Insulin action on activity and cell surface disposition of human HepG2 glucose transporters expressed in Chinese hamster ovary cells

Complementary DNA encoding a facilitative glucose transporter was isolated from a human hepatoma cell line (HepG2) cDNA library and subcloned into a metal-inducible mammalian expression vector, pLEN (California Biotechnology) containing human metallothionein gene II promoter sequences. Chinese hamster ovary (CHO) cells transfected with this transporter expression vector, pLENGT, exhibited a 2-17-fold increase in immunoreactive HepG2-type glucose transporter protein, as measured by protein immunoblotting with antipeptide antibodies directed against the HepG2-type glucose transporter C-terminal domain. Expression of the human glucose transporter was verified by protein immunoblotting with a mouse polyclonal antiserum that recognizes the human but not the rodent HepG2-type transporter. 2-Deoxy-D-glucose uptake was increased 2-7-fold in transfected cell lines. Polyclonal antisera directed against purified red blood cell glucose transporter were raised in several rabbits. Antiserum from one rabbit, delta, was found to bind to the surface of intact red cells but not to inside-out red cell ghosts. Using this delta-antiserum in intact cell-binding assays, 1.6-9-fold increases in cell surface expression of the human glucose transporter were measured in CHO-K1 cell lines transfected with the transporter expression vector. Measurements of total cellular glucose transporter immunoreactive protein using anti-HepG2 transporter C-terminal peptide serum, cell surface glucose transporter protein using delta-antiserum and 2-deoxyglucose uptake revealed proportional relationships among these parameters in transfected cell lines expressing different levels of transporter protein. Insulin increased 2-deoxyglucose uptake 40% in control CHO-K1 cells and in CHO-K1 cells expressing modest levels of the human glucose transporter protein. However, stimulation of sugar-uptake by insulin was only 10% in cells overexpressing human glucose transporter protein 9-fold, and no effect of insulin on sugar uptake was detected in several cell lines expressing very high levels (12-17-fold over controls) of human HepG2 glucose transporter protein. No insulin stimulation of anti-cell surface glucose transporter antibody binding was detected in any control or transfected CHO-K1 cell lines. These data indicate that a glucose transporter protein that is insensitive to insulin in HepG2 cells is regulated by insulin when expressed at low but not at high levels in insulin-response CHO-K1 cells. Additionally, the results suggest that insulin does not increase 2-deoxyglucose uptake by increasing the number of cell surface HepG2-type glucose transporters in CHO-K1 fibroblasts.     

Prophage mutation causing heat inducibility of defective Bacillus subtilis bacteriophage PBSX.

A mutant of Bacillus subtilis 168 has been isolated in which the defective phage PBSX was heat inducible, whereas another phage, phi105, was not so induced. A culture of the mutant grown at 30 degrees C, when shifted to 45 degrees C, began to lyse after 45 min; cell viability began to decrease after 10 min. Heat-induced lysis of the mutant was prevented by chloramphenicol. DNA, RNA, protein, and peptidoglycan synthesis were normal at the nonpermissive temperature up to the time of lysis. The site of xhi-1479 mutation causing this phenotype was linked (50%) in phage PBS1-mediated transduction to the host marker metC and to another PBSX marker xtl and was thus thought to map within the PBSX prophage. The order of markers was argC-thiB-metA-xhi-metC. The xhi mutation was thus distinct from another mutation, tsi-23, causing a similar heat inducibility of PBSX (Siegel and Marmur, 1969), which was unlinked to the metC marker. tsi-23 is therefore thought to be a host mutation, and the available evidence for a scattered phage genome being the cause of the defective nature of PBSX is thus less tenable. It was shown that the mutant, besides carrying the xhi mutation, also carried another closely linked mutation, xki-1479, which caused the PBSX produced to have no killing activity on the sensitive strain W23. The xki mutation was separated from xhi by recombination.     

Changes in invertebrate and macroalgal populations in Tasmanian marine reserves in the decade following protection

Densities of macrobenthic invertebrates and macro-algae in four Tasmanian ‘no-take’ marine protected areas (MPAs) were monitored annually for 10 years following MPA establishment, with changes compared to those at external (fished) reference locations. Fishing substantially influenced the population characteristics of many species, including altering the mean size and abundance of rock lobsters and the abundance of prey species such as urchins and abalone. Strong declines in abundances of purple urchins and abalone within the largest MPA at Maria Island indicate likely indirect effects related to protection of predators from fishing. The two smallest MPAs (ca. 1 km coastal span) generated few detectable changes. Our results affirm the importance of long-term monitoring and the value of MPAs, when sufficiently large, as reference areas for determining and understanding ecosystem effects of fishing in the absence of historical baseline data.     

Induction of nonmuscle myosin heavy chain II-C by butyrate in RAW 264.7 mouse macrophages

RAW 264.7 macrophages express nonmuscle myosin heavy chain II-A as the only significant nonmuscle myosin heavy chain isoform, with expression of nonmuscle myosin heavy chain II-B and II-C low or absent. Treatment of the cells with sodium butyrate, an inhibitor of histone deacetylase, led to the dose-dependent induction of nonmuscle myosin heavy chain II-C. Trichostatin A, another inhibitor of histone deacetylase, also induced nonmuscle myosin heavy chain II-C. Induction of nonmuscle myosin heavy chain II-C in response to these histone deacetylase inhibitors was attenuated by mithramycin, an inhibitor of Sp1 binding to GC-rich DNA sequences. Bacterial lipopolysaccharide alone had no effect on basal nonmuscle myosin heavy chain II-C expression, but attenuated butyrate-mediated induction of nonmuscle myosin heavy chain II-C. The effects of lipopolysaccharide were mimicked by the nitric oxide donors sodium nitroprusside and spermine NONOate, suggesting a role for nitric oxide in the lipopolysaccharide-mediated down-regulation of nonmuscle myosin heavy chain II-C induction. This was supported by experiments with the inducible nitric-oxide synthase inhibitor 1400W, which partially blocked the lipopolysaccharide-mediated attenuation of nonmuscle myosin heavy chain induction. 8-Bromo-cGMP had no effect on nonmuscle myosin heavy chain induction, consistent with a cGMP-independent mechanism for nitric oxide-mediated inhibition of nonmuscle myosin heavy chain II-C induction.     

Craniofacial development in the talpid3 chicken mutant

The talpid(3) chicken mutant has a pleiotropic phenotype including polydactyly and craniofacial abnormalities. Limb polydactyly in talpid(3) suggests a gain of Hedgehog (Hh) signaling, whereas, paradoxically, absence of midline facial structures suggests a loss of Hh function. Here we analyze the status of Shh signaling in the talpid(3) mutant head. We show that Shh expression domains are lost from the talpid(3) head--in hindbrain, midbrain, zona limitans intrathalamica, and stomodeal ectoderm--and that direct targets of Hedgehog signaling, Ptc1, Ptc2, and Gli1, are also absent even in areas associated with primary Shh expression. These data suggest that the talpid(3) mutation leads to defective activation of the Shh pathway and, furthermore, that tissue-to-tissue transduction of Shh expression in the developing head depends on Hh pathway activation. Failure to activate the Shh pathway can also explain absence of floor plate and Hnf-3beta and Netrin-1 expression in midbrain and hindbrain and absence of Fgf-8 expression in commissural plate. Other aspects of gene expression in the talpid(3) head, however, suggest misspecification, such as maintenance of floor plate-like gene expression in telencephalon. In branchial arches and lower jaw, where Shh is expressed, changes in expression of genes involved in patterning and mesodermal specification suggest both gain and loss of Hedgehog function. Thus, analysis of gene expression in talpid(3) head shows that, as in talpid(3) limb, expression of some genes is lost, while others are ectopically expressed. Unlike the limb, many head regions depend on Hh induction of a secondary domain of Shh expression, and failure of this induction in talpid(3), together with the inability to activate the Shh pathway, explain the loss-of-function head phenotype. This gene expression analysis in the talpid(3) head also confirms and extends knowledge of the importance of Shh signaling and the balance between activation and repression of Shh targets in many aspects of craniofacial morphogenesis.