A nucleoside triphosphate-dependent deoxyribonuclease from Bacillus laterosporus. Purification and characterization of the enzyme.

A deoxyribonuclease, which requires nucleoside triphosphate for reaction, has been purified about 150-fold from extracts of Bacillus laterosporus. Potassium phosphate and ethylene glycol stabilize the purified enzyme. The enzyme degrades double-stranded DNA about 100 times faster than heat-denatured DNA in the presence of nucleoside triphosphate. Double-stranded DNA is not degraded to any measurable extent in the absence of ATP, but the enzyme exhibits activity toward denatured DNA in the absence of nucleoside triphosphate, and this activity seems to be an intrinsic property of this enzyme protein. The optimum pH is 8.5 and the maximum activity is obtained in the copresence of Mg2+ (8.0 X 10(-3)M) and Mn2+ (7.0 X 10(-5)M). ATP and dATP are most effective and nucleoside di- or monophosphates are ineffective. ATP is converted to ADP and inorganic phosphate during the reaction and the ratio of the amount of ATP cleaved to that of hydrolyzed phosphodiester bonds of DNA is about 3:1. An inhibitor of the enzyme was observed in bacterial extracts prepared by sonic disruption; the inhibitory substance is produced in the bacteria in the later stages of cell growth. Preliminary results show that the inhibitor emerged near the void volume of a Sephadex G-200 column, and was relatively heat-stable, RNase-resistant, and DNase-sensitive.     

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.     

Microbial Characterization of Microbial Ecosystems Associated to Evaporites Domes of Gypsum in Salar de Llamara in Atacama Desert

The Central Andes in northern Chile contains a large number of closed basins whose central depression is occupied by saline lakes and salt crusts (salars). One of these basins is Salar de Llamara (850 m a.s.l.), where large domed structures of seemingly evaporitic origin forming domes can be found. In this work, we performed a detailed microbial characterization of these domes. Mineralogical studies revealed gypsum (CaSO₄) as a major component. Microbial communities associated to these structures were analysed by 454 16S rDNA amplicon sequencing and compared between winter and summer seasons. Bacteroidetes Proteobacteria and Planctomycetes remained as the main phylogenetic groups, an increased diversity was found in winter. Comparison of the upper air-exposed part and the lower water-submerged part of the domes in both seasons showed little variation in the upper zone, showing a predominance of Chromatiales (Gammaproteobacteria), Rhodospirillales (Alphaproteobacteria), and Sphingobacteriales (Bacteroidetes). However, the submerged part showed marked differences between seasons, being dominated by Proteobacteria (Alpha and Gamma) and Verrucomicrobia in summer, but with more diverse phyla found in winter. Even though not abundant by sequence, Cyanobacteria were visually identified by scanning electron microscopy (SEM), which also revealed the presence of diatoms. Photosynthetic pigments were detected by high-performance liquid chromatography, being more diverse on the upper photosynthetic layer. Finally, the system was compared with other endoevaporite, mats microbialite and Stromatolites microbial ecosystems, showing higher similitude with evaporitic ecosystems from Atacama and Guerrero Negro. This environment is of special interest for extremophile studies because microbial life develops associated to minerals in the driest desert all over the world. Nevertheless, it is endangered by mining activity associated to copper and lithium extraction; thus, its environmental protection preservation is strongly encouraged.     

Feedback inhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2alpha

Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha) on serine 51 integrates general translation repression with activation of stress-inducible genes such as ATF4, CHOP, and BiP in the unfolded protein response. We sought to identify new genes active in this phospho-eIF2alpha-dependent signaling pathway by screening a library of recombinant retroviruses for clones that inhibit the expression of a CHOP::GFP reporter. A retrovirus encoding the COOH terminus of growth arrest and DNA damage gene (GADD)34, also known as MYD116 (Fornace, A.J., D.W. Neibert, M.C. Hollander, J.D. Luethy, M. Papathanasiou, J. Fragoli, and N.J. Holbrook. 1989. Mol. Cell. Biol. 9:4196-4203; Lord K.A., B. Hoffman-Lieberman, and D.A. Lieberman. 1990. Nucleic Acid Res. 18:2823), was isolated and found to attenuate CHOP (also known as GADD153) activation by both protein malfolding in the endoplasmic reticulum, and amino acid deprivation. Despite normal activity of the cognate stress-inducible eIF2alpha kinases PERK (also known as PEK) and GCN2, phospho-eIF2alpha levels were markedly diminished in GADD34-overexpressing cells. GADD34 formed a complex with the catalytic subunit of protein phosphatase 1 (PP1c) that specifically promoted the dephosphorylation of eIF2alpha in vitro. Mutations that interfered with the interaction with PP1c prevented the dephosphorylation of eIF2alpha and blocked attenuation of CHOP by GADD34. Expression of GADD34 is stress dependent, and was absent in PERK(-)/- and GCN2(-)/- cells. These findings implicate GADD34-mediated dephosphorylation of eIF2alpha in a negative feedback loop that inhibits stress-induced gene expression, and that might promote recovery from translational inhibition in the unfolded protein response.     

Key Golgi factors for structural and functional maturation of bunyamwera virus

Several complex enveloped viruses assemble in the membranes of the secretory pathway, such as the Golgi apparatus. Among them, bunyaviruses form immature viral particles that change their structure in a trans-Golgi-dependent manner. To identify key Golgi factors for viral structural maturation, we have purified and characterized the three viral forms assembled in infected cells, two intracellular intermediates and the extracellular mature virion. The first viral form is a pleomorphic structure with fully endo-beta-N-acetylglucosaminidase H (Endo-H)-sensitive, nonsialylated glycoproteins. The second viral intermediate is a structure with hexagonal and pentagonal contours and partially Endo-H-resistant glycoproteins. Sialic acid is incorporated into the small glycoprotein of this second viral form. Growing the virus in glycosylation-deficient cells confirmed that acquisition of Endo-H resistance but not sialylation is critical for the trans-Golgi-dependent structural maturation and release of mature viruses. Conformational changes in viral glycoproteins triggered by changes in sugar composition would then induce the assembly of a compact viral particle of angular contours. These structures would be competent for the second maturation step, taking place during exit from cells, that originates fully infectious virions.     

ER stress-regulated translation increases tolerance to extreme hypoxia and promotes tumor growth

Tumor cell adaptation to hypoxic stress is an important determinant of malignant progression. While much emphasis has been placed on the role of HIF-1 in this context, the role of additional mechanisms has not been adequately explored. Here we demonstrate that cells cultured under hypoxic/anoxic conditions and transformed cells in hypoxic areas of tumors activate a translational control program known as the integrated stress response (ISR), which adapts cells to endoplasmic reticulum (ER) stress. Inactivation of ISR signaling by mutations in the ER kinase PERK and the translation initiation factor eIF2alpha or by a dominant-negative PERK impairs cell survival under extreme hypoxia. Tumors derived from these mutant cell lines are smaller and exhibit higher levels of apoptosis in hypoxic areas compared to tumors with an intact ISR. Moreover, expression of the ISR targets ATF4 and CHOP was noted in hypoxic areas of human tumor biopsy samples. Collectively, these findings demonstrate that activation of the ISR is required for tumor cell adaptation to hypoxia, and suggest that this pathway is an attractive target for antitumor modalities.     

Population genetic structure of rock ptarmigan in the 'sky islands' of French Pyrenees: implications for conservation

Expected consequences of global warming include habitat reduction in many cool climate species. Rock ptarmigan is a Holarctic grouse that inhabits arctic and alpine tundra. In Europe, the Pyrenean ptarmigan inhabits the southern edge of the species' range and since the last glacial maximum its habitat has been severely fragmented and is restricted to high-alpine zones or 'sky islands'. A recent study of rock ptarmigan population genetic in Europe found that the Pyrenean ptarmigan had very low genetic diversity compared with that found in the Alps and Scandinavia. Habitat fragmentation and reduced genetic diversity raises concerns about the viability of ptarmigan populations in the Pyrenees. However, information on population structuring and gene flow across the Pyrenees, which is essential for designing a sound management plan, is absent. In this study, we use seven microsatellites and mitochondrial control region sequences to investigate genetic variation and differentiation among five localities across the Pyrenees. Our analyses reveal the presence of genetic differentiation among all five localities and a significant isolation-by-distance effect that is likely the result of short dispersal distances and high natal and breeding philopatry of Pyrenean ptarmigan coupled with severe habitat fragmentation. Furthermore, analysis of molecular variance, principal component analysis and Bayesian analysis of genetic structuring identified the greatest amount of differentiation between the eastern and main parts of the Pyrenean chain separated by the Sègre Valley. Our data also show that the Canigou massif may host an isolated population and requires special conservation attention. We propose a management plan which includes the translocation of birds. If a sky island structure affects genetic divergence in rock ptarmigan, it may also affect the genetic structure of other sky island species having low dispersal abilities.     

Isolation of Vibrio alginolyticus and Vibrio splendidus from Aquacultured Carpet Shell Clam (Ruditapes decussatus) Larvae Associated with Mass Mortalities

Two episodes of mortality of cultured carpet shell clams (Ruditapes decussatus) associated with bacterial infections were recorded during 2001 and 2002 in a commercial hatchery located in Spain. Vibrio alginolyticus was isolated as the primary organism from moribund clam larvae that were obtained during the two separate events. Vibrio splendidus biovar II, in addition to V. alginolyticus, was isolated as a result of a mixed Vibrio infection from moribund clam larvae obtained from the second mortality event. The larval mortality rates for these events were 62 and 73%, respectively. Mortality was also detected in spat. To our knowledge, this is the fist time that these bacterial species have been associated with larval and juvenile carpet shell clam mortality. The bacterial strains were identified by morphological and biochemical techniques and also by PCR and sequencing of a conserved region of the 16S rRNA gene. In both cases bacteria isolated in pure culture were inoculated into spat of carpet shell clams by intravalvar injection and by immersion. The mortality was attributed to the inoculated strains, since the bacteria were obtained in pure culture from the soft tissues of experimentally infected clams. V. alginolyticus TA15 and V. splendidus biovar II strain TA2 caused similar histological lesions that affected mainly the mantle, the velum, and the connective tissue of infected organisms. The general enzymatic activity of both live cells and extracellular products (ECPs), as evaluated by the API ZYM system, revealed that whole bacterial cells showed greater enzymatic activity than ECPs and that the activity of most enzymes ceased after heat treatment (100°C for 10 min). Both strain TA15 and strain TA2 produced hydroxamate siderophores, although the activity was greater in strain TA15. ECPs from both bacterial species at high concentrations, as well as viable bacteria, caused significant reductions in hemocyte survival after 4 h of incubation, whereas no significant differences in viability were observed during incubation with heat-killed bacteria.