Hepatitis B Virus Pre-S2 Mutant Induces Aerobic Glycolysis through Mammalian Target of Rapamycin Signal Cascade

Hepatitis B virus (HBV) pre-S2 mutant can induce hepatocellular carcinoma (HCC) via the induction of endoplasmic reticulum stress to activate mammalian target of rapamycin (MTOR) signaling. The association of metabolic syndrome with HBV-related HCC raises the possibility that pre-S2 mutant-induced MTOR activation may drive the development of metabolic disorders to promote tumorigenesis in chronic HBV infection. To address this issue, glucose metabolism and gene expression profiles were analyzed in transgenic mice livers harboring pre-S2 mutant and in an in vitro culture system. The pre-S2 mutant transgenic HCCs showed glycogen depletion. The pre-S2 mutant initiated an MTOR-dependent glycolytic pathway, involving the eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1), Yin Yang 1 (YY1), and myelocytomatosis oncogene (MYC) to activate the solute carrier family 2 (facilitated glucose transporter), member 1 (SLC2A1), contributing to aberrant glucose uptake and lactate production at the advanced stage of pre-S2 mutant transgenic tumorigenesis. Such a glycolysis-associated MTOR signal cascade was validated in human HBV-related HCC tissues and shown to mediate the inhibitory effect of a model of combined resveratrol and silymarin product on tumor growth. Our results provide the mechanism of pre-S2 mutant-induced MTOR activation in the metabolic switch in HBV tumorigenesis. Chemoprevention can be designed along this line to prevent HCC development in high-risk HBV carriers.     

Protein glycosylation defects in the Saccharomyces cerevisiae mnn7 mutant class. Support for the stop signal proposed for regulation of outer chain elongation

Total cell mannoprotein was isolated from Saccharomyces cerevisiae X2180 mutants that have defects in elongation of the outer chain attached to the N-linked core oligosaccharides (mnn7, mnn8, mnn9, and mnn10) (Ballou, L., Cohen, R. E., and Ballou, C. E. (1980) J. Biol. Chem. 255, 5986-5991). Comparison of the oligosaccharides released by endoglucosaminidase H digestion confirmed that the mnn9 mutation eliminates all but two mannoses of the outer chain, whereas the mnn8 and mnn10 strains produce outer chains of variable but similar lengths. The isolate designated mnn7 was found to be allelic with mnn8. Haploid mutants of the type mnn8 mnn9 or mnn9 mnn10 had the mnn9 phenotype, which established that the mnn9 defect is dominant and presumably acts at a processing step prior to the steps affected by mnn8 and mnn10. Analysis of the mnn1 mnn2 mnn10 oligosaccharides revealed that the heterogeneous outer chain contained 6-16 alpha 1----6-linked mannose units and each was terminated by a single alpha 1----2-linked mannose unit, whereas the core lacked one such unit that was present in the mnn9 oligosaccharide. The results are consistent with and support the hypothesis (Gopal, P. K., and Ballou, C. E. (1988) Proc. Natl. Acad. Sci. U.S.A. 84, 8824-8828) that addition of such a side-chain mannose unit is associated with termination of outer chain elongation in these mutants and may serve as a stop signal that regulates outer chain synthesis in the parent wild-type strain.     

Lipid Content and Composition of Oocytes from Five Coral Species: Potential Implications for Future Cryopreservation Efforts

Given the previously documented importance of lipid concentration and composition in the successful cryopreservation of gorgonian corals, these parameters were assessed in oocytes of five species of scleractinian coral; Platygyra daedalea, Echinopora gemmacea, Echinophyllia aspera, Oxypora lacera and Astreopora expansa. Wax esters, phosphatidylethanolamine, phosphatidylcholine, and fatty acids were all measured at detectable levels, and the latter were produced at significantly elevated quantities in E. gemmacea, E. aspera, and O. lacera. On the other hand, phosphatidylethanolamine, phosphatidylcholine, and wax ester were found at significantly higher concentrations in A. expansa oocytes. Triacylglycerol was not present in any species. Interestingly, the total lipid content of oocytes from all five scleractinians was significantly lower than that of oocytes of two gorgonian species, Junceella juncea and Junceella fragilis. As higher total lipid concentrations may be correlated with greater degrees of cellular membrane fluidity at lower temperatures, it stands to reason that gorgonian coral oocytes may be more likely to survive the cryopreservation process than oocytes of scleractinian corals.     

Cyclic AMP-induced inhibition of collagen lattice contraction by fibroblasts may be attenuated by both cyclic AMP dependent and independent mechanisms

The contraction of collagen lattices made with forskin fibroblasts in medium containing 1% fetal bovine serum was inhibited by intracelluar cyclic AMP raising drugs including cholera toxin (CT), forskolin, and dibutyryl-cAMP. The inhibition by CT was attenuated by insulin, acidic fibroblast growth factor (aFGF), and transforming growth factor-β (TGF-β). All three peptide factors have previously been reported to promote collagen lattice contraction by arterial smooth muscle cells and/or fibroblasts. Incubation of cells suspended in collagen gels with CT and forskolin resulted in a transient rise of the intracellular cyclic AMP levels, which peaked at 2 hr and 30 min, respectively, after drug exposure. Cholera toxin-induced intracellular cyclic AMP increase was attenuated by TGF-β, but not by aFGF and insulin, when added simultaneously. Thus, TGF-β may attenuate CT's inhibition on collagen lattice contraction by attenuating CTinduced intracellualr cyclic AMP increse, whereas the attenuation by insulin and aFGF on the inhibition of lattice contraction may be mediated by a cyclic AMPindependent mechannism. © 1993 Wiley-Liss, Inc.     

Comparative studies of glutathione reductase and lipoamide dehydrogenase

1. Glutathione reductase and lipoamide dehydrogenase are structurally and mechanistically related flavoenzymes catalyzing various one and two electron transfer reactions between NAD(P)H and substrates with different structures. 2. The two enzymes differ in their coenzyme and functional specificities. Lipoamide dehydrogenase shows higher coenzyme preference while glutathione reductase displays greater functional specificity. 3. Binding preference of the two flavoenzymes for nicotinamide coenzymes is demonstrated by 31P-NMR spectroscopy. 4. The presence of arginines in glutathione reductase which is inactivated by phenyl glyoxal, is likely to be responsible for the NADPH-activity of glutathione reductase. 5. The substrate binding sites of the two enzymes are similar, though their functional details differ. 6. The active-site histidine of glutathione reductase functions primarily as the proton donor during catalysis. While the active-site histidine of lipoamide dehydrogenase stabilizes the thiolate anion intermediate and relays a proton in the catalytic process.     

Heterogeneity and variation among Neisseria meningitidis lipopolysaccharides.

Eight immunotype lipopolysaccharides (LPSs) of Neisseria meningitidis were prepared by the phenol-water procedure and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and sugar analyses. By SDS-PAGE and a highly sensitive silver strain. N. meningitidis LPSs from cells grown in tryptic soy broth were shown to contain one or two predominant components and a few minor, somewhat higher-molecular-weight components. The molecular sizes of the two predominant components were approximately the same as those of two E. coli rough-type LPSs, one with a complete core and the other with an incomplete core. The molecular weight of the major LPS component varied somewhat among different immunotypes but was estimated to be in the range of 4,200 to 5,000. By sugar analyses, the eight immunotype LPSs were different in their monosaccharide compositions. All contained glucose, galactose, heptose, glucosamine, and 2-keto-3-deoxyoctonate, but in different molar ratios. The growth of N. meningitidis in tryptic soy broth under different levels of aeration resulted in a change in the two major LPS components seen on the SDS-PAGE gel. High aeration increased the amount of the smaller component, whereas low aeration increased the amount of the larger component. Sugar analyses of LPSs from high and low aeration indicated that the larger LPS component contained more galactose residues per molecule. Use of different media for cell growth may also result in small, but noticeable, variations in the LPS components and in the galactose content of the LPS. The observed heterogeneity of N. meningitidis LPS may explain why many strains of N. meningitidis appear to possess more than one immunotype.     

The behavior of human neutrophils during flow through capillary pores

The passage times of individual human neutrophils through single capillary-sized pores in polycarbonate membranes were measured with the resistive pulse technique, and results were compared to those obtained from the micropipette aspiration of entire cells. Pore transit measurement serves as a useful means to screen populations of cells, and allows for protocols that measure time dependent changes to the population. Neutrophils exhibited a highly linear pressure/flow rate relationship at aspiration pressures from 200 Pa to 1,500 Pa in both the pore and pipette systems. Cellular viscosity, as determined by the method of Hochmuth and Needham, was 89.0 Pa.s for the pore systems and 134.9 Pa.s for the pipette systems. These results are in general agreement with recent values of neutrophil viscosity published in the literature. Extrapolation of the observed linear flow response revealed an apparent minimum pressure for whole cell aspiration significantly above the threshold pressure predicted by Evans' liquid drop model. However, whole cell aspiration was achieved in both the pore and pipette systems at pressures below this extrapolated minimum, although the calculated cellular viscosity was greatly increased. The implications of these two regimes of cell deformation is unclear. This behavior could be explained by shear thinning of the material in the cell body. However the origin of this phenomenon may be in the cortical region of the cell, which exhibits an elastic tension that may be deformation rate dependent.     

miR-196a Ameliorates Phenotypes of Huntington Disease in Cell,Transgenic Mouse,and Induced Pluripotent Stem Cell Models

Huntington disease (HD) is a dominantly inherited neurodegenerative disorder characterized by dysregulation of various genes. Recently, microRNAs (miRNAs) have been reported to be involved in this dysregulation, suggesting that manipulation of appropriate miRNA regulation may have a therapeutic benefit. Here, we report the beneficial effects of miR-196a (miR196a) on HD in cell, transgenic mouse models, and human induced pluripotent stem cells derived from one individual with HD (HD-iPSCs). In the in vitro results, a reduction of mutant HTT and pathological aggregates, accompanying the overexpression of miR-196a, was observed in HD models of human embryonic kidney cells and mouse neuroblastoma cells. In the in vivo model, HD transgenic mice overexpressing miR-196a revealed the suppression of mutant HTT in the brain and also showed improvements in neuropathological progression, such as decreases of nuclear, intranuclear, and neuropil aggregates and late-stage behavioral phenotypes. Most importantly, miR-196a also decreased HTT expression and pathological aggregates when HD-iPSCs were differentiated into the neuronal stage. Mechanisms of miR-196a in HD might be through the alteration of ubiquitin-proteasome systems, gliosis, cAMP response element-binding protein pathway, and several neuronal regulatory pathways in vivo. Taken together, these results show that manipulating miR-196a provides beneficial effects in HD, suggesting the potential therapeutical role of miR-196a in HD.