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Biomedical subjects

D A Walsh

Publications and source records attributed to D A Walsh.

At least 163 records · Page 9Linked to original sources
PubMed1984

Detection of methicillin-resistant Staphylococcus aureus by microdilution and disk elution susceptibility systems.

To determine whether methicillin-resistant (MR) Staphylococcus aureus from different geographic areas are detected reliably by various commercially available microdilution broth and disk elution systems, 73 such isolates obtained from hospitals in 13 cities were tested by a reference method (agar dilution) and by the Microscan, API 3600S, Autobac I, and MS-2 systems. Both Eugonic broth and Low Thymidine Eugonic broth were used in the evaluation of the Autobac I, and two versions of the MS-2 were used. The proportions of isolates categorized as MR by the various methods were: agar dilution method, 99%; Microscan, 100% (if the suggested cut-off of the manufacturer was used); API 3600S, 96%; Autobac I, 84 to 93%; and MS-2, 54 to 68%. With the MS-2 system, isolates from Jackson, Miss., were classified as susceptible to methicillin more often than were strains from other cities. With the Autobac I (Eugonic broth), only 55% of isolates from Houston, Tex., were classified as MR, whereas 89% of isolates from all other cities were correctly classified as MR. With the API 3600S, strains from some cities were categorized as nafcillin susceptible, whereas strains from other cities were classified as resistant to nafcillin. The results of this study suggest that future evaluations of antimicrobial susceptibility testing systems should include MR strains of S. aureus from several geographic areas.

Humans↗
PubMed1983

Autophosphorylation of phosphorylase kinase. Divalent metal cation and nucleotide dependency.

This study reports on the divalent metal ion specificity for phosphorylase kinase autophosphorylation and, in particular, provides a comparison between the efficacy of Mg2+ and Mn2+ in this role. As well as requiring Ca2+ plus divalent metal ion-ATP2- as substrate, both phosphorylase kinase autoactivation and phosphorylase conversion are additionally modulated by divalent cations. However, these reactions are affected differently by different ions. Phosphorylase kinase-catalyzed phosphorylase conversion is maximally enhanced by a 4- to 10-fold lower concentration of Mg2+ than is autocatalysis and, whereas both reactions are stimulated by Mg2+, autophosphorylation is activated by Mn2+, Co2+, and Ni2+ while phosphorylase a formation is inhibited. This difference may be due to an effect of free Mn2+ on phosphorylase rather than the inability of phosphorylase kinase to use MnATP as a substrate when catalyzing phosphorylase conversion since Mn2+, when added at a level which minimally decreases [MgATP], greatly inhibits phosphorylase phosphorylation. The interactions of Mn2+ with phosphorylase kinase are different from those of Mg2+. Not only are the effects of these ions on phosphorylase activation opposite, but they also provoke different patterns of subunit phosphorylation during phosphorylase kinase autocatalysis. With Mn2+, the time lag of phosphorylation of both the alpha and beta subunits of phosphorylase kinase in autocatalysis is diminished in comparison to what is observed with Mg2+, and the beta subunit is only phosphorylated to a maximum of 1 mol/mol of subunit. With both Mg2+ and Mn2+ the alpha subunit is phosphorylated to a level in excess of 3 mol/mol, a level similar to that obtained for beta subunit phosphorylation in the presence of Mg2+. The support of autophosphorylation by both Co2+ and Ni2+ has characteristics similar to those observed with Mn2+. Although Mn2+ stimulation of autophosphorylation occurs at levels much higher than normal physiological levels, the possible potential of phosphorylase kinase autophosphorylation as a control mechanism is illustrated by the 80- to 100-fold activation that occurs in the presence of Mn2+, a level far in excess of the enzyme activity change normally seen with covalent modification. Autophosphorylation of phosphorylase kinase demonstrates a Km for Mg X ATP2- of 27.7 microM and a Ka for Mg2+ of 3.1 mM. The reaction mechanism of autophosphorylation is intramolecular. This latter observation may indicate that phosphorylase kinase autocatalysis could be of potential physiological relevance and could occur with equal facility in cells containing either constitutively high or low levels of this enzyme.

Adenosine Triphosphate↗
PubMed1983

Hormonal regulation of the phosphorylation of glycogen synthase in perfused rat heart. Effects of insulin, catecholamines, and glucagon.

32P-labeled perfused rat hearts were used to study the hormonal regulation of glycogen synthase. Following equilibration of perfused hearts with inorganic [32P]phosphate for 30 min, there was an incorporation of approximately 200 pmol of [32P]phosphate/unit of enzyme activity that arose from an exchange of [32P] phosphate with the endogenous [31P]phosphate. Maximum insulin-induced activation (10 milliunits/ml for 5 min), which promoted an I/D activity ratio change from 25% I to 40% I, was associated with a 22% decrease in phosphate content of the enzyme. With hearts from alloxan-induced diabetic animals, there was a 17% higher level of phosphate incorporation and a 4-fold decrease in % I glycogen synthase activity compared to normal animals, but with the diabetic tissue, insulin added to the perfusate had no effect on either the phosphate content or the activity ratio of the enzyme. In perfused hearts from normal animals, DL-isoproterenol and glucagon caused an increase in glycogen synthase phosphorylation of 85-100 pmol/unit of enzyme activity, while L-phenylephrine increased the phosphate content by only 20-35 pmol, but all three hormones caused the same degree of inactivation. The increase in cardiac glycogen synthase phosphorylation induced by DL-isoproterenol, glucagon, and L-phenylephrine was identical, with or without insulin pretreatment; this despite the fact that these three hormones promoted a 3- to 4-fold larger decrease in the enzyme activity ratio with the insulin-treated tissue. In perfused diabetic hearts, DL-isoproterenol, glucagon, and L-phenylephrine caused increased phosphorylation of glycogen synthase without affecting the albeit already low activity ratio of the enzyme. These results show that in the intact perfused heart the same degree of glycogen synthase inactivation can occur as a consequence of differing degrees of phosphorylation, presumably due to phosphorylation at different sites promoted by different second messengers. Conversely, the data indicate that the same extent of phosphorylation, as stimulated by the same second messenger, can inactivate glycogen synthase by different amounts depending upon the prior phosphorylation state of other sites in the protein.

Animals↗
PubMed1983

Mg X ATP2-dependent interaction of the inhibitor protein of the cAMP-dependent protein kinase with the catalytic subunit.

The interaction between the inhibitor protein and the catalytic subunit of the cAMP-dependent protein kinase has been investigated by steady state kinetics and by an assessment of the requirement of this interaction for ATP. By analysis for tightly bound inhibitors, inhibition by the inhibitor protein was shown to be competitive versus peptide substrate and uncompetitive versus Mg X ATP2-. This, together with the observations of Gronot et al. (Gronot, J., Mildvan, A.S., Bramson, H. N., Thomas, N., and Kaiser, E.T. (1981) Biochemistry 20, 602-610) and those given in the accompanying paper (Whitehouse, S., Feramisco, J.R., Casnellie, J.E., Krebs, E.G., and Walsh, D.A. (1983) J. Biol. Chem. 258, 3693-3701), would indicate that the probable reaction mechanism of the protein kinase is ordered with the nucleotide binding first and that the inhibitor protein blocks catalysis by interaction with the catalytic subunit-Mg X ATP complex. The Ki for this interaction at saturating Mg X ATP and zero peptide substrate is 0.49 nM. Multiple inhibition analysis in the presence of 5'-adenylimidodiphosphate (AMP X PNP) indicates that the inhibitor protein does not interact with a catalytic subunit-AMP X PNP complex. The requirement for ATP for the inhibitor protein-catalytic subunit interaction has also been demonstrated by direct binding measurements and by the observation that the efficiency of the inhibitor protein is increased by preincubation of the inhibitor protein, catalytic subunit, and ATP in the absence of peptide substrate. By either measurement, the catalytic subunit in the presence of the inhibitor protein, was shown to exhibit an apparent Kd of 20 approximately 60 nM for ATP; this value is two orders of magnitude higher than the affinity for ATP by the catalytic subunit alone. This high apparent affinity of the catalytic subunit for ATP (in the presence of the inhibitor) does not require that there be a specific binding site on the inhibitor protein for some moiety of the ATP but may simply be a reflection of the formation of a catalytic subunit-Mg X ATP X inhibitor protein complex with resultant displacement of the equilibrium of ATP binding to the protein kinase.

Adenosine Triphosphatases↗
PubMed1983

Studies on the kinetic mechanism of the catalytic subunit of the cAMP-dependent protein kinase.

The kinetic mechanism of the catalytic subunit of the cAMP-dependent protein kinase has been investigated employing the heptapeptide Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) as substrate. Initial velocity measurements performed over a wide range of ATP and Kemptide concentrations indicated that the reaction follows a sequential mechanistic pathway. In line with this, the results of product and substrate inhibition studies, the patterns of dead end inhibition obtained employing the nonhydrolyzable ATP analogue, AMP X PNP (5'-adenylylimidodiphosphate), and equilibrium binding determinations, taken in conjunction with the patterns of inhibition observed with the inhibitor protein of the cAMP-dependent protein kinase that are reported in the accompanying paper (Whitehouse, S., and Walsh, D.A. (1983) J. Biol. Chem. 258, 3682-3692), are best fit by a steady state Ordered Bi-Bi kinetic mechanism. Although the inhibition patterns obtained employing the synthetic peptide analogue in which the phosphorylatable serine was replaced by alanine were apparently incompatible with this mechanism, these inconsistencies appear to be due to some element of the structure of this latter peptide such that it is not an ideal dead end inhibitor substrate analogue. The data presented both here and in the accompanying paper suggest that both this substrate, analogue and the ATP analogue, AMP X PNP, do not fully mimic the binding of Kemptide and ATP, respectively, in their mechanism of interaction with the protein kinase. It is proposed that, as with some other kinase reactions, the configuration of the terminal anhydride bond of ATP assumes a conformation once the nucleotide is bound to the protein kinase that assists in the binding of either Kemptide or the inhibitor protein but not the alanine-substituted peptide and that AMP X PNP, because of its terminal phosphorylimido bond, cannot assume this conformation which favors protein (or peptide) binding.

Adenylyl Imidodiphosphate↗
PubMed1983

Fetal alcohol syndrome. A great paediatric imitator.

Twenty children affected by prenatal exposure to alcohol are described. Their mothers either suffered from chronic alcoholism, or were binge or heavy drinkers while pregnant. Only in nine mothers (45%) was alcohol abuse recognized and associated with their babies' anomalies in the first months after birth; in some, the diagnosis of fetal alcohol syndrome was delayed for many years. Alcohol abuse in pregnancy is probably a significant cause of birth and developmental defects in Australian children.

Alcoholism↗
PubMed1983

Teratogenesis after acute alcohol exposure in cultured rat embryos.

In order to investigate whether alcohol has teratogenic properties, rat embryos were cultured in vitro during their organogenetic period and exposed to ethanol at 200-800 mg% culture medium for the 48 hours of culture (0-24 somite stage) or to 600-800 mg% for 24 hours, or 6-hour periods. Exposure to alcohol throughout the entire 48-hour culture period or the first 24-hour period (0-12 somites) produced marked growth retardation, particularly of the head region in a dose-dependent manner, but did not prevent neural tube closure. Exposure to high levels of ethanol during specific 6-hour periods of early organogenesis (three to nine somites) prevented closure of the neural tube in 30% of cultured rat embryos, indicating a direct teratogenic action of ethanol. These results implied an effect of ethanol on embryonic development, independent of maternal metabolism. The 6-hour exposure experiments demonstrated that high doses of ethanol during specific periods of organogenesis can be teratogenic.

Animals↗
PubMed1983

Some teratogenic properties of ethanol and acetaldehyde in C57BL/6J mice: implications for the study of the fetal alcohol syndrome.

To investigate the teratogenic effect of acute alcohol exposure, pregnant C57BL/6J mice were exposed to 25% ethanol (either two doses of 2.9g/kg or one dose 5.8g/kg) during the organogenic period either by intraperitoneal injections or by intubation. The incidence of malformations varied according to (1) the stage of embryonic development at the time of exposure, (2) the route of administration of the alcohol, and (3) the amount of alcohol given and the time period over which it was administered. Oral doses of alcohol were teratogenic although less so than the same dose given intraperitoneally, and two intraperitoneal doses four hours apart produced significantly more malformation than the same two doses six hours apart. The primary metabolite of alcohol, acetaldehyde, was also investigated for its teratogenicity. It was found that one or two doses of four percent acetaldehyde (0.32g/kg), administered intraperitoneally were teratogenic. A further attempt was made to raise blood acetaldehyde levels by exposing mice to disulfiram, an inhibitor of acetaldehyde dehydrogenase, prior to administration of alcohol. The disulfiram pretreatment did not increase the malformation rate. Treatment with alcohol on day 7 or 8 caused a variety of facial abnormalities, some of which were comparable to those seen in children with fetal alcohol syndrome. Exposure on day 9 or 10 resulted in limb defects. The results suggest that one or more episodes of heavy maternal drinking at critical periods in pregnancy may severely damage the embryo and may produce many features of the fetal alcohol syndrome.

Acetaldehyde↗
PubMed1983

Regulation of cardiac glycogen synthase.

In perfused rat hearts insulin can activate, and catecholamines can inactivate, glycogen synthase (EC 2.4.1.11); the magnitude of each hormonal response is magnified if tissue glycogen levels are depleted. Both beta-adrenergic and alpha-adrenergic agonists inactivate insulin-stimulated and basal glycogen synthase, with each promoting the same extent of inactivation in both circumstances. In this system beta-adrenergic agonists act via cyclic AMP (cAMP), and alpha-adrenergic agonists via Ca2+, whereas insulin action appears to be independent of either cAMP or Ca2+. The action on cardiac glycogen synthase by the physiological catecholamine epinephrine is apparently mediated by the concomitant interaction with both alpha and beta receptors; interaction with each is mediated by their separate second messenger systems, which combine to produce the end physiological response.

Animals↗
PubMed1982

Cardiac phosphorylase kinase. Deactivation by selective dephosphorylation of alpha' and beta subunits.

We have documented previously that although the phosphorylation of the beta subunit of cardiac phosphorylase kinase plays a major role in activation of the enzyme, enzyme activity is also modulated by either cAMP-dependent or cAMP-independent phosphorylation of the alpha' subunit (Sul, H. S., Cooper R. H., Whitehouse S., and Walsh D. A. (1982) J. Biol. Chem. 257, 3484-3490). This paper reports that deactivation of 32P-labeled cardiac phosphorylase kinase by protein phosphatase occurs concomitantly with the dephosphorylation of either the alpha' or beta subunits. However, enzyme activation attributable to alpha' subunit phosphorylation is still apparent after dephosphorylation of the beta subunit and, similarly, that caused by beta subunit phosphorylation is maintained after alpha' subunit dephosphorylation. These data support out previously stated conclusion that both alpha' and beta subunit phosphorylation regulates the activity of cardiac phosphorylase kinase.

Adenosine Triphosphate↗
PubMed1982

Purification of a physiological form of the inhibitor protein of the cAMP-dependent protein kinase.

The inhibitor protein of the cAMP-dependent protein kinase exists in two forms which can be distinguished by separation techniques that characterize proteins on the basis of size (McPherson, J. M., Whitehouse, S., and Walsh, D. A. (1979) Biochemistry 18, 4835-4845). To date, only one of these forms has been purified and the form obtained was not the species that is present in the initial extracts of tissues. This paper reports the purification to homogeneity of the form of the inhibitor that is present in initial cell extracts and which, in consequence, appears to be a physiological size species of this protein.

Amino Acids↗
PubMed1982

Cyclic AMP-dependent and cyclic AMP-independent antagonism of insulin activation of cardiac glycogen synthase.

The hormonal regulation of glycogen synthase has been studied with isolated perfused hearts that were depleted of 85% of their endogenous glycogen. Glycogen depletion alone promoted a 3-fold activation of glycogen synthase and magnified by 3-fold the response to insulin. Glycogen depletion also facilitated the detection of epinephrine-promoted glycogen synthase inactivation. Hormonal effects on glycogen synthase have been correlated with changes in phosphorylase, phosphorylase kinase, and tissue cAMP levels. Insulin activation of glycogen synthase was observed within 90 s of hormone addition and was maximal by 4 min. A half-maximum effect was obtained at an insulin concentration of 100 microunits/ml. Insulin-dependent activation is reversed by beta-adrenergic agonists, alpha-adrenergic agonists, and glucagon. Each promote the same degree of inactivation and the maximum extent of inactivation produced by each is independent of whether or not the tissue has been stimulated with insulin. beta-Adrenergic agonists and glucagon act via cAMP, alpha-agonists most likely act via intracellular Ca2+ translocation, and insulin action would appear to be independent of either cAMP or Ca2+. The action of epinephrine on cardiac glycogen synthase is mediated by interaction with both alpha- and beta-receptors. As indicated by dose-response curves, receptor occupancy of each occurs to an almost equal extent at suboptimal epinephrine concentrations. Regulation of cardiac glycogen synthase by epinephrine thus is mediated by two second messenger systems which converge to produce the end physiological response.

Animals↗