Insulin resistance in denervated skeletal muscle. Inability of insulin to stimulate dephosphorylation of glycogen synthase in denervated rat epitrochlearis muscles

We have investigated the effects of insulin and motor denervation on the phosphorylation of glycogen synthase in skeletal muscle. Rat epitrochlearis muscles were denervated in vivo 3 days before the contralateral and denervated muscles were incubated in vitro with 32Pi to label sites in glycogen synthase. The 32P-labeled synthase was rapidly immunoprecipitated from extracts under conditions which prevented changes in the phosphorylation state of the enzyme. When 32P-labeled synthase from contralateral muscles was cleaved with CNBr, essentially all of the 32P was recovered in two fragments, denoted CB-1 and CB-2. Incubating these muscles with insulin decreased the 32P content of each fragment by approximately 25%, indicating that the hormone stimulated dephosphorylation of at least two sites. Peptide mapping by reverse phase high performance liquid chromatography was performed to resolve phosphorylation sites more completely. The results suggest that the enzyme was phosphorylated in sites 1a, 1b, 2, 3(a+b+c), and 5. Insulin stimulated dephosphorylation of sites in peptides presumed to contain sites 1b, 2, and 3(a+b+c). Synthase from denervated muscles appeared to contain the same amount of phosphate as enzyme from contralateral muscles, and denervation did not detectably affect the distribution of 32P within the subunit. However, denervation abolished the effect of insulin on decreasing the 32P content of synthase. The results indicate that the insulin resistance induced by denervation involves a loss in the ability of insulin to stimulate dephosphorylation of glycogen synthase.     

BCNU-induced protection from hyperbaric hyperoxia: role of glutathione metabolism

To explore the role of the glutathione oxidation-reduction cycle in altering the sensitivity of rats to the effects of hyperbaric hyperoxia, we administered N,N-bis(2-chloroethyl)-N-nitrosourea (BCNU) to decrease tissue glutathione reductase activity. We then exposed these animals and their matched vehicle-treated controls to 100% O2 at 4 ATA. Animals that received BCNU and were immediately exposed to hyperbaric O2 showed enhanced toxicity by seizing earlier in the exposure than controls. Animals that received BCNU 18 h before the hyperbaric O2 exposure were paradoxically protected from the effects of the exposure with a prolongation of their time to initial seizure and a marked increase in their survival time during the exposure. Tissue glutathione concentrations were also measured in the various groups and the hyperbaric O2 exposure produced marked decreases in hepatic glutathione levels in all control animals. In animals treated with BCNU 18 h before exposure, hepatic glutathione concentrations also decreased, but the concentrations had significantly increased during the 18-h waiting period, allowing these animals to maintain hepatic levels in the normal range even during their hyperbaric exposures. We conclude that treatment of rats with BCNU 18 h before exposure to hyperbaric hyperoxia results in enhanced protection of the animals during the exposure.     

Growth of H-35 rat hepatoma cells in unsupplemented serum-free media: Effect of transferrin,insulin and cell density

Summary Serum-free tissue culture medium consisting of a 1∶1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium is herein shown to support growth of Reuber H-35 cells over several days in culture. Cells were initially plated in serum containing DMEM medium for 3 h. After cell attachment, serum is removed and replaced with a serum-free 1∶1 mixture of these two commercially available tissue culture media. The doubling time of cell growth in this unsupplemented serum-free medium was 46 h in lightly plated cultures over the first 5 d. The presence of transferrin (5 μg/ml) and insulin (3.3 nM) results in a cell doubling time of 17 h, which equaled the growth rate in medium containing 10% fetal bovine serum. In the absence of transferrin, growth rates in serum-free medium were correlated with the cell density of cultures. Conditioned medium from dense, serum-free cultures has growth-stimulating activity in recipient lightly plated cultures. This simple, serum-free culture medium will facilitate studies on the growth regulation of H-35 rat hepatoma cells. This work was funded by a feasibility grant from the American Diabetes Association, as well as by the National Institutes of Health grants CA 24604-09 and CA 16463-14.     

Definition and identification of homology domains

A method is described for identifying and evaluating regionsof significant similarity between two sequences. The notionof a ‘homology domain’ is employed which definesthe boundaries of a region of sequence homology containing noinsertions or deletions. The relative significance of differentpotential homology domains is evaluated using a non-linear similarityscore related to the probability of finding the observed levelof similarity in the region by chance. The sensitivity of themethod is demonstrated by simulating the evolution of homologydomains and applying the method to their detection. Severalexamples of the use of homology domain identification are given. Received on July 29, 1987; accepted on November 15, 1987     

Non-coordinate expression of collagen mRNAs during carbon monoxide-induced cardiac hypertrophy

Collagen gene expression during volume overload-induced cardiac hypertrophy was investigated in adult male rats. Hypertrophy of the left ventricle (22%) and right ventricle (37%) occurred following 27 days continuous exposure to 700 ppm carbon monoxide; hematocrit increased nearly 47%. To examine potential cellular and molecular control of restructuring in the heart, we investigated the expression of two specific procollagen mRNAs for collagen types which have different structural-functional roles [Type I (alpha-1) & Type IV]. Type I (interstitial) mRNA levels increased at least 100% relative to controls within 3 days of initial exposure to 700ppm CO, then declined afterwards; type IV(basement membrane) mRNA levels increased more modestly at first, and increased further afterwards. The ratio of type I/type IV RNA's also increased initially, then later declined, with the greatest differences in the relative responses of type I and IV mRNAs seen in the right ventricle. These data suggest that types I and IV collagen mRNA expression is not coordinately expressed during this type of volume overload-induced hypertrophy in rat heart.     

The response of a bumblebee goby,Brachygobius sabanus,to chemical stimuli from injured conspecifics

Synopsis Brachygobius sabanus move less often and spend less time swimming when they detect chemicals released from injured conspecifics. This resembles the alarm response found in ostariophysan fishes, darters, and at least one other gobiid. Chemicals from injured Poecilia reticulata do not induce an alarm response in B. sabanus.     

Genetic mapping of three human homologues of murine t-complex genes localizes TCP10 to 6q27, 15 cM distal to TCP1 and PLG

Human homologues of mouse t-complex genes have been cloned and localized physically to chromosome 6p or 6q. TCP1, TCP10, and PLG are human homologues of genes located in the proximal portion of the t-complex on mouse chromosome 17. We present here results of genetic mapping of these human t-complex homologues previously localized to 6q25-q27, 6q21-q27, and 6q26-q27, respectively, by physical techniques. TCP1 and PLG do not recombine with each other and are separated from TCP10 by about 15 cM, while the corresponding mouse genes are no more than 4 cM apart. Genetic mapping with markers well localized cytogenetically places TCP1 and PLG proximal to TCP10 and localizes the latter to the cytogenetic band 6q27. It is likely that the organization of human t-complex homologues on 6q is similar to that of t haplotypes rather than that of wildtype murine chromosome 17.     

Trolox C, a lipid-soluble membrane protective agent, attenuates myocardial injury from ischemia and reperfusion

The lipophilic antioxidant Trolox C, a vitamin E analog, was administered to isolated, buffer-perfused rabbit hearts subjected to 25 min of global stop-flow ischemia and 30 min of reperfusion. In six hearts, Trolox C (200 microM) was infused for 15 min immediately prior to ischemia and for the first 15 min of reperfusion. Six control hearts received only vehicle. Gas chromatography analysis confirmed that effective myocardial levels of Trolox were attained. At 30 min reperfusion, the recovery of left ventricular developed pressure was 56 +/- 3% of baseline in control hearts versus 70 +/- 4% in Trolox-treated hearts (p < .01). There was also significant improvement in recovery of Trolox-treated hearts in diastolic pressure and both maximum and minimum values of the first derivative of left ventricular pressure (dP/dt). Creatine phosphokinase release into the coronary effluent at 30 min of reperfusion was 16.5 +/- 8.4 IU/min in untreated and 6.3 +/- 1.0 IU/min (p < .05) in Trolox-treated hearts. Thus Trolox C, a lipophilic antioxidant, attenuated myocardial injury during stop-flow ischemia and reperfusion.     

When Epstein-Barr virus persistently infects B-cell lines, it frequently integrates.

In this study we used Gardella gel analysis of intact DNA, Southern blotting of digested DNA, and fluorescence in situ hybridization to provide complementary and unequivocal information on the state of the Epstein-Barr virus (EBV) genome in persistently infected cells. The fluorescence in situ hybridization technique allowed us to directly visualize both integrated and episomal EBV DNA at the single-cell level. We show here that circularization of the EBV genome is rarely detected upon infecting activated normal B cells. The virus can persist upon infection of a different proliferating B-cell target, EBV-negative Burkitt's lymphoma tumor cell lines. Analysis of 16 such lines reveal again, that the virus infrequently persists as covalently closed episomes; rather, the virus preferentially persists by integrating into the host DNA (10 of 16 clones). The integrated virus is linear and usually intact, although 3 of 10 isolates have deletions from the left-hand end including the latent origin of replication. At the level of our analysis, no obvious relationship was seen between the integration sites. These studies provide, for the first time, a reproducible in vitro model system to study integration by EBV.     

A mechanistic perspective on bacterial metabolism of chlorinated methanes

Chlorinated methanes are important environmental pollutants, which can be metabolized by bacteria. The biotransformation of chlorinated methanes by bacteria has been shown to be due either to gratuitous metabolism (cometabolism) or their use as a source of carbon and energy. The reactions which result in carbon-halogen bond cleavage include substitutive, reductive, oxygenative, and gem-elimination mechanisms. Certain methylotrophic bacteria can use dichloromethane as a source of carbon and energy. Dichloromethane dehalogenase catalyzes the first substitutive reaction in this metabolism. The enzyme shows a 10¹⁰-fold rate enhancement over the reaction of the bisulfide anion with dichloromethane in water. Pseudomonas putida G786 synthesizes cytochrome P-450_CAM which catalyzes the gratuitous reduction of chlorinated methanes. These studies with purified enzymes are beginning to reveal more detailed mechanistic features of bacterial chlorinated methane metabolism.Abbreviations DNA deoxyribonucleic acid - k_cat catalytic first order rate constant for an enzyme catalyzed reaction - K_M Michaelis constant for an enzyme catalyzed reaction - MNDO modified neglect of diatomic overlap - PIMA pattern induced multialignment - DCMD dichloromethane dehalogenase