibitor effect of some unknown metabolite on the HMP. The availability of erythrocytes from subjects recovering from haemolysis with high reticulocyte counts and increased G6PD activity, provides new information on the rate of synthesis as well as on the in vivo decay of the mutant enzyme. Correlation of G6PD activity to reticulocyte count and extrapolation to an ideally homogenous population of reticulocytes reveal that the mutant enzyme is synthesized at a nearly normal rate. Furthermore, the present correlation allows an estimate of the in vivo half-life of Mediterranean G6PD. The rate of decline of about 8 d observed in this study well correlates to the intracellular metabolic aspects of G6PD Mediterranean erythrocytes.


Solubilization and reconstitution of the catecholamine transporter from bovine chromaffin granules. The catecholamine transporter from bovine chromaffin granules has been solubilized by using low concentrations of sodium cholate in the presence of phospholipids. The functional solubilized protein has been incorporated into liposomes after removal of the detergent either by gel filtration or by dialysis. Reserpine-sensitive accumulation against a concentration gradient is achieved by artifically imposing a pH gradient across the membrane. In the reconstituted system adenosine 5'-triphosphate (ATP) serves as an energy source only at higher detergent concentrations. The proton-translocating adenosine triphosphatase (ATPase) is solubilized in parallel with the increasing efficiency of ATP as an energy source. Several criteria are proposed to distinguish between carrier-mediated (reserpine sensitive) and unmediated transport in the reconstituted system. The reserpine-sensitive process shows affinity and ss presented in this communication provide further support for the contention that concentrative uptake in biogenic amine storage vesicles is driven by a transmembrane pH gradient, which, in the native system, is generated by a proton-translocating ATPase. Moreover, the assays described provide a tool for the isolation and purification of the transport protein.


Development of a method for the incorporation of substitution-inert metal ions into proteins. Site-specific modification of arsanilazotyrosine-248 carboxypeptidase A with cobalt(III). This investigation demonstrates the use of substitution-inert metal ions as site-specific amino acid modifying reagents. The approach involves the production of a chelating agent at the site of interest with the subsequent in situ oxidation of substitution-labile cobalt(II) to exchange-inert cobalt(III) with H2O2. We have produced the chelate complex ethylenediamine-N,N'-diacetato(arsanilazotyrosinato-248 carboxypeptidase A)cobalt(III) [CoIII(EDDA)(AA-CPA-Zn)]. Model CoIII(EDDA)(azophenolate) complexes have helped to define the reaction conditions necessary to produce the enzyme derivative and have proved invaluable in the spectral analysis of the cobalt(III)-enzyme complex. The modified enzyme contains one active-site zinc and one externally bound cobalt per enzyme monometer. Circular dichroism and visible spectra of the derivative and apoenzyme substantiate the site-specific nature of the incorporation. Concimitant with CoIIIEDDA incorporation, the enzyme loses its peptidase activity yet maintains with FeIIEDTA returns the original properties of the arsanilazotyrosine-248 enzyme.


The respiratory chain of Paramecium tetraurelia in wild type and the mutant Cl1. II. Cyanide-insensitive respiration. Function and regulation. 1. The cyanide-insensitive respiration in Paramecium tetraurelia was found to be located in mitochondria. 2. Sensitivity of the mitochondrial respiration to cyanide depended on growth conditions. Under standard conditions of growth, 15--20% of respiration was insensitive to 1 mM cyanide. Full resistance to 1 mM cyanide was observed by growing cells in the presence of erythromycin (100--400 microgram/ml) 0.2 mM cyanide. The mitochondrial respiration of the mutant Cl1 harvested during the exponential phase of growth was largely insensitive to cyanide (more than 80%). 3. Pyruvate was oxidized at the same rate by wild type mitochondria and mitochondria of the mutant Cl1. In contrast, succinate oxidation was 2--3 times faster in mitochondria of the mutant Cl1 than in wild type mitochondria. 4. The cyanide-insensitive respiration was inhibited by 1 mM salicylhydroxamic acid to nearly 100%. Other efficient respiratory inhibitors included amytal and heptylhydroxyquinoline. Antimycin was not inhibitory even at concentrations as high as 5 microgram/mg protein, a finding consistent with the lack of antimycin binding sites.


Electron acceptors of bacterial photosynthetic reaction centers. II. H+ binding coupled to secondary electron transfer in the quinone acceptor complex. The photoreduction of ubiquinone in the electron acceptor complex (QIQII) of photosynthetic reaction centers from Rhodopseudomonas sphaeroides, R26, was studied in a series of short, saturating flashes. The specific involvement of H+ in the reduction was revealed by the pH dependence of the electron transfer events and by net H+ binding during the formation of ubiquinol, which requires two turnovers of the photochemical act. On the first flash QII receives an electron via QI to form a stable ubisemiquinone anion (QII-); the second flash generates QI-. At low pH the two semiquinones rapidly disproportionate with the uptake of 2 H+, to produce QIIH2. This yields out-of-phase binary oscillations for the formation of anionic semiquinone and for H+ uptake. Above pH 6 there is a progressive increase in H+ binding on the first flash and an equivalent decrease in binding on the second flash until, at about pH 9.5, the extent of H+ binding is the same on all flashes. The semiquinone oscillations, however, are undiminished up to pH 9. It is suggested that a non-chromophoric, acid-base group undergoes a pK shift in response to the appearance of the anionic semiquinone and that this group is the site of protonation on the first flash. The acid-base group, which may be in the reaction center protein, appears to be subsequently involved in the protonation events leading to fully reduced ubiquinol. The other proton in the two electron reduction of ubiquinone is always taken up on the second flash and is bound directly to  QII-. At pH values above 8.0, it is rate limiting for the disproportionation and the kinetics, which are diffusion controlled, are properly responsive to the prevailing pH. Below pH 8, however, a further step in the reaction mechanism was shown to be rate limiting for both H+ binding electron transfer following the second flash.


Blue and red shifts of bacteriochlorophyll absorption band around 880 nm in Rhodospirillum rubrum. The redox potential dependence of the light-induced absorption changes of bacteriochlorophyll in chromatophores and subchromatophore pigment-protein complexes from Rhodospirillum rubrum has been examined. The highest values of the absorption changes due to the bleaching of P-870 and the blue shift of P-800 in chromatophores and subchromatophore complexes are observed in the 360-410mV redox potential range. At potentials below 300 mV (pH 7.0), the 880 nm band of bacteriochlorophyll shifts to shorter wavelengths in subchromatophore complexes and to longer wavelengths in chromatophores. The data on redox titration show that the red and blue shifts of 880-nm bacteriochlorophyll band represent the action of a non-identified component (C340) which has an oxidation-reduction midpoint potential close to 340 mV (n=1) at pH 6.0--7.6. The Em of this component varies by 60 mV/pH unit between pH 7.6 and 9.2. The results suggest that the red shift is due to the transmembrane, and the blue shift to the local intramembrane electrical field. The generation of both the transmembrane and local electrical fields is apparently governed by redox transitions of the component C340.


Surface potential and reaction of membrane-bound electron transfer components. I. Reaction of P-700 in sonicated chloroplasts with redox reagents. Salt- or pH-induced change of the rate of reduction of the photoxidized membrane bound electron transfer components, P-700, by ionic and nonionic reductants added in the outer medium was studied in sonicated chloroplasts. The rate with the negatively charged reductants increased with the increase of salt concentration at a neutral pH or with the decrease of medium pH. Salts of divalent cations were much more effective than those of monovalent cations. A trivalent cation was even more effective. The rate with a nonionic reductant was little affected by salts. The change of the reduction rate was analysed using the Guoy-Chapman theory, which explains the change of reduction rate by the changes of activities of ionic reductants at the charged membrane surface where the reaction takes place. This analysis gave more useful parameters and explained more satisfactorily the case with high-valence cation salts than the Brönsted type analysis. The values for the surface charge density and the surface potential of the membrane surface in the vicinity of P-700 estimated from the analysis were lower than those estimated for the surface in the vicinity of Photosystem II primary acceptor, suggesting the heterogeneity of the thylakoid surface. The salt-induced surface potential change was shown to affect the activation energy of the reaction between P-700 and the ionic reagent.


The effect of pH on the oxygen kinetics of cytochrome c oxidase proteoliposomes. The effect of pH on the oxygen kinetics of cytochrome c oxidase incorporated into phospholipid vesicles is studied. The pH profiles of the oxygen kinetics of energized and deenergized oxidase vesicles are similar. An effect of pH on the slope of the reciprocal plot of rate against oxygen concentration is observed, and this may indicate that protons are involved in the rate limiting step of the reaction between oxygen and reduced oxidase. In contrast to the pH dependence of the oxygen kinetics, the binding of CO to the oxidase is not pH dependent.


Studies on purine transport and on purine content in vacuoles isolated from Saccharomyces cerevisiae. The transport of purine derivatives into vacuoles isolated from Saccharomyces cerevisiae was studied. Vacuoles which conserved their ability to take up purine compounds were prepared by a modification of the method of polybase-induced lysis of spheroplasts. Guanosine greater than inosine = hypoxanthine greater than adenosine were taken up with decreasing initial velocities, respectively; adenine was not transported. Guanosine and adenosine transporting systems were saturable, with apparent Km values 0.63 mM and 0.15 mM respectively, while uptake rates of inosine and of hypoxanthine were linear functions of their concentrations. Adenosine transport in vacuoles appeared strongly dependent on the growth phase of the cell culture. The system transporting adenosine was further characterized by its pH dependency optimum of 7.1 and its sensitivity to inhibition by S-adenosyl-L-methionine. In the absence of adenosine in the external medium, [14C]adenosine did not flow out from preloaded vacuoles. However, in the presence of external adenosine, a very rapid efflux of radioactivity was observed, indicating an exchange mechanism for the observed adenosine transport in the vacuoles. In isolated vacuoles the only purine derivative accumulated was found to be S-adenosyl-L-homocysteine.


A ribonuclease from yeast associated with the 40 S ribosomal subunit. 1. Autodegradation of yeast ribosomes is due to a 'latent' ribonuclease which is associated with the 40 S ribosomal subunit. 2. The ribonuclease was extracted in the presence of EDTA from ribosomes and purified 118-rold by protamine sulphate precipitation, (NH4)2SO4 fractionation and chromatography on DEAE-cellulose. 3. The optimum pH for this enzyme is 5 to 6.5 while the optimum temperature is 45 to 50 degrees C. Incubation for 10 min at 60 degrees C caused a reduction in enzyme activity of 70%. 4. The ribonuclease has an endonucleolytic activity against rRNA, tRNA, poly(A), poly(U) and poly(C) but does not degrade poly(G) or DNA. It hydrolyzes the homopolymers to nucleoside 3'-phosphates. 5. Zn2+, Mn2+, heparin, glutathione and p-chloromercuribenzoate inhibit the ribonuclease, while Na+, K+, EDTA and sermidine have only little or no effect. 6. It binds tightly to yeast ribosomes but only loosely to ribonuclease-free wheat germ ribosomes. 7. Polyribosomes possess less autodegradation activity than monoribosomes, isolated from the same homogenate.


Properties of cyclic AMP-dependent protein kinase in normal and goitrous rat thyroid gland. Most of the cyclic AMP-dependent protein kinase activity in propylthiouracil-induced goiters and control rat thyroid glands was found in the soluble fraction. The activity in the particulate fractions was cyclic AMP-independent. Protein kinase activity was 2--3-fold higher in all the subcellular fractions of goitrous tissue than of control tissue. In the presence of Triton X-100, both groups showed a significant increase in kinase activity in all subcellular fractions, and the kinase activity in the particulate fractions could now be slightly stimulated by cyclic AMP. Again, enzyme activity in fractions from goiters was significantly higher than in control tissue. Two major peaks, Types I and II, of soluble cyclic AMP-dependent protein kinase activity could be separated by DEAE-cellulose chromatography. Chronic in vivo stimulation by TSH was associated with a selective increase in Type II isoenzyme activity. Elution and pH profiles, dissociation of subunits with 0.5 M NaCl, and activity ratios (-cyclic AMP/+cyclic AMP) for various substrates for Type II isoenzyme in goitrous and control tissue were similar. The elevated activity in goitrous tissue was manifested by an increase in V for histone, ATP, Mg2+ and cyclic AMP, with no change in the apparent Km.


1,3-beta-D-glucanases from Pisum sativum seedlings. I. Isolation and purification. Two buffer-soluble endo-1,3-beta-D-glucanases (EC 3.2.1.6) have been purified to within 1% of electrophoretic homogeneity from etiolated Pisum sativum stem tissues. Purified glucanase I and II differ in physical properties, such as electrophoretic mobility in sodium dodecyl sulfate polyacrylamide gels (Mr values were 22 000 and 37 000, respectively) and isoelectric focusing, (pI values were 5.4 and 6.8, respectively). Although the enzymes have similar pH optima (5.5--6.0), Km values for various substrates (0.6--7.4 mg/ml) and thermal inactivation profiles, they are localized in different tissues and they differ markedly in the rates with which they attack the internal linkages of long- vs. short-chain substrates. Glucanase I is concentrated in apical regions of the stem and is most effectively assayed reductometrically (as laminarinase), while glucanase II is localized in mature regions and is relatively more active in viscometric assays (as carboxymethyl-pachymanase).


Alpha and beta-fibrinogenases from Trimeresurus gramineus snake venom. By means of DEAE-Sephadex A-50 Column chromatography, Trimeresurus gramineus venom was separated into twelve fractions. The fibrinogenolytic activities were distributed in Fractions 1 and 10. These enzymes were further purified by gel filtration and were homogeneous as judged by cellulose acetate membrane, sodium dodecyl sulfate polyacrylamide gel electrophoresis and ultracentrifugal analysis. Both of them were single peptide chains. The sedimentation constants of alpha- (Fraction 1) and beta-fibrinogenases (Fraction 10) were 2.20 and 3.60, respectively. The molecular weights of alpha- and beta-fibrinogenases were 23 500 and 25 000 respectively. The contents of proline and glycine were higher in beta-fibrinogenase than in alpha-fibrinogenase. The isoelectric points of alpha-fibrinogenase and beta-fibrinogenase were pH greater than 10 and 4.5, respectively. The optimal pH of alpha-fibrinogenase was approx. 7.4 and that of beta-fibrinogenase was approx. 9.0. The activity of alpha-fibrinogenase was completely destroyed after 30 min at 60 degrees C, pH 5.4, 7.4 and 9.0, while that of beta-fibrinogenase was much less affected by the same treatment. The specific fibrinogenolytic activity alpha-fibrinogenase was 31 mg fibrinogen/min per mg protein, while that of beta-fibrinogenase was 9 mg fibrinogen/min per mg protein. alpha-Fibrinogenase cleaved specifically the alpha(A) chain of monomeric fibrinogen without cleaving the beta(B) chain and gamma-chain. beta-fibrinogenase preferentially cleaved the beta(B) chain, and the alpha(A) chain was also partially cleaved by beta-fibrinogenase, if the incubation time was prolonged. Both enzymes showed proteolytic activities toward fibrinogen, fibrin and casein, but were devoid of phospholipase A, alkaline phosphomonoesterase and phosphodiesterase activities found in the crude venom. The tosyl-L-arginine methylester esterase activity of beta-fibrinogenase was about 14 times that of crude venom, while alpha-fibrinogenase was completely devoid of this activity. The fibrinogenolytic activity of alpha-fibrinogenase was markedly inhibited by EDTA and cysteine, while that of beta-fibrinogenase was inhibited markedly by phenylmethanesulfonylfluoride. alpha- and beta-fibrinogenases exert their fibrinogenolytic activity by a direct action on fibrinogen or fibrin without activation of plasminogen.


Electric field effects on immobilized urease activity. The behavior of an enzyme/membrane system containing urease is studied when an external electric field is applied. The device using a potential difference across the enzyme/membrane system is first described. Optimal operating conditions with respect to substrate concentration, ionic strength and pH are studied. Possible mechanisms of the change in membrane activity by electric field are discussed.


Purification of an alkaline nuclease from Physarum polycephalum. An alkaline nuclease was purified from microplasmodia of Physarum polycephalum. The nuclease, active on denatured DNA and RNA and free of contamination by other nucleolytic activities, appeared to be a zinc-metallo protein. The enzyme was only active under conditions, where Zn2+ were retained in the enzyme. Loss of zinc occurred by the chelating action of EDTA, EGTA or ampholines, by acid of highly alkaline pH conditions or by high ionic strength. The addition of ZnCl2 to compensate losses, restored all activity, while all other divalent cations caused inhibition. The nuclease, with a molecular weight of 32 000, was stable at neutral pH at high temperatures with a half-life of 20 min at 80 degrees C. It was inhibited by any salt of buffer concentration above the level of zero ionic strength and showed a special sensitivity towards phosphate ions. The possible similarity of this enzyme to nuclease S1 from Aspergillus oryzae is pointed out.


Immunochemical aspects, molecular and kinetic properties of multiple forms of acetyl-CoA acetyltransferase from rat liver mitochondria. Acetyl-CoA acetyltransferase (EC 2.3.1.9) from rat liver mitochondria, which catalyzes the first step in the biosynthesis of ketone bodies, exists in two forms, designated transferase A and transferase B. Both transferases showed immunochemical cross-reactivity, but are immunologically unrelated to cytosolic acetyl-CoA acetyltransferase activity and the mitochondrial acetyl-CoA acyltransferase from rat liver. The transferases A and B were estimated to have molecular weights of 151 000 in the absence and 40 000 in the presence of sodium dodecyl sulfate. They differ with respect to charge states and multiplicity of forms as indicated by isoelectric focusing. Transferase A appeared in two forms with isoelectric points of 8.4 and 9.1, whereas transferase B represents a stable protein state with an isoelectric point of 9.0. Kinetic analysis of the reactions leading to acetoacetyl-CoA synthesis revealed saturation curves with multiple intermediary plateaus, indicating a complex kinetic behaviour. The data presented are interpreted as representing a microheterogeneity of forms of the mitochondrial acetyl-CoA acetyltransferase. The kinetic properties exhibited suggest a role for this microheterogeneity in the regulation of ketogenesis.


Monoacylglycerol hydrolase in human platelets. In the present paper we show for the first time monoacylglycerol hydrolase in human platelets. No monoacylglycerol hydrolase activity could be demonstrated in the other blood cells. The monoacylglycerol hydrolase of platelets could not be released from the cells by heparin, thus the enzyme is distinct from the postheparin plasma lipases. The enzyme could be solubilized by a non-ionic detergent, Triton X-100. The solubilized monoacylglycerol hydrolase from platelets was optimally active at pH between 7 and 8 and at ionic strength corresponding to [NaCl] between 0.1 and 0.3 M. The optimal assay temperature was 37 degrees C. The enzyme activity was sensitive to HgCl2 but not to NaF. Accordingly, it was stabilized by 2-mercaptoethanol.


Characterization of the forward and reverse reactions catalyzed by CDP-diacylglycerol:inositol transferase in rabbit lung tissue. CDPdiacylglycerol:inositol transferase activity in rabbit lung tissue has been characterized and the optimum conditions for assaying this enzyme in vitro were determined. Rabbit lung tissue CDPdiacylglycerol:inositol transferase activity was found primarily in the microsomal fraction. The pH optimum of the enzyme activity was between 8.8 and 9.4, and the reaction was dependent on either Mn2+ or Mg2+. Detergents and Ca2+ inhibited the activity of the enzyme. The apparent Km values of the enzyme for CDPdioleoylglycerol and myoinositol were 0.18 mM and 0.10 mM, respectively. The reversibility of the reaction catalyzed by CDPdiacylglycerol:inositol transferase in microsomes prepared from rabbit lung tissue was demonstrated by the synthesis of [3H]CMPdiacylglycerol when [3H]CMP and phosphatidylinositol were present in the incubation mixture. The reverse reaction was characterized and its importance in the regulation of the acidic phospholipid composition of surfactant during lung development is discussed. The pH optimum for the reverse reaction was 6.2, and the reverse reaction was also dependent on Mn2+ or Mg2+. The apparent Km value of CDPdiacylglycerol:inositol transferase for CMP was found to be 2.8 mM.


Distinct effects of three bile salts on cholesterol solubilization by oleate-monoolein-bile salt micelles. Micellar cholesterol solubilities in bile salt-monoolein-oleic acid systems have been determined. Whatever the bile salt/oleyl compounds ratio, taurochenodeoxycholate solubilizes more cholesterol than taurocholate and much more than tauroursodeoxycholate. At pH 6.7, the cholesterol solubility limit is about the same with either oleate or monoolein. Cholesterol solubility falls in oleate-bile acid mixtures as the pH is raised. The capacity for supersaturation with cholesterol is greater for bile salt-monoolein than for bile salt-oleate micelles. For the latter it decreases as pH increases.


Skeletal keratan sulfate from different tissues. Characterization and alkaline degradation. Keratan sulfate-rich peptides were isolated after digestion of proteoglycans from bovine nasal cartilage and bovine nucleus pulposus with chondroitinase ABC, trypsin and chymotrypsin. The keratan sulfate enriched peptides from nucleus pulposus were larger than those from nasal cartilage. Keratan sulfate chains were isolated after treatment of the keratan sulfate-rich peptides under alkaline, reductive conditions. Proteoglycans from nucleus pulposus contain longer keratan sulfate chains, as is shown primarily by gel chromatography of the keratan sulfate-rich peptides and the keratan sulfate chains, but also from end-group analyses of the keratan sulfate chains.


Stoichiometry of the reaction of oxyhemoglobin with nitrite. During the reaction of oxyhemoglobin (HbO2) with nitrite, the concentration of residual nitrite, nitrate, oxygen, and methemoglobin (Hb+) was determined successively. The results obtained at various pH values indicate the following stoichiometry for the overall reaction: 4HbO2 + 4NO2- 4H+ leads to 4Hb+ + 4NO3- + O2 + 2H2 O (Hb denotes hemoglobin monomer). NO2- binds with methemoglobin noncooperatively with a binding constant of 340 M-1 at pH 7.4 and 25 degrees C. Thus, the major part of Hb+ produced is aquomethemoglobin, not methemoglobin nitrite, when less than 2 equivalents of nitrite is used for the oxidation.


Co-binding studies on Hb M Iwate. Allostery of a T state haemoglobin. The mutant haemoglobin Hb M Iwate alpha 2Mmet87His leads to Tyr beta 2, is characterized by a stable T structure and a low ligand affinity. Sigmoidal CO-binding isotherms of symmetrical shape with Hill coefficients of n = 1.4 at pH 6 to n = 1.9 at pH 10 and the differences in the mean affinity (PCO(1/2)) and the affinity of the first ligand-binding beta subunit (1/L1 greater than Pco(1/2)) are the evidence for the cooperativity. The comparison of the Bohr effects of the two valency hybrid states (alpha 2Mmet beta met beta deoxy alpha 2Mmet beta 2deoxy) in the absence of and in the presence of polyphosphates leads to an indirect proof of pH-dependent subunit-subunit interaction. Inositol hexaphosphate-binding suppresses cooperativity in the pH range 5.5-8 (n = 1). Above pH 8 hte cooperativity increases to a final value of n = 1.9 at pH greater than 10, which is identical to that of stripped Hb M Iwate. The CO binding to the first binding site exhibits a Bohr effect. Polyphosphate anions have no influence on the CO binding of the first binding site. The heterotropic effects are discussed as intrachain effects (Bohr effect of the first binding site) and interchain effects (Bohr effect of Pco(1/2); influence of polyphosphates).


Carp hemoglobin. V. Effects of pH and P6-inositol on autooxidation. Autooxidation of carp hemoglobin has been measured from 4--25 degrees C with and without P6-inositol. The rate was accelerated by the increase of proton concentration and/or the addition of P6-inositol. As the rate increases the kinetics become more complex. In acidic media with P6-inositol, the autooxidation is initially rapid which becomes slower subsequently but eventually proceeds very fast. The activation energy for autooxidation is about 19 kcal. mol-1. The complicated kinetics are partly attributable to the fact that carp hemoglobin is incompletely oxygenated under these conditions and that various liganded molecules including partially oxidized species have different susceptibilities toward autooxidation.


Characterization of rat testicular guanylate cyclase during development. The biochemical characteristics of rat testicular guanylate cyclase were investigated and the activity and subcellular distribution of the enzyme was determined during testicular development. Examination of the effects of metal ions, nucleotides, detergents and other in vitro activators on the activity of guanylate cyclase revealed that the testicular enzyme is similar in most respects to guanylate cyclase isolated from other mammalian tissues. Changes in the total activity of guanylate cyclase during testicular development paralleled changes in the tissue concentration of cyclic GMP; i.e. guanylate cyclase activity and tissue cyclic GMP were highest during the early stages of development. Subcellular fractionation revealed that the activity of the soluble form of guanylate cyclase was best correlated with tissue cyclic GMP. Biochemical analysis of the soluble enzyme prepared from testes of neonatal and adult rats did not reveal any significant differences in the characteristics of the enzyme during ontogeny with the exception of a 2.5 fold increase in V noted in the neonatal testis. The results of this study are consistent with a molecular mechanism that allows independent regulation of the different forms of guanylate cyclase.


Studies on the mobilization of iron from ferritin by isolated rat liver mitochondria. Rat liver mitochondria and rat liver mitoplasts mobilize iron from ferritin by a mechanism which depends on a respiratory substrate (preferentially succinate), a small molecular weight electron mediator (FMN, phenazine methosulphate or methylene blue) and (near) anaerobic conditions. The release process under optimized conditions (approx. 50 mumol/1 FMN, 1 mmol/l succinate, 0.35 mmol/1 Fe(III) (as ferritin iron), 37 degrees C and pH 7.40) amounts to 0.9--1.2 nmol iron/mg protein per min. The results suggest that ferritin might function as an intermediate in the cytosolic transport of iron to the mitochondria.


[Mitochondrial transhydrogenase: general principles of functioning]. A new mechanism for the functioning of mitochondrial transhydrogenase has been proposed. This mechanism makes it possible, without additional postulates, to explain the generation of delta muH+ of different signs in the forward and reverse transhydrogenase reactions and why this generation is not accompanied by the membrane uncoupling. It is suggested that the reduced nicotinamide rings of NADH and NADPH participate in a relay transfer of H+ ions across the membrane, while the oxidized nicotinamide rings of NAD+ and NADP+ block the H+-transporting paths in the transhydrogenase.


[Comparative study of effects of the tyrosine-copper(II) complex on xenobiotic hydroxylation and lipid peroxidation]. It has been found that NADPH-dependent hydroxylation of dimethylaniline, aniline, p- and o-nitroanisol and lipid peroxidation is inhibited by the tyrosine-copper (II) complex (low molecular weight analog of superoxide dismutase), which is indicative of a possibility of superoxide radicals formation in these reactions. The inhibition of the above-mentioned reactions with Tyr2-Cu2+ is less pronounced or absent, if cumole hydroperoxide is used as cosubstrate instead of NADPH. Differences in the Tyr2-Cu2+ complex effects on the cumule hydroperoxide-dependent xenobiotics hydroxylation and lipid peroxidation catalyzed by various forms of cytochrome P-450, e. g. microsomal, soluble and incorporated into liposomes, have been found. The data obtained suggest that the efficiency of the inhibitory effect of the Tyr2-Cu2+ complex depends on the type of cosubstrates (NADPH, cumole hydroperoxide) and substrates used as well as on the form of cytochrome P-450.


[Shifts of the bacteriochlorophyll absorption band at 880 nm in chromatophores and subchromatophore pigment-protein complexes from Rhodospirillum rubrum]. The redox potential dependency of the light-induced absorption changes of bacteriochlorophyll in the chromatophores and subchromatophore particles from Rhodospirillum rubrum has been studied. The highest values of the absorption changes due to the bleaching of P870 and the blue shift of P800 are observed within the redox potential range of 360--410. At the potential values below 300 mV the 880 nm band of bacteriochlorophyll shifts to shorter wavelengths in the subchromatophore particles and to longer wavelengths in the chromatophores. Redox titration revealed that the red and blue shifts of 880 nm bacteriochlorophyll band are caused by the functioning of a non-identified component (X) which has an oxidation -- reduction midpoint potential close to 340 mV (n = 1) within the pH range of 6,0--7,6. The Em for this component decreases by 60 mV/pH unit within the pH range of 7.6--9,2. The results obtained suggest that the red shift is due to the transmembrane, while the blue shift -- to the local intramembrane electric field. The generation of both the transmembrane and local intramembrane electric fields apparently depends on redox transitions of the component X.


[Kinetic properties of highly purified luciferase from fireflies Luciola mingrelica]. Luciferase of the fireflies Luciola mingrelica was isolated from dried lanterns of fireflies and purified by chromatography on DEAE-Sephadex. The homogeneity of the preparation was determined by polyacrylamide gel disc electrophoresis. The molecular weight of the enzyme equal to 45000 was determined by disc electrophoresis in the presence of sodium dodecyl sulfate. The kinetic properties of the enzyme (V and Km for luciferin and ATP) within the pH-range of 7,0--8,5 were studied. The kinetic curves of the pH-dependences of log V and log Km for both substrates are bell-shaped, with a slope equal to 2. At pH optimum (7,7--7,9) the Km values for luciferin and ATP are 6,6 mkM and 0,3 mM, respectively. The properties of luciferase L. m. were compared to those of luciferase from fireflies Phophinus pyralis previously described in literature.


[Modelling the effect of photosynthetic enhancement and chromatic transitions]. A simple method is proposed and analysed which explains characteristic manifestation of the enchancement effect (2nd Emerson effect) and of chromatic transients effect (Blinks effect). The model is based on the hypothesis concerning the ATP/NADP concentrations ratio with a change of falling light wavelength. The model is suggested to reflect the behaviour of the reduction pentosophosphate cycle (Calvin cycle) under low illumination intensity, when the photosynthesis rate is determined only by ATP and NADP.H concentrations formed in the course of different photochemical reactions.


[Comparative kinetic properties of structure-bound and solubilized L-threonine dehydratase from brewer's yeast]. It has been shown that L-threonine dehydratase (EC 4.2.1.16) of brewer's yeast Saccharomyces carlsbergensis is localized in the mitochondrial fraction. The enzyme is easily solubilized from the mitochondria by changing the pH and ionic strength of the buffer. Some kinetic properties of structure-bound and solubilized L-threonine dehydratase have been compared at pH 6,5. The kinetic plots of the initial rate of the reaction versus initial substrate concentration for both enzymes have a hyperbolic shape; the affinities of both enzymes for the substrate appear to be similar (Km = 20 mM). Both enzymes are inhibited by L-isoleucine, the shape of the kinetic plots being thereby changed into sigmoidal. Solubilization results in a decrease of the mitochondral enzyme sensitivity to the inhibition by L-isoleucine and in an appearance of cooperative interactions between the allosteric sites.


[Chromatography of carboxylic proteinases on sorbents containing the 2,4-dinitrophenyl group. Role of ionic interactions]. The chromatography of porcine pepsin on biospecific sorbents (Sepharose-4B-epsilon-DNP-aminocapronylhydrazide and Sepharose-4B-N-DNP-N'-acetylhexamethylenediamine) was studied. The sorbents in question differ from the previously used hydrophobic sorbent Sepharose-4B-DNP-hexamethylenediamine by the lack of strongly basic groups in the site of the ligand binding to the polymeric matrix. No qualitative differences in the pepsin chromatography on the three sorbents were observed. Presumably the decrease of the pepsin binding to the sorbents, containing the dinitrophenyl group, at pH values above the isoelectric point may be due to the effects of the salt on the binding site in the enzyme molecule rather than to the screening of the positive charges of the sorbent by chlorine ions. A commercial preparation of pepsin was purified 2-fold on the sorbent Sepharose-4B-epsilon-DNP-animocapronylhydrazide. The synthesis of sorbents is described.


Accumulation of N,N-dimethyltryptamine in rat brain cortical slices. 14C-DMT accumulates in rat brain cortical slices incubated at 37 C. This process has many of the properties of an active uptake mechanism. It is temperature-sensitive, sodium-dependent, saturable, and is inhibited by metabolic inhibitors. Tryptamine derivatives were more effective than the catecholamines in competing for 14C-DMT accumulation. A number of psychotropic drugs were inhibitors of 14C-DMT accumulation. In general, irrespective of pharmacologic class, the tertiary amines were more potent than the secondary or primary amines, although there were some exceptions. Most of the accumulated 14C-DMT was associated with the cytoplasmic fraction. Of the portion associated with the crude mitochondrial fraction, 54.4% was associated with nerve-ending fraction.


The utilization of hypnotics in chronic schizophrenics: some critical remarks. Of 23 hospitalized chronic schizophrenic patients, all under neuroleptic medication, hypnotics taken previously for a long time could be totally withdrawn in 16 cases, and in 7 cases, the dosage was diminished by 30%, without any sleep impairment. The gradual reduction of hypnotics was accompanied by a shift of neuroleptic dosage to the evening and bedtime, with reduction of the morning and midday dose, without change of the total daily dose. A significant improvement in the psychic state was observed in 16 patients after withdrawal of the hypnotic; 7 patients showed a slight improvement after reduction of the hypnotic. Monthly or bimonthly reassessment of insomnia in the hospitalized population of chronic schizophrenics is indispensable to avoid the deleterious effects and abuse of hypnotic drugs.


Role of vasa vasorum in nourishment of the aorta. The media of arteries is virtually avascular. Thus, oxygen and nutrients must reach the cells of the media by diffusion from the lumen of the vessel and from adventitial vessels. The thickness of the thoracic aorta of man and dog exceeds the effective diffusion distance of oxygen, but nutrition is supplemented by vasa vasorum which enter into the outer layers of the media. Occlusion of vasa vasorum in dogs produces medial necrosis, which indicates that these vessels are essential for the nourishment of the aorta. Recently the microsphere method has been used to provide the first measurements of blood flow through vasa vasorum. There is substantial flow to the outer layers of media of the thoracic aorta in dogs, with virtually no blood flow in the inner layers. The vessels are very responsive to physiological stimuli: they dilate during infusion of adenosine and constrict during stimulation of sympathetic nerves. During acute increases in arterial pressure, blood flow to the media decreases probably from distortion of vasa vasorum. Vasa vasorum may play role in disease states. Insufficient blood flow through vasa vasorum may contribute to medial necrosis of the aorta and to aortic atherosclerosis. A role of vasa vasorum in aortic changes in hypertension and other vascular diseases merits further study.


Monoamine mechanisms in chronic schizophrenia: post-mortem neurochemical findings. Dopamine and its metabolites homovanillic acid and dihydroxyphenylacetic acid, noradrenaline, serotonin and its metabolite 5-hydroxyindoleacetic acid, and tryptophan and its metabolite kynurenine have been assayed in 9 schizophrenic and 10 control brains, together with the monoamine-related enzymes tyrosine hydroxylase monoamine oxidase, dopamine-beta-hydroxylase, and catechol-o-methyl-transferase. In schizophrenic brains dopamine, noradrenaline and serotonin were significantly increased in some areas of corpus striatum, but there were no significant changes in enzyme activity or monoamine metabolite concentrations in any of the brain areas examined. The findings are not consistent with theories that serotonin or noradrenaline stores are grossly depleted or noradrenaline neurones have degenerated, or that monoamine oxidase activity is abnormal, in schizophrenia, and provide no direct support for the hypothesis that dopamine neurones are overactive.


Studies on synaptic vesicles in mammalian brain characterization of highly purified synaptic vesicles from bovine cerebral cortex. Synaptic vesicles have been isolated from bovine cerebral cortex by sequential differential and density gradient centrifugations followed by chromatography on a Sepharose 6B column. We have studied the morphology, enzymatic markers, neurotransmitter and ATP contents and protein composition of the vesicles. The specific contents of acetylcholine, gamma-aminobutyric acid, aspartate, glutamate and catecholamines were 4--8-fold higher in the vesicle fraction compared to the crude synaptosomal pellet. Electron micrographs of the vesicle preparation showed enrichment of vesicular material with an average diameter of 50 nm. The purity of the preparation was assessed by the very low activities of enzymatic markers of cellular membranes and cytosol components. Some Ca--Mg-activated ATPase activity was detected in the vesicle preparations, but its content relative to the neurotransmitters fell on chromatography, suggesting that this activity may be partially contributed by non-synaptic vesicle components, such as small microsomes. The isolated synaptic vesicles were solubilized with 1% sodium dodecyl sulfate and subjected to polyacrylamide gel electrophoresis. The major Coomassie blue stained bands observed with apparent molecular weights of 160,000 and 55,000 were enriched in parallel to the increase in purity of the preparation.


In vivo receptor occupation by benzodiazepines and correlation with the pharmacological effect. The existence of specific receptor sites for benzodiazepines has been well documented by in vitro binding studies. In this study, using a highly radiolabelled [3H]-flunitrazepam, we investigated the binding of benzodiazepines to their receptor sites under in vivo conditions. Tracer doses of [3H]flunitrazepam (0.001 mg/kg) were injected i.v. into mice and the concentration of the drug in the brain was monitored. The accumulation of [3H]flunitrazepam 20 min after injection was found to be highest in the hippocampus, cortex, hypothalamus; to be intermediate in the striatum, medulla oblongata/pons and midbrain and to be lowest in the cerebellum. This corresponds well with the different densities of benzodiazepine receptors which we found in in vitro studies, with the exception of medulla oblongata/pons and cerebellum. When increasing doses (0.01--10 mg/kg) of non-labelled benzodiazepine derivatives (flunitrazepam, clonazepam, the 3S and 3R enantiomers of 5-(o-fluorophenyl)-1,3-dihyrdo-1,3-dimethyl-7-nitro-2H-1,4-benzodiazepine-2-one, and chlordiazepoxide) were injected simultaneously with [3H]flunitrazepam, a dose-dependant, saturable and and stereo-specific decrease of [3H]flunitrazepam concentration in the mouse hippocampus was observed. The dose range in which the unlabelled benzodiazepines decreases the levels of [3H]flunitrazepam in the hippocampus corresponds closely to that which inhibited pentylenetetrazol- or picrotoxin-induced seizures, indicating that this in vivo method determines the occupation of pharmacologically relevant receptors.


The resolution of dopamine and beta 1- and beta 2-adrenergic-sensitive adenylate cyclase activities in homogenates of cat cerebellum, hippocampus and cerebral cortex. The stimulation of adenylate cyclase by dopamine and various beta-adrenergic agonists has been investigated in homogenates from 3 areas of cat brain: the cerebral cortex, cerebellum and hippocampus. The purpose of the study was to determine whether the beta-arenergic receptors coupled to adenylate cyclase could be classified as either beta 1 and beta 2 subtypes in the different regions studied. The stimulation of adenylate cyclase by the beta-adrenergic agonist, (-)isoproterenol (5 X 10(-6) M), was completely blocked by the specific beta-adrenergic antagonist, (p)alprenolol (1-(-5) M), but not by the dopaminergic antagonist, fluphenazine (10(-5) M), whereas the stimulation of adenylate cyclase by (-)epinephrine (10(-4) M) was blocked to varying extents by these two drugs in each of the 3 regions studied. The (-)epinephrine effect was always blocked in the combined presence of (p)alprenolol and fluphenazine. The adenylate cyclase stimulation by (p)epinephrine which is not blocked by (p)alprenolol was due to interaction of (p)epinephrine with a dopaminergic-sensitive adenylate cyclase which has been characterized in cerebral cortex, hippocampus and cerebellum. Regional differences in the affinity of beta-adrenergic-sensitive adenylate cyclase for various agonists were investigated in the presence of fluphenazine (10(-5) M). In the cerebellum the potency order was (+/-)protokylol greater than (+/-)hydroxybenzylisoproterenol greater than (+/-)isoproterenol greater than (-)epinephrine greater than (+/-)salbutamol greater than (-)norepinephrine, indicating the presence of a beta 2-adrenergic receptor. In the cerebral cortex the potency order was (-)isoproterenol greater than +/-)protokylol greater than (+/-)hydroxybenzylisoproterenol greater than (-)epinephrine = (-)norepinephrine ((+/-)salbutamol being inactive). A similar pattern was found in the hippocampus indicating the presence of a beta 1-adrenergic receptor in these two regions. (+/-)Salbutamol was a partial agonist in the cerebellum and a competitive antagonist in the cerebral cortex. The ratio of the antagonist potencies of (+/-)practolol and (+/-)butoxamine preferential beta 1- and beta 2-adrenergic antagonists respectively, to block the stimulation of adenylate cyclase was 25 in the cerebellum, compared to 0.5 in the cerebral cortex and 1.6 in the hippocampus. These results confirm the presence of a beta 2 subtype of receptor coupled to adenylate cyclase in the former and beta 1 subtypes in the latter two regions. The comparison between the affinities of a series of beta-adrenergic agonists and antagonists for the beta-adrenergic receptors coupled with an adenylate cyclase in cerebral cortex and cerebellum with their affinities for well characterized beta 2-adrenergic receptors in lung and beta 1-adrenergic receptor in heart substantiated this conclusion.


[Adenylate cyclase and guanylate cyclase activity in the isolated kidney glomerulus of the rat]. Isolated rat renal glomeruli contain an adenylate cyclase system and guanylate cyclase system. Adenylate cyclase was strikingly activated by purified parathyroid hormone, epinephrine, prostaglandin I2 and histamine. The demonstration of PTH activated adenylate cyclase in glomeruli raises the possibility of a role of this hormone in regulation of glomerular filtration rate. Guanylate cyclase was strikingly activated by CA2+, nitrate derivatives such as sodium nitroprusside. Its role remained still unknown.


The influence of blood flow and arterial blood pressure during cardiopulmonary bypass on deltoid muscle gas tensions and body temperature after bypass. The results of this study demonstrate that skeletal muscle perfusion during bypass requires high flows and mean arterial pressures and that use of vasopressors during bypass impairs skeletal muscle blood flow. Our findings also indicate that skeletal muscle perfusion during bypass decreases metabolic acidosis after operation and prevents decreases in body temperature and poor skeletal muscle blood flow in the post-bypass and early postoperative periods. Our results suggest that perfusing to maintain normal Pmo2 during extracorporeal circulation is superior to more conventional techniques of conducting bypass.


Anaesthetic management of phaeochromocytoma. The incidence, mortality, physiology, clinical findings and diagnosis of phaeochromocytoma are reviewed. Treatment, after adequate medical stabilization, must be surgical because of the high incidence of malignancy. Alpha-adrenergic receptor blockade and beta-adrenergic receptor blockade in the preoperative period was discussed. Anaesthetic management of patients with phaeochromocytoma requires close monitoring. Virtually all inhalational anaesthetic agents have been used in cases of phaeochromocytoma. Recent reports have favored enflurane. The merits of neuroleptanaesthesia and the various muscle relaxants are also discussed. Most authors favour lidocaine over propranolol for management of dysrhythmias during operation. Phentolamine or sodium nitroprusside are used for hypertension during operation. Hypotension is treated by fluid replacement with nor-epinephrine if a vasopressor becomes necessary. Close monitoring is necessary in the postoperative period. Adequate urinary output is of more importance than actual blood pressure levels.


Neurotransmitter receptors on gill muscle fibers and the gill peripheral nerve plexus in Aplysia. Isolated pinnules of the gill of Aplysia contract when dopamine (DA) is perfused through the bath. The contraction is not blocked by high-Mg2+ seawater, and reflects excitatory receptors for DA on the smooth muscle cells of the gill. The pinnule often shows irregular, spontaneous contractions which are blocked by high-Mg2+ seawater and 30 mM CoCl2. These contractions reflect spontaneous activity of a peripheral nerve plexus. No other transmitter was found to be directly excitatory on the muscle fibers, although there are inhibitory receptors for serotonin (5-HT). Tactile stimulation of the pinnule evoked a two-component contractile reflex contraction due to activation of the peripheral nerve plexus. Acetylcholine, octopamine, and 5-HT but not several other transmitters depressed these responses, presumably due to inhibitory receptors on the neurons of the peripheral plexus.


Noradrenaline inhibition of transmitter efflux in a renal artery preparation and the presynaptic receptor theory. The effect of noradrenaline on the stimulation-induced efflux of tritium in cattle renal arteries preincubated with [3H]noradrenaline was determined. Preparations were stimulated transmurally, with 300 shocks over a range of frequencies (1--15 Hz) in the presence and absence of noradrenaline (3 X 10(-6) M). The agonist inhibited the efflux most at 1 Hz but the extent of the inhibition did not vary with frequency between 2, 5, and 15 Hz. It is concluded that a negative feedback system, modulating neurotransmitter release, and increasingly activated by endogenously released noradrenaline as the frequency of stimulation rises, cannot account for the pattern of efflux inhibition induced by exogenous noradrenaline.


Induction of foci of altered hepatocytes by a single injection of azaserine to rats. The induction of foci of altered, gamma-glutamyltranspeptidase (GGT)-positive hepatocytes by azaserine was investigated. After injection of a single dose of azaserine, many foci developed in male Wistar rats fed a choline-devoid (CD) diet containing acetylaminofluorene (AAF), but only a few in rats fed a choline-supplemented (CS) diet containing AAF. Similar results were obtained in rats fed a plain CD diet or a plain CS diet and injected with a single dose of azaserine after a partial hepatectomy. These findings indicate that azaserine is an effective initiator of liver carcinogenesis in rats, and that a CD diet acts as a strong promoter of the evolution initiated liver cells to foci of altered, GGT-positive hepatocytes.


APUD-type recepto-secretory cells in the chicken lung. The epithelium of the intrapulmonary airways of the chicken lung has been studied by fluorescence and electron microscopy. Numerous intensely yellow-fluorescent cells occur in the epithelium of the primary and secondary bronchi. The cell cytoplasm contains characteristic granular vesicles with an electron-dense central core. The vesicles react positively to chromaffin and argentaffin treatment, indicating that they are possible storage sites for amines. Synapse-like junctions occur between the granular cells and the intraepithelial nerve endings, filled with numerous mitochondria, suggesting that these granular cells may have a dual function as both receptor and endocrine cell.


Affinity of human leukocyte interferon for polyribonucleotides. Human leukocyte interferon (HL-IF)binds to AGPOLY(A)TM, AGPOLY(U)TM and AGPOLY(I)TM. The bound interferon could be displaced from all three polyribonucleotides by including sodium chloride in the eluant. The nature of interaction of HL-IF with polyribonucleotides is electrostatic and not hydrophobic since its binding was not prevented in the presence of 50% ethylene glycol. The binding of HL-IF on AGPOLY(I)TM is stronger at lower pH since an increase in ionic strength is required to displace it.


Novel 125I-labeled nortriptyline derivatives and their use in liquid-phase or magnetizable solid-phase second-antibody radioimmunoassays. Nortriptyline derivatives prepared by reaction with fluorescein isothiocyanate or conjugation to N-acetyl-L-histidine were radioiodinated and the products purified with Sephadex LH-20 columns to obtain two novel nortriptyline radioligands. Antisera were raised in rabbits by immunization with nortriptyline conjugated to succinylated ovine albumin. By use of the iodinated fluorescein derivative we developed a liquid-phase second-antibody radioimmunoassay that gives results correlating closely (r = 0.98) with those by an established radioimmunoassay of similar specificity in the assay of apparent total amitriptyline and its metabolite nortriptyline in serum or plasma from patients being treated with these drugs. With the iodinated N-acetyl-L-histidine derivative we developed a magnetizable solid-phase second-antibody radioimmunoassay. The cross reactivities of amitriptyline and nortriptyline could be made equal by performing the assay at pH 9.0, which makes it possible to measure true total active drug concentrations in patients receiving amitriptyline.


Gas-liquid chromatography of undervatized drugs after chromatographic extraction from blood. We have developed an integrated method that overcomes the two main procedural difficulties of gas-liquid chromatography, namely, solvent-solvent extraction and chemical derivatization. Drugs are extracted from serum by column chromatography on granular diatomaceous earth (kieselguhr). Subsequent gas-liquid chromatography of underivatized samples can be performed on either of two liquid phases. A mixed liquid phase, used for quantitative gas-chromatographic assay on patients with a known therapeutic regimen, has enabled quantitation of 12 drugs in serum. Alternatively, a single liquid phase, used with the mixed liquid phase, permits the gas-chromatographic identification of unknown drugs on the basis of the characteristic pattern of the two relative retention times; by this approach more than 40 drugs have been identified in cases of suspected intoxication, both in serum and in gastric aspirate. Besides providing ease of performance and wide applicability, the proposed procedure offers a degree of precision and accuracy that compares favorably with established methods.


A new assay for diaphorase activity in reagent formulations, based on the reduction of thiazolyl blue. This new assay procedure for diaphorase eliminates problems of high blank rates and nonlinear kinetics associated with other methods. The dye thiazolyl blue tetrazolium bromide is reduced in the presence of NADH and diaphorase to yield a colored formazan, which as maximum absorbance at 560 nm.


Physical properties of human serum hexosaminidases A and B: studies in normal and cancer patients. The major forms of human serum hexosaminidases A and B (Hex A and Hex B) were isolated from normal subjects and cancer patients using DEAE-cellulose. In normal serum, Hex A was heat-labile and had an apparent KM of 1.13 mM and Vmax of 0.51 mumol/ml/h; Hex B was heat-stable and had an apparent KM of 0.85 mM and Vmax of 0.22 mumol/ml/h. Both forms had the same pH optimum, at 4.3. Hex A and Hex B from the sera of cancer patients resembled their normal counterparts in heat stability, pH optimum, and apparent KM (1.07 mM for Hex A, 0.88 mM for Hex B). In contrast, the Vmax values for the cancer sera isozymes were greater than those of normal sera (0.70 and 0.40 mumol/ml/h for Hex A and Hex B, respectively).


Human leucocyte cerebroside sulphate sulphatase. Some of the properties of the cerebroside sulphate sulphatase of human leucocyte extracts have been studied. The enzyme has an apparent KM of 2.9 mmol/l and is inhibited by the products of the reaction, sulphate and galactocerebroside. A number of divalent metal ions including manganese and magnesium stimulated the reaction only slightly at 5 mmol/l but inhibited strongly at 20 mmol/l. Triton X-100 present in leucocyte extracts also inhibited, increasing both the apparent KM and Vmax. The enzyme activity was dependent on the presence of anionic detergents. At low substrate concentrations a crude taurocholate preparation was the most active of all bile acids examined. Pure taurocholate and sodium cholate were considerably less active. At highter substrate concentrations however, sodium cholate produced the greatest stimulation of enzyme activity. These data suggest that the bile acid/substrate ratio may be a critical factor in determining cerebroside sulphate sulphatase activity at different substrate concentrations.


Enzymatic microdetermination of plasma lipoprotein cholesterol without ultracentrifugation. The lipoprotein fractions of 15 microliter capillary plasma were separated by electrophoresis on agarose gel. The samples were dried, dissolved in HCl, and neutralized with a saturated Tris solution producing a Tri-buffer system at pH 7. This improvement of sample neutralizing made the system highly suitable for enzymatic determination of cholesterol. The kinetic properties and linear range of this reaction system, the precision (coefficient of variation 1--3%) and the recovery of cholesterol (100%) were found to be optimal.


Factors affecting the solubility of ammonium acid urate. Evidence is presented that sodium acid urate exists in aqueous solution in a non-colloidal state. The levels of ammonium and urate ions required to precipitate ammonium acid urate have been established for some aqueous media. The effect of pH on the formation product of ammonium acid urate is described. Evidence is presented that there is no epitaxial relationship between this salt and calcium oxalate monohydrate.


Glycosaminoglycans as inhibitors of calcium oxalate crystal growth and aggregation. Evidence is presented that the glycosaminoglycans, chondroitin 6-sulphate and chondroitin 4-sulphate, are the major inhibitors of calcium oxalate crystal growth and aggregation in dilute normal urine.


Human dihydropteridine reductase: a method for the measurement of activity in cultured cells, and its application to malignant hyperphenylalaninemia. 1. The kinetic, assay and culture variables involved in the determination of dihydropteridine reductase activity in human cultured cells have been investigated, and a modified assay is described which allows for accurate determination of activity. Unlike previously used assays, this assay utilizes saturating concentrations of substrates and its increased sensitivity allows for excellent replication on a smaller number of cells than was previously required. 2. The modified assay was used to investigate the levels of dihydropteridine reductase in patients with malignant hyperphenylalaninemia; results of assay for the enzyme in cultured cells from affected infants and parents are presented. These are compared with control data for dihydropteridine reductase activity in fibroblasts and continuous lymphoid cells from normal controls and phenylketonuric patients, in normal amniotic cells and in liver obtained at autopsy.


A clinical score for predicting the level of respiratory care in infants with respiratory distress syndrome. A scoring system was developed to predict the need for transferring infants with respiratory distress syndrome (RDS) from community hospitals to specialized respiratory care centers. Five clinical and laboratory determinations (birthweight, clinical RDS score, FI02, PCO2 and pH) recorded from 100 infants with RDS during one year were utilized in a score with values ranging from 0 to 10. Application of the score to 159 infants with RDS during the following year showed that: (1) 73 per cent of infants scoring less than or equal to 3 received only oxygen by hood; (2) 75 per cent of infants scoring 4--5 required continuous positive airway pressure (CPAP); and (3) 87 per cent of infants scoring greater than or equal to 6 needed mechanical ventilation (7V). Mean scores were significantly different (p less than 0.02) for each type of respiratory therapy employed: oxygen by hood (2.30 +/- 0.19 S.E.M.); CPAP (4.27 +/- 0.16 S.E.M.); MV (6.72 +/- 0.25 S.E.M.). The accuracy and simplicity of the score make it valuable for the physician in the community hospital to assist in deciding when to transfer a neonate with RDS for more intensive respiratory therapy.


Stress lesions of the lower leg and foot. Technetium 99 methylene diphosphonate scans were performed on 32 patients with suspected stress fractures. Twenty-six patients had abnormal scans, 13 had abnormal X-rays and on these only nine had either fresh periosteal new bone and/or a fracture line. A logical approach of the use of X-ray and radionuclide techniques in patient management is suggested.


The adenylate cyclase system in human liver: characterization, subcellular distribution and hormonal sensitivity in normal or cirrhotic adult, and in foetal liver. 1. Adenylate cyclase (EC 4.6.1.1) activity was characterized in human liver, and its subcellular distribution compared with that of three other potential enzyme markers of the pericellular membrane: leucine aminopeptidase (EC 3.4.11.1), gamma-glutamyltransferase (EC 2.3.2.2) and 5'-nucleotidase (EC 3.1.3.5). Although these three enzyme activities were detected in each of the subcellular fractions studied, 85% of the total adenylate cyclase activity was found in the 1000 g pellet ('nuclear' fraction) with a threefold increase in specific activity as compared with the homogenate. No adenylate cyclase activity existed in the 150 000 g supernatant fraction. 2. In the 'nuclear' fraction, adenylate cyclase activity was increased in a dose-dependent fashion by glucagon with a half-maximal stimulation at 10 nmol/l and a maximal four- to seven-fold increase at 1 mumol/l. Catecholamines activated adenylate cyclase 2.5- to three-fold, with an order of potency (protokylol greater than isoprenaline greater than adrenaline greater than noradrenaline) typical of a beta 2-adrenoreceptor. Prostaglandin E1 and NaF also stimulated cyclase two- and four-fold respectively. Insulin, serotonin, dopamine, thyroid-stimulating hormone and ACTH had no effect. Adenosine provoked a weak inhibition at 0.1 mmol/l. Finally guanosine triphosphate and 5'-guanylyl imidodiphosphate induced a marked increase in basal activity, four- and eight-fold respectively, but both reduced the relative increase in enzyme activity due to glucagon or adrenaline. 3. Cyclase from foetal liver (12--16 weeks old) and cirrhotic adult liver appeared to behave similarly to that from normal liver; however, foetal cyclase was more active, and cirrhotic enzyme less active than normal adult liver. Both systems responded to catecholamines via a beta 2-adrenoreceptor. 4. These results validate the use of rat liver adenylate cyclase as a tool for pharmacological and physiological studies.


Characteristics of a renin-binding substance for the conversion of renin into a higher-molecular-weight form in the dog. 1. Renal cortical homogenates of the dog were subjected to sieve separation, a Nucleopore Filter being used to separate the renin granules. 2. The molecular weight of renin in the granules was estimated to be about 40 000 by gel filtration. Renin was converted into a higher-molecular-weight form (60 000) by mixing with cytosol in the presence of sodium tetrathionate, a thiol inhibitor. 3. When cytosol was pretreated with acid (pH 3.0) or heating (100 degrees C), the molecular-weight conversion did not occur. 4. Cytosol was separated into three parts by gel filtration. Fraction A included substances with a molecular weight of over 47 000, fraction B from 47 000 to 32 000, and fraction C from 32 000 to 15 000. The mixture of renin in the granules with fraction A and sodium tetrathionate resulted in the formation of a higher-molecular-weight form of the enzyme, but no change in molecular weight was detected when renin was mixed with fractions B or C and sodium tetrathionate.


Role of the lung in total body clearance of circulating drugs. Attention in recent years has focused upon the ability of lung to accumulate and/or to metabolise circulating hormones, drugs and other xenobiotic agents. Although often studied in broken cell preparations, such functions must be examined in intact lung before extrapolation to the situation in vivo is possible. The role of lung in total body clearance of xenobiotic agents is often considered to be small, as compared with the liver it usually has much lower concentrations of degradative enzymes and a smaller mass. However, consideration of such factors as organ blood flow or stimulation of drug metabolising enzymes suggests that the contribution of the lung to total body clearance of some drugs is greater than previously recognised. This may explain some of the alterations in drug clearance seen clinically. For example, cigarette smoking may increase the clearance of certain xenobiotic compounds by stimulating pulmonary drug metabolising enzymes. Pulmonary drug disposition may also be altered by conditions affecting cardiac output, such as exercise, hypoxia, and circulatory shock, or those affecting acid-base balance, such as hyperventilation. In addition, pneumotoxicants may affect pulmonary clearance of circulating drugs and xenobiotics.


Activation of inactive plasma renin by plasma and tissue kallikreins. 1. Normal human plasma contains a proactivator of inactive renin. The pro-activator is activated at physiological pH in plasma that has been pretreated with acid. This activation in vitro leads to the conversion of inactive renin into the active form with simultaneous generation of kallikrein activity. 2. The endogenous activator of inactive renin has the same pH profile and inhibitor spectrum as plasma kallikrein. 3. Inactive renin can also be activated by exposure of plasma to exogenous trypsin, and in normal plasma the quantities of inactive renin that are activated after acidification and with trypsin are identical. Prekallikrein (Fletcher factor)-deficient plasma, however, has much lower renin activity after acidification than with trypsin. Thus acid activation of inactive renin depends on plasma prekallikrein, whereas the action of trypsin is independent of prekallikrein. 4. Highly purified tissue (pancreatic) kallikrein, in a concentration of less than 2 X 10(-8) mol/l, activates inactive renin that has been isolated from plasma by ion-exchange chromatography. In this respect it is at least 100 times more potent than trypsin. 5. It is therefore possible that plasma and/or tissue (renal) kallikreins are also involved in the activation of inactive renin in vivo.


New experiments of biotin enzymes.

New experiments of biotin enzymes. The objects of structural studies on biotin-enzymes were acetyl CoA-carboxylase and pyruvate carboxylase of Saccharomyces cerevisiae and beta-methylcrotonyl CoA-carboxylase and acetyl CoA-carboxylase of Achromobacter IV S. It was found that these enzymes can be arranged in three groups. In the first group, as represented by acetyl CoA-carboxylase of Achromobacter, the active enzyme could be resolved in three types of functional components: (1) the biotin-carboxyl carrier protein, (2) the biotin carboxylase, and (3) the carboxyl transferase. In the second group, as represented by beta-methylcrotonyl CoA-carboxylase from Achromobacter only two types of polypeptides are present. The one carries the biotin carboxylase activity together with the biotin-carboxyl-carrier protein, the other one carries the carboxyl transferase activity. In this third group, as represented by the two enzymes of yeast, all three catalytic functions are incorporated in one multifunctional polypeptide chain. The evolution of the different enzymes is discussed. The animal tissues acetyl CoA-carboxylase is under metabolic control, as known from previous studies. It thus has to be expected that the levels of malonyl CoA in livers of rats in all states of depressed fatty acid synthesis are much lower than under normal conditions because the carboxylation of acetyl CoA is strongly reduced and cannot keep pace with the consumption of malonyl CoA by fatty acid synthetase. A new highly sensitive assay method for malonyl CoA was developed which uses tritiated NADPH and measures the incorporation of radioactivity into the fatty acids formed from malonyl CoA in the presence of purified fatty acid synthetase. The application of this method to liver extracts showed that the level of malonyl CoA which amounts to about 7 nmoles per gram of wet liver drops to less than 10% within a starvation period of 24 hr and even further if the starvation period is extended to 48 hr. A low malonyl CoA concentration is also found in the alloxan diabetic animals and in animals being fed a fatty diet after starvation. On the other hand, feeding a carbohydrate rich diet leads to malonyl CoA levels surpassing the levels found after feeding a balanced diet. These observations reconfirm the concept that fatty acid synthesis is principally regulated by the carboxylation of acetyl CoA.


Hormonal regulation of acetyl-CoA carboxylase activity in the liver cell. Chick liver cell monolayers synthesize fatty acids at in vivo rates and are responsive to insulin and glucagon. High rates of fatty acid synthesis are maintained with insulin present and lost slowly without insulin. Glucagon or 3',5'-cyclic AMP cause immediate cessation of fatty acid synthesis. The site of inhibition appears to be cytoplasmic acetyl-CoA carboxylase which catalyzes the first committed step of fatty acid synthesis. Liver carboxylase exists either as catalytically inactive protomers or active filamentous polymers. Citrate, an allosteric activator of the enzyme, is required for both catalysis and polymerization. Glucagon and cAMP cause an immediate decrease in the cytoplasmic citrate concentration of chick liver cells apparently by inhibiting the conversion of glucose to citrate at the phosphofructokinase reaction. Since fatty acid synthesis and citrate level are closely correlated, citrate appears to be a feed-forward activator of the carboxylase in vivo. Compelling evidence indicates that carboxylase filaments are present in the intact cell when citrate levels are high and depolymerize when citrate levels fall. Hence, carboxylase activity and fatty acid synthetic rate appear to be determined by cytoplasmic citrate level.


The significance of 2,3-DPG in red blood cell transfusions. This review will begin by giving the highlights of the history and explain development of the basic science knowledge of hemoglobin chemistry, function, and physiology. The necessary involvement of red cell metabolism, as it pertains to the maintenance of 2,3-diphosphoglycerate (2,3-DPG) levels, both normally and under the perturbed and experimental conditions of blood storage, will be given as part of the basic science data. The clinical science and transfusion data will comprise the main critical aspects of the paper. Analysis and comment of over 20 studies will be given on the effects of animal and human transfusions with altered 2,3-DPG levels. Decreased survival and organ function have been demonstrated with transfusion of low 2,3-DPG red cells, with or without anemia, in the conditions of exercise, shock, hypotension, ischemia, cardiac surgery, hypoxia, sepsis, and acidosis. By critical analysis of these studies, recommendations on general and specific patient needs for red cell transfusions with normal or high 2,3-DPG levels are given.


Preparation of high-fructose syrup from the tubers of the Jerusalem artichoke (Helianthus tuberosus L. Fructose has recently received much attention due to renewed interest in natural sweeteners. In addition, fructose has some advantages to sucrose in sweetness, solubility, viscosity, and dental health characteristics. Fructose is deposited as storage fructans of the inulin (beta-1,2) type in tubers and rhizomes of the Compositae family. The utilization of the Jerusalem artichoke (Helianthus tuberosus) tuber as a source of fructose syrup is discussed. This plant has the potential to produce more sugar per acre than corn or sugar beets. In addition, the artichoke has higher frost resistance and lower heat unit requirements than corn and is somewhat more tolerant to low moisture conditions than sugar beets. A high quality fructose syrup can be produced from artichoke tubers. The extraction step was found to be particularly important since development of adverse colors and flavors must be prevented. The fructans may be acid or enzyme hydrolyzed but the latter method gave a higher quality syrup. Ion-exchange resins and activated charcoal were effective in removing coloring and flavoring materials, and also reduced other noncarbohydrate constituents. Since the enzymatic hydrolysis of the fructans is an attractive alternative to acid hydrolysis, a process was developed for producing and purifying a special beta-fructofuranosidase (inulase) from Saccharomyces fragilis. Inulase has a much higher specificity for fructans than commerically available beta-fructofuranosidase (invertase).


[Gastroesophageal reflux: a factor eliciting esophageal variceal hemorrhage?]. Following a hemorrhage, ten patients with esophageal varices were examined by means of gastroscopy and functional analysis of the esophagus with special regard to gastroesophageal reflux. A pathological gastroesophageal reflux was not confirmed. These results plus previously published findings lead to the assumption that reflux is of not importance in the initiation of esophageal variceal bleeding.


Isolation and purification of large quantities of DNA replication intermediates by pH step alkaline elution. The alkaline elution technique has been modified to be used in the isolation of DNA replication intermediates and in the study of the process of DNA replication. In this procedure pulse labeled CHO cells are layered onto a membrane filter, lysed with detergent, and the nascent DNA eluted in step-wise fashion with tetrapropylammonium hydroxide at pH 11.0, 11.3, 11.5 and 12.1. Alkaline sucrose sedimentation of the eluted DNA shows that the pH 11.0 material consists of less than 9S fragments consistant with those described by Okazaki and others. DNA eluting at pH 11.3 has a molecular weight of 8-12 million daltons, DNA which elutes at pH 11.5 sediments with a molecular weight of 20-30 million daltons. Two independent lines of evidence suggest that the pH 11.3 material includes DNA sequences synthesized at replicon origins. (1) Exposure of cells to low doses of X-ray prior to pulse labeling reduces the pH 11.3 fraction by 40-50% while there is little change in the other fractions. (2) Synchronization of cells by inhibiting DNA synthesis with FdU, followed by a 2 min pulse label, yields approximately 50% of the incorporated 3H-thymidine in the pH 11.3 fraction. The pH step elution technique has the following advantages: 1. Intermediates of high specific activity can be isolated from 10(6) cells per filter; 2. By lysing cells on a filter, proteins, nucleases, and other cellular materials are eliminated; 3. DNA in the lysate is never handled, thus eliminating shearing; 4. Eluted DNA may be instantaneously neutralized by collecting into a buffer to protect it from alkaline degradation.


Crohn's disease associated with Takayasu's arteritis. In a young woman with Takayasu's arteritis, standard roentgenographic studies suggested a diagnosis of Crohn's ileocolitis. Mesenteric angiography was performed and revealed specific abnormalities diagnostic of Crohn's disease, namely, the zoning sign in the cecum with a hypervascular inner layer and a hypovascular outer layer, and paraintestinal inflammatory neovasculature adjacent to the ascending colon. In addition, several helpful but nonspecific findings were demonstrated, and these features are reviewed. This patient represents another example of large vessel lesions associated with inflammatory bowel disease.


Screening in diabetes mellitus: report of the Atlanta workshop. A Diabetes Screening Workshop was held in Atlanta, Georgia, May 15--17, 1978, which was sponsored by the Center for Disease Control, the American Diabetes Association, and the National Institute of Arthritis, Metabolism, and Digestive Diseases. The workshop formulated the following recommendations for the use of screening procedures in diabetes mellitus from a community control viewpoint: (1) screening for asymptomatic glucose intolerance should be done among pregnant women as part of a well-coordinated program to decrease perinatal morbidity and mortality; (2) screening programs to detect asymptomatic glucose intolerance per se are not recommended as health services in nonpregnant populations; (3) screening for diabetes or its complications for research purposes should be done only as part of well-designed studies focusing on identification of predictive factors, implementation and effectiveness of preventive and therapeutic measures, descriptive epidemiology in selected populations, dynamic and economic factors of the medical care system related to case detection and management, and the nature and effects of screening processes; (4) information and education programs for health care providers, parents, and the general public should be implemented to bring about increased awareness of the clinical signs and symptoms of diabetes; and (5) all persons known to have diabetes should be evaluated regularly for the detection and management of the common chronic complications of the disease.


Physical dependence on ethanol: conceptual considerations. The mechanism of physical dependence is defined as a chain of events that begins at the physical level of membrane organization and that progresses through the various levels of biological organization to the behavioral level. An ethanol-induced alteration may be (1) cause, (2) effect, (3) an unrelated covariate of physical dependence, or (4) an experimental artifact. A variety of criteria in addition to correlation must be met before the significance of an ethanol-induced effect can be assessed. The reasons are summarized for assuming that physical effects of ethanol on membranes are the primary initiators of the cascade of events that leads to physical dependence.


[Inhibition of pentagastrin-stimulated gastric secretion by ranitidine (author's transl)]. The dose-effect relationship of ranitidine, a recently developed selective histamine H2 receptor antagonist was tested on six healthy subjects after oral administration. Ranitidine gave dose-dependent inhibition of pentagastrin-stimulated acid secretion. Half-maximal inhibition occurred at 75 mg. The antisecretory effect was still demonstrable five hours after oral intake. The drug is thus more powerful and has more lasting antisecretory action, even after oral intake, than cimetidine.


Role of calcium in ethanol-membrane interactions: a model for tolerance and dependence. The calcium content of membranes may regulate a number of key neuronal processes. Studies are described in which acute and chronic administration of ethanol was shown to alter calcium binding to synaptosomal plasma membranes.


Progress toward a voluntary oral consumption model of alcoholism. With the goal of obtaining a suitable animal model for voluntary oral consumption of ethanol, the investigators selectively bred lines of alcohol-preferring and alcohol-nonpreferring rats, with preference considered as a function of the concentration of ethanol ingested. Studies with these animals showed that drinking is voluntary and not contingent on caloric restriction; that they will work to obtain ethanol even when food and water are freely available, and in so doing, show psychological or behavioral tolerance; that the amount of ethanol voluntarily consumed approaches their apparent maximum capacity for ethanol elimination. This amount of ethanol was capable of altering brain neurotransmitter content, thus exerting a CNS pharmocologic effect. In addition, the rats will bar-press for intravenous administration of ethanol, and with prolonged, free-choice consumption, ethanol intake increases to as much as 12 g per kg body weight per day without producing behavioral deficits, suggesting the development of tolerance.


[Gonadotropin secretion following pernasal stimulation with synthetic gonadotropin releasing hormone (GnRH) and a highly effective GnRH-analog in healthy men and prepubertal boys]. Potent long acting analogs of GnRH are of great interest especially in view of pernasal (p.n.) treatmen of hypogonadism of hypothalamic origin and of cryptorchidism. To find the necessary p.n. dosage of such a substance, serum LH and FSH were measured in 6 normal adult human males after p.n. application of various doses of D Leu6-des-Gly10-GnRH ethylamide. 50 microgram of the GnRH analog were necessary to obtain increased serum gonadotropins over a period of at least 8 hours. By repeated p.n. application of 200 microgram of synthetic GnRH every 2 hours in 6 normal adult males a considerable increase of serum gonadotropins could be demonstrated as well. Pernasal application of 200 microgram GnRH repeated at an interval of 1 hour in 3 cryptorchid boys produced a distinct increase of the serum gonadotropins. The intraindividual comparison of 200 microgram GnRH and 20 microgram of the GnRH analog in one boy showed equivalent net increases of the gonadotropins. With the analog the gonadotropin increase lasted for about 6 hours.


Semiautomated enzymic microassay for plasma L-alanine. Plasma L-alanine is deaminated by bacterial alanine-dehydrogenase; the resulting ammonia is dialyzed out and measured by use of a continuous flow phenol-hypochlorite colorimetric microassay. Concentrations in the range of 10-1,000 mumol alanine/l can be determined in a 50-microliter sample. The optimal conditions for the assay are specified. Study of the analytical qualities of the technique shows high specificity, good reproducibility , and a detection limit of 6 mumol/l. Usual values in human plasma from arterial or venous blood are respectively 296 +/- 166 and 376 +/- 214 mumol alanine/l (x +/- 2 SD). The usual values in rats are close to those found in man.


Involvement of an arginyl residue in the catalytic activity of myosin heads. 1. Phenylglyoxal reacts rapidly with isolated myosin heads (subfragment 1) and induces two successive and distinguishable effects on their enzymic properties: first, a twofold activation of the Ca2+ and Mg2+-dependent ATPases with no effect onthe K+-ATPase followed by inhibition of the K+, Ca2+ and actin-activated Mg2+-ATPases. A specific protein-reagent reagent complex is formed during the second phase of the modification reaction (Ki approximately 5 x 10(-3) M). 2. ADP and ATP with or without cations provide efficient protection only against the loss of ATPase activities, suggesting that the second inhibitory process is occurring at or close to the active site. 3. On the basis of [14C]phenylglyoxal-labelling experiments and the composition of modified subfragment-1 derivatives, it is demonstrated that the sequential modification of two reactive arginyl residues is responsible for the observed activation-inhibition phenomena. Blocking of the first reactive residue produces a shift in the pH/activity curves related to the Ca2+ and Mg2+-dependent ATPases with an apparent activation effect. Modification of the second guanidino group does not destroy the affinity of the protein for the nucleotide substrates but does alter the nucleotide binding site as reflected in the inability of Mg2+. ATP to dissociate the modified subfragment-1--actin complex. It is concluded that electrostatic interactions between this positively charged group and the negatively charged ATP and ADP molecules may be critical for the hydrolytic efficiency of myosin heads. 4. After dissociation and separation of the polypeptide constituents of the protein in acetic acid medium, both labelled sites are found to reside in the heavy chain.


Bovine serum chitinase.

Bovine serum chitinase. 1. A glycol-chitin-splitting enzyme without lysozyme (muramidase) activity has been found in calf serum. The enzyme also degrades colloidal chitin and is thus a true chitinase, 1,4-beta-poly-N-acetylglucosaminidase, without exo-beta-N-acetylglucosaminidase effect. 2. The enzyme is purified 1000-fold by ion-exchange chromatography and gel filtration. Its optimal activity is between pH 1.5-2.0 with glycol chitin and between pH 3-6 in a rather broad optimum with colloidal chitin as substrate. The optimal stability of the enzyme is in the pH interval 3.0-6.5 when tested by incubation with glycol chitin at 50 degrees C for 60 min. The optimal temperature for the degradation of glycol chitin is 40 degrees C when assayed at pH 1.5 and 51 degrees C when assayed at pH 3.5. 3. The enzyme is activated by moderate heating at pH 6.5. The highest relative activity, 135% is reached after 45 min incubation at 30 degrees C, pH 5 or after 30 min at 40, pH 2.4. By incubation with small amounts of trypsin at pH 6.5 at 3m degrees C the enzyme was temporarily activated. 4. The isoelectric point, pH 5.3, and the molecular weight, 47,000 +/- 3,000 were determined by respectively isoelectric focusing and gel filtration. 5. The Michaelis-Menten constant, Km = 0.76 +/- 0.05 (S.E.) mg/ml, was measured with glycol chitin as substrate.


The pH dependence of the binding of D-glycerate 2,3-bisphosphate to deoxyhemoglobin and oxyhemoglobin. Determination of the number of binding sites in oxyhemoglobin. The number of Bohr protons released upon oxygenation has been measured over a large range of human hemoglobin concentrations (0.02 to 4.5 mM) in the presence of equimolar amounts of D-glycerate 2,3-bisphosphate. From these data the association constants for the binding of this organic phosphate to deoxyhemoglobin and oxyhemoglobin were calculated at different pH values. The maximum number of protons absorbed upon binding to oxyhemoglobin was determined as well. The maximum number of protons bound to deoxyhemoglobin upon binding of D-glycerate 2,3-bisphosphate was measured independently. From the pH dependence of the association constants and the maximum number of protons absorbed it could be concluded that only one D-glycerate 2,3-bisphosphate can be bound to both deoxyhemoglobin and oxyhemoglobin.


Proton-nuclear-magnetic-resonance/pH-titration studies of the histidines of pancreatic phospholipase A2. The study by means of 1H nuclear magnetic resonance (NMR) of the histidines of phospholipase A2 isolated from porcine, bovine and equine pancreas is reported. Assignment of the histidine resonances was achieved by comparison of different enzymes and the use of paramagnetic probes. pH titration curves for various histidyl resonances were obtained and compared in the presence and absence of calcium. Calcium is shown to lower the pKa of the active site histidine. The NMR results are compared with the known X-ray three-dimensional structure for the bovine enzyme.


Procollagen processing. Limited proteolysis of COOH-terminal extension peptides by a cathepsin-like protease secreted by tendon fibroblasts. An enzymatic activity, capable of removing the COOH-terminal extensions of type I chick procollagen, has been demonstrated in embryonic chick tendons and in cultured tendon fibroblasts utilizing two new methods of analysis. The protease was purified by a combination of ultrafiltration concanavalin A affinity chromatography and gel filtration. The isolated protein has an apparent Mr of 43,000 by gel filtration and sodium dodecyl sulfate gel electrophoresis. The enzyme shows a major pH optimum at 4.2 and is susceptible to inhibitors such as pepstatin and leupeptin; it therefore seems related to the cathepsins. The possibility that this enzyme plays a role in the limited proteolytic processing of procollagen is discussed.


Metabolic acidosis in patients receiving anticonvulsants. Blood pH, bicarbonate, PCO2, serum calcium, alkaline phosphatase and red cell carbonic anhydrase were measured in 37 selected patients receiving anticonvulsants. Patients with metabolic acidosis showed a high incidence of hypocalcemia with increased alkaline phosphatase and a significant reduction of carbonic anhydrase-B activity. High iPTH levels were found in 13 patients, but this was not correlated with acid-base balance status. Anticonvulsant drugs seemed to inactive carbonic anhydrase-B activity. Metabolic acidosis might be one of the factors causing a disturbance of calcium metabolism in these patients.


Role of the renin-angiotensin system in the blood pressure rebound to sodium nitroprusside in the conscious rat. Intravenous infusions of sodium nitroprusside (SNP) at doses of 20, 40 or 80 micrograms/kg min-1 for 30 min produced dose-related decrements in blood pressure in conscious rats fitted with indwelling aortic and vena caval catheters. Immediately upon termination of SNP infusions, blood pressure rebounded to levels which were significantly above pre-SNP control values. The following evidence indicates that the rebound increase in blood pressure was due to increased activity of the renin-angiotensin system: (1) plasma renin activity was increased approximately four-fold by SNP, (2) rebound did not occur in nephrectomized rats, (3) rebound was markedly attenuated in animals treated with an angiotensin converting enzyme inhibitor, SQ14225, (D-3-mercapto-2-methylpropanoyl-L-proline) and (4) beta-adrenergic receptor blockade with propranolol reduced the rebound response. In addition, the magnitude of the rebound following SNP infusions was directly related to the dose of SNP infused. These results are consistent with the hypothesis that renin accumulates during SNP infusion more rapidly than it is metabolized. Consequently, the accumulated renin elicits a hypertensive response when SNP treatment is withdrawn.


A methodological weakness in the use of neuroleptic antagonism as a sole criterion for DAergic mediation of drug-induced behavioural effects. Contralateral rotational behavior following unilateral injections of the GABA agonist muscimol (100 ng) or the GABA analogue baclofen (1 microgram) into the zona reticulata of the rat substantia nigra was moderately attenuated by pretreatment with haloperidol (0.4 mg/kg i.p.), but not by destruction of ipsilateral dopamine (DA) neurons induced with 6-hydroxydopamine. Antagonism by neuroleptics can erroneously indicate DAergic mechanisms in the behavioural effects of drugs if used as a sole criterion.


The renal effects of clonidine in unanesthetized rats. Clonidine s.c. (0.01-0.3 mg/kg), in unanesthetized rats, caused an initial rise (+20 mm Hg), followed by a continuous fall of BP and a dose-dependent natriuresis and diuresis for up to 2 h. Glomerular filtration rate (GFR) (CIn) increased during the first 20 min, while effective renal plasma flow (ERPF) (CPAH) remained normal. Subsequently, between 20 and 60 min after injection, ERPF (CPAH) decreased considerably while GFR had reverted to its normal value. In saline-infused rats clonidine diuresis was accompanied by an "inappropriate" positive free water clearance. Pentobarbital anesthesia suppressed the initial BP peak and the diuresis. Phenoxybenzamine (1 mg/kg i.v.) was antinatriuretic in saline diuresis; the effect of phenoxybenzamine + clonidine on diuresis and salt excretion represented the sum of the effects of both drugs, but phenoxybenzamine enhanced the clonidine-induced increase of GFR. Neither haloperidol (1 mg/kg i.v.) nor bulbocapnine (3 mg/kg i.v.) interfered with the renal effects of clonidine. Clonidine s.c. caused hyperglycemia and glucosuria which did not account for the natriuresis. Clonidine thus appears to increase the GFR and "filtration fraction" (FF) by a phenoxybenzamine-insensitive rise of glomerular ultrafiltration, to depress ERPF by alpha-adrenergic afferent vasoconstriction, to induce natriuresis by a tubular action not blocked by phenoxybenzamine and to exert an antivasopressin effect, either by depressing pituitary vasopressin secretion or the renal response to vasopressin.


Antiulcer activity of hypertonic solutions in the rat: possible role of prostaglandins. The effects of hypertonic solutions on gastric acidity and on experimental gastric and duodenal ulcers as well as on gastric prostaglandins (PGs) were studied in the rat. The oral administration of a 10% NaCl solution resulted in complete absence of free acidity and very significant reductions in total acidity 24 h after pyloric ligation. The antiulcer effect of hypertonic saline was studied in three experimental models. In pyloric-ligated rats, both the incidence and the severity of gastric ulcers were remarkably reduced by hypertonic saline treatment. Indomethacin-induced gastric erosions were significantly reduced by hypertonic NaCl or sorbitol and completely prevented by hypertonic xylitol. Cysteamine-induced duodenal ulcers were also significantly reduced by hypertonic solutions of NaCl, xylitol or sorbitol. In the latter model, indomethacin potentiated the ulcerogenic effect of cysteamine and also reduced the efficacy of the hypertonic NaCl gavage. The possible contribution of PGs to these effects was further investigated by analysing PGE in the gastric mucosa and juice. Rats treated orally with hypertonic NaCl solutions had several-fold higher PGE contents in their gastric mucosa as well as higher PGE levels in the gastric juice. It is concluded that hypertonic solutions stimulate endogenous PGE biosynthesis and also exert profound antiulcer effects in the rat. A causal relationship between the two phenomena is suggested.


Tricyclic antidepressants: potent blockade of histamine H1 receptors of guinea pig ileum. Six tricyclic antidepressants were tested for their ability to antagonize histamine actions at histamine H1 receptors in a bioassay for these receptors (histamine-induced contractions of guinea pig ileum). All compounds were competitive antagonists with equilibrium dissociation constants in the range of 5.6 x 10(-11) M to 1.5 x 10(-7) M. Doxepin hydrochloride and amitriptyline hydrochloride were the most potent compounds of the series and may be the most potent antihistamines known. Antagonism at histamine H1 receptors by these compounds may explain their sedative effects.


Correlation between the increase in tyrosine hydroxylase activity and the decrease in serotonin content in the rat locus coeruleus after 5,6-dihydroxytryptamine. A correlation is demonstrated between the increase in tyrosine hydroxylase activity and the decrease in serotonin concentration in the rat locus coeruleus after various doses of 5,6-dihydroxytryptamine and gradual protection of serotonin neurons by pretreatment with two serotonin uptake inhibitors: fluoxetine or citalopram.