Psychometric assessment of the therapeutic efficiency of antidepressant agents. A clinical trial of four weeks duration was conducted involving a total of thirty depressed patients, of both sexes, aged between twenty and thirty-four years. The total number of patients was divided into three groups of ten patients each. One group received amitriptyline, the second group was administered noxiptyline and the third group was given dibenzepine. All drugs were administered orally. Patients were submitted to psychometric testing before and after drug administration. The tests used included the 'Hamilton Rating Scale for depression', the 'Hildreth Feeling Scale' and the 'D Scale' and the 'Trail Making Test' for the evaluation of psychomotor retardation. It was concluded that the Hamilton Rating Scale was the most relatively sensitive test utilized in assessing the depressive state and its improvement. Amitriptyline was found to be mostly anxiolytic; noxiptyline controlled both depression and anxiety to approximately the same extent; and dibenzepine was found to be a mood-elevating drug with an energizing action.


Some aspects of the pharmacology of analgesia. This paper makes comparisons of some of the pharmacological properties of the opiate analgesics and the tricyclic antidepressants in order to justify the clinical examination of the tricyclics as potential analgesic or proanalgesic agents in man. Preliminary animal data suggest that the tricyclic agent clomipramine (Anafranil, Geigy) will potentiate the effects of morphine, pentazocine and cyclazocine.


The use of psychotropic drugs in rheumatology. The possible role of various psychotropic drugs in the management of rheumatic diseases is considered--tranquillizers, sedatives, tricyclic antidepressants and monoamine oxidase inhibitors. The dilemma of which anti-depressant for which patient is considered. The possible association between rheumatoid disease and disorders of the brain, brain-stem and autonomic nervous system is discussed. Psychotropic drugs may effect the disease itself as well as the patient's mood. The author is opposed, however, to the idea that rheumatoid arthritis may be regarded as a psychosomatic disease.


The use of psychotropic drugs in a pain clinic. The paper is based on experiences gained at a hospital pain clinic. Patients attending such a pain clinic can be divided into three categories: (1) Those with chronic organic lesions in whom the mental state was little affected. (2) Those with organic lesions who had mental changes which influenced their experience of the pain. (3) Patients who had primary mental changes in whom the complaint of pain was a symptom of their mental state. The importance of full history taking, ab initio, is stressed. Much can be learned from patients' ideas of causation and these should be considered. Phychiatric diagnoses should be reached positively and not by a process of exclusion. Distinction must be made between depression and normal sadness and it is important to establish the relationship between the onset of depression and the painful symptoms. When psychopathology is evident a variety of treatments are available. There is, however, a 'hard core' of patients who display no depression and little anxiety. In such cases the manipulative value of pain should be considered.


Nefopam HCl interaction study with eight other drugs. A controlled clinical trial was undertaken to determine potential for drug interaction of nefopam HCl, a new analgesic, with eight other widely used compounds. Nefopam HCl was administered in combination with these drugs and placebo to forty-five healthy volunteer subjects in nine groups of five subjects each. Possible drug interactions were detected by the occurrence of side-effects and interference with bioavailability of the new analgesic, also by changes in vital signs or in various laboratory tests. Results indicated no statistically significant differences in these parameters between the nefopam HCl-placebo regimen and the other eight combinations. Despite this, there were substantial clinical differences in the intensity and incidence of side-effects with combinations of codeine, pentazocine and propoxyphene. These differences warrant further study. Serum nefopam HCl levels were significantly higher on Day 3 than on Day 1, indicating no defect in bioavailability due to drug interaction. Overall, results of this study support the feasibility of concomitant use of these eight drugs with nefopam HCl for short treatment periods.


A comparative study of antidepressants in the treatment of depressive states. The effect of six antidepressants has been investigated in six groups of twenty patients suffering from depressive states. The over-all response of the six groups, as well as the response of the individual target symptoms of depression according to the Hamilton Rating Scale are reported.


Preference studies of triazolam with standard hypnotics in out-patients with insomnia. One hundred and four patients suffering from insomnia took part in four different two-night double-blind crossover trials of triazolam. In three separate studies, triazolam 0-5 mg was compared to placebo, flurazepam 30 mg and chloral hydrate 500 mg. Triazolam 0-5 mg was found to be preferred and to be superior to placebo, flurazepam and chloral hydrate in the treatment of insomnia. Analysis of sleep questionnaire data showed triazolam to be superior to the other treatments on the following: How much did the medication help you sleep, onset of sleep, duration of sleep and number of awakenings. Additionally, triazolam was superior to chloral hydrate on the feeling in the morning parameter. In another comparison of triazolam 0-25 mg to flurazepan 15 mg, triazolam was not significantly better than flurazepam on any of the efficacy parameters except that the patients felt more alert the morning following triazolam that following flurazepam. On all efficacy endpoints, trends for all parameters favoured triazolam 0-25 mg over flurazepam 15 mg. Untoward side-effects in these four studies were minimal.


The nocturnal intragastric pH in EEG sleep stages in peptic ulcer patients. In 10 peptic ulcer patients, 5 duodenal ulcer and 5 gastric ulcer patients, the relation of nocturnal intragastric pH to EEG sleep stages was examined. The nocturnal intragastric pH was monitored by pH-measuring telemeter and sleep stages were recorded simultaneously by electroencephalography throughout the night. The EEG sleep stages were classified after the criteria of Dement and Kleitman. The average of intragastric pH level at each sleep stage was higher in gastric ulcer than the duodenal ulcer. The intragastric pH level became low as the patients' sleep advanced to deep stage. This tendency was more likely in duodenal ulcer patients than the gastric ulcer patients, which indicated that the cephalic phase might play an important role in the pathogenesis of hyperacidity in duodenal ulcer.


Comparison of an H2 receptor antagonist and a neutralizing antacid on postprandial acid delivery into the duodenum in patients with duodenal ulcer. Measurement of the postprandial rate of acid delivery into the duodenum directly assessed the efficacy of two radically different acid-reducing therapies for duodenal ulcer disease. Cimetidine, 400 mg, with an ordinary solid meal decreased the 4-hr delivery of titratable acid and hydrogen ion into the duodenum by 63 and 86%, respectively (P less than 0.01 versus control). Liquid Maalox, 30 ml, 1 and 3 hr after an identical meal reduced 4-hr delivery of acid by 47 and 74%, respectively (P less than 0.01 versus control). During the study period, the H2 receptor antagonist effected a continuous reduction in gastric acidity and the delivery of acid into the duodenum. The liquid neutralizing antacid produced a more fluctuating decrease in these parameters. However, given in these dosages, the magnitude and duration of the acid-reducing effect were similar for both treatments.


Aspirin esterase of gastric mucosal origin. The enzyme, aspirin esterase, which converts acetylsalicyclic acid to the less toxic salicyclic acid, was found to be present in gastric mucosal specimens obtained from surgically resected tissue. The enzyme was found to be stable to storage and active at two pH optima. Alcohol in the reaction mixture produced a net effect of slowing the rate of aspirin hydrolysis; this was attributable to a marked inhibitory effect on aspirin esterase activity which was not completely counteracted by the increased rate of spontaneous breakdown of aspirin by alcohol. Age, sex, or gastric disease state of the patient from whom the tissue was obtained, did not significantly alter the level of enzyme activity measured, nor were different levels obtained from body or antral mucosa. In patients with gastric ulcer, those with a previous history of regular aspirin consumption did not show significantly different levels from those without such a history. It is concluded that aspirin esterase activity of gastric mucosa is not alone a significant factor in any role acetylsalicyclic acid may play in the etiology and natural history of chronic peptic ulcer.


Ditazole and platelets. I. Effect of ditazole on human platelet function in vitro. Ditazole (4,5-diphenyl-2-bis-(2-hydroxyethyl)-aminoxazol) has been  shown to be a strong in vitro inhibitor of human platelet aggregation brought about by release reaction inducers; in contrast, it did not significantly affect primary ADP-induced aggregation. Ditazole strongly inhibited the release of platelet-bound 14C-serotonin under the influence of Thrombofax, whereas it did not interfere with the transport and storage of serotonin in nonstimulated platelets. The effect of ditazole was not potentiated by acetylsalicylic acid. Ditazole also inhibited ADP-reptilase clot retraction and modified thrombin-induced clot formation. The inhibition of platelet aggregation exerted by ditazole in plasma could be removed following gel filtration of platelets on Sepharose 2-B gel. This would indicate that ditazole does not act on platelets by a 'hit and run' mechanism.


[Histamine 2 receptor antagonists in the treatment of gastric ulcer]. Eight patients with a clinical, radiology and endoscopic diagnosis of benign peptic ulcer were studied in a prospective fashion using Cimetidine (H2-Receptor antagonist). With this outgoing form of therapy the need for antiacid diminished greatly and pain totally disappeared. We discuss the possible etiological pathways of gastric ulcer and finally propose the simultaneous use of H2-Receptor antagonist and colesteramine with the goal of eliminating the two predominant factors that cause the lesion.


[Glucocorticoid action of beclomethasone and its propionate ester derivatives]. In the present pharmacokinetic studies on beclomethasone dipropionate in rats, it was suggested that most of the beclomethasone-17, 21dipropionate is transformed rapidly into beclomethasone-17-monopropionate or beclomethasone in the blood or tissues and exerts the glucocorticoid effect in the form of beclomethasone. Glucocorticoid activity of beclomethasone was revealed to be 1-10% that of dexamethasone both in the hypothalamopituitaryadrenocortical suppressive effect and liver tyrosine amino transferase inducing efect in rats. Beclomethasone and its propionate derivatives were shown to have higher binding affinity to glucocorticoid receptors in rat liver, thymus and hypophysis than that of dexamethasone. The experimental results herein may serve to explain why beclomethasone and its propionate derivatives showed such a weak glucocorticoid action in comparison with that of dexamethasone. The amounts of receptor-beclomethasone (or its propionate derivatives) complexes which bound specifically to liver nuclei were only 10% or less than those of receptordexamethasone complexes and the in vivo metabolism of beclomethasone and beclomethasone-dipropionate was more rapid than that of dexamethasone.


A comparative clinical study for the evaluation of antianxiety agents, (lorazepam, diazepam and placebo), in neurosis. To evaluate the clinical effects of lorazepam on neurosis, a double-blind controlled trial by group comparison procedure was carried out using diazepam and placebo as control drugs. The clinical improvement and aggravation rates by means of a variety of assessment of change were compared between each of the two active drugs and placebo. The superiority, characteristics of therapeutic effects and minor side effects of lorazepam which is similar to those of diazepam, a standard benzodiazepine anxiolytic for the treatment of neurosis, were demonstrated. From this study, many important problems which might be influenced upon the outcome of the pharmacotherapy on neurotics, such as positive and negative placebo effects, aggravation and drop-out during the trial, were also evaluated.


[The regulation of FSH release by the testis. Studies on inhibin]. The FSH release from the hypophysis is suggested to be particularly regulated by a testicular hormone called inhibin. Origin, structure and target organs of inhibin are unknown. Experiments to test some hypotheses in this field are described. Adult male rats, prenatally treated with busulfan, show only Sertoli cells in the semiferous tubules. Experimental cryptorchidism and orchidectomy, however, leads to an increase in FSH levels as observed in normal animals. This indicates the role of Sertoli cells in FSH regulation. Ligation of efferent ducts of testes leads to an increase of FSH levels, too, indicating that an FSH-inhibiting principle cannot be absorbed. Interstitial testis fluid (ITF) of normal rats was applicated to immature female rats. Their FSH release is inhibited, visible in the lower ovarian weight gain following additional hCG-administration. Orchidectomized animals react with a decrease of FSH levels to the application of ITF. Therefore ITF seems to contain a FSH-inhibiting factor. Androgen binding protein-content of epididymes, however, is increased after repeated injections of ITF. It is concluded that testis (probably the Sertoli cells) produces a FSH-inhibiting factor, but ITF contains only small amounts of inhibin.


Suppression of ACTH secretion by synthetic MSH-release inhibiting factor Pro-Leu-Gly-NH2 in Addison's disease. The regulation of release of MSH and ACTH seems to be closely related. The effect of Pro-Leu-Gly-NH2, the most active MSH-release inhibiting factor (MIF), was investigated on ACTH-hypersecretion in six patients suffering from primary adrenocortical insufficiency. MIF was infused at constant rates with stepwise increase in concentration (0.25 mg, 2.5 mg, 25.0 mg/30 min). Plasma ACTH-levels were measured radioimmunologically at 10 minutes intervals. In four cases there was a remarkable decrease (P less than 0.005) in the ACTH plasma level of more than 50% as compared to the elevated starting levels of 800 to 1400 pg/ml. It is concluded that under certain circumstances MIF could play a role in the regulation of ACTH-secretion.


Isolation and partial characterization of plasma membranes from the livers of control and Streptococcus pneumoniae-infected rats. Plasma membranes were isolated from the livers of control and Streptococcus  pneumoniae-infected rats. This work, therefore, represents the first isolation of plasma membranes from infected actron microscopy and by the use of enzyme markers for microsomes (glucose-6-phosphatase), mitochondria (glutamate and malate dehydrogenases), and lysosomes (acid phosphatase). Plasma membranes from infected cells banded at the same sucrose density as plasma membranes from uninfected cells. Moreover, equivalent amounts of plasma membranes could be isolated from control and infected rat livers. There were, however, significant alterations in the enzyme complement of the plasma membrane after infection. 5'-Nucleotidase activity was significantly decreased, whereas alkaline phosphatase activity was significantly increased. Kinetic analysis demonstrated that only the Vmax and not the Km of these two enzymes was changed, suggesting that the altered affinity of the enzymes for substrate was not the mechanism responsible for the observed alterations. No change in the mitochondrial enzyme markers was observed after infection, but the specific activity of microsomal glucose-6-phosphatase decreased significantly. Possible explanations for the observed alterations are discussed.


Gamma-irradiated chymotrypsin-like proteins. II. Connection between inactivation and structural changes. The radiation yeilds of unfolding (Gconf) determined by the method of tryptophan fluorescence coincide with the radiation yields of proteolytic inactivation (Gin) for chymotrypsin-like (CT-like) enzymes on irradiation in air, both in solution and in the dry state with futher dissolution at pH7. It can be supposed that the unfolding is the main process determining the proteolytic gamma-inactivation of CT-like enzymes. It was also shown that the transition of chymotrypsin and trypsin gamma-irradiated at acid pH to neutral pH is an additional action, leading to unfolding of part of the molecules.


Effect of changes in Pco2 on intraocular tension. Elevated levels of inspired CO2 and blood Pco2 resulted in moderate elevation of IOT. A marked rapid decrease in IOT to a level less than baseline value was noted when inspired CO2 was suddenly decreased to ambient levels. Decrease in IOT was more pronounced than the decrease in Pco2 and increase in blood pH. Changes in IOT appeared related to the rate of change of Pco2 rather than the actual level of Pco2. Increased ventilatory excursions with constant inspired CO2 levels did not cause any elevation of IOT, but a minimal compensatory drop in IOT below resting values occurred when increased ventilatory excursions were discontinued. It is postulated that the changes in IOT noted are the result of sudden changes in aqueous production or ocular blood volume.


Loss of histochemically demonstrable catecholamines in the glomus cells of the carotid body after alpha-methyl-para-tyrosine treatment. A statistically significant decrease in the intensity of catecholamine fluorescence of some carotid body glomus cells was observed after inhibition of the enzyme tyrosine hydroxylase by injection of 80 mg/kg alpha-methyl-paratyrosine. The intensity of the formaldehyde-induced fluorescence was measured in individual glomus cells. The maximum decrease in the intensity was observed 4 to 6 hr after the alpha-methyltyrosine injection. This suggests a rapid turnover in the catecholamines of the carotid body.


A new glucose 6-phosphate dehydrogenase variant (GDTrinacria) in two unrelated families of Sicilian ancestry. A new G6PD variant in two unrelated families of Sicilian extraction is described. It is characterized by an enzyme activity amounting to 20% of normal, by a slow electrophoretic mobility, decreased Km values for both TPN and G6P, increased utilization of substrates analogues, thermolability and biphasic pH curve. It has been named G6PD Trinacria.


Endopolygalacturonase, an extracellular pectic enzyme of Rhizoctonia fragariae. A pectolytic enzyme from culture filtrates of Rhizoctonia fragariae was purified approx. 29-fold. The enzyme exhibited maximal depolymerizing activity on Na-polypectate rather than pectin at pH 5.0 and was inhibited at different extent by divalent cations Mg++, Mn++, Ca++. The analysis by paper chromatography of the products of enzyme hydrolysis suggested that the enzyme attacks the substrate by a random mechanism. The absorption spectrum of the chromogen formed by the hydrolysis products and thiobarbituric acid suggested that the enzyme is a hydrolytic enzyme. On the basis of these results the enzyme is classifiable as endo-polygalacturonase (poly alpha-1,4-galacturonide glycanohydrolase E.C. 3.2.1.15).


Volume characteristics of extra- and intraparenchymal segments of the canine pulmonary artery. The volumes of the extraparenchymal segment (VpaEP) and intraparenchymal segment (VpaIP) of the pulmonary arterial tree were determined in intact anesthetized dogs during room air breathing and acute hypoxia. Total pulmonary arterial blood volume (Vpatotal) was calculated as the product of pulmonary blood flow and pulmonary arterial circulation time. An angiographic technique was used to estimate VpEP. VpaIP was calculated by subtracting VpaEP from Vpatotal. During room air breathing at functional residual capacity, mean +/- SD of VpaEP was 17.1 +/- 5.1 ml and of VpaIP was 31.7 +/- 20.8 ml, representing 40% and 60%, respectively, of Vpatotal. Vpatotal increased 22.2 +/- 10.5 ml during lung inflation, with proportional increases in VpaIP and VpaEP. VpaEP was found to be influenced equally by changes in transmural pulmonary arterial and transpulmonary pressures. Acute hypoxia was accompanied by an increase in pulmonary vascular resistance and a decrease in volume distensibility of the extraparenchymal segment. Vpatotal increased 76% without changes in the relative volume distribution of VpaEP and VpaIP. These findings can be best explained by active vasomotion with an increase in down-stream pulmonary vascular resistance.


Comparison of arm versus leg work in induction of acute episodes of asthma. The severity of exercise-induced asthma varies with the type of exercise performed. To determine whether such variation could be attributed to the use of different muscle groups, we exercised arms separately from legs using a bicycle ergometer. First, arms were exercised to exhaustion, then legs were exercised at the same load for the same duration. Arm work resulted in greater ventilation, heart rate, hydrogen ion concentration, and airway obstruction than did leg work. Later, legs were exercised to exhaustion using a load more than twice that of the arm work. Both the exhausting leg work and exhausting arm work resulted in significant bronchospasm and acidosis, whereas the nonexhausting leg work did not. These data suggest that, in arm and/or leg exercise, the relationship of work load to muscle mass is a determinant of airway obstruction.


Ventilatory response in the fetal lamb following peripheral chemodenervation. Carotid infusions of sodium cyanide solution and perfusions of hypoxemic or hypercapnic fetal blood were done before and after peripheral chemoreceptor denervation. Step changes in PaO2 ranged from -11 to -22 Torr; step changes in PaCO2 ranged from +17 to +42 Torr. The cyanide dose was 0.2 mg/kg per loop system. Control perfusions consisted of 25 ml of fetal blood without changes in pH and blood gases. A ventilatory response occurred in the majority of all experimental perfusions regardless of innervation status of the peripheral chemoreceptors. No response occurred with control perfusions. There was a marked variability in the time of onset of ventilatory activity with a delay of greater than 10 s occurring following most perfusions. These studies demonstrate that the fetus has an attenuated ventilatory response to chemical stimuli and that hypoxia stimulates ventilation in the absence of peripheral chemoreceptors.


H+ transport from CNS in hypercapnia and regulation of CSF [HCO3-]. CSF HCO3- increases more than plasma HCO3- in hypercapnia, and there are at least two sources for the CSF HCO3- increase--one derived from the simultaneous increase in plasma HCO3-, and the other, HCO3-formed from hydration of CO2 in the choroid plexus and glia and susceptible to inhibition by acetazolamide (J. Appl. Physiol. 38: 504-512, 1975). It was proposed that the H+ formed in the CNS in CO2 hydration is actively exchanged for plasma Na+ utilizing the Na-K ATPase pump. H+ transport from the CNS was therefore studied in four groups of dogs breathing 5% CO2 at constant VA for 4 h with repeated injections of saline, acetazolamide 5 mg/ml, ouabain 0.1 mg/ml, and acetazolamide and ouabain together into lateral cerebral ventricles. Arterial HCO3-increased 2.5 meq/l at 4 h of hypercapnia in all groups. CSF HCO3-increased 5.8 meq/l in the saline-injected animals, but it increased only about 2 meq/l and equaled plasma HCO3- rise in the other three groups. Therefore CNS HCO3- formation in hypercapnia can be blocked by inhibiting the CO2 hydration reaction with acetazolamide or by blocking H+ removal by inhibiting Na-K ATPase with ouabain. The data support the thesis of active H+ removal from the CNS in exchange for plasma Na+ in hypercapnia.


Hematology, viscosity, and respiratory functions of whole blood of the lesser mouse deer, Tragulus javanicus. Blood samples from the lesser mouse deer were examined for hematology, viscosity, oxygen dissociation curve, and magnitude of the Bohr effect. Red corpuscle dimensions, determined under oil immersion with an ocular micrometer, averaged 2.2 micron while the cell counts averaged 53 million/micronl blood, and the packed cell volume averaged 31.2%. Blood hemoglobin concentration averaged 11.2 g/100 ml and the calculated mean cell hemoglobin concentration was 38 g/100 ml. The relative viscosity of the mouse deer plasma was 1.97 and increased in a nonlinear manner with hematocrit to 100 at 80% packed cell volume. Oxygen-hemoglobin equilibrium curves, determined with a mixing technique at 37 degrees C and 10, 36 and 71 Torr PCO2, have the same configuration observed in blood from mammals in general. The P50 of the mouse deer blood at pH = 7.40 is 34 Torr and the Bohr effect (deltalog P50/deltapH) is -0.483. The mouse deer have blood hematocrits which are well below the hematocrits observed in mammals with larger erythrocytes, but similar to the blood hematocrits observed in other mammals with small erythrocytes. We suggest that the low hematocrit is an adaptation which circumvents the hemodynamic problems associated with a high blood viscosity and that, in the mouse deer, the expected concomitantly low total blood hemoglobin concentration is compensated by a higher than average mean cell hemoglobin concentration.


Effects of potassium ions on the electrical and pH gradients across the membrane of Streptococcus lactis cells. Bacteria transduce and conserve energy at the plasma membrane in the form of an electrochemical gradient of hydrogen ions (deltap). Energized cells of Streptococcus lactis accumulate K+ ions presumably in exchange for H+. We reasoned that if the movement of H+ is limited, then an increase in H+ efflux, effected by potassium transport inward, should result in changes in the steady-state deltap. We determined the electrical gradient (deltapsi) from the fluorescence of a membrane potential-sensitive cyanine dye, and the chemical H+ gradient (deltapH) from the distribution of a weak acid. The deltap was also determined independently from the accumulation levels of the non-metabolizable sugar thiomethyl-beta-galactoside. KCl addition to cells fermenting glucose or arginine at pH 5 changed the deltap very little, but lowered the deltapsi, while increasing the deltapH. At pH 7, the deltapH only increased slightly; thus, the decrease in deltapsi, effected by addition of potassium ions, resulted in a lowered steady-state deltap. These effects were shown not to be due to swelling or shrinking of the cells. Thus, in these nongrowing cells, under conditions of energy utilization for the active transport of K+, the components of deltap can vary depending on the limitations on the net movement of protons.


Purification and properties of cis-toluene dihydrodiol dehydrogenase from Pseudomonas putida. The purification of (+)-cis-1(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene dehydrogenase from cells of Pseudomonas putida grown with toluene as the sole source of carbon and energy is reported. The molecular weight of the enzyme is 104,000 at pH 9.7. The enzyme is composed of four apparently identical subunits with molecular weights of 27,000. The enzyme is specific for nicotinamide adenine dinucleotide and oxidizes a number of cis-dihydrodiols. Both enantiomers of a racemic mixture of cis-1,2-dihydroxyl-1,2-dihydronaphthalene dihydrodiol are oxidized by the enzyme. No enzymatic activity is observed with trans-1,2-dihydroxyl-1,2-dihydronaphthalene dihydrodiol.


Purification and characterization of a neutral protease from Saccharomycopsis lipolytica. Saccharomycopsis lipolytica 37-1 produced two inducible extracellular proteases, one under neutral or alkaline growth conditions and the second under acid conditions. Secretion of the neutral protease was repressed in the presence of glycerol or glucose, both of which supported rapid growth of the organism. Ammonium ions also repressed the secretion of the enzyme. The neutral protease activity copurified with esterase activity during ammonium sulfate fractionation, chromatography on diethylaminoethyl-cellulose, and gel filtration on Sephadex G-150. The molecular weight of the enzyme was estimated to be 42,000 by sucrose density gradient centrifugation and 38,500 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified enzyme had a pH optimum of 6.8. Phenylmethylsulfonylfluoride inhibited both protease and esterase activities, indicating the presence of a serine residue in the active center. Protease, but not esterase, activity was sensitive to ethylenediaminetetraacetate and was significantly activated by divalent ions. Dithiothreitol inhibited both protease and esterase activities, indicating the presence of a critical disulfide bridge. The enzyme hydrolyzed casein (K(m) = 25.6 muM) and hemoglobin as well as the nitrophenyl esters of tyrosine (K(m) = 2.4 mM), glycine, tryptophan, and phenylalanine.


6-Phospho-D-gluconate:NAD+ 2-oxidoreductase (decarboxylating) from slow-growing Rhizobia. 6-Phospho-D-gluconate:NAD+ 2-oxidoreductase (decarboxylating) (NAD+-6PGD) was detected in several slow-growing strains of rhizobia, and no activity involving NADP+ was found in the same extracts. By contrast, fast-growing strains of rhizobia had NADP+-6PGD activity; most of them also had NAD+-6PGD activity. NAD+-6PGD was partially purified from the slow-growing strain Rhizobium japonicum 5006. The reaction was shown to be an oxidative decarboxylation.


Regulation of enzyme synthesis by the glutamine synthetase of Salmonella typhimurium: a factor in addition to glutamine synthetase is required for activation of enzyme formation. In Klebsiella aerogenes but not in Salmonella typhimurium glutamine synthetase can function during nitrogen-limited growth to increase the rate of synthesis of histidase from the hut genes of S. typhimurium 15-59 (hutS. 15-59). Formation of proline oxidase is also not increased in nitrogen-limited cultures of S. typhimurium. However, in hybrid strains of Escherichia coli or K. aerogenes, the glutamine synthetase of S. typhimurium activates synthesis of histidase from the hutS. 15-59 genes. Apparently, glutamine synthetase is necessary but not sufficient for activation of transcription of the hut genes; another factor must also be present. This factor is active in both K. aerogenes and E. coli but is missing or altered in S. typhimurium.


Alkaline serine proteinases D and E of Streptomyces griseus K-1. Two DFP-sensitive alkaline proteinases with strong esterase activity toward Ac-(Ala)3-OMe, designated as alkaline serine proteinases D and E, were purified pronase, a protease mixture from St. griseus K-1. Each was shown to be homogeneous by acrylamide disc gel electrophoresis. The molecular weights of these enzymes were estimated to be about 27,000 be gel filtration. Studies on their actions on acyl-tl-amino acid methyl or ethyl esters indicated that proteinases D and E both exhibited a broad substrate specificity and hydrolyzed the ester bonds of esters containing Trp, Tyr, Phe, Leu, and Ala. The esterase activities of both enzymes toward Ac-(Ala)3-OMe were the highest among proteinases so far isolated from various sources. Proteinases D and E also lacked cystine residues in their molecules, being entirely different from alkaline serine proteinases A, B, and C in pronase. Some differences were , however, observed between them as regards pH stability, behavior on CM-cellulose, mobility on polyacrylamide electrophoresis, and amidase activity toward Suc-(Ala)3-pNA.


Amino acid sequence of a cyanogen bromide fragment containing the two tryptophanyl residues of lobster arginine kinase (Homarus vulgaris). Lobster arginine kinase [EC 2.7.3.3] contains 2 tryptophanyl residues and 9 methionyl residues. The whole carboxymethylated protein was first subjected to CNBr cleavage and the resulting fragments were isolated by gel filtration and other experimental approaches. One fragment, CB5, which contains 60 residues including the two tryptophanyl residues and two of the five cysteinyl residues of the protein, was characterized and the results are reported inthis paper. The overall strategy for the establishment of the complete sequence of this fragment was based on the use of three types of peptides: (a) whole cyanogen bromide peptide CB5 which was partially characterized by automatic Edman degradation using a sequencer: 42 steps were performed out of 60 residues, (b) tryptic peptides of CB5, (c) peptides formed by cleavage of S-carboxymethylated arginine kinase (whole protein) at the two tryptophanyl residues with BNPS-skatole. The complete amino acid sequence of the CNBr polypeptide (CB5) which contains the two tryptophanyl residues of the whole protein was established.


Purification and characterization of arylamidase from monkey brain. Arylamidase [EC3.4.11.2] was isolated from monkey brain extract and purified about 2100-fold in approximately 11% yield by a six-step procedure comprising extraction from monkey brain homogenate, ammonium sulfate fractionation, first hydroxylapatite chromatography, DEAE-cellulose chromatography, Sephadex G-200 gell filtration and second hydroxylapatite chromatography. The enzyme showed a single band on polyacrylamide disc electrophoresis and consisted of a single polypeptide chain, as judged by disc electrophoresis in the presence of sodium dodecyl sulfate. The enzyme was strongly inhibited by PCMB, TPCK, and puromycin. Puromycin competitively inhibited the enzyme and the Ii value was about 5 x 10(-7)M. Treatment with EDTA resulted in a loss of enzyme activity. The enzyme activity was restored by addition of Zn2+, Co2+, Mn2+. Among various amino acid beta-naphthylamides, L-alanine beta-naphthylamide was most rapidly hydrolyzed and N-carbobenzoxyl-L-leucine beta-naphthylamide was not hydrolyzed by this enzyme preparation. The molecular weight of the enzyme was 92,000 as determined by gel filtration on Sephadex G-200.


Anhydrotrypsin: new features in ligand interactions revealed by affinity chromatography and thionine replacement. Anhydrotrypsin was isolated in high purity from the product of base elimination from phenylmethanesulfonyl-trypsin, by a single operation of affinity chromatography. The adsorbent used for the chromatography was an agarose derivative coupled with peptides containing C-terminal arginine residues. As the affinity of the adsorbent for anhydrotrypsin was high compared with that for trypsin, purification of the enzyme derivative was easily achieved without the prior inactivation of trypsin which had been regenerated during the elimination reaction. Comparative studies of the ligand interaction specificities with anhydrotrypsin and trypsin confirmed the stronger interaction of the former protein with product-type ligands such as Bz-Arg-OH. No marked differences were observed between them in affinities toward substrate-type ligands such as Bz-Arg-NH2. The higher affinity of anhydrotrypsin was found to be limited to product-type ligands of L-configuration, i.e., the protein displayed an ability to discriminate the L-ligand from its optical isomer. THE PKa value for the ionization form of anhydrotrypsin responsible for the interaction with Bz-Arg-OH was estimated to be 7.60+/-0907


Glutathione reductase in the sea urchin egg. III. Activation of the complex form by proteinases. The G-200 flow-through fraction of the extract of sea urchin eggs contained a complex form of glutathione reductase (GR) [EC 1.6.4.2]. The complex was unstable and gradually dissociated with ain increase in GR activity. The activation was facilitated by high concentrations of EDTA, KCI or (NH4)2SO4. The rate of activation by salts was apparently dependent on the ionic strength. The complex form was also activated rather quickly by treatment with proteinases such as trypsin [EC 3.4.21.4], alpha-chymotrypsin [EC 3.4.21.1] or subtilisin [EC 3.4.21.14]. Trypsin caused the complex to release the free form of GR. Even after trypsin treatment, little change was observed in the dependence of the GR activity on GSSG or NADPH concentration. The GR activity of the complex form was not inhibited at all by 0.2 mM N-ethylmaleimide (NEM) in the presence of GSSG, but was reduced to 3% in the presence of NADPH. When excess NEM was sequestered with GSH, the NEM-treated complex form was strikingly activated by trypsin, while no activation was detected with the free form of enzyme pretreated with NEM. These results suggest that the active site of GR in the complex form is largely masked by a polypeptide moiety of theinhbitiory component.


A new enzyme, NADPH-dihydropteridine reductase in bovine liver. An enzyme designated as NADPH-dihydropteridine reductase was found in the extract of bovine liver and partially purified. In contrast to NADH-dpendent dihydropteridine reductase [EC 1.6.99.7], the enzyme catalyzes the reduction of quinonid-dihydropterin to tetrahydropterin in the presence of NADPH. The two enzymes were separated by column chromatography on DEAE-sephadex. Tyrosine formation in the phenylalanine hydroxylation system was also stimulated by NADPH-dihydropteridine reductase. The existence of these two dihydropteridine reductases suggests that the tetrahydro from ofpteridine cofactor may be regenerated in two different ways in vivo.


Glycolysis of red cells suspended in solutions of impermeable solutes. Intracellular pH and glycolysis. The glycolytic rate human red cells suspended in a sucrose medium of low or physiological pH was higher than that of the cells suspended in Ringer's medium of the same. pH. The medium pHP-glycolytic rate curve of red cells suspended in soucrose media shifted to the acidic side by about one unit compared with that of cells suspended in Ringer's medium. Similarly, the pattern of glycolytic intermediates in red cells suspended in a sucrose medium resembled that in cells suspended in Ringer's solution of about one unit higher pH. These phenomena could be ascribed to the change of intracellular pH, which was measured by the 5,5'-dimethyl-oxazolidine-2,4-dione method. A similar stimulation of glycolysis was observed when sodium citrate was added to red cells suspended in Ringer's solution at constant pH. These observations indicate that membrane-impermeable non-electrolytes or anions stimulate glycolysis of red cells by elevation ofthe intracellular pH. Red cell glycolysis is influenced mainly by the intracellular pH rather than by the pH of the suspending medium.


Actin-induced local conformational change in the myosin molecule. I. Effect of metal ions and nucleotides on the conformational change around a specific thiol group (S2) of heavy meromyosin. As previously reported when a specific thiol group, S2, of myosin reacts with N-ethylmaleimide (NEM), its Ca2+-ATPase activity is decreased. Therefore, the reactivity of S2 can be estimated by measuring the decrement of the enzymatic activity. Using the change in the reactivity as a structural probe, we investigated whether F-actin affects the conformation around the region containing S2 under physiological conditions (at neutral pH and low ionic strength). 1. Experiments were carried out with heavy meromyosin (HMM), S1 of which had heen blocked with NEM, to observe the reactivity of S2 alone. In the experiments done in the presence of F-actin, the Ca2+-ATPase activity was measured using the heavy meromyosin fraction after actin had been removed by centrifugation and gel filtration. 2. ATP and other nucleotides activated the reactivity of S2 in the presence of Mg2+. On the other hand, F-actin markedly activated the reactivity of S2 which had been increased by ATP, but not by the other nucleotides. 3. The above cooperative action of F-actin with ATP was not observed in the presence of Ca2+ instead of Mg2+, or above 0.2 M KCl. These results suggest that the S2 region of the myosin molecule is a key region in the molecular interaction of the actin myosin-ATP system under physiological conditions.


Ciliary dynein from sea urchin embryos.

Ciliary dynein from sea urchin embryos. Axonemal dynein ATPase [EC 3.6.1.3] was extracted from cilia of sea urchin embryos for a study of its enzymatic properties. Sedimentation analysis on a sucrose density gradient revealed that ATPase activity was associated with the 12S particles. The partially purified 12S enzyme was characterized mainly with regard to the optimum pH, divalent cation and ionic strength requirments and substrate specificity. Comparative investigation of the data obtained indicates that the properties of the present dyneine ATPase resemble those of other dynein(-like) ATPase hitherto reported. In addition, the possible relationship among dyneins within a single species, in particular between the ciliary dynein and cytoplasmic dynein-like ATPase, is discussed.


Amino acid sequences around 1, 2-epoxy-3-(p-nitrophenoxy)propane-reactive residues in rhizopus chinensis acid protease: homology with pepsin and rennin. Two different peptides containing an aspartyl residue reactive with 1, 2-epoxy-3-(p-nitrophenoxy)propane (EPNP) in the acid protease from Rhizopus chinensis were isolated from a peptic digest of the EPNP-modified enzyme. One of the peptides was sequenced as Asp-Thr-Gly-Ser-Asp. The amino acid sequence had very high homology with those around the EPNP-reactive aspartyl residues in rennin (chymosin) [EC 3.4.23.4] and pepsin [EC 3.4.23.1]. The other peptide contained no methionine residue and gave the sequence: Asp-Thr-Gly-Thr-Thr-Leu. The N-terminal aspartyl residue of each peptide was deduced to be the EPNP-reactive site.


Phosphoenolypyruvate synthetase of Escherichia coli: molecular weight, subunit composition, and identification of phosphohistidine in phosphoenzyme intermediate. Phosphoenolypyruvate synthetase of Escherichia coli has been shown to be a dimer of molecular weight 150,000. The constituent subunits appear to be identical. The enzyme tends to dissociate to monomers at low protein concentration, but the tendency is much diminished in the phosphoenzyme form, suggesting that enzyme phosphorylation is accompanied by a structural rearrangement in the subunit contact domain. The enzyme appears to show half of the sites reactivity with respect to its phosphorylation by ATP. Several lines of evidence, including identification of 3-phosphohistidine in alkaline digests of the phosphoenzyme, indicate that a histidyl residue is the site of phosphorylation.


Source of residual Bohr effect in hemoglobin oxidation. The hemoglobin oxidation Bohr effect is larger than the ligation Bohr effect, even when the former is corrected for any ionization of the water molecule bound to the ferric iron of methemoglobin. This residual oxidation Bohr effect is here shown to be solely caused by the influence of the positively charged ferriheme, and is abolished when the oxidized heme binds an anion. This result frees the formal equivalence of hemoglobin ligation and oxidation from the last apparent experimental discrepancy.


Magnetic resonance studies of concanavalin A: assignment of histidine resonances in 220 MHz proton spectrum of complexes with Co2+ and Zn2+. The low field regions of the 220 MHz proton magnetic resonance spectra of concanavalin A (Con A) complexes with metal ions show well resolved resonances from the C2 protons of histidine side chains. Shifts of these resonances are observed when Zn2+ ions at the transition metal ion binding site, S1, are replaced by CO2 ions. The magnitude of these shifts can be used to determine the orientation of axis of anisotropy of the Co2+ ligand field since the distances from the C2 protons of the histidines to S1 can be computed from the crystal structure coordinates. Assignment of the separate peaks in the spectrum of the Con A-Co2+-Ca2+ complex and of the Con A-Zn2+-Ca2+ complex, to particular histidines in the amino acid sequence of Con A then follows. The refined crystal coordinates of both Reeke et al. (Reeke, G. N., Jr., Becker, J. W., and Edelman, G. M. (1975) J. Biol. Chem, 250, 1525-1547) and of Hardman and Ainsworth (private communication) have been used. These two sets of coordinates both yield orientations for the axis of anisotropy which are approximately in the direction of the His 24 nitrogen ligand.


Bovine adrenal tyrosine hydroxylase: purification and properties. Bovine adrenal tyrosine hydroxylase has been obtained in a form that is 85 to 90% pure. Sodium dodecyl sulfate-gel electrophoresis and density gradient centrifugation studies have established that the subunit molecular weight of the chymotrypsin-solubilized enzyme is 34,000. The presence of iron in the purified enzyme (0.50 to 0.75 mol of iron/mol of enzyme) has been established. Crude particulate tyrosine hydroxylase can be activated by the phospholipid, phosphatidyl-L-serine, or by exposure to enzymatic phosphorylating conditions. Both forms of activation lower the Km of the enzyme for its 2-amino-4-hydroxypteridine cofactor. By contrast, tyrosine hydroxylase that has been solubilized by chymotrypsin cannot be activated by either of these methods.


Isolation of glutamic acid methyl ester from an Escherichia coli membrane protein involved in chemotaxis. We have isolated glutamic acid 5-methyl ester from an Escherichia coli protein that is involved in chemotaxis. The bacteria were first incubated with [methyl-3H]methionine under conditions which are known to result in methylation of the protein. The protein, isolated by gel electrophoresis, was then digested by successive treatment with three proteolytic enzymes. One of the products was [methyl-3H]glutamic acid 5-methyl ester, identified by comparison with an authentic sample in the following studies: (a) chromatography on an automatic amino acid analyzer, (b) chromatography on paper in two solvent systems, (c) chromatography on paper of the N-acetyl derivatives, and (d) stability of the ester bond to various pH conditions. No aspartic acid 4-methyl ester was found in the enzymatic digest. Treatment of the methylated protein with alkali released the radioactivity as [3H]methanol, which was identified by gas chromatography and by preparation of the 3,5-dinitrobenzoate.


Induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase in HeLa cells by glucocorticoids. The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) has been demonstrated both in homogenates and microsomes of the S3G strain of HeLa cells. It was increased 8- to 10-fold by the removal of serum from the growth medium. The presence of steroids, specifically of the glucocorticoid series, in the serum-less growth medium elicited an additional 100 to 345% increase over the serum-less control, whereas the addition of N6,O2'-dibutyryl adenosine 3':5'-monophosphate to the medium or dexamethasone to the assay mixture was without any stimulatory effect. Both inductions were blocked by cycloheximide and actinomycin D, suggesting a protein synthesis-dependent elevation of enzyme activity. Glucocorticoids were effective in the induction at concentrations ranging from 10(-6) to 10(-8) M and there was a demonstrated parallel between the magnitude of enzyme induction and glucocorticoid potency. The HMG-CoA reductase activities from steroid-induced and control cultures had identical assay characteristics (pH optima and apparent Km values for both NADPH and HMG-CoA). This induction of the rate-controlling enzyme of cholesterogenesis occurred despite the observation that glucocorticoids specifically depress the rate of acetate or water, but not mevalonate, incorporation into cholesterol.


Purification and properties of a plant endonuclease specific for apurinic sites. An endonuclease which hydrolyzes depurinated DNA has been isolated from Phaseolus multiflorus enbryos; it has a molecular weight around 40,000. The enzyme is specific for apurinic sites; it has no action on normal DNA strands or on alkylated sites, and is without exonulcease activity. The rate of phosphoester bond hydrolysis near apurinic sites is far greater in native than in denatured DNA. The endonuclease is not inactivated by 10 mM EDTA, but is activity is however stimulated by Mg2+ or Mn2+. Its optimum pH is 7.5 to 8.0, and its optimum temperature 40degrees although, at this temperature, it is rapidly denatured; even low NaCl concentrations inhibit the enzyme activity. The endonuclease for apurinic sites of P. multiflorus is a non-histone protein of chromatin; the properties (like thermosensitivity of susceptibility to ionic strength) of the enzyme in situ, working on chromatin DNA, might be different from those described for the isolated endonuclease in homogenous aqueous solution.


Estrogen-binding protein from rat preputial gland: purification and characterization. Cytosol from the rat preputial gland has been shown to contain a protein which binds both estrone and estradiol. The protein, after a 26-fold purification from the cytosol of female Sprague-Dawley rats, migrated as one band during electrophoresis in sodium dodecyl sulfate on acrylamide gel. The electrophoretic mobility indicated a molecular weight of 15,000. The association constant for estrone as determined by equilibrium dialysis was 1.2 X 10(7) M-1, while that for 17beta-estradiol was 3.3 X 10(6) M-1. Progesterone, cortisol, testosterone, or diethylstilbestrol did not bind to the purified protein, whereas 17alpha-estradiol or estriol bound only slightly. In the presence of retinoic acid, but not retinol, the binding of estrone was reduced. Optimum binding for estrone was at pH 6.5 to 8.5.


Properties of chitin synthetase in isolated chitosomes from yeast cells of Mucor rouxii. Chitin synthetase was isolated and purified 120-fold from the supernatant fraction (54,500 X g) of broken yeast cells of Mucor rouxii. The purified preparations consisted mainly of chitin synthetase particles (chitosomes) with an average size larger than 7 X 10(6) daltons (by gel filtration) and an average sedimentation coefficient of 105 S. The samples also contained other enzyme complexes (fatty acid synthetase, pyruvate dehydrogenase, and, depending on method, ribosomes). Nearly all of the chitosomal chitin synthetase occurred in a zymogenic form that required proteolytic activation. In most properties, the chitosomal enzyme was similar to crude enzyme (54,000 X g sediment): kinetics, activation by proteases, response to metals, stimulation by N-acetylglucosamine, and inhibition by polyoxin or UDP. One mamor difference was the much greater stability of the chitosomal chitin synthetase zymogen against spontaneous activation and destruction. Product (chitin microfibril) and enzyme (chitin synthetase) remained associated in a complex that was readily separated by centrifugation.


DNA polymerase of mitochondria is a gamma-polymerase. Mitochondria isolated from rat liver cells or mycoplasma-free HeLa cells contain a single DNA polymerase activity which is closely related to, or identical to, the DNA polymerase gamma activity found in the homologous cell. In rat liver cells, about 16% of the total cytoplasmic gamma-polymerase activity is found associated with mitochondria and in HeLa cells about 20% of the total cellular gamma-polymerase is mitochondria associated. Since mitochondria possess no unique DNA polymerase activity, the number of DNA polymerases now known in mammalian cells is reduced, from the previously proposed four enzymes, to three--DNA polymerases alpha, beta, and gamma.


Characterization of plasma membrane adenosine triphosphatase of Neurospora crassa. It has been proposed (Slayman, C.L., Long W.S., and Lu, C.Y.-H. (1973) J. Membr. Biol. 14, 305--338) that in Neurospora crassa, a plasma membrane ATPase functions to pump H+ ions out of the cell, thereby generating an electrochemical gradient that can drive transport processes. Using the concanavalin A method of Scarborough (Scarborough G.A. (1975)J. Biol. Chem. 250, 1106--1111), we have prepared plasma membranes of Neurospora and have deomonstrated that they do contain a distinct ATPase activity with the following properties. It has a pH optimum of 6.0, is highly specific for ATP (hydrolyzing other nucleoside triphosphates less than 6% as rapidly), requires Mg2+ at concentrations approximately equimolar to the concentration of ATP, is weakly stimulated by certain monovalent cations (K+ and NH4+) and anions (SCN- and acetate), is inhibited by N,N'-dicyclohexylcarbodiimide, but is not affected by oligomycin or ouabain. The plasma membrane fraction also contains residual mitochondrial contamination, which can be determined quantitatively by assaying oligomycin-sensitive ATP-ase activity, at pH 8.25, and succinic dehydrogenase activity.


Identification of folate binding macromolecule in rabbit choroid plexus. A macromolecular binder of folic acid and folic acid derivatives has been identified in the particulate fraction of homogenates of rabbit choroid plexus. Within the choroid plexus, there are 2.3 nmol of folate-binding activity (binder) per g of tissue. The molecular weight of the folate binder complex, separated from the particulate fraction after solubilization with Triton X-100, was 340,000 to 400,000 by Sephadex gel filtration. The partially purified binder, when freed of endogenous folates, bound equivalent amounts of both [3H]folic acid and [methyl-14C]methyltetrahydrofolic acid per mg of protein. Folic acid, homofolic acid, 5-methyltetrahydrofolic acid, and to a lesser degree, methotrexate, inhibited the binding of both [3H]folic acid and [14C]methyltetrahydrofolic acid. Binding activity, which decreased below pH = 7.0, was unaffected by pretreatment with ribonuclease but was eliminated completely by papain and a protease (Streptomyces griseus). Although dihydrofolate reductase was present in choroid plexus, the binder was distinct from dihydrofolate reductase as judged by gel filtration and methotrexate sensitivity. This high affinity binder of folates may be responsible, in part, for the rapid, saturable uptake of folic acid and methyltetrahydrofolic acid by rabbit choroid plexus in vitro.


Repressible alkaline phosphatase from Thermus aquaticus: associated phosphodiesterase activity. A repressible alkaline phosphatase has been isolated from the extreme bacterial thermophile. Thermus aquaticus, and has been purified to homogeneity as judged by disc acrylamide electrophoresis and sodium dodecyl sulfate electrophoresis. Upon investigation, the purified enzyme was shown to hydrolyze certain phosphodiesters in addition to a wide variety of phosphomonoesters. The diesters included bis-p-nitro-phenyl phosphate and thymidine 3'-monophospho-p-nitro-phenyl ester. The temperature optimum for the diesterase activity was 80--85 degrees at pH 7.2. Orthophosphate competitively inhibited both activities. Nucleotides such as AMP, ADP, and ATP also inhibited both esterase activities as did alpha-D-glucose 1-phosphate and alpha-sodium glycerol phosphate. The isoelectric point of the enzyme was determined to be 8.4.


Specificity of 2-keto-3-deoxygluconate-6-P aldolase for open chain form of 2-keto-3-deoxygluconate-6-P. 2-Keto-3-deoxygluconate-6-P exists as an euqilibrium of three forms at 25 degrees measurable by 13C NMR: alpha-furanose anomer (41%), beta-furanose anomer (50%), and open chain keto (9%). The three forms are interconverted rapidly (greater than 0.5 s-1) so that the unidirectional rates of furanose ring opening and closing can be quantitated by an NMR line broadening method. The 2-keto-3-deoxygluconate aldolase is specific for only one of these forms, the open chain keto form. The rates for ring opening calculated from the rapid kinetic enzyme system compare closely with those obtained with the NMR method.


Effect of covalent attachment of polyethylene glycol on immunogenicity and circulating life of bovine liver catalase. Methoxypolyethylene glycols of 1900 daltons (PEG-1900) or 5000 daltons (PEG-5000) were covalently attached to bovine liver catalase using 2,4,6-trichloro-s-triazine as the coupling agent. Rabbits were immunized by the intravenous and intramuscular routes with catalase modified by covalent attachment of PEG-1900 to 43% of the amino groups (PEG-1900-catalase). The intravenous antiserum did not yield detectable antibodies against PEG-1900-catalase or native catalase, as determined by Ouchterlony and complement fixation methods, whereas the intramuscular antiserum contained antibodies to both PEG-1900-catalase and catalase. PEG-1900 did not react with either antiserum. Catalase was prepared in which PEG-5000 was attached to 40% of the amino groups (PEG-5000-catalase). This catalase preparation did not react with either antiserum. PEG-1900-catalase retained 93% of its enzymatic activity; PEG-5000-catalase retained 95%. PEG-5000-catalase resisted digestion by trypsin, chymotrypsin, and a protease from Streptomyces griseus. PEG-1900-catalase and PEG-5000-catalase exhibited enhanced circulating lives in the blood of acatalasemic mice during repetitive intravenous injections. No evidence was seen of an immune response to injections of the modified enzymes. Mice injected repetitively with PEG-5000-catalase remained immune competent for unmodieied catalase, and no evidence of tissue or organ damage was seen.


Human thrombins. Production, evaluation, and properties of alpha-thrombin. Human alpha-thrombin, the thromboplastin activation product of prothrombin with high clotting and esterase activity, was produced from Cohn Fraction III paste. The procedure started with 0.4 to 3.2 kg of frozen paste and was completed in 2 or 3 days. Some 23 g of thrombin were recorded for 65 quantitated preparations made from 11 lots of Fraction III paste. These preparations were obtained at protein concentrations of 3.9 +/- 1.3 mg/ml with a yield of 340 +/- 110 mg/kg of paste, which represented 48 +/- 14% of the clotting potential extracted as prothrombin. They had specific clotting activities of 2.8 +/- 0.4 U.S. (NIH) units/microng of protein and titrated to 88 +/- 8% active with p-nitrophenyl-p'-guanidinobenzoate (NPGB). Those (N - 29) examined by labeling with [14C]diisopropyl phosphorofluoridate (iPr2P-F) and electrophoresing in sodium dodecyl sulfate (SDS)-polyacrylamide gels were found to contain only (N = 4) or predominantly alpha-thrombin (97 +/- 3%) and corresponding amounts of ists degradation product, beta-thrombin (2.6 +/- 3.1%). No plasmin(ogen), prothrombin complex factors (II, VII, IX, IXalpha, X, Xalpha), or prothrombin fragments were detected in representative preparations. As produced in 0.75 M NaCl, pH approximately 6, thrombin was stable for approximately 1 week at 4 degrees and for greater than 1 year at less than or equal to 50 degrees; freeze-dried thrombin stored at 4 degrees for greater than 1 year displayed stable clotting activity and no vial to vial variation, permitting its use for reference purposes. Human thrombin generated by Taipan snake venom activation was compared with that produced by rapid thromboplastin activation: after treatment with [14C]iPr2P-F, greater than 95% of the label in both thrombins migrated at the same rate during electrophoresis in SDS; identical pairs of NH2-terminal residues were released in three consecutive Edman degradation cycles.


Interaction of apoA-II from human high density lipoprotein with lysolecithin. The effects of lysolecithin and hexadecyltrimethylammonium bromide on the structure and stability of apoA-II from human high density lipoprotein have been evalued by circular dichroism and fluorescence measurements. There is a profound enhancement in the stability of apoA-II to guanidinium hydrochloride denaturation when it forms a phospholipid complex with lysolecithin micelles. This complex is not only resistant to guanidinium hydrochloride denaturation, but it can be formed in a 6 M solution of this denaturant. The behavior of apoA-II in the native human high density protein is much closer to that of the lysolecithin apoA-II complex than to that of the free apoA-II.


Liver microsomal epoxide hydrase.

Liver microsomal epoxide hydrase. 1. The substrate specificity of membrane-bound and purified epoxide hydrase from rat liver microsomes has been studied. Both enzyme preparations catalyzed the hydration of a variety of alkene oxidase as well as arene oxides of several polycyclic aromatic hydrocarbons. 2. Unlike the membrane-bound enzyme, the rate of hydration for most of the substrates catalyzed by the purified epoxide hydrase was constant for only 1 or 2 min. The addition of dilauroyl phosphatidylcholine or heated microsomes to the incubation mixture extended the linearity of the reaction. 3. When rat liver microsomes were used as the source of the enzyme, the apparent Km values for many of the substrates were dependent on the amount of microsomes used. When purified epoxide hydrase was used as the enzyme source and benzo(a)pyrene 11,12-oxide as substrate, the apparent Km for benzo(a)pyrene 11,12-oxide was independent of enzyme concentration but dependent on added lipid concentration. Thus, in the absence of added dilauroyl phosphatidylcholine or in the presence of this lipid at a concentration below its critical micelle concentration, the observed Km for benzo(a)pyrene 11,12-oxide remained constant. However, when the lipid concentration was greater than the critical micelle concentration, the apparent Km value increased linearly with lipid concentration. These results are consistent with a model based on the partition of lipid-soluble substrate between the lipid micelle and the aqueous medium.


Acidic dissociation constants of folic acid, dihydrofolic acid, and methotrexate. The acidic dissociation constants in the range HO--1.5 to pH 7 of folic acid, dihydrofolic acid, methopterin (N(10)methylfolic acid), and methotrexate have been measured by potentiometric and spectrophotometric titrations. Assignment of these dissociations was made by comparison to model compounds, by proton magnetic resonance measurements, and by examination of associated ultraviolet absorbance changes. For folic acid, the dissociation constants are as follows: N(1), pK' 2.35; N(10), pK' 0.20; N(5), pK' greater than -1.5. For dihydrofolic acid: N(5), pK' 3.84; N(1), pK' 1.38; N(10), pK' 0.28. For methotrexate: N(1), pK' 5.71; gamma-carboxyl, pK' 4.70; alpha-carboxyl, pK' 3.36; N(10), pK' 0.50; N(5), boxyl, pK' 4.70; alpha-carboxyl, pK' 3.36; N(10), pK' 0.50; N(5) pK' greater than -1.5. For methopterin: acidic ionization of amide, pK' 7.68; gamma-carboxyl, pK' 4.62; N(1), pK' 2.40; N(10), pK; 0.36; N(5), pK' greater than -1.5. The pK' values were determined directly for the four compounds at 25 degrees near 0.1 ionic strength, or in 0.1 to 4 M HCl for pK ln 0.1 M NaCl.


Protein modification enzymes associated with the protein-synthesizing complex from rabbit reticulocytes. Protein kinase, phosphoprotein phosphatase, and acetyltransferase. A number of protein modification activities are present in the protein-synthesizing complex isolated from rabbit reticulocytes. These enzymes are solubilized by sedimentation of the ribosomes through buffered sucrose containing 0.5 M KCl, and have been partially purified from the high salt wash fraction by chromatography on DEAE-cellulose and phosphocellulose. The ribosomal-associated enzymatic activities include cyclic AMP-regulated and cyclic nucloetide-independent protein kinase, phosphoprotein phosphatase, and acetyltransferase activities. These enzymatic activities have been shown to modify specific ribosomal and ribosomal-associated proteins. The cycli c AMP-regulated protein kinase phosphorylate the 40 S ribosomal subunit from rabbit reticulocytes. One of the cyclic nucleotide-independent protein kinase catalyzes the phosphorylation of two different factors involved in the initiation of hemoglobin synthesis. A single phosphoprotein phosphatase activity is shown to remove phosphate from 40 S ribosomal subunits. The major acetyltransferase activity associated with ribosomes acetylates a 60 S ribosomal protein.


Purification and properties of a membrane-associated, folate-binding protein from Lactobacillus casei. A folate-binding protein has been solubilized from Lactobacillus casei by treatment of membrane preparations with Triton X-100 in the presence of [3H]folate. The protein-folate complex was purified 100-fold and recovered in a 22% yield by adsorption and elution from microgranular silica (Quso G-32), followed by passage through Sephadex G-150. When subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the purified preparations showed only a single, protein-staining band whose molecular weight was 25,000. Bound folate (34 nmol/mg of protein) corresponded to 0.85 mol/mol of protein. Analyses of the protein revealed relatively few charged or polar amino acids, an unusually high content of hydrophobic residues and methionine, and the absence of cysteine. The purified protein-folate complex was contained within a Triton micelle (molecular weight, 220,000; about 340 mol of detergent per mol of protein). Bound folate was retained when the micelle was exposed at 4 degrees to solutions whose pH values ranged between 3 and 12; at 23 degrees, however, stability was decreased, especially above pH 8. Folate could be released by treatment of the micelle with ethanol or with chaotropic agents such as guanidinium chloride, perchlorate, or thiocyanate.


Purification and properties of NADPH-dependent aldehyde reductase from human liver. An aldehyde reductase (EC 1.1.1.2) from human liver has been purified to homogeneity. The enzyme is NADPH-dependent, prefers aromatic to aliphatic aldehydes as substrates, and is inhibited by barbiturates and hydantoins. The following physicochemical parameters were determined: molecular weight, 36,200; sedimentation coefficient, 2.9 S; Stokes radius, 2.65 nm; isoelectric point, pH 5.3; extinction coefficient at 280 nm, 54,300 M-1 cm-1. Results from polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate, gel filtration, and ultracentrifugation suggest a monomeric structure. On molecule of NADPH binds to the enzyme causing a red shift of the coenzyme absorption maximum from 340 to 352 nm. The amino acid composition has been determined and a partial specific volume of 0.74 was computed from these data. An alpha-helicity of 7 and 18% was estimated from the ellipticities at 208 and 222 nm, respectively. Combination of the most reactive thiol group with p-mercuribenzoate does not cause loss of catalytic activity. Inactivation occurs when more than one thiol group is modified. The presence of NADPH or NADP+ prevents loss of activity by thiol modification. The comparison of structural features of aldehyde reductase with other monomeric and oligomeric dehydrogenases suggest similarities of aldehyde reductase with octopine dehydrogenase.


Species-specific aggregation factor in sponges. Sialyltransferase associated with aggregation factor. The sialyltransferase (= glycoprotein-sialic acid transferase) was studied in the sponge Geodia cydonium, a mesozoan organism. The experiments were performed both in intact cellular and in isolated enzyme systems. It is shown, that desialylated cells show a lower aggregation potency than the controls. During aggregation enzymic sialylation of desialylated sponge cells occurs in the presence of an aggregation factor, which is associated with a high molecular weight particle. The sialylation process is temperature-dependent and can be inhibited by N-ethylmaleimide. Sialylation occurs predominantly at a distinct cell surface component, the aggregation receptor. The sialyltransferase was isolated and purified by the following steps: Sepharose 4B, CM-cellulose, Nonidet treatment, and Sephadex G-100. By this procedure the enzyme was purified 680-fold with a 31% yield. The sialyltransferase is originally associated with the high molecular weight particle also carrying the aggregation factor. In the last step the aggregation factor was separated from the sialyltransferase. The enzyme catalyzes the transfer of sialic acid from CMP-sialic acid to the desialylated aggregation receptor. The molecular weight of the sialyltransferase has been determined to be 52,000. Kinetic studies revealed no lag phase and a dependence on enzyme concentration. The purified transferase has a pH optimum of 7.75 and requires 200 mM NaCl for activity. No requirement for Mg2+ or Ca2+ could be observed. The reaction is inhibited by 10 micronM N-ethylmaleimide.


Compartmentalization of adenosine 3':5'-monophosphate and adenosine 3':5'-monophosphate-dependent protein kinase in heart tissue. In rabbit heart homogenates about 50% of the cAMP-dependent protein kinase activity was associated with the low speed particulate fraction. In homogenates of rat or beef heart this fraction represented approximately 30% of the activity. The percentage of the enzyme in the particulate fraction was not appreciably affected either by preparing more dilute homogenates or by aging homogenates for up to 2 h before centrifugation. The particulate enzyme was not solubilized at physiological ionic strength or by the presence of exogenous proteins during homogenization. However, the holoenzyme or regulatory subunit could be solubilized either by Triton X-100, high pH, or trypsin treatment. In hearts of all species studied, the particulate-bound protein kinase was mainly or entirely the type II isozyme, suggesting isozyme compartmentalization. In rabbit hearts perfused in the absence of hormones and homogenized in the presence of 0.25 M NaCl, at least 50% of the cAMP in homogenates was associated with the particulate fraction. Omitting NaCl reduced the amount of particulate-bound cAMP. Most of the particulate-bound cAMP was probably associated with the regulatory subunit in this fraction since approximately 70% of the bound nucleotide was solubilized by addition of homogeneous catalytic subunit to the particulate fraction. The amount of cAMP in the particulate fraction (0.16 nmol/g of tissue) was approximately one-half the amount of the regulatory subunit monomer (0.31 nmol/g of tissue) in this fraction. The calculated amount of catalytic subunit in the particulate fraction was 0.18 nmol/g of tissue. Either epinephrine alone or epinephrine plus 1-methyl-3-isobutylxanthine increased the cAMP content of the particulate and supernatant fractions. The cAMP level was increased more in the supernatant fraction, possibly because the cAMP level became saturating for the regulatory subunit in the particulate fraction. The increase in cAMP was associated with translocation of a large percentage of the catalytic subunit activity from the particulate to the supernatant fraction. The distribution of the regulatory subunit of the enzyme was not significantly affected by this treatment. The catalytic subunit translocation could be mimicked by addition of cAMP to homogenates before centrifugation. The data suggest that the regulatory subunit of the protein kinase, at least that of isozyme II, is bound to particulate material, and theactive catalytic subunit is released by formation of the regulatory subunit-cAMP complex when the tissue cAMP concentration is elevated. A model for compartmentalized hormonal control is presented.


Interaction of glucocorticoid hormones and cyclic nucleotides in induction of tyrosine aminotransferase in cultured hepatoma cells. Reproducible induction of the enzyme tyrosine aminotransferase by dibutyryl cAMP (Bt2cAMP) in a line of HTC hepatoma cells in suspension culture requires that the cells be preinduced with dexamethasone, a synthetic glucocorticoid which itself induces tyrosine aminotransferase. Concentrations of dexamethasone that do not induce tyrosine aminotransferase fail to support Bt2cAMP induction, removal of the steroid from the medium leads to a loss of the Bt2cAMP effect, and an HTC cell line whose aminotransferase is not steroid-inducible does not respond to the cyclic nucleotide. We show that the further induction of tyrosine aminotransferase by Bt2cAMP in dexamethasone-treated cells is due to an increased rate of enzyme synthesis. The cyclic nucleotide has no effect on aminotransferase synthesis in cells grown in the absence of steroid. Several lines of evidence suggest that dexamethasone acts at a step beyond the activation of protein kinase by cAMP: (a) basal levels of cAMP are not altered by growth of HTC cells in dexamethasone; (b) accumulation of cAMP from the medium is not enhanced; (c) the glucocorticoid does not induce cAMP-dependent protein kinase in HTC cells; and (d) there is no augmentation of cAMP binding to the regulatory protein, nor is there any change in cAMP activation of protein kinase caused by growth in dexamethasone. These results help define a system that should be useful in studying the interaction of cyclic nucleotides and steroid hormones.


Effect of hydrostatic pressure on ligand binding to hemoglobin. Increase in hydrostatic pressure to 1000 atm increased the affinity of human and menhaden (Brevoortia tyrannus) hemoglobins for oxygen. With necessary assumptions about the form of the equilibrium curve, and after correction for changes in pH and volume due to pressure, the increase in affinity is about 2-fold for both hemoglobins. At pH 6.5, Hill's n for menhaden hemoglobin is near 1, and it is believed to remain in the T state, whereas human hemoglobin undergoes a T to R transition. This suggests that the R-T equilibrium is not disturbed by pressure. In direct experiments the binding of a fluorescent effector (8 hydroxy-1,3,6-pyrene (trisulfonic acid) to deoxyhemoglobin was not changed by pressure. The binding of n-butylisocyanide to hemoglobin and to myoglobin is also greater at high pressures, similarly suggesting that the R-T transition is not involved in the pressure effect.


Charges of nicotinamide adenine nucleotides and adenylate energy charge as regulatory parameters of the metabolism in Escherichia coli. Methods for measurements of catabolic reduction charge (defined as NADH/(NADH+NAD+)) and anabolic reduction charge (defined as NADPH/(NADPH + NADP+)) are described using [14C]nicotinamide labeling of Escherichia coli cultures. Together with these parameters the adenylate energy charge (ATP + 1/2ADP)/(ATP + ADP + AMP) was measured using labeling with [2-3H]adenine. These three charges were found under different exponential growth conditions to have values independent of the growth conditions: catabolic reduction charge, 0.05; anabolic reduction charge, 0.45; and adenylate energy charge, 0.9. The charges were examined during interruption of growth primarily affecting catabolism, respiration, or anabolism, leading to changes of the charges. The changes of charges are evaluated as a possible regulation of the metabolic rates utilizing or producing the nucleotides by their respective charges.


Phosphoprotein phosphatase activity associated with the cytoplasmic membrane of Salmonella typhimurium and Escherichia coli. Membrane-associated phosphoprotein phosphatase activity was demonstrated in extracts of Salmonella typhimurium and Escherichia coli. The active protein could be extracted from the membrane as a large water-soluble complex (Mr greater than 150,000). Maximal activity was observed at pH 6 to 7 in the presence of a divalent cation. The enzyme appears to be distinct from previously described phosphatases.


In vitro activation of the in vivo colony-forming units of the mouse yolk sac. Experiments were performed to investigate the presence of colony-forming units (CFU) in the mouse embryonic yolk sac during the developmental period in which the yolk sac is the sole hemopoietic organ. Injection of yolk sac cell suspensions from normal embryos into syngeneic, lethally irradiated adult recipients evoked a very low number of spleen colonies. However, prior cultivation of yolk sacs in vitro caused a dramatic increase in the spleen colony-forming capacity--as high as 84-fold--following 48 hours in culture. The yolk sac origin of the spleen colonies was confirmed by: (a) Chromosomal marker analysis; (b) dose-response analysis; (c) demonstrating that the above colonies were not of endogenous origin induced by the mere injection of grafted cells. We conclude that the yolk sac contains many precursors of colony-forming cells which though undetectable by immediate grafting apparently become activated in culture by an as yet unknown induction process.


The diverse effects of 5'-bromodeoxyuridine on enzyme activities in cultured H35 hepatoma cells. Reuber (H35) hepatoma cells were grown in medium containing 10(-5)M bromodeoxyuridine (BrdU), which was incorporated into their DNA. Cell growth rate was unaffected by BrdU for the first two generations, after which it was reduced by about 50%. The effect of BrdU incorporation on the activities of several enzymes with rapid turnover rates was examined to test the hypothesis that the synthesis of such enzymes will be preferentially inhibited by BrdU. Tyrosine amino-transferase (TAT) activity decreased by 70% within two generations whereas thymidine kinase activity remained at control values. PEP carboxykinase activity was unchanged during the first generation in BrdU-containing medium but, during the second, its activity increased by at least 30%. Ornithine decarboxylase levels decreased by about 50% only after two generations in the presence of BrdU. There appeared to be no simple relationship between turnover rates and the effect of BrdU on enzyme activity. Incorporation of BrdU was found to inhibit the induction of both TAT and PEP carboxykinase by dexamethasone and to enhance the inhibition of cell growth by this steroid. These results are discussed with respect to possible mechanisms of gene expression and development in both normal and neoplastic cells.


The relation of cycling of intracellular pH to mitosis in the acellular slime mould Physarum polycephalum. The relation between intracellular pH and the mitotic cycle of Physarum polycephalum was studied by two-independent techniques. Both techniques revealed a long term cycling of intracellular pH which has the same period as the mitotic cycle, Qualitative detection of the changes in intracellular pH was made by measuring the changes in fluorescence of 4-methylesculetin which had been absorbed by the plasmodium. Quantitative measurements of intracellular pH were made throughout the mitotic cycle with antimony micro pH electrodes. The cycle of intracellular pH is sinusoidal in appearance. The maximum intracellular pH (pH 6.6) occurred at, or very near to, mitosis, and was approximately 0.6 pH units higher than the minimum pH, which occurred near the middle of the mitotic cycle.


Evaluation of the Hall electrolytic conductivity detector for the analysis of narcotic alkaloid and phenothiazine drugs extracted from urine. The electrolytic conductivity detector can be used for the rapid and selective determination of narcotic alkaloid and phenothiazine drugs in the presence of urine coextractants. The high selectivity of the detector enables drug concentrations as low as 0.1 mg/dl to be analyzed without extensive sample cleanup or derivatization.


Studies on the pathogenesis of an immune defect in multiple myeloma. The reduced capacity of patients with multiple myeloma to respond to antigen challenge is well recognized. Response to antigen involves antigen recognition, cell proliferation, and synthesis and secretion of antibody. This study examines this sequence of events in peripheral blood lymphocytes from untreated and treated patients with myeloma, from individuals with benign monoclonal gammopathy, and from normal healthy donors. Antigen-binding capacity was assessed by testing the ability of lymphocytes to bind radio-labeled pneumococcal polysaccharide, tetanus toxoid, or diptheria toxin. The in vitro proliferative response to these antigens as well as to pokeweed mitogen and streptokinase-streptodornase was evaluated. The secretion of immunoglobulin in response to pneumococcal polysaccharide, tetanus toxoid, and pokeweed mitogen by 2-4 x 10(6) lymphocytes in 7-day cultures was determined. The effects of coculture of myeloma peripheral blood lymphocytes and normal peripheral blood lymphocytes on immunoglobulin production and mixed leukocyte reactions were explored. All myeloma patients had normal numbers (3-8/5,000 cells) of antigen-binding cells. However, most showed a diminished antigen-induced blast transformation as measured by uptake of [(125)I]5-iodo-2'-deoxyuridine in culture. Immunoglobulin production in response to specific antigen in myeloma lymphocytes was 30-80% less than in normal lymphocytes. Immunoglobulin synthesis and mixed leukocyte responses by normal peripheral blood lymphocytes could be suppressed by myeloma lymphocytes. Multiple suppressor populations were present. Thus, the immune defect in myeloma is beyond the antigen recognition step and involves both the proliferation of antigen-sensitive cells and immunoglobulin production. Further suppressive effects are imposed on normal cells, implying defects in immunoregulation in this disease.


Aminoglycoside antibiotics and renal function: changes in urinary gamma-glutamyltransferase excretion. The urinary excretion of the renal proximal tubular enzyme, gamma-glutamyltransferase (gamma-GT), has been studied in 41 patients receiving tobramycin, gentamicin or streptomycin for a variety of infections. All patients receiving tobramycin or gentamicin have shown increased excretion of gamma-GT in the urine. Only 46% of those receiving streptomycin have shown an increase in gamma-GT excretion and this is of a lesser degree. A change in creatinine clearance which could only be explained by antibiotic administration was detected in three patients (2 on gentamicin, 1 on streptomycin). The degree of elevation of urinary gamma-GT activity was greater when the initial creatinine clearance was lower, and it is therefore suggested that those patients with pre-existing renal dysfunction should be monitored particularly carefully for signs of nephrotoxicity from these antibiotics. Urinary gamma-GT is a useful enzyme in the investigation of renal drug effects.


Effect of beta adrenergic agonist, prostaglandins, and cortisol on lymphocyte levels of cyclic adenosine monophosphate and glycogen: abnormal lymphocytic metabolism in asthma. Decreased beta adrenergic regulation of cyclic adenosine monophosphate (cAMP) in lymphocytes has been described in asthma. We investigated adrenergic stimulation of glycogenolysis and responses to prostaglandin E1 (PGE1). Lymphocytes from 24 normal and 24 mild asthmatic subjects who had no drugs for at least 2 weeks were separated on Ficoll-hypaque and incubated in medium 199 with Hepes buffer. Beta adrenergic stimulation of cAMP and glycogenolysis was reduced in the asthmatics (p less than 0.05). PGE produced less of a rise in cAMP in asthmatics than in normals, but the difference was not significant (p greater than 0.05) and glycogenolysis was normal. Cortisol added in vitro potentiates the effect of isoproterenol and PGE1--but in the presence of cortisol the response of the asthmatic cells to isoproterenol is still lower than that of normal cells. This observation would support that "beta adrenergic blockade" is the major defect of asthmatic cells. The conclusion is further supported by the observation that the degree of the blockade is associated with a pathologic condition.


Further studies on the mechanism of human histamine-induced asthma: the effect of an aerosolized H1 receptor antagonist (diphenhydramine). This study was designed to better define the mechanism of histamine-induced bronchoconstriction in humans by pharmacologic manipulation of the postulated bronchial histamine receptor sites. Histamine challenges were performed on a heterogeneous group of adult asthmatic subjects. The cumulative units of histamine required for induction of a sustained 20% or greater decrease in FEV1 from baseline were determined. The effect of pretreatment with an aerosolized H1 receptor antagonist, diphenhydramine hydrochloride, was then studied. Analysis of the data showed that the administration of an H1 receptor antagonist prior to histamine challenge significantly blocked the bronchial response to histamine (p less than 0.005). This effect was considered to be due to specific competitive antagonism at the H1 receptor site and suggests the presence of H1 receptors in human bronchial mucosa.


[Acid-base equilibrium during acute fetal hypoxia. Experimental study]. The authors studied the effects of acute hypoxia of short duration in eight pregnant ewes that had been anaesthetised. They showed that during the experiment the E.E.G. as well as the pH and pCO and the maternal lactate levels were not changed and that therefore changes that can be found in the fetus are due solely to its own hypoxia and not due to transmission from the mother. In the fetus the trace flattens in about 30 minutes on an average. After this interval a moderate drop in pH occurs at the same time as an increase in lactacidemia and the relationship between lactate and pyruvate. The heart rate is changed in a variable manner: it accelerates if the basal rate is less than 180 beats a minute and slows if it is above 180. Blood pressure rises. Changes in the cardio-vascular system always precede those in the electroencephalograms.


Enhancement of murine cytomegalovirus infection during graft-vs.-host reaction. A mouse model was used for elucidation of the role of a graft-vs-host reaction in promoting cytomegalovirus infection. F1 (DBA/2 X C3H/He) hybrid mice were infected with murine cytomegalovirus. Five weeks later, a graft-vs.-host reaction was produced in the chronically infected animals by the administration of multiple doses of DBA/2 parental splenocytes. Cytomegalovirus was recovered more often from the organs of mice undergoing graft-vs.-host reaction than from those of control animals (P less than 0.001). These results indicate that the graft-vs.-host reaction alone can enhance murine cytomegalovirus infection in a chronically infected host and may help explain the high incidence of cytomegalovirus infection after bone marrow allograft transplantation in man.


Protein binding to monosodium urate monohydrate, calcium pyrophosphate dihydrate, and silicon dioxide crystals. I. Physical characteristics. The protein adsorptive properties of monosodium urate monohydrate (MSU), silica (Si), and calcium pyrophosphate dihydrate crystals were studied by qualitative and quantitative techniques. Immunoglobulin G (IgG) was adsorbed preferentially by MSU crystals from normal human serum and demonstrated high-affinity binding isotherms when compared with several isolated proteins in solution. The physical characteristics of this reaction suggest principally an ionic mechanism, since adsorption was enhanced by decreasing pH or ionic strength. Weaker physical forces also were suggested by studies showing enhanced adsorption at lower temperatures. The following order of affinity for Si or MSU crystals was found when equal concentrations of proteins were compared: Cohn fraction II greater than lysozyme greater than beta lactoglobulin greater than bovine serum albumin greater than ovalbumin. IgG adsorption to the crystals studied may explain certain features of their biological activity. It is suggested that this phenomenon blocks the membranolytic properties of crystals and stimulates their phagocytosis through interaction with Fc receptors on the surface of the phagocytic cells.


Effect of CO2 concentration on phosphatidylcholine and phosphatidylglycerol metabolism in surfactant and residual lung fractions. An investigation of the effect of change of total CO(2) concentration from 7 to 43 mM at pH 7.35 in the medium perfusing isolated rat lungs on [U-(14)C]glucose incorporation into lung phospholipids has been carried out. The incorporation of [U-(14)C]glucose into phosphatidylcholine and phosphatidylglycerol of the surfactant fraction and of the remaining lung tissue (residual fraction) was observed. Increased CO(2) concentration increased [U-(14)C]glucose incorporation into phosphatidylcholine of the surfactant fraction and residual fraction by 43 and 50%, respectively, during a 2 hr perfusion. Likewise, incorporation of [U-(14)C]glucose into phosphatidylglycerol was increased 22 and 34% into the surfactant and residual fractions, respectively. The percentage of [U-(14)C]glucose incorporated into the fatty acid moieties of phosphatidylcholine of both fractions increased as a result of increased CO(2) concentration. The increase in the incorporation of [U-(14)C]glucose into the fatty acid moieties of phosphatidylcholine was confirmed by an average increase of 56 and 77% in the specific activity of palmitic acid isolated from phosphatidylcholine of the surfactant and residual fraction, respectively, as a result of increased CO(2) concentration. The results suggest that alteration in extracellular CO(2) concentration affects the de novo synthesis from glucose of phosphatidylcholine and phosphatidylglycerol of the surfactant-lipoprotein fraction of lung.


The significance of lipoprotein lipase in rat skeletal muscles. Lipoprotein lipase was assayed in extracts of acetone-ether powders of rat skeletal muscles. Enzyme activity in soleus had typical characteristics of lipoprotein lipase in other tissues: inhibition by molar NaCl and protamine sulfate and activation by the human apolipoprotein, R-glutamic acid. Activity in muscles with predominantly red fibers (soleus, diaphragm, lateral head of gastrocnemius and anterior band of semitendinosus) was higher than in those with predominantly white fibers (body of gastrocnemius and posterior band of semitendinosus). No effect of a 24 hour fast upon enzyme activity was observed in ten skeletal muscles, but activity decreased substantially in four adipose tissue depots and increased slightly in heart muscle with fasting. Four minutes after intravenous injection of labeled lymph chylomicrons, skeletal muscles with predominantly red fibers incorporated several times more chylomicron triglyceride fatty acids than thos with predominantly white fibers. Estimated lipoprotein lipase activity in total skeletal muscle was about two-thirds that in total adipose tissue of rats fed ad libitum. After a 24 hour fast, total activity in skeletal muscle was about twice that in adipose tissue. These data suggest that a substantial fraction of lipoprotein lipase is in skeletal muscle of rats and that this tissue, especially its red fibers, is an important site of removal of triglycerides from the blood.


Endocrine regulation of sex-dependent hydroxysteroid dehydrogenase activities in rat kidney: NADP-dependent microsomal 3alpha- and 20beta-hydroxysteroid dehydrogenase. The NADP-dependent microsomal kidney enzymes, 3alpha- and 20beta-hydroxysteroid dehydrogenase (HSDH), which exhibit considerable sex differences in their activities (male:female activity ratios, 16:1 and 30:1 respectively), were investigated after interference with the pituitary-gonad and pituitary-adrenal systems. Prepubertal gonadectomy as well as hypophysectomy of mature male rats led to a decline in HSDH activity to almost that found in the normal female rat, whereas activities in female rats were unaffected. Testosterone induced typical male 3alpha-HSDH activity in both gonadectomized and hypophysectomized rats of either sex. Administration of 5alpha-dihydrotestosterone (5alpha-DHT) or 5alpha-androstane-3alpha, 17beta-diol to hypophysectomized male rats was equally effective in restoring full 3alpha- and 20beta-HSDH activities whereas 5alpha-androstane-3beta, 17beta-diol was less effective and dehydroepiandrosterone was ineffective. Simultaneous administration of cyproterone acetate did not block the inductive action of 5alpha-DHT. Administration of chorionic gonadotrophin, pregnant mare serum gonadotrophin or a combination of luteinizing hormone and follicle-stimulating hormone to hypophysectomized male rats all led to parallel increases in the weight of the seminal vesicles and in both renal enzyme activities; administration of growth hormone, prolactin or thyroid-stimulating hormone was ineffective. Adrenalectomy of gonadectomized, but not of hypophysectomized male rats, caused a further drop in activity to the normal female level. Adrenalectomy of otherwise intact rats did not affect either enzyme activity. The hypophysis was involved in the regulation of the two NADP-dependent renal HSDH activities through its gonadotrophic function in male rats; adrenal secretions were of little physiological significance.


[A comparision of different methods and reagents for the determination of gamma-glutamyl transferase in serum (author's transl)]. Four commercially available kinetic photometric tests for the determination of gamma-glutamyl transferase were compared. Test A is based on the substrate L-gamma-glutamyl-4-nitroanilide; test B uses this same substrate dissolved in hydrochloric acid. C1 and C2 are tests from different suppliers; both use the substrate L-gamma-glutamyl-3-carboxy-4-nitroanilide, and all other constituents are the same as quoted by the suppliers. Parallel determinations were performed on 1017 native sera and 12 control sera. Compared with values from test A, two of the three other tests showed statistically significant differences in the diagnostically important range 10 to 50 U/l. Significant differences were also observed between the results from the apparently identical tests, C1, and C2. The values obtained for control sera showed a dependence on the test procedure and/or composition.


Exchange diffusion of dopamine induced in planar lipid bilayer membranes by the ionophore X537A. The ionophore X537A causes a large increase in the [(14)C]dopamine (a catecholamine) permeability of planar bilayer membranes. Dopamine transport increases linearly with the ionophore concentration. At relatively high concentrations in the presence of dopamine, the ionophore omdices a conductance which is nearly ideally selective for the dopamine cation. However, the total dopamine flux as determined in tracer experiments is not affected by an electric field and is over 10(5) times larger than predicted from the estimated dopamine conductance. Increasing the dopamine concentration on the side containing radioactive dopamine (the cis side) saturates the dopamine transport. This saturation is relieved by trans addition of nonradioactive dopamine, tyramine, H(+), or K(+). With unequal concentrations of dopamine cis and trans (49 and 12.5 mM), the unidirectional dopamine fluxes are equal. Increasing H(+) cis and trans decreases dopamine transport. It is concluded that at physiological pH, the X537A-induced transport of dopamine occurs via an electrically silent exchange diffusion of dopamine cation with another cation (e.g., dopamine(+), H(+), or K(+)). X537A induces a Ca(++)-independent release of catecholamines from sympathetic nerves by interfering with intracellular storage within storage vesicles (R.W. Holz. 1975. Biochim. Biophys. Acta. 375:138-152). It is suggested that X537A causes an exchange of intravesicular catecholamine with a cytoplasmic cation (perhaps K(+) or H(+)) across the storage vesicle membrane.


The rumen flagellate Piromonas communis: its life-history and invasion of plant material in the rumen. The rumen flagellat Piromonas communis is the zoospore of a phycomycete fungus inhabiting the rumen. Zoosporogenesis was stimulated by a dietary component (the inducer), and inhibited by compounds affecting membrane structure and function, but not by inhibitors of protein synthesis. The zoospores showed taxis towards the tissues surrounding the inflorescence of Lolium perenne L. in the rumen, invading principally the stomata and damaged tissues. The zoospores germinated on this substratum and the rhizoids of the developing vegetative stage penetrated the tissue, taking up C14 from labelled plant material, which was incorporated into the fungal cells. The conditions for maximum flagellate production (39 degrees C, pH 6-0 to 7-0, high concentration of CO2, absence of O2) resembled those found in the rumen. The organism was cultured in an undefined medium in vitro in the absence of other flagellates.


The effects of beta adrenergic blockade on spinal cord autoregulation in the monkey. Blood flow in the spinal cord was measured in a group of monkeys over a wide range of artificially varied blood pressures after the administration of propranolol, a beta adrenergic blocker. Spinal cord blood flow was found to be constant and in the normal range between a mean system arterial blood pressure of 50 to 150 mm Hg. From 150 to 180 mm Hg spinal cord blood flow decreased. There was no breakthrough of autoregulation, previously seen in the untreated animal. It is suggested, therefore, that the previously observed breakthrough of autoregulation is a beta adrenergic-mediated phenomenon.


In vivo effects of glucagon on fatty acid synthesis in fasted and refed rats. Since altered nutritional states evoke compensatory changes in systemic levels of several hormones, the present study was conducted to determine in vivo effects of glucagon and insulin on hepatic and adipose tissue lipogenesis in fed, fasted (3-days) and refed (3-days) rats. Compared to the fed controls, glucagon reduced hepatic fatty acid synthesis and acetyl CoA carboxylase activity by 62% and 65% in fed rats, and by 51% and 48%, respectively, in refed rats. In contrast, glucagon had no effect on fatty acid synthesis or acetyl CoA carboxylase activity in adipose tissue of any of the three experimental groups. Exogenous insulin antagonized the glucagon effects and restored hepatic fatty acid synthesis and enzyme activity in fed or refed rats. No glucagon or insulin effects were observed in fasting rats. In addition, glucagon reduced in vivo incorporation of acetate into hepatic cholesterol by about 33% and into fatty acids of the liver, and heart and the kidney by 33%, 77% and 30%, respectively. The hormone had no effect on fatty acid synthesis in the muscle.


Effects of putative neurotransmitters on the motor activity of Spirometra mansonoides. 5-Hydroxytryptamine (5-HT), epinephrine, and dopamine strongly stimulated the motor activity of larval Spirometra mansonoides. By contrast, a cholinomimetic agent, arecoline, paralyzed the worms. There was some pharmacological specificity among the agonists but not with various antagonists. Acetylcholinesterase activity was present in both larval and adult Spirometra.