Insulin activation of phosphatidylinositol 3-kinase in human skeletal muscle in vivo

Hickey, Matthew S., Charles J. Tanner, D. Sean O'Neill,Lydia J. Morgan, G. Lynis Dohm, and Joseph A. Houmard. Insulin activation of phosphatidylinositol 3-kinase in human skeletal muscle invivo. J. Appl. Physiol. 83(3):718-722, 1997.The purpose of this investigation was to determinewhether insulin-stimulated phosphatidylinositol 3-kinase (PI3-kinase)activity is detectable in needle biopsies of human skeletal muscle.Sixteen healthy nonobese males matched for age, percent fat, fastinginsulin, and fasting glucose participated in one of two experimentalprotocols. During an intravenous glucose tolerance test (IVGTT)protocol, insulin-stimulated PI3-kinase activity was determined frompercutaneous needle biopsies at 2, 5, and 15 min post-insulinadministration (0.025 U/kg). In the second group, a 2-h, 100 mU · m² · min¹euglycemic hyperinsulinemic clamp was performed, and biopsies wereobtained at 15, 60, and 120 min after insulin infusion was begun.Insulin stimulated PI3-kinase activity by 1.6 ± 0.2-, 2.2 ± 0.3-, and 2.2 ± 0.4-fold at 2, 5, and 15 min, respectively, duringthe IVGTT. During the clamp protocol, PI3-kinase was elevated by 5.3 ± 1.3-, 8.0 ± 2.6-, and 2.7 ± 1.4-fold abovebasal at 15, 60, and 120 min, respectively. Insulin-stimulatedPI3-kinase activity at 15 min post-insulin administration wassignificantly greater during the clamp protocol vs. the IVGTT(P < 0.05). These observations suggest that insulin-stimulated PI3-kinase activity is detectable inneedle biopsies of human skeletal muscle, and furthermore, that theeuglycemic, hyperinsulinemic clamp protocol may be a useful tool toassess insulin signaling in vivo.

     

Degradation of 4,4'-dichlorobiphenyl, 3,3',4,4'-tetrachlorobiphenyl, and 2,2',4,4',5,5'-hexachlorobiphenyl by the white rot fungus Phanerochaete chrysosporium.

The white rot fungus Phanerochaete chrysosporium has demonstrated abilities to degrade many xenobiotic chemicals. In this study, the degradation of three model polychlorinated biphenyl (PCB) congeners (4,4'-dichlorobiphenyl [DCB], 3,3',4,4'-tetrachlorobiphenyl, and 2,2',4,4',5,5'-hexachlorobiphenyl) by P. chrysosporium in liquid culture was examined. After 28 days of incubation, 14C partitioning analysis indicated extensive degradation of DCB, including 11% mineralization. In contrast, there was negligible mineralization of the tetrachloro- or hexachlorobiphenyl and little evidence for any significant metabolism. With all of the model PCBs, a large fraction of the 14C was determined to be biomass bound. Results from a time course study done with 4,4'-[14C]DCB to examine 14C partitioning dynamics indicated that the biomass-bound 14C was likely attributable to nonspecific adsorption of the PCBs to the fungal hyphae. In a subsequent isotope trapping experiment, 4-chlorobenzoic acid and 4-chlorobenzyl alcohol were identified as metabolites produced from 4,4'-[14C]DCB. To the best of our knowledge, this the first report describing intermediates formed by P. chrysosporium during PCB degradation. Results from these experiments suggested similarities between P. chrysosporium and bacterial systems in terms of effects of congener chlorination degree and pattern on PCB metabolism and intermediates characteristic of the PCB degradation process.     

Inhibition of protein synthesis by Cl-

Optimum K+ concentration for protein synthesis in four eukaryotic cell-free systems is obtained with 70 to 80 mM added KCl or with 110 to 150 mM added K(OAc). The different K+ optima are due to inhibition of protein synthesis by Cl- at concentrations higher than those present in the cytoplasm of eukaryotic cells. Initiation of protein synthesis is severely inhibited with 150 mM added KCl. This inhibition results from an impairment of mRNA binding to ribosomes. The binding of initiator Met-tRNAt, however, is only slightly inhibited by 150 mM KCl.     

Gap junction structures: Analysis of the x-ray diffraction data

Models for the spatial distribution of protein, lipid and water in gap junction structures have been constructed from the results of the analysis of X-ray diffraction data described here and the electron microscope and chemical data presented in the preceding paper (Caspar, D. L. D., D. A. Goodenough, L. Makowski, and W.C. Phillips. 1977. 74:605-628). The continuous intensity distribution on the meridian of the X-ray diffraction pattern was measured, and corrected for the effects of the partially ordered stacking and partial orientation of the junctions in the X-ray specimens. The electron density distribution in the direction perpendicular to the plane of the junction was calculated from the meridional intensity data. Determination of the interference function for the stacking of the junctions improved the accuracy of the electron density profile. The pair-correlation function, which provides information about the packing of junctions in the specimen, was calculated from the interference function. The intensities of the hexagonal lattice reflections on the equator of the X-ray pattern were used in coordination with the electron microscope data to calculate to the two-dimensional electron density projection onto the plane of the membrane. Differences in the structure of the connexons as seen in the meridional profile and equatorial projections were shown to be correlated to changes in lattice constant. The parts of the junction structure which are variable have been distinguished from the invariant parts by comparison of the X-ray data from different specimens. The combination of these results with electron microscope and chemical data provides low resolution three- dimensional representations of the structures of gap junctions.     

A cytochemical investigation of the host-parasite interface inPisum sativum infected by the downy mildew fungusPeronospora pisi

Summary A cytochemical study involving enzymic digestions, chemical extractions and specific staining methods was made of the host-parasite interface in downy mildew infected pea plants. Enzymic hydrolysis revealed both the penetration and extrahaustorial matrices to have a proteinaceous component, possibly glycoprotein, while the extrahaustorial matrix had cellulose as an additional constituent. Intense silver proteinate staining of both matrices following a prolonged incubation in thiocarbohydrazide indicated the presence of complex carbohydrates. Carbohydrates were confirmed as constituents of both matrices following the complete suppression of silver staining using the aldehyde blocking agents dimedone and sodium borohydride. Both matrices also exhibited a marked affinity for phosphotungstic acid. Following acetylation a complete elimination of phosphotungstate staining resulted, again indicating carbohydrate as a constituent of both matrices. Digestion of the fungal cell wall using an enzyme which hydrolyses -1,3-glucans showed that these carbohydrates are important in its construction. However such enzyme treatment did not affect the structural integrity of either the penetration or extrahaustorial matrix. The extrahaustorial membrane exhibited negligible staining with phosphotungstic-chromic acid treatment, while the host plasmalemma showed a positive but variable staining reaction. A very intense staining reaction characterized the fungal plasmalemma after staining with either phosphotungstic-chromic acid, phosphotungstic acid or silver proteinate.     

Long term effect on mortality of stopping smoking after unstable angina and myocardial infarction.

Subjects who stop smoking cigarettes after myocardial infarction have an improved rate of survival compared with those who continue, but to date it was not known whether the benefit persisted for more than six years. A total of 498 men aged under 60 years who had survived a first episode of unstable angina or myocardial infarction by two years were followed up by life table methods for a further 13 years. Mortality in those who continued to smoke was significantly higher (82.1%) than in those who stopped smoking (36.9%). These differences increased with time. Mortality in those who were non-smokers initially and who continued not to smoke was intermediate (62.1%). The adverse effect of continued smoking was most pronounced in those with unstable angina. Continuing to smoke increased the rate of sudden death to a greater degree in those with less severe initial attacks, while the effect of smoking on fatal reinfarctions was most apparent in those with a more complicated presentation. These findings suggest that stopping cigarette smoking is the most effective single action in the management of patients with coronary heart disease.     

Enhanced thermodynamic stabilities of yeast iso-1-cytochromes c with amino acid replacements at positions 52 and 102.

We have determined the structures and thermodynamic stabilities of the wild type Asn-52 and unusually thermostable mutant Ile-52 yeast iso-1-cytochromes c (Das, G., Hickey, D. R. McLendon, D., McLendon, G., and Sherman, F. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 496-499). Although both structures were similar, Water-166, buried within the wild type protein, is excluded from the Ile-52 mutant, which substantially reorganizes the local hydrogen bonding. Wild type Cys-102 was replaced with alanine or serine to eliminate dimerization in vitro. The Cys-102 (wild type), Ala-102, and Ser-102 proteins were equally stable, whereas the chemically modified Cys-102-SCH3 was less stable. The order of stability observed with replacements at positions 52 and 102 was as follows: Ile-52 Ala-102 greater than Ala-52 Ala-102 greater than Asn-52 Ala-102 ("normal") greater than Gly-52 Ala-102. No significant stabilization was attributed to potential energy interactions expressed as helix-forming propensities of replacements at position 52. A high correlation between differences in free energy changes and transfer free energies suggests hydrophobic interactions are the main factor for enhancing stability in the Ile-52 mutant. Additional possible contributions to the thermostability of the Ile-52 variant are energetic effects due to packing and hydrogen bonding changes surrounding position 52.     

Precipitation and 13C-NMR relaxation enhancement measurements of the interactions of bile acids with synthetic cationic bile acid derivatives, and with spin labelled fatty acids.

In an investigation of novel potential bile acid sequestrants, the affinities of the sodium salts of the glycine and taurine conjugates of naturally occurring bile acids (cholate, deoxycholate, chenodeoxycholate and lithocholate) for several cationic ammonium bile acid derivatives have been investigated by measurements of the extent to which the derivatives are able to precipitate the bile acids. This is roughly proportional to the lipophilicity of the interacting species. Thus, amino and ammonium derivatives of cholic acid do not precipitate taurocholate or glycocholate to any great extent, whereas ammonium derivatives of deoxycholate and lithocholate are much more effective. To complement the precipitation measurements, high resolution 13C-NMR has been applied to investigate the weaker interactions between the ammonium cholate derivative and glycocholate, glycodeoxycholate and glycochenodeoxycholate. Addition of either of the latter two bile acids to the cationic ammonium compound results in considerable broadening of the 13C resonances of both species, indicating the formation of relatively rigid structures. In addition, we have used T2 relaxation enhancement induced by spin-labelled fatty acids to examine the mechanism of interaction with bile acids of amphiphilic anions, which might compete with bile acids for sites on bile acid sequestrants. Low concentrations of 16-DOXY L-Stearate dramatically broaden the 13C-NMR resonances of deoxycholate carbons 19, 18 and 7 in particular, while 5-DOXY L-Stearate exerts much less specific effects. These results have been incorporated into a snapshot model of bile acid-fatty acid interactions.     

The structure of human mitochondrial manganese superoxide dismutase reveals a novel tetrameric interface of two 4-helix bundles.

The 2.2 A resolution crystal structure of recombinant human manganese superoxide dismutase, a homotetrameric enzyme that protects mitochondria against oxygen-mediated free radical damage, has been determined. Within each subunit, both the N-terminal helical hairpin and C-terminal alpha/beta domains contribute ligands to the catalytic manganese site. Two identical 4-helix bundles, symmetrically assembled from the N-terminal helical hairpins, form novel tetrameric interfaces that stabilize the active sites. Structurally altered polymorphic variants with reduced activity, such as tetrameric interface mutant Ile-58 to Thr, may produce not only an early selective advantage, through enhanced cytotoxicity of tumor necrosis factor for virus-infected cells, but also detrimental effects from increased mitochondrial oxidative damage, contributing to degenerative conditions, including diabetes, aging, and Parkinson's and Alzheimer's diseases.