[The myocardiopathies of dermatomyositis and periarteritis nodosa]. Myocardial involvement was indicated by isolated findings (especially on the ECG) without corresponding functional disorders in one third of cases of dermatomyositis. Rapidly fatal heart failure occurred rarely in this disease, and was noteworthy for a QS complex in the central precordial leads, and for the presence of intracardiac thromboses which could give rise to emboli. Terminal cardiac failure was the rule in cases of periarteritis nodosa, which is a much rarer disease; this occurrence was secondary to hypertension. The most characteristic lesions were nodular coronary arteritis and disseminated myocarditis due to the arteriolar lesions.


Experiments on the preparations and testing of associated vaccine against foot and mouth disease and vesicular disease in swine. Tests for associated immunization of swine against Foot and Mouth Disease (FMD) and Vesicular Disease (SVD) of swine were carried out. As a result of this investigation, it was established that the prepared and tested inactivated oil vaccine is harmless and immunogenic in sensitive animals. In investigating the course of immunity, the presence of antibody against both antigens was demonstrated in vaccinated animals. All once-vaccinated animals were defended against the virus of SVD during challenge, and 75% of them were defended against FMD. After revaccination, all immunized swine were defended against infection with both viruses. The question of the quality of the associated vaccine and the possibilities of its massive use in industrial swine rearing was discussed.


[Properties and isozymes of acid and alkaline phosphatase in cattle testes and endometrium]. The activity of acid phosphatase in the testicular parenchyma of cattle was found to be higher than that in the endometrium. The acid phosphatase of either tissue broke up p-nitrophenylphosphate at a rate twice as high as that reached for di-sodium phenylphosphate. Hydrolysis of phenolphthalein diphosphate was slower and that of beta-glycerophosphate very slow. Two optimum pH values and two isozymes were recordable from acid phosphatase in testicular supernatant and three optimum pH values as well as three isozymes from that in endometrial supernatant. Even concentrations pf 0.2 mM of copper and mercury ions were strongly inhibiting the acid phosphatase, whereas lead and cadmium ions proved less effective. The activity of acid phosphatase from testes dropped by 24 per cent and that from the endometrium by 16 per cent over eight weeks of storage at -20 degrees C. The average activity of alkaline phosphatase in the endometrium was much higher than that in testicular parenchyma. Alkaline phosphatase from testes exhibited the shortest break-up time for p-nitrophenylphosphate, whereas disodium-phenylphosphate was broken up at the highest rate by endometrial alkaline phosphatase. Two isozymes of alkaline phosphatase were recordable each from the testes and endometrium.


[Relationship between the limbic system and gonadal function. 3. Quantifiable prepuberal morphokinesis of the basolateral amygdaloid body and the ventral hippocampus formation]. In juvenile female rats the neurons of the limbic system which are attached to the basolateral amygdaloid nucleus and to a circumscribed area of ventral hippocampal formation are characterised by markedly quantifiable morphokinesis between the 21st day of age and onset of the first oestrus. This morphokinesis will take the form of consecutively occurring highly significant volume alterations of the cell nuclei which, in turn, reflect variation in neuronal activity. Some of such variation represents a expression of the general maturation and differentiation of central nervous neurons. The time of occurrence of such variation as well as its peculiar nature also appear to suggest the existence of some special relationship to sexual maturation. This seems to support the conclusion that the basolateral amygdaloid nucleus and the ventral hippocampal formation are attached to the extrahypothalamic central nervous structures of the cerebro-hypophyseal-gonadal axis, in addition to the medial and cortical amygdaloid nuclei. However, the very nature of relationships that may exist between the two investigated components of the limbic system, on the one hand, and sexual maturation as well as gonadal function, on the other, cannot be derived from the results reported in this paper.


[Blood acid-base status in fattening pigs in exhausting physical stress of high intensity]. Reported in this paper are changes in the acid-base state and lactic concentration of blood and muscle samples taken from 28 pigs prior to, during, and after high-intensity exhausting stress applied at ambient temperatures between 17 degrees C and 21 degrees C and at 35 degrees C. Close correlations were found to exist between acid-base states, on the one hand, and lactic acid concentrations, on the other. The lactic acid concentrations reached in blood plasma were between 30 and 40 mHol/1, pH being below 7.0 and BE values being smaller than -22 mHol/1. Close relationships were established between the lactic acid concentration of venous blood plasma, blood pH, and the pH of the musculus longissimus dorsi. Respiratory compensation was of minor importance with ambient temperatures between 17 degrees C and 21 degrees C, but it was of considerable effectiveness at 35 degrees C.


[Studies on diarrhea of young calves. 1. Quantitative analysis of the gastrointestinal flora in clinically healthy calves and in calves with diarrhea]. Quantitative composition of gastro-intestinal-flora was determined in 23 clinically healthy calves and the course of germ spreading was followed up. In comparison with these healthy animals in 25 diarrhoea affected calves at the age of 4 to 17 days there could be observed signigicant differences only in few parts of the gastro-intestinal-tract concerning the quantitative composition of germ flora. Only in 3 animals enterotoxin producing E coli strains could be detected.


Stabilization of human fibroblast interferon purified on concanavalin A-agarose. Human fibroblast interferon, obtained by chromatography on concanavalin A-agarose, was stable for at least a month in 30--50 per cent ethylene glycol at 4 degrees, --20 degrees, and --70 degrees C. The succinct point of the present finding is that human fibroblast interferon may be stabilized by ethylene glycol alone without the addition of bovine serum albumin and 'back-contamination' of the interferon preparation.


Inhibition of thermogenic drinking by beta-adrenergic antagonists. The thirst induced by transfer of rats from air at 5 degrees C to air at 25 degrees C (thermogenic drink) was inhibited by d,1-, but not d-propranolol (6 mg/kg body weight) administered 0.5 h prior to removal from cold. The thermogenic drink was not inhibited by a similar administration of the beta1-adrenergic antagonist, practolol (50 and 150 mg/kg body weight), but was inhibited by the beta2-adrenergic antagonist, butoxamine (35 mg/kg body weight). The results are consistent with the suggestion that beta2-adrenergic receptors play a role in mediation of the thermogenic drink in rats.


The effects of acridine orange on deoxyribonucleic acid in Escherichia coli. 1. Acridine Orange inhibits growth of Escherichia coli K12 when incubated at pH 7.9, but not at pH 7.4.2. At a non-permissive temperature for DNA polymerase I, Acridine Orange inhibits growth of a temperature-sensitive strain and also increases the rate of elimination of the F'-Lac plasmid. 3. DNA isolated from cells treated with Acridine Orange under conditions that inhibit growth contains material of low molecular weight, which is absent from DNA isolated from cells treated under conditions in which growth is not impaired. 4. Cells incubated with Acridine Orange at both pH 7.4 and 7.9 suffer degradation of DNA, as shown by loss of labelled DNA from the acid-insoluble fraction, which is not observed with untreated cells at either pH. 5. The results suggest that elimination of the F'-Lac plasmid by Acridine Orange requires inactivation of repair processes.


Electron-paramagnetic-resonance studies on nitrate reductase from Escherichia coli K12. Nitrate reductase was purified from anaerobically grown Escherichia coli K12 by a method based on the Triton X-100 extraction procedure of Clegg[(1976) Biochem. J.153, 533-541], but hydrophobic interaction chromatography was used in the final stage. E.p.r. spectra obtained from the enzyme under a variety of conditions are well resolved and were interpreted with the help of the computer-simulation procedures of Lowe [(1978) Biochem. J.171, 649-651]. Parameters for five molybdenum(V) species from the enzyme are given. The low-pH species (g(av.) 1.9827) is in pH-dependent equilibrium with the high-pH species (g(av.) 1.9762), the pK for interconversion of the species being 8.26. Of a variety of anions tested, only nitrate and nitrite formed complexes with the enzyme (in the low-pH form), giving modified molybdenum(V) e.p.r. spectra. These complexes, as well as the low-pH form of the free enzyme, showed interaction of molybdenum with a single exchangeable proton. The fifth molybdenum(V) species, sometimes detected in small amounts, appears not to be due to functional nitrate reductase. After full reduction of the enzyme with dithionite, addition of nitrate caused reoxidation of molybdenum to the quinquivalent state, in a time less than the enzyme turnover. Activity of the enzyme in the pH range 6-10 is controlled by a pK of 8.2. It is suggested that the low-pH signal-giving species is the form of the enzyme involved in the catalytic cycle. Iron-sulphur and other e.p.r. signals from the enzyme are briefly described and the enzymic reaction mechanism is discussed.


Oxygen-binding characteristics of three extracellular haemoglobins of Artemia salina. The oxygen-binding characteristics of the three extracellular haemoglobins of brine shrimp (Artemia salina) were studied in vitro by using highly purified preparations. Haemoglobin I is induced last in the development of brine shrimps when functional gills are formed. It has the lowest oxygen affinity (p(50) 5.34mmHg), an intermediate Bohr effect (ø -0.09 at 20 degrees C) above pH8 and a temperature-sensitivity (DeltaH -44.8 to -45.6kJ/mol at pH8-9) comparable with those observed with other invertebrate haemoglobins [Weber & Heidemann (1977) Comp. Biochem. Physiol. A57, 151-155]. Haemoglobin II, which is the first to be induced, soon after hatching of nauplius larvae, persists generally throughout the whole adult life. It has an intermediate oxygen affinity (p(50) 3.7mmHg), the highest Bohr effect (ø -0.21 at 20 degrees C) above pH8 and a similar temperature-sensitivity (DeltaH -46.0 to -54.8kJ/mol at pH8-9) as haemoglobin I. However, haemoglobin III, which is induced second several hours after the induction of haemoglobin II but disappearing from the haemolymph in the middle of adult life, has the highest oxygen affinity (p(50) 1.8mmHg), the lowest Bohr effect (ø -0.03 at 20 degrees C) above pH8.5 and a high resistance against temperature variation between 10 and 25 degrees C at pH8.5-9 (DeltaH -22.6 to -23.0kJ/mol). At pH7.5-8, haemoglobin III exhibits a similar temperature-sensitivity under 30 degrees C as do other haemoglobins. All three haemoglobins have a rather low co-operativity, with Hill coefficients (h 1.6-1.9 at pH8.5), which are dependent on both pH and temperature. The highest co-operativity was observed at 20 degrees C and pH9 for haemoglobins I and II, whereas it was at 27 degrees C and pH8.5 for haemoglobin III. Thus the oxygen-binding behaviour of haemoglobin III in vitro is significantly different from those of haemoglobins I and II and indicates possibly its specific physiological role in vivo in the adaptive process in the natural environment.


Equilibrium binding of coenzymes and substrates to nicotinamide-adenine dinucleotide phosphate-linked isocitrate dehydrogenase from bovine heart mitochondria. 1. The stoicheiometries and affinities of ligand binding to isocitrate dehydrogenase were studied at pH 7.0, mainly by measuring changes in NADPH and protein fluorescence. 2. The affinity of the enzyme for NADPH is about 100-fold greater than it is for NADP+ in various buffer/salt solutions, and the affinities for both coenzymes are decreased by Mg2+, phosphate and increase in ionic strength. 3. The maximum binding capacity of the dimeric enzyme for NADPH, from coenzyme fluorescence and protein-fluorescence measurements, and also for NADP+, by ultrafiltration, is 2 mol/mol of enzyme. Protein-fluorescence titrations of the enzyme with NADP+ are apparently inconsistent with this conclusion, indicating that the increase in protein fluorescence caused by NADP+ binding is not proportional to fractional saturation of the binding sites. 4. Changes in protein fluorescence caused by changes in ionic strength and by the binding of substrates, Mg2+ or NADP+ (but not NADPH) are relatively slow, suggesting conformation changes. 5. In the presence of Mg2+, the enzyme binds isocitrate very strongly, and 2-oxoglutarate rather weakly. 6. Evidence is presented for the formation of an abortive complex of enzyme-Mg2+-isocitrate-NADPH in which isocitrate and NADPH are bound much more weakly than in their complexes with enzyme and Mg2+ alone. 7. The results are discussed in relation to the interpretation of the kinetic properties of the enzyme and its behaviour in the mitochondrion.


Transient kinetics of nicotinamide-adenine dinucleotide phosphate-linked isocitrate dehydrogenase from bovine heart mitochondria. Pre-steady-state studies of the isocitrate dehydrogenase reaction show that the rate constant for the hydride-transfer step is above 990s-1, and that both subunits of the enzyme are simulataneously active. After the fast formation of NADPH in amounts equivalent to the enzyme subunit concentration, the rate of NADPH formation is equal to the steady-state rate if the enzyme has been preincubated with isocitrate and Mg2+. If the enzyme has been preincubated with NADP+ and Mg2+, in 0.05 M-triethanolamine chloride buffer, pH 7.0, with the addition of 0.1 M-NaCl, the amount of NADPH formed in the fast phase is only 60% of the enzyme subunit concentration, and the turnover rate is at first lower than the steady-state rate. In 0.05 M-triethanolamine chloride buffer, pH 7.0, if the enzyme is preincubated with NADP+ or NADPH, the turnover rate increases 3-fold to reach the steady-state rate after about 5 s. Preincubation of the enzyme with isocitrate and Mg2+ abolishes this lag phase, the steady-state rate being reached at once. It is suggested that the enzyme exists in at least two conformational forms with different activities, and that the lag phase represents the transition (k = 0.4s-1) from a form with low activity to the fully active enzyme, induced by the binding of isocitrate and Mg2+.


Purification and some properties of an alkaline proteinase from rat skeletal muscle. 1. Rat skeletal muscle was homogenized in 0.05M-Tris/HCl, pH 8.5, containing 1M-KCl. Myofibrillar proteins were precipitated by addition of (NH4)2SO4 (33% saturation). 2. The alkaline proteolytic activity that was precipitated with the myofibrillar proteins was solubilized with trypsin (conjugated to Sepharose) and further purified by affinity chromatography, ion-exchange chromatography and gel filtration. 3. The purified enzyme migrates as a single band in polyacrylamide-disc electrophoresis, and has optimum hydrolytic activity with azocasein and [14C]haemoglobin as substrates at pH 9.4 and 9.6 respectively. Its apparent molecular weight, as determined by gel filtration on Sephadex G-75, is 30800. 4. The purified alkaline proteinase is strongly inhibited by equimolar amounts of soya-bean trypsin inhibitor and ovomucoid, whereas di-isopropyl phosphorofluoidate and alpha-toluenesulphonyl fluoride have no effect. On the other hand N-ethylmaleimide and p-chloromercuribenzoate have inhibitory effects on the enzyme activity. 5. Bivalent metal ions (Fe2+, Co2+, Zn2+, Mg2+, Mn2+) diminish the proteolytic activity, at 1mM concentrations. Ca2+ ions and the metal-ion-chelating agent EDTA are without effect on enzyme activity. 6. The enzyme is part of the alkaline proteolytic activity that appears to be associated with myofibrillar proteins.


Association of defective feedback suppressor T cell activity with autoimmunity in NZB mice. A "feedback suppressor T cell" highly dependent on signals from Ly 1 T helper cells for activation is described. The signal from the Ly 1 cell binds to Fc-like receptors on macrophage membranes. The feedback suppressor cell expresses all three Ly antigens as well as the Qa 1 antigen on its surface, is very sensitive to low doses of cyclophosphamide, disappears relatively rapidly after adult thymectomy, and cannot be demonstrated in NZB mice 6 weeks or older.


Some clinical pharmacological studies with terfenadine, a new antihistamine drug. 1 A double-blind cross-over trial between placebo, chlorpheniramine, and terfenadine, a new antihistamine drug, was performed in healthy male volunteers to determine and compare their CNS and autonomic effects. 2 Terfenadine and chlorpheniramine were administered orally in therapeutic doses. 3 In objective tests of critical flicker frequency, pursuit rotor, reaction time, salivary volume and pupillary diameter, no statistically significant difference was observed between the treatments. 4 On analogue rating scales, chlorpheniramine produced a statistically significant (P less than 0.05) degree of sedation and impaired concentration as compared to placebo and terfenadine. 5 The results obtained in analogue rating scales were not normally distributed and, therefore, use of non-parametric statistical methods for analysis of such data is strongly advocated.


Performance studies with antihistamines. 1 Effect of four antihistamines, chlorpheniramine (4 mg), clemastine (1 mg), promethazine (10 mg) and terfenadine (60 mg), on visuo-motor coordination and on subjective assessments of performance and well-being were compared with placebo in six healthy females from 0.5--7.0 h after morning ingestion of each drug. The study was double-blind, and the doses used were believed to be equally potent in their antihistaminic activity. 2 There was impaired performance 1.5 h (P less than 0.01) after chlorpheniramine, 3.0 h (P less than 0.05) and 5.0 h (P less than 0.01) after clemastine, and 3.0 h (P less than 0.01) and 5.0 h (P less than 0.001) after promethazine. It was not possible to establish effects on performance after ingestion of terfenadine. Subjective assessments of performance were not altered. 3 The subjects as a group reported improved alertness (P less than 0.05) and improved wakefulness (P less than 0.05) 0.5 h and 3.5 h respectively after ingestion of terfenadine, and were less energetic (P less than 0.05) 7.0 h after ingestion of chlorpheniramine. There were not other consistent changes in assessments of well-being.


The acute effects of the administration of rimiterol aerosol in asthmatic children. 1 Ten asthmatic children received on two separate occasions rimiterol (500 microgram) and orciprenaline (1.5 mg) as aerosols in a double-blind, crossover design trial. Respiratory parameters (FEV1, FVC and PEFR) and pulse rate were recorded for 60 min after each administration. 2 Rimiterol produced effective bronchodilation in children with negligible cardiac stimulation. It differed from orciprenaline in that the peak bronchodilator effect was not maintained during the 60 min observation period. 3 Rimiterol is a selective, short-acting, sympathomimetic bronchodilator in children.


Periarteritis nodosa and thrombotic thrombocytopenic purpura with serous retinal detachment in siblings. Periarteritis nodosa and thrombotic thrombocytopenic purpura in siblings is reported. In both patients a localised serous retinal detachment and lesions of the retinal pigment epithelium had developed owing to choroidal vascular obstruction. These cases support the suggested possible relationship between the two conditions.


Gastric secretion in suckling pigs and early-weaned pigs given a dry cow's-mild formula ad lib. 1. Twelve gastric-cannulated litter-mate pigs were used to study secretion and proteolytic activity in the stomach of suckling and early-weaned pigs in relation to age and food intake. 2. Results demonstrate that from the first observation at day 8, piglets were able to secrete acid. pH and acid concentration did not change during the first 4 weeks of life. 3. Proteolytic activity was low during the first 2--3 weeks of life and rapidly increased thereafter. 4. Two phenomena differentiated suckling pigs from pigs given dry cow's milk: (1) a low buffering capacity the gastric contents, beginning 1 hr after feeding the dry cow's-milk formula, results in a low total acid concentration in the weaned pigs and (2) the increase in proteolytic activity in relation to the age is much more pronounced in the artificially-reared pigs. 5. These two phenomena are discussed and related to the formation of a hard casein clot in the stomach of the cow's-milk-fed pigs, which has a long retention time and stimulates gastrin release.


Studies on the microsomal mixed function oxidase system: redox properties of detergent-solubilized NADPH-cytochrome P-450 reductase. Hepatic microsomal NADPH-cytochrome P-450 reductase was solubilized from rabbit liver microsomes in the presence of detergents and purified to homogeneity by column chromatography. The purified reductase had a molecular weight of 78 000 and contained 1 mol each of FAD and FMN per mol of enzyme. On reduction with NADPH in the presence of molecular oxygen, an 02-stable semiquinone containing one flavin free radical per two flavins was formed, in agreement with previous work on purified trypsin-solubilized reductase. The reduction of oxidized enzyme by NADPH, and autoxidation of NADPH-reduced enzyme by air, proceeded by both one-electron equivalent and two-electron equivalent mechanisms. The reductase reduced cytochrome P-450 (from phenobarbital-treated rabbits) and cytochrome P-448 (from 3-methylcholanthrene-treated rabbits). The rate of reduction of cytochrome P-450 increased in the presence of a substrate, benzphetamine, but that of cytochrome P-448 did not.


Reactions of reduced nicotinamide adenine dinucleotide in acid: studies by reversed-phase high-pressure liquid chromatography. Reversed-phase high-pressure liquid chromatography was used to isolate acid breakdown products of reduced nicotinamide adenine dinucleotide (NADH) and products produced when NADH breakdown is catalyzed by glyceraldehyde-3-phosphate dehydrogenase (G-3-PD). Chromatographic and UV spectral data on these and related products support a mechanism for NADH acid degradation involving hydroxy addition at the nicotinamide C-6 followed by cyclization of the ring and the adjacent ribose moiety. G-3-PD is shown to catalyze a reaction in which two products are formed which are also intermediates in the acid degradation of NADH (alpha- and beta-6-hydroxynicotinamide products). Formation of the major acid products fits a three-step, first-order mechanism curve, making it possible to calculate the rate constants k2 and k3 as well as the previously determined k1.


Affinity chromatography of H+-translocating adenosine triphosphatase isolated by chloroform extraction of Rhodospirillum rubrum chromatophores. Modification of binding affinity by divalent cations and activating anions. 1. ATPase isolated from Rhodospirillum rubrum by chloroform extraction and purified by gel filtration or affinity chromatography shows three bands (alpha, beta and gamma) upon electrophoresis in sodium dodecyl sulphate. 2. Ca2+-ATPase activity of the preparation is inhibited by aurovertin and efrapeptin but not by oligomycin. Activity may be inhibited by treatment with 4-chloro-7-nitrobenzofurazan and subsequently restored by dithiothreitol. 3. The enzyme fails to reconstitute photophosphorylation in chromatophores depleted of ATPase by sonic irradiation. 4. Most of the active protein from the crude chloroform extract binds to an affinity chromatography column bearing an immobilised ADP analogue but not to a column bearing immobilised pyrophosphate. 5. In the absence of divalent cations, a component with a very high specific activity for Ca2+-ATPase is eluted from the column by 1.6 mM ATP. This protein migrates asa single band on 5% polyacrylamide gel electrophoresis and only possesses three subunits. At 12 mM ATP an inactive protein is eluted which does not run on acid or alkali polyacrylamide gels and shows a complex subunit structure. 6. ATPase preparations prepared by acetone extraction or by sonic irradiation of chromatophores may also be purified 10-fold by affinity chromatography. 7. The inclusion of 5 mM MgCl2 or CaCl2 during affinity chromatography of chloroform ATPase increases the capacity of the column for the enzyme and demands a higher eluting concentration of ATP. 8. When the enzyme is more than 90% inhibited by efrapeptin or 4-chloro-7-nitrobenzofurazan, the binding characteristics of the enzyme are not affected. 9. 10 mM Na2SO3, which greatly stimulates the Ca2+- and Mg2+-dependent ATPase activity of the enzyme and increases Ki (ADP) for Ca2+-ATPase from 50 to 850 micron, prevents binding to the affinity column. Binding may be restored by the addition of divalent cations. 10. Na2SO3 increases the rate of ATP hydrolysis, ATP-driven H+ translocation and ATP-driven transhydrogenase in chromatophores. 11. It is proposed that anions such as sulphite convert the chromatophore ATPase into a form which is a more efficient energy transducer.


Synergism of triggered luminescence by simultaneous treatment of pH transition and potassium addition in chloroplasts. KCL-induced luminescence in relation to slow delayed light emission (greater than 3 s) and pH shift-triggered luminescence was studied in preilluminated chloroplasts. An activation pathway for KCl-induced luminescence similar to that for acidbase-triggered luminescence but different from that for delayed light emission is suggested. When the chloroplasts were subjected to a small amount of pH transition together with a simultaneous addition of KCl, a synergistic enhancement of triggered luminescence was observed. The synergism was not observed when the pH transition was increased. The results are interpreted according to the protonation model for stimulated luminescence.


Interaction of fluorinated ether anesthetics with artificial membranes. Fluorine-19 nuclear magnetic resonance spectroscopy is applied to the study of the environment of dipalmitoyl phosphatidylcholine-bound fluorinated ether anesthetics (enflurane, fluoroxene and methoxyflurane) both below and above the lipid gel to liquid crystal phase transition temperature. Line widths and spin-lattice relaxation time (T1) measurements are consistent with substantial immobilization of the lipid-bound anesethetic molecules. Heating anesthetic/lipid mixtures above the lipid transition temperature leads to narrowing of the lipid-bound anesthetic fluorine resonances accompanied by little or no change in anesthetic fluorine-19 chemical shifts, suggesting that although the mobility of the bound anesthetic increases at the higher temperature, the nature of the anesthetic-lipid interaction changes little as a result of this phase change. Differential scanning calorimetric studies of the effects of these anesthetics on the phase transition behavior of the phospholipid indicate that the regions of the bilayer in which volatile anesthetics partition at lower concentrations are different from the regions in which they partition at higher concentrations.


Characterization of two poly(A) polymerases from cultured hamster fibroblasts. The enzymatic and physiochemical properties of poly(A) polymerases IIA and IIB from cultured hamster fibroblasts were investigated. The enzymes show an absolute requirement for Mn2+ as the divalent ion. Although Mg2+ alone is inactive, maximum activity is observed in the presence of both Mn2+ and Mg2+. An optimal pH of approx. 8 is found for polymerases IIA and IIB. The enzymes, however, differ somewhat in the pH curves as well as in the Mn2+ and Mg2+ concentration curves. Poly(A) polymerases IIA and IIB have an isoelectric point of about 6 and a sedimentation coefficient of 3.5--4 S. The molecular weights, obtained by gel filtration chromatography, are 145 000 and 155 000 for enzymes IIA and IIB, respectively. Poly(A) polymerases IIA and IIB can utilize a variety of natural and synthetic RNAs as well as DNA as primers. Poly(A) polymerase IIA is saturated at much lower concentrations of primer than enzyme IIB. On the other hand, the chain length of the product synthesized by polymerase IIA is independent of the primer concentration, whereas, with polymerase IIB, the length of the product decreases when the concentration of RNA is increased.


The phosphorylation of ribosomal proteins L14 and S3 in Krebs II ascites cells. Krebs II ascites cells incubated in Earle's saline (lacking glucose and amino acids) contain ribosomes with proteins S6 and Lgamma phosphorylated, as do ascites cells grown in the peritonea of mice or hamster fibroblasts grown in Eagle's medium. When ascites cells were incubated in Eagle's medium (containing glucose and amino acids) there was extensive glycolysis, producing very acidic conditions, and ribosomal proteins S3 and L14 became phosphorylated whereas Lgamma became dephosphorylated. This altered pattern of phosphorylation could not be produced merely by incubating ascites cells in Earle's saline at a decreased pH, but a rather similar pattern was produced when Earle's saline was supplemented with amino acids (but with glucose still omitted). These results suggest that depriving ascites cells of glucose may induce the synthesis of a protein (or proteins), necessary for alteration of the pattern of phosphorylation of the ribosomal proteins.


Protein methylation in animal cells. I. Purification and properties of S-adenosyl-L-methionine:protein (arginine) N-methyltransferase from Krebs II ascites cells. 1. A protein methylase which specifically transfers methyl groups from S-adenosyl-L-methionine to arginine residues of histones has been substantially purified from Krebs II ascites cells. The purified enzyme was obtained free of contamination by other protein methyl transferases specific for carboxyl and lysine residues. This latter activity copurified with the present enzyme until advanced stages of purification. 2. The purified enzyme does not require any divalent cation for maximum activity. It is inhibited by ionic strength, N-ethylmaleimide and S-adenosyl-L-homocysteine. It has an apparent molecular weight on gel filtration of approx. 5 . 10(5). A Km value for S-adenosyl-L-methionine of 2.5 . 10(-6) M was determined, while the dissociation constant Ki for S-adenosyl-L-homocysteine, which acts as a competitor, was 1.4 . 10(-6) M.


Poly(A) polymerases of rat liver nuclei. Purification and specificity. Two poly(A) polymerases were isolated from rat liver nuclei and purified more than one thousand times by ion exchange chromatography on DEAE-Sephadex and phosphocellulose columns as well as affinity chromatography on a chromosomal RNA-Sepharose column. One of the two enzymes is bound to chromatin and uses as primer chromosomal RNA, while the second one is localized in the nucleoplasm and uses as primer poly(A) and hnRNA isolated from chromatin. The two enzymes seem to participate in the polyadenylation of chromosomal RNA in vitro, by a coupled mechanism. According to this mechanism, the chromatin bound enzyme adds 120-130 adenosine nucleotides to chromosomal RNA and consequently the nucleoplasmic enzyme completes the poly-adenylation by adding 80-90 more AMP units to the polyadenylated end of chromosomal RNA.


Inhibition of ribonuclease. Efficacy of sodium dodecyl sulfate, diethyl pyrocarbonate, protein ase K and heparin using a sensitive ribonuclease assay. The effectiveness of several commonly used inhibitors of ribonuclease (RNAase) has been studied using the removal of radio-labelled leucine from leucyl-tRNA as a sensitive assay for RNAase activity. The inhibitors were tested under a variety of conditions, varying the temperature, the pH, and the source of RNAase. When each inhibitor is udes separately in the presence of pancreatic RNAase, sodium dodecyl sulfate (SDS) is the most effective; but during long exposures to temperatures above 0 degrees C considerable amounts of RNA  are still degraded. Combination of inhibitors are more effective in preserving RNA; with this assay, a combination of SDS with diethyl pyrocarbonate is the most effective. Proteinase K acts as an inhibitor when used in combination with SDS; however, it has RNAase activity when used by itself. Diethyl pyrocarbonate, when used at the high range of concentrations employed by others for RNAase inhibition, reacts with RNA changing its charge. However, when diethyl pyrocarbonate is used in smaller amounts the effects on RNA are minimal, and when used in combination with SDS it effectively inhibits RNAase.


Intramembraneous localization of rat liver microsomal hexose-6-phosphate dehydrogenase and membrane permeability to its substrates. A method for purifying hexose-6-phosphate dehydrogenase (beta-D-glucose: NAD(P) -oxidoreductase, EC 1.1.1.47) from rat liver microsomes is described. The purified enzyme was shown to be homogeneous by sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis. It is shown that the enzyme is bound to the inner surface of microsomal membranes, and that glucose 6-phosphate, but not NADP, penetrates almost freely into the membranes at 37 degrees C.


Binding of m-nitrobenzeneboronic acid to the active site of subtilisin BPN. m-Nitrobenzeneboronic acid as a possible transition-state analog for serine proteases was found to cause absorption spectral change from 250 nm 350 nm upon binding with subtilisin BPN' (EC 3.4.21.14) at pH 6.5. Similar difference spectral changes of m-nitrobenzeneboronic acid were also observed at alkaline pH or upon addition of N-methylimidazole at pH 6.5. A characteristic circular dichroism spectrum of m-nitrobenezeneboronic acid was induced upon binding with subtilisin BPN' not only at pH 6.5, but also at alkaline pH. Circular dichroism spectral titration confirmed the stoichiometry of 1 : 1 for the m-nitrobenzeneboronic acid - subtilisin complex. m-Nitrobenzeneboronic acid was shown to be useful as a reversible chromophoric probe for the catalytic site of serine proteases.


Excitation of indole-3-acetic acid (an auxin) in a linoleate-lipoxygenase system. The weak luminescence that accompanies the linoleate-lipoxygenase reaction was greatly enhanced by the addition of indole analogues, and especially indole acetic acid. The main emitting species in the indole acetic acid-linoleate-lipoxygenase system was analysed spectrophotometrically in the visible region and ascribed to the transition of excited indole acetate in triplet state to its ground state. Such an excited indole acetate could be generated by transfer of energy from the excited CO2 and excited carbonyl (generated by the linoleate-lipoxygenase reaction) to indole acetate in the ground state, but not by cleavage of the dioxetane analog (positions 2 and 3 on the indole ring).


Lipid metabolism by The gall-bladder. II. The in vitro conversion of lysophosphatidylcholine to phosphatidylcholine. Lysophosphatidylcholine acyltransferase, which catalyzes the acylation of lysophosphatidylcholine with fatty acid coenzyme A to form phosphatidylcholine, was assayed in gall-bladder mucosa. In guinea pig gall-bladder the activity parallels that of the microsomal enzyme, glucose-6-phosphatase with 3--4-fold enrichment of the activity in the microsomes. Studies with saturated and unsaturated substrates demonstrated highest activity when oleoyl coenzyme A and palmitoyl lysophosphatidylcholine were used and the lowest activity when palmitoyl coenzyme A and palmitoyl lysophosphatidylcholine were used. This activity was demonstrated in the dog, rabbit, cat, calf and human gall-bladder mucosa; however, a wide variation in the amount was observed. Lysophospholipase, which catalyzes the hydrolysis of lysophosphatidylcholine to glycerophosphorylcholine and fatty acid, was also demonstrated in gall-bladder mucosa.


Sites of direct and indirect halogenation of albumin. The sites of radiohalogenation in proteins vary with the labeling method and the pH of the labeling reaciton. We have directly halogenated albumin with carrier-free radioiodide by three methods (pH range 2.2--9.3), and with carrier-free radiobromide by the chloroperoxidase method (pH range 2.2--4.6). Albumin was also indirectly halogenated by attaching a radioiodinated acylating agent, N-succinimidyl-3-(4-hydroxyphenyl) propionate (SHPP). The labeled proteins were proteolyzed enzymatically at neutral pH and the labeled amino acids produced were analyzed by liquid chromatography. Iodination at pH 7 yielded predominantly monoiodotyrosine, but at lower pH, fewer tyrosyl residues are labeled and a greater number of unstable sulfur-iodine bonds are formed at cysteinyl residues. Bromination with chloroperoxidase resulted in a high degree of labeling of cysteinyl residues at pH 2.8, the condition for optimum activity of this halogenating enzyme. Indirect halogenation with SHPP resulted in labeling of mid-chain lysyl, histidyl and tyrosyl residues.


Characteristics and conversion of high molecular weight forms of renin in plasma and their incomplete activation by the current acid treatment. Two distinctly different high molecular weight forms of renin are present in mouse plasma in addition to the well-recognized active 40 000 dalton form. The biggest form has a molecular weight of about 800 000, and is stable in 4 M urea, but can be converted to the active 40 000 dalton form, by storage, exposure to acid and limited proteolysis. The 70 000 dalton form can be activated by acid and limited proteolysis. However, the 70 000 dalton form does not change molecular weight with activation. By measuring renin, not only by its enzymatic activity, but also by the direct radioimmunoassay for the renin molecule, which measures enzymatically active as well as inactive renin, it was found that both forms were activated but neither of them completely. The validity of the currently used term "total" renin as the enzymatic renin activity after acid activation, is, therefore, questionable. The quantitative significance of this must await methods which can ensure complete conversion or activation of the high molecular weight forms of renin in plasma.


Effect of pH on bovine liver catalase as determined by electron paramagnetic resonance. Two major rhombic high-spin ferric heme signals are observed during the pH titration of bovine liver catalase. The less rhombic signal if dominant above pH 6.0 and the more rhombic signal below pH 6.0. Ethanol in high concentration enhances the relative intensity of the less rhombic signal. These data demonstrate the sensitivitiy of the ligand field to changes in catalase solvent and, furthermore, suggest that both rhombic configuration posses identical spectral and catalytic properties.


Studies on erythrocruorin. VII. Reconstitution of earthworm erythrocruorin from the apoprotein. Apoerythrocruorin prepared from the giant respiratory hemoprotein of the earthworm (60 S, Mr = 3 X 10(-6)) is an electrophoretically homogeneous molecule which sediments as a single peak of low molecular weight (3.5 S) and has a lower alpha-helical content (approx. 30%) than the native protein. Titration of globin with ferric heme indicates the presence of different binding sites; however, after purification by ion exchange chromatography, the reconstitution product contains 1 haem/23 000 g of protein as the native molecule. Reconstituted ferric erythrocruorin is a low molecular weight hemichrome with the same optical and physicochemical properties of the hemichrome formed by natural ferric erythrocruorin. Reconstituted ferrous erythrocruorin reacquires the alpha-helical content and the quaternary structure of the native molecule. Reassociation into 10-S speices (1/12 of the whole molecules) is fast and easy, while that into whole molecules is slow and somewhat erratic. The functional properties of reconstituted ferrous erythrocruorin (oxygen affinity, cooperativity in oxygen binding, magnitude of Bohr effect) are very similar to those of the "stable" low cooperativity form of the undissociated protein.


Oxygen equilibrium analyses of isolated hemoglobins A2, Lepore-Washington and P-nilotic. Oxygen equilibrium studies have been carried out on hemoglobins A2 (alpha2delta2), Lepore-Washington (alpha2(deltabeta)2) and P-Nilotic (alpha2(beta2delta)2) using the beta chain containing hemoglobins A and S as controls. This investigation was initiated mainly because of controversial data that have been published on the oxygen affinity of hemoglobin (Hb) A2 and because samples containing the rare Hb P-Nilotic became available. Each hemoglobin was isolated in pure form by anion  exchange chromatography; the samples used in the equilibrium analyses contained 100 mg Hb/dl with less than 5% ferrihemoglobin and no 2,3--diphosphoglycerate. Oxygen equilibrium analyses were made at 37 degrees C with the method of Benesch et al. (1965) Anal. Biochem. 11, 81--87; Anal. Biochem. 55, 245--248 (1973). A slight, but definite increase in oxygen affinity was observed for Hb A2 as well as for Hb P-Nilotic while the increase for the Hb Lepore-Washington was somewhat greater. The values for n, the Hill coefficient, and the Bohr effects were the same for all hemoglobin types. The differences in oxygen affinity of these hemoglobins apparently result from the differences in primary structure that are characteristic for those proteins.


The cleavage and adsorption of parathyroid hormone at high dilution: implications for receptor binding studies. Like other polypeptide hormones, purified intact parathyroid hormone (1-84)parathyroid hormone is notoriously unstable and is subject to large adsorptive losses in routine laboratory manipulation. The present studies were undertaken with 125I-labeled hormone to quantitate the problem and to develop preventative measures, particularly with concentrations of physiological interest, 1 . 10(-10) M. It was found that spontaneous cleavage of the hormone takes place upon its incubation in air or oxygen. This can be prevented by the presence of mercaptoethanol or by plasma levels of cysteine and ascorbate. Under non-cleavage conditions, adsorption was found to be extensive on all materials tested. This adsorption increased with time up to 2 h, was independent of ionic strength, increased with increasing temperature and was presumed to involve hydrophobic interactions. Under given conditions, adsorption was proportional to concentration (constant percentage). However, at very high concentrations, 1 . 10(-6) M, adsorption was markedly reduced. Adsorption was minimized at low pH (2). Bovine serum albumin reduced adsorption under all conditions when present at concentrations of 2 mg/ml or more. Coating laboratory ware with cetyl alcohol also was helpful. Using optimal conditions, cleavage is prevented and losses are less than 5% at neutral pH, and under 2% at pH 2.


An evaluation of respiration chain-associated functions during initiation of germination of Bacillus megaterium spores. In Bacillus megaterium QM B1551, spore germination could be initiated by glucose in the absence of detectable oxygen consumption, ATP synthesis or a pH decrease in the external media, suggesting that none of those reactions were mandatory. In addition, initiation of germination was insensitive to a variety of inhibitors of energy production or protonmotive force uncouplers. Therefore the respiratory chain-associated functions are not prerequisites for initiation of germination but these functions may be necessary to drive energy-dependent transport systems and other biosynthetic reactions during outgrowth.


Mediated transport and metabolism of lactate in rat aorta. 1. Under appropriate conditions L- and D-lactate enter the cells of rat aorta and are metabolized. Oxidation of lactate to CO2 occurs under aerobic conditions. 2. L- and D-lactate are taken up into the cells when oxygen, glucose, or both oxygen and glucose are present in the incubation medium. Both L- and D-lactate are excluded from the cells when neither oxygen nor glucose is present. 3. D,L-Glyceraldehyde prevents the uptake of L-lactate. The effect is apparently not due to the inhibition of glucose metabolism by L-glyceraldehyde. 4. L-lactate (20 mM) markedly inhibits the uptake of 5 mM D-lactate, but 20 mM D-lactate fails to inhibit the uptake of 5 mM L-lactate. 5. Raising the pH of the incubation medium markedly depresses the uptake of L-lactate. 6. The results provide evidence that L- and D-lactate enter the cells of rat aorta by a mediated transport system.


Changes in collagen cross-linking and lysyl oxidase by estrogen. Dermal collagen solubility and lysyl oxidase activity of bones were measured in DDD mice of advancing age. Insoluble fractions of the dermal collagen increased more rapidly in females than in males after 5 weeks of age. Activity of the lysyl oxidase extracted from bones was higher in females than in males after 4 weeks of age. After sexual maturation, such sex differences were always observed in skin as well as in bone tissue. In other experimental animals, dermal collagen solubility was markedly decreased by estrogen treatment and lysyl oxidase was remarkably activated by estrogen in both skin and bone. Thus it is clear that estrogen stimulates the enzyme activity and accelerates the maturation of collagen and elastin in extracellular space.


Effects of catecholamines and glucagon on glycogen metabolism in human polymorphonuclear leukocytes. Addition of 10 micron of the alpha-adrenergic agonist phenylephrine to polymorphonuclear leukocytes suspended in glucose-free Krebs-Ringer bicarbonate buffer (pH 6.7) activated phosphorylase, inactivated glycogen synthase R maximally within 30 s, and resulted in glycogen breakdown. Phenylephrine increased 45Ca efflux relative to control of 45Ca prelabelled cells, but did not affect cyclic adenosine 3',5'-monophosphate (cAMP) concentration. The effects of phenylephrine were blocked by 20 micron phentolamine and were absent in cells incubated at pH 7.4. The same unexplained dependency of extracellular pH was observed with 2.5 nM--2.5 micron glucagon, which activated phosphorylase and inactivated synthase-R, but in addition caused a 30-s burst in cAMP formation. 25 nM glucagon also increased 45Ca efflux. The activation of phosphorylase by phenylephrine and possibly also by glucagon are thought mediated by an increased concentration of cytosolic Ca2+ activating phosphorylase kinase. The effects of 5 micron isoproterenol or 5 micron epinephrine were independent of extracellular pH 6.7 and 7.4 and resulted in a sustained increase in cAMP, an activation of phosphorylase and inactivation of synthase-R within 15 s, and in glycogenolysis. The effects of both compounds were blocked by 10 micron propranolol, whereas 10 micron phentolamine had no effect on the epinephrine action. The efflux of 45Ca was not affected by either isoproterenol or epinephrine. The beta-adrenergic activation of phosphorylase is consistent with the assumption of a covalent modification of phosphorylase kinase by the cAMP dependent protein kinase. Phosphorylation of synthase-R to synthase-D can thus occur independently of increase in cAMP, but the evidence is inconclusive with respect to the cAMP dependent protein kinase also being active in this phosphorylation.


Properties and partial purification of the detergent-solubilized insulin receptor: a demonstration of negative cooperativity in micellar solution. Turkey erythrocytes possess insulin receptors with binding properties very similar to those of mammalian insulin receptors. In the present study, the insulin receptor of the avian erythrocyte has been solubilized in Triton X-100, extensively characterized and partially purified, and its properties compared to those of the membrane-bound receptor. The solubilized insulin receptor has a Stokes radius of 70 A and an apparent molecular weight of 300 000 in 0.05% Triton. The binding of insulin to the soluble receptor was very similar to the binding observed with the membrane-bound receptor. Thus, binding was markedly temperature dependent for both the soluble and membrane-bound forms, although the kinetics of binding were slower with the soluble receptor. Both forms of the receptor also showed a sharp pH optimum; however, solubilization produced a shift from maximal binding at pH 7.8 to pH 7.3. The soluble receptor also retained insulin analog specificity, ion sensitivity and negative cooperativity. The soluble receptor did not appear to degrade either bound or free insulin. On DEAE-cellulose chromatography the receptor eluted as a single peak. The specific activity of this partially purified preparation was 25--30 pmol/mg protein (about 500-fold enrichment over crude extract and 5-fold over highly purified membranes). Extensive attempts to purify further the receptor by gel filtration, carboxymethyl-cellulose chromatography and affinity chromatography resulted in either a very low yield or only modest enrichment. Purification was also complicated because the receptor was easily denatured; about 40% of the activity was lost after a 90-min exposure to 3 M urea or pH 4.5. These data suggest that the insulin receptor retains its properties in the absence of the lipid bilayer of the membrane. Complete purification will be difficult due to a lack of stability under a number of conditions.


Properties of partially purified bovine brain dopamine beta-hydroxylase. Dopamine beta-hydroxylase has been partially purified from bovine brain. A 140-fold purification factor was achieved using solubilization with Triton X-100, ammonium sulphate fractionation between 20-50 per cent saturation, affinity chromatography on concanavalin A-Sepharose 4 B and then filtration through Sephadex G200. The specific activity at the end was 51 nmoles/h/mg protein. The majority of endogenous inhibitors were lost. Immunological studies, kinetic studies, studies on the interaction with lectins and the effect of carboxylic acids on enzyme activity were carried out. Our data are in favour of the close similarity between the bovine brain and adrenal enzymes. No major differences could be found, at least with the characterization experiments using in the present study.


[Effect of pH on the permeability and mechanical properties of intercellular contacts in the small intestinal epithelium]. It is shown that acid site population controlling ion permeation through cell contacts is localized in the region of tight junction. The isoelectric point of cell surface in the region to tight junction changed from mean pH = 2.37 +/- 0.1 for duodenum to pH =2.80 +/-  +/- 0.1 for ileum. The maximum of cell adhesion in the tissue reached at mean pH that corresponding the isoelectric point. It seems possible, that this acid site population take part in interaction of plasma membranes of neighbouring cells.


New trends in cryoenzymology: I.-Supercooled aqueous solutions. A water in oil emulsion technique is proposed to investigate enzyme catalyzed reactions at sub-zero temperatures in the supercooled liquid state to avoid some reversible effects of the usual cosolvents on kinetics. Some results are listed: potentialities and technical problems of the procedure are discussed.


[Phosphorylation in acid shocked chloroplasts]. ADP phosphorylation up to 2.5 nmol per mg of chlorphyll has been found in chloroplasts from Vicia faba plants when the suspension was sharply acidified from pH 8 to pH 1.6 with excess of HC1/KC1 solution. This fact appears to be due to confromation relaxation of the coupling factor under acid-induced denaturation. Heat pretreatment inhibits this effect.


Capacity for biosynthesis of prostaglandin-related compounds: distribution and properties of the rate-limiting enzyme in hydrocorals, gorgoniants, and other coelenterates of the Caribbean and Pacific. A convenient and reliable assay is described for PGEP synthetase, the rate-limiting enzyme determining the total capacity for biosynthesis of prostaglandin-related compounds. Results of such assays, performed withe fresh specimens under both field and laboratory conditions, newly identify several marine coelenterate species as potentially important resources of PGRCs for research and possible development. Properties of the typical marine coelenterate PGEP synthetase, and the reaction which this enzyme catalyzes, have been further characterized.


[Kinetics and mechanism of nucleophilic effect on the oxidation of o-dianisidine catalyzed by horseradish peroxidase]. The effects of benzimidazole and 4-nitroimidazole on the reaction of o-dianisidine peroxidase oxidation within the pH range of 3.7--9.0 were studied. Both substituted imidazoles activate the reaction at less than 0.6. In the presence of 4-nitroimidazole the activation is non-competitive, whereas in the presence of benzimidazole it is of a mixed type, which is close to the non-competitive one. The kinetic parameters (kAcat, alpha, KA) for the reaction activated by both imidazoles were determined. It was assumed that the activators interact with the protein group (pK approximately to 6.5), which limits the enzyme activity. This results in the increase of pKapp of the protein group in question, resulting in the appearance of the maximal peroxidase activity in the alkaline region of pH. It was shown that the intermolecular interactions involved in the peroxidase-induced oxidative catalysis are largely due to electrostatic rather than to hydrophobic factors.


[Properties of cyclic nucleotide phosphodiesterase from lingual taste papillae]. Phosphodiesterase activity is estimated in extracts and partially purified preparations from functionally different parts of bovine tongue. The enzyme activity varied from 4.0 to 10.4 nmole/mg of protein/min. Properties of phosphodiesterase from circumvallate papillae are studied, the pH optimum being 8.0--8.5, Km for cAMP--1.5.10(-4) M and for cGMP--6.5.10(-5) M. The enzyme activity did not change after the treatment with trypsin, protamine sulphate (0.01--1.0%), heparin (0.01--1.0) and taste agents: L-leucine (from 1.10(-2) M to 1.10(-5) M), quinine (from 4.10(-3) M to 4.10(-8) M) and D-glucose (from 1.10(-1) M to 1.10(-4) M). The protein inhibitor of the enzyme, isolated from retina external rod-cell segments considerably suppressed phosphodiesterase activity, and the protein activator from brain tissue stimulated it insignificantly. Thermostable protein modulators, which inhibit or activate (depending on experimental conditions) phosphodiesterase activity, are isolated from circumvallate papillae.


[Coenzyme-induced slow transitions of NADP-sorbitol dehydrogenase from Gluconobacter oxydans]. The kinetic properties of NADP-dependent sorbitol dehydrogenase from G. oxydans cell extract were studied at pH 8.8 and 9.3 in the direction of D-sorbitol oxydation. It was shown that the shape of the kinetic curves of NADPH accumulation in time is characterised by initial burst whose magnitude depends on the concentration of the enzyme extract used. Preincubation of the enzyme with NADP or D-sorbitol eliminated the initial burst on these curves and transformed them into straight lines coming from the start of co-ordinates. The dependence of the stationary reaction rate on the enzyme extract concentration is not a linear one. The kinetic dependences of stationary rate of the reaction catalysed by the enzyme on the concentration of D-sorbitol and NADP at pH 8.8 and 9.3 were examined under all conditions studied; the shape of these kinetic curves altered to considerable extent with the alteration of the enzyme extract concentration in the reaction mixture and pH. At pH 9.3 several intermiediate plateaux were found on the curves of the D-sorbitol concentration dependent stationary rate of the reaction. The preincubation of the enzyme extract with NADP during 1.5 h removed the intermediate plateau on these curves and made them hyperbolic. Disk-electrophoresis of the enzyme extract in PAAG concentration gradient showed that at pH 8.8 the enzyme exists in one active form, while at pH 9.3 it exists in three major and three minor active forms of the enzyme differing in their molecular weights are found. It is assumed that the enzyme from G. oxydans cell extract can exist in a great number of molecular equilibrium forms, the rate of quilibrium being comparable or significantly less than that of the enzymatic reaction. NADP significantly influences on the equilibrium of the molecular forms of the enzyme.


[Presence of SH-groups and histidine in the active site of Chlorella glutamine synthetase]. The presence of two cysteine residues per each six monomers comprising the oligomer of Chlorella glutamine synthetase (E.C.6.3.1.2) is demonstrated using homogenous enzyme preparation. p-Chloromercuribenzoate (p-CMB) is found to inhibit glutamine synthetase activity, the degree of inhibition depending on the inhibitor concentration. The following enzyme reactivation by dithiotreitol (10(-2) M) was observed only when the enzyme was inactivated with 10(-5) M p-CMB under 15 min. preincubation. Preincubation of the enzyme with 10(-4) M p-CMB for 45 min. did not result in its reactivation. Gel filtration of glutamine synthetase treated with 10(-4) M p-CMB has revealed the dissociation of the enzyme into inactive monomers. Incubation of glutamine synthetase with p-CMB at various pH values, incubation after pre-treatment with urea and experiments with HgCl2 indicate the presence of free and masked inside the globula SH-groups in the enzyme molecule. Competitive character of the enzyme inhibition with p-CMB with respect to ATP indicates that SH-groups of the active site participate in the ATP binding, probably, as Mg-ATP or Mn-ATP complexes. Data on the estimation of ionization constant of glutamate-binding group and experiments on the effect of histidine photooxidation on the enzyme activity indicate the presence of histidine residue in the enzyme active site, which participates in glutamate binding.


[Isolation, purification and characterization of regulatory properties of adenylate cyclase from rabbit heart]. Rabbit heart membranes possessing the adenylate cyclase activity were isolated and purified by extraction with high ionic strength solutions and centrifugation in the sucrose density gradient. It was shown that the membranes are characterized by a high percentage of cholesterol (molar ratio cholesterol/phospholipids is 0.24) and an increased activity of Na, K-ATPase, which suggests the localization of adenylate cyclase in the sarcolemma. During centrifugation in the sucrose density gradient the activities of andenylate cyclase and Na,K-ATPase are not separated. Treatment of heart sarcolemma with a 0.3% solution of lubrol WX results in 10--20% solubilization of adenylate cyclase. Purification of the enzyme in the membrane fraction is accompanied by a decrease in the activity of phosphodiesterase; however, about 2% of the heart diesterase total activity cannot be removed from the sarcolemma even after its treatment with 0.3% lubrol WX. Epinephrine and NaF activate adenylate cyclase without changing the pH dependence of the enzyme. The alpha-adrenergic antagonist phentolamine has no effect on the adenylate cyclase activation by catecholamines, glucagon and histamine; the beta-adrenergic antagonist alprenolol competitively inhibits the effects of isoproterenol, epinephrine and norepinephrine, having no effect on the enzyme activation by glucagon and histamine. There is no competition between epinephrine, glucagon and histamine for the binding site of the hormone; however, there may occur a competition between the hormone receptors for the binding to the enzyme. A combined action of several hormones on the membranes results in the averaging of their individual activating effects. When the hormones were added one after another, the extent of adenylate cyclase activation corresponded to that induced by the first hormone; the activation was insensitive to the effect of the second hormone added. It is assumed that the outer membrane of myocardium cells contains a adenylate cyclase and three types of receptors, each being capable to interact with the same form of enzyme. The activity of adenylate cyclase is determined by the type of the receptor, to which it is bound and by the amount of the enzyme-receptor complex.


[Properties of DNA-methylase reaction in isolated nuclei of normal and regenerating rat liver]. Evidence from comparative determination of DNA radioactivity methylation degree of acidic extraction and chlorophormic deproteination of the samples suggest that the former technique is a more efficient one. The properties of the DNA-methylase reaction in isolated rat liver nuclei were studied. The DNA-methylase activity is found to be considerably stable during incubation of the nuclei at 37 degrees C; a broad pH-optimum in the alkaline region is observed (pH 8.6--9.8); this activity is inhibited by Mn2+, nucleotides, actynomycin and S-adenosyl methionine analogs and is activated by Mg2+; the incorporation of methyl groups into DNA is reversible. The data suggest that the DNA-methylase activities of the nuclei isolated at different stages of regeneration do not show substantial variations. No differences in DNA methylation before and after DNA synthesis in the regenerating nuclei were observed. Inhibition of DNA synthesis in the course of regeneration does not decrease the level of DNA methylation. The interrelationship between methylation and replication of DNA is discussed.


[Effect of cortisol on the content of mRNA coding inducible tyrosine aminotransferase isoenzyme in rat liver]. In vitro estimation of synthesis of inducible tyrosine aminotransferase isoenzyme, directed by poly-A-containing RNA from liver of intact and corticol treated rats, is carried out. Total poly-A-containing RNA from liver polyribosomes of intact and induced rats was translated in cell-free system from wheat germs. Two antibodies immunoprecipitation was used to identify the translocation product (tyrosine aminotransferase). It was found that a synthesis of a specific protein product, precipitated by antibodies to tyrosine aminotransferase, takes place in cell-free system under translation of polysomic poly-A-containing liver RNA. The amount of immunoprecipitated product indicates, that the content of individual poly-A-containing mRNA for inducible tyrosine aminotransferase isoenzyme in liver of cortisol-induced rats is considerably higher than in intact animals.


Evaluating clustering methods for psychiatric diagnosis. This report represents an empirical evaluation of the major clustering approaches on psychiatric diagnostic data. Experienced psychiatrists, using 17 psychopathological variables, developed 88 archetypal psychiatric patients to represent four diagnostic categories (manic-depressive depressed, manic-depressive manic, simple schizophrenic, and paranoid schizophrenic). Ten computerized methods representative of the major clustering approaches and using various measures of similarity between patients were applied to this data set to develop de novo patient groupings. Evaluative criteria included the concordance of clustering output to the structure of the original data, and clustering replicability. Considerable differences were obtained among clustering methods. The best-ranked procedures were nearest centroid sorting methods and complete and centroid linkage hierarchical methods. The overall poorest ranking were obtained for multivariate normal mixture analysis and facial representation of multidimensional points. Further evaluation of cluster analytic methods on real biological and psychosocial data sets yielded similar rankings.


Difficulties in comparing catecholamine-related enzymes from the brains of schizophrenics and controls. The catecholamine-forming and metabolizing enzyme tyrosine hydroxylase, dopa decarboxylase, dopamine-beta-hydroxylase, phenylethanolamine N-methyltransferase, and catecholamine-O-methyltransferase, as well as the endogenous inhibitor of dopamine-beta-hydroxylase were compared in the brains of schizophrenics and controls. While there were no statistically significant differences in the enzyme or inhibitor activity between groups, tbre was a decided trend toward a decreased enzyme activity in the brains of the schizophrenics. From another set of control brains it was found that changes in human enzyme activity following death are variable and may be dependent on how the brains were handled. Thus, it is unclear whether the apparent differences between schizophrenics and controls were present when they were alive or occurred after death.


Effect of anticonvulsant drugs on alkaline phosphatase and gamma-glutamyltransferase activity in human leukocytes. The authors compared the alkaline phosphatase and gamma-glutamyl-transferase activities in leukocytes from presumably healthy subjects and from epileptic patients treated with anticonvulsants, matched for age and sex. Intraleukocyte alkaline phosphatase activity was higher in women than in men and was greatly reduced by the anticonvulsant drugs. Intraleukocyte gammaglutamyltransferase activity did not vary with sex and was unaffected by the anticonvulsant drugs contrary to findings for the activity of this enzyme in plasma and liver.


Thermodynamic investigations of proteins. IV. Calcium binding protein parvalbumin. The conformational transitions of calcium binding protein parvalbumin III from carp muscle were studied by scanning calorimetry, potentiometric titration and isothermal calorimetric titration. Changes of Gibbs energy, enthalpy and partial heat capacity were determined. The removal of calcium ions by EDTA is accompanied by 1) a heat absorption of 75 +/- 10 kJ per mole of the protein, 2) a decrease in the Gibbs energy of protein structure stabilisation of about 42 kJ mol-1 and 3) a decrease in thermostability by more than 50 K. The protonation of the acidic groups leads to a loss of calcium followed by denaturation, while the pH of the transition strongly depends on calcium activity. The enthalpy and heat capacity changes at denaturation are comparable with the values observed for other compact globular proteins.


Immunosuppressive activity of two plant steroidal lactones withaferin A and withanolide E. Withaferin A and withanolide E, two steroidal lactones of plant origin were demonstrated to have specific immunosuppressive effects on human B and T lymphocytes as well as on mice thymocytes. E rosettes and EAC rosette formation by normal human T and B lymphocytes were inhibited by the two compounds at very low concentrations. The formation of mouse red blood rosettes by chronic lymphatic leukemic cells were only inhibited by withaferin A. The functional activity of normal human T lymphocytes as assessed by a local xenogeneic graft versus host reaction was also affected by these two plant steroidal lactones. These experiments demonstrate a specific action of the compound on antigen recognition as well as proliferative capacity of T lymphocytes as well as B lymphocytes.


Temperature dependence of the interfacial behavior of uracil derivatives. The effect of temperature on the interfacial behavior of uracil, thymine and 1,5-dimethyluracil has been used to characterize the thermodynamics of adsorption of these compounds at a mercury electrode-aqueous electrolyte interface. Between 4.5 degrees C and 40 degrees C all three compounds exhibit two adsorption regions. The first, a dilute layer where the molecules adsorb in a flat orientation on the electrode surface. The second, a compact layer where the molecules adsorb in a perpendicular orientation. The thermodynamic constants characterizing formation of these surface layers have been deduced.


Action of beta-adrenoceptor blocking agents on hemodynamic variables and on baroreceptor and postganglionic sympathetic activities in rabbits. Fifty-six experiments were performed in rabbits anesthetized by urethane or sodium pentobarbital. Baroreceptor (aortic nerve) and post-ganglionic sympathetic (renal nerve) activities were recorded, and peripheral resistances were determined after infusion of beta-adrenoceptor blocking agents (propranolol, pindolol, timolol). A progressive vasodilation was observed after the onset of infusion of the 3 drugs; at the same time arterial pressure decreased, but aortic flow did not vary significantly. Baroreceptor activity was enhanced, whereas efferent renal nerve activity was decreased.


Volume changes during enzyme reactions. The influence of pressure on the action of invertase, dextranase and dextransucrase. The pressure dependence of the maximum velocities and the Michaelis constants for the enzymes invertase and dextranase was measured up to 1400 bar. The corresponding activation volumes deltaV not equal to c and deltaV not equal to Km proved to be independent of pressure. Together with data from other sources the meaning of deltaV not equal to c and deltaV not equal to Km is established and the volume profiles of the reactions are constructed. These profiles are similar in contour to the volume profile of the dextran formation catalyzed by the enzyme dextransucrase, but the amount of the volume changes is very much larger for dextransucrase. The evaluation of salt effects shows, that for all three enzymes solvent interactions are not important in explaining the results. The reaction mechanisms seem to be governed by conformation changes of the enzymes. The larger effects in dextransucrase are explained by the produced dextran chain remaining tightly bound to the enzyme and being transported relative to the enzymes position in each reaction cycle.


[Effect of the alpha-adrenoblocker pyrroxan on systemic hemodynamics in puppies and dogs with vasorenal hypertension]. Vasorenal hypertension was induced in 2--3-month-old puppies and adult dogs by stricture of both renal arteries. 1.5 mg/kg of pyrroxan was injected intravenously 3 and 14 days later. A reduction of increased arterial pressure was noted both in adult dogs and in puppies, to the subnormal level in the latter. Hypotensive effect of the preparation was connected in adult animals with diminution of the general peripheral vascular resistance and in puppies, besides, with reduction of cardiac output. Pyrroxan injection was accompanied in all the animals with tachycardia, reduction of the phase of isometric contraction and activation of myocardial contractility.


[Pharmacology of azidomorphine].

[Pharmacology of azidomorphine]. As revealed, in acute experiments on rabbits, cats, and rats, a new morphine-like synthetic analgesic drug azidomorphine exceeded 20--100-fold the morphine ability to inhibit synaptic transmission in the thalamic structures, cerebral cortex, and the spinal cord during the nociceptive stimulation, and also by its analgesic activity.


Platelet storage at 22 degrees C: effect of type of agitation on morphology, viability, and function in vitro. Recovery in vivo after 51Cr labeling, platelet morphology, and platelet aggregation were studied with platelet concentrates (PC) stored for transfusion under carefully controlled conditions. PC were prepared to a final volume of 50 ml from whole blood anticoagulated with citrate-phosphate-dextrose (CPD). The platelet count was kept between 0.8 and 1.6 X 10(12) platelets/liter. The PC were stored in bags constructed of polyvinylchloride (PVC) or polyethylene (PE) at 22 degrees C for 72 hr. The bags were placed on a horizontal shaker or a ferris wheel for agitation during storage. No significant changes in pH or platelet count were observed during storage. PC stored on the wheel showed moderate loss of viability and a marked deterioration of platelet morphology and aggregation compared to the shaker. PC stored on the shaker in bags made of PE showed better aggregation with ADP and thrombin but had the same viability and morphology as PC in bags constructed of PVC. Maintenance of normal platelet morphology as determined by phase-contrast microscopy, extent of shape change response, and the size distribution according to the Coulter Counter correlated with recovery in vivo.


Neurochemical anatomy of the neuroendocrine hypothalamus. Neurochemical anatomy of the hypothalamus. Microdissection techniques for isolated removal of the various regions of the hypothalamus as well as the individual hypothalamic nuclei are detailed. Recent development of biochemical microassays have made it possible that the concentrations of neurohormones, neuropeptides, neurotransmitters and their related enzymes could be detected in such a small volume of brain tissue than the hypothalamic nuclei. Data available of the hypothalamic distribution of above substances are summarized. The possible role and origin of intra- and extrahypothalamic neurohormones as well as the existence of the so-called "hypophysiotrophic area" are discussed.


Dopaminergic control of prolactin secretion. A brief summary is given about present-day knowledge and views on the control of prolactin secretion in mammals. This is followed by the presentation of the evidence that dopamine is the inhibitory hypothalamic transducer controlling hormone release by prolactin cells. Most of the evidence is pharmacological in nature. It is only very recently that dopamine has been found in a meaningful concentration in the blood flowing from the median eminence to the pituitary. Although the dopamine concept of an inhibitory control of prolactin secretion seems quite satisfactory, the possibility of another unrelated inhibitory control system is not excluded.


Hypothalamic regulatory hormones: physiological and clinical implications. The hypothalamic regulatory hormones used for clinical studies are TRH, Gn-RH and somatostatin. In addition, as dopamine appears to be a physiological PIF, the dopamine agonists such as bromocriptine, could be considered as functional analogues of PIF. Gn-RH can be used to study the hypothalamic-pituitary gonadal relationship and to test the secretory reserve capacity of the gonadotrophs in disease states. Unfortunately Gn-RH testing discrimulates between pituitary and hypothalamic diseases only poorly. However gonadotrophin deficient men or women may be successfully treated with long-term Gn-RH with induction of puberty, potency, spermatogenesis and ovulation. Somatostatin has multiple actions in inhibiting endocrine and exocrine secretion but its actions are still being explored in diabetes. Bromocriptine, a long acting dopamine agonist (a functional analogue of PIF), suppresses prolactin and is highly effective in treating many hypogonadal states since hyperprolactinaemia is common. It also lowers growth hormone in acromegaly. TRH has provided a major, accurate, sensitive and safe test of thyroid function.


[Pathophysiology of sulfatide metabolism in metachromatic leukodystrophy]. Metachromatic leucodystrophies (MLD) comprise a small group of heredodegenerative disorders of the nervous system. Deficiency of sulfatide-sulfatase or arylsulfatase A is the common defect in all forms of MLD leading to lysosomal sulfatide storage in the nervous tissue and in the kidney. On the basis of animal experiments, experiments with cultured fibroblasts of the patients as well as ultrastructural studies in a case of prenatal MLD, the following pathomechanism is proposed: 1. Lysosomal degradation of a large portion newly synthetised sulfatide normally regulating the net synthesis and incorporation of sulfatide into myelin, causes early accumulation of sulfatide in lysosomes of myelinating cells and in neurons in the genetic deficiency of arylsulfatase A. 2. Early accumulation of sulfatide does not lead to disturbance in myelination. Demyelination occurs possibly by storage of a cytotoxic compound, psychosin sulfate, also a substrate for the missing enzyme. Prevention of MLD is possible by prenatal diagnosis of arylsulfatase A deficiency in cultured amniotic cells. Enzyme substitution of the missing arylsulfatase A is possible by exogenous uptake of the enzyme in cultured fibroblasts. Thereby the defect of sulfatide degradation can be corrected. Although principles of enzyme substitution have been demonstrated, the problems of treating patients with MLD with arylsulfatase A infusions have yet to be overcome.


Alteration of medullary respiratory unit discharge by iontophoretic application of putative neurotransmitters. 1 Cats with midcollicular decerebration were vagotomized, paralyzed and artificially ventilated. Phrenic nerve activity was recorded as an index of central respiratory rhythm. Medullary respiratory neurones and non-respiratory cells located in approximation to the ventral respiratory nucleus were tested for their responsiveness to iontophoretically applied gamma-aminobutyric acid (GABA), acetylcholine (ACh) and glutamate. 2 GABA tended to inhibit, whereas ACh and glutamate excited activity both of respiratory and non-respiratory units. Some phase-spanning respiratory unit activities were converted to phasic discharge patterns linked to either inspiration or expiration concomitant with application of low GABA doses. Appropriate applications of GABA also resulted in a complete cessation of the respiratory or non-respiratory neuronal activities. 3 While application of ACh or glutamate induced continuous firing in phasic, phase-spanning respiratory neurones, the periodic discharge patterns of inspiratory or expiratory units was not altered by ACh or, in many instances, by glutamate. Only at high doses of glutamate was the phasic discharge of some inspiratory or expiratory units converted to tonic activity. 4 These observations suggest that strong inhibitory processes serve to maintain the phasic firing pattern of respiratory units. These data also support the concept that active-inhibitory phase-switching mechanisms serve to define respiratory rhythmicity.


Factors limiting the rate of termination of the neuromuscular blocking action of fazadinium dibromide. 1 Reasons for the termination of the neuromuscular blocking action of fazadinium dibromide have been investigated. 2 In the anaesthetized cat, maximum neuromuscular block of tibialis anterior muscle twitches following rapid intravenous injection of fazadinium was obtained as the injection bolus passed through the muscle, indicating the fazadinium very readily crosses capillary membranes. 3. The half-life of plasma clearance of fazadinium was about 1 min in both cat and man, despite a 10 fold difference in duration of action in these species. Plasma samples were bioassayed for neuromuscular blocking activity on an isolated, superfused phrenic nerve-diaphragm preparation of the rat. 4 In the anaesthetized cat, occlusion of the renal or hepatic circulations did not prolong the neuromuscular blocking action of single doses of fazadinium. Repeated doses accumulated slowly when the hepatic circulation was occluded but not when the renal circulation was occluded. 5 Fazadinium is eliminated from the body by both the liver and kidneys but the rates of these processes are insufficient to account for the initial rapid plasma clearance. 6 The rate-limiting step for the termination of the neuromuscular blocking action of fazadinium is most likely to be the rate of drug-receptor dissociation


Evidence for the participation of beta1-adrenoceptors in isoprenaline-induced renin release from rat kidny slices in vitro. 1. The inhibitory effects were studied of 4 beta-adrenoceptor antagonists against renin release induced by isoprenaline (0.5 mumol/1) in rat kidney slices. Additionally the pA2 values of these 4 drugs were measured against isoprenaline in guinea-pig isolated atria and trachea (against beta1- and beta2-adrenoceptors respectively). 2 When employed at a concentration of 2 mumol/1 propranolol and atenolol significantly inhibited renin release (P less than 0.001 and P less than 0.01) whereas practolol and IPS 339 [t-butyl-amino-3 ol-2 propyl) oximino-9 fluorene] had little effect. 2 A positive correlation was shown between the degree of inhibition of renin release and the pA2 of the antagonists at the beta1-adrenoceptors. 4 When practolol and IPS 339 were used in equipotent molar concentrations to propranolol for the beta1-adrenoceptors they inhibited renin release. 5 The results suggest that the adrenoceptor involved in the renin release induced by isoprenaline in the rat kidney is of the beta1-type.


The effects of isoprenaline and a new beta-sympathomimetic amine upon spontaneous activity, diastolic depolarization and plateau height in cardiac Purkinje fibres. 1 In spontaneously active Purkinje fibres of young cows the dose-response curves of the action of isoprenaline upon different electrophysiological parameters were measured.2 The increase in slope of diastolic depolarization could roughly be described by a one-for-one binding curve with a half maximum effect near 10(-8) M and the increase in the height of the plateau level by a two-for-one binding curve with a half maximum effect near 10(-7) M (-)-isoprenaline.3 These dose-response curves were similar to those of two parameters measured under voltage clamp conditions by other authors. The increase in slope of diastolic depolarization behaved like the shift of the activation curve for the pacemaker potassium current towards positive potentials and the growth in plateau height like the increase in the slow inward current mainly carried by Ca ions. From this conformity we propose that the parameters evaluated by us from action potential records could be used for a qualitative analysis of the action of catecholamines on pacemaker potassium current and Ca influx.4 The effects of the isomers of a new drug, 1-isopropylamino-3(4'hydroxyphenoxy)-propan-2-ol (IHP), were evaluated in the same way as those of isoprenaline. The (-)-isomer was at optimal concentrations (10(-5) M) nearly half as effective as isoprenaline in increasing frequency and slope of diastolic depolarization but caused no increase in plateau height. An identical relationship, but at 5 to 10 times higher concentrations, was obtained with the (+)-isomer.5 When 10(-4) M(-)-IHP was added to a preparation equilibrated with a maximum dose of (-)-isoprenaline (10(-6) M), frequency and plateau height declined. This result together with the observation that the effects of IHP could be blocked by the specific beta-antagonist propranolol, revealed the beta-agonistic nature of the new drug. Its inefficiency in increasing the plateau height and thus the slow (Ca) inward current was explained by its relatively low potency and intrinsic activity.


Enhancement by an antagonist of transmitter release from frog motor nerve terminals. 1 The effect of Ba2+ on the synchronous release of acetylcholine from frog motor nerve terminals was studied by conventional electrophysiological techniques. 2 When Ca2+ and Ba2+ were the only divalent cations in the bathing fluid, Ba2+ caused a presynaptic reduction in the amplitude of the endplate potential (e.p.p.). This effect was surmountable by increasing the Ca2+ concentration. 3 The affinity constant (KA) for Ba2+, calculated on the assumption that Ba2+ is a competitive inhibitor of the agonist, Ca2+, was 1.1 +/- 0.4 mM-1 (mean +/- s.e. mean, n = 8). 4 When e.p.ps were depressed by the addition of 1 mM Mg2+, addition of Ba2+ (1 to 3 mM) caused either a further presynaptic depression of moderate magnitude or had no additional effect. 5 When e.p.p.s were depressed with [Mg2+] greater than or equal to 2 mM, addition of Ba2+ greater than or equal to 0.9 mM enhanced the e.p.p. amplitude by a presynaptic mechanism. 6 The interaction of the divalent cation antagonists Mg2+ and Ba2+ with the agonist, Ca2+ is discussed. It is demonstrated that a model which considers the nonequilibrium, kinetic properties of binding can be used to describe interactions between divalent cations at the external surface of the motor nerve ending.


Rat cortical neurons in cell culture: culture methods, cell morphology, electrophysiology, and synapse formation. Rat cortical neurons from 15 day embryos are grown in dissociated cell culture and maintained in vitro for 8--12 weeks. The neurons develop into forms which resemble mature cortical neurons in situ, stain with silver and exhibit passive and active electrophysiological properties similar to those of cortical neurons. Extensive chemical excitatory and inhibitory synapses develop de novo. These cultures can provide a model for future studies of mammalian CNS neuronal physiology, transmitter pharmacology, pathophysiology and mechanisms of drug action.


The use of kainic acid in the localization of enzymes in the substantia nigra. The selective destruction of neuronal perikarya via intracerebral injections of kainic acid was used to elucidate the cellular location of four neurotransmitter-related enzymes in the substantia nigra (SN). Two weeks after intranigral injections of kainic acid, dopamine-sensitive adenylate cyclase, glutamic acid decarboxylase (GAD), choline acetyltransferase (CAT) and acetylcholinesterase (AChE) were measured in the SN. Histological examination of the SN, and a reduction of striatal tyrosine hydroxylase (TH) activity by 94%, confirmed the extensive loss of neuronal cell bodies in the SN. Dopamine stimulation of adenylate cyclase was not reduced in the lesioned SN, supporting the view that dendritically-released dopamine can regulate cyclic AMP synthesis in afferent terminals to these dendrites. Nigral GAD activity was significantly reduced by the lesions, suggesting that there are GAD-containing perikarya in the SN. CAT activity was not affected by the kainic injections, indicating the absence of cholinergic perikarya in the SN. Nigral AChE activity was significantly decreased after kainic injections, thus confirming the presence of AChE within the nigral perikarya. The results suggest that dopamine-sensitive adenylate cyclase and CAT are located within afferents to the SN, while GAD and AChE are found, to some extent at least, in neuronal soma of the SN. The differentail effects of kainic acid on these enzymes suggest that this compound may be a useful neurochemical tool with which to determine the cellular distribution of enzyme systems in the central nervous system.


Evidence for glutamate as a neurotransmitter in the corticofugal fibres to the dorsal lateral geniculate body and the superior colliculus in rats. The high-affinity uptake of L-glutamate, D-aspartate and GABA were examined in homogenates from the dorsal lateral geniculate body and the superior colliculus after removal of the right visual cortex of adult rats. The high-affinity uptake of D-aspartate and L-glutamate were reduced by 53 to 75% respectively in the dorsal lateral geniculate body and by 46 and 53% respectively in the superior colliculus ipsilateral to the lesion. The uptake on the contralateral side was unaffected. The reductions were detected 3 days after the lesion and were maximally developed after 7 days. Subcellular fractionation showed that the main part of the uptake was confined to the synaptosomal fraction of both regions and that the reductions were most prominent in this fraction. The lesion was not accompanied by significant changes in high-affinity uptake of GABA nor in changes of choline acetyltransferase and glutamate decarboxylase activities. The high-affinity L-glutamate uptake on the contralateral visual cortex was unchanged from control values. After ablation of the visual cortex the level of L-glutamate was reduced by 32 and 17% in the ipsilateral dorsal lateral geniculate body and superior colliculus, respectively. The levels of the other amino acids examined, including L-aspartate, were unchanged. Enucleation had no effect on the uptake of L-glutamate and of GABA in the dorsal lateral geniculate body or in the superior colliculus.


Receptor-linked cyclic AMP systems in rat neostriatum: differential localization revealed by kainic acid injection. Various receptor-linked cyclic AMP systems were measured in rat neostriatum 2--14 days after selective destruction of neuronal cell bodies and dendrites by micro-injection of 3 microgram of kainic acid. Basal adenylate cyclase activity was reduced by up to 56% in the injected side and the sensitivity to dopamine was abolished. Up to 84% of cyclic nucleotide phosphodiesterase activity, hydrolyzing either cyclic AMP or cyclic GMP, was destroyed by kainic acid injection. Specific binding of [3H]etorphine and [3H]spiroperidol was reduced by up to 62% in the injected side, while non-specific binding was unchanged. All of these changes were time-dependent, and were greatest 7--14 days after kainic acid treatment. On the other hand, intrastriatal kainic acid injection caused no change in the steady-state concentration of cyclic AMP in striatal slices, or in the in vivo cyclic AMP content in the striatum of rats killed by microwave irradiation. Receptor-mediated increases in cyclic AMP accumulation in striatal slices were either unchanged or markedly potentiated by kainic acid treatment. The maximum response to adenosine was unchanged, while the response to isoprenaline was increased up to 3.7-fold, the response to dopamine increased up to 6.7-fold, and the response to PGE1 increased up to 30-fold. The effect of dopamine in kainic acid-treated striatal slices was no longer blocked by fluphenazine, but was blocked by propranolol, suggesting an interaction of dopamine with a beta-adrenoceptor in kainic acid-treated slices. The results suggest differential cellular localizations of the various receptor-linked cyclic AMP systems in rat neostriatum. Some dopamine and opiate receptors, as well as most of the phosphodiesterase activity, are associated with local neuronal elements, while beta-adrenoceptor, adenosine and PGE1 alterations in cyclic AMP are not. The potentiation of the beta-adrenoceptor and PGE1 responses suggests that they may occur in glial cells. In addition, the pool of adenylate cyclase destroyed by kainic acid appears to make little contribution to normal levels of cyclic AMP in the tissue.


Effects of estrogen and aminergic drugs on thresholds of medial basal hypothalamic axons in the median eminence of the rat. Antidromic responses were induced in the medial basal hypothalamus (MBH) of female rats by median eminence (ME) stimulation, and thresholds were measured under various endocrine conditions. Two different types of antidromic response were recognized. In 83 out of 97 responses, repetitive shocks to the ME at 20--40 Hz induced a fractionation of antidromically-driven spikes into A- and B-components. In the remainder, the antidromically activated spikes were stable to stimulation at high frequency. The threshold tended to show higher values in proestrus than in diestrus-I in both types of response. However, only in the former type of response, application of conditioning electrical shocks to the ME was effective in decreasing the threshold for successive test pulses. A decrease in the threshold lasted for 150--200 msec following the conditioning, with the lowest threshold at about 60 msec after the shock. Effects of the conditioning persisted in animals with chronic complete deafferentation of the MBH but were abolished in intact rats by reserpine treatment. Dopamine, but not norepinephrine or serotonin, mimicked the threshold-decreasing effect of the conditioning when applied to the ME. The results suggest the possibility of a depolarizing effect of some dopaminergic neural mechanism projecting from the MBH to the ME and acting on the axons of a particular type of MBH neuron characterized by fractionating spikes.


Calcium-binding properties of cardiac and skeletal troponin C as determined by circular dichroism and ultraviolet difference spectroscopy. Calcium titration of the conformational change in cardiac and skeletal troponin C (TN-C) was followed by circular dichroism (CD) at pH values in the range from 5.2 to 7.4. Computer analysis was used to resolve the contributions from the different classes of Ca2+ -binding sites. At pH 6.94 in skeletal TN-C, apparent affinity constants for calcium of 1.8 x 10(7) and 4.5 x 10(5) M-1 were determined for the two classes of binding sites. The more sophisticated computer analysis of the data has revealed a substantial CD contribution from the low-affinity sites (approximately 30% of the high affinity contribution at pH 6.94) and suggests that skeletal TN-C with Ca2+ bound at the low-affinity sites is in a different conformation from that when just the high-affinity sites are occupied, in agreement with a recent nuclear magnetic resonance (NMR) study on this system (Seaman, K. B., Hartshorne, D. J. & Bothener-By, A. A. (1977) Biochemistry 16,4039-4046). With the cardiac protein at pH 7.07, an apparent affinity constant for calcium of 2.0 x 10(7) M-1 was calculated while no low-affinity site at this pH was detected by CD. On the other hand, at lower pH values, such as 6.05, a CD contribution from the cardiac low-affinity Ca2+ -binding site is detected with an apparent binding constant of 3.7 +/- 0.7 x 10(4) M-1. At the lower pH values, protonation of a class of carboxyl groups in each protein which possesses a high pKa (6.2-6.3) elicits the conformational change at the high-affinity sites with a corresponding decrease in the overall magnitude of the Ca2+  -evoked changes. The expression of a conformational change upon Ca2+ binding at the level of the low-affinity sites is enchanced by protonation of a class of carboxyls with a pKa of 6.3 in cardiac TN-C and 6.7-6.8 with the skeletal homologue. In both cases, this contribution is reduced upon protonation of carboxyls with pKa less than or equal to 5.5. It was also observed that the low-affinity sites of skeletal TN-C have a much larger role to play in the total conformational change than the low-affinity sites of cardiac TN-C, a finding probably related to the inability of site 1 in the cardiac protein to bind calcium. In the cardiac protein, the Ca2+ -induced tyrosine difference-spectrum maximum is reduced from deltaepsilonM,287nm =330M-1.cm-1 to 20M-1.cm-1 by protonation of a class of groups with a pKa of 6.4, presumably the same carboxyl groups as those invoved in the CD conformational contribution from the high-affinity binding sites. No such effect was observed for the skeletal protein where deltaepsilonM,287nm was constant at 110M-1 .cm-1 over the pH range studied. The dramatic alterations in the tyrosine environment of cardiac TN-C with pH are attributed to either or both of the tyrosines located in the two high-affinity Ca2+ -binding sites (sites 3 and 4)...


Guanosine triphosphate catabolism in human and rabbit erythrocytes: role of reductive deamination of guanylate to inosinate. The reductive deamination of guanylate to inosinate was demonstrable but occurred at low rates in human and rabbit erythrocytes incubated in vitro with or without glucose. However, the process was considerably accelerated in erythrocytes incubated with deoxyglucose. In human erythrocytes incubated with deoxyglucose, deamination was the major pathway of catabolism of guanylate; little or no guanylate was dephosphorylated. In rabbit erythrocytes, guanylate was both deaminated and dephosphorylated. Inosinate formed from guanylate was metabolized only by dephosphorylation in human erythrocytes, but in rabbit erythrocytes, it was also converted to xanthylate.


Some properties of a Ca2+- and (or) Mg2+-requiring nucleoside di- and tri-phosphatase(s) associated with the membranes of rat pancreatic zymogen granules. Membranes of rat pancreatic zymogen granules have been purified and found to be essentially free of contamination by mitochondria, microsomes, and plasma membranes. They possessed an acid phosphatase activity which derived probably from lysed lysosomes contaminating the purified zymogen granules from which the membranes were prepared. These membranes were found to contain a strong Ca2+- and (or) Mg2+-requiring activity toward all nucleoside di- and tri-phosphates. Various data support the tentative conclusion that a single protein catalyzes the hydrolysis of the nucleoside di- and tri-phosphates. This protein appears to be intrinsic with its active site localized on the internal face of the membranes.


The control of pyruvate kinases of Escherichia coli: further studies of the enzyme activated by ribose-5-phosphate. The pyruvate kinase activated by ribose-5-phosphate from Escherichia coli has been purified to homogeneity, taking advantage of the stabilization of the enzyme by its inhibitor phosphate and by thiol reagents. The native enzyme has a tetrameric quaternary structure which, while prone to dissociation under many conditions, remains intact in the presence of the above reagents. The enzyme was found to reactivate on dilution out of 8 M urea. Interestingly, the recovery of activity is greatly increased by phosphate, an allosteric inhibitor, but markedly reduced by the allosteric activator, ribose-5-phosphate, implying that it is harder for the enzyme to refold to a 'relaxed state.' Proteolysis studies indicate a more open structure in the presence of the activator.


Effect of pH on the antimicrobial activity of some triphenylmethane dyes. Four common dyes were tested as inhibitors of four types of bacteria over the pH range 5.0-9.0. Inhibition of the gram-negative types, Salmonella anatum and Enterobacter aerogenes, was markedly affected by the pH of the medium. These organisms tolerated concentrations of crystal violet and ethyl violet about 100-fold higher at pH 5.0 than at pH 9.0. Above pH 7.0 brilliant green (BG) and malachite green (MG) were precipitated as their respective carbinols and lost their inhibitory properties with these two organisms. Two gram-positive types, Staphylococcus aureus and Bacillus cereus, were more sensitive to dyes and results were less affected by pH. The carbinol forms of MG and BG were nearly as inhibitory to these organisms as the ionized forms.