p110beta is up-regulated during differentiation of 3T3-L1 cells and contributes to the highly insulin-responsive glucose transport activity

Activation of p85/p110 type phosphatidylinositol kinase is essential for aspects of insulin-induced glucose metabolism, including translocation of GLUT4 to the cell surface and glycogen synthesis. The enzyme exists as a heterodimer containing a regulatory subunit (e.g. p85alpha) and one of two widely distributed isoforms of the p110 catalytic subunit: p110alpha or p110beta. In the present study, we compared the two isoforms in the regulation of insulin action. During differentiation of 3T3-L1 cells into adipocytes, p110beta was up-regulated approximately 10-fold, whereas expression of p110alpha was unaltered. The effects of the increased p110 expression were further assessed by expressing epitope tagged p110beta and p110alpha in 3T3-L1 cells using adenovirus transduction systems, respectively. In vitro, the basal lipid kinase activity of p110beta was lower than that of p110alpha. When p110alpha and p110beta were overexpressed in 3T3-L1 adipocytes, exposing cells to insulin induced each of the subunits to form complexes with p85alpha and tyrosine-phosphorylated IRS-1 with similar efficiency. However, whereas the kinase activity of p110beta, either endogenous or exogeneous, was markedly enhanced by insulin stimulation, only very small increases of the activity of p110alpha were observed. Interestingly, overexpression of p110beta increased insulin-induced glucose uptake by 3T3-L1 cells without significantly affecting basal glucose transport, whereas overexpression of p110alpha increased both basal and insulin-stimulated glucose uptake. Finally, microinjection of anti-p110beta neutralizing antibody into 3T3-L1 adipocytes abolished insulin-induced translocation of GLUT4 to the cell surface almost completely, whereas anti-p110alpha neutralizing antibody did only slightly. Together, these findings suggest that p110beta plays a crucial role in cellular activities evoked acutely by insulin.     

Partial purification of 6-(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihydropterin triphosphate synthetase from chicken liver.

An enzyme that catalyzes the formation of 6-(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihydropterin triphosphate (D-erythrodihydroneopterin triphosphate) and formic acid from GTP has been purified about 3700-fold from homogenates of chicken liver. The molecular weight of the enzyme, D-erythrodihydroneopterin triphosphate synthetase (GTP cyclohydrolase), has been estimated to be 125,000 by gel filtration on Ultrogel AcA-34. The enzyme functions optimally between pH 8.0 and 9.2 and is considerably heat-stable. No cofactors or metal ions have been demonstrated to be required for activity; however, the reaction is strongly inhibited by Cu2+ and Hg2+. GTP is the most efficient substrate, with GDP being 1/17 as active and guanosine, GMP, and ATP being inactive. The Km for GTP has been found to be 14 micrometer. Although the overall reaction catalyzed by D-erythrodihydroneopterin triphosphate synthetase from chicken liver is identical with that from Escherichia coli GTP cyclohydrolase, immunological studies show no apparent homology between the two enzymes.     

Detection of anaerobic ammonium-oxidizing bacteria in Ago Bay sediments

We identified 16S rRNA gene sequences in sediment samples from Ago Bay in Japan, forming a new branch of the anammox group or closely related to anaerobic ammonium oxidizing (anammox) bacterial sequences. Anammox activity in the sediment samples was detected by (15)N tracer assays. These results, along with the results of fluorescence in situ hybridization (FISH) analysis, suggest the presence of anammox bacteria in the marine sediments.     

Interaction of surfactants with bilayer of negatively charged lipid: effect on gel-to-liquid-crystalline phase transition of dilauroylphosphatidic acid vesicle membrane

The interaction of surfactants with the vesicle membrane of the negatively charged lipid, dilauroylphosphatidic acid, was investigated through their effect on the gel-to-liquid-crystalline phase transition of the lipid bilayer. Three types of surfactants (anionic, cationic and non-ionic) with different hydrocarbon chain length were examined. (i) Anionic sodium alkylsulfates affected the phase transition temperature, Tm, only weakly. (ii) Non-ionic alkanoyl-N-methylglucamides decreased Tm monotonously with increasing concentration. The depression of Tm induced by these surfactants was analyzed by applying the van't Hoff model for the freezing-point depression, and the partition coefficients of the surfactants between bulk water and lipid membrane were estimated. (iii) Cationic alkyltrimethylammonium bromides affected Tm in a complex manner depending on the hydrocarbon chain length of the surfactants. Octyl-/tetradecyl-trimethylammonium bromide depressed/elevated Tm monotonously with increasing concentration, whereas the change in Tm induced by decyl- and dodecyltrimethylammonium bromides was not monotonous but biphasic. This complex behavior of the phase transition temperature was well explained, based on the statistical mechanical theory presented by Suezaki et al. (Biochim. Biophys. Acta, 818 (1985) 31-37), which takes into account the interaction between surfactant molecules incorporated in the lipid membrane.     

The GTPase effector domain sequence of the Dnm1p GTPase regulates self-assembly and controls a rate-limiting step in mitochondrial fission

Dnm1p belongs to a family of dynamin-related GTPases required to remodel different cellular membranes. In budding yeast, Dnm1p-containing complexes assemble on the cytoplasmic surface of the outer mitochondrial membrane at sites where mitochondrial tubules divide. Our previous genetic studies suggested that Dnm1p's GTPase activity was required for mitochondrial fission and that Dnm1p interacted with itself. In this study, we show that bacterially expressed Dnm1p can bind and hydrolyze GTP in vitro. Coimmunoprecipitation studies and yeast two-hybrid analysis suggest that Dnm1p oligomerizes in vivo. With the use of the yeast two-hybrid system, we show that this Dnm1p oligomerization is mediated, in part, by a C-terminal sequence related to the GTPase effector domain (GED) in dynamin. The Dnm1p interactions characterized here are similar to those reported for dynamin and dynamin-related proteins that form higher order structures in vivo, suggesting that Dnm1p assembles to form rings or collars that surround mitochondrial tubules. Based on previous findings, a K705A mutation in the Dnm1p GED is predicted to interfere with GTP hydrolysis, stabilize active Dnm1p-GTP, and stimulate a rate-limiting step in fission. Here we show that expression of the Dnm1 K705A protein in yeast enhances mitochondrial fission. Our results provide evidence that the GED region of a dynamin-related protein modulates a rate-limiting step in membrane fission.     

Structural basis for defects of Keap1 activity provoked by its point mutations in lung cancer

Nrf2 regulates the cellular oxidative stress response, whereas Keap1 represses Nrf2 through its molecular interaction. To elucidate the molecular mechanism of the Keap1 and Nrf2 interaction, we resolved the six-bladed beta propeller crystal structure of the Kelch/DGR and CTR domains of mouse Keap1 and revealed that extensive inter- and intrablade hydrogen bonds maintain the structural integrity and proper association of Keap1 with Nrf2. A peptide containing the ETGE motif of Nrf2 binds the beta propeller of Keap1 at the entrance of the central cavity on the bottom side via electrostatic interactions with conserved arginine residues. We found a somatic mutation and a gene variation in human lung cancer cells that change glycine to cysteine in the DGR domain, introducing local conformational changes that reduce Keap1's affinity for Nrf2. These results provide a structural basis for the loss of Keap1 function and gain of Nrf2 function.     

Preserved postischemic heart function in sucrose-fed type 2 diabetic OLETF rats

Cardiovascular disease is one of the most important causes of morbidity and mortality in diabetes mellitus, but there has been controversy over functional impairment of diabetic hearts and their tolerance to ischemia. We studied ischemic heart function in type 2 diabetic rats with different degrees of hyperglycemia and its relationship with cardiac norepinephrine release. Otsuka Long-Evans Tokushima Fatty rats (OLETF) and age-matched Long-Evans Tokushima Otsuka normal rats (LETO) were used. One group of OLETF rats was given 30% sucrose in drinking water (OLETF-S). Hearts were isolated and perfused in a working heart preparation and subjected to 30 min ischemia followed by 40 min reperfusion at age of 12 months. Hemodynamics and coronary norepinephrine overflow were examined. Fasting plasma glucose in OLETF increased markedly at 12 months and sucrose administration exacerbated hyperglycemia in diabetic rats (LETO 6.6 +/- 0.5, OLETF 8.3 +/- 0.7, OLETF-S 15.0 +/- 1.7 mmol/L, P < 0.01). Basic cardiac output in OLETF was decreased as compared with LETO and OLETF-S (LETO 29.4 +/- 2.5, OLETF 24.0 +/- 2.4, OLETF-S 27.0 +/- 0.9 ml/min/g, P < 0.05) and remained very low after ischemia, while in OLETF-S it was well preserved (OLETF 4.2 +/- 2.1, OLETF-S 13.7 +/- 2.6 ml/min/g, P < 0.01). Correspondently, cardiac norepinephrine released during ischemia and reperfusion was lower in OLETF-S (OLETF 2.3 +/- 1.0, OLETF-S 0.7 +/- 0.1 pmol/ml, P < 0.01). Thus, OLETF hearts were more vulnerable to ischemia but sucrose feeding rendered their hearts resistant to ischemia. Less norepinephrine release may play a role in preventing postischemic functional deterioration in sucrose-fed diabetic hearts.     

Replacement of a terminal cytochrome c oxidase by ubiquinol oxidase during the evolution of acetic acid bacteria

The bacterial aerobic respiratory chain has a terminal oxidase of the heme-copper oxidase superfamily, comprised of cytochrome c oxidase (COX) and ubiquinol oxidase (UOX); UOX evolved from COX. Acetobacter pasteurianus, an α-Proteobacterial acetic acid bacterium (AAB), produces UOX but not COX, although it has a partial COX gene cluster, ctaBD and ctaA, in addition to the UOX operon cyaBACD. We expressed ctaB and ctaA genes of A. pasteurianus in Escherichia coli and demonstrated their function as heme O and heme A synthases. We also found that the absence of ctaD function is likely due to accumulated mutations. These COX genes are closely related to other α-Proteobacterial COX proteins. However, the UOX operons of AAB are closely related to those of the β/γ-Proteobacteria (γ-type UOX), distinct from the α/β-Proteobacterial proteins (α-type UOX), but different from the other γ-type UOX proteins by the absence of the cyoE heme O synthase. Thus, we suggest that A. pasteurianus has a functional γ-type UOX but has lost the COX genes, with the exception of ctaB and ctaA, which supply the heme O and A moieties for UOX. Our results suggest that, in AAB, COX was replaced by β/γ-Proteobacterial UOX via horizontal gene transfer, while the COX genes, except for the heme O/A synthase genes, were lost.     

Forward mutation assay of V79 cells to 6-thioguanine resistance in a soft agar technique that eliminates effects of metabolic co-operation

A technique involving culture in soft agar was used for the assay of forward mutation of V79 cells to 6-thioguanine (6TG) resistance. The main reason for the use of soft agar was to prevent reduction in recovery of mutants depending on the cell density plated for mutation selection, which is the chief problem in the liquid method, and which results mainly from metabolic co-operation due to cell-to-cell contact.V79 cells grew well in fortified soft agar medium (DMEM + 20% FBS) showing cloning efficiencies (>80%) as high as in liquid culture. Therefore, V79/HGPRT mutagenesis could be assayed quantitatively in soft agar culture.The frequency of 6TG-resistant colonies in agar selective medium increased linearly with increase in concentration of EMS. Toxicity and mutagenic responses were greater in soft agar than in liquid culture.In cultures of untreated and EMS-treated cells, more than 95% of the 6TG-resistant colonies isolated were aminopterin-sensitive.Use of soft agar for selection prevented the reduction in the number of mutants with increase in the size of incula on plating up to 1?2 × 10⁶ cells per 9-cm dish: in liquid culture, even with a lower plating number (2 × 10⁵ cells per 9-cm dish), a notable reduction in numbers of mutants was observed. This character was re-examined in a reconstruction experiment. The results show that, when up to 2 × 10⁶ cells were plated per 9-cm dish, 6TG-resistant cells were almost completely recovered from the soft agar medium, whereas only 10% were recovered from liquid culture.