[Mycetoma in Somalia - results of a survey done from 1959 to 1964]. The Pasteur Institute studied 103 mycetoma patients in Somalia between 1959 and 1964. Grains were seen in 94 of them and this, added to cultural features, allowed the diagnosis of 60 pathogens as follows: 44 Madurella mycetomi, 1 Leptosphaeria senegalensis, 7 Pyrenochaeta romeroi (or Madurella grisea), 3 Allescheria boydii, 1 Fusarium sp., 3 Neotestudina (Zopfia) rosatii, and 1 unidentified; 34 were actinomycetes: 24 Streptomyces somaliensis, 4 Actinomadura madurae, 3 A. pelletieri and 3 Nocardia spp. The patients delayed too long in consulting their doctors and health education is vital if amputations are to be avoided. The geographical distribution is related to climate and fungal species. In central Somalia the association of M. mycetomi and S. somaliensis, organisms characteristic of desert conditions, was found; white grain mycetomata and those caused by Nocardia spp. occurred in more humid areas. The study revealed 2 new fungi. One, obtained in culture was called Neotestudina (Zopfia) rosatii. The 3 patients affected, lived in Mudugh (2 in El Bur). The other fungus was not identified. It also was recovered from El Bur and one with similar microscopic characters  has been seen in Chad and also in "territoire français des Afars et des Issas". Both fungi are desert species.


[Tropical entomophthoromycoses].

[Tropical entomophthoromycoses]. A review was made about human entomophthoromycosis observed in tropical areas due to fungi of the order Entomophthorales. Rhinophycomycosis is caused by Entomophthora coronata, a cosmopolitan fungus which is only pathogen in wet tropical and subtropical climates. The disease, a muco-cutaneous infection of the face, induces monstruous deformations. Basidiobolomycosis is caused by Basidiobolus meristosporus, a saprophyte of different amphibians and reptiles very often present in the family Agamidae. The disease interests the subcutaneous tissues and is mainly located at the trunk, shoulders and upper part of the limbs. The epidemiology, clinic and therapeutic were reported. Histopathological lesions and morphology of both fungi were studied. The etiology of the casual agents of these mycoses was discussed.


Tryptophan metabolism in baboons: effect of riboflavin and pyridoxine deficiency. Seven urinary metabolites of tryptophan-kynurenine pathway were measured in riboflavin-deficient, pyridoxine-deficient, pair-fed and natural diet baboons. The most significant changes in the pyridoxine-deficient baboons was a mean sevenfold increase in the excretion of xanthurenic acid and a threefold decrease in 3-hydroxy anthranilic acid. The riboflavin-deficient baboons showed a twelvefold increase in the excretion of anthranilic acid, and a tenfold decrease in 3-hydroxy kynurenine. Red blood-cell pyridine nucleotides decreased only in the pyridoxine-deficient baboons, while plasma 11-hydroxy corticosteroids increased only in the riboflavin-deficient baboons. These results are discussed in relation to enzymatic changes that may be expected during these deficiencies.


On the determination of free L-tryptophan. The determination of free L-tryptophan depends on several analytical well checked points e.g. absorbing effects, pH of the serum, dependence of the throughput, concentration dependence of retention, volume ultrafiltrated, cut-off, temperature dependence of the binding rate, purification on ion-exchange column, interfering substances of the fluorometric method. Ultrafiltration was compared to an ultracentrifugation technique. In normal human serum the concentration of free L-tryptophan is 3,1 +/- 0,6 microgram/ml and of the bound plus free form 12,2 +/- 1,12 microgram/ml. Both values are dependent on the albumin concentration.


Studies on the function of tryptophan-108 on lysozyme. Chemical studies of selective modification of Trp-108 of lysozyme gave ambiguous results concerning its function on the catalytic activity, since the oxyndole derivative obtained with N-bromosuccinimide is inactive, whereas the kynurenine derivative obtained by oxidation with ozone is fully active. In order to explain this discrepancy, lysozyme has been modified with 2-nitro-4-carboxyphenylsulfenyl chloride (NCPS-Cl). This reagent reacts with the indole ring of tryptophan giving a 2-thioaryl-derivative. By chromatographic fractionation of the reaction mixture, a lysozyme derivative was isolated, that by sequence studies was proved to be modified only at Trp-108 retaining 10% of the lytic activity. Physico-chemical as well as kinetic studies indicate that the large decrease in activity following modification could be related to minor effects in the microenvironment of the active site, with a concomitant modification of the ionization constants of the groups involved in catalysis.


T antigen synthesis and resistance to interferon in human adenovirus type 12 infected chick cells. Tye specific T antigen formation has been demonstrated in primary and secondary chick embryo cells (CEC) infected with adenovirus type 12. The frequency of cells synthesizing T antigen was closely dependent on the multiplicity of infection (MOI). At a MOI of 2.85 TCD50 per cell, T antigen was formed in 50% of cells. Cycloheximide inhibited T antigen formation while cytosine arabinoside had no such effect. CEC infected with adenovirus 12 produced interferon and T antigen, both appearing early and at about the same time of infection Exogenous chick interferon had no inhibitory effect on the formation of T antigen. In adenovirus 12-infected CEC, virus specific structural antigen could not be detected.


Scanning electron microscopy of L-929 cells exposed to interferon and reovirus. L-929 cells were studied under the scanning electron microscope (SEM) in the course of reovirus infection with and without prior interferon treatment. Two major stages in the cytopathic effect (CPE) were identified on the basis of fine surface morphology as revealed by SEM. Uninfected control cells were spindle-shaped with microvilli and numerous filopodia and were firmly attached to the substratum. In stage 1 of CPE, the cells lose filopodia and develop large blebs. Stage 2 is characterized by undulating surface and pits on the nearly spherical cells which are devoid of microvilli and filopodia. At all time intervals observed post infection, interferon-treated reovirus-infected cells showed more advanced CPE than the non-interferon-treated reovirus-infected counterpart controls.


The effect of supraoptimal temperature on the formation of pseudorabies virus particles. Replication of virulent and attenuated strains of pseudorabies virus (PRV) at supraoptimal temperature was inhibited in L cells non-adapted to 40 degrees C. Electron microscopy revealed the prevalence of hollow core particles without nucleoid; both the nuclei and cytoplasm of L cells were markedly altered at supraoptimal temperature. In BHK-21 cells adapted to 40 degrees C only replication of attenuated strains was inhibited whereas the virulent ones reached high titres at this temperature. The particles produced at 40 degrees C by attenuated strains were non-infectious but morphologically normal. The ability of attenuated PRV strains to reproduce in adapted BHK-21 cells at 40 degrees C was in correlation with the degree of their attenuation. Changes in the nuclei and cytoplasm of adapted BHK-21 cells at supraoptimal temperature were not as marked as in L cells.


The course of influrenza infection in mice with graft-versus-host reaction. The course of influenza infection in mice with a developed graft-versus-host reaction (GVHR) was changed. Due to disturbances in the inflammatory process the pneumonia was delayed and less marked. Consequently, the infected mice died later than controls. Influenza virus reproduction in the lungs was more intensive and its persistence more prolonged. Interferon production in the lungs of mice with GVHR was similar to that in the controls.


Evaluation of the effectiveness of receptor destroying enzyme preparations. A procedure has been developed for testing receptor destroying enzyme (RDE) preparations used to remove nonspecific inhibitors before carrying out haemagglutination inhibition tests with influenza virus. Four criteria should be taken into account: (1) titre of RDE, usually used to indicate the activity of the preparation; (2) neuraminidase activity of the preparation, determined biochemically, which partially corresponds to the RDE titre; (3) direct demonstration of the complete removal of nonspecific inhibitors; and (4) determination that the RDE preparation does not affect specific antibody.


Induction of interferon in mice and cell cultures by Mycoplasma pneumoniae. Inoculation of mice and L and human embryonic lung (HEL) cell cultures with Mycoplasma pneumoniae failed to induce the production of interferon. M. pneumoniae multiplied in these cell cultures without a marked cytopathic effect. M. pneumoniae induced interferon in human peripheral blood leukocytes with maximum titres of 32 units/ml at 48 hours after infection.


Electron microscopic studies of the replication of mouse cytomegalovirus in mouse embryo cells. During the replication a pathogenic and a non-pathogenic strain of mouse cytomegalovirus in mouse embryo cells, three types of progeny virus particles were observed in both cases; the virus yields of the two strains differed considerably.


Autoantibodies in sera of rabbits immunized with influenza virus. Rabbits immunized with purified influenza A (H3N2) virus within 4 weeks developed autoantibodies of SMA type (smooth muscle antibodies). In some of them also ABBA (antibodies against brush border of proximal renal tubuli) and ANA (anti-nuclear antibodies) were detected. This autoimmune response was found to be unrelated to either the virus dose or the adjuvant used for immunization. Autoantibodies were not parallel in titre with influenza antibody.


Ability of human polymorhonuclear blood cells to produce interferon after induction with phage double-stranded RNA. The ability of human peripheral blood leukocytes to produce interferon in response to phage double-stranded (ds) RNA was studied. Under the conditions used, interferon was produced not only by lymphocytes but also by polymorphs and monocytes. These "pure" cultures showed no marked differences in the degree of interferon production as compared with the mixed leukocyte culture. The involvement of polymorphs in the production of interferon induced by phage ds RNA is discussed.


Biochemical studies of pancreatic juice collected by duodenal aspiration and endoscopic cannulation of the main pancreatic duct. Biochemical studies of pancreatic juice collected by duodenal aspiration and endoscopic cannulation of the main pancreatic duct were performed and compared in the same fasted normal subjects in seven cases. The comparison of the results shows that under the same stimulation (0.5 CU/kg/hr of GIH secretin and 75 ng/kg/hr of cerulein) the endoscopic cannulation of the main pancreatic duct provides the best values for the maximal secretory capacity. On the other hand, this technique is no better than our current method of duodenal aspiration for the exploration of the enzyme secretion.


Eastern equine encephalitis in Massachusetts, 1957-1976. A prospective study centered upon analyses of mosquitoes. Reappearance of eastern equine encephalitis (EEE) in Massachusetts residents in the 1970's provided an opportunity to assess the predictive value of data on rainfall, EEE in horses, and carriage of EEE virus (EEEV) by mosquitoes, factors which had been studied annually since the last EEE outbreak in 1955-1956. The cycle of multiple cases during 1973-1975 started in a second consecutive year of rainfall that exceeded the annual mean by more than 20 cm, conditions recapitulating the 1955-1956 experience. In 1973, widespread EEE fatalities in horses presaged human cases, another recapitulation of the 1955-1956 experience. However, in 1974, when horses were immunized extensively, no equine cases were seen even though three human fatalities occurred. An unseasonably early appearance of EEEV in mosquitoes was the only basis upon which the threat to humans could have been recognized. These changes in the recognition and distribution of EEEV activity from season to season illustrate the difficulty in making rational decisions regarding widespread aerial insecticide applications for mosquito control.


The oxygen affinity of concentrated human hemoglobin solutions and human blood. The log P50 of normal human blood at 37 degrees C, PCO2 = 0, 21, 42, AND 57 MM Hg in the absence and in the presence of 2,3-DPG and ATP, has been determined in the pH range 7.0 to 7.6. Similar data have been obtained for human hemoglobin isotonic solutions at different protein concentrations in the presence of various amounts of each of the cofactors which are known to affect hemoglobin oxygen affinity in blood. It has been found that the addition of KC1, organic phosphates, magnesium ions, and CO2 confers to a 32% human hemoglobin solution the same oxygen affinity (over the entire physiological pH range) of whole blood. Thus there is no room for significant effects caused by some other unidentified molecules or ions.


The effect of drugs on fetal heart rate variability. Baseline FHR variability has been quantitated into the mathematical indices of differential index (short-term variability) and interval index (long-term variability). Normal values have been determined. The effect that drugs used during labor have on FHR baseline variability has been evaluated. A clinically meaningful change in variability has been observed following low-dose administration of Demerol, morphine, Nisentil, Phenergan, and Vistaril. A statistically significant increase in variability has been observed following administration of magnesium sulfate.


Aerobic glycolysis in bone: lactic acid production by rat calvaria cells in culture. Considerable data have been accumulated on aerobic glycolysis of intact bone preparations. To test whether aerobic glycolysis is a feature of the bone cells themselves or of the localized conditions within an intact bone, cells isolated from rat calvaria were cultured and the effect of several factors on lactate production was determined. These cells drastically decreased lactate production when the pH in the culture medium was lowered, changing from 100 to 20 percent for a pH shift from 7.4 to 6.75. L-lactate inhibited its own formation by 40 percent at 20 mM. Parathyroid hormone (PTH) (820 U/mg) at a concentration ranging from 0.2 to 5.0 U/ml stimulated slightly the lactate production in a log-linear response, the ratio treated over control changing from 1.1 to 1.3. The maximal stimulation is observed at pH 7.0. Isolated cells respond qualitatively the same as did intact calvaria. Quantitatively, there were significant differences: notably, a smaller response to parathyroid hormone and a higher sensitivity to lowered pH.


Time course of the cerebral circulatory response to metabolic depression. Baboons anesthetized with halothane and N2O/O2 were given an intravenous steroid anesthetic (Althesin; Glaxo Laboratories Ltd., U.K.). The drug bolus was labeled with 99mTc, and the time from central venous injection to peak radioactivity in the brain was designated drug brain arrival (DBA-peak). The electroencephalogram slowed 1.2 +/- 0.9 s after DBA-peak (P greater than 0.2), and approximately 2 s after DBA-peak, internal carotid blood flow (ICarBF) decreased and calculated internal carotid vascular resistance (ICarVR) rose. During this 2-s delay in the cerebrovascular response to the arrival of a cerebral metabolic depressant in the brain, the decrease in mean cortical Pco2 was calculated to be less than 0.26 mmHg from cortical CO2 solubility, and less than 0.32 mmHg from cortical CO2 diffusivity, which indicated that mean cortical Pco2 changes do not control cerebral blood flow (CBF). The unaltered time course of the changes in EEG, ICarBF, and ICarVR after acute cervical sympathectomy and alpha-adrenergic receptor blockade excluded the involvement of the sympathetic nervous system in the vasoconstrictor response. Intracarotid Althesin showed that the cerebral vasoconstriction was not a direct effect of the drug. The postulated link between the effects of Althesin on CBF and cerebral metabolism remains to be elucidated but is probably indirect, involving the brainstem.


Antipsychotic drugs, neurotransmitters, and schizophrenia. Inhibition of central dopamine functions appears to be a common basic property of antipsychotic drugs. The mesolimbic and nigrostriatal portions of the dopaminergic system are probably the main targets for the mental and the extrapyramidal actions, respectively, of these drugs. The fact that dopaminergic hyperfunction induced by amphetamines or dopa may lead to a disturbance mimicking paranoid schizophrenia lends further support for a key role of dopamine in mental functions. Although a primary disturbance in dopamine function in schizophrenia cannot be ruled out, the intimate relationship between dopaminergic and other neuronal systems must be emphasized. The possible involvement of other amine, amino acid, or peptide transmitters in schizophrenia cannot be disregarded.


Quality assessment of a telephone care system utilizing non-physician personnel. An approach to providing medical care by telephone is described and its content and quality assessed by means of an outcome study. Pediatric health assistants have been trained to evaluate, triage and offer home management advice in lieu of an office visit for medical problems presented by parents via telephone. During a four-week study period, data were collected on all calls (N = 2520) using a telephone encounter form. Fifty-nine per cent of all calls involved requests for care of illness, 30 per cent of which were independently handled by the health assistant. Quality of care was evaluated for those cases who received advice in home management by means of a seven day follow-up interview with parents (N = 247). Access, parent satisfaction, residual symptoms, and the need for further care were ascertained. Greater than 90 per cent of parents expressed satisfaction and 92 per cent of problems had resolved. Results indicate that this telephone care system can effectively sort out and advise parents in home management for the many minor problems which occur in a pediatric practice, thereby increasing time for direct contact with patients.


Physicians and non-physician health practitioners: the characteristics of their practices and their relationships. Six primary care practices which utilize both physician and non-physician practitioner types were studied to measure differences between practitioner types in the care of patients. By chart review 1,369 patient-practitioner encounters were examined. Physicians identified less symptoms and signs in their patients and prescribed less non-drug therapies than did non-physicians. Likewise, at follow-up visits, physicians tended to document less follow-up of these types of problems and therapies than non-physicians. When examining the interaction between practitioners, the highest rates of follow-up of all types of problems and therapies were found when the same practitioner saw the patient at two successive visits to the same clinic. When a physician saw a patient following a previous visit to a nurse practitioner, there was a significant drop-off in the follow-up rate of problems and therapies. However, when a nurse practitioner saw the patient following a previous visit to a physician, the drop-off in follow-up rates was not as striking. These findings indicate that the skills of physician and nonphysician practitioners are potentially complementary. However, this potential is not fully exploited, particularly by physicians.


Relationship of transmural electrical potential difference to changes in gastric mucosal permeability to H+ and blood flow. Chambered in vivo wedges of proximal canine gastric mucosa were used to evaluate the relationship between PD, net H+ and Na+ fluxes, and gastric mucosal nutrient blood flow, as adjudicated by the ability of the mucosa to clear aminopyrine. Assuming that steady state conditions existed during the study, the results indicate that (1) the mean PD/15 minute period is a significant linear function of both net H+ and net Na+ flux and therefore, presumably, of changes in gastric mucosal permeability to cations, and (2) the mean PD/15 minute period is a significant exponential function of gastric mucosal nutrient blood flow, marked reductions in PD occurring at flows of 1 to 1.5 ml or less. If the thesis that acute posttraumatic hemorrhagic gastritis is a consequence of concomitant alterations in gastric mucosal permeability to H+ and reductions in mucosal blood flow is correct, the PD might represent a useful clinical tool for detecting, in susceptible patients, the evolutionary stages of this devastating complication of trauma.


Secretory state of gastric mucosa and its resistance to aspirin injury. The influence of the secretory status on gastric mucosal tolerance to aspirin injury was assessed in a canine ex vivo model, which provided two segments of mucosa supplied by a single vascular pedicle in the same dog. Acid secretion was stimulated by intravenous infusion of histamine in one mucosa, whereas the secretory response to histamine was significantly impaired by topical treatment with 16,16-dimethyl prostaglandin E2 in the other. As expected, the secreting mucosa exhibited a twofold greater blood flow than the inhibited mucosa. Exposure of both mucosae to 20 mM aspirin for 30 minutes resulted in the appearance of small multiple superficial erosions. The degree of mucosal injury was not significantly different between the secreting and inhibited mucosae. The extent of mucosal damage was similar to that seen when aspirin in acid was placed on resting mucosa in the same model. These data do not support the hypothesis that histamine-stimulated acid secretion and accompanying increase in gastric mucosal blood flow protects the stomach against aspirin injury.


[Social-gerontological theories of the situation in the lives of the aged--a discussion on geroprophylaxis (author's transl)]. The contribution discusses the most important theories which chiefly refer in a mainly sociologically orientated way to the explanation of the various forms of the process of aging: activity- and disengagement-theory. Both theories have had an exceptionally strong influence on scientific soziological research since they had become known and the result of this research has made a modification of the theoretical onset necessary. The conclusions to be drawn under socio-economical points of view under, respect of the theory of socialisation, sociology of knowledge and socioeducational points of view will be explained at the end of the contribution.


[The possible use of sociological onsets in theory on the problem of age and surroundings (author's transl)]. The possibility of using sociological theories on practical problems is a main view point for judging their usefulness. From a scientific-theoretical point of view activity- and disengagement-theory are adequately examined in this contribution. By this it is shown that these theories are lacking mainly in precision and consistency; moreover there is a limitation concerning their relevancy for daily use by the lack of the possibility of producing the variables used in the onsets.


[Survey on the gerontological research in the Federal Republic of Germany (author's transl)]. This survey on the gerontological research shows the topical main accents of work in this field in the Federal Republic of Germany: Social services for aged, old-age-security, social and health situation, and older workers' problems are the most important items of the essay. Empirical examinations are dealt with separately. Moreover, there is a prospect on the different types of projects, research institutions and where they are distributed in the different areas and the different ways of financing this research.


[Problems of retirement and their mastering (author's transl)]. Based on four functions (assignment of status and role, making one's living, reasons for social contacts, and structure of time) the importance of work and profession in a society mainly orientated on performance are shown. From this we can conclude that with retirement from the profession a lability of the level of satisfaction can be found. Based on the analysis some measures are discussed which could assist to restabilisation by strengthening the individual competences for overcoming problems connected with loosing professional tasks.


[Psycho-social problems in rehabilitation (author's transl)]. This contribution contains the results of a study on rehabilitation of patients undergoing treatment in the Alpen-Sanatorium in Bad Tölz, Germany, which was planned as a prestudy for an extensive examination of psycho-social problems of this group of patients and their chances for rehabilitation. The especially important age-group of those between 50- and 60-years-old for a successful geroprophylaxis was chosen. In this essay the connections between graded seriousness of the illness and the social classes of the patients as well as the following problems: difficulties, situated in the personality, general satisfaction with place in life, real worries, disturbances in childhood and youth, and decisive turning points in life, are worked out.


[Aging and the risk of illness (author's transl)]. The necessity to conform with the fact of being ill leads to some psycho-social stress which can increase the risk of falling ill in aging persons in numerous ways. Different tendencies in theory (concepts in activity, disengagement and continuity) contain hints on those conditions which guarantee an individually successful taking over the role of old age and this adds to the decrease of the risk of falling ill. Aimed at health-education carried out in time adds to the chance of an adequate behaviour of old people concerning health and illness.


[Determinants of the Knowledge of the human aging process (author's transl)]. 1800 man and women were interviewed to get informations on there knowledge of the human aging process. The results showed, that the validity and the amount of these informations correlated with education and socio-economic status.


Hypoxaemia in elderly patients suffering from fractured neck of femur. The purpose of this study was to assess the degree and frequency of hypoxaemia in elderly patients with fractured neck of femur and to note the effect of manipulation and internal fixation on these measurements. Twenty-seven patients aged between 44 and 93 were studied for 5 days after the fracture and for 5 days after surgery. The fractures were treated by internal fixation under either general anaesthesia or spinal analgesia. The arterial oxygen tension following the fracture but before surgery was low relative to the ages of the patients, and the arterial carbon dioxide tension was low in twenty-five out of twenty-seven patients. There was also a significant increase in the dead space/tidal volume ratio (VD/VT) and the alveolar-arterial oxygen tension difference, (A--a)DO2 in eight out of nine patients in the first 5 days following the fracture. The mean arterial oxygen tension was still reduced up to 5 days postoperatively. The VD/VT ratio returned to normal postoperatively although the (A-a)DO2 remained elevated. The haemoglobin and platelet levels following the fracture and before surgery were low, although not remarkably so, and there was no significant change post-operatively. There were no significant changes in the electrocardiograph or the chest radiographs. These results and their implications are discussed.


The effect of diazepam and lorazepam on awareness during anaesthesia for Caesarian section. One hundred-and-eighty-three patients having general anaesthesia for Caesarian section were given lorazepam or diazepam intravenously immediately following delivery. The incidence of unpleasant recall was comparable with the reports of other workers. There was no evidence of a specific retrograde amnesic effect by either drug. Neither agent appeared to affect cardiovascular stability, uterine contraction, the reversal of muscle relaxant or the time to recovery of protective reflexes. The patients tended to be drowsier after lorazepam but as safe as after diazepam so far as circulation, respiration and protective reflexes were concerned.


APUD cells and the apudomas. A concept relevant to anaesthesia and endocrinology. A variety of cells found in the pituitary and pineal glands, sympathetic nervous system and adrenal glands, the gut, pancreas, thyroid (C-cells), chemoreceptors (type I-Cells), lungs (P-cells), skin (melanocytes) and the urogenital tract have a common origin from the neural crest. These cells are programmed for neuro-endocrine function and, as a group, can be regarded as one of the physiological control systems. They secrete a variety of amine and peptide hormones and have common cytochemical characteristics from which the term APUD cell is derived. Tumours of these cells are referred to as 'apudomas' and may synthesise not only their own hormones but also those which are normally produced by other APUD cells. The relevant physiological properties of some of the peptides which have been described relatively recently are discussed and the principal clinical syndromes produced by the APUDomas are described.


[Anaphylactoid reactions following administration of plasma substitutes in man. Prevention of this side-effect of haemaccel by premedication with H1- and H2-receptor antagonists (author's transl)]. In a randomized controlled single blind trial in 50 volunteers the problem was investigated whether a combination of dimethpyrindene (0.1 mg/kg b.w.) and cimetidine (10 mg/kg b.w.) could prevent anaphylactoid or allergoid reactions following Haemaccel infusion. In the control group 6 anaphylactoid and 9 allergoid reactions were observed, in the H1 + H2-group, however, none of those reactions occured. No single wheal could be detected. It is considered appropriate to recommend the general use premedication of an H1 + H2-blocker before all anaesthetics and operations. This mainly depends on further clinical trials with the antihistaminic drugs concerning their side-effects under various conditions.


[Morphokinetic reactions in the thyroid, parathyroid, and tracheal glands of the albino rat after anaesthesia with thiobutabarbitone sodium and diethyl ether (author's transl)]. A significant decrease was observed in nuclear volume of the follicular cells of the thyroid gland and the cells of the parathyroid of albino rats subjected to ether anaesthesia, in contrast to generally unchanged nuclear size in animals anaesthetized by thiobutabarbitone sodium. It is suggested that ether exerts its depressant influence mainly by direct action on the function of the follicular cells of the thyroid. Thiobutabarbitone Sodium probably influences thyroid function especially by its antithyroid-drug effects, which have been reported by other authors. The highly significant increase of nuclear volume in the tracheal glands during ether narcosis is referred to the mucous secretion activating stimulus of ether vapors.


Chest pain: coronary or esophageal?

Chest pain: coronary or esophageal? Although it is often relatively easy for the clinician to differentiate between the heart and the esophagus as sources of chest pain, there remains a substantial minority of patients in whom this task is difficult. We have attempted to review points of differential significance which can be elicited through assessment of the patient's symptoms. Diagnostic studies for the detection of esophageal disorders have been reviewed, and the relative usefulness of these studies has been emphasized. As the result of advancements in diagnostic techniques as well as better understanding of esophageal pathophysiology, the clinician is now capable of accurately identifying the esophagus as the source of chest pain.


[Optimal conditions of alpha-amylase production by Aspergillus oryzae in liquid media]. The alpha-amylase secretion in a mineral culture medium containing starch and glucose follow the lysis of mycelium. This lysis seems to result from the hydrolysing action of dextranase and levulanase on cell wall. Cell lysis and amylase secretion are greatly enhanced by pH elevation of culture medium (optimal pH 8,8). In such conditions of production the amylase is not stable but can be stabilized by addition of starch. A method is described using pH and starch content modifications, which allows to obtain an amylase production three times greater than in standard culture medium.


[Study of E. coli penicillin amidase. The pH-dependence of the enzymatic inactivation kinetics]. The pH-dependence of the inactivation rate constant of penicillin amidase at a temperature of 40 degrees C was studied. It was shown that in all cases the enzyme inactivation corresponded to the kinetics of the reaction of the 1st order. The pH-dependence profile was found to be bell-shaped, the effect of transfer from the highest to the lowest values of the inactivation rate constants increasing more than 100 times. On the basis of the data obtained and published earlier it was concluded that the enzyme inactivation proceeded in accordance with the scheme in which out of 3 equilibrium ionic forms of penicillin amidase, i.e. "acid", "neutral" and "alkaline" the neutral form of the active enzyme was most stable. Kinetic analysis of the scheme was carried out and it was shown that the dependence found was in accordance with the theoretical curve in which the pK values of the ionogenic groups controlling the interconvertions between the penicillin amidase forms were equal to 2.4 and 10.1 at a temperature of 40 degrees C. The value of the inactivation rate constant of the "acid" or "alkaline" form was equal to 5.95 min-1, while the "neutral" form of the enzyme was characterized by the inactivation rate constant equal to 5.1.10(-4) min-1. A mechanism for the enzyme inactivation was proposed. According to this mechanism, destruction of the salt bridge in the native structure of penicillin amidase resulted in production of extremely labile forms of the enzyme as compared to the native form.


Intercellular junctions in the organ of Corti. The intercellular junctions between adjacent supporting cells and between apposed hair and supporting cells in the organ of Corti of cat and human were studied. At the endolymphatic surface, the intercellular space was closed by a series of tight junctions (zonula occludens), whereas there were no tight junctions at the basilar membrane surface of the neuroepithelium. Beneath the adlumenal zonula occludens between adjacent supporting cells, a zonula adherens (intermediate junction, "desmosone") was found. Many gap junctions joined apposed supporting cells both within apposed hair and supporting cells, membrane specialization sharing the morphological characterics of both the macula adherens and zonula adherens was found. Between cells, there were short areas of parallel limiting membranes separated by a 20A intercellular space. These areas were suggestive but not characteristic of gap junctional specialization. The functional significance of the junctional specialization between cells in the organ of Corti is discussed.


A pleiotropic mutant of Rhodopseudomonas capsulata defective in nitrogen metabolism. Wild type strains of Rhodopseudomonas capsulata typically can use N2, NH+4, or various nitrogenous organic compounds as N sources for photosynthetic growth. One class of mutants selected for inability to grow on N2 (Nif-) also shows simultaneous loss of capacity to obtain N from numerous organic substrates. When supplied at relatively high concentrations, ammonia can be used as the sole N source for growth of such strains. Enzymatic analysis of one mutant (W11) indicates that the pleiotropic effect on N nutrition is neither due to detectable alteration in the activities of nitrogenase or the initial enzymes responsible for bulk assimilation of ammonia (glutamine synthetase and glutamate synthase) nor to absence of systems required for catabolism of organic N sources. The phenotype of W11 (Nit-; defective in N metabolism) appears to result from loss of ability to grow using low concentrations of ammonia (supplied externally or generated in vivo).


Some properties of formate dehydrogenase, accumulation and incorporation of 185W-tungsten into proteins of Clostridium formicoaceticum. Formate dehydrogenase of Clostridium formicoaceticum used only methyl and benzyl viologen, but not NAD as electron acceptor. The S0.5 values were 0.9 X 10(-4) M for formate and 5.8 X 10(-3) M for methyl viologen. Using potassium phosphate buffer a pH-optimum of 7.9 was observed. The initial velocity of the formate dehydrogenase activity reached a maximum at 70 degrees C, whereas the activity was stable only up to 50 degrees C. The level of formate dehydrogenase in C. formicoaceticum was increased to its maximum when 10(-6) M selenite and 10(-7) M tungstate were added to a synthetic medium. Addition of molybdate instead of tungstate did not increase the level of formate dehydrogenase. 185W-tungsten was concentrated about 100-fold by C. formicoaceticum; molybdate had no major effect on the uptake of tungsten. 185W-tungsten was found almost exclusively in the soluble fluid and was predominantly recovered after chromatography in a protein of about 88000 molecular weight. Occasionally a labelled protein of low molecular weight was observed. Again molybdate added even in high molar excess did not influence the labelling pattern. No radioactivity peak could be obtained at the elution peak of formate dehydrogenase activity. The extreme instability of formate dehydrogenase prevented further purification.


[Metabolic products of microorganisms. 166. Optimization of the desferri-ferricrocin production by Aspergillus viridi-nutans Ducker & Thrower (author's transl)]. At low iron(III)-concentrations (less than 10(-5) M) the fungus Aspergillus viridi-nutans Ducker & Thrower excretes desferri-ferricrocin as the main sideramine into the culture medium. While this compound accounts for 95% of the sideramines produced, small amounts of additional sideramines may also be detected. In a search for an inexpensive nutrient medium for optimum production of desferri-ferricrocin, experiments using shake flasks with good aeration were undertaken initially. The best medium conditions were then employed in a fermentor system. In a 20-1 fermentor with "intensor" system, it was shown that at certain growth rates there was an inverse correlation between rate of growth and rate of sideramine production. A defined nutrient medium of glucose plus acetate as carbon sources, and urea or ammonium acetate as nitrogen sources was used. Two different feeding regimens were used in response to changes of pH or to changes of partial pressure of oxygen in the submerged culture: acetic acid/urea or acetic acid/ammonium acetate additions regulated these conditions. The rate of sideramine production under such feeding achieved a maximum of 20 mg 1(-1) h-1 over a period of several days.


Control of the chemotactic behavior of Bacillus subtilis cells. The effects of nigericin, valinomycin and some lipophilic cations on the motile behavior of non-starved and methionine-straved Bacillus subtilis cells were studied. For valinomycin and nigericin a quantitative relationship between the flux in the proton-motive force and the duration of the twiddle response was found. Lipophilic cations bind to the ion gate controlling the twiddle frequency and thereby cause the cells to swim smoothly. To explain the transmission of the chemotactic signal a model is given in which receptors, a hyperpolarizing wave, an ion gate and two methylation sites, viz. methyl-accepting chemotaxis proteins and a further methylation site (MT), play a role. For the transmission of the signal caused by an attractant both the hyperpolarizing wave and an interaction between receptor and methylation site (MT) are needed. The methyl-accepting chemotaxis proteins are involved in the adaptation/deadaptation to altered levels of attractant. Artificial changes in the proton-motive force act directly on the ion gate, which finally controlls the twiddle frequency of the cells.


Purification and properties of beta-ketothiolase from Zoogloea ramigera. beta-Ketothiolase from Zoogloea ramigera I-16-M was purified 140-fold to electrophoretic homogeneity. The bacterium appeared to contain a single isoenzyme of beta-ketothiolase with a molecular weight of 190 000, as determined by Sephadex G-200 gel filtration. The monomer molecular weight was 44 000, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The native enzyme thus appeared to be a tetramer with identical subunits. The enzyme showed a pH optimum of 7.5 in the condensation reaction, and 8.5 in the thiolysis reaction. The enzyme employed a Bi Bi ping pong mechanism for the forward thiolysis reaction. The apparent Km value for acetoacetyl coenzyme A in the thiolysis reaction was 10 micron, and that for coenzyme A was 8.5 micron. The apparent Km value for acetyl coenzyme A in the condensation reaction was 0.33 mM. The condensation reaction was inhibited by coenzyme A concentrations lower than 0.1 mM. The enzyme was stable in the presence of dithiothreitol and other SH-compounds, but was strongly inhibited by 0.4 mM p-chloromercuribenzoate.


Glucose-6-phosphate dehydrogenase of Anabaena sp. Kinetic and molecular properties. The kinetic and molecular properties of cyanobacterial glucose-6-phosphate dehydrogenase, partly purified from Anabaena sp. ATCC 27893, show that it undergoes relatively slow, reversible transitions between different aggregation states which differ in catalytic activity. Sucrose gradient centrifugation and polyacrylamide gel electrophoresis reveal three pincipal forms, with approximate molecular weights of 120 000 (M1), 240 000 (M2) and 345 000 (M3). The relative catalytic activities are: M1 less than M2 less than M3. In concentrated solutions of the enzyme, the equilibrium favors the more active, oligomeric forms. Dilution in the absence of effectors shifts the equilibrium in favor of the M1 form, with a marked diminution of catalytic activity. This transition is prevented by a substrate, glucose-6-phosphate, and also by glutamine. The other substrate, nicotinamide adenine dinucleotide phosphate (NADP+), and (in crude cell-free extracts) ribulose-1,5-diphosphate are negative effectors, which tend to maintain the enzyme in the M1 form. The equilibrium state between different forms of the enzyme is also strongly dependent on hydrogen ion concentration. Although the optimal pH for catalytic activity is 7.4, dissociation to the hypoactive M1 form is favored at pH values above 7; a pH of 6.5 is optimal for maintenance of the enzyme in the active state. Reduced nicotamide adenine dinucleotide phosphate (NADPH) and adenosine 5'-triphosphate (ATP), inhibit catalytic activity, but do not significantly affect the equilibrium state. The relevance of these findings to the regulation of enzyme activity in vivo is discussed.


The role of vanadium in green plants. III. Influence on cell division of Chlorella. Vanadium, although essential for growth and chlorophyll formation in unicellular green algae, reveals toxic influences on cell division of Chlorella pyrenoidosa, these disturbances arising in the same range of V-concentrations as the known positive effects of the trace metal. In permanent light, as documented by cell volume statistics, vanadium (4-10(-7) g-at/1 as NH4VO3) causes a significant shift of the distribution maxima to higher values of the algal cell volume, the shift having its optimum at 10(-5) g-at V/1. It is documented in pH-constant liquid culture that this effect is not due to a change of pH in the nutrient medium. Under synchronous conditions of algal cultivation (16:8h), vanadium causes a total arrest of cell division after 3 periods; this stop lasts for the next 3 cycles. Afterwards, asynchronous divisions newly occur and lead to generally larger autospores. Staining of algal cell nuclei revealed an inhibitory V-effect on nuclear division, yielding giant nuclei with multiple sets of chromosomes, and thereby limiting cell division. Under these conditions, Chlorella pyrenoidosa is not synchronizable in presence of vanadium.


[Investigations of carbromal metabolism (author's transl)]. Erythro-2-Brom-2-ethyl-3-hydroxybutyramide has been isolated from the urines of patients with Carbromal intoxications as well as from the urine of rat, mouse and dog treated with either Carbromal or with 2-Brom-2-ethyl-acetamide. The identification of the excreted and synthetized metabolite was carried out by means of IR, Mass and NMR spectroscopy. Along with the erythro-2-Brom-2-ethyl-3-hydroxybutyramide one other bromine containing compound was isolated after Carbromal application only, its mass spectrum and rel. RF values having the same characteristics as the synthetized erythro-2-Brom-2-ethyl-3-hydroxybutyrylcarbamide. However, the purification of this metabolite so far has not been successful.


Partial purification of somatomedin from human plasma. Fractional precipitation of human plasma using ethanol, followed by chromatography on S.P. Sephadex, yielded a somatomedin-enriched fraction freed from substantial amounts of inhibitory substances. Heat coagulation of the proteins present in this fraction allowed the recovery of appreciable amounts of active components which were then chromatographed on Sephadex G-75 and S.P. Sephadex. A major part of the activity was associated with components less than 4,000 daltons, suggesting that somatomedin, or an active fragment thereof, had been dissociated from a carrier protein by the heat treatment. The range of pH employed throughout was 5.3-9.8. Recoveries of about 30% of biological activity with fold-purification up to 38, as measured by radioactive sulphate uptake in the chick pelvic cartilage assay, were higher than those obtained using acid-ethanol extraction.


Isolation of Sindbis (alphavirus) and Leanyer viruses from mosquitoes collected in the Northern Territory of Australia, 1974. Mosquitoes collected near Darwin, Northern Territory, in 1974 yielded two virus strains. One was identified as Sindbis virus, not previously isolated from the Northern Territory. The other is antigenically distinct from viruses previously isolated from arthropods in Australia, and the name "Leanyer" is proposed for it. Its properties suggest that it may be a togavirus serologically unrelated to available alphaviruses and flaviviruses.


Effects of 6 hours hypoxic and cold exposure on urinary electrolyte and catecholamine excretion. Eleven young male Indian volunteers fasted overnight and were exposed to 6 h to cold at 8 degrees C (I), hypoxia at 4267 m at 28 degrees C (II), and cold plus hypoxia of 4267 m at , degrees C (III), in a walk-in climatic chamber, and excretion of some urinary constituents was measured. Urine output was significantly decreased in (II) and increased in (I) and (III). Urine pH significantly increased only in (II). Catecholamine excretion significantly increased only in (I). Ca++ excretion was significantly raised in (I) and (III) and lowered in (II). Na+ excretion was significantly decreased and K+ excretion remained unchanged in all three stress conditions. Cold seemed to be a greater stressor than hypoxia, under stated experimental conditions.


Detection after electrophoresis of enzymes involved in ammonia metabolism using L-glutamate dehydrogenase as a linking enzyme. The use of L-glutamate dehydrogenase (GLUD) as a reagent in staining mixtures to detect the isozymes of enzymes which catalyze the production of ammonia has been investigated. Methods have been devised for the electrophoresis and detection, using GLUD, of seven enzymes: cytidine deaminase, adenosine deaminase, adenosine monophosphate deaminase, arginase, argininosuccinase, D-amino acid oxidase, and D-aspartate oxidase. GLUD-linked staining methods appear to be sensitive, specific, and of general application.


Chemical modification of cellulase from Aspergillus niger. N-Bromosuccinimide completely inactivated the cellulase, and titration experiments showed that oxidation of one tryptophan residue per cellulase molecule coincided with 100% inactivation. CM-cellulose protected the enzyme from inactivation by N-bromosuccinimide. The cellulase was inhibited by active benzyl halides, and reaction with 2-hydroxy-5-nitrobenzyl bromide resulted in the incorporation of 2.3 hydroxy-5-nitrobenzyl groups per enzyme molecule; one tryptophan residue was shown to be essential for activity. Diazocarbonyl compounds in the presence of Cu2+ ions inhibited the enzyme. The pH-dependence of inactivation was consistent with the reaction occurring with a protonated carboxyl group. Carbodi-imide inhibited the cellulase, and kinetic analysis indicated that there was an average of 1 mol of carbodi-imide binding to the cellulase during inactivation. Treatment of the cellulase with diethyl pyrocarbonate resulted in the modification of two out of the four histidine residues present in the cellulase. The modified enzyme retained 40% of its original activity. Inhibition of cellulase activity by the metal ions Ag+ and Hg2+ was ascribed to interaction with tryptophan residues, rather than with thiol groups.


Changes in apparent pH on freezing aqueous buffer solutions and their relevance to biochemical electron-paramagnetic-resonance spectroscopy. Changes in apparent pH occurring during fast freezing of aqueous buffer solutions and cooling to -196 degrees C were studied by various semiquantitative methods, including simple visual measurements of colour changes with pH indicators, as well as measurements of pH-dependent changes in the e.p.r. (electron paramagnetic resonance) spectra of solutions of three different metalloenzymes. It is concluded that apparent pH changes of up to about 3pH units may occur under particular conditions. Such changes were independent of the time taken to freeze the samples, when this was varied from about 3ms t0 20s, but were affected by the presence of some proteins in solution. Recommendations on the buffers that should be used to avoid such apparent pH changes in e.p.r. spectroscopy and other low-temperature biochemical work are made. Phosphate and pyrophosphate buffers, which gave large decreases (2-3 pH units), and Tris, which under some conditions gave increases of about the same magnitude, are to be avoided. Certain zwitterionic buffers such as Bicine [NN-bis-(2-hydroxyethyl)glycine] are satisfactory. Apparent pH effects were found to depend on buffer and protein concentration. It is therefore recommended that as a prelude to future detailed low-temperature biochemical work, appropriate tests with an indicator system should be performed.


Fungal degradation of aromatic nitriles. Enzymology of C-N cleavage by Fusarium solani. 1. A strain of the fungus Fusarium solani able to use benzonitrile as sole source of carbon and nitrogen was isolated by elective culture. 2. Respiration studies indicate that the nitrile, after degradation to benzoate, is catabolized via catechol or alternatively via p-hydroxybenzoate and 3,4-dihydroxybenzoate. 3. Cell-free extracts of benzonitrile-grown cells contain an enzyme mediating the conversion of benzonitrile into benzoate and ammonia. 4. The nitrilase enzyme was purified by DEAE-cellulose chromatography, (NH(4))(2)SO(4) precipitation and gel filtration on Sephadex G-200. The homogeneity of the purified enzyme preparation was confirmed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and isoelectric focusing on polyacrylamide gel. 5. The enzyme showed a broad pH optimum between pH7.8 and 9.1 and a K(m) with benzonitrile as substrate of 0.039mm. The activation energy of the reaction deduced from an Arrhenius plot was 48.4kJ/mol. 6. The enzyme was susceptible to inhibition by thiol-specific reagents and certain heavy metal ions. 7. Gel filtration gave a value of 620000 for the molecular weight of the intact enzyme. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis demonstrated that the enzyme was composed of eight subunits of mol.wt. 76000. 8. Rates of enzymic attack on various substrates indicated that the nitrilase has a fairly broad specificity and that the fungus probably plays an important role in the biodegradation of certain nitrilic herbicides in the environment.


The separation and characterization of marmoset kidney beta-D-galactosidase and beta-D-glucosidase. beta-D-Galactosidase and beta-D-glucosidase activities were determined in homogenates of marmoset kidney by using the appropriate 4-methylumbelliferyl glycoside, beta-D-Galactosidase activity was separated into two main components by ion-exchange chromatography on DEAE-cellulose, starch-gel electrophoresis, isoelectric focusing and gel filtration on Sephadex G-200. One form designated A had a pI of 5.1, was loosely bound to DEAE-cellulose at pH7.0, remained near the origin on starch-gel electrophoresis at pH 7.0 and had an apparent molecular weight of 160000. The second beta-D-galactosidase component, designated B, was associated with the total beta-D-glucosidase activity, had a pI of 4.3, was firmly bound to DEAE-cellulose, migrated rapidly towards the anode on starch-gel electrophoresis and had an apparent molecular weight of 50000. The optimum pH values of beta-D-galactosidase A and B were 4.5 and 6.0 respectively. beta-D-Galactosidase A was activated by 0.1 M-NaC1 but the activity of the B form was inhibited by 1 M-NaC1 at pH 4.5. beta-D-galactosidase had a bimodal distribution, the A form being recovered in the lysosomal fraction whereas the B form was present in the soluble fraction, as was the major portion of the beta-D-glucosidase activity. The lysosomal and soluble forms were further characterized by DEAE-cellulose chromatography.


Proteoglycan-degrading enzymes. A radiochemical assay method and the detection of a new enzyme cathepsin F. 1. Polyacrylamide beads containing entrapped 35S-labelled proteoglycan molecules have been prepared. 2. The measurement of release of radioactivity provides an extremely sensitive assay for proteoglycan-degrading enzymes, including proteinases and hyaluronidase. 3. The amount of label released is a logarithmic function of enzyme concentration or time of incubation. Experiments were made in an attempt to explain this. 4. Assays were made by the new method at several pH values, and with the inclusion of inhibitors to identify the proteoglycan-degrading enzymes of rabbit ear cartilage. 5. A previously undescribed proteinase active against proteoglycan at pH4.5 but unaffected by pepstatin, was discovered. The enzyme was named cathepsin F, and was partially purified and characterized; it was detected in human articular cartilage.


Steady-state kinetic studies of the negative co-operativity and flip-flop mechanism for Escherichia coli alkaline phosphatase. 1. A study of variations in experimental error of velocity measurement with substrate concentration for alkaline phosphatase reveals that the standard error is not constant or strictly proportional to velocity, but obeys a more complex dependence. 2. By using an approach based on error estimates at each individual substrate concentration, we show that the double-reciprocal plots in general are curved, necessitating a high-degree rate equation. The curves are analysed according to a recent classification of possible curve shapes for the 3:3 function, which is shown to be the lowest-degree rate equation satisfying the experimental data. 4. Other workers have supposed the enzyme to follow Michaelis-Menten kinetics, and it is shown that this assumption is approximately true at low temperatures in the absence of phosphate. 5. A study of the effects of phosphate concentration, pH and temperature on the kinetics shows that there is a gradual alteration in curve shape with these experimental variables, resulting in an apparent reduction in degree under certain special conditions, and particularly at low temperature. 6. It is shown that the steady-state kinetics do not require a flip-flop or half-of-sites reactivity mechanism as claimed, and a mechanism is proposed, a rate equation calculated and an analysis attempted. 7. An analysis of the product-inhibition effects for a linked two-sited Uni Bi enzyme is given. Alterations of asymptotic double-reciprocal slopes and limiting (1/nu) intercepts with products is discussed, and it is shown how the theory of product inhibition can be extended to complex kinetic situations to extract information as to molecular mechanism. 8. Deviations from Michaelis-Menten kinetics are expressed in terms of the magnitude of the appropriate Sylvester resultants.


Benzofuroxan as a thiol-specific reactivity probe. Kinetics of its reactions with papain, ficin, bromelain and low-molecular-weight thiols. 1. The characteristics of benzofuroxan (benzofurazan 1-oxide, benzo-2-oxa-1,3-diazole N-oxide) that relate to its application as a reactivity probe for the study of environments of thiol groups are discussed. 2. To establish a kinetic and mechanistic basis for its use as a probe, a kinetic study of its reaction with 2-mercaptoethanol was carried out. 3. This reaction appears to proceed by a rate-determining attack of the thiolate ion on one of the electrophilic centres of benzofuroxan (possibly C-6) to provide a low steady-state concentration of an intermediate adduct; rapid reaction of this adduct with a second molecule of thiol gives the disulphide and o-benzoquinone dioxime. 4. The effects of the different types of environment that proteins can provide on the kinetic characteristics of reactions of thiol groups with benzofuroxan are delineated. 5. Benzofuroxan was used as a thiolspecific reactivity probe to investigate the active centres of papain (EC 3.4.22.2), ficin (EC 3.4.22.3) and bromelain (EC 3.4.22.4). The results support the concept that the active centres of all three enzymes either contain a nucleophilic thiolate ion whose formation is characterized by a pKa of 3-4 and whose reaction with an electrophile can be assisted by interaction of a site of high electron density in the electrophile with active-centre imidazolium ion of pKa 8-9, or can provide such ions by protonic redistribution in enzyme-reagent or enzyme-substrate complexes.


Degradation of myofibrillar proteins by cathepsins B and D. 1. The procedure of Barrett [(1973) Biochem. J.131, 809-822] for isolating cathepsins B and D from human liver was modified for use with rat liver and skeletal muscle. The purified enzymes appeared to be similar to those reported in other species. 2. Sephadex G-75 chromatography of concentrated muscle extract resolved two peaks of cathepsin B inhibitory activity, corresponding to molecular weights of 12500 and 62000. 3. The degradation of purified myofibrillar proteins by cathepsins B and D was clearly demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. After incubation with enzyme, the polypeptide bands representing the substrates decreased in intensity and lower molecular weight products appeared. 4. Cathepsins B and D, purified from either rat liver or skeletal muscle, were shown to degrade myosin, purified from either rabbit or rat muscle. Soluble denatured myosin was degraded more extensively than insoluble native myosin. Degradation by cathepsin B was inhibited by lack of reducing agent, or by myoglobin, iodoacetic acid and leupeptin, but not by pepstatin. The same potential modifiers were applied to cathepsin D, and only pepstatin produced inhibition. 5. Rat liver cathepsin B had a pH optimum of 5.2 on native rabbit myosin. The pH optimum of cathepsin D was 4.0, with a shoulder of activity about 1pH unit above the optimum. 6. Rat liver cathepsins B and D were demonstrated to degrade rabbit F-actin at pH5.0, and were inhibited by leupeptin and pepstain, respectively. 7. The degradation of myosin and actin by cathepsin D was more extensive than that by cathepsin B.


The active site of beta-glucosidase from Botryodiplodia theobromae. Effects of pH and dioxan on enzyme-catalysed reactions. 1. The hydrolysis of o-nitrophenyl beta-D-glucopyranoside by the high-molecular-weight beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) of Botryodiplodia theobromae Pat in the absence or presence of added dioxan was found to be dependent on the ionization of two groups, which appeared to be a carboxyl group and an imidazole group. 2. Dioxan increased the Michaelis constant, Km, but decreased the maximum velocity, V.


Phospholipase A activity associated with membranes of human polymorphonuclear leucocytes. Homogenates of human polymorphonuclear leucocytes (granulocytes) contain a Ca2+-dependent phospholipase A with optimal activity pH7.0. This enzyme is membrane-bound and is enriched in crude cytoplasmic-granule fraction. Ratezonal centrifugation of the cytoplasmic-granule fraction demonstrates that the phospholipase A is associated not only with specific- and azurophilic-granule populations but also with an 'empty' vesicular fraction containing 85% of the total alkaline phosphatase activity of whole homogenate. Thus this phospholipase is associated with granule as well as with other cellular membranes of human granulocytes.


The pH-dependence of second-order rate constants of enzyme modification may provide free-reactant pKa values. 1. Reactions of enzymes with site-specific reagents may involve intermediate adsorptive complexes formed by parallel reactions in several protonic states. Accordingly, a profile of the apparent second-order rate constant for the modification reaction (Kobs., the observed rate constant under conditions where the reagent concentration is low enough for the reaction to be first-order in reagent) against pH can, in general, reflect free-reactant-state molecular pKa values only if a quasi-equilibrium condition exists around the reactive protonic state (EHR) of the adsorptive complex. 2. Usually the condition for quasi-equilibrium is expressed in terms of the rate constants around EHR: (formula: see text) i.e. k mod. less than k-2. This often cannot be assessed directly, particularly if it is not possible to determine kmod. 3. It is shown that kmod. must be much less than k-2, however, if kobs. (the pH-independent value of kobs.) less than k+2. 4. Since probable values of k+2 greater than 10(6)M-1.S-1 and since values of kobs. for many modification reactions less than 10(6)M-1.S-1, the equilibrium assumption should be valid, and kinetic study of such reactions should provide reactant-state pKa values. 5. This may not apply to catalyses, because for them the value of kcat./Km may exceed 5 X 10(5)M-1.S-1. 6. The conditions under which the formation of an intermediate complex by parallel pathways may come to quasi-equilibrium are discussed in the Appendix.


[Effect of the beta-adrenergic blocking agent bupranolol on the plasma renin activity in normotensive rats]. The effects of the beta-adrenergic blocking agent 3-tert.-butyl-amino-1-(6'-chloro-3'-methylphenoxy)-propan-2-ol hydrochloride (bupranolol; KL 255; Betadrenol) on the plasma renin activity (PRA) of normotensive rats were studied in comparison to propranolol. Bupranolol (10--100 microgram/kg) reduced basal PRA and inhibited the isoproterenol as well as dihydralazine induced increase of PRA in a dose dependent fashion. Bupranolol exhibited an effect 2--4 times stronger than that of propranolol. The PRA inhibiting effects of bupranolol seem to be linked to its beta-receptor blocking properties.


Effect of halopredone acetate on rat liver tryptophan oxygenase and tyrosine aminotransferase activities. The possible inductive effect of a new local antiinflammatory steroid, 17,21-bis(acetyloxy)-2-bromo-6beta,9-difluoro-11beta-hydroxypregna-1,4-diene-3,20-dione (halopredone acetate; Topicon), on the rat liver enzymes tryptophan oxygenase and tyrosine aminotransferase was evaluated. The new steroid, when present in the implanted cotton-pellets or when s.c. injected, unlike the reference compound, fluocinolone acetonide, which induced a large increase in both enzymes, did not modify either tryptophan oxygenase or tyrosine aminotransferase. The results confirm that halopredone acetate elicits potent local antiinflammatory activity in the absence of systemic effects.


Drugs affecting the release of rheumatoid factor in a plaque-forming cell assay. Addition of propranolol to the agarose phase of a plaque-forming cell (PFC) assay for rheumatoid factor (RF) caused reduction in the number of plaques seen. This reduction in rheumatoid factor plaque-forming cell (RF PFC) did not depend upon an effect at the beta-adrenergic receptor, since d- and 1-propranolol reduced equally well. Furthermore, in a series of polycyclic compounds with varying beta-receptor blocking capabilities there was no agreement between plaque reduction and blocking. When propranolol was tested in the agarose in an anti-sheep erythrocyte (SRC) plaque assay (anti-SRC PFC), it had no inhibitory effect, but it was capable of inhibiting the generation of new anti-SRC PFC in an in vitro culture. Propranolol is thought to exert these effects through its membrane stabilizing (anesthetic) properties.


[Studies on Xipamide (4-chloro-5-sulfamoyl-2',6'-salicyloxylidide). Part 1: Physico-chemical and chemical properties (author's transl)]. The saluretic agent xipamide (4-chloro-5-sulfamoyl-2',6'-salicyloxylidide; Aquaphor) is a lipophilic substance as demonstrated by solubilities and partition coefficients. In the physiological pH-range, however, xipamide forms an anion which has an octanol-water partition coefficient of approx. 1. Two steps of ionization were investigated photometrically. The first one gives rise to a phenolate anion; pKa1 = 4.75. In the second one, ionization of the sulfamoyl group occurs; pKa2 = 10. Xipamide is stable in acidic as well as in basic media at room temperature. But when xipamide is acted upon by strong alkali and elevated temperature, 2,6-xylidine is formed by hydrolysis. For analytic use UV-photometric, colorimetric and TLC data of xipamide are given.


Camazepam versus placebo. A double-blind clinical study on geriatric patients suffering from psychic complaints. Short Communication. Camazepam, a dimethylcarbamic derivative of benzodiazepines, was studied on forty geriatric in-patients suffering from psychic disorders concerning anxiety and depression. Patients were treated with placebo and 30 mg of camazepam per day for 20 days using the double-blind method. The patients were assessed using a simplified rating scale with target symptoms of anxiety and depression. Statistical evaluation was performed with general Student's t-test for paired samples and chi2 test. Both groups of patients improved with camazepam and placebo. Camazepam 30 mg per day was more effective than placebo to a statistically significant degree.


Synthetic MIF analogues. Part I: synthesis by four-component condensation (4 CC) and classical methods. Analogues of melanocyte stimulating hormone/release inhibiting hormone (MIF), H-Pro-N-isobutyl-Gly-Gly-NH2, H-Pro-MeLeu-Gly-NH2 (L,L) and H-Pro-MeLeu-Gly-NH2 (L,D) were synthesized by the four-component condensation (4 CC). In addition compounds H-Pro-MeLeu-Ala-NH2 (L,L,D) and H-Pro-MeLeu-Ala-NH2 (L,D,D) were prepared by classical methods.


Synthetic MIF analogues. Part II: Dopa potentiation and fluphenazine antagonism. A comparison is described between the activity of the melanocyte-stimulating hormone release inhibiting factor (MIF) and those of five synthetic tripeptide analogues of MIF. The comparison was based upon the ability of the compounds to potentiate the behavioral effects of L-dopa in mide and to antagonize fluphenazine-induced catalepsy in rats. Three of the tripeptides potentiated L-dopa in a manner similar to that of MIF. All of the synthetic analogues antagonized fluphenazine after a single dose although their potency and their duration of action differed. MIF was active in the latter test only following chronic administration. The possible mechanism of action and the potential clinical applicability of the tripeptides are briefly discussed.


Synthetic melanocyte stimulating hormone release-inhibiting factor (MIF). Part III: effect of L-prolyl-N-methyl-D-leucyl-glycinamide and MIF on biogenic amine turnover. The effect of the factor that inhibits the release of melanocyte stimulating hormone (MSH), i.e., L-prolyl-L-leucyl-glycinamide (MIF), and L-prolyl-N-methyl-D-leucyl-glycinamide, an analog, on brain norepinephrine (NE), dopamine (DA) and serotonin (5-HT) turnover was examined in rats. The analog (40 mg/kg i.p.), in a fashion similar to MIF (40 and 5 mg/kg i.p.), increased brain DA turnover; only MIF (40 mg/kg i.p.) increased endogenous DA levels. The analog (40 and 5 mg/kg i.p.) decreased brain NE turnover; MIF at the same doses was ineffective. Neither MIF nor the analog affected rat brain 5-HT turnover or the 5-HTP-induced behavioural syndrome in the mouse. These results indicate that the analog, like MIF, exerts effects on central catecholamine turnover. The different biochemical profile of the analog compared to MIF may be importance with regard to potential clinical use in the treatment of Parkinson's disease and depression.


beta-adrenoceptor blockade with diphenylhydantoin (DPH). Diphenylhydantoin (DPH) (20 x 10(-6) to 80 x 10(-6) M/kg) blocked the depressor responses to isoproterenol in spinal, bilaterally vagotomized and atropine pretreated cats; depressor responses to histamine were unaffected; DPH shifted the isoproterenol concentration-response curve to the right in isolated guinea pig tracheal chain preparation, isolated rabbit ileum and isolated perfused heart of frog. The results suggest that DPH has a specific beta-adrenoceptor blocking action and the blockade appears to be competitive in nature as shown by PA2 and PA10 values.


Experiments in animals on the pharmacological effects of metipranolol in comparison with propranolol and pindolol. The beta-blocking agent 1-(4-acetoxy-2,3,5-trimethylphenyloxy)-3-isopropylamino-propan-2-ol (metipranolol) was compared with propranolol and pindolol. The beta-blocking activity on isoproterenol induced tachycardia was determined in rabbits (ED250bpm). The following doses in microgram/kg i.v. were required to produce the same inhibition: 410 propranolol; 160 metipranolol; 130 pindolol. When intrinsic sympathomimetic activity was determined in reserpinized rats metipranolol was found to produce less than 10% of the increase in heart rate induced by a standard isoproterenol dose (1 mg/kg i.p.), whereas propranolol caused less than 20% and pindolol ca. 60%. The membrane stabilising activity, determined by the elevation of the fibrillation threshold (ED delta100muA) in rabbit hearts, was found to be greatest with propranolol (0.98 mg/kg i.v.) and least with metipranolol (1.68 mg/kg), with pindolol occupying an intermediate position (1.10 mg/kg). The cardioprotective action to hypoxia stress of metipranolol in rats was found to be the best, requiring a dose of only 5 microgram/kg i.p. compared with values of 28 microgram/kg for pindolol and 250 microgram/kg for propranolol. These results show that metipranolol has a high beta-sympatholytic activity which is not accompanied by either marked intrinsic activity or membrane-stabilising properties. The relatively marked cardioprotective action, which is probably due to a metabolic action, is particularly noteworthy.


[Elucidation of the mechanism of nicotinic acid flush in the animal experiment (author's transl)]. The reddening of the ears of guinea pigs following the administration of nicotinic acid and its derivatives is regarded as an animal experiment for the human flush-reaction. In agreement with the observations in humans we found that the reddening effects decreased after repeated administration of xantinol nicotinate (Complamin). The results demonstrate that the reddening of the ears of guinea pigs--and presumably flushing in humans as well--is a complex event. The liberation of prostaglandins, histamine and serotonin, and first of all, cholinergic reactions, seem to be involved.


Metabolic fate of carteolol hydrochloride, (OPC-1085) VIII, a new beta-adrenergic blocking agent. Pharmacokinetic studies of carteolol in man. The pharmacokinetics of 5-(3-tert.-butylamino-2-hydroxy)-propoxy-3,4-dihydrocarbostyril hydrochloride (carteolol hydrochloride, OPC-1085) have been investigated in man following single or repetitive oral administration. The plasma half-lives. The plasma half-lives of carteolol at single doses of 10, 15 and 30 mg were 5.4, 5.5 and 5.0 h, respectively. The amounts of carteolol excreted into urine within 24 h at the same dose levels accounted for 64, 70 and 76% of the respective doses. The half-lives obtained by the Sigmaminus method were 5.6, 5.6 and 5.4 h, respectively, being essentially consistent with the aforementioned plasma half-lives of carteolol after administration at 15 mg daily for 7 successive days were determined to be 5.54 h on the 1st day and 6.91 h on the 7th day, displaying the increase in half-life value with the repetitive dosing. While, the predicted value determined using the experimental value on the 1st day agreed with the experimental value on the 7th day. Furthermore, the amounts of carteolol excreted in the urine were not significantly different between the 1st and 7th days. The 7-day repetitive administration with carteolol brought about the steady state of plasma levels. It was concluded from these results that carteolol has little ability to accumulate in man.


Bioavailability from various galenic formulations of flunitrazepam. The absolute and relative bioavailability of flunitrazepam (Rohypnol) tablet, oral solution and suppository formulations was determined in a cross-over study on 6 healthy volunteers. The tablet and the oral solution were shown to be bioequivalent, absorption being at least 80%. For flunitrazepam suppositories the bioavailability was found to be about 50%. The plasma concentrations are described on the basis of a three-compartment model, the adjustment of the curves from the same subject being made simultaneously. The central compartment and the second compartment are about equal in size and rapid exchange takes place between them. The third compartment is about four times as large as the central one. The slow reflux of the compound from the third to the first compartment is the rate determining factor for the overall elimination in the beta-phase.


[Importance of ventilation in metabolic acidosis]. The study included ten patients with severe metabolic acidosis (pH under 7.0) and clinical signs of shock. In four of them assisted ventilation was indicated as an urgency. In the other six patients assisted ventilation was used since the beginning, simultaneously with reposition of blood volume and administration of sodium bicarbonate. There were much better results with the latter management, with faster correction of the acidosis, without any complications and good survivorship.


Severe hypercapnia associated with a non-respiratory alkalosis. A case of hypoventilation in response to a non-respiratory alkalosis is presented. It is postulated that the degree of hypoventilation encountered was a normal response and that a fall in intracellular hydrogen ion concentration was responsible for the hypoventilation. This explains why the alkalosis associated with potassium deficiency is not associated with hypoventilation since the intracellular hydrogen ion concentration then remains constant. The renal response in this condition is responsible for maintaining the alkalosis and seems to be aimed at sodium conservation and hence plasma volume control rather than defence of acid-base balance.


Personnel radiation dosimetry in drug photosensitivity: field study of patients on phenothiazine therapy. A lapel badge dosimeter sensitive to short wave UVR has been used in a preliminary trial to survey photosensitivity in psychiatric patients on phenothiazine therapy. The badge was found easy to use and the results suggest that chlorpromazine is more photosensitizing than other phenothiazines.


Intraerythrocyte pH, pCO2 and the hexose monophosphate shunt. We have documented a rise in pCO2 when erythrocytes are haemolysed by freeze-thaw using solid CO2 and methanol. This is partially corrected by preventing leakage of CO2 into the system. The remaining increment is partially explained by stimulation of the hexose monophosphate shunt when erythrocytes are haemolysed by this technique.


Interaction of ribonuclease A with aqueous 2-methyl-2,4-pentanediol at pH 5.8. The interactions between ribonuclease A and solvent components in aqueous 2-methyl-2,4-pentanediol (MPD) have been investigated by differential refractometry and light scattering at pH 5.8, i.e., conditions similar to those used to crystallize the protein from this solvent system. Application of multicomponent thermodynamic theory shows that, at all solvent compositions up to 50% (v/v) MPD, the protein is preferentially hydrated; i.e., addition of ribonuclease to the mixed solvent leads to an increase in the chemical potential of MPD. This unfavorable thermodynamic interaction leads to phase separation, probably caused by local salting out of the MPD by the charges on the surface of the protein molecule. A parallel examination by circular dichroism (CD) has shown that the CD spectrum of ribonuclease in 50% MPD is indistinguishable from that in dilute buffer.


31P nuclear magnetic resonance spectra of the thiophosphate analogues of adenine nucleotides; effects of pH and Mg2+ binding. The 31P nuclear magnetic resonance (NMR) spectra of the adenine nucleotide thio analogues, AMPS, ADPalphaS, ADPbetaS, ATPalphaS, ATPbetaS, and ATPgammaS, have been studied. Of primary interest were the increased sensitivity of chemical shifts to protonation and to magnesium binding of these analogues compared with the corresponding effects on AMP, ADP, and ATP. The usefulness of the characteristic NMR parameters of the thio analogues as probes in enzymatic reactions is discussed. The A2 diastereoisomers of ADPalphaS and ATPalphaS and the A and B isomers of ATPbetaS were enzymatically synthesized and the diasterioisomers of ADPalphaS and ATPbetaS were distinguished by their 31P NMR parameters. The stereospecificity of the enzymatic reactions involving the thio analogues of nucleotides can therefore be determined by 31P NMR. The difficulty involved in assigning phosphate ligands of Mg in MgADP and MgATP and their analogues on the basis of the magnitude of chemical shift changes (deltadelta) induced by Mg binding upon each 31P is discussed in the context of the anomalies in deltadelta of each 31P observed upon protonation of the terminal phosphate group. It is concluded that chemical shift data cannot yield unequivocal information concerning the absolute structure of metal complexes of nucleotides but can be used to monitor changes in metal chelation, for example, upon binding to enzyme.


Studies of the control of luminescence in Beneckea harveyi: properties of the NADH and NADPH:FMN oxidoreductases. Highly purified NADH and NADPH:FMN oxidoreductases from Beneckea harveyi have been characterized with regard to kinetic parameters, association with luciferase, activity with artificial electron acceptors, and the effects of inhibitors. The NADH:FMN oxidoreductase exhibits single displacement kinetics while the NADPH:FMN oxidoreductase exhibits double displacement or ping-pong kinetics. This is consistent with the formation of a reduced enzyme as an intermediate in the reaction of catalyzed by the NADPH:FMN oxidoreductase. Coupling of either of the oxidoreductases to the luciferase reaction decreases the apparent Kms for NADH, NADPH, and FMN, supporting the suggestion of a complex between the oxidoreductases and luciferase. The soluble oxidoreductases are more efficient in producing light with luciferase than is a NADH dehydrogenase preparation obtained from the membranes of these bacteria. The soluble enzymes use either FMN or FAD as substrates for the oxidation of reduced pyridine nucleotides while the membrane NADH dehydrogenase is much more active with artificial electron acceptors such as ferricyanide and methylene blue. FMN and FAD are very poor acceptors. The evidence indicates that neither of the soluble oxidoreductases is derived from the membranes. Both enzymes are constitutive and do not depend on the synthesis of luciferase.


Purification and properties of deoxyribonuclease from human urine. The DNAase in human urine was purified about 30-fold with a recovery of 28%. This involved DEAE-cellulose and phosphocellulose chromatography steps and gel filtration on Sephadex G-75. The enzyme required divalent cations such as Co2+, Mg2+, Mn2+ and Zn2+ for activity, but Ca2+, Cu2+ and Fe2+ were ineffective. EDTA and G-actin inhibited the reaction. The maximum activity was observed at pH 5.5 in acetate buffer plus Co2+ or Mg2+ and Ca2+. It had a molecular weight of approximately 38 000, estimated by gel filtration on Sephadex G-75 and isoelectric point of around pH 3.9. The enzyme is an endonuclease which hydrolyzes native, double-stranded DNA about 3 to 4 times faster than thermally denatured DNA to produce 5'-phosphoryl- and 3'-hydroxyl-terminated oligonucleotides. The final preparation was free of non-specific acid and alkaline phosphatases, phosphodiesterase and ribonuclease activities.


Study on plant RNAases. Isolation and properties of several activities from Vicia faba root cells. Vicia faba root cells contain several nucleolytic activities: phosphomonoesterase and phosphodiesterase (which however were not studied in details), one nuclease and four ribonucleases. These results were obtained by separating the extracted proteins into anionic and cationic species by chromatography on CM-cellulose at pH 5.5 and analysing each kind of proteins. Anionic species were subjected to chromatography on DEAE-cellulose which lead to isolation of one nuclease (A1) and two RNAases (A2, A3), the properties of which were studied. It was shown that the RNAases pH optima are near 6; A2 is more thermolabile than A3; both are endonucleases unable to attack double-stranded structure; studies with homopolymers, i.e. poly(A), poly(I), poly(C), poly(U), showed that their base specificities were analogous to that of already known plant RNAases. The cationic proteins, analysed with CM-cellulose, contain two RNAases (C1, C2). The pH optima were near 6 and 7, respectively; C1 is much more thermolabile than C2; both were endonucleases inactive on double-stranded structures. C1 and C2 hydrolysed poly(C) and poly(U) but not poly(A) and poly(U).


Studies of the catalytic properties of an endonuclease isolated from Tetrahymena pyriformis. An acid endonuclease hydrolyzing both DNA and RNA was purified from Tetrahymena pyriformis, strain E. The enzyme is distributed in all major subcellular compartments and is excreted into the growth medium towards the middle of the logarithmic phase. It hydrolyzes DNA to penta or hexanucleotides, on the average, bearing the monoesterified phosphate at the 3'-position. Particularly in early phases of the reaction it shows a very pronounced specificity for bases with a keto group at position 4 of the pyrimidine ring, such as guanine and thymine.


Purification and properties of a cyclic AMP-independent protein kinase from calf thymus nuclei. A phosphoprotein kinase (ATP : protein phosphotransferase, EC 2.7.1.37) from calf thymus nuclei was purified by DEAE-cellulose chromatography, hydroxyapatite, and Sepharose 6B gel filtration. The enzyme is a cyclic AMP-independent protein kinase by the following criteria: (a) the protein kinase did not bind cyclic AMP; (b) no inhibition of activity was obtained with the heat-stable protein kinase inhibitor from rabbit skeletal muscle; (c) the regulatory subunit of cyclic AMP-dependent protein kinase had no effect on activity; and (d) no inhibition was obtained with antibody to cyclic AMP-dependent protein kinase. The nuclear cyclic AMP-independent protein kinase readily phosphorylated protamine on serine and to a lesser extent on threonine. Homologous nucleoplasmic RNA polymerase (EC 2.7.7.6) is a better substrate than arginine-rich histone, phosvitin or casein. Physical characteristics of the enzyme are described.


Methylamine dehydrogenase of Pseudomonas sp. J. Purification and properties. Methylamine dehydrogenase was purified in a homogeneous form from methylamine-grown Pseudomonas sp. J. The specific activity of the purified enzyme was 5.19 at 19 degrees C. The molecular weight was estimated to be 105 000, and the enzyme was composed of two kinds of subunit with molecular weights of 40 000 and 13 000, respectively. The enzyme contained little phosphorus, iron and copper. The enzyme had absorption maxima at 278, 330, 430 and 460 nm (shoulder). On addition of methylamine, the peaks at 430 and 460 nm decreased, while that at 330 nm increased. Primary amines served as substrates, but secondary and tertiary amines did not. Phenazine methosulfate was the most effective electron acceptor and oxygen was ineffective. The enzyme was inhibited by carbonyl reagents, cuprizone and HgCl2 but not by other chelators or sulfhydryl reagents. Some of other physical and biochemical properties of the enzyme were studied. These results show that the enzyme purified from Pseudomonas sp. J is essentially similar to the enzyme obtained from Pseudomonas AM1, although it differs slightly in some properties.


Methylamine dehydrogenase of Pseudomonase sp. J Isolation and properties of the subunits. Two kinds of subunits, light subunit (Mr =1300) and heavy subunit (Mr=40 000), were isolated from a methylamine dehydrogenase (Mr=105 000) of Pseudomonas sp. J. The isolation of the subunits was carried out by gel chromatography after the enzyme had been treated with 3M guanidine-HCl. Coexistence of both of the subunit exhibited an absorption maximum only at 278 nm but in addition to the peak at 278 nm. The results indicate that the prosthetic group, assumed to be a derivative of pyridoxal, was bound to the light subunit. The spectral changes of the light subunit were observed by addition of methylamine. Various physical and biochemical parameters of the subunits are reported.


Methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase from porcine liver. Interaction between the dehydrogenase and cyclohydrolase activities of the multifunctional enzyme. Methylenetetrahydrofolate dehydrogenase (5,10-methylenetetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.5) one of the activities of a trifunctional folate-dependent enzyme from porcine liver, uses an ordered kinetic mechannism as determined from initial velocity, product inhibition and dead-end inhibition studies. The final product released from the dehydrogenase is methenyltetrahydrofolate. However, from the time course of appearance of products it is observed that the methenyltetrahydrofolate, rather than equilibrating with the solution, is converted preferentially to formyltetrahydrofolate by the cyclohydrolase, (5,10-methenyletrahydrofolate 5-hydrolase (decyclizing), EC 3.5.4.9) demonstrating a functional interaction between these two enzymic activites.


Multiple forms of gamma-glutamyltransferase in normal human liver, bile and serum. A study of the multiple forms of gamma-glutamyltransferase ((gamma-glutamyl)-peptide:amino acid gamma-glutamyltransferase, EC 2.3.2.2) in normal human liver, bile and serum are reported. An amphiphilic form of the enzyme was demonstrated in all three samples. When solubilized with detergent, estimated values for Stoke's radius of 48 A and sedimentation coefficient of 5 S were obtained for this form. A hydrophilic form was also present in serum and bile, which showed identical properties to the enzyme form obtained after papain-treatment of the three samples. The Stoke's radius was found to be 37 A, and the sedimentation coefficient 5 S. It was concluded that the heterogeneity of enzyme activity found both on gel filtration and on electrophoresis was due to aggregates of the amphiphilic form with lipids and other proteins, and could not be ascribed to the presence of isoenzymes.


Biosynthesis of glycogen in Neurospora crassa. Purification and properties of the UDPglucose:glycogen 4-alpha-glucosyltransferase. The Neurospora crassa glycogen synthase (UDPglucose:glycogen 4-alpha-glucosyltransferase, EC 2.4.1.11) was purified to electrophoretic homogeneity by a procedure involving ultracentrifugation, DEAE-cellulose column chromatography, (NH4)2SO4 fractionation and 3-aminopropyl-Sepharose column chromatography. The final purified enzyme preparation was almost entirely dependent on glucose-6-P and had a specific activity of 6.9 units per mg of protein. The subunit molecular weight of the glycogen synthase was determined by electrophoresis in sodium dodecyl sulfate-polyacrylamide gel to be 88 000--90 000. The native enzyme was shown to have a molecular weight of 270 000 as determined by sucrose density gradient centrifugation. Thus, the glucose-6-P-dependent form of the N. crassa glycogen synthase can exist as trimer of the subunit. Limited proteolysis with trypsin or chymotrypsin converted the glucose-6-P-dependent form of the enzyme into an apparent glucose-6-P-independent form. The enzyme was shown to catalyze transfer of glucose from UDPglucose to glycogen as well as to its phosphorylase limit dextrin, but not to its beta-amylase limit dextrin. Moreover, glucose, maltose and maltotriose were not active as acceptors.


Purification and properties of 4-aminobutyrate 2-ketoglutarate aminotransferase from pig liver. 4-Aminobutyrate-transaminase (4-aminobutyrate: 2-oxoglutarate amino-transferase, EC 2.6.1.19) from pig liver has been purified to electrophoretic homogeneity. It has a molecular weight of about 110 000 and is composed of two subunits of the same molecular weight but of different charges. Two forms of pig liver 4-aminobutyrate-transaminase were isolated by DEAE-cellulose chromatography and designated as 4-aminobutyrate-transaminase I and 4-aminobutyrate-transaminase II, corresponding to a cationic and anionic form. Some physical and kinetic properties of liver enzyme were compared to those of brain enzyme and no significant difference were found, except for their sedimentation coefficients and the charges of their subunits. The role of 4-aminobutyrate-transaminase in liver remains a matter of speculation, but could be related to a metabolic function.


Purification and properties of polynucleotide kinase of calf thymus. Polynucleotide kinase (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) has been purified approx. 1500-fold from calf thymus. This enzyme phosphorylates 5'-hydroxyl termini in DNA using ATP as phosphate donor. RNA is phosphorylated at a much lower rate than DNA. The reaction requires the presence of a divalent cation, preferably Mg2+ or Mn2+ and is sensitive to sulfhydryl antagonists. The optimum pH for enzyme activity is 5.5. Enzyme activity is inhibited by low concentrations of inorganic sulfate and by some sulfate polymers. The kinase-catalyzed incorporation of the terminal phosphate of ATP into polynucleotides is inhibited by other nucleoside and deoxynucleoside triphosphates. The enzyme molecule has a molecular weight of about 70 000 and a Stokes radius of 4.3 nm. It has a frictional ratio of 1.44 indicating an asymmetrical structure. Calf thymus tissue should provide a useful alternative source for preparation of mammalian polynucleotide kinase.


Purification and characterization of two isozymes of 3-phosphoglycerate kinase from the mouse. Two isozymes of 3-phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3), designated PGK-A and PGK-B, were purified from separate extracts of muscle and testicular tissue of DBA/2J mice, respectively. A similar procedure was used to purify the corresponding isozymes from C57BL/6J mice in order to make inter-strain comparisons. The purification involved the use of affinity chromatography with an 8-(6-aminohexyl)amino-ATP-Sepharose column and DEAE-Sephadex chromatography. Lactate dehydrogenase isozyme LDH-X was also co-purified from extract of mouse testes by this two-step procedure. The same isozyme isolated from either mouse strain was found to be identical in physical and biochemical properties. Both isozymes are monomeric as determined by gel filtration chromatography and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Furthermore, the isozymes have similar molecular weights, of 47 000 +/- 2000 and exhibit similar Km values for both coenzymes and substrate, as well as temperature dependence of enzyme activity. However, it was observed that the B isozyme is more labile than the A isozyme by denaturation at high temperature, urea and acidic pH.


Solubilization and partial characterization of a phosphoprotein phosphatase from human myelin. The phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) solubilized from human central nervous system myelin has been shown to possess a comparatively high degree of specificity towards myelin basic protein, a constituent of the membrane and most likely its natural substrate, rather than the mixed histones. The enzyme has a pH optimum of 7.5. Hydrolysis of both the substrates is stimulated by dithiothreitol and is almost completely dependent upon the presence of divalent metal ions. The maximum rate of dephosphorylation of basic protein is attained in the presence of 125 micrometer Mn2+ whereas a much higher concentration of Mg2+ (50--100 mM) is required for the optimal dephosphorylation of histones. The dephosphorylation of basic protein was also stimulated by Triton X-100 (0.15%, v/v) and was shown to result from a 3-fold increase in the V of the reaction catalyzed by the phosphatase. The apparent Km values for basic protein and histones were unaffected by the presence of Triton X-100 and were found to be approx. 1 and approx. 160 micrometer, respectively. Under optimal conditions of assay, the phosphatase cleaved approx. 32 and approx. 0.7 nmol of orthophosphate.min-1.mg-1 of protein from basic protein and histones, respectively.


Acid glycohydrolase in Chinese hamster with spontaneous diabetes. I. Depressed levels of renal alpha-galactosidase and beta-galactosidase. The activites of alpha-and Beta-galactosidases (alpha-D-galactoside galactohydrolase, EC 3.2.1.22; beta-D-galactoside galactohydrolase, EC 3.2.1.23) were significantly lower in the kidneys of diabetic XA line than those in the nondiabetic M line Chinese hamsters. The depression of these enzymes was found only in the kidney but not in liver, spleen, hind leg muscle, cheek pouch or spinal cord. In young XA animals before onset of glycosuria, renal alpha-galactosidase level was similar to that in age-matched M animals; whereas, their renal beta-galactosidase activity was about 90% of those in the M animals. Partial purification and separation of these enzymes were achieved by chromatography on DEAE-Sepharose CL-6B columns. beta-galactosidase was separated into two isozymes and depression of activity in the XA kidneys was evident in both. alpha-galactosidase was recovered in a single peak. The pH optima of these enzymes from XA and M animals were identical. With p-nitrophenyl glycosides as substrates, the Michaelis constants of these enzymes were also the same of XA and M animals. Molecular weight estimation by gel filtration on Sepharose 6B yielded similar results between M and XA samples: 2.4-10(5) for alpha-galactosidase and 1.6.10(5) and 1.9.10(5) for beta-galactosidase isozymes. The data suggest that the diabetic animals had lower concentrations of alpha-and beta-galactosidase in their kidneys, probably as a consequence of hyperglycemia.


Specificity and inhibition studies of Armillaria mellea protease. The action of Armillaria mellea protease has been evaluated on a number of polypeptide substrates. It has been shown to split the Pro7-Lys8 bonds in both native and oxidised lysine-vasopressin and the Ser11-Lys12 bond in glucagon. No other splits were detected in these substrates. The enzyme also caused extensive degradation of S-carboxymethyl lysozyme, S-carcoxymethyl pepsinogen and oxidised ribonuclease. A. In each case the only new amino-terminal residue to appear was lysine. A. mellea protease was inhibited by the chelating agents 1,10-phenanthroline, alpha, alpha'-bipyridine and imidazole. The pK1 values (negative log10 of concentration required for 50% inhibition) for these three inhibitors were 3.9, 3.4 and 1.1, respectively. Lysine, S-2-aminoethylcysteine and short chain aliphatic amines also proved to be relatively good inhibitors of A. mellea protease while arginine was a poor inhibitor.


Purification and partial characterization of rat brain acid proteinase (isorenin). 1. Isorenin was purified 2000-fold from rat brain by a simple 3-step procedure involving affinity chromatography on pepstatinyl-Sepharose, The preparation appears as a homogenous protein in analytical polyacrylamide gel electrophoresis. Sodium dodecyl sulfate gel electrophoresis indicated an apparent molecular weight of 45 000. Isoelectric focusing separated isoenzymes with isoelectric points at pH 5.45, 5.87, 6.16 and 7.05. 2. The enzyme generates antiotensin I from tetradecapeptide (pH optimum 4.7) and from sheep angiotensinogen (pH optima 3.9 and 5.5). The rate of angiotensin I formation from tetradecapeptide was 30 000 times higher than that from sheep angiotensinogen. The enzyme has acid protease activity at pH 3.2 with hemoglobin as the substrate and pepstatin is a potent inhibitor of the enzyme with a Ki of less than 10(-9) M. 3. The properties of the enzyme strongly suggest that it is identical with cathepsin D.