Biomedical subjects
M S Johnson
Publications and source records attributed to M S Johnson.
Confidence limits on the branching order of phylogenetic trees.
We describe a confidence test for branching order that can aid protein phylogeny reconstruction as well as the evaluation of the optimal tree. It is proposed that the process resulting in the observed amino acid residue differences, which is the basis for the identification of the order and relative times of divergence events, is appropriately described by a modification of the negative binomial distribution. The relative total numbers of mutations (accepted and nonaccepted), which result in a given number of amino acid differences, may be obtained as the expectation of this distribution. The associated variances enable significant differences in tree branching order to be established. If the total rates of mutation of the genes encoding the compared proteins are equal, the expected total mutations and their associated variances map identically to their relative times of divergence. In addition, significantly different rates of change (due to differences in total mutation rate and/or acceptance rate) may be identified without the requirement of outlying reference group. The method is equally applicable to phylogenies derived from DNA or RNA sequence information.
The involvement of dihydropyridine-sensitive calcium channels in phorbol ester-induced luteinizing hormone and growth hormone release.
We examined the role of voltage-activated, L-type, Ca2+ channels in phorbol ester-induced luteinizing hormone (LH) and growth hormone (GH) release from rat anterior pituitary tissue. The L-type Ca2+ channel inhibitor, nimodipine (NMD), inhibited phorbol 12,13-dibutyrate (PDBu)-induced GH release but had no significant effect on LH release. The L-type Ca2+ channel activator BAY K 8644 had no effect on PDBu-induced GH release but potentiated PDBu-induced LH release. In contrast, 60 mM K(+)-induced LH and GH release were inhibited by NMD, whereas BAY K 8644 had no effect. When PDBu and either K+ or BAY K 8644 were used together, they acted synergistically to evoke levels of LH release greater than addition of release caused by each secretagogue alone. However, the release of GH was additive with PDBu and either K+, BAY K 8644. The protein kinase C (PKC) inhibitor staurosporine inhibited both PDBu-induced LH release and GH release. A structurally different PKC inhibitor, H7, significantly inhibited PDBu-induced LH release but had no effect on PDBu-induced GH release. Both staurosporine and H7 inhibited LH release induced by PDBu and BAY K 8644 together. In contrast, although staurosporine inhibited GH release induced by PDBu and BAY K 8644, H7 significantly potentiated this response. A difference in the action of these two inhibitors was also apparent on K(+)-induced hormone release where staurosporine partially blocked K(+)-induced LH and GH release but H7 had no effect on the release of either hormone. Data obtained in 45Ca2+ influx experiments further suggested that a staurosporine-sensitive, but H7-resistant, PKC-like kinase may tonically maintain L-channels in a voltage-sensitive state, as down-regulation of PKC in dispersed anterior pituitary cells by long term PDBu treatment caused a significant reduction in K(+)-induced 45Ca2+ influx. We conclude that phorbol ester-induced GH release, but not LH release, is a result of L-type Ca2+ channel activation which may occur by means of alterations in the channel itself to increase its responsiveness to a given depolarisation.
The differential effects of protein kinase C activators and inhibitors on rat anterior pituitary hormone release.
We investigated the possibility that various protein kinase C (PKC) activators and inhibitors may differentially affect luteinizing hormone (LH) and growth hormone (GH) release from rat anterior pituitary tissue, incubated in vitro. Activators of PKC induced LH release with the following order of potency: mezerein > phorbol 12,13-dibutyrate (PDBu). Mezerein and PDBu were equipotent on GH release. A range of PKC inhibitors (including compounds highly selective for PKC) potently and completely inhibited PKC activator-induced LH and GH release. Chelerythrine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7) were less potent inhibitors of PDBu-induced GH release than of LH release. A component of PDBu- and mezerein-induced LH release was inhibited by H7 with high potency, but a second H7-insensitive component was detected. Mezerein- and PDBu-induced GH release consisted of an H7-resistant component only. When the regulatory domain of PKCs from different sources was investigated by displacement of [3H]PDBu binding, the affinity for mezerein was 3-5-fold greater than that for PDBu at PKCs from cerebral cortex, lung and alpha and beta isoforms extensively purified from brain. Anterior pituitary PKCs were unusual in showing closely matched affinity for mezerein and PDBu, reminiscent of their equivalent potency on GH release. In order to investigate the potency of the catalytic domain inhibitor H7 on PKCs from different sources, enzyme activity assays were carried out on partially purified cytosolic PKCs from midbrain and anterior pituitary and on extensively purified PKC alpha and PKC beta. The Ca(2+)-independent component of PDBu-induced (phosphatidylserine-dependent) activity from anterior pituitary alone showed unusually low potency of inhibition by H7 but was potently inhibited by staurosporine and Ro 31-8220. In contrast, the Ca(2+)-dependent PKC activity in anterior pituitary was inhibited by H7, staurosporine and Ro-31-8220 with high potency as in all other preparations. These results are consistent with the presence and active role in secretion of pharmacologically distinct forms of PKC (or PKC-like kinases) in rat anterior pituitary cells.
Molecular recognition in protein families: a database of aligned three-dimensional structures of related proteins.
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Biochemical characterisation of an apparently novel isoform of protein kinase C in pituitary.
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Modelling of the lignin peroxidase LIII of Phlebia radiata: use of a sequence template generated from a 3-D structure.
A model of the lignin peroxidase LIII of Phlebia radiata was constructed on the basis of the structure of cytochrome c peroxidase (CCP). Because of the low percentage of amino acid identity between the CCP and the lignin peroxidase LIII of Phlebia radiata, alignment of the sequences was based on the generation of a template from a knowledge of the 3-D structure of CCP and consensus sequences of lignin peroxidases. This approach gave an alignment in which all the insertions in the lignin peroxidase were placed at loop regions of CCP, with a 21.1% identity for these two proteins. The model was constructed using this alignment and the computer program COMPOSER, which assembles the model as a series of rigid fragments derived from CCP and other proteins. Manual intervention was required for some of the longer loop regions. The alpha-helices forming the structural framework, and especially the haem environment of CCP, are conserved in the LIII model and the core is close packed without holes. A possible site of the substrate oxidation at the haem edge of LIII is discussed.
Comparison of methods for specific depletion of ATP in Salmonella typhimurium.
Three methods of ATP depletion in Salmonella typhimurium were compared. ATP concentrations were lowest after arsenate treatment. Arsenate or alpha-methylglucoside-plus-azide treatment nonspecifically lowered all nucleotide triphosphate levels. Histidine starvation in a hisF mutant was relatively specific for ATP depletion and therefore has potential in distinguishing ATP-dependent processes from processes dependent on other nucleotides.
Oestradiol-17 beta modulates the actions of pharmacologically distinct forms of protein kinase C in rat anterior pituitary cells.
Phorbol ester-induced release of LH and GH from rat anterior pituitary tissue in vitro is differentially inhibited by some, but not other, inhibitors of protein kinase C (PKC), suggesting that pharmacologically distinct species of PKC may have different functional roles in these cells. Since stimulus-induced anterior pituitary hormone release can be enhanced by oestradiol-17 beta (OE2) pretreatment, we investigated the effect of OE2 treatment of long-term (4 weeks) ovariectomized rats on the amount, activity and cellular actions of pharmacologically distinct PKC species in rat anterior pituitary tissue. Here we report that OE2 treatment enhanced phorbol 12,13-dibutyrate (PDBu)-induced LH but not GH release measured in vitro. This effect of OE2 on LH release may involve synthesis of additional PKCs that are not targeted by the synthetic diacylglycerol, 1,2-dioctanoyl-sn-glycerol (DOG). Measurements of anterior pituitary PKC activity and [3H]phorbol ester-binding studies suggested that the facilitatory action of OE2 on LH release may occur, at least in part, by altering the quantity and activity of PKC(s). Our results also demonstrate that the OE2-induced PKC(s) which facilitate LH release may be of the type that are not dependent upon raised intracellular Ca2+ for their activation and display distinct pharmacological properties (being readily activated by PDBu, but not by DOG, and are staurosporine-sensitive but H7-insensitive). This facilitatory action of OE2 on PKC-induced LH release does not appear to involve OE2-induced changes in the affinity of existing PKC(s) for PDBu, or changes in the amount of releasable LH in the pituitary prior to the stimulus.
New radio service targets EMS communications.
The EMS community should commend the FCC for attempting to help relieve some of the frequency-congestion problems by adopting the EMRS. EMS users, however, should continue to advocate cost-effective solutions to EMS frequency-congestion problems.
Environment-specific amino acid substitution tables: tertiary templates and prediction of protein folds.
The local environment of an amino acid in a folded protein determines the acceptability of mutations at that position. In order to characterize and quantify these structural constraints, we have made a comparative analysis of families of homologous proteins. Residues in each structure are classified according to amino acid type, secondary structure, accessibility of the side chain, and existence of hydrogen bonds from the side chains. Analysis of the pattern of observed substitutions as a function of local environment shows that there are distinct patterns, especially for buried polar residues. The substitution data tables are available on diskette with Protein Science. Given the fold of a protein, one is able to predict sequences compatible with the fold (profiles or templates) and potentially to discriminate between a correctly folded and misfolded protein. Conversely, analysis of residue variation across a family of aligned sequences in terms of substitution profiles can allow prediction of secondary structure or tertiary environment.
Trace metal accumulation by the shrew Sorex araneus. I. Total body burden, growth, and mortality.
A laboratory feeding trial is described in which a known trace metal accumulator, the shrew Sorex araneus, was presented with an artificial diet containing high levels of copper and cadmium. Methodological difficulties relating to feeding trials on first generation offspring of insectivorous small mammals caught in the wild are described. There was no relationship between copper or cadmium intake and mortality during the trials. Sex differences in growth were apparently not influenced by metal intake. At the end of the trials there was a significant negative correlation between body weight and total cadmium burden. Whole body concentrations and burdens of cadmium increased with estimated intake. There was a significant negative correlation between percentage of cadmium retained as body burden and estimated cadmium intake. There was no discernible relationship between copper burden and intake.
Trace metal accumulation by the shrew Sorex araneus. II. Tissue distribution in kidney and liver.
The distributions of trace metals in tissues of shrews in a series of feeding trials are described. The relationship between body cadmium burden and organ weight was directly related to differences in body weight. Organ weight expressed as a percentage of body weight was constant in the kidney, but was correlated with cadmium burden for the liver. Liver and kidney accounted for up to 80% of total body cadmium. The proportion of the body cadmium burden found in the kidney ranged from 8 to 17%. Copper and zinc concentrations in the kidney increased in response to cadmium accumulation. Hepatic zinc concentrations increased with cadmium accumulation. Exposure to a copper-contaminated diet resulted in increased liver copper levels. High liver concentrations were correlated with high kidney concentrations. At the lowest levels of exposure to metals liver and kidney concentrations were similar, but as exposure increased so did liver concentrations and much more so than in the kidney.
Mercury in flounder (Platichthys flesus L.) from estuaries and coastal waters of the north-east Irish Sea.
This paper reports concentrations of mercury in muscle tissue of flounder (Platichthys flesus L.) from various estuaries and coastal waters of north Wales and north-west England that encompass the north-east Irish Sea. Mercury concentrations were highest within and offshore the Ribble Estuary (0.476 +/- 0.037 mg kg(-1)) but were also high within the Mersey (0.389+/-0.042 mg kg(-1)) and Dee Estuaries (0.295+/-0.065) and at coastal sites in the vicinity. Values for all three estuaries and their near coastal sites were significantly higher than for sites in the north and central sectors of the study area. Regressions of mercury concentration against fish length showed significant relationships and age accumulation for the majority of sites, particularly within or in close proximity to the Mersey, Dee and Ribble Estuaries.
The priming effect of luteinizing hormone-releasing hormone (LHRH) but not LHRH-induced gonadotropin release, can be prevented by certain protein kinase C inhibitors.
The priming effect of LHRH in vitro (which results in increased responsiveness of gonadotropes to both LHRH receptor-mediated and receptor-independent stimuli) is brought about by an unknown mechanism. The present results indicate that induction of the LHRH priming effect is inhibited in a concentration-dependent manner by the protein kinase C (PKC) inhibitors staurosporine, K252a, H7 and by the novel highly-selective PKC inhibitor, Ro 31-8220. In contrast, a range of other compounds that are relatively selective inhibitors of other kinases such as tyrosine kinases and Ca2+/calmodulin-dependent kinases were unable to prevent priming. The PKC inhibitors prevented priming without affecting initial LHRH-induced gonadotropin secretion. Thus, the priming-elicited increment in secretion was selectively removed, restoring hormone release to the level measured during an initial response to LHRH. Similar results were obtained on different days of the estrous cycle where the magnitude of the priming effect varies. Experiments on the time course of PKC inhibitor action revealed that the critical period was in the induction of the priming effect, not its expression. The PKC inhibitors had neither acute nor delayed effects on gonadotropin secretion induced by ionomycin. Staurosporine, K252a and Ro 31-8220 inhibited LHRH priming with identical potencies to their inhibition of phorbol ester-induced gonadotropin secretion. The reduced potency of H7 seen on LHRH priming compared to phorbol ester-induced gonadotropin release parallels results seen with this inhibitor on phorbol ester-induced secretion of growth hormone (Johnson and Mitchell (1989) Biochem. Soc. Trans. 17, 751-752) and on the pharmacological characteristics of PKCs partially purified from anterior pituitary tissue. In all aspects of this study, effects on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion appeared to be entirely similar.
Properties and [32P] phosphorylation targets of a novel form of protein kinase C in pituitary.
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EMS in a new world order. Visiting the Commonwealth of Independent States.
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The FCC may be listening. An update on EMS communications.
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