[Effects of nucleophils on kinetic parameters of peroxidase-catalyzed oxidative reactions]. The effect of imidazole on the kinetic parameters of reactions of potassium ferrocyanide and o-dianizidine peroxidation by hydrogen peroxide within a wide range of pH was studied. It was shown that imidazole activates the reaction of o-dianizidine peroxidation at pH greater than or equal to 6.5, but does not affect the oxidation of potassium ferrocyanide. It was also found that imidazole causes a similar increase in the kkat and Km, i. e. non-competitive activation occurs. The data obtained suggest a possible mechanism of the activator effect. Differences in the mechanism of interaction of various substrates uith peroxidase are discussed.


[Dependence of thrombin- and trypsin-catalyzed hydrolysis of N-alpha-arylsulfonyl-L-arginine methyl esters on the structure of acylamide part of substrates]. For comparative studies on the esterase activities of thrombin and trypsin N(alpha)-arylsulfonyl-L-arginine methyl esters were synthetised containing in aromatic ring substituents of different polar nature, size and hydrophobicity. The kinetics of their hydrolysis by thrombin and trypsin were measured. Values of Km and kcat in steady-state conditions were determined. It was shown, that thrombin-catalysed hydrolysis was more sensitive than that of trypsin to the nature of substituents of arylsulfonyl group and determined by their polar and steric effects. A line correlation between specificity constants (kcat/Km) and sigma and Es of substituents were demonstrated. The difference in reactivity of compounds under investigation is suggested to depend on alterations of stability of hydrogen bond between arylsulfonylamide nitrogen atom of substrate and the active center of the enzyme due to changes in the acidity of the arylsulfonylamide group affected by substituent of the benzene ring.


[Characterization of dextranase from Penicillium purpurogenum (Ftoll)]. An extracellular dextranase (E. C. 3.2.1.11) was purified from cell-free culture filtrates of Penicillium purpurogenum (Ftoll). The enzyme was most active at pH 5,5. The dextranase was endo-type, it split quickly isomaltotetraose into two isomaltose molecules, slowly degraded isomaltotriose, and did not act on isomaltose. The rate of isomaltooligosaccharides hydrolysis was increased with the increase of the polymerization degree. Polyols obtained from isomaltooligosaccharides were split more slowly than the respective sugars. The isomaltopentaitol was split at two glucosidic linkages, 38% of hydrolyzed linkages being the second linkage from the sorbitol end of the molecule and 62% being the third one. The degree of degradation of dextrans depended on amount of 1,6 linkages. Isomaltose and tetrasaccharides of two types, 2(2)-alpha-D-glucosylmaltotriose and linear tetrasaccharide(s), are the lowest molecular weight products of exhaustive hydrolysis of branched dextrans.


[Effect of environment on allosteric properties of acetylcholinesterase]. In the presence of organophosphorus inhibitors (OPI) AChE inhibition is initiated at a lower concentration of ACh; the plot reaction rate versus substrate concentration shows two maxima with a distinct minimum between them. It was shown that extremely mild conditions (short-term heating up to 50 degrees C; acidic or alkaline pH shift by 0.5 units; high concentrations of bivalent cations; erythrocyte storage) which do not affect substrate inhibition, remove this effect. The data obtained suggest that OPI effect is not directed to the site of AChE responsible for enzyme inhibition by ACh excess ("substrate inhibition site"), but to some other area. This results in a change in the conformation of the substrate inhibition site and a pronounced inhibition of the AChE activity takes place at lower substrate concentration.


[Proteins of Bacillus thuringiensis delta-endotoxin crystals]. Pure crystals (at least 99% purification) of sigma-endotoxin were isolated from Bac. thuringiensis var. galleriae. The complete dissolution of crystals might be achieved by the increase of pH up to 12 and higher or by a combined action of S = S-reducing and denaturing agents. Electrophoresis of the solubilized crystal proteins in 5% polyacrylamide gels containing 0,1% sodium dodecyl sulfate and 8 M urea reveals two major bands corresponding to molecular weights of 120000--140000 (65%) and 65000 (8-10%), and some minor components whose molecular weights varied from 65000 to 340000. Urea (3--8 M) causes to partial dissolution of the crystals; the component with molecular weight of 65000 is mainly found in the solution (component A). In dithioerythritol extracts at pH 9 the major component of the crystal is the protein with molecular weight 120000--140000 (component B). The crystals, alkali-soluble components and proteins isolated from crystals by selective extraction (3--8 M urea or 0.01 M dithioerythrytol, pH 9) were found toxic for the larvae of Galleria mellonella.


The gas chromatography mass spectrometry of the major metabolites of flurazepam. Mass spectra and gas chromatographic data are presented for flurazepam and its metabolites; monodesethylflurazepam, didesethylflurazepam, hydroxyethylflurazepam, N1-desalkylflurazepam, N1-desalkyl-3-hydroxy-flurazepam, and flurazepam-N1-acetic acid. The on-column thermal degradation of didesethylflurazepam, N1-desalkyl-3-hydroxyflurazepam and flurazepam-N1-acetic acid is reported and discussed. Mass spectrometric and gas chromatographic data are also presented for the benzophenones obtained by acid hydrolysis of flurazepam and its metabolites. The occurrence of flurazepam metabolites in urine from five forensic cases after various treatments has been investigated. A possible new 'metabolite' of flurazepam was detected in two of these cases.


[Changes in the electrical activity of myocardial fibers following heart transplantation to under the skin of the ear in mice]. Intracellular rest and action potentials (RP and AP, respectively) of mouse heart graft were reduced on the 3rd--4th days after the transplantation as compared with the intracellular potentials of newborn mice. Beginning from the 5th--6th days there occurred a gradual increase of the intracellular potentials of amplitude. The firt 7--8 days, both in the case of allograft and heterograft, the changes in the intracellular activity were the same. Then in heterograft there occurred a repeated reduction of the RP and AP amplitude of the graft myocardial fibers, this apparently being connected with the development of the rejection reaction. In the allograft samples the amplitude of the intracellular potentials approached by the end of the first month after the graft with RP and AP values of the recipient's myocardial fibers.


[Inhibitory processes in the cerebral cortex and the anticonvulsive action of benzodiazepine derivatives]. A comparative study of the effect of some benzodiazepine deprivatives (chlonazepam, lorazepam, diazepam, and medazepam) on the recovery cycles of the interzonal response was carried out on unanesthetized curare-immobilized cats. These drugs proved to selectively inhibit the testing potential within the range of 20 to 100 msec. between the conditioning and the testing stimuli. This indicates that potentiation of GABA-ergic inhibition in the cerebral cortex. The threshold doses of the drugs inducing the depression of the test response and of ED50, preventing the development of convulsions, caused by GABA deficiency or by GABA-ergic receptor block, were compared; a correlation between the mentioned effects was demonstrated. The significance of GABA-positive effect of benzodiazepines in the mechanism of their anticonvulsive activity is suggested.


[Changes in the H-alloantigen-recognizing function of mouse lymphoid cells following hydrocortisone administration]. The capacity of spleen, thymus, and bone marrow cells of intact (control) and of hydrocortisone-treated mice CBA to induce the lymph node type of graft-v-host reaction (GVHR) in hybrids F1 (CBA X c57bl) was studied. After hydrocortisone injection (2.5 mg per mouse) the donor spleen cells became more active in GVHR, considering the value of lymph node indices and immunoblast content in the regional lymph node as compared with a control group. Following transplantation of thymus cells taken from the hydrocortison-treated donors the immunoblast count was higher, although the lymph node weight remained the same as in the control group. On the contrary, following the transfer of the bone marrow cells from the hydrocortisone-treated mice the lymph nodes enlarged, while the immunoblast count remained as low as in control. Consequently, exogenously conditioned increase in the hydrocortisone level was accompanied by an enrichment of the spleen and thymus cell populations with T-lymphocytes, proliferating in response to H-alloantigens.


[Effect of aminazin on the enzymatic oxidation of lipids]. An inhibitory effect of chlorpromazine on the enzymatic NADPH-dependent lipid peroxidation in rat liver microsomal fraction was found. This inhibition was caused by the 1) antioxidative effect of hydroxy-derivatives appearing during the oxidative metabolism of chlorpromazine with NADPH-dependent microsomal oxygenases, and by the 2) competition for reduced components of electron-carriers between the NADPH-dependent processes: chlorpromazine metabolism and lipids peroxidation.


The dependence of the lipid bilayer membrane: buffer partition coefficient of pentobarbitone on pH and lipid composition. 1 The membrane/buffer partition coefficient of [14C]-pentobarbitone has been determined as a function of the lipid composition of bilayer membranes. 2 A new technique based on ultrafiltration gave comparable results to conventional techniques but required less time for equilbration. 3 The membrane/buffer coefficient was independent of pentobarbitone concentration in the range studies. 4 The apparent partition coefficient varied with pH and was a linear function of the degree of dissociation of pentobarbition. 5 Both the charged and uncharged forms of pentobarbitone partitioned into the membrane, the latter to a much greater extent than the former. 6 At low pH the highest partition coefficient observed was in egg phosphatidylcholine bilayer membranes. 7 Incorporation of cholesterol or phosphatidic acid into phosphatidylcholine membranes greatly reduced the partition coefficient. 8 High pressures do not greatly change these partition coefficients.


Assessment of the selectivity of OPC-2009, a new beta2-adrenoceptor stimulatn, by the use of the blood-perfused trachea in situ and of the isolated blood-perfused papillary muscle of the dog. 1 The potency and selectivity of 5-(1-hydroxy-2-isopropylamino)butyl-8-hydroxy carbostyril hydrochloride hemihydrate (OPC-2009), a new beta(2)-adrenoceptor stimulant, was compared with those of isoprenaline, trimetoquinol and salbutamol by the use of blood-perfused tracheal preparations in situ and of blood-perfused papillary muscle preparations of the dog. All drugs were injected intra-arterially.2 All the four drugs decreased tracheal intraluminal pressure (tracheal relaxation) and increased tracheal blood flow in a dose-dependent manner. The four drugs produced a dose-dependent increase in developed tension of papillary muscles. In both preparations the duration of action of isoprenaline and salbutamol was short, whereas that of OPC-2009 and trimetoquinol was long. These effects were antagonized by propranolol.3 Dose-response curves to the four drugs for tracheal relaxation were almost parallel. OPC-2009 was 2.4 times more potent, and trimetoquinol and salbutamol were 2.2 and 6.2 times less potent than isoprenaline in causing tracheal relaxation.4 Dose-response curves to the four drugs for tracheal vasodilatation were also parallel. OPC-2009, trimetoquinol and salbutamol were 3.9, 6.7 and 23 times less potent than isoprenaline.5 Slopes of the dose-response curves to the four drugs for increased developed tension were not parallel; that of OPC-2009 was the least steep, whereas that of isoprenaline was the steepest. Trimetoquinol, salbutamol and OPC-2009 were about 18, 570 and 2400 times less potent than isoprenaline.6 Selectivity calculated from relative potencies indicate that OPC-2009 was about 6000 times, salbutamol about 92 times and trimetoquinol about 8.2 times more selective than isoprenaline for tracheal smooth muscle as compared to ventricular muscle.7 The high potency and selectivity of OPC-2009 for tracheal smooth muscle and its long duration of action suggest its potential usefulness for treatment of bronchial asthma.8 The present results are also compatible with the concept that beta(1)-adrenoceptors in cardiac muscle and beta(2)-adrenoceptors in tracheal and vascular smooth muscle can be distinguished. Furthermore, the results revealed that the beta-adrenoceptors mediating tracheal relaxation and vasodilatation may also be different.


Attitude change during behavioural treatment of sexual inadequacy. Attitudes towards 'self' and 'partner' were studied in couples undergoing three different behavioural treatments for sexual inadequacy: systematic desensitization with counselling; guided practice with counselling; and practice with minimal counselling. Factor analysis of semantic differential scales identified five components--general evaluation, anxiety, and three factors relevant to sexual evaluation designated as 'loving', 'sexually attractive' and 'easy to arouse'. Differences in derived factor scores were found which related to sex of rater, identity of complainant, and treatment received; with the treatment combining guided practice with counselling being followed by significantly greater attitude changes.


Histochemical and biochemical characterization of the rat paracervical ganglion. The nature and identity of the catecholamines in the paracervical ganglion and superior cervical ganglion small, intensely fluorescent (SIF) cells were investigated using fluorescence histochemical and immunohistochemical techniques. The paracervical ganglion SIF cells were found to contain norepinephrine and the superior cervical ganglion SIF cells, dopamine. The norepinephrine content of the paracervical ganglion SIF cells averaged about 72 ng/ganglion and did not change during the rat estrus cycle. The activity of the enzyme tyrosine hydroxylase in the PCG was very low (about 0.48 nmoles DOPA formed/h/mg protein) and was about 1/50th of the activity of the enzyme in the SCG, where it averaged about 23.90 nmoles DOPA formed/h/mg protein. These experiments suggested that the paracervical ganglion has large numbers of norepinephrine containing SIF cells with a relatively slow turnover of their catecholamine content.


[Guanyl cyclase activity in the EF-T elongation factor of Escherichia coli]. Highly purified EF-Ts from E. coli does contain guanylate cyclase activity, which is absent from other purified transfer factors, such as EF-Tu and EF-G. Guanylate cyclase activity has been characterized by its sensitivity to inhibitors and substrate specificity. Although the physicochemical properties of guanylate cyclase are closely related to those of EF-Ts, it does not appear to be a contaminant of this transfer factor, but a specific enzyme. The possible role of guanylate cyclase in protein synthesis is discussed.


Relationship between urine acidification and intracellular pH in respiratory acidosis. The renal net acid excretion (NAE), the blood pH (pHe), the total body intracellular pH (pHi) and the urinary pH (pHu) were calculated in 10 patients with chronic obstructive lung disease and hypercapnia and in 5 normocapnic subjects. The mean value of NAE was significantly higher in hypercapnic subjects than in normocapnic ones. pHe was significantly lower in hypercapnic than in normocapnic subjects. The differences of pHi and pHu between normo and hypercapnic subjects were not significant. NAE is significantly correlated with PaCO2, pHe, pHu and pHi in all the subjects considered together. H+-secretion probably depends on the H+-availability and pHi of tubular cells, but from our results it is not possible to confirm this relationship, because the method used for pHi is fundamentally a measure of muscle-pHi.


Optimum pH and ionic strength for the assay of cytochrome c oxidase from pea cotyledon mitochondria. Cytochrome c oxidase (EC 1.9.3.1) has been solubilized by use of the nonionic detergents were determined for the oxidation of ferrocytochrome c in air. The results indicate that the plant cytochrome c oxidase resembles mammalian preparations in its sensitivity towards ionic strength and pH of the assay buffer.


Effect of metabolic alkalosis on respiratory function in patients with chronic obstructive lung disease. Eleven instances of a mixed acid-base disorder consisting of chronic respiratory acidosis and metabolic alkalosis were recognized in eight patients with chronic obstructive lung disease and carbon dioxide retention. Correction of the metabolic alkalosis led to substantial improvement in blood gas values and clinical symptoms. Patients with mixed chronic respiratory acidosis and metabolic alkalosis constitute a common subgroup of patients with chronic obstructive lung disease and carbon dioxide retention; these patients benefit from correction of the metabolic alkalosis.


Structural studies on the specific type-14 pneumococcal polysaccharide. The structure of the Pneumococcus type-14 capsular polysaccharide has been reinvestigated by using methylation analysis, different specific degradations, and n.m.r. spectroscopy. It is concluded that the polysaccharide is composed of tetrasaccharide repeating-units having the structure: (formula: see text).


Left internal mammary--left ventricular fistula after Vineberg operation. This communication presents an unusual complication in a patient who underwent the Vineberg procedure with the formation of an internal mammary to left-ventricular fistula, which caused a new apical diastolic murmur. This represents a previously unreported etiology for the appearance of an apical blowing diastolic murmur.


Clinical usefulness of alkaline phosphatase isoenzyme determinations. 1. We report on the clinical usefulness of alkaline phosphatase isoenzyme determinations using a combined chemical inhibition method on 731 patient serum specimens exhibiting elevated (greater than 350 U/L) alkaline phosphatase (AP) activity. 2. The relative percentages of the organ-specific alkaline phosphatase activities were computed on the basis of three independent assays: total activity, activity in the presence of 10 mMl-phenylalanine, and activity in the presence of 3.1 M urea. 3. Gamma-glutamyl transferase (GGT) activity assays were also performed on the same specimens. Using an upper reference limit of 30 U/L for GGT and comparing the GGT results with the percent liver AP, we found that the GGT results were 91% sensitive and 60% specific. 4. We conclude that AP isoenzyme determinations are very useful in identifying the organ source(s) responsible for elevated AP values. 5. The reference ranges for several age groups in relation to the adult population and their significance are presented.


Questionable usefulness of gamma-glutamyl transpeptidase test in legal medicine. Because of the high level of GGT activity in semen, the suggestion was made that this enzyme could serve as a test in forensic medical practice; especially in rape cases. Comparisons of GGT and ACP tests revealed, however, that GGT is not sensitive enough and did not show acceptable specificity. One can conclude that GGT cannot be recommended either as a confirmatory test or as a substitute for ACP determinations in rape cases.


[Enzymatic and serologic study of human trichinosis. Apropos of a recent epidemic in a suburb south of Paris]. An enzymatic and immunologic study of 18 patients with trichinosis leads to the following conclusions: The stage of muscular invasion in trichinosis is accompanied by a release of cellular enzymes representative of striated muscle fibres in nearly all the cases. This release can be observed by a study of the LDH iso-enzymes at a time when immunological techniques are not always significantly positive. The specific aspect of this phenomenon can be proposed with reservations since there does not exist any interference with other enzymatic systems such as the gamma-GT and furthermore no other evident cause of muscular lysis is present. The existence of a blood hypereosinophilia completes the biological picture. These early modifications of the enzymatic activities are most probably transient.


Use of different chemical methods for acid phosphatase in cases of rape. Generally acid phosphatase (ACP) assay is used for testing cases of alleged rape. Comparison of three different chemical methods for Acid Phosphatase (Andersch's method with p-nitrophenyl-phosphate substrate and tartrate inhibitor (A-Tart), Roy's method with thymolphthalein phosphate substrate (R-TMP), and Babson's method with alphanaphthyl phosphate substrate) indicated that Roy's method (R-TMP) should be the preferred one. This method had both acceptable sensitivity and confirmed specificity. Our data confirmed that the vaginal wash of normal healthy women has a very low level of ACP activity. Because of inconclusive data in the literature regarding this ACP level, a normal and equivocal range of ACP was suggested until more is known about causes and interferences. Possible sources of normal ACP activity in the wash fluids were also indicated.


Value of alkaline phosphatase, 5'-nucleotidase, gamma-glutamyltransferase, and glutamate dehydrogenase activity measurements (single and combined) in serum in diagnosis of metastasis to the liver. We assessed, in 98 patients with cancer, the diagnostic value of measuring serum alkaline phosphatase, 5'-nucleotidase, gamma-glutamyltransferase, and glutamate dehydrogenase activities as an aid to detection of liver metastases. All four enzymes showed diagnostic value, but 5'-nucleotidase appeared to have the greatest. It showed the lowest false-positive results (7.4%) with the highest predictive value of a positive test (85.7%) and agreement (81.3%).. gamma-Glutamyltransferase showed the lowest proportion of false-negative results (2.8%), but was the least specific 35% false-positive results). Analysis of various test combinations showed that the best agreement (77.5%) was obtained when the patients were divided into those who had no or only one abnormal test result, and those who had two or more abnormal test results. However, this was not better than the agreement for 5' nucleotidase alone (81.3%). The agreement of 5'-nucleotidase and gamma-glutamyltransferase (i.e., both tests were positive or negative) was excellent (91.4%), but such agreement included only 67% of the patients with liver metastases.


Activity and interference effects in measurement of ionized calcium with ion-selective electrodes. We assessed the magnitude of the errors in ionized-calcium measurements resulting from changes in electrolyte composition for three different ion-selective electrodes: the Orion Model 93-20, the Radiometer F2112, and the Simon neutral carrier membrane electrode. We attempted to distinguish between errors arising from changes in calcium ion activity and those due to interferences in the electrode response. Variation in sodium ion concentration over the range 100--180 mmol/liter produces changes in apparent ionized-calcium concentration that are largely attributable to activity effects for the Radiometer and Simon electrodes. The Orion electrode is subject to an additional sodium-interference effect. Apparent ionized calcium concentration measurements are independent of pH for the Radiometer electrode but not for the Orion electrode; the Simon electrode exhibits intermediate pH response, which is probably clinically negligible. Magnesium and potassium ions have little effect on ionized calcium concentration measurements, particularly when these ions are incorporated into calibration standards.


Effects of ethanol (0.75 g/kg body weight) on the activities of selected enzymes in sera of healthy young adults: 2. Interindividual variations in response of gamma-glutamyltransferase to repeated ethanol challenges. We measured individual temporal changes in gamma-glutamyltransferase activity in serum after four consecutive ethanol challenges in each of eight apparently healthy volunteers. Each challenge consisted of ingesting 0.75 g of ethanol per kilogram of body weight on two evenings, followed by five days of abstention during which the activity was assessed at regular intervals. A nine-day abstention period preceded the study, and initial baseline activity was determined. The highest values for the group mean activity were +12%, +11%, +22%, and +18% greater than baseline after the first, second, third, and fourth ethanol challenges, respectively. We noted marked intra-individual and interindividual variation in the enzymic response; peak values ranged from +1% in one subject to +57% in another. The possible causes for these biological variations are discussed.


Albumin/calcium association at different pH, as determined by potentiometry. Calcium binding by albumin was determined potentiometrically at physiological ionic strength and temperature as a function of pH. The binding data indicate at least 30 different binding sites with different association constants and different H+ interaction. One site appears to be responsible for the major binding at physiological pH and substance concentration of free calcium, together with three other sites that bind with less affinity.


The use of tears for diagnosis of GM1 gangliosidosis. The properties of beta-galactosidase of tears were investigated and the standard assay system was accomplished. The pH optimum was 4.2. The enzyme had a KM of 8.3 X 10(-4) M. The activity was stimulated by chloride ions and slightly stabilized by bovine serum albumin. The activities of normal individuals were 205 +/- 80 (S.D.) nmol/h/ml. The activity in the tears of the patient with GM1 gangliosidosis decreased to about 20% of normal control and this disease could be diagnosed by the assay of beta-galactosidase in tears.


Assay of serum free fatty acids by extraction-photometric procedure. Reaction conditions for extraction-photometric determination of free fatty acids in serum were studied. The results were found to be influenced by (a) sodium chloride concentration in the copper reagent, (b) the kind of standard solution used, (c) centrifugation of the reaction mixture and (d) serum phospholipids. A micromethod based on the formation of free fatty acids-copper soaps with a stable copper reagent and sensitive indicator for copper, 2-(2-thiazolylazo)-4-methoxyphenol, is described.


Sweat testing for cystic fibrosis: errors associated with the in-situ sweat test using chloride ion selective electrodes. The in-situ sweat test is prone to errors from various sources. This paper examines errors due to evaporation, absorption of water into the skin, and pressure of the electrode on the skin. Only evaporation caused serious errors. The accuracy and precision when measuring small chloride/sweat samples on the skin are not significantly worse than when measuring bulk solutions.


Influence of ions on the antigen-antibody complex formation as measured by radioimmunoassay. In this study, using radioimmunoassay techniques, we found that ions at concentrations in the order of 0.1 molar influence the antigen-antibody complex formation. The angiotensin I/anti-angiotensin I reaction was studied in detail. Particularly bivalent cations and anions with a strong chaotropic effect (SCN-, I- and ClO4-) were found to influence strongly the specific immunological reaction. However, NO3- had also a remarkably strong influence. We found that the equilibrium constant, rather than the number of binding sites of the antibody, is influenced by the ions. It should be borne in mind that relatively high concentrations of electrolyte (as compared with the concentrations of antigen and antibody) show this effect. Consequently, this effect is of less practical importance for routine radioimmunoassay than is, for example, the effect of pH. However, this phenomenon shows that the radioimmunoassay technique might be valuable not only for quantization of very low hormone concentrations in biological fluids, but has also important potential applications in physical and protein chemistry. Particularly, the high sensitivity of this technique and the possibility of studying a homogeneous reaction system might give it advantages over other techniques.


Comparison of soluble and membrane-bound neutral arylamidases from renal cell carcinoma. Soluble and membrane-bound neutral arylamidases from renal cell carcinoma were partially purified and their properties were compared. Soluble neutral arylamidase was a heat labile and SH-dependent metalloenzyme. Membrane-bound neutral arylamidase was a rather heat stable metalloenzyme and was activated by Co2+ with changing KM and VMAX. KM values for soluble and membrane-bound neutral arylamidases were different. L-Methionine (5mM) did not affect the enzymic activity of soluble neutral arylamidase, but inhibited 82 percent of the enzymic activity of membrane-bound neutral arylamidase. Molecular weights of soluble and membrane-bound neutral arylamidases by Sephadex G-200 gel filtration were 140000 and 240000, respectively. Soluble and membrane-bound neutral arylamidases from renal cell carcinoma appear to be distinct enzymes.


Comparative activity of sulphatases in human liver on two synthetic substrates. Ion-exchange chromatography and gel chromatography manifested the presence of three different forms of sulphatase activity in human liver using 4-methylumbelliferyl sulphate as substrate. Studies with the substrate 4-nitrocatechol sulphate and inhibition experiments with AgNO3 were also performed on these three different forms of sulphatases. This makes it possible to identify the three forms as arylsulphatase A, B, and C. The conclusion is drawn that the 4-methylumbelliferyl sulphate substrate is inadequate to measure the lysosomal arylsulphatases A and B, but it could be used to measure the microsomal arylsulphatase C.


Some kinetic properites of liver ornithine carbamoyl transferase (OCT) in a patient with OCT deficiency. Some kinetic properties of liver OCT from a patient with OCT deficiency were studied. Contrary to controls, in which two pH optima were observed (pH 7.7 and pH 8.5), only the pH optimum of 8.5 could be demonstrated in our patient. From KM studies at pH 7.7 and pH 8.5, the most striking abnormalities in comparison with human controls were (a) a strongly increased KM (ornithine) at pH 7.7, but less pronounced at pH 8.5, (b) a higher VMAX at pH 8.5 compared with the VMAX at pH 7.7 and (c) the absence of substate inhibition at pH 8.5 to ornithine was elevated up to a concentration above approximately 1.5 mM.


The effect of anti-inflammatory and antirheumatic drugs on inflammation in the rat. The anti-inflammatory effects of flurbiprofen and a number of other antirheumatic agents were studied in the rat using a model of inflammation, which involved the subcutaneous implantation of polyurethane cubes impregnated with an irritant. Results of acute and chronic studies indicated that flurbiprofen was extremely effective in suppressing both fluid and cellular phases of inflammation and, in this model, was comparable in potency to prednisolone.


The effects of urine pH and plasma protein binding on the renal clearance of disopyramide. To ascertain whether the renal clearance of disopyramide (pKa = 8.36) is affected by urine pH, the disposition kinetics of disopyramide were compared during excretion of acidic and alkaline urine following both single dose intravenous (2mg/kg) and oral (5 mg/kg) administration to 4 healthy male volunteers. No significant difference was observed in the plasma concentration-time curve of disopyramide. The mean 72 hour recovery of disopyramide and its N-deisopropyl metabolite (MND) in urine was 55.1 and 20.3% of the dose respectively, with no apparent difference between the two routes of administration or pH of urine. Renal clearance of disopyramide was found to vary with time, which is partly the result of a concentration dependent change in plasma protein binding. The unbound fraction of drug in plasma varied from 0.32 to 0.72 between 0.4 to 4microgram/ml concentration. However, time-dependent change in renal clearance of disopyramide persists even after correction for plasma protein binding.


Use of EMG biofeedback in behavioral treatment of an obsessive-phobic-depressive syndrome. A 25 year old woman who suffered from depression (associated with obsessive, phobic thoughts of animal abuse or neglect) was treated with systematic desensitization in which EMG biofeedback was used to achieve relaxation. EMG responses were also used to aid both the therapist and the patient in the pairing of hierarchical imagery and muscular relaxation during desensitization, EMG responses were particularly useful in detecting rising muscular tension, indicating when visualization of a depression-provoking situation should be terminated. Ratings concerning frequency and intensity of depressive thoughts (made by the patient before and after therapy), as well as 6-month follow-up information, attest to the success of the therapeutic program.


The permissive role of glucocorticoids in the development of ethanol dependence and tolerance. In mice chronically treated with ethanol (in a liquid diet containing 6% ethanol ad libitum for 2 weeks), brain tryptophan hydroxylase (TPH) activity was increased (by 30-45% in whole brain), while brain tyrosine hydroxylase activity remained unchanged. Such chronic ethanol treatment also induced susceptibility to audiogenic seizures during withdrawal (60% incidence). When ethanol treatment was given to adrenalectomized (Adx) mice, the increase of brain TPH activity and the development of withdrawal audiogenic seizures were both prevented. In Adx mice receiving daily injections of corticosterone (0.5 mg/mouse), the ethanol-induced increase of brain TPH activity and the occurrence of withdrawal audiogenic seizures were both restored. Similarly, the ethanol-induced increase of liver alcohol dehydrogenase activity (by 60%) was prevented in Adx mice and restored by corticosterone replacement. It was noted that in all three cases replacement with such large doses of the corticoid did not enhance the ethanol effects, but merely restored the effects to the levels observed in intact mice. Apparently, glucocorticoids are required in a permissive role in order for the ethanol effects to occur.


Alclofenac: a review of its pharmacological properties and therapeutic efficacy in rheumatoid arthritis and allied rheumatic disorders. Alclofenac is a non-steroidal anti-inflammatory agent advocated for use in rheumatoid arthritis, degenerative joint disease and ankylosing spondylitis. Published data to date, suggest that alclofenac 3g daily is comparable in efficacy with aspirin 4.8g daily, phenylbutazone 300 to 600mg daily and indomethacin 150mg daily. In Welsh patients, gastro-intestinal side-effects have generally been less frequent and milder than with the standard comparison drugs, but in other populations differences in the overall incidence of these side-effects have been less marked. Results of a long-term trial, as evidenced by alterations in certain biochemical indications of disease activity, suggest that alclofenac may possibly reduce the severity of the disease itself, but further studies will be needed to confirm this. However, at present alclofenac should be considered along with the other drugs of its type in the initial treatment of the arthritic patient. Skin rash is the most frequent side-effect, which in a small proportion of affected patients may be associated with systemic effects. A cutaneous reaction appears to be more likely in patients with a history of previous allergy to penicillin and other drugs.


Treatment of chronic schizophrenia.

Treatment of chronic schizophrenia. The comprehensive treatment of schizophrenia requires the full resources of a clinical team that is able to offer treatment for the acute psychotic state in a hospital environment, and appropriate rehabilitation following resolution of the florid symptoms. Following a second or subsequent relapse, maintenance therapy with long-acting injections of depot antipsychotics will be required for an unknown period. Although drugs form an essential part of all treatments, it is essential to examine the environment for precipitating factors and to involve the patient's family in the rehabilitation. Recent studies have reported that some patients still have a relatively poor prognosis; although this proportion may be in the minority, the strain on the whole family of an even moderately handicapped patient can be enormous, and it is important to examine the needs of the whole family in evaluating care within the community. The effect of antipsychotic drugs is much wider that the mere control of acute symptoms and can influence the patterns of social behaviour and rehabilitation, in addition to offering protection against stress. The proper use of depot injections requires that they be kept under constant review. The current widespread practice of prescribing anti-cholinergic drugs on a prophylactic basis, or even as the inital treatment of extrapyramidal side-effects, needs revision.


Physical studies on the conformation of ribosomal protein S4 from Escherichia coli. Proton magnetic resonance, circular dichroism and infrared spectroscopy were used to investigate the secondary and tertiary structure of the 16-S RNA binding protein S4 from Escherichia coli ribosomes. The proton magnetic resonance spectra of protein S4 in ribosomal reconstitution and low-salt buffers were identical and showed little dipolar broadening of the peaks, suggesting that the protein had an open extended structure. A ring-current-shifted apolar methyl resonance in the high-field region of the spectrum, together with a perturbation of the tyrosine ring proton resonance in the low-field region, indicated the existence of a specific tertiary fold in the polypeptide chain. This structure disappeared on lowering the pH below 5 or on heating above 30 degrees C, both processes being reversible. Circular dichroism measurements on protein S4 showed an alpha-helix content of 32% in reconstitution buffer compared with 26% in low-salt buffer. Heating the protein solution in reconstitution buffer above 35 degrees C reversibly disrupted this extra helix. Infrared studies on both solid films and solutions of protein S4 indicated the presence of little or no beta-structure. These results correlate well with the known RNA binding properties of protein S4.


Influence of 1,2,3-benzene-tricarboxylate on pyruvate metabolism in rat-liver mitochondria. 1,2,3-Benzene-tricarboxylate, a known inhibitor of the mitochondrial tricarboxylate carrier, was found to inhibit pyruvate carboxylation as well as the transport of citrate out of the matrix in rat liver mitochondria incubated with pyruvate. The inhibition of pyruvate carboxylation was observed with both intact mitochondria and with the solubilized pyruvate carboxylase. The inhibition of the pyruvate carboxylase by 1,2,3-benzene-tricarboxylase was not mediated via one of the parameters known to regulate the activity of the enzyme and therefore a direct inhibition of the enzyme by the tricarboxylate was assumed. Since the pyruvate carboxylase is exclusively localized in the mitochondrial matrix space it was concluded that 1,2,3-benzene-tricarboxylate penetrates into this compartment.


Prostatic binding protein. A steriod-binding protein secreted by rat prostate. Rat prostatic cytosol contains a high concentration of a prostatic binding protein with peculiar steroid-binding properties. Indeed, in spite of a relatively low affinity, charcoal adsorption can be used for its measurement. Furthermore, the binding is not specific for particular steroids and increases very strongly after delipidation. In delipidated cytosol the concentration of the binding site is 3.1 micronmol/g protein and the apparent affinity for pregenolone 1.7 X 10(6) M-1. The high concentration of prostatic binding protein in prostatic fluid shows that this substance is secreted by the prostate. Prostatic binding protein has the following physicochemical characteristics: it is precipitated by ammonium sulfate between 50 and 70% saturation; the elution position from a Sephadex G-100 column corresponds to a molecular weight of 51000; it sediments in sucrose density gradients at 3.7 S and is eluted from DEAE-cellulose columns at about 0.25 M KCl. On polyacrylamide gel electrophoresis the binding activity coincides with the major cytosolic protein band. This band has the same mobility as serum albumin in 7% gels, but a higher mobility in more concentrated gels.


Yeast pyruvate kinase: a mutant from catalytically insensitive to fructose 1,6-bisphosphate. The paper describes some of the characteristic properties of an altered form of pyruvate kinase from a mutant of Saccharomyces cerevisiae. The partially purified enzyme does not require fructose 1,6-bisphosphate for activity but is stabilised in its presence both at low and at high temperatures. The enzyme displays in the absence of fructose 1,6-bisphosphate hyperbolic kinetics with phosphoenolpyruvate (Km, 0.11 mM), ADP (Km, 0.12 mM) and K+ (Km, 11 mM). Sedimentation velocity experiments indicate that the mutated enzyme and the wild type enzyme have S20,w values of 8.9 and 8.6 S respectively. The mutant with the pyruvate insensitive to fructose 1.6-bisphosphate is capable of growing on synthetic media with alcohol or malate as the sole carbon source. The steady-state intracellular levels of phosphoenolpyruvate in the mutant suggest mechanisms that prevent depletion of this metabolite despite an active pyruvate kinase. Spontaneous reversion of this mutant yields clones with normal enzyme activated by fructose 1,6-bisphosphate.


A new factor from enteric bacteria of rats amplifying induction of liver enzyme by glucocorticoid. 1. Purification, properties and biological action. 1. A factor, which amplifies the inductions of several liver enzymes by glucocorticoid, was partially purified from Proteus mirabilis from rat intestine. The factor (amplifier) was completely inactivated by alpha-glucosidase, but not by other glycoside hydrolases, proteases, nucleases or phosphatases tested; it was also hydrolysed by HCl with liberation of reducing sugars. Thus the oligosaccharide in this factor seems to be essential for the amplification. 2. In adrenalectomized rats the amplifier increased the inductions of several liver enzymes, such as tyrosine aminotransferase and leucine aminotransferase, by glucocorticoid. But it did not amplify the induction of tyrosine aminotransferase by glucagon or insulin or the activities of enzymes that are not induced by glucocorticoid. The amplifier by itself did not have any glucocorticoid-like action in adrenalectomized rat. These results show that the amplifier specifically increases the inductions of liver enzymes by glucocorticoid. 3. Since similar amplification was also observed in isolated perfused liver and cultured hepatoma cells in vitro, the amplifier seems to act directly on the target organ or cells.


A new factor from enteric bacteria of rats amplifying induction of liver enzyme by glucocorticoid. 2. Mechanism of action. 1. An amplifier of the action of glucocorticoid was purified from Proteus mirabilis as described previously. It was found that it amplified the induction of liver tyrosine aminotransferase by dexamethasone markedly with doses of dexamethasone that caused minimal enzyme induction, but had little effect with doses that caused maximal induction. Thus the amplification may represent a saving of glucocorticoid. The amplification of enzyme activity was brought about by increase in amount of enzyme. 2. The amplification was observed when the amplifier was administered before or with dexamethasone, but not when it was given 2 h after dexamethasone. These results and the finding that actinomycin D inhibited the amplification indicate that the amplifier does not act on the translational level of enzyme induction. 3. It was found that the amplifier increased both incorporation of [3H]dexamethasone into the cytosol and binding of [3H]dexamethasone of cytosol protein and that it decreased decay of the [3H]dexamethasone-protein complex.


gamma-Glutamyl transpeptidase in rat ascites tumor cell LY-5. Lack of functional correlation of its catalytic activity with the amino acid transport. gamma-Glutamyl transpeptidase activity was detected in rat ascites tumor cells (LY-5) suspended in Hanks' balanced saline solution using L-gamma-glutamyl-p-nitroanilide as a substrate. Whole-cell suspension of the tumor cells exhibited full activity of the enzyme without detectable cell disruption under the conditions examined. Various amino acids, transported through specific membrane carriers, did not affect the accessibility of substrate for the enzyme. An inhibitor of sodium-dependent transport systems of amino acids caused no significant change in the rate of enzyme catalysis. Like glutathione or S-methylglutathione, S-acetyldextran (mol. wt 215000) derivative of glutathione, which is believed to be unable to penetrate into intact cells, caused marked inhibition of the rate of p-nitroaniline release from the synthetic substrate by the tumor cells. These data indicated that the active site of the enzyme faced to the outer surface of the cells. gamma-Glutamyl transpeptidase of the tumor cells was successfully affinity-labeled by 6-diazo-5-oxo-L-norleucine, a glutamine analog, without causing detectable change in the viability of the cells under the conditions examined. The rate of transport of alanine, leucine, glycine and glutamine into cells was not affected by the inactivation of this enzyme with the affinity label. Thus, the activity of gamma-glutamyl transpeptidase located on the outer surface of tumor cell membrane does not seem to be requisite for the transport process of amino acids.


Hydrogen-ion binding by tobacco-mosaic-virus protein polymers. Hydrogen ion titration curves of tobacco mosaic virus protein have been measured in various conditions of protein concentration, temperature, ionic strength, and rate of pH change. The polymers present at each stage are deduced from turbidity and sedimentation data, plus published information. A simple semi-quantitative analysis of the curves is given, and the pK values of the two abnormal carboxylates in single helix are estimated as 6.4 and about 7.0. Disks, and some faster-forming unknown polymers in the same size range, have been abnormal carboxylate with pK 6.9. These results are most easily interpreted in terms of electrostatic interactions between carboxylates, probably at the axial ends of the protein subunits.


Ionization behaviour of native apolipoproteins and of their complexes with lecithin. 1. Calorimetric and potentiometric titration of the native apoA-I protein and of the apoA-I protein-dimyristoyl lecithin complex. The ionization behaviour of native apoA-I protein is compare to that of its complex with synthetic dimyristoyl lecithin in studies using calorimetric, potentiometric and spectrophotometric titration. In the presence of phospholipids, 10 out of 21 lysines together with 22 acidic residues are masked in the complex. All tyrosines remain accessible to titration below pH 13. The apparent ionization enthalpy of the 11 lysine residues is not affected by the presence of phospholipids. These data are consistent with discrete binding sites located in the apoprotein helical segments as suggested by the model of Segrest et al. [FEBS Lett. 38, 247-253 (1974)]. A tentative localisation of lysine, arginine, aspartic acid and glutamic acid residues directly involved in phospholipid binding is suggested, assuming that such helical regions are involved in apoprotein-phospholipid association.


Ionization behaviour of native apolipoproteins and of their complexes with lecithin. 2. Potentiometric titration of the native apo-A-II, apoC-I, apoC-III proteins and of their complexes with dimyristoyl lecithin. A comparison of the ionization behaviour of the human apoA-II, apoC-I, apoC-III proteins and of their complexes with dimyristoyl lecithin is based on potentiometric titration of the basic and acidic residues and spectrophotometric titration of the phenolic groups. Experimental data suggest that a number of lysine, arginine, aspartic acid and glutamic acid residues are masked in the complexes. For each of these amino acids and in all three proteins the number of masked residues is consistent with the content of those regions predicted to be involved in lipid binding by the model of Segrest et al. [FEBS Lett. 38, 247-253 (1974)]. These data taken together with the results of calorimetric and titration experiments with the apoA-I protein reported in the accompanying article [Rosseneu et al. (1977) Eur. J. Biochem. 79, 251-257] strongly support the general nature of the proposed model and further suggest that ionic interactions have some role in the formation of the dimyristoyl lecithin/apolipoprotein complexes.


Substrate specificity via ternary complex formation with glutamate dehydrogenase. Very littly discrimination is observed in the binary binding of dicarboxylic acid substrate analogues to glutamate dehydrogenase as monitored by proton nuclear magnetic resonance. Variation in length, charge, bulkiness and conformational rigidity resulted in only a factor of five variation in KD and apparent relaxation time, T2. Upon titration of the binary enzyme-ligand complex with coenzyme to form the ternary enzyme-ligand-coenzyme complex strong discrimination is observed. Coenzyme binds tightly only when the correct substrate is present, otherwise it binds 10 to 150 times more weakly.


Reversible graft versus host reaction as cause of erythrophagic splenomegaly in a child? The case history of a 9 months old infant with hepatosplenomegaly, pancytopnaenia and disturbances of clotting and cellular immune reactivity is reported. The spleen was removed and showed striking erythrophagocytosis by proliferating histiocytes, typical of "familial erythrophagocytic reticulosis" (Farquhar). A graft-versus-host reaction is discussed as a possible underlying cause. The favourable clinical course and full recovery point to an interrelation with primary hypersplenism.


Interstitial cystitis. Observations on diagnosis and on treatment with anti-inflammatory drugs, particularly benzydamine. Interstitial cystitis is often missed on "routine" cystoscopy. If the disease is suspected, the bladder must be observed twice during complete filling. An anti-inflammatory drug, Benzydamine, has given mixed results but in view of its low toxicity it is worth trying in any patient with severe symptoms.


Prune belly syndrome: treatment of terminal renal failure by hemodialysis and renal transplantation. The second case of successful renal transplantation in a patient with "prune belly" syndrome is reported. In spite of early aggressive surgical approach in the management of this disease terminal renal failure frequently ensues. Hemodialysis and renal transplantation have offered new possibilities of prolonging life in these patients. The success of renal transplantation depends on the anatomic and functional state of the lower urinary tract. Pretransplant urologic examination is extremely important for the evaluation of urinary tract abnormalities.


Acid mucopolysaccharides in fibroblast cultures. 4. 35S-sulfate incorporation in dependence on pH-value cell density and lactate. Cultures of embryonic rat fibroblasts were incubated with 35S-sulfate at pH 6.6 and 7.4 (Eagle medium, Hepes buffer) for 48 hours. The MPS were isolated and fractionated. Determination of hexuronic acid was done according to BITTER and MUIR, measuring of sulfate incorporation by liquid scintillation counting. 1. HS, Ch-6-S and DS showed different synthesis rates. They rised at increasing cell density and were lower at pH 6.6 than at 7.4. 2. At pH 6.6 the sulfate incorporation rates (de novo-synthesis) of HS, Ch-6-S and DS are lowered. The percent increase of the MPS fractions at pH 6.6 and their specific activities are explained by individual differences in the unlabelled MPS pools as well as by differences in the rates of synthesis and catabolism. The cell density also has an influence on that. Increase of the MPS at pH 6.6 is by mainly due to an inhibition of catabolism. 3. Contrary to CH-6-S and DS heparan sulfate is catabolized more slowly at high cell densities. 4. The volume of the unlabelled MPS pool is modified by lactate at pH 6.6, e.g. it effectuates enhancement of the DS pool at low cell densities. De novo-synthesis and specific activity are scarcely influenced. CH-6-S and DS catabolism are considerably inhibited, their total amount is increased. The relative synthesis rate of DS is enhanced by lactate. 5. The possible importance of the results for inflammatory processes and wound healing is mentioned.


[Analysis of the effect of bicarbonate on taste receptor sensitivity to organic acids]. Addition of NaHCO3 or NaOH to the solutions of lactic or pyruvic acids decreased threshold concentrations of H+ ions and increased intensity of afferent impulsation in the cat chroda tympani nerve. In spite of the fact that the concentrations of both lactate and pyruvate anions with the presence of NaOH and NaHCO3 are the same at equal pH values the observed changes of the receptors responses were more intensive if NaHCO3 had been added. Therefore the influence of NaHCO3 on the perception of organic acids depends not only on its buffer properties but, probably, on direct action of CO2 appearing as the result of interaction of NaHCO3 and acids, as well. This assumption was confirmed by the data on high sensitivity of the cat tongue receptors for the carbon dioxide.


Degradation and liquefaction effect of streptokinase-Streptodornase and stabilized trypsin on necroses, crusts of fibrinoid, purulent exudate and clotted blood from leg ulcers. Necrotic materials most frequently found in leg ulcers, including crusts with fibrinogen, pus and blood clots, were exposed to solutions of streptokinase-streptodornase and stabilized crystalline trypsin respectively. The investigations were performed at the temperatures of 26 degrees and 33 degrees C, representing the extreme values of the temperature range found in leg ulcers (arteriosclerotic ulcers, stasis ulcers) and at the pH corresponding to that of the enzyme preparations in wet dressings. Streptokinase-streptodornase demonstrated in vitro a more potent proteolytic activity than crystalline trypsin on necroses, crusts of fibrinoid and purulent exudate, which were more rapidly and thoroughly broken up. Both enzyme preparations were, however, equally effective on three-day-old blood clots.


Anxiety states in family practice: an evaluation of the need for antidepressant as well as anxiolytic therapy. Patients attending their family practitioner with emotional disturbance manifesting predominantly as anxiety were treated once daily for 4 weeks with either a pure anxiolytic, potassium clorazepate, or a formulation of a specific antidepressant together with an anxiolytic, fluphenazine/nortriptyline, in accordance with a double-blind, completely randomized design. After the first week the patients receiving fluphenazine/nortriptyline were showing a better response in terms of total symptomatology as well as anxiety, tension and depression taken separately, and after 4 weeks treatment this trend reached statistically significant levels on both the physicans' ratings and the patients' self-ratings for overall symptomatology (p less than 0-05) as well as anxiety and tension on the physicians' scale (p less than 0-01). Side-effects were infrequent, with the exception of drowsiness which was complained of by 42% of the patients receiving clorazepate. Although simple and convenient to take, a once daily benzodiazepine formulation of fixed dose is likely to be too inflexible to achieve optimal therapeutic effect in many patients. These results are in accord with accumulating evidence for the importance of a depressive aetiology underlying the majority of so-called anxiety states in family practice. Anxiolytic, in the absence of specific antidepressant, therapy is unlikely to be adequate for these patients, and may lead to long-term palliative use of benzodiazepines incurring a risk of dependence.


Therapy of alveococcosis in man.

Therapy of alveococcosis in man. Diagnostic, therapeutic and prognostic aspects are interpreted on the basis of two cases of Echinococcus alveolaris. Both patients had undergone partial hepatectomy and were subsequently treated chemotherapeutically. In one case dehydroemetine and mebendazole (Vermox, Janssen), and in the other case metrifonate (Bilarcil, Bayer A.G.) were used. The diagnosis of alveococcosis of the liver was demonstrated in one case histologically on the occasion of appendectomy, and in the other case by the indirect immunofluorescence test and passive haemagglutination. In case of suspected alveococcosis these serological are imperative, as they are the most reliable methods of demonstrating this disease. The latest trend in theraphy aims at a combination of surgery and chemotherapy. Mebendazole seems to be promising as an anthelmintic agent for the treatment of alveococcosis. Remission of the disease was obtained in either case.


[The orthostatic hypotension in patients treated with antihypertensive drugs (author's transl)]. Occasional or recurrent episodes of orthostatic hypotension were observed in 58 out of 584 hypertensive patients treated with alpha-methyldopa, beta-blockers and clonidine alone or associated with diuretics and/or hydralazine and/or reserpine. They occurred more frequently in the elderly. In none dicardial ischaemia. In only 3 cases it caused drowsiness and in 2 transient T wave inversion. These results suggest that postural hypotension does not contraindicate the continuation of antihypertensive treatment.


[Management and monitoring of multiple pregnancies (author's transl)]. The fetal prognosis in multiple pregnancies can be improved by a multifaceted antenatal program which includes early diagnosis prior to 28 weeks gestation, follow-up of multiple pregnancies in the high risk antenatal clinic, early discontinuation of work, treatment of pre-eclampsia, bed rest in hospital between 28 and 33 weeks and sometimes cerclage, prophylactic and therapeutic administration of labour inhibiting drugs, speedy delivery of the second twin and immediate pediatric care. Bed rest and administration of labour inhibiting drugs are the most important points of this program. With this combination, the utero-placental perfusion can be improved. The gestation can be prolonged and the incidence of small weight neonates and the incidence of the perinatal mortality can be reduced. Since even a large antenatal clinic only cares for a small number of multiple pregnancies, a multicentre study to determine the optimal management of multiple pregnancies is urgently required. Multiple pregnancies had too little attention in modern perinatal medicine and deserve all our attention for an improvement of their outcome.


[Clinical polymorphism and ceruloplasmin variants in hepatolenticular degeneration]. The relationship between differences in the clinical polymorphism of hepatolenticular degeneration (Wilson's disease) and characteristics of CP (ceruloplasmin) structural changes were investigated. The comparative study of Wilson's disease patients revealed two forms of clinical development of this disease which differ from each other by the expression of the visceral symptoms preceding the establishment of the typical neurological picture. The peptide map analysis of tryptic hydrolysates of the CP from individual patients has demonstrated the altered peptide patterns in five cases. Clinical and genetic heterogeneity of Wilson's disease is discussed.


Ditazole and platelets III. Effect of ditazole on tumor-cell induced thrombocytopenia and on bleeding time in mice. Ditazole (4,5-diphenyl-2-bis-(2-hydroxyethyl)-aminoxazol) inhibits in vivo platelet aggregation induced in mice by intravenous injection of cells derived from an experimental tumor, the Lewis lung carcinoma. Such a protective effect of ditazole could not be observed when the number of circulating platelets dropped slowly following intramuscular implantation and spontaneous dissemination of the same cancer cells. These results support previous observations suggesting a different mechanism for the thrombocytopenia observed after intravenous and intramuscular injection of cancer cells. Tail transection bleeding time of normal mice is significantly prolonged by ditazole, a finding at variance with that reported in rats.


[Effects of a new minor tranquilizer, 10-chloro-3-methyl-1 1b-(2-chlorophenyl)-2,3,5,6,7,11b-hexahydrobenzo [6,7]-1,4-diazepino [5,4-b]-oxazol-6-one (CS-386), on the after-discharge and behavior induced by electrical stimulation of the amygdala in freely-moving cats (author's transl)]. Effects of a new minor tranquilizer, CS-386, on the after-discharge(AD) and behavior induced by amygdaloid electrical stimulation in freely-moving cats were compared with those of cloxazolam, oxazolam, diazepam, chlordiazepoxide, phenobarbital and chlorphromazine. Effects on change of the AD threshold and duration and on facial twitching, salivation and tonic-clonic convulsion were investigated. CS-386, cloxazolam and oxazolam inhibited amygdaloid AD. CS-386 had the most potent inhibitory effect. These drugs depressed all behavior described above. Diazepam had no effects on the AD threshold, but decreased the AD duration and inhibited the behavior. Chlordiazepoxide had no apparent effects on amygdaloid AD and on facial twitching. Salivation was inhibited with high doses of administration. Phenobarbital shortened the AD duration and at a high dose elevated the AD threshold. This drug also inhibited salivation, but inhibitory effects on other behavior required doses as high as 90 mg/kg. These results suggest that CS-386, cloxazolam and oxazolam are compounds belonging to a classification different from that of chlorpromazine. CS-386 in particular, is a more potent drug chlordiazepoxide, diazepam and phenobarbital and acts on the amygdala itself.


[Studies on monoamine oxidase. (Report 37) Effects of oxygen concentration on rat liver and brain monoamine oxidase (author's transl)]. MAO activity in rat brain mitochondria with tyramine as substrate at 100% oxygen concentration was three times as much as that at 20%. When serotonin served as substrate, difference in activities between the two oxygen concentrations was not significant. Similar results were obtained when rat liver MAO was used as the enzyme source. At 100% oxygen concentration, pargyline showed the most potent inhibition of MAO activity in liver mitochondria with tyramine as substrate, but inhibitions caused by pheniprazine and harmaline were not remarkable. At 100% oxygen concentration, harmaline showed the most potent inhibition of MAO activity in the liver when serotonin served as substrate, while inhibitions of the MAO activity by pargyline and pheniprazine were weak. At 20% oxygen concentration, harmaline showed the most potent inhibition of MAO activity in the brain when serotonin was used as substrate. These inhibitions were studied using Lineweaver-Burk plots. Pargyline revealed a noncompetitive inhibition to MAO activity in liver and brain with tyramine and serotonin as substrate, harmaline a competitive inhibition to MAO activity in liver and brain with tyramine as substrate, while noncompetitive inhibition to MAO activity in liver and brain was evident when serotonin was used as the substrate.


[Effects of minor tranquilizers and neuroleptics on open-field behavior in rats (author's transl)]. Minor tranquilizers (diazepam, nitrazepam, oxazepam, bromazepam, medazepam, fludiazepam, meprobamate) at low doses increased ambulation score to 145 approximately 288% of control rats. Nitrazepam, diazepam and bromazepam which are potent, clinically prescribed minor tranquilizers increased the ambulation at lower doses than was seen with the other drugs. Fludiazepam and nitrazepam showed a maximum increase in ambulation at the same dose. Fludiazepam, nitrazepam and diazepam proved to have potent inhibitory effects on defecation. Trifluperidol, haloperidol and ID-4708 (a new butyrophenone derivative) and chlorpromazine when given at low doses reduced ambulation, while at higher doses defecation was inhibited. These four drugs reduced ambulation and elicited a recover in rates of defecation in methamphetamine treated rats. Clozapine, thioridazine and floropipamide inhibited defecation at nearly the same doses which reduced ambulation in rats not given the methamphetamine tratment. These three durugs reduced ambulation, but did not produce a recovery in the defecation rates in methamphetamine-treated rats. These results indicate that neuroleptics such as clozapine which rarely induce extrapyramidal side-effects when clinically prescribed, inhibit defecation at nearly the same doses which reduce ambulation. In methamphetamine-treated rats, haloperidol was 31 times more potent than chlorpromazine in inhibiting activity noted with ambulation. This ratio in open-field test was close to the ratio of potency of these drugs as antipsychotic clinically prescribed agents.


[Calcium containing antacids and acid rebound. Testing with intragastric titration and extragastric measurement of pH]. Intragastric titration with the pH being measured extragastrically is a method both precise and highly qualified for clinical trials of antacids and quantitative analysis of gastric secretion. In 12 patients with duodenal ulcer, acid secretion was measured by intragastric titration both before and after adminstration of a Ca-Mg-containing antacid. Acid secretion was found to be significantly lower after administration of the Ca-Mg-containing antacid, a so-called acid rebound could not be demonstrated.


[The clinical picture of lactate acidosis. 5. Lactatemia without acidosis. Conclusions]. Several inherited metabolic diseases are accompanied by a greater or lesser increase in blood lactate concentration under certain metabolic conditions. These diseases are glycogenosis type I (glocuse-6-phosphate deficiency), fructose-1,6-diphosphatase deficiency, glucose-induced hyperlactate emia, idiopathic lactate acidosis. The conditions are discussed when hyperlactate emia develops. Very large increases in blood lactate concentration are found during muscular activity, lactate concentrations can be as much as 20 mmol/l under these conditions. Regarding these values, the increase in blood lactate concentration during intravenous carbohydrate infusion is minimum, even in the case of fructose infusions (1-4 mmol/l). Therapeutical measures for treatment of increased lactate concentration are discussed. A causal therapy is optimum; however, the precondition is a definite diagnosis. Besides bicarbonate infusions (or infusions of other alkalizing substances) dialysis seems to be a favourable therapy in certain cases. In future, prognosis of lactate emia should be better if the diagnostic measures and differential diagnosis are improved.


[Neurological-psychiatrical and muscular manifestations of vasculitis nodosa (author's transl)]. A general review on vasculitis nodosa of report in literature since the work of Strammler 1958 is given. The neurological and psychiatric manifestations of the disease are completely dealt with, whereas dermatological, ophthalmological and otological aspects are brieftly mentioned. Varieties of diffuse and localized cerebral, as well as peripheral syndromes are described. The neuropathological findings are also discussed extensively.


Primary care and physician extenders in affluent countries. The worldwide growth of specialization in medicine has led to a perceived shortage of primary care. A major response in the United States has been the training of physician extenders (both physician assistants and nurse practitioners). Other industrialized countries have rejected this approach, in favor of strengthening general medical practice through continuing education, provision of ancillary personnel, use of health centers, and by other methods. Developing countries use doctor-substitutes as a reasonable adjustment to their lack of economic resources. All countries use ancillary personnel for selected procedures, such as midwifery, which involve only limited judgment and decision making. The American strategy on use of doctor-substitutes for primary care, however, follows from unwillingness to train greater numbers of primary care physicians and to require them to serve in places of need. This results in an inequitable concentration of doctor-substitutes on service to the poor in both urban and rural areas.


Is the alkaline cleavage of disulfide bonds in peptides an alpha-beta elimination reaction or a hydrolysis? The cleavage of oxidized glutathione by alkali has been studied as representative of the cleavage of protein disulfides. This process is quite different when studied in 10-4N or 2-10-1N NaOH. At low alkali concentration no spectral changes are noted; at higher hydroxyl concentration the appearance of persulfide groups (followed at 335 nm), the formation of thiocyanate (arising from cold cyanolysis of persulfide groups) and the absorbance at 240 nm follow the same kinetics. The amount of half-cystine, recovered as cysteic acid after a 3-h reaction, is significantly lower than calculated. These results confirm that oxidized glutathione undergoes beta-elimination at high pH values, and that persulfide groups absorb not only at 335 nm (as already known) but also at 240 nm where, under our conditions, the contribution of other absorbing species is not very high.


Modification of lipoxygenase by hydrogen peroxide and photooxidation. The kinetic study of fluorescence stopped-flow method suggested that the interaction between lipoxygenase and H2O2 is consistent with a simple irreversible one-step mechanism. The activation energy of the reaction was 7.2 kcal/mol. Participation of an ionizable group with pK about 8.8, possibly a histidine residue, was suggested from the pH-dependence of the rate constant. No further fluorescence quenching of lipoxygenase was observed when the product was added to the lipoxygenase solution before mixing the lipoxygenase and H2O2 solutions. The fluorescence quenching of lipoxygenase by H2O2 was in parallel with the inactivation of the enzyme. Hydroperoxylinoleic acid strongly protects the inactivation of lipoxygenase caused by H2O2. These results are consistent with an interpretation that OH- and/or O- - are produced when the iron of the enzyme is oxidized by H2O2, which in turn will attack some amino acid essential for the enzyme activity. The pH-dependence of the inactivation rate constant of photooxidation of lipoxygenase sensitized by methylene blue indicated that an ionizable group with pK about 8.8 is concerned with the enzymatic activity. In contrast to the inactivation of lipoxygenase by H2O2, the product protected the inactivation of the enzyme by photooxidation only at high concentration.


The effect of additives on the free radical formation in aqueous solutions of ascorbic acid. The relative rate constants for the decay of ascorbate free radical in aqueous solutions in the presence of heavy metal ions, hydrogen peroxide and sulphite were measured the ESR-spectroscopy. The oxidation of ascorbic acid showed a strong pH dependence, reaching a maximum rate at pH 9.6. It was shown that the ascorbic acid radical decay generally follows overall second order kinetics, being however first order in the presence of hydrogen peroxide. The protective effect of sulphur dioxide is proposed to have nutritional implications.


Comparison of the effects of timolol and other adrenergic agents on intraocular pressure in the rabbit. The effect of timolol, propranolol, epinephrine, and isoproterenol on intraocular pressure (IOP) (measured by tonometry) were compared after topical administration in conscious rabbits. Epinephrine and isoproterenol decreased IOP in normotensive rabbits, whereas propranolol had no effect. Timolol produced only a slight and inconsistent lowering of IOP in normotensive rabbits. All four agents reduced IOP elevated by an oral water load; the adrenergic agonists were substantially more active than the two beta-adrenergic blocking agents. In alpha-chymotrypsin-induced ocular hypertension, epinephrine, isoproterenol, and timolol were essentially equally effective, whereas propranolol exhibited only weak activity. In this latter model, timolol did not lose its effectiveness after multiple instillations (three/day) over an 8-day period. The concentration of timolol in the acqueous humor after topical application of effective hypotensive doses was relatively high as compared to that found in plasma. In addition, topical doses of timolol required to lower IOP were considerably greater than those needed to reduce or block the ocular hypotensive activity of isoproterenol. The mode of action and therapeutic implications of beta-adrenergic blocking agents in glaucoma are discussed.


Studies on avian erythrocyte metabolism. VII. Effect of inositol pentaphosphate and other organic phosphates on oxygen affinity of the embryonic and adult-type hemoglobins of the turkey embryo. The effects of 2, 3-diphosphoglyceric acid (2, 3-DPG), adenosine triphosphate (ATP), inositol tetraphosphate (ITP), inositol pentaphosphate (IPP), and inositol hexaphosphate (IHP) on oxygen affinity of whole stripped hemoglobin (WSH), hemoglobin H (Hb-H; hatching hemoglobin), hemoglobin A (Hb-A), and hemoglobin D (Hb-D) isolated from erythrocytes (RBC) of the 25-day turkey embryo have been studied. The order of the decrease in oxygen affinity induced by these organic phosphates, at molar ratios of phosphate compound to hemoglobin (tetramer) between 2 and 4, is 2, 3-DPG less than ATP less than ITP less than IPP less than IHP. 2, 3-DPG shows a slightly greater effect on reducing oxygen affinity of Hb-H than on either adult-type hemoglobin. The effect of IPP upon lowering the oxygen affinity of either WSH, Hb-H, Hb-A, or Hb-D is approximately 20 percent less than IHP. The effects of the various organic phosphates upon the Hill constant, n, of these purified hemoglobins is variable but appears to reach a maximum when the molar ratio of organic phosphate to hemoglobin (tetramer) is 2 or greater. None of the physiologically occurring organic phosphates has a significant preferential interaction with any specific hemoglobin. These experiments strengthen and support our earlier conclusion, that the changes in whole blood oxygen affinity which occur during avian development result from the changes in composition of the intraerythrocytic organic phosphates.


Studies on the physico-chemical properties of alhagain. The protease isolated jawasee shrub was found to hydrolyze egg albumin, casein, haemoglobin and gelatin optimally near neutral pH. Fibrin, bovin serum albumin, skin albumin and skin mucoids were hydrolyzed at slightly alkaline pH, while skin globulins were hydrolyzed at slightly acidic pH. The enzyme had no effect of fibrous collagen. The optimum conditions for the hydrolysis of 50 mg of egg albumin were found to be 50 mg of alhagain at pH 6.0 and 45 degrees C for 30 minutes. A Km value of 4.4 X 10(-3) M was obtained from the Lineweaver-Burk plot for the hydrolysis of egg albumin. The enzyme was found to be comparatively thermostable and was most stable at pH 4.7. Ultraviolet irradiation exhibited no appreciable effect on the enzyme activity. The ultraviolet absorption spectrum of alhagain in bi-distilled water resembles those of bromelain and trypsin. The sugar-containing enzyme was found to have a molecular weight of 20,650. The enzymeconsists of 189 amino acid residues per molecule, neutral and acidic amino acids being present in high concentrations. The partial specific volume of alhagain was calculated to be 0.743 ml/g from its amino acid composition. Phenylalnine and arginine formed the amino terminal amino acids of alhagain, while aspartic acid and serine were identified as its carboxy terminal amino acids. Results are discussed with relation to other plant proteases.


Stearoyl-CoA desaturase in mitochondrial membrane fractions. Several arguments suggested the possibility that the stearoyl-CoA desaturase might be located in the outer mitochondrial membrane in addition to its well presence in the microsomes. In the present investigation, preparations of rat liver microsomes and submitochondrial fractions (outer and inner membranes) were comparatively studied with respect to their stearoyl-CoA desaturase activities. Each fraction has been characterized by determinations of enzymic and chemical markers. This study revealed that while the activity of the stearoyl-CoA desaturase in microsomes was comparable to that found in other laboratories, a very low level of activity was detected in the mitochondrial outer membrane. The possible implications of the lack of stearoyl-CoA desaturase in mitochondria are discussed with respect to the lipid metabolism of these organelles.


Comparison of the activities of some dehydrogenases in the juxtaglomerular complex of kidneys of Wistar rats and desert rats (Meriones unguiculati). The authors compared the enzyme histochemical activities of some dehydrogenases in the macula densa, the Goormaghtigh cells and the epithelioid (or juxtaglomerular cells in the kidneys of desert rodents (Mongolian Gerbils) with those of the Wistar rats. The macula cells (Table 1), which in the Wistar rats are clearly distinct from the non specific epithelial cells of the distal convolution, show, in the desert rats, noticeable fluctuations. Their enzyme histochemical reactions are often weaker than those of the distal convolution cells, with the exception of the NAD-tetrazolium-reductase activity. The Goormaghtigh cells (Table 2) in the kidneys of the Meriones have a much larger enzymatic spectrum than in the Wistar rats. Here also, we find functional variations in the examined desert species. In the epithelioid cells (Table 3) we observed a somewhat weaker enzymatic activity in the Meriones. These cells contain no secretion granules, this making their diagnosis difficult.


Influence of temperature on hemoglobin-ligand interaction in whole blood. Temperature-dependent change in hemoglobin-oxygen affinity was measured as a function of hemoglobin-oxygen saturation. In addition, the CO2 Bohr factor and fixed acid Bohr factor were measured as a function of saturation of temperatures of 23, 30, 37, and 44 degrees C. Measurements were made on normal blood and blood with reduced 2,3-diphosphoglycerate (DPG). The influence of temperature is greatest at low saturation and is enhanced slightly by DPG depletion. The CO2 Bohr factor is increased at high temperatures; this is primarily due to increased carbamino formation with rising temperature, especially at lower oxygen saturation. The effect of DPG on oxygen affinity is reduced at a high temperature and elevated at low temperature. These diverse effects of temperature on hemoglobin-ligand interaction require consideration in assessing oxygen delivery when temperature is increased or decreased.


Regulatory mutations in the Klebsiella aerogenes structural gene for glutamine synthetase. Glutamine synthetase could be repressed several hundredfold rather than 6- to 10-fold as previously reported. Ammonia was not the primary repression signal for glutamine synthetase. Repression appeared to be mediated by a high level of glutamine and probably by a high ratio of glutamine to alpha-ketoglutarate. Mutations in glnA (the structural gene for glutamine synthetase) were seen to fall into three phenotypic groups: glutamine auxotrophs that produced no detectable glnA product; glutamine auxotrophs that produced a glnA product lacking enzymatic activity (and hence repressibility by ammonia) but were repressible under appropriate conditions; and glutamine synthetase regulatory mutants, whose glnA product was enzymatically active and not repressible under any conditions.


Autogenous regulation of the synthesis of glutamine synthetase in Klebsiella aerogenes. We isolated an F' episome of Escherichia coli carrying the glnA+ gene from K. aerogenes and an F' episome of E. coli carrying the glnA4 allele from K. aerogenes responsible for the constitutive synthesis of glutamine synthetase. Complementation tests with these episomes showed that the glnA4 mutation (leading to the constitutive synthesis of active glutamine synthetase) was in the gene identified by mutations glnA20, glnA51, and glnA5 as the structural gene for glutamine synthetase. By using these merodiploid strains we were able to show that the glnA51 mutation lead to the synthesis of a glutamine synthetase that lacked enzymatic activity but fully retained its regulatory properties. Finally, we discuss a model that explains the several phenotypes associated with mutations such as glnA4 located within the structural gene for glutamine synthetase leading to constitutive synthesis of active glutamine synthetase.


Carbon monoxide oxidation by methanogenic bacteria. Different species of methanogenic bacteria growing on CO(2) and H(2) were shown to remove CO added to the gas phase. Rates up to 0.2 mumol of CO depleted/min per 10 ml of culture containing approximately 7 mg of cells (wet weight) were observed. Methanobacterium thermoautotrophicum was selected for further study based on its ability to grow rapidly on a completely mineral medium. This species used CO as the sole energy source by disproportionating CO to CO(2) and CH(4) according to the following equation: 4CO + 2H(2)O --> 1CH(4) + 3CO(2). However, growth was slight, and the growth rate on CO was only 1% of that observed on H(2)/CO(2). Growth only occurred with CO concentrations in the gas phase of lower than 50%. Growth on CO agrees with the finding that cell-free extracts of M. thermoautotrophicum contained both an active factor 420 (F(420))-dependent hydrogenase (7.7 mumol/min per mg of protein at 35 degrees C) and a CO-dehydrogenating enzyme (0.2 mumol/min per mg of protein at 35 degrees C) that catalyzed the reduction of F(420) with CO. The properties of the CO-dehydrogenating enzyme are described. In addition to F(420), viologen dyes were effective electron acceptors for the enzyme. The apparent K(m) for CO was higher than 1 mM. The reaction rate increased with increasing pH and displayed an inflection point at pH 6.7. The temperature dependence of the reaction rate followed the Arrhenius equation with an activation energy (DeltaHdouble dagger) of 14.1 kcal/mol (59.0 kJ/mol). The CO dehydrogenase activity was reversibly inactivated by low concentrations of cyanide (2 muM) and was very sensitive to inactivation by oxygen. Carbon monoxide dehydrogenase of M. thermoautotrophicum exhibited several characteristic properties found for the enzyme of Clostridium pasteurianum but differed mainly in that the clostridial enzyme did not utilize F(420) as the electron acceptor.


Efficient sporulation of yeast in media buffered near pH6. Diploid cells of Saccharomyces cerevisiae underwent meiosis and sporulation when placed in 1% potassium acetate sporulation medium. In unbuffered sporulation medium the pH rose very rapidly, reaching pH 8.4 after 2 h of sporulation. Under these conditions, the uptake of radioactive adenine and lysine was extremely limited, and ascus formation was insensitive to inhibitors such as 5-fluorouracil and canavanine. By using several different buffers, we showed that an increase in the pH of sporulation media was not necessary for sporulation to occur. Spore viability and the kinetics of ascus and prototroph formation were normal for cells sporulated in several types of media buffered as low as pH 5.5. Incubation of sporulating cells below pH 6.5 did cause separation of small but viable buds from their mother cells. With sporulating cells buffered below pH 6.5, the incorporation of radioactive adenine and lysine was greatly enhanced and cells became sensitive to inhibition by 5-fluorouracil and canavanine.


Deoxyribonucleic acid synthesis in permeabilized spheroplasts of Saccharomyces cerevisiae. Osmotically shocked spheroplasts from Saccharomyces cerevisiae incorporated deoxynucleoside triphosphates specifically into double-stranded nuclear and mitochondrial deoxyribonucleic acid (DNA). Results with this in vitro system for cells with and without mitochondrial DNA were compared. Strains lacking mitochondrial DNA were used to study nuclear DNA replication. With a temperature-sensitive mutant defective in DNA replication in vivo, DNA synthesis in vitro was temperature sensitive as well. The product of synthesis with all strains after very short labeling times consisted principally of short fragments that sedimented at approximately 4S in alkali; with longer pulse times or a chase with unlabeled nucleotides, they grew to a more heterogenous size, with an average of 6 to 8S and a maximum of 15S. There was little, if any, integration of these DNA fragments into the high-molecular-weight nuclear DNA. Analysis by CsCl density gradient centrifugation after incorporation of bromodeoxyuridine triphosphate showed that most of the product consisted of chains containing both preexisting and newly synthesized material, but there was also a small fraction (ca. 20%) in which the strands were fully synthesized in vitro. (32)P-label transfer ("nearest-neighbor") experiments demonstrated that at least a part of the material synthesized in vitro contained ribonucleic acid-DNA junctions. DNA pulse-labeled in vivo in a mutant capable of taking up thymidine 5'-monophosphate, sedimented in alkali at 4S, as in the case of the in vitro experiments.


Location and properties of glucose dehydrogenase in sporulating cells and spores of Bacillus subtilis. Late during sporulation, Bacillus subtilis produces glucose dehydrogenase (GlcDH; EC 1.1.1.47), which can react with D-glucose or 2-deoxy-D-glucose and can use nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) as a cofactor. This enzyme is found mainly in the forespore compartment and is present in spores; it is probably made exclusively in the forespore. The properties of GlcDH were determined both in crude cell extracts and after purification. The enzyme is stable at pH 6.5 but labile at pH 8 or higher; the pH optimum of enzyme activity is 8. After inactivation at pH 8, the activity can be recovered in crude extracts, but not in solutions of the purified enzyme, by incubation with 3 M KCl and 5 mM NAD or NADP. As determined by gel filtration, enzymatically active GlcDH has a molecular weight of about 115,000 (if the enzyme is assumed to be globular). GlcDH is distinct from a catabolite-repressible inositol dehydrogenase (EC 1.1.1.18), which can also react with D-glucose, requires specifically NAD as a cofactor, and has an electrophoretic mobility different from that of GlcDH.


Purification and characterization of the membrane-bound ferrochelatase from Spirillum itersonii. The membrane-bound enzyme ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1) was purified from isolated membrane fragments of Spirillum itersonii approximately 490-fold. Purification was achieved by solubilization with chaotropic salts followed by ammonium sulfate fractionation, diethylaminoethyl-cellulose chromatography, and gel filtration on Sephadex G-200. The purified enzyme has an apparent minimum molecular weight of approximately 50,000, as determined by gel filtration in the presence of 0.1% Brij 35 and 1 mM dithiothreitol but forms high-molecular-weight aggregates in the absence of detergent. Purified ferrochelatase is strongly stimulated in the presence of copper. The apparent Km for Fe2+ is 20 micrometer in the absence of copper and 9.5 micrometer in the presence of 20 micrometer CuCl2. The apparent Km for protoporphyrin is 50 micrometer, and it is unaltered by copper. Ferrochelatase has a single pH optimum of 7.50, and it is inhibited 50% by 20 micrometer heme. Certain divalent cations and sulfhydryl reagents also inhibit the enzyme.


Regulation of superoxide dismutase synthesis in Escherichia coli: glucose effect. Growth of Escherichia coli, based upon the fermentation of glucose, is associated with a low intracellular level of superoxide dismutase. Exhaustion of glucose, or depression of the pH due to accumulation of organic acids, causes these organisms to then obtain energy from the oxidative degradation of other substances present in a rich medium. This shift in metabolism is associated with a marked increase in the rate of synthesis of superoxide dismutase. Depression of the synthesis of superoxide dismutase by glucose is not due to catabolite repression since it is not eliminated by cyclic adenosine 3',5'-monophosphate and since alpha-methyl glucoside does not mimic the effect of glucose. Moreover, glucose itself no longer depresses superoxide dismutase synthesis when the pH has fallen low enough to cause a shift to a non-fermentative metabolism. It appears likely that superoxide dismutase is controlled directly or indirectly by the intracellular level of O2- and that glucose depressed the level of this enzyme because glucose metabolism is not associated with as rapid a production of O2- as is the metabolsim of many other substances. In accord with this view is the observation that paraquat, which can increase the rate of production of O2- by redox cycling, caused a rapid and marked increase in superoxide dismutase.


Obligatory coupling between proton entry and the synthesis of adenosine 5'-triphosphate in Streptococcus lactis. Proton influx was measured after imposition of an electrochemical potential difference for protons (delta muH+) across the cell membrane of the anaerobe, Streptococcus lactis. As delta muH+ was increased, there was an approximately parallel increase in proton entry, until delta muH+ attained 175 to 200 mV. At this point, a new pathway became available for proton entry, allowing an abrupt increase in both the rate and extent of H+ influx. This gated response depended upon the value of delta muH+ itself, and not upon the value of either the membrane potential or the pH gradient. For delta muH+ above 175 to 200 mV, elevated proton entry occurred only in cells having a functional membrane-bound Ca2+-stimulated, Mg2+stimulated adenosine 5'-triphosphatase (EC 3.6.1.3). When present, elevated proton entry coincided with the appearance of net synthesis of adenosine 5'-triphosphate catalyzed by this adenosine 5'-triphosphatase. These observations demonstrate that membrane-bound adenosine 5'-triphosphatase catalyzes an obligatory coupling between the inward movement of protons and synthesis of adenosine 5'-triphosphate.


Transformation in pneumococcus: existence and properties of a complex involving donor deoxyribonucleate single strands in eclipse. Donor deoxyribonucleic acid (DNA) single strands exist in a complex during the eclipse phase in pneumococcal transformation. This eclipse complex exhibited specific physical properties distinct from those of both pure DNA single strands and native DNA. These included a lower affinity for diethylaminoethyl-cellulose and hydroxylapatite than that of single-strand DNA, faster sedimentation than the DNA chains that it contains, and a buoyant density in Cs2SO4 lower than that of native DNA. The complex was dissociated by treatments with sodium dodecyl sulfate, NaOH, guanidine-hydrochloride, chloroform, and proteinase K but was insensitive to ribonuclease.


Nitrogen and ammonia assimilation in the cyanobacteria: purification of glutamine synthetase from Anabaena sp. strain CA. Glutamine synthetase was purified from the cyanobacterium Anabaena sp. strain CA, a newly isolated marine organism. This organism grows rapidly under nitrogen-fixing conditions and therefore is ideally suited for studies concerning cyanobacterial nitrogen metabolism. Studies were conducted to optimize the production of glutamine synthetase by Anabaena CA. The highest specific activities were obtained from cells grown in the presence of atmospheric N(2) or KNO(3) (13 mM); when NH(4)Cl was used as the nitrogen source, the specific activity was reduced by approximately 40%. Furthermore, through the use of a whole-cell gamma-glutamylhydroxamate transferase assay, it was found that the maximum number of enzyme units is obtained in the late logarithmic stage of growth. Glutamine synthetase purification requires only three steps and results in a preparation that is electrophoretically homogeneous. The transferase specific activity (units per milligram of protein) of the purified enzyme is 78, whereas the biosynthetic specific activity is 2.2. The molecular weight of the native protein was found to be approximately 590,000, and the subunit molecular weight was determined to be about 50,000. Thus, this cyanobacterial enzyme closely resembles the enzyme obtained from other procaryotic sources, at least with regard to size. The purification of glutamine synthetase from Anabaena CA should stimulate a more detailed study of this enzyme and its role in cyanobacterial nitrogen metabolism.


Secretion of lipids induced by inhibition of peptidoglycan synthesis in streptococci. Inhibition of peptidoglycan synthesis causes an immediate and massive secretion of both newly synthesized and "old" lipids from several species of bacteria, including streptococci, Staphylococcus epidermidis, and Bacillus subtilis. Lipid secretion occurs in the absence of detectable bacterial lysis. This novel phenomenon was examined in more detail in three strains of streptococci: S. sanguis (group H), S. pyogenes (group A), And S. pneumoniae. The secretion of lipids is specifically induced by inhibitors of peptidoglycan synthesis; it is not caused by inhibitors of protein, ribonucleic acid, or deoxyribonucleic acid synthesis. The occurrence appears to be reversible since penicillin-induced secretion comes to a halt upon the timely addition of penicillinase, correlating with resumption of culture growth. All cellular lipids are secreted in essentially the same proportions as those found in the drug treated bacteria. It is suggested that continued peptidoglycan synthesis may be essential for the integration and retention of lipid material in the plasma membrane.


Isolation and characterization of NADP+-specific isocitrate dehydrogenase from the pupa of Bombyx mori. NADP+-specific isocitrate dehydrogenase was found in several tissues of the pupa of the silkworm, Bombyx mori. This enzyme was highly purified from the whole bodies of pupae. This is the first isolation of the enzyme from insect materials. The purified enzyme gave a single protein band on polyacrylamide gel electrophoresis. The reaction catalyzed by the purified enzyme was readily reversible. The pH optimum for the forward reaction (reduction of NADP+) was 7.8, and that for the reverse reaction (oxidation of NADPH) was 6.6. The enzyme had a molecular weight of 86,000 and was found to be composed of two identical subunits, which have a molecular weight of 44,000. The activity of the enzyme in the forward reaction was slightly inhibited by citrate, oxaloacetate, alpha-ketoglutarate, and others. Citrate stabilized the activity over a wide pH region.


Stimulatory effect of n-alkanes on CTP: Cholinephosphate cytidyltransferase activity in rat liver microsomal membranes in vitro. To assess the effect of alteration of membrane structure on the enzymic activities related to phospholipid synthesis in microsomal membrane, the effects of several organic solvents have been studied in an in vitro system, in which the cytoplasmic extract prepared from rat liver incorporated [14C]choline or [14C]CDP-choline into phosphatidycholine (lecithin). The optimum conditions for the incorporation were determined. Among several organic solvents examined, n-alkanes such as n-hexane, n-octane, and n-tetradecane stimulated the incorporation. It was shown that n-alkanes stimulated one of three enzymic steps of lecithin biosynthesis from choline; that is, the formulation of CDP-choline catalyzed by CTP: cholinephosphate cytidyltransferase [EC 2.7.7.15], an enzyme on the microsomal membrane. It was further shown that the same enzyme was also stimulated by preincubation of microsomes in the absence of substrate. It is suggested that alteration of the lipid environment of the microsomal membrane induced by n-alkanes caused activation of this enzymic step.


H+ translocation in lysozyme-treated cells of the blue-green alga Plectonema boryanum. Difference between isotonically and hypotonically treated preparations and effects of divalent cations. Lysozyme-treated cells of a blue-green alga, Plectonema boryanum, had an internal pH of 7.3+/-0.2 under isotonic and hypotonic conditions. This value was similar to that of untreated cells. The CCCP-induced biphasic H+ change seen in the isotonic cells was not observed in the hypotonically treated cells. The biphasic time course remained in the hypotonic preparation if CaCl2 or MgCl2 was added prior to the osmotic shock. It is suggested that the cells have two compartments of H+ concentration. The outer region may be more acidic than the inner region. A light-induced H+ efflux was observed under isotonic conditions and an influx of H+ under hypotonic conditions. The H+ influx was not observed when lysozyme-treated cells were incubated with CaCl2 or MgCl2 prior to the hypotonic treatment. Two types of effects of divalent cations, one on the rigidity of the outer membrane and another on the permeability characteristics of the inner photosynthetic membrane, are indicated. Rearrangement of the photosynthetic membranes and an apparent inversion of the H+ pump by hypotonic shock are also suggested.