Characterization and expression analysis of three cold shock protein (CSP) genes under different stress conditions in the Antarctic bacterium Psychrobacter sp. G

Low temperature is one of the major environmental challenges that Antarctic bacteria must face. Detailed studies of cold shock responses of cold-adapted microorganisms are still insufficient. Here, we cloned three cold shock protein (CSP) genes (Csp1137, Csp2039, and Csp2531) in the Antarctic bacterium Psychrobacter sp. G and their regulatory sequences were identified. The three CSPs were highly conserved with other known CspAs. qRT-PCR was performed to evaluate their expression characteristics under stress conditions, and the potential influence of regulatory sequences also was analyzed. The expression of Csp1137 was enhanced both by low (0, 10?°C) and high temperature (30?°C). The expression of Csp2039 was enhanced by low temperature (0?°C), but was lower than that of Csp1137. This can be explained by the absence in Csp2039 of the AT-rich UP element. Different from Csp1137, the expression of Csp2531 was inhibited by low temperature (0?°C), even with the presence of AT-rich UP element, and it was not sensitive to high temperature (30?°C). The expression of Csp1137 was enhanced by high salinity (90, 120), whereas that of Csp2531was enhanced by low salinity (0, 15). At 0?°C and a salinity of 15, the expression of Csp1137 was repressed initially, but then it increased greatly during the next 10?h. The expressions of Csp2039 and Csp2531 were repressed significantly under four different combinations of stress conditions. Our results showed that the role of the upstream regulation sequences were much more complex than previously thought. Also, gene expressions were also affected by the environmental salinity. These are helpful in further clarification of the adaptation mechanism of Psychrobacter sp. G.     

Design of drug-resistant alleles of type-III phosphatidylinositol 4-kinases using mutagenesis and molecular modeling

Molecular modeling and site directed mutagenesis were used to analyze the structural features determining the unique inhibitor sensitivities of type-III phosphatidylinositol 4-kinase enzymes (PI4Ks). Mutation of a highly conserved Tyr residue that provides the bottom of the hydrophobic pocket for ATP yielded a PI4KIIIbeta enzyme that showed greatly reduced wortmannin sensitivity and was catalytically still active. Similar substitutions were not tolerated in the type-IIIalpha enzyme rendering it catalytically inactive. Two conserved Cys residues located in the active site of PI4KIIIalpha were found responsible for the high sensitivity of this enzyme to the oxidizing agent, phenylarsine oxide. Mutation of one of these Cys residues reduced the phenylarsine oxide sensitivity of the enzyme to the same level observed with the PI4KIIIbeta protein. In search of inhibitors that would discriminate between the closely related PI4KIIIalpha and -IIIbeta enzymes, the PI3Kgamma inhibitor, PIK93, was found to inhibit PI4KIIIbeta with significantly greater potency than PI4KIIIalpha. These studies should aid development of subtype-specific inhibitors of type-III PI4Ks and help to better understand the significance of localized PtdIns4P production by the various PI4Ks isoforms in specific cellular compartments.     

Regulation of insulin exocytosis by Munc13-1

The slower kinetics of insulin release from pancreatic islet beta cells, as compared with other regulated secretory processes such as chromaffin granule secretion, can in part be explained by the small number of the insulin granules that are docked to the plasma membrane and readily releasable. In type-2 diabetes, the kinetics of insulin secretion become grossly distorted, and, to therapeutically correct this, it is imperative to elucidate the mechanisms that regulate priming and secretion of insulin secretory granules. Munc13-1, a synaptic protein that regulates SNARE complex assembly, is the major protein determining the priming of synaptic vesicles. Here, we demonstrate the presence of Munc13-1 in human, rat, and mouse pancreatic islet beta cells. Expression of Munc13-1, along with its cognate partners, syntaxin 1a and Munc18a, is reduced in the pancreatic islets of type-2 diabetes non-obese Goto-Kakizaki and obese Zucker fa/fa rats. In insulinoma cells, overexpressed Munc13-1-enhanced green fluorescent protein is translocated to the plasma membrane in a temperature-dependent manner. This, in turn, greatly amplifies insulin exocytosis as determined by patch clamp capacitance measurements and radioimmunoassay of the insulin released. The potentiation of exocytosis by Munc13-1 is dependent on endogenously produced diacylglycerol acting on the overexpressed Munc13-1 because it is blocked by a phospholipase C inhibitor (U73122) and abrogated when the diacylglycerol binding-deficient Munc13-1H567K mutant is expressed instead of the wild type protein. Our data demonstrate that Munc13-mediated vesicle priming is not restricted to neurotransmitter release but is also functional in insulin secretion, where it is subject to regulation by the diacylglycerol second messenger pathway. In view of our findings, Munc13-1 is a potential drug target for therapeutic optimization of insulin secretion in diabetes.     

Inscuteable-independent apicobasally oriented asymmetric divisions in the Drosophila embryonic CNS

Inscuteable is the founding member of a protein complex localised to the apical cortex of Drosophila neural progenitors that controls their asymmetric division. Aspects of asymmetric divisions of all identified apicobasally oriented neural progenitors characterised to date, in both the central and peripheral nervous systems, require inscuteable. Here we examine the generality of this requirement. We show that many identified neuroblast lineages, in fact, do not require inscuteable for normal morphological development. To elucidate the requirements for apicobasal asymmetric divisions in a context where inscuteable is not essential, we focused on the MP2 > dMP2 + vMP2 division. We show that for MP2 divisions, asymmetric localisation and segregation of Numb and the specification of distinct dMP2 and vMP2 identities require bazooka but not inscuteable. We conclude that inscuteable is not required for all apicobasally oriented asymmetric divisions and that, in some cellular contexts, bazooka can mediate apicobasal asymmetric divisions without inscuteable.     

The 25-kDa synaptosome-associated protein (SNAP-25) binds and inhibits delayed rectifier potassium channels in secretory cells

Delayed-rectifier K(+) channels (K(DR)) are important regulators of membrane excitability in neurons and neuroendocrine cells. Opening of these voltage-dependent K(+) channels results in membrane repolarization, leading to the closure of the Ca(2+) channels and cessation of insulin secretion in neuroendocrine islet beta cells. Using patch clamp techniques, we have demonstrated that the activity of the K(DR) channel subtype, K(V)1.1, identified by its specific blocker dendrodotoxin-K, is inhibited by SNAP-25 in insulinoma HIT-T15 beta cells. A co-precipitation study of rat brain confirmed that SNAP-25 interacts with the K(V)1.1 protein. Cleavage of SNAP-25 by expression of botulinum neurotoxin A in HIT-T15 cells relieved this SNAP-25-mediated inhibition of K(DR). This inhibitory effect of SNAP-25 is mediated by the N terminus of K(V)1.1, likely by direct interactions with K(Valpha)1.1 and/or K(V)beta subunits, as revealed by co-immunoprecipitation performed in the Xenopus oocyte expression system and in vitro binding. Taken together we have concluded that SNAP-25 mediates secretion not only through its participation in the exocytotic SNARE complex but also by regulating membrane potential and calcium entry through its interaction with K(DR) channels.     

Isolation and characterization of a new bacterium capable of biotransforming cis -epoxysuccinic acid to D(-) -tartaric acid

A bacterial strain 1-3 capable of enantioselectively hydrolyzing cis-epoxysuccinic acid to D(-)-tartaric acid was isolated from soil. Phenotypic characteristics, Biolog GN test and phylogenetic analysis of strain 1-3 indicated that it belongs to the genus Bordetella. cis-Epoxysuccinic acid could be used as a carbon source and inducer for production of cis-epoxysuccinic acid hydrolase. After the purification of the enzymatic biotransformed product, a colorless and scentless crystal was obtained, which was confirmed to be the tartaric acid with laevorotatory optical rotation by the (1)H nuclear magnetic resonance spectrum and optical rotation analysis.     

RanGAP‐mediated nucleocytoplasmic transport of Prospero regulates neural stem cell lifespan in Drosophila larval central brain

By the end of neurogenesis in Drosophila pupal brain neuroblasts (NBs), nuclear Prospero (Pros) triggers cell cycle exit and terminates NB lifespan. Here, we reveal that in larval brain NBs, an intrinsic mechanism facilitates import and export of Pros across the nuclear envelope via a Ran‐mediated nucleocytoplasmic transport system. In rangap mutants, the export of Pros from the nucleus to cytoplasm is impaired and the nucleocytoplasmic transport of Pros becomes one‐way traffic, causing an early accumulation of Pros in the nuclei of the larval central brain NBs. This nuclear Pros retention initiates NB cell cycle exit and leads to a premature decrease of total NB numbers. Our data indicate that RanGAP plays a crucial role in this intrinsic mechanism that controls NB lifespan during neurogenesis. Our study may provide insights into understanding the lifespan of neural stem cells during neurogenesis in other organisms.     

Insights into the Role of the Unusual Disulfide Bond in Copper-Zinc Superoxide Dismutase

The functional and structural significance of the intrasubunit disulfide bond in copper-zinc superoxide dismutase (SOD1) was studied by characterizing mutant forms of human SOD1 (hSOD) and yeast SOD1 lacking the disulfide bond. We determined x-ray crystal structures of metal-bound and metal-deficient hC57S SOD1. C57S hSOD1 isolated from yeast contained four zinc ions per protein dimer and was structurally very similar to wild type. The addition of copper to this four-zinc protein gave properly reconstituted 2Cu,2Zn C57S hSOD, and its spectroscopic properties indicated that the coordination geometry of the copper was remarkably similar to that of holo wild type hSOD1. In contrast, the addition of copper and zinc ions to apo C57S human SOD1 failed to give proper reconstitution. Using pulse radiolysis, we determined SOD activities of yeast and human SOD1s lacking disulfide bonds and found that they were enzymatically active at ∼10% of the wild type rate. These results are contrary to earlier reports that the intrasubunit disulfide bonds in SOD1 are essential for SOD activity. Kinetic studies revealed further that the yeast mutant SOD1 had less ionic attraction for superoxide, possibly explaining the lower rates. Saccharomyces cerevisiae cells lacking the sod1 gene do not grow aerobically in the absence of lysine, but expression of C57S SOD1 increased growth to 30–50% of the growth of cells expressing wild type SOD1, supporting that C57S SOD1 retained a significant amount of activity.