[Covalent binding of peroxidase to CH- and AH-Sepharose 4 B]. The properties of peroxidase insolubilized by covalent binding to CH- and AH-Sepharose 4 B in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) are described. CH-Sepharose 4 B bound peroxidase yields an enzyme preparation with a residual specific activity of 60.6%. When bound to AH-Sepharose 4 B, the residual specific activity is to 78%. The reasons of these differences in the catalytic activity of the two insolubilized enzyme preparations are discussed. By covalent binding on CH- and AH-Sepharose 4 B, peroxidase exibits no changes in its pH optimum; it virtually keeps the same activity after being used ten times. Insolubilized peroxidase preparations, dried and reimbibed after being stored for 6 weeks at room temperature still display 50% of the initial specific activity of the insolubilized enzyme. Stored in acetate buffer, the enzyme preparations maintain their activity during all this interval.


Aerobacter aerogenes PRL-R3 urease. Purification and properties. A method for the preparation of a 150-fold purified and homogenous A. aerogenes urease is reported. The enzyme exhibited two pH optima at pH 7.0 and 7.5 in triethanolamine and phosphate buffer, respectively. The affinity of the enzyme toward its substrate increased with the increase of pH. No effect of the pH was observed on the measured temperature coefficient (Q10). There was no discontinuity in the Arrhenius plots at pH 5.4 and 7.5 but an upward discontinuity at pH 6.15 and 8.7 with transition temperature at 30 degrees C. Also, the calculated activation energies are greatly affected by the pH of the enzyme reaction mixture.


Further characterization of the association of glyceraldehyde-3-phosphate dehydrogenase with reticulocyte membranes. 1. The behaviour and properties of membrane-bound GAPDH of rabbit reticulocytes were investigated. 2. The bound GAPDH is more resistant to inactivation by KCl than the soluble enzyme (allotopy). 3. The bound enzyme is released by electrolytes. This effect does not only depend on the ionic strength but additionally on the kind of ions, pH-value and protein concentration. 4. A comparison of the releasing effect of NAD analogues shows the necessity of the 5'-AMP moiety in the structure of the effector. 5. The represented results demonstrate the specifity of the GAPDH-membrane binding in rabbit reticulocytes.


[Effect of the vagus nerve on isolated rabbit atria in ganglionic blockade due to hexamethonium]. By quantitative stimulation of the vagus nerves of isolated rabbit atria frequency-response relations were obtained for both the electrotropic effect (reduction of the area of the monophasic action potential) and the inotropic response. An addition of hexamethonium in a final concentration of 10(-5) g/ml resulted in a diminution of vagal effectivity in the range of lower and medium frequencies of stimulation, and was connected with a shift of the frequency-response characteristic to the right. At higher frequencies vagal effectivity was increased. In contrast to the inhibitory effect of hexamethonium the facilitating action is irreversible. By raising the concentration up to 4-10(-5) g/ml the vagal effects were reduced to a large extent, and the frequency dependence of the response was abolished at medium frequencies. In the range of 20 sec(-1) to 100 sec(-1) this dependence was re-established and may be considered as a part of a normal frequency-response relation extremely shifted to the right. The time courses of both types of effect are characterized by a steep rise and a decay of the response during the stimulation period. A mathematical handling of the frequency-response characteristics provides quantitative evidence for the extent of the hexamethonium blockade of vagal ganglion cells in the atria; furthermore it leads to the conception of these cells to act as a distributing system for a homogeneous innervation by a widespread divergency of postganglionic fibres.


Chemical relaxation studies on the system liver alcohol dehydrogenase, NADH and imidazole. Several years ago, Theorell and Czerlinski conducted experiments on the system of horse liver alcohol dehydrogenase, reduced nicotinamide adenine dinucleotide and imidazole, using the first version of the temperature jump apparatus with detection of changes in fluorescence. These early experiments were repeated with improved instrumentation and confirmed the early experiments in general terms. However, the improved detection system allowed to measure a slight concentration dependence of the relaxation time of around 3 ms. Furthermore, the chemical relaxation time was smaller than the one determined earlier (by factor 2). The data were evaluated much more rigorously than before, allowing an appropriate interpretation of the results. The observed relaxation time is largely due to rate constants in an interconversion of ternary complexes, which are faster than three (of the four) dissociation rate constants, determined previously by Theorell and McKinley-McKee.1,2 This fact contributed to earlier difficulties of finding any concentration dependence. However, the binding of imidazole to the binary enzyme-coenzyme complex can be made to couple kinetically into the interconversion rate of the two ternary complexes. The observed signal derives largely from the ternary complex(es). A substantial fluorescence signal change is associated with the observed relaxation process, suggesting a relocation of the imidazole in reference to the nicotinamide moiety of the bound coenzyme. Nine models are considered with two types of coupling of pre-equilibria (none-all). Quantitative evaluations favor the model with two ternary complexes connected by an interconversion outside the four-step (bimolecular) cycle. The ternary complex outside the cycle has much higher fluorescence yield than the one inside. The interconversion equilibrium is near unity for imidazole. If it would be shifted very much to the side of the "dead-end" complex (as in isobutyramide?!), stimulating action could not take place.


Comparative study of virological infections in asthmatic and nonasthmatic children. The author shows complex analyses: clinical, laboratory, X-rays, bronchoscopical, bronchographical and measuring lung function tests as well as the serological examinations in blood serum of both groups of asthmatic and nonasthmatic children with virological infection. The calculation of statistically significant differences between the various diagnostical results of both groups has confirmed that in asthmatic children virological infection of the respiratory tract, pathological findings in X-ray and lung function tests, bronchiectasis and secondary bacteriological invasion occurs statistically significantly more often than in nonasthmatic children.


Multiple forms of staphylococcal alpha-toxin. A group of proteins was readily extracted at neutrality from trichloroacetic acid precipitates of staphylococcal culture filtrate supernatants, while alpha-toxin was dissolved and activated by treating the precipitate with 8 M urea, with acidic buffers or by heating to 90-100 degrees C at neutrality. Heat activation of the precipitate produced a relatively pure alpha-toxin with a molecular weight of 39,000. alpha-Toxin was eluted together with three other proteins on hydroxyl apatite chromatography, and evidence was obtained for an association between the four proteins. On isoelectric focusing a haemolytic fraction was obtained at pH 6.2, probably due to acid activation of the precipitate formed at the cathodic end of the column. The alpha-haemolytic fractions with pI's of 7.4 and 8.6 were shown to consist of alpha-toxin only when analyzed by acrylamide electrophoresis in the presence of sodium dodecyl sulphate. The haemolytic component with a pI of 9.2 contained two additional components of molecular weights of 27,500 and 18,000. Chromatography of this material on Sephadex G-200 showed that alpha-toxin and the two proteins appeared as a high molecular complex.


Acute otitis media. A clinical bacteriological and serological study of children with frequent episodes of acute otitis media. A series of episodes of acute otitis media was studied with reference to bacterial findings and specific serological responses in 48 children with histories of frequent episodes before. D. pneumoniae and H. influenzae were the most frequently isolated pathogens. Re-isolations after therapy were often made in episodes with slow healing or therapeutic failure. Most children harboured pathogens in nasopharynx even when they had no signs of respiratory tract infections. Homologous relapses were seen only in few cases and never with pneumococcus type 3 and only once with H. influenzae type b. Specific serological responses were demonstrable generally in children over 2 years of age. D. pneumococcus type 3 and H. influenzae type b generally provoked antibody response. No levels indicating immunoglobulin deficiencies could be found in the children.


Endosymbiosis and cellular tolerance in the Hawaiian soft coral Sarcothelia edmondsoni verrill. The relationship between the soft coral Sarcothelia edmondsoni Verrill and its symbiotic algae is considered as an early instance of cellular tolerance which can be disturbed by a variety of adverse conditions. The algal cells lie in vesicles deep within the endodermal cells of the host and are not subject to digestion. Their expulsion appears to be a reverse translocation to the distal end of the host cell and escape by a form of reversed phagocytosis resembling secretion. The cellular mechanisms involved are not clear.


Epidemiologic investigations of the 1969 epidemic of Venezuelan encephalitis in Ecuador. An epizoodemic of Venezuelan equine encephalitis (VEE), subtype I variant B, occurred in Ecuador during the rainy and hot season of 1969. In this paper, a general description of the epidemic is given and virus isolations from humans are reported. A serologic survey was performed in order to determine the extension of the epidemic along the coastal zone of the country. It is not clear whether the higher antibody rate in older people was because they were at greater risk of infection or was the result of an accumulated immunity with time. The latter could be an indication of endemic virus activity, not yet proven for the VEE IB virus variant. Mosquito surveillance and attempted control by aerial spraying were carried out.


Congenital malformations of the central nervous system produced by narcotic analgesics in the hamster. Maternal dose--fetal teratogenic response data were obtained for a variety of narcotic and related compounds by single subcutaneous injections of the drugs into pregnant hamsters during the critical periods of central nervous system organogenesis. The number of abnormal fetuses from females injected with diacetylmorphine (heroin), thebaine, phenazocine, pentazocine, propoxyphene, and methadone increased as the maternal dose of the compounds was increased. By contrast, morphine, hydromorphone, and meperidine produced an increase in the number (per cent) of fetal anomalies only up to a certain maternal dose level. Further increases in maternal dose levels did not produce additional fetal anomalies. Comparative studies of single and multiple maternal doses indicated that diacetylmorphine (heroin) and methadone produced a four- to sixfold increase in fetal anomalies with repetitive doses whereas the percentage of malformed fetuses remained the same with hydromorphone (Dilaudid). The narcotic antagonists nalorphine, naloxone, levallophan, and cyclazocine blocked the teratogenic effects of both single and multiple doses of the narcotics.


Evaluation of a non-professional visual screening method. A screening method designed for administration by lay personnel is evaluated and compared to Modified Clinical Technique screening results for a screening of 1600 elementary school children.


Ionic requirements of proximal tubular sodium transport. II. Hydrogen ion. Simultaneous perfusion to proximal convoluted tubules and peritubular capillaries was used to study the effects of different perfusion fluids on sodium reabsorption and hydrogen secretion, which was calculated as bicarbonate reabsorption and titratable acid. Results show that sodium reabsorption was not tightly coupled to hydrogen secretion. Bicarbonate stimulates both sodium reabsorption and hydrogen secretion, but Tris stimulates only sodium reabsorption. Imposing an adverse chloride gradient across the proximal tubule (C1- peritubular greater than C1- luminal) decreased sodium reabsorption but did not diminish hydrogen secretion. Diamox inhibited both net sodium and hydrogen transport. It is concluded that there is not firm linkage between sodium reabsorption and hydrogen secretion and that bicarbonate probably stimulates sodium transport by a number of mechanisms, including an effect on the sodium transport unrelated to its ability to increase hydrogen ion secretion.


ECS, intracellular pH, and electrolytes of cardiac and skeletal muscle. The extracellular space (ECS) of muscle from each ventricle of the heart (RV and LV), the atria, diaphragm, and quadriceps was estimated in the anesthetized rabbit from the distribution volumes of [14C]insulin, [14C]sucrose, [51Cr]EDTA, and C1--. Whole-tissue electrolytes were measured and intracellular electrolytes calculated. The ECS of the tissues varied, increasing in the order quadriceps less than LV less than RV less than atria. The volume of distribution of [14C]inulin was always less than that of either [14C]sucrose or [51Cr]EDTA which agreed closely, whereas that of C1-- was always greater. There was no difference in intracellular K+ in muscle from each of the cardiac chambers, whereas intracellular Na+ and C1-- varied, increasing in the order quadriceps less than LV less than RV less than atria. Intracellular pH, measured with [14C]DMO did not differ in any of the tissues studied. It is concluded that, in vivo, the estimated ECS incardiac muscle is lower than that reported in vitro, that [51Cr]EDTA is a satisfactory ECS marker, and that differences in intracellular Na+ and C1-- but not K+ or pH exist between muscle from the cardiac chambers.


Intracellular pH and K+ of cardiac and skeletal muscle in acidosis and alkalosis. The effects of a metabolic and respiratory acidosis and alkalosis on intracellular pH (pHi) and K+ have been compared in cardiac and skeletal muscle from the anesthetized rabbit. The extracellular space and pHi were calculated from the distribution volumes of [51Cr] EDTA and [14C]DMO, respectively. When pHe was varied by altering PCO2, the slope of the line relating pHi to the extracellular pH (pHe) was greater (P less than 0.05--0.001) than that obtained during metabolic changes of pHe in right and left ventricles, atria, diaphragm, and quadriceps. During metabolic acidosis and alkalosis, the slope of pHi/pHe line did not vary between tissues. During respiratory acidosis, there was no difference in slope between cardiac tissues, but it was less in left ventricle than quadriceps (P less than 0.001). In left ventricle intracellular K+ increased in a metabolic (P less than 0.05) or respiratory acidosis (P less than 0.02), whereas in diaphragm it decreased (P less than 0.02). Intracellular K+ correlated with pHe and pHE-PHi. Changes in pHi but not intracellular K+ could explain known differences in myocardial function in respiratory and metabolic acidosis.


Effects of autonomic neurohumors on transmembrane potentials of atrial plateau fibers. Isolated canine atrial plateau fibers were treated with acetylcholine or norepinephrine to note the effects on the transmembrane potential. Acetylcholine, 1.0 or 2.0 mug/ml, consistently reduced the slope of inherent phase 4 depolarization. Increases in maximum diastolic potential and rising velocity occurred along with a decrease in overshoot. The plateau phase disappeared. Pretreatment with atropine, 1.0 mug/ml, prevented these responses, and alone this drug had no discernible effect. Norepinephrine consistently increased the slope of phase 4 depolarization. Frequently plateau fibers generated action potentials by the normal pacemaker mechanism. "Arrhythmias" characterized by spontaneous excitations were induced in 92% of the norepinephrine experiments. Norepinephrine also enhanced the plateau phase of the action potential and decreased the rising velocity and overshoot. Racemic propranolol, 1.0 mug/ml, blocked all the above effects including arrhythmias. Dextropropranolol, 1.0 mug/ml, did not block effects produced by norepinephrine. Acetylcholine, applied to fibers under treatment with norepinephrine, reduced the slope of norepinephrine-induced phase 4 depolarization and terminated induced arrhythmias.


Interstitial fluid pressure and alkaline gastric secretion. The interstitial fluid pressure of the submucosa of the gastric fundus was monitored by means of Guyton's capsules in dogs anesthetized with pentobarbital. The intracapsular pressure (ICP) was measured during secretion produced by: a) hypertonic solutions placed inside the stomach; b) arterial hypertension (200 mmHg) applied during intra-arterial infusion of histamine, and c) intra-arterial infusion of acetylcholine. The first procedure did not modify the ICP. On the other hand, whenever interstitial fluid appeared in the gastric lumen during hypertension plus histamine, the mean ICP increased, mostly due to augmented capillary filtration. The hydraulic coefficient measured in these experiments was at least 4 orders of magnitude larger than the respective osmotic coefficient. The action of acetylcholine was complex: large doses enlarged the net capillary filtration, but small doses increased the mean ICP by muscle stimulation only. Contraction of the muscularis mucosae might be the most important mechanism underlying bulk flow of interstitial fluid in physiological conditions. It is concluded that hydraulic gradients across the epithelium might account for the "secretion" of "alkaline" juice.


Overview: maintenance therapy in psychiatry: I. Schizophrenia. Hospital psychiatry has evolved from long-term "treatment" programs that were primarily custodial to the successful pharmacological treatment of acute psychotic episodes. Unfortunately, many patients still return to the hospital with relapses. This so-called revolving door syndrome draws attention to the critical importance of preventing as well as treating acute episodes. In the first part of this overview, the author reviews the clinical literature on prophylactic treatment of schizophrenia with maintenance antipsychotic drugs. The second part will review the literature on prophylactic treatment of affective disorders with lithium and tricyclics. In the opinion of the author these drugs provide the potential for truly preventive psychiatry.


Inpatient and outpatient patterns of psychotropic drug prescribing by nonpsychiatrist physicians. The authors found that among 228 general hospital patients, minor tranquilizers were prescribed most often and with the least justification and that major tranquilizers were prescribed sparingly and by and large judiciously. Antidepressants were given less often than would be justified by the incidence of depressive illness among these patients. Nonrecognition of depression in patients with somatic complaints and autonomic signs of depression contributed to this lack of treatment.


[Metabolic acidosis as a side-effect of methoxyflurane anesthesia (author's transl)]. In 14 patients undergoing surgery with methoxyflurane anaesthesia the development of metabolic acidosis was observed. As mechanisms causing such acidosis regional ischaemia and inhibition of lactic acid metabolism in the liver by breakdown products of methoxyflurane are discussed.


Protective effect of hypothermia in cerebral oxygen deficiency caused by arterial hypoxia. To study the cerebral protective effects of hypothermia in arterial hypoxia, anesthetized (70% N2O), mechanically ventilated rats were cooled to a body temperature of 27 C. Hypoxia was induced by decreasing the oxygen content in the inspired gas mixture either to 6-7 per cent or to 2.5-3 per cent. This reduced mean PaO2 to about 25 and 11-12 torr, respectively. At PaO2 torr, there was no change in cerebral blood flow (CBF), cerebrla oxygen consumption (CMRO2), or labile tissue metabolites. The absence of signs of cerebral hypoxia could be attributed to an effect of temperature and pH on the hemoglobin-oxygen dissociation curve. Thus, at 27 C with a PaO2 of 25 torr the total oxygen content (TO2) of arterial blood remained greater than 15 ml (100 ml)-1, about three times the value obtained at this PO2 in normothermic rats. At PaO2 11-12 torr, arterial TO2 was reduced to about 5 ml (100 ml) (-1). The hypoxia induced no change in CMRO2, a threefold increase in CBF, a moderate lactacidosis in the tissue, and a small decrease in phosphocreatine content, but no change in ATP, ADP, or AMP. These changes are less marked than those occurring at the same arterial TO2 in normothermic rats. It is concluded that hypothermia exerts a pronounced protective effect on the brain in hypoxic hypoxia, and that two mechanisms are involved. First, since hypothermia shifts the oxyhemoglobin-dissociation curve towards the left, and prevents or minimizes a rightward shift due to acidosis, it maintains a high TO2 in arterial blood at a given PaO2. Second, by reducing CMRO2, and thereby presumably also cellular energy requirements, hypothermia exerts a protective effect at the cellular level.


Characterization of a caprine herpesvirus. A virus, which was isolated from kids (Capra hircus) affected with a relatively severe generalized infection, was found to contain DNA and to have a buoyant density of 1.2820 g/cm3. The virus was sensitive to the action of lipid solvents and trypsin and was rapidly inactivated at pH 3.0 and at temperatures of 50 and 56 C. The virion, an icosahedron consisting of a nucleoid surrounded by a double membrane, measured approximately 135 nm in diameter. On the basis of its chemical and physical properties, the virus is considered a herpesvirus.


Transfer of drugs across the ruminal wall in goats. The rates at which pentobarbital, salicylate, antipyrine, and quinine were transferred from the rumen of intact, conscious goats were measured. The rates at which the same drugs diffused from the blood plasma (under conditions of constant drug concentration) into the ruminal solution were also evaluated. These compounds were absorbed by simple diffusion, and the rates of transfer were a function of pH of the intraruminal solution. The diffusion of drugs from plasma into the reticulorumen allowed steady-state distributions to be established in some goats. The theoretical and observed steady-state distributions were compared. There were good correlations for pentobarbital and antipyrine, but not for salicylate and quinine. These findings confirm in vivo the general principles of drug transfer across ruminal epithelium that were derived from previous studies conducted in vitro.


Preservation of hypoxic pulmonary pressor response in canine pneumococcal pneumonia. To determine the role of hypoxic pulmonary vasoconstriction in pneumococcal pneumonia, hemodynamic measurements were made in 16 dogs before, and within 36 hours after, intrapulmonary administration of type III pneumococcus. Ten dogs with one lobe or more of pneumonia increased their pulmonary vascular resistances and slightly decreased their arterial O2 tensions. Hypoxia increased and hyperoxia decreased their pulmonary vascular resistances. During O2 breathing, arterial PO2 was less during than before the pneumonia and increased when pulmonary perfusion was diverted away from the diseased lung. In 2 dogs breathing air, forcing the cardiac output through the diseased lung caused an increase in vascular resistance that could clearly be reduced by O2 breathing. In 5 dogs, lung mast cell counts showed no decrease in the lobes with pneumonia. In pneumococcal pneumonia, the hypoxic pulmonary pressor mechanism serves to decrease blood flow to the diseased lobes and, thus, to maintain the arterial PO2. Lung mast cells could participate in this response.


Value of capillary blood gas analyses in the management of acute respiratory distress. A comparative study of blood gases and acid-base parameters, obtained simultaneously from arterial and finger capillary samples, was performed in 45 patients in acute respiratory distress without circulatory shock. Although small and significant differences were found between the 2 sample pH, Po2, Pco2, and bicarbonate values, the correlations between the 2 were greater than or equal to 0.97 for each variable. It was concluded that although the arterial blood is the preferred sample for evaluation of blood gases and acid-base status of patients in acute respiratory distress, capillary blood appears to be a valid substitute in the management of these patients. This technique is particularly valuable in pediatric practice, where repeated arterial samples are less easily obtained.


Bone marrow transplantation in man.

Bone marrow transplantation in man. Bone marrow transplantation is emerging as a viable therapeutic approach to a number of diseases that are usually or uniformly fatal. We review here recent experiences in bone marrow transplantation in man at UCLA and in various other institutions throughout the world. We examine marrow transplantation in immunodeficiency diseases, acute leukemia, and aplastic anemia and consider the problems of infection in the transplant recipients. The applications of tissue typing to marrow transplantation and immunologic manipulations, which may influence engraftment and graft-versus-host disease, are also reported.


The virus hypothesis in systemic lupus erythematosus. Type-C viruses are currently the prime etiologic candidates in systemic lupus erythematosus. On the basis of knowledge gained from studies of experimental and human models of chronic viral disease, there are possible pathogenetic roles of a virus in systemic lupus erythematosus. Experimental attempts at implicating specific viruses have been predominantly negative, but evidence for enhanced type-C-virus expression has recently been reported.


The membrane transport of ascorbic acid. A system for measuring the rate of transport of dehydroascorbate into human red blood cells shows Michaelis-Menten type kinetics with substrate inhibition at levels above 150 muM DHA. The addition of sugars impairs this transport in the diminishing hierarchy D-glucose, D-mannose, D-xylose, D-galactose, L-lyxose, D-araboascorbate, L-sorbose and 2-deoxy-D-ribose. The effect of glucose on transport of ascorbate is marked at physiological levels. Transport of DHA is accelerated by copper ion and allows dehydroascorbate to move against a concentration gradient. The evidence supports the hypotheses proposing that hyperglycemia will impair the intracellular availability of vitamin C.


Carcinofetal alterations in glucosamine-6-phosphate synthetase. The levels of glucosamine-6-phosphate synthetase in various rat tissues including those undergoing differentiation or regeneration revealed that the enzyme is related to tissue proliferation and differentiation. In the liver upon neoplastic transformation, the level of glucosamine 6-phosphate synthetase rises and the liver form of the enzyme having a pI at 5.0 is replaced by a form with a pI of 4.1. Since the latter form has also been found present in whole embryos (12- and 14-day) and brain, the molecular alterations of glucosamine-6-phosphate synthetase in liver neoplasia can be considered to be carcinofetal.


A hepatoma-associated alkaline phosphatase, the Kasahara isozyme, compared with one of the isozymes of FL amnion cells. It was found that a human hepatoma-associated ALP (orthophosphoric monoester phosphohydrolase, E.C. 3.1.3.1) shared electrophoretic mobility, inactivation by urea, inhibition by inorganic phosphate, ethylenediaminetetraacetate, and amino acids (L-phenylalanine, L-leucine and L-homoarginine), heat stability, sensitivity to neuraminidase, pH optimum, Km value, and antigen site with fast moving ALP isozymes of FL cell strain derived from human amniotic membrane. However, 40-week-old fresh amniotic membrane lacked this isozyme. Instead, it had a placental type ALP consisting of minor components. The other ALP isozyme of FL cells had properties common to hepatoma ALP with regard to L-phenylalanine sensitivity, inhibition by ethylenediaminetetraacetate, inactivation by urea, and antigen site, but differed from it in electrophoretic mobility, sensitivity to L-leucine and L-homoarginine, and the presence of another antigen site. It was more heat stable and more sensitive to inhibition by inorganic phosphate than Hepatoma AP. The possible regulatory mechanism between the hepatoma-type ALP and the placental type ALP in the amnion cells is considered.


Gamma-glutamyl transpeptidase. A sensitive indicator  of renal ischaemic injury in experimental animals and renal homograft rejection in man. The sites of ischaemic injury within the kidney are reviewed and the diagnostic value of measurements of plasma and urinary enzymes in renal ischaemic injury and in renal homotransplant rejection in experimental animals and man is examined. Gamma-glutamyl transpeptidase (gamma-GT) is an enzyme primarily located in the brush border of the proximal convoluted tubule of the kidney. Its unique localization in the cells most easily damaged by ischaemia and its ease of assay provide the rationale for its use in the measurement and diagnosis of renal ischaemic injury. gamma-GT activity was measured in dogs undergoing varying periods of renal ischaemia and under conditions of local renal hypothermia and was shown to be a sensitive indicator of ischaemic injury. Twenty consecutive patients undergoing renal homotransplantation were studied by daily estimation of their 24-h urinary gamma-GT activity; excellent correlation was obtained between raised levels of this enzyme and the clinical diagnosis of transplant rejection.


Influence of pH on the heat inactivation of staphylococcal enterotoxin A as determined by monkey feeding and serological assay. The effect of pH on the thermal inactivation of staphylococcal enterotoxin A was investigated. Analysis of heated toxin by immunodiffusion in gel indicated that enterotoxin A in beef bouillon was inactivated faster at pH 5.3 than at pH 6.2. The z values (slopes) for the heat inactivation curves at pH 6.2 and 5.3 were 49.5 and 55 F (about 27 and 30 C), respectively. Enterotoxin produced and heated in dialyzed Casamino Acids medium and assayed by monkey feeding was more easily inactivated by heat at pH 5.3 than at pH 7.8. Thermal inactivation curves for enterotoxin A in beef bouillon (5 mug/ml, pH 5.3) were determined by two methods, monkey feeding and serological assay. The z values for the curves obtained by these two methods were both 55 F, although loss of biological or toxic activity of the enterotoxin occurred before loss of serological activity.


Survival of salmonellae during pepperoni manufacture. Survival of salmonellae in artificially contaminated beef-pork mixtures (approximately 10(4) salmonellae/g) was studied in pepperoni prepared by either a natural flora or lactic starter culture fermentation or in nonfermented sausages. The pepperoni did not become salmonellae free during the usual commercial 15 to 30-day drying period. Salmonella dublin was present in all products, fermented or unfermented, after 42 to 43 days of drying. At a lower level of contamination, 10(3)/g, S. dublin could not be recovered from starter culture-fermented pepperoni after 14 days of drying but persisted in the natural flora-fermented sausage. S. typhimurium (initial count, 10(4)/g) was absent after 42 days of drying when starter culture was used to ferment the pepperoni, but was still present in the natural flora-fermented and unfermented products. S. dublin, host adapted to cattle, or S. choleraesuis, host adapted to swine, had similar  survival patterns in beef pork, or beef-pork pepperoni. Heating salmonellae contaminated beef-pork pepperoni (after fermantation but before drying) to an internal temperature of 60 C (trichinae inactivating) eliminated the food-borne pathogen from the sausage product.


Growth of Staphylococcus and Salmonella on frankfurters with and without sodium nitrite. Conventional and nitrite-free frankfurters in loosely wrapped packages were compared as to their ability to support growth of Salmonella, Staphylococcus, and their naturally occurring spoilage flora at 7 C (simulating refrigerated  storage) and 20 C (simulating possible temperature abuse). At 7 C Salmonella did not grow in either type of frankfurter; Staphylococcus and the natural spoilage flora sometimes grew more rapidly in the absence of nitrite, but the difference was not significant. At 20 C growth of Salmonella, Staphylococcus, and of the spoilage flora was, at most, only slightly faster on nitrite-free frankfurters. Salmonella was not suppressed in broth culture experiments the pH and nitrite content found in frankfurters. Although either type of frankfurter can become hazardous due to growth of Salmonella or Staphylococcus, no unusual or additional hazard resulted from the omission of nitrite from frankfurters.


Characterization of an extracellular dextranase from Fusarium moniliforme. An extracellular dextranase (EC 3.2.1.11) was purified approximately 75-fold from cell-free culture filtrates of Fusarium moniliforme. The purified dextranase was of the endo type, and isomaltose was identified as the primary end product of dextran hydrolysis. The molecular weight of the dextranase was determined to be 39,000 by gel permeation chromatography. The enzyme was most active at pH 5.5, and the temperature optimum was near 55 C. Activity was not inhibited by either ethylenediaminetetraacetic acid or iodoacetate. The Km for dextran with an average molecular weight of 10,000 was estimated to be 1.1 X 10(-4) M. The electrophoretic mobility of the dextranase was distinctly different from that of a Penicillium-derived commercial dextranase. The F. moniliforme dextranase was also found to differ from the commercial preparation by its greater relative activity against glucans isolated from Streptococcus mutans.


Nonautotrophic Thiobacillus in acid mine water. Nonautotrophic thiobacilli were isolated from the acidic water of a coal mine. Based on their mixotrophic physiology, the isolates are regarded as strains of Thiobacillus perometabolis.


Graft versus host reaction. An ultrastructural study. Graft versus host reaction (GvH) following leukocytic transfusions occurred in a 34-year-old man with a generalized lymphosarcoma. Histologic and ultrastructural studies were performed, with special reference to dyskeratotic cells scattered in the epidermis. These cells are usually considered to be a constant and important feature of GvH reaction. Dense aggregation of tonofilaments, including cytoplasmic organelles and loss of desmosomes, were seen in dyskeratotic cells. Several intracellular desmosomes with tonofilaments bound to their attachment plaque were observed. Some of these cells were able to reach the horny layer; some others were phagocytosed by neighboring keratinocytes. These cells could be the result of a toxic damage to the epidermis, provoked by the immunologic phenomenon implicated in GvH reaction. Later in the clinical course, bullae formations occurred, showing some features of toxic epidermal necrolysis (TEN), as well as features of GvH reaction.


Nephrotic syndrome in indian children.

Nephrotic syndrome in indian children. A clinicopathological study of 206 Indian children with nephrotic syndrome showed a primary renal cause in 195 (96%), of which 77% were boys. In 126 children (96 boys, 30 girls) onset of the disorder occurred before the age of 5 years. Renal biopsy showed minimal lesions in 150 patients (77%); in 85 of these biopsy was done 3 months to 16 years after onset of the nephrotic syndrome. Significant renal histological abnormalities in 45 cases were labelled as mesangiocapillary 8, mesangioproliferative 4, proliferative with extensive crescents 2, membranous 3, focal segmental glomerulosclerosis 9, focal global glomerulosclerosis 2, advanced nonspecific 8, and mild proliferative 9. Nephritic manifestations were mainly associated with significant renal lesions, which were more frequently encountered when the onset of disease was after the age of 5 years. Clearance of proteinuria with corticosteroid therapy was practically confined to patients with minimal or mild renal histological changes. Our findings suggest that the pattern of idiopathic nephrotic syndrome in Indian children is similar to that reported from Western countries.


Separation of deoxyribonucleases (DNases) of normal human stratum corneum and psoriatic scales by micro-disc-electrophoresis. Normal stratum corneum and psoriatic scales were homogenized and a differential centrifugation was performed. The DNase activity of the individual fractions was investigated by micro-dis-electrophoresis. At pH 5 only in the 600xg pellet and 105.000xg supernatant of normal keratin DNase activity could be observed. However, all psoriatic fractions showed distinct enzyme activities. At pH 7.4 little psoriatic DNase activity could only be demonstrated in the 105.000xg supernatant. Except from the 15.000xg pellet all fractions of normal stratum corneum displayed marked activities. In addition the 105.000xg supernatant showed two different DNase bands.


Subcellular distribution of phosphatases, proteinases, and ribonucleases in normal human stratum corneum and psoriatic scales. The subcellular distribution of phosphatases, proteinases, and ribonucleases of normal human stratum corneum and psoriatic scales was determined after differential centrifugation. All psoriatic enzymes showed much increased activities as compared to the normal stratum corneum enzymes. The highest activities of alkaline phosphatase from psoriatic scales could be detected in the nuclear fraction. The main activities of all other tested phosphatases and proteinases were present in the cytoplasmatic fraction. The subcellular distribution of the ribonucleases varied according to the pH value.


A reinvestigation of the sites of transcription and translation of Euglena chloroplastic phenylalanyl-tRNA synthetase. An attempt was made to determine the sites of chloroplast phenylalanyl-tRNA synthetase transcription and translation. Inhibitors of bacterial RNA and protein synthesis were added to logarithmic and stationary phase cultures of Euglena gracilis wild-type B. Logarithmic phase cultures were sensitive to both types of inhibitors. In stationary phase cultures plastid synthetase was reduced by RNA but not by protein synthesis inhibitors. The effect of the antibiotics on the mitochondrial enzyme was also noted. Several possible explanations of these resuults are discussed.


[Hyperthermia in the rabbit. II. Studies on influencing respiration with exogenic hyperthermia]. Rectal temperature, respiratory rate, arterial and venous pH and arterial and venous pCO2 were recorded at intervals of 15-30 minutes in female broiler rabbits exposed an environmental temperature of 35 degrees C, until they died. Respiratory rate and blood pH rose, and pCO2 fell, until a rectal temperature of 42 degrees C was reached. Upon further increase in rectal temperature the respiratory rate and pH began to fall, while pCO2 began to rise. The rabbits died when rectal temperature reached 43 degrees C. Features of respiratory function peculiar to the rabbit were discussed.


Outpatient phenothiazine use and bone marrow depression. A report from the drug epidemiology unit and the Boston collaborative drug surveillance program. Phenothiazine-induced bone marrow depression (BMD) was evaluated in three separate but complementary data bases: (1) Among 1,048 patients admitted to psychiatric hospitals, there was no evidence of subclinical depression of the white blood cell (WBC) count attributable to phenothiazines used before admission. (2) Among 18,587 medical inpatients, there were 34 patients admitted for BMD in the absence of neoplasia or prior cytotoxic drug therapy; one of the latter reported using chlorpromazine hydrochloride, but it is doubtful whether this drug was the cause of the BMD. (3) Among 24,795 medical, surgical, and gynecological patients surveyed over a ten-month period in 1972, there were four who were admitted for BMD; one of the latter had a reversible leukopenia attributed to trifluoperazine hydrochloride.


[Tumour hyperacidulation through intravenous glucose infusion enhanced by amygdalin and beta-glucosidase application (author's transl)]. Tumour peracidity in otherwise moderately hyperacidulated tumours or tumour regions of DS carcinosarcoma-bearing Wistar rats attained by glucose infusion was substantially increased by simultaneous infusion of amygdalin and intratumoral i.m. or i.v. application of beta-glucosidase. Here the pH value of healthy tissue, measured at the sceletal muscle, remained unchanged. By means of the said process, tumour hyperacidulation has been raised to a level of deltapH =0.97; attaining a pH difference between tumourous and normal tissue of up to deltapH = 1.6. In one case, the slope of pH reduction in the tumour increased to 870%. Moreover, combined administration of glucose, amygdalin and beta-glucosidase evoked a significant cancerostatic effect hypogenesis, tumour regression) being comparable with the action of an Ifosfamid dosage of 150 mg-kg-1. However, i.m. and i.v. application of beta-glucosidase under narcosis results in an overall process that still remains somewhat too toxic. Hence optimizing studies are intended with the particular aim to further improve the comparability of this process.


Studies of the pain produced by mafenide acetate preparations in burns. In a double-blind triple cross-over clinical study, 37 patients were exposed to several formulations of mafenide acetate (Sulfamylon Cream) and their pain responses were recorded and converted to a semiquantitative pain index. The 11.2% concentration in cream was two to three times more painful than the 5% concentration. Hypertonicity  and not the pH level appears to be the cause of the pain produced by the high (11.2%) concentration. The tonicity of the cream carrier and 11.2% mafenide acetate are 1,080 mOsm/kg and 1,100 mOsm/kg, respectively, for a total of 2,180 mOsm/kg. The carrier cream without glycerol and a 5% concentration of mafenide cream were much less painful than the 11.2% concentration of mafenide. Both afforded a great deal of relief to the patients who received the medications.


Microbiol growth in lipid emulsions used in parenteral nutrition. Parenteral nutrition via central venous catheterization is associated with serious risks, especially that of sepsis. Lipid emulsion (Intralipid[Sweden]), which may be administered peripherally, was evaluated for its potential to support microbial growth. Washed cultures of Staphylococcus aureus, Candida albicans, and three species of Gram-negative rods were all capable of multiplying in the emulsion at room temperature. Variations in inoculum size did not affect the growth rate. Studies comparing the emulsion to amino acid-glucose solutions (total parenteral nutrition [TPN])confirmed other reports that TPN inhibits the growth of certain bacteria but merely retards fungal multiplication. When human serum was added to the lipid emulsion in an attempt to simulate in vivo conditions at the catheter tip, Escherichia coli was inhibited while the growth of S aureus and C albicians was unaltered.


Propoxyphene and norpropoxyphene tissue concentrations in fatalities associated with propoxyphene hydrochloride and propoxyphene napsylate. Propoxyphene and its major metabolite norpropoxyphene have been determined in blood and liver in 29 cases of death in which propoxyphene, either as the hydrochloride or as the napsylate salt, was involved. The use of propoxyphene napsylate (Darvon-N) contributed to the deaths of 4 persons, 3 of whom were former heroin addicts receiving large amounts of this drug in connection with propoxyphene substitution programs. In the majority of cases the norpropoxyphene blood concentrations exceed the propoxyphene concentrations, although brain determinations in several instances indicate that norpropoxyphene does not cross the blood-brain barrier with the same ease as propoxyphene. On the basis of the comparative toxicities of propoxyphene and norpropoxyphene in animals and the high tissue concentrations of norporpoxyphene in man after propoxyphene administration, it is conceivable that norpropoxyphene contributes to the toxic effects of propoxyphene.


[Morphological manifestations and morphogenesis of the graft vs. host reaction in the brain of F1 hybrids following the administration of parental lymphoid cells and its effect on tumor inoculation]. It was established that intracerebral introduction of 10 min parental cells of the spleen brought about in hybrids (CBAXC57Bl/6) F1 infiltrative-destructive changes (development of lymphocytic infiltrations, dystrophy in the nerve cells, myelin fibres and neurogliacytes) the intensity of which was found to be considerably higher in cases of injection of parental spleen cells from specifically sensitized donors. Spleen cells of mice CBA produced much greater changes as compared with those produced by spleen cells of mice C57Bl/6. Inoculation into the mice brain of 10 mln of spleen cells together with tumour in a dose of 1500 cells produced a clear cut inhibitory effect on the tumour growth, this effect being more pronounced following introduction of sensitized cells of the spleen of mice CBA.


Metachromatic leukodystrophy. Ultrastructural and enzymatic study of a case of variant O form. A variant of metachromatic leukodystrophy (MLD), Austin disease, is characterized by a multiple isozyme deficiency of arylsulfatase. A 3 1/2-year-old girl with progressive mental and physical deterioration had decreased activities of arylsulfatases A and B in the leukocytes, shown by acylamide gel electrophoresis. Under the electron microscope, biopsy specimens of the brain and the peripheral nerve showed lamellar structures with socalled zebra bodies in the cytoplasmic processes of glial cells, granulo-membranous inclusions with fingerprint configurations in neurons, and myelinlike material in Schwann cells. Results from our study suggest an intricate nature of this dysmetabolic disorder, which shows ultrastructural changes usually seen in classic MLD, a deficiency of arylsulfatase A only, concomitant with those seen in mucopolysaccharidoses such as Hurler and Sanfilippo syndromes.


Changes in the acid base status of sheep anaesthetised with a combination of atropine sulphate acepromazine and ketamine hydrochloride. pH, PaCO2, PaO2, standard bicarbonate, base excess and reduced pH were measured in sheep before and at regular intervals after administration of ketamine with and without atropine and acepromazine premedication. A decrease in pH and PaO2 and a rise in PaCO2 was observed 15 minutes after administration of ketamine. Administration of atropine with and without acepromazine had no significant effect on pH, PaCO2 and PaO2. The values for standard bicarbonate, base excess and reduced pH were not significantly affected. This indicates that minor changes observed in pH, PaCO2 after ketamine administration are compensated for by the healthy animal's blood buffer system.


Molecular nature of beta-galactosidase from different tissues in two strains of the house mouse. One inbred mouse strain, C57BL/Kl, has high galactosidase activities in all tissues while another strain, DBA/2/Kl, has low activities determined by the Bgs locus. Beta-Galactosidase from these two strains was partly purified by a five-step procedure: acidification, ammonium sulfate precipitation, gel filtration at two pHs, and isoelectric focusing. No qualitative differences were found between the enzyme preparations from the two strains. They had identical heat inactivation curves, pH optima, molecular weight, and isoelectric points, and the Km values were very similar. It thus seems that this genetic difference in enzyme activity probably cannot be explained by a variation of the galactosidase-specific activity but rather reflects a difference in number of enzyme molecules. Eight different isoenzymes were separated from liver, kidney, and spleen. Each isoenzyme has a different electrophoretic mobility and there is a stepwise increase in molecular weight from 143,000 to 380,000 beginning with the protein having the lowest isoelectric point. A likely interpretation is that the isoenzymes bind a smaller polypeptide in varying numbers in addition to the enzymatic polypeptide per se.


The effects of acute and chronic nicotine hydrogen (+)-tartrate administration and subsequent withdrawal on rat liver tryptophan pyrrolase activity and their comparison with those of morphine, phenobarbitone and ethanol. Acute administration of nicotine hydrogen (+)-tartrate enhances the activity of rat liver tryptophan pyrrolase by a hormonal mechanism. Chronic nicotine treatment inhibits, and subsequent withdrawal enhances, the pyrrolase activity. The inhibition during chronic treatment is not due to a defective apoenzyme synthesis nor a decreased cofactor availability. Regeneration of liver NADP+ in vitro and in vivo reverses the inhibition. Chronic nicotine administration increases the liver NADPH concentration. The above effects of nicotine resemble to a remarkable degree those previously shown for morphine, phenobarbitone and ethanol. All effects are compared, and their possible significance in relation to drug dependence is discussed.


Plastid development in primary leaves of Phaseolus vulgaris. Development of plastid adenosine triphosphatase activity during greening. The etioplasts of dark-grown bean leaves showed ATPase (adenosine triphosphatase) activity which had a pH optimum of 8.5, was stimulated by dithiothreitol and unaffected by light-triggering. Bean chloroplasts showed a low activity of dark-induced ATPase with a pH optimum of 8.5 and a substantial amount of light-triggered activity with a pH optimum of 8.0. The light-triggered activity depended on dithiothreitol and Mg2+ and was promoted by phenazine methosulphate. Light-triggered ATPase activity was completely inhibited by 20mum-dicyclohexylcarbodi-imide. Etioplasts developed light-triggered ATPase activity in response to 30 min illumination of the etiolated leaves. During the 48 h of light-induced greening of dark-grown leaves there was a 70% increase of the chloroplast ATPase activity found after light-triggering and a 30% fall in the dark-induced activity, both expressed on a per leaf basis. As the larger part of these changes occurred during the first 30 min of illumination, it is concluded that most or all of the chloroplast ATPase was present in the etioplast, a conclusion identical with that of Lockshin et al. (1971) for maize. During 48 h of greening there was a tenfold increase in the amount of thylakoid membrane in the leaf together with an 83% fall in the ATPase activity per m2 of thylakoid membrane, measured after light-triggering.


Influence of some physical factors on survival of Marek's disease vaccine virus. Cell-associated Marek's disease (MD) vaccine was suspended at dilutions normally used for vaccination in seven commercially available diluents and in tryptose phosphate broth. The stability of diluted vaccines was determined by assay in cell cultures subjected to 0 to 37 C for 0 to 90 minutes. Optimum holding temperatures for MD vaccine virus survival varied with the specific diluents employed. Some diluents afforded greatest survival when dilution was at 0 C and held at 0 C, while others performed best when dilution was at 25 C followed by cooling and holding at 0 C. Diluents which allowed greatest survival when tested at 37 C also performed well under other temperature regimes. Spectinomycin dihydrochloride pentahydrate and various buffering compounds were added to commercial diluents used for diluting MD vaccine. Additives producing osmolality of 745 mOsm/kg and higher markedly reduced vaccine virus survival. The adverse effects of high osmotic pressure were accentuated by extended holding time, elevated incubation temperature, and physical manipulations including mechanical mixing or expressing through a syringe and needle. Satisfactory MD vaccine virus survival was afforded by a commercial diluent especially formulated to accommodate the pH osmolality changes produced by adding spectinomycin dihydrochloride pentahydrate.


Tricarboxylic acid-cycle and related enzymes in restricted facultative methylotrophs. The isolation is described of pure cultures of three non-methane-utilizing methylotrophic bacteria which, together with the previously described Bacillus PM6, have a very limited range of growth substrates; these organisms are designated "restricted facultative' methylotrophs. Two of these isolates, W6A and W3A1, grow only on glucose out of 50 non-C1 compounds tested, whereas the third isolate S2A1 and Bacillus PM6 grow on betaine, glucose, gluconate, alanine, glutamate, citrate and nutrient agar, but not on any of a further 56 non-C1 compounds. Crude sonic extracts of trimethylamine-grown and glucose-grown W6A and W3A1 isolates, and of trimethylamine-grown C2A1 (an obligate methylotroph) contain (i) no detectable 2-oxogltarate dehydrogenase activity, (ii) very low or zero specific activities of succinate dehydrogenase and succinyl-CoA synthetase and (iii) NAD+-dependent isocitrate dehydrogenase activity. Extracts of trimethylamine-grown PM6 and S2A1 methylotrophs have (i) very low 2-oxoglutarate dehydrogenase specific activities, (ii) comparatively high specific activities of succinate dehydrogenase, malate dehydrogenase and succinyl-CoA synthetase and (iii) NADP+-dependent isocitrate dehydrogenase activity but no NAD+-dependent isocitrate dehydrogenase activity. The activities of most of these enzymes are increased during growth on glucose, alanine, glutamate or citrate, but only very low 2-oxoglutarate dehydrogenase activities are present under all growth conditions. The restricted facultative methylotrophs grow on certain non-C1 compounds in the absence of 2-oxoglutarate dehydrogenase and, in some cases, of other enzymes of the tricarboxylic acid cycle; these lesions cannot therefore be the sole cause of obligate methylotrophy.


Desaturation of stearic acid by liver and adipose tissue from obese-hyperglycaemic mice (ob/ob). Stearic acid desaturase activity was assayed in preparations from perigenital adipose tissue and liver from lean and genetically obese female mice (ob/ob). The total activity in the perigenital adipose tissue from obese mice was threefold greater than in the tissue from lean mice, but per g of adipose tissue the activity was twofold greater in tissue from lean mice. In liver, the activity in obese mice was elevated at 8 weeks of age, remained elevated up to 24 weeks and then decreased by half at 48 weeks, but at all ages was higher than that in lean mice. The decrease in desaturase activity of liver from obese mice at 48 weeks corresponded to a change in the fatty acid composition of liver lipids toward that found in lean mice. Whereas in adipose tissue much of the increased enzyme activity may be due to tissue hyperplasia, in liver it is mainly an increased activity per cell.


The stimulation by synaptic transmitters of the incorporation of oleate into the phospholipid of synaptic membranes. Noradrenaline stimulated the incorporation of oleate into choline glycerophospholipids of guinea-pig brain synaptic membranes incubated in sodium phosphate buffer. In the presence of 1 mm-NaF, noradrenaline stimulated the incorporation of oleate into the choline glycerophospholipids, phosphatidylinositol, ethanolamine glycerophospholipids, phosphatidylserine and phosphatidic acid of synaptic membranes incubated in 10 mm-Tris-HCl buffer. In Tris-CHl containing 1 mm-NaF, stimulation of incorporation of oleate into choline glycerophospholipids by noradrenaline was enhanced by ATP, CaCl2, MgCl2 and CoA plus dithiothreitol. The optimum concentration of CaCl2 for stimulation by 10 mum-noradrenaline was 10 mum. In the presence of CaCl2, the optimum concentration of ATP-2MgCl2 was in the range 0.1-1 mm. Acetylcholine, carbamoylcholine, 5-hydroxytryptamine, dopamine, histamine and gamma-aminobutyric acid also stimulated the incorporation of oleate into choline glycerophospholipids of synaptic membranes. Sigmoidal dose-response curves were obtained, similar to those obtained previously for stimulation by the same agonists of the hydrolysis of phosphatidylcholine by phospholipase A2 (Gullis & Rowe, 1975a). The initial rate of transfer of oleate from oleoyl-CoA to choline glycerophospholipid was similar to the initial rate of transfer from oleate-albumin, stimulated by noradrenaline. Transfer of oleate from oleoyl-CoA was not appreciably stimulated by noradrenaline, but was stimulated by ATP and MgCl2.


The interaction of magnesium ions with teichoic acid. The binding of Mg2+ to the wall teichoic acid of Lactobacillus buchneri N.C.I.B. 8007 was measured by equilibrium dialysis at controlled ionic concentration and pH. In an aqueous solution containing 10mM-NaCl at pH 5.0 one Mg2+ ion was bound for every two phosphate groups of the teichoic acid, with an apparent association constant, Kassoc. = 2.7 x 10(3) M-1. On lowering the pH below the pKa of the phosphate groups the amount of bound Mg2+ decreased concomitantly with decreasing ionization of the phosphate groups. Both the amount of Mg2+ bound to the teichoic acid and the apparent association constants were similar in the presence of 10 mM concentrations of NaCl or KCl but decreased markedly in the presence of 10 mM-CaCl2 because of competition between Ca2+ and Mg2+ for the binding sites. A similar effect was found when the concentration of NaCl was increased from 0 to 50 mM. The results are discussed in relation to the function of teichoic acid in the walls of Gram-positive bacteria.


The mechanism of hydrolysis of beta-glycerophosphate by kidney alkaline phosphatase. 1. To identify the functional groups that are involved in the conversion of beta-glycerophosphate by alkaline phosphatase (EC 3.1.3.1) from pig kidney, the kinetics of alkaline phosphatase were investigated in the pH range 6.6-10.3 at substrate concentrations of 3 muM-30 mM. From the plots of log VH+ against pH and log VH+/KH+m against pH one functional group with pK = 7.0 and two functional groups with pK = 9.1 were identified. These groups are involved in substrate binding. Another group with pK = 8.8 was found, which in its unprotonated form catalyses substrate conversion. 2. GSH inhibits the alkaline phosphatase reversibly and non-competitively by attacking the bound Zn(II). 3. The influence of the H+ concentration on the activation by Mg2+ ions of alkaline pig kidney phosphate was investigated between pH 8.4 and 10.0. The binding of substrate and activating Mg2+ ions occurs independently at all pH values between 8.4 and 10.0. The activation mechanism is not affected by the H+ concentration. The Mg2+ ions are bound by a functional group with a pK of 10.15. 4. A scheme is proposed for the reaction between enzyme, substrate, Mg2+ and H+ and the overall rate equation is derived. 5. The mechanism of substrate binding and splitting by the functional groups of the active centre is discussed on the basis of a model. Mg2+ seems to play a role as an autosteric effector.


The pH-dependence and group modification of beta-lactamase I. The pH-dependence of the kinetic parameters for the hydrolysis of the beta-lactam ring by beta-lactamase I (penicillinase, EC 3.5.2.6) was studied. Benzylpenicillin and ampicillin (6-[D(-)-alpha-aminophenylacetamido]penicillanic acid) were used. Both kcat. and kcat./Km for both substrates gave bell-shaped plots of parameter versus pH. The pH-dependence of kcat./Km for the two substrates gave the same value (8.6) for the higher apparent pK, and so this value may characterize a group on the free enzyme; the lower apparent pK values were about 5(4.85 for benzylpenicillin, 5.4 for ampicillin). For benzylpenicillin both kcat. and kcat./Km depended on pH in exactly the same way. The value of Km for benzylpenicillin was thus independent of pH, suggesting that ionization of the enzyme's catalytically important groups does not affect binding of this substrate. The pH-dependence of kcat. for ampicillin differed, however, presumably because of the polar group in the side chain. The hypothesis that the pK5 group is a carboxyl group was tested. Three reagents that normally react preferentially with carboxyl groups inactivated the enzyme: the reagents were Woodward's reagent K, a water-soluble carbodi-imide, and triethyloxonium fluoroborate. These findings tend to support the idea that a carboxylate group plays a part in the action of beta-lactamase I.


Oxidase-peroxidase enzymes of Datura innoxia. Oxidation of formylphenylacetic acid ethyl ester. An enzyme system from Datura innoxia roots oxidizing formylphenylacetic acid ethyl ester was purified 38-fold by conventional methods such as (NH4)2SO4 fractionation, negative adsorption on alumina Cy gel and chromatography on DEAE-cellulose. The purified enzyme was shown to catalyse the stoicheiometric oxidation of formylphenylacetic acid ethyl ester to benzoylformic acid ethyl ester and formic acid, utilizing molecular O2. Substrate analogues such as phenylacetaldehyde and phenylpyruvate were oxidized at a very low rate, and formylphenylacetonitrile was an inhilating agents, cyanide, thiol compounds and ascorbic acid. This enzyme was identical with an oxidase-peroxidase isoenzyme. Another oxidase-peroxidase isoenzyme which separated on DEAE-chromatography also showed formylphenylacetic acid ethyl ester oxidase activity, albeit to a lesser extent. The properties of the two isoenzymes of the oxidase were compared and shown to differ in their oxidation and peroxidation properties. The oxidation of formylphenylacetic acid ethyl ester was also catalysed by horseradish peroxidase. The Datura isoenzymes exhibited typical haemoprotein spectra. The oxidation of formylphenylacetic acid ethyl ester was different from other peroxidase-catalysed reactions in not being activated by either Mn2+ or monophenols. The oxidation was inhibited by several mono- and poly-phenols and by catalase. A reaction mechanism for the oxidation is proposed.


Purification of 2-oxoaldehyde dehydrogenase and its dependence on unusual amines. 1. 2-Oxoaldehyde dehydrogenase was purified from sheep liver and gave one band on polyacrylamide-gel electrophoresis. 2. The enzyme was completely dependent for its activity on the presence of Tris or one of a number of related amines, all of general structure: (See article). When more than one R group was hydrogen no enzyme activity was observed. 3. Only one of these amines is known to exist in living tissues and large concentrations of all amines were required for maximum activity. L-2-Aminopropan-1-ol was the most effective amine on the basis of substrate Km and Vmax. values and the amine Km values. 4. The enzyme was activated by phosphate which lowered the Km values for methylglyoxal, amine and NAD+. 5. The pH optimum of the enzyme was 9.3 and there was no activity at pH values below 7.8. A search for activators that might produce activity at pH 7.4 proved unsuccessful. 6. The enzyme was inhibited by rather large concentrations of barbiturates (6-46 mM) and nitro-alcohol analogues of the activating amines (66-139 mM).


Determination of polyadenylate-rich ribonucleic acid in the nucleus and in the cytoplasm of plasmacytoma cells. A number of parameters affecting the adsorption of rRNA and poly(A)-containing RNA to Millipore filters were investigated separately. Binding of both types of RNA to the filter was dependent on the concentration of RNA, pH and Mg2+ concentration of the reaction mixture. Both types of RNA bound to the filter optimally at slightly acid pH values. The binding of poly(A)-containing RNA to the filter exhibited a broad pH-dependence compared with that of rRNA. The ratio of poly(A)-rich RNA/rRNA retained by the filter was maximal between pH7 and 8. The presence of 1 mM-EDTA or a high concentration of NaCl (over 0.5M) decreased the affinity of RNA for the filter. The amount of poly(A)-containing RNA in the nucleus and in the cytoplasm of a plasmacytoma cell line (MPC-11) labelled with [32P]Pi was determined by the Millipore-filter technique under conditions that minimized contamination by rRNA. These data were compared with the estimations made by oligo(dT)-cellulose chromatography. The results obtained by these two methods were in good agreement for RNA labelled for short periods (up to 2h). In long labelling and pulse-chase experiments, however, contamination of the filter by rRNA of increasing specific radioactivity in the cytoplasm gave an erroneous value for poly(A)-containing RNA by the Millipore-filter technique. Determinations made on the nuclear fraction by these two methods did not show significant variation in short- and long-term labelling experiments.


The amino acid sequence of Neurospora NADP-specific glutamate dehydrogenase. The tryptic peptides. The NADP-specific glutamate dehydrogenase of Neurospora crassa was digested with trypsin, and peptides accounting for 441 out of the 452 residues of the polypeptide chain were isolated and substantially sequenced. Additional experimental detail has been deposited as Supplementary Publication SUP 50052 (11 pages) with the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies may be obtained under the terms given in Biochem J. (1975) 145, 5.


The amino acid sequence of Neurospora NADP-specific glutamate dehydrogenase. Peptides from digestion with a staphylococcal proteinase. The extracellular proteinase of Staphylococcus aureus strain V8 was used to digest the NADP-specific glutamate dehydrogenase of Neurospora crassa. Of 35 non-overlapping peptides expected from the glutamate content of the polypeptide chain, 29 were isolated and substantially sequenced. The sequences obtained were valuable in providing overlaps for the alignment of about two-thirds of the sequences found in tryptic peptides [Wootton, J. C., Taylor, J, G., Jackson, A. A., Chambers, G. K. & Fincham, J. R. S. (1975) Biochem. J. 149, 739-748]. The blocked N-terminal peptide of the protein was isolated. This peptide was sequenced by mass spectrometry, and found to have N-terminal N-acetylserine by Howard R. Morris and Anne Dell, whose results are presented as an Appendix to the main paper. The staphylococcal proteinase showed very high specificity for glutamyl bonds in the NH4HCO3 buffer used. Partial splits of two aspartyl bonds, both Asp-Ile, were probably attributable to the proteinase. No cleavage of glutaminyl or S-carboxymethylcysteinyl bonds was found. Additional experimental detail has been deposited as Supplementary Publication SUP 50053 (5 pages) with the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K, from whom copies may be obtained under the terms given in Biochem. J. (1975) 1458 5.


The amino acid sequence of Neurospora NADP-specific glutamate dehydrogenase. Peptic and chymotryptic peptides and the complete sequence. Peptic and chymotryptic peptides were isolated form the NADP-specific glutamate dehydrogenase of Neurospora crassa and substantially sequenced. Out of 452 residues in the polypeptide chain, 265 were recovered in the peptic and 427 in the chymotryptic peptides. Together with the tryptic peptides [Wootton, J. C., Taylor, J. G., Jackson, A. A., Chambers, G. K. & Fincham, J. R. S. (1975) Biochem. J. 149, 749-755], these establish the complete sequence of the chain, including the acid and amide assignments, except for seven places where overlaps are inadequate. These remaining alignments are deduced from information on the CNBr fragments obtained in another laboratory [Blumenthal, K. M., Moon, K. & Smith, E. L. (1975), J. Biol. Chem. 250, 3644-3654]. Further information has been deposited as Supplementary Publication SUP 50054 (17 pages) with the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies may be obtained under the terms given in Biochem. J. (1975) 145, 5.


Activities of citrate synthase, NAD+-linked and NADP+-linked isocitrate dehydrogenases, glutamate dehydrogenase, aspartate aminotransferase and alanine aminotransferase in nervous tissues from vertebrates and invertebrates. 1. The activities of citrate synthase and NAD+-linked and NADP+-linked isocitrate dehydrogenases were measured in nervous tissue from different animals in an attempt to provide more information about the citric acid cycle in this tissue. In higher animals the activities of citrate synthase are greater than the sum of activities of the isocitrate dehydrogenases, whereas they are similar in nervous tissues from the lower animals. This suggests that in higher animals the isocitrate dehydrogenase reaction is far-removed from equilibrium. If it is assumed that isocitrate dehydrogenase activities provide an indication of the maximum flux through the citric acid cycle, the maximum glycolytic capacity in nervous tissue is considerably greater than that of the cycle. This suggest that glycolysis can provide energy in excess of the aerobic capacity of the tissue. 2. The activities of glutamate dehydrogenase are high in most nervous tissues and the activities of aspartate aminotransferase are high in all nervous tissue investigated. However, the activities of alanine aminotransferase are low in all tissues except the ganglia of the waterbug and cockroach. In these insect tissues, anaerobic glycolysis may result in the formation of alanine rather than lactate.


Properties of 5-aminolaevulinate synthetase and its relationship to microsomal mixed-function oxidation in the southern armyworm (Spodoptera eridania). 1. Activity of 5-aminolaevulinate synthetase was measured in the midgut and other tissues of the last larval instar of the southern armyworm (Spodoptera eridania Cramer, formerly Prodenia eridania Cramer). 2. Optimum conditions for measuring the activity were established with respect to all variables involved and considerable differences from those reported for mammalian enzyme preparations were found. 3. Maximum activity (20 nmol/h per mg of protein) occurs 18-24 h after the fifth moult and thereafter decreases to trace amounts as the larvae age and approach pupation. 4. Synthetase activity was rapidly induced by oral administration (in the diet) of pentamethylbenzene, phenobarbital, diethyl 1,4-dihydro-2,4,6-trimethylpyridine-3, 5-dicarboxylate, and 2-allyl-2-isopropylacetamide. 5. Puromycin inhibited the induction of synthetase by pentamethylbenzene. 6. Induction of 5-aminolaevulinate synthetase correlated well with the induction of microsomal N-demethylation of p-chloro-N-methylaniline, except for phenobarbital, which induced the microsomal oxidase relatively more than the synthetase.


Nitrosation of phenacetin. Formation of N-nitroso-2-nitro-4-ethoxyacetanilide as an unstable product of the nitrosation in dilute aqueous-acidic solution. Reaction of phenacetin with N2O4 in glacial acetic acid at 10(0) C gives N-nitroso-2-nitro-4-ethoxyacetanilide. This N-nitrosoacylarylamine is stable at low temperatures (--30 degress C) but unstable at ambient temperature. No intact N-nitroso-2-nitro-4-ethoxyacetanilide can be detected when phenacetin is nitrosated under conditions simulating those in the stomach (37 degrees C, pH 1). Instead, 2-nitro-4-ethoxybenzenediazonium chloride is the main reaction product found. Under the conditions applied, the N-nitrosoacylarylamine rapidly rearranges by 1,3-migration of the acetyl group. The resulting diazoester dissociates into the corresponding diazonium salt. Trapping of the diazonium ion with 1-naphthol as an azo dye provides a useful means to identify the parent N-nitroso compound and to measure colorimetrically its rate of formation. The yields obtained in dilute aqueous nitrosation mixtures are lower than expected; the reasons for this finding are discussed. Preliminary results of animal experiments show that the N-nitroso compound is a directly acting carcinogen.


[Thermographic and histologic studies of the antiinflammatory effect of benorilate by means of the cotton pellet test in the rat (author's transl)]. 1. The effect of (4-acetamido-phenyl)-2-acetoxy-benzoate (benorilate, Benortan) on the inflammatory process was studied thermographically and histologically in cotton-pellet tests on rats. 2. Following implantation of the cotton-pellet, thermography shows a clear inhibition of the local inflammation due to treatment with benorilate. 3. Histological examination shows a corresponding influence of benorilate upon the proliferative phase of the inflammation. 4. The success of antiphlogistic therapy is in correlation with the time of medication.


Acidic antiinflammatory agents--correlations of some physical, pharmacological and clinical data. Fifteen acidic antiinflammatory agents, for which some clinical data have previously been published, have been examined for their potency in the carrageenan-induced rat foot edema test, and for their acidity (pKa) and partition coefficients. Published serum half-life data and daily clinical (anti-arthritic) dose have been tabulated for these drugs and correlations between these various parameters are discussed. The rat foot edema carrageenan test has proved to be a fairly reliable predictor of clinical dose for most acidic antiinflammatory agents of moderate serum half-life.


[Clinical trial of a novel benzodiazepine derivative in a double-blind study using the Wittenborn psychiatric rating scale (author's transl)]. From testing a new benzodiazepine derivative, 8-chloro-1-phenyl-2,3,4,5-tetrahydro-1H-1,5-benzodiazepin-2-one (Bu 1014), as measured against a placebo in a double-blind trial, the following conclusions can be drawn. The test was carried out over two periods of a fortnight each with a change-over between the two periods. 1. The change-over method has proven suitable to reveal side effects of the substance which last for at least two weeks. Owing to the substance's sequelae, however, statistical analysis of the second treatment period's information is not possible with this experimental design. 2. The statistical methods used proved more effective than the usual methods as they allow clearer statements to be made on the efficacy of the substance. 3. Within the first period of 14 days both the group receiving the placebo and the drug treated group showed a decrease in the intensity of anxiety. 4. The sequelae of Bu 1014 can be described as an increase in restiveness and anxiety in those patients who received the placebo in the second treatment period.


Flavoxate and 3-methylflavone-8-carboxylic acid. Assay methods in blood and urine, plasma-red cells repartition and stability. The following assay methods for pharmacokinetic studies on flavoxate (F) and on its main metabolite, i.e. 3-methylflavone-8-carboxylic acid (A), are described. 1. Spectrophotometry for the assay of F and of A in plasma, 2. TLC-Spectrodensitometry and GLC for the assay of A in urine after acid hydrolysis, 3. TLC-Spectrodensitometry for determining the F : A ratio in plasma or in urine. It was found that F hydrolyzes into A. This process depends on the pH and on the medium. In water, at pH 5.0, F is stable, while in phosphate buffer at pH 7.4 the semi-hydrolysis time is 60 min. In a solution with bovine serum albumin, in rat, rabbit, dog or human plasma the semi-hydrolysis times are between 5 and 60 min. Finally the plasma-red cells repartitions of F and of A were studied in vitro in rat, rabbit, dog and human blood and found between 0.8 and 2.0 for F and between 2.1 and 4.6 for A.


[On the pharmacology of 9,10-dihydro-10-(1-methyl-4-piperidylidene)-9-anthrol (WA 335), a histamine and serotonin antagonist (author's transl)]. The substance 9,10-dihydro-10-(1-methyl-4-piperidylidene)-9-anthrol (WA 335) was examined for its antagonistic effects against histamine and serotonin, for its atropine-like properties as well as for a series of other qualities in comparison with cyproheptadine and pimethixene. The anti-histamine and anti-serotonin activities of compound WA 335 on the smooth muscle and the capillary do not only exceed that of cyproheptadine but also that of pimethixene. WA 335 shows an extremely strong binding to histamine and serotonin receptors. In the dose range in which it causes already an antamine effect, its oral absorption is very good. The anti-anaphylactic effect is much stronger than that of cyproheptadine. Like pimethixene and cyproheptadine, WA 335 has no distinct antagonistic qualities against bradykinin. The anticholinergic effects of WA 335 are dependent on the test object. In examinations on the bronchus of the guinea pig and the pupil of the mouse, the atropine-like efficiency corresponds to that of cyproheptadine; it is stronger on the stimulated vagus of the cat and less efficient than cyproheptadine on the stomach of the rat. WA 335 has distinct central atropine-like properties. It possesses a strong surface anesthetic activity. The effects of WA 335 on circulation are dependent on species. In contrast to pimethixene, compound WA 335 like cyproheptadine potentiates the effects of norepinephrine in cats. The reduction of the carotid sinus reflex in the cat is more distinct after WA 335 than after pimethixene and corresponds to that produced by cyproheptadine. Higher doses of WA 335 than are necessary to demonstrate antaminic effects are needed to provoke central nervous effects. WA 335 shows no analgesic potency in mice. The influence on body temperature in the rat is similar to that of cyproheptadine. WA 335 is equally efficient as pimethixene with regard to the inhibition of spontaneous motility and prolongation of barbiturate sleep in mice, and shows the same anti-emetic activity as does chlorpromazine in dogs. In contrast to chlorpromazine the behaviour of dogs and cats is distinctly altered already by doses of WA 335 which cause a slight sedation.


[Central action of WA-335-BS, a substance with peripheral antiserotonin and antihistaminic activity]. In rats and mice the serotonin and histamine antagonistic drug 9,10-dihydro-10-(1-methyl-4-piperidylidene)-9-anthrol (WA 335-BS) caused stronger central sedative effects than did cyproheptadine. WA 335-BS also displayed stronger activity against reserpine- and central tremorine-induced effects than did cyproheptadine and it slightly enhanced d-amphetamine-induced effects: therefore it may have antidepressant properties. WA 335-BS proved to be very effective against isolation-induced aggression in male mice. The comparatively small anxiolytic effects may have been caused in part by the central antiserotonin properties. Like cyproheptadine, WA 335-BS increased food consumption in cats. In EEG-experiments in the conscious rabbit the serotonin-antagonistic drugs WA 335-BS and cyproheptadine exerted stronger depressant activity on the arousal reactions than did the neuroleptic chlorpromazine. The results of our animal studies suggest WA 335-BS to be an antidepressant with sedative properties.


The distribution and elimination of radioactivity in the rat after administration of 14C-4-acetamidophenyl-2-acetoxybenzoate (benorylate). Following oral administration of 4-acetamido-phenyl-2-acetoxybenzoate (carboxyl-14C) (benorylate) to rats, no gross differences were detected 7.5 h after administration with respect to the distribution of 14C in various tissues, including the upper sections of the small intestine. A high concentration of 14C was found in the lower sections of the intestine 4 h after administration. The 14C in the intestine was present as unchanged benorylate, as detected by thin-layer chromatography, suggesting that benorylate absorption was slow. Intravenous injection of 14C-benorylate to rats showed that the drug had a relatively high elimination rate from the blood with a half-life of 1.9 h. In blood benorylate must be rapidly hydrolysed enzymatically since no 14C-metabolites, other than salicylic acid, could be detected.


Comparative studies of parasympatholytic drugs on stomach (double blind test) and analysis of relation between gastric form and tonus. Effects of 3 kinds of parasympatholytic drugs (timepidiumbromide, hyoscine-N-butylbromide and prifinium-bromide) and placebo (physiological saline solution) on the gastrointestinal tract were evaluated roentgenographically by double blind technique in a total of 101 male human subjects. The results may briefly be summarized as follows: 1. There were significant differences on hypotonic rate being one of the indexes of the gastric tonus between timepidium-bromide and placebo. The effects of 3 experimental drugs were significantly high as compared with that of placebo on peristaltic movement of the stomach. The effect of timepidium-bromide was significantly different from that of placebo on the site of arrival of barium. 2. The comparison of degrees of the gastric tonus between main and control tests revealed that the effect of placebo obtained in the main test was significantly inferior to that of hyoscine-N-butylbromide obtained in the control test, whereas there existed no significant differences of the effects among 3 active drugs. 3. Effects of each drug on the gastric tonus which was scored by hypertonic, normotonic and hypotonic state were evaluated by stratification. The result showed that the effect of timepidium-bromide was significantly greater than that of placebo. 4. All active drugs were not significantly different in terms of 7 observed values each on the gastric form.


[Treatment of exocrine pancreatic insufficiency with fungal lipase (author's transl)]. 7 patients suffering from severe exocrine pancreatic insufficiency have been treated with a lipolytic enzyme extracted from Rhizopus arrhizus. Comparing the fungal lipase with a placebo the drug lowered the daily stool weight from 809 g to 443 g on an average, i.e. by 45.2%. The steatorrhea was reduced from 75.6 g/24 h to 32.9 g, i.e. by 56.5%. When incubating the enzyme in vitro in saline solutions of different pH for 1 h the loss of lipolytic activity is 13% at pH 5, 15% at pH 4, and 19% at pH 3. So the enzyme, like fungal proteases, is almost not altered by hydrochloric acid at concentrations found physiologically in the stomach.


Study of the sporulation of Bacillus thuringiensis var. thuringiensis. During the submerged cultivation of Bacillus thuringiensis var. thuringiensis in 300- and 3000-liter fermentors, lysis occurred at the end of the exponential phase of growth. New vegetative cells were subsequently formed which usually sporulated. At time of lysis, the amount of soluble sugar was 1-12 g/liter, pH value dropped to 5.3-5.8 from the original PH 6.8 and started to rise after all the cells had lysed. The proteolytic activity was low during the lysis and increased as the sporulation commenced.


Some observations on the effects produced in white mice following the injection of certain suspensions of corroding bacilli. Strictly anaerobic and facultatively anaerobic strains of "corroding bacilli" failed to produce any pathological symptoms when injected into white mice and no viable organisms could be recovered after 7 days. However, when these same strains were coupled with certain other living bacteria or certain sterile bacterial extracts, lesions developed from which corroding bacilli could be isolated even after 21 days.


Lysosomal hydrolases of the epidermis. 3. Peptide hydrolases. Four distinct peptide hydrolases (EC 3-4) have been characterized in guinea-pig epidermis; these are cathepsin B1, cathepsin C, cathepsin D and arylamidase. Their properties are consistent with those of lysosomal enzymes. Cathepsin E was not detected.


Micellar solubilization of fatty acids in aqueous media containing bile salts and phospholipids. 1. The solubility of fatty acids in aqueous media containing bile salts alone and in admixture with either lecithin (phosphatidylcholine) or phosphatidylethanolamine was determined. 2. Over the pH range 2-0-7-4, the order of fatty acid solubility in aqueous solutions containing bile salts was linoleic greater than oleic greater than elaidic greater than palmitic greater than stearic. The solubility of each fatty acid increased as the pH of the miceus solutions of bile salts greatly increased the solubility of palmitic acid and stearic acid. 4. In the presence of bile salts and lecithin, the solubility of oleic acid and elaidic acid decreased with increasing pH of the micellar solution, indicating a competitive effect between the fatty acid anions and lecithin. The solubility of linoleic acid increased linearly with lecithin concentration. 5. Phosphatidylethanolamine as an additive to bile salts increased the solubility of both saturated and unsaturated fatty acids in the pH range 2-3-7-4. The effectiveness of phosphatidylethanolamine as an amphiphile was similar to that of lecithin, although at pH 3.0 fatty acid solubility was greater in the presence of phosphatidylethanolamine. 6. The significance of these findings is discussed in relation to the intestinal absorption of fatty acids in sheep.


Influence of the products of phospholipolysis of phosphatidylcholine on micellar solubilization of fatty acids in the presence of bile salts. 1. The solubility of fatty acids in aqueous solutions containing bile salts and lysolecithin at pH values between 2-0 and 7-4 was studied. Both the 1-acyl and 2-acyl isomers of lysolecithin increased the solubility of fatty acids to the same extent, the order of solubility being linoleic greater than oleic greater than elaidic greater than palmitic greater than stearic. 2. The influence of the products of phospholipolysis of lecithin on palmitic acid solubility was determined. On a molar basis, lysolecithin was more effective than were bile salts in promoting the solubilization of the fatty acid. 3. In bile salt solutions in which the phospholipid concentration was constant on a molar basis, in solubility of palmitic acid decreased linearly with the progressive replacement of lecithin by lysolecithin. Palmitic acid was solubilized to the same extent on replacing lecithin with lysolecithin on a constant weight basis. 4. In bile salt solution containing lysolecithin and oleic acid in equimolar amounts, the solubility of palmitic acid was similar to that in bile salt solution containing lecithin in equivalent proportion. 5. The results are discussed in relation to the action of phospholipolytic activity on the intestinal absorption of fatty acids in sheep.


The purification and characterization of the low molecular weight human folate binding protein using affinity chromatography. The low molecular weight folate binding protein (FABP) has been purified 1000-fold to a specific activity of 7.2 gamma g of pteroylglutamic acid (PGA) bound per mg of protein. This purified FABP represents two protein bands that bind PGA on polyacrylamide disc gel electrophoreis, elutes from DEAE-cellulose in 0.001 M phosphate buffer, stains positive with PAS, Elutes from concanavalin A Sepharose affinity columns with methyl alpha-mannoside, and shows three major peaks (pl =6.8, 7.5, 8.2) by isotric focusing. The binding of PGA to purified FABP dependent on pH and is inhibited by urea...


Studies on IgA and IgA monoclonal proteins derived from a single patient. Evidence for identical light chains and variable regions of the heavy chain. Two immunoglobulins, IgA(K) and IgG(K), were isolated from the serum of a single patient with two monoclonal components (biclonal proteins). After chain separation, the light chains from each molecule were found to be identical by the following criteria: electrophoretic mobilities under various pH and dissociating conditions, amino acid compositon, fingerprint analysis of tryptic peptides and of 14C-succinylated chymotryptic peptides, and amino acid sequence of the N-terminal 40 residues. The heavy chains were indistinguishable for the N-terminal 45 amino acid residues. These data are consistent with the hypothesis that a single heavy chain variable (VH) region may be associated with two different heavy chain constant (CH) genes.


Conformational properties of bovine plasma albumin with a cleaved internal peptide bond. As shown previously, proteinases frequently associated with plasma albumin samples catalyze a very limited and specific cleavage of the albumin molecule when it exists in the F conformational state near pH 3.7. The primary proteolytic product, BPA, has a molecular weight similar to or identical with that of the parent protein but yields two large fragments of molecular weight approximately 46000 and 23000 on reduction. Evidence is presented here that cleavage occurs within the disulfide loop between Cys390 and Cys434 with no detectable loss of small peptides, the amino acid composition of BPA being identical with that of the parent protein within experimental error. Cleavage exposes a new amino-terminal phenylalanine residue and may occur at the Glx392-Phe393 bond although the possibility exists that it occurs at another X-Phe bond in the unsequenced region of residues 400-402. The damaged protein has a somewhat altered secondary structure as judged from optical rotatory dispersion and circular dichroism measurements, probably an approximate 15% loss in helicity. The hydrodynamic volume is increased by approximately 20%. However, various physical studies indicate the tertiary structure to be strikingly similar to that of the native protein. Of most significance is the fact that the protein still undergoes the N-F and N-B transitions, although in both cases they occur at somewhat more moderate pH than in the parent protein. Moreover a sensitivity of the N-B transition to Ca2+ is still seen and binding behavior toward the dye 8-anilino-1-naphthalenesulfonic acid is essentially unaltered. The results are best understood in terms of the concept of a multidomain structure which has been suggested frequently for plasma ablumin. Bond cleavage damages one domain but leaves the overall structure essentially unaltered except for some weakening of the interaction between domains.


Detection and partial characterization of lipoprotein lipase in bovine aorta. Extracts of acetone-ether powders of bovine thoracic aorta contain lipase activity which has an alkaline pH maximum (7.8-8.4) and is stimulated 4-10-fold by adding serum or isolated apolipoprotein-glutamate to the assay mixture. Serum activation is completely reversed by isolated apolipoprotein-serine or apolipoprotein-alanine. Lipolysis is strongly inhibited by NaCl (0.5 M) and protamine sulfate (1 mg/ml) and partially inhibited by heparin. Based on these characteristics, the lipase is identified as lipoprotein lipase.


Purine nucleotide pyrophosphotransferase from Streptomyces morookaensis, capable of synthesizing pppApp and pppGpp. Purine nucleotide pyrophosphotransferase was purified to apparent homogeneity from a culture filtrate of Streptomyces morookaensis. It is a monomeric protein with a molecular weight of 24 000-25 000, and its isoelectric point is 6.9. The enzyme synthesizes purine nucleoside 5'-phosphate (mono, di, or tri) 3'-diphosphates such as pppApp, ppApp, pApp, pppGpp, ppGpp and pppIpp by transferring a pyrophosphoryl group from the 5'-position of ATP, dATP and ppApp to the 3'-position of purine nucleotides. The purified enzyme catalysed the formation of 435 mumol of pppApp and 620 mumol of pppGpp from ATP and GTP per min mg protein under the standard conditions. The enzyme requires absolutely a divalent cation for activity, and optimum pH for the enzyme activity lay above 10 for Mg2+, for Co2+ and Zn2+ from 9 to 9.5, and for Fe2+ from 7.5 to 8. The following Michaelis constants were determined: AMP, 2.78 mM; ADP, 3.23 mM; GMP, 0.89 mM; GDP, 0.46 mM and GTP, 1.54 mM, in the case of ATP donor. The enzyme is inhibited by guanine, guanosine, dGDP, dGTP, N-bromosuccinimide, iodacetate, sodium borate and mercuric acetate.


Protein kinase in cultured plant cells.

Protein kinase in cultured plant cells. A protein kinase (EC 2.7.1.37) which phosphorylates histones was purified partially from the soluble fractions of cultured plant cells. The optimum pH was 7.5 to 9.0. The activity wasnot stimulated by exogeneous cyclic AMP. It was thermolabile and completely dependent on the presence of Mg2+ or Mn2+ for activity. p-Chloromercuribenzoate inactivated this enzyme and this inactivation was overcome by mercaptoethanol.


Adenosine triphosphate: nicotinamide mononucleotide adenylyltransferase of pig liver. Purification and properties. Adenosine triphosphate : nicotinamide mononucleotide adenylyltransferase (EC 2.7.7.1) has been purifiec approximately 3500-fold from an extract of pig liver nuclei to a specific activity of 40 mumol of NAD+ per min per mg protein. The enzyme was found to have a molecular weight of 203 000, a frictional ratio of 1.6 and an isoelectric point of approximately 5. Michaelis constants for ATP and NMN were 0.11 mM and 0.12 mM, respectively.


Phospholipase A2 isoenzyme from porcine pancreas. Purification and some properties. Porcine pancreas synthesizes a prephospholipase A2 which occurs in a 5 : 95 ratio compared with the more abundant zymogen of the same enzyme (phosphatide-acylhydrolase; EC 3.1.1.4). These two prephospholipases could be well separated by CM-cellulose chromatography. Both the active and the zymogen form of the isoenzyme were isolated and purified. The activation peptides of both prephospholipases appeared to be identical, while the active enzymes showed a few interesting differences. The most striking differences were the loss of one histidine and one methionine in the isoenzyme, corresponding to residues 24 and 27, respectively, in alpha-phospholipase A2. The positional and stereo specificity of both enzymes are the same, but the specific activity of the beta-phospholipase A2 is lower. The molecular weight of the isoenzyme was estimated to be about 14 000, while the isoelectric points were 5.1 and 5.9 for the isoprecursor and active isoenzyme, respectively.


Cylic nucleotide phosphodiesterase in silkworm. Characterization of cyclic GMP phosphodiesterase. The existence of cyclic GMP phosphodiesterase (EC 3.1.4.-) was demonstrated in silkworm larva by gel filtration of the homogenate. The cyclic GMP phosphodiesterase was separated from cyclic AMP phosphodiesterases by column chromatography on hydroxyapatite and Sephadex G-200. The enzyme has a molecular weight of approx. 260 000, and optimum pH of 8.3 and a Km value of 2 muM. The enzyme is activated by 5 mM of Mg2+ and 2 mM of Mn2+. The cyclic GMP phosphodiesterase activity was greatly inhibited by low concentrations of cyclic IMP but to a lesser extent by cyclic AMP even at a high concentration. The activity was also inhibited by caffeine and theophylline.


Glucanases in Schizosaccharomyces. Isolation and properties of an exo-beta-glucanase from the cell extracts and culture fluid of Schizosaccharomyces japonicus var. versatilis. (11 Cell extracts and extracellular culture fluids of species of the yeast genus Schizosaccharomyces exhibited exo-beta-(1 leads to 3)- and exo-beta-(1 leads to 6)-glucanase (EC 3.2.1.-) activities. (2) Using a combination of Sephadex G-100 and DEAE-cellulose chromatography, the exo-beta-(1 leads to 3)-glucanases from the cell extracts and culture fluid of Schizosaccharomyces japonicus var. versatilis were purified extensively. The enzymes from either location exhibited similar purification and other properties. (3) The purified enzymes hydrolysed the beta-(1 leads to 6)-glucosidic linkage in addition to the beta-(1 leads to 3) linkage. Heat denaturation, inhibition and electrophoretic studies indicated that both hydrolytic activities were properties of a single protein. Laminarin and pustulan hydrolysis followed Michaelis-Menten kinetics. The Km and V for laminarin hydrolysis were 6.25 mg/ml and 350 mumol of glucose released/min/mg protein, and for pustulan they were 166 mg/ml and 52 mumol of glucose released/min/mg protein. (4) The exo-beta-glucanase was assigned a molecular weight of 43 000. (5) the purified enzyme failed to hydrolyse isolated cell walls from either baker's yeast or Schizosaccharomyces pombe or to induce protoplast formation from intact cells of S. japonicus var. versatilis or Saccharomyces cerevisiae.


Purification and some properties of a novel maltohexaose-producing exo-amylase from Aerobacter aerogenes. Maltohexaose producing amylase (EC 3.2.1.-) is the fourth known exo-amylase, the three previously known being glucoamylase, beta-amylase and Pseudomonas stutzeri maltotetraose producing amylase. The enzyme after release from Aerobacter aerogenes cells by 0.1% sodium lauryl sulfate extraction was purified by ammonium sulfate precipitation, DEAE-Sephadex column chromatography and Sephadex G-100 gel filtration to 80-fold of the original sodium lauryl sulfate extract activity, It gave a single band on disc electrophoresis, and the molecular weight by gel filtration was 54 000. This amylase showed maximal activity at 50 degrees C and pH 6.80. The pH stability range was relatively wide, the enzyme retaining more than 90% of its initial activity in the range of 6.50-9.0. 80% of the activity was retained after 15 min at 50 degrees C. This enzyme produced maltohexaose from starch, amylose and amylopectin by exo-attack, but did not act on alpha- or beta-cyclodextrin, pullulan or maltohexaitol. Also the enzyme acted on beta-limit dextrins of amylopectin and glycogen to form branched oligosaccharides. The unusual reaction of this enzyme on beta-limit dextrin is discussed from the standpoint of the stereochemistry of 1,4-alpha- and 1,6-alpha-glucosidic bonds. This is the anomalous amylase for which it is recognized that 1,6-alpha-glucosidic linkages in the substrates can mimic the effect of 1,4-alpha-bonds, as previously observed in pseudo-priming reactions of E. coli phosphorylase.