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M Johnston

Publications and source records attributed to M Johnston.

At least 253 records · Page 14Linked to original sources
PubMed1986

Purification and properties of the bovine liver mitochondrial dihydroorotate dehydrogenase.

Dihydroorotate dehydrogenase has been purified 6,000-fold from bovine liver mitochondria to apparent homogeneity in six steps. Electrophoretic migration of the homogeneous enzyme on sodium dodecyl sulfate-polyacrylamide gels reveals a subunit Mr of 42,000. By contrast to the well-characterized, cytosolic dihydroorotate oxidases (EC 1.3.3.1), the purified bovine dehydrogenase is a dihydroorotate:ubiquinone oxidoreductase. Maximal rates of orotate formation are obtained using coenzymes Q6 or Q7 as cosubstrate electron acceptors. Concomitant with substrate oxidation, the enzyme will reduce simple quinones, such as benzoquinone, but at significantly lower rates (10-15%) than that obtained for reduction of coenzyme Q6. Enzyme-catalyzed substrate oxidation is not supported by molecular oxygen. The specificity of the purified enzyme for dihydropyrimidine substrates has also been explored. The methyl-, ethyl-, t-butyl-, and benzyl-S-dihydroorotates are substrates, but 1- and 3-methyl and 1,3-dimethyl methyl-S-dihydroorotates are not. Competitive inhibitors include product orotate, 5-methyl orotate, and racemic cis-5-methyl dihydroorotate.

Animals↗
PubMed1986

Mechanisms of action of chloroalanyl antibacterial peptides. Identification of the intracellular enzymes inactivated on treatment of Escherichia coli JSR-O with the dipeptide beta Cl-LAla-beta Cl-LAla.

The dipeptide beta Cl-LAla-beta Cl-LAla is an antibacterial agent designed to utilize bacterial peptide transport for intracellular delivery of the alanine racemase inactivator beta Cl-LAla. The minimum inhibitory concentrations (MICs) for the peptide against Gram-negative species grown on enriched agar medium range from 1.56 to 12.5 micrograms/ml; MICs are increased to greater than 100 micrograms/ml when D-alanine is included in the medium, indicating that alanine racemase is, in fact, inhibited in sensitive species. When susceptible Gram-negative cells are grown on a minimal medium, D-alanine supplementation alone does not increase the MICs for beta Cl-LAla-beta Cl-LAla, but complete protection is afforded by supplementation with D-alanine, L-valine, L-leucine, and L-isoleucine. In liquid culture, the peptide is: bactericidal and lytic against Escherichia coli JSR-O growing in enriched medium or in minimal medium supplemented with the branched-chain amino acids; only inhibitory against these cells growing in minimal medium supplemented with D-alanine; and ineffective against these cells in minimal medium containing the branched-chain amino acids plus D-alanine. Cells exposed to beta Cl-LAla-beta Cl-LAla (with the protection of the four amino acids) have specific activities of both alanine racemase and transaminase B that are lower than those of cultures not treated with the peptide. Finally, E. coli JSR-O alanine racemase experiences time-dependent loss of activity when exposed to the dipeptide in the presence of aminopeptidases; the dipeptide alone is not an inactivator of the racemase in vitro. These results suggest the following mechanism of action for beta Cl-LAla-beta Cl-LAla: transport of the dipeptide into the cell; intracellular hydrolysis to give accumulation of beta Cl-LAla; and subsequent inactivation of targeted enzymes. Whether inactivation of the racemase or of the transaminase determines the pathophysiologic effects of the peptide depends on the composition of the growth medium.

Alanine Racemase↗
PubMed1986

Reactions of Escherichia coli TEM beta-lactamase with cephalothin and with C10-dipeptidyl cephalosporin esters.

Two novel C10-(dipeptidyl)cephalosporin esters (3-(beta-chloro-L-alanyl-beta-chloro-L-alanyloxymethyl)-7 beta-(2-thienylacetamido)-3-cephem-4-carboxylic acid (7) and sodium 3-(L-alanyl-L-alanyloxymethyl)-7 beta-(2-thienylacetamido)-3-cephem-4-carboxylate, toluene-sulfonic acid salt (18] were synthesized, and their reactions with Escherichia coli TEM beta-lactamase were examined. Kinetic parameters determined for the enzymatic reactions of 7 (Km = 0.32 mM; Vmax = 338 mumol min-1 (mg protein)-1) and of 18 (Km = 0.33 mM, Vmax = 338 mumol min-1 (mg protein)-1) demonstrate that both of the peptidyl esters are good substrates for the lactamase. In fact, the Vmax rates for 7 and 18 are each more than 4-fold greater than that obtained for cephalothin, 1 (Vmax = 78 mumol min-1 (mg protein)-1), a well characterized substrate for the lactamases. Analysis of the enzymatic reactions by high field (500 MHz) 1H NMR revealed similar patterns for fragmentation of the cephem nucleus of 1, 7, and 18. However, while hydrolysis of 1 produces acetate, cleavage of 7 and 18 releases beta Cl-LAla-beta Cl-LAla and LAla-LAla, respectively, from the dipeptidyl cephalosporin esters. Based on these findings, a strategy for co-opting the beta-lactamases of Gram-negative bacteria for "delivery" of bactericidal agents is described, and an explanation for the previously reported (Mobashery, S., Lerner, S.A., and Johnston, M. (1986) J. Am. Chem. Soc. 108, 1685) antibacterial activity of 7 is offered.

Cephalothin↗
PubMed1986

Determinants of beliefs about illness: a study of parents of children with diabetes, asthma, epilepsy, and no chronic illness.

Social psychological models of health care behaviour frequently take as their starting point perception of the illness. This paper seeks to examine factors that may influence these perceptions. Beliefs about the seriousness of eleven childhood illnesses are compared in four groups of parents. Significant differences were evident between the groups of parents depending upon their experiences with illness. For three conditions (diabetes, asthma and epilepsy) the lowest rating of seriousness was made by parents with a child with that condition. For parents of children with diabetes, having a relative with diabetes in good health was associated with a perception of the illness as significantly less serious than those with a relative in average or poor health, or those with no relatives with diabetes. These results are interpreted within an information processing model. Implications of the findings for social psychological models of health care behaviour are discussed.

Adaptation, Psychological↗
PubMed1986

Frequency and mechanism of neonatal thrombocytopenia.

We performed a 1-year prospective study of 807 consecutive infants admitted to a regional neonatal intensive care unit to determine the frequency, natural history, mechanism(s), and cause of thrombocytopenia. Thrombocytopenia developed in 22% of the infants. The platelet count nadir usually occurred by day 4 and resolved by day 10. Possible mechanisms responsible for the thrombocytopenia were assessed by comparing mean platelet volume, platelet-associated IgG (PAIgG), and coagulation test results in those infants whose platelet count fell below 100 X 10(9)/L (n = 97) with values in age-, weight-, and disease-matched control infants without thrombocytopenia (n = 80). In some thrombocytopenic infants, 111In-labeled-platelet survival, an estimate of megakaryocyte number in bone marrow biopsy specimens obtained at autopsy, and response to platelet infusions were also assessed. The thrombocytopenia was caused by increased platelet destruction, as shown by short 111In-labeled-platelet survival (12 to 128 hours), a rising mean platelet volume during the first week of life, normal numbers of megakaryocytes, and a poorer than predicted response to platelet infusions. A potential cause for the thrombocytopenia could be found in the majority of infants: 52% had elevated levels of PAIgG, 21% had laboratory evidence of disseminated intravascular coagulation, and 12% had had exchange transfusions. In contrast, the control infants had normal coagulation assay results, and only 15% had elevated levels of PAIgG. Birth asphyxia was identified as an associated risk factor for thrombocytopenia. This study demonstrates that transient, destructive thrombocytopenia develops in a large proportion (22%) of infants admitted to a neonatal intensive care unit, and that birth asphyxia is an important risk factor.

Asphyxia Neonatorum↗
PubMed1986

Chloroalanyl antibiotic peptides: antagonism of their antimicrobial effects by L-alanine and L-alanyl peptides in gram-negative bacteria.

A large number of structurally diverse di- and tripeptides containing the alanine racemase inactivator beta-chloro-L-alanine (beta-Cl-LAla) have been synthesized, and their antibacterial properties in vitro have been evaluated. The dipeptides 1, 3-6, and 8-17 and the tripeptide 20 are all broad-spectrum antibacterial agents with considerable potency against both Gram-positive and Gram-negative species, but none of these peptides improves dramatically on the antibiotic efficacy of the previously described beta-Cl-LAla-beta-Cl-LAla, 9 (Cheung, K. S.; Wasserman, S. A.; Dudek, E.; Lerner, S. A.; Johnston, M. J. Med. Chem. 1983, 26, 1733). Gram-negative microorganisms, such as Escherichia coli, Hemophilus influenzae, Shigella flexneri, and Enterobacter species are consistently resistant to any haloalanyl peptide containing an alanyl residue, such as the dipeptide LAla-beta-Cl-LAla (2) and the tripeptides LMet-LAla-beta-Cl-LAla (7), LAla-LAla-beta-Cl-LAla (18), and LVal-LAla-beta-Cl-LAla (19). Correspondingly, these same organisms are protected from the bactericidal effects of 9 by supplementation of the growth medium with LAla or LAla-LAla. Escherichia coli JSR-O exposed to 9, but protected from lysis by sucrose stabilization, has only about 10% the normal level of intracellular alanine racemase activity. But when these cells are cultured in the presence of 9 with LAla supplementation, or in the presence of 2 with no supplementation, the alanine racemase levels are only about 20-30% below control values. These findings suggest that the resistance of Gram-negative species to chloroalanyl peptides containing alanyl units arises from the ability of LAla to protect the targeted racemase from inactivation by beta-Cl-LAla in vivo, an event which otherwise leads to cell death and lysis. Inactivation of alanine racemase in Gram-positive organisms appears not to be the cellular event that confers sensitivity of these species to a haloalanyl peptide.

Alanine↗
PubMed1986

Facilitating comprehension of discharge medication in elderly patients.

Provision of adequate information is well established as an important determinant of patient compliance with taking medication. Elderly patients, who constitute a large proportion of patients discharged from hospital on medication, may be particularly at risk of unintentional noncompliance due to inadequate or forgotten information. This study demonstrates that counselling provided to the elderly by a clinical pharmacist significantly improved knowledge about medication and, therefore, the opportunity for compliance at home.

Aged↗
PubMed1986

Use of a medical 'schema' in facilitating access to understanding in psychology.

Cognitive models in psychology suggest that knowledge can only be fully comprehended and recalled within the framework of existing schemata. Two experiments are reported which demonstrate how psychological principles may be taught within the schemata available to preclinical medical students. It is suggested that the provision of appropriate schemata makes psychological knowledge more accessible to medical students, and more easily recalled as they move into clinical practice. Increasing the impact of psychology in this way may also encourage greater interest and integration of the subject in the medical curriculum.

Adult↗
PubMed1986

Doctors taking blood from children: a suitable case for treatment?

Despite an intention to take blood samples regularly from children with diabetes, only 31 per cent of 60 required samples were collected. Following an intervention which required the doctors to complete an 'explanation slip' each time a request was not met, the collection rate of blood samples rose from 31 to 69 per cent. These results draw attention to the role of the doctors' behaviour in achieving health care objectives.

Attitude of Health Personnel↗
PubMed1985

Purification and properties of a secondary alcohol dehydrogenase from the parasitic protozoan Tritrichomonas foetus.

A novel secondary alcohol dehydrogenase has been isolated from Tritrichomonas foetus, the protozoan parasite which is responsible for bovine trichomonal abortion. The enzyme has been obtained in apparently homogeneous form after a 120-fold purification from cell homogenates, thus indicating that this activity constitutes an unusually high 1% of the total cytosolic protein. The native Mr = 115,000, determined by polyacrylamide gel electrophoresis. Mobility on sodium dodecyl sulfate gels suggests that the enzyme is composed of 6-8 subunits, identical as to molecular size (Mr = 17,000). The enzyme catalyzes the reversible oxidation of 2-propanol to acetone, using NADP+ (and not NAD+) as the redox-active co-substrate. Other small secondary alcohols, such as 2-butanol, 2- and 3-pentanol, cyclobutanol, and cyclopentanol are substrates, as are the corresponding ketones of these alcohols. Primary alcohols, such as ethanol and 1-propanol, are oxidized at rates less than 5% of that observed for 2-propanol. Product inhibition studies demonstrate an ordered kinetic mechanism, wherein the co-substrate (NADP+/NADPH) binds to the enzyme prior to binding of the substrate (alcohol/ketone).

Acetaldehyde↗
PubMed1985

The immunodepletion of factor VII from human plasma using a monoclonal antibody.

A murine hybridoma cell line which secretes monoclonal antibody to factor VII has been prepared to facilitate the immunodepletion of this clotting factor from plasma. Specific monoclonal antibody was purified from mouse ascites tumours by protein A-Sepharose chromatography and shown to be of the IgG1 immunoglobulin subclass. On immunoblotting, this antibody reacted with a single protein band identical to purified factor VII. The purified monoclonal antibody was coupled to Sepharose 4B and was used to immuno-deplete factor VII from pooled normal human plasma. The prothrombin time of plasma immunodepleted in this way was 35 s compared to 12 s for the starting plasma. Specific factor assays of the immunodepleted plasma showed factor VII activity to be less than 1% while the levels of the other clotting factors were unchanged. The immunodepleted plasma was equivalent to severe congenital factor VII deficient plasma as a substrate for factor VII assays. Bound factor VII could be eluted from the immunoaffinity column with citrate buffer, pH 6.0, with good recovery.

Animals↗
PubMed1984

Educational package on hypertension for primary care physicians.

The usefulness of an educational package on hypertension that provides clinically important, up-to-date medical information and office "aids" to primary care physicians was tested in a randomized controlled trial. Fifty-six physicians completed a pretest multiple-choice questionnaire and were allocated at random either to a group that received the educational package (the "study group") or to a control group. There was a highly significant correlation between the pretest scores and the number of years since graduation (r = -0.55, p less than 0.0001), which indicated that younger physicians are more likely than older physicians to have an up-to-date knowledge of the management of hypertension. The increase in knowledge in the study group (17.5%) was significantly greater than that in the control group (2.7%). Furthermore, although the post-test scores in the control group were still significantly correlated with the number of years since graduation, those in the study group were not. It was concluded that although the older physicians knew less than their younger colleagues about hypertension, the use of the educational package significantly increased knowledge, and the increase was not limited by the physician's age.

Age Factors↗
PubMed1984

A microprocessor-based system for measurement of gas exchange.

The basic physical measurements for determining gas exchange are difficult to make accurately even in a well-equipped, human-performance laboratory with experienced personnel. A fully automated system has been developed to achieve the accuracy of standard laboratory measurements. The application of this instrument extends from critical care to stress-testing. Real-time, multitasking software integrates the data collected from several transducers and analyzers and calculates up to several dozen physiological variables, which are range-checked for reasonableness. The operator is provided with user-friendly means to tailor the data-reporting and- collection functions of the system to his own needs and requirements. Because the instrument is controlled by software, the functions of calibration, measurement, timing, reporting, plotting, and data quality assurance are highly cost-effective. Extensive use of formal test procedures permits verifying all systems and data reliability; it also assures meeting the desired specifications. The ease of operation and high-quality results inherent in this system make it unsurpassed in gas-exchange measurements.

Computers↗
PubMed1984

Molecular cloning of the GAL80 gene from Saccharomyces cerevisiae and characterization of a gal80 deletion.

An integrated GAL1-lacZ fusion provided a useful phenotypic marker for the gal80- regulatory mutation in Saccharomyces cerevisiae. On minimal glucose plates containing a beta-galactosidase indicator, a GAL80 strain containing the fusion gave white colonies, whereas a gal80- strain gave blue colonies. This color difference was used to isolate the GAL80 gene from a plasmid bank by complementation of the gal80- mutant. The putative GAL80 gene was located on a 2.6-kb HindIII-SalI fragment and has been subcloned into an integrating vector. Genetic analysis showed that the clone integrated at the GAL80 locus. A deletion that covered the entire GAL80 region was constructed in vitro and transplaced into the yeast genome to give an isogenic pair of GAL80 and gal80 deletion strains. Glucose repression of a GAL1-lacZ fusion was normal in the gal80 deletion strain, implying that the GAL80 gene product is not involved in glucose repression.

Chromosome Deletion↗
PubMed1984

Failure of weight reduction to reduce mildly elevated blood pressure: a randomized trial.

To determine the value of weight reduction on blood pressure, we randomly allocated 60 untreated, mildly hypertensive, obese individuals to a no-treatment control group or to a behaviourly-oriented weight loss ('diet') programme administered by professional dietitians. Behavioural techniques included self-monitoring, shaping, reinforcement and modelling. Subjects were reassessed after six months by an observer who was unaware of their study group. Fifty-four subjects (90%) completed the study. Diet subjects lost 4.1 kg and controls only 0.8 kg (P = 0.018). However, neither systolic nor diastolic blood pressures differed. The chance that we missed a clinically important diastolic difference of 6 mmHg (our pre-study target) is less than 1%. We conclude that our weight loss programme was successful in reducing weight but that weight loss is not useful in lowering blood pressure in mild, otherwise untreated hypertensives.

Blood Pressure↗