Identification of the Site of Inhibition of Oncogenic ras–p21-Induced Signal Transduction by a Peptide from a ras Effector Domain

We have previously found that a peptide corresponding to residues 35–47 of the ras-p21 protein, from its switch 1 effector domain region, strongly inhibits oocyte maturation induced by oncogenic p21, but not by insulin-activated cellular wild-type p21. Another ras–p21 peptide corresponding to residues 96–110 that blocks ras–jun and jun kinase (JNK) interactions exhibits a similar pattern of inhibition. We have also found that c-raf strongly induces oocyte maturation and that dominant negative c-raf strongly blocks oncogenic p21-induced oocyte maturation. We now find that the p21 35–47, but not the 96–110, peptide completely blocks c-raf-induced maturation. This finding suggests that the 35–47 peptide blocks oncogenic ras at the level of raf; that activated normal and oncogenic ras–p21 have differing requirements for raf-dependent signaling; and that the two oncogenic-ras-selective inhibitory peptides, 35–47 and 96–110, act at two different critical downstream sites, the former at raf, the latter at JNK/jun, both of which are required for oncogenic ras-p21 signaling.     

A-protein from achromogenic atypical Aeromonas salmonicida: molecular cloning, expression, purification, and characterization.

Achromogenic atypical Aeromonas salmonicida is the causative agent of goldfish ulcer disease. Virulence of this bacterium is associated with the production of a paracrystalline outer membrane A-layer protein. The species-specific structural gene for the monomeric form of A-protein was cloned into a pET-3d plasmid in order to express and produce a recombinant form of the protein in Escherichia coli BL21(DE3). The induced protein was isolated from inclusion bodies by a simple solubilization-renaturation procedure and purified by ion exchange chromatography on Q-Sepharose to over 95% pure monomeric protein. Recombinant A-protein was compared by biochemical, immunological, and molecular methods with the A-protein isolated from atypical A. salmonicida bacterial cells by the glycine and the membrane extraction methods. The recombinant form was found to be undistinguishable from the wild type when examined by SDS-PAGE and gel filtration chromatography. The immunological similarity of the protein samples was demonstrated by employing polyclonal and monoclonal antibodies in ELISA and Western blot techniques. All forms of A-protein were found to activate the secretion of tumor necrosis factor alpha from murine macrophage. To date, this represents the first large-scale production of biologically active recombinant A-protein.     

ER-localized phosphatidylethanolamine synthase plays a conserved role in lipid droplet formation

The asymmetric distribution of phospholipids in membranes is a fundamental principle of cellular compartmentalization and organization. Phosphatidylethanolamine (PE), a nonbilayer phospholipid that contributes to organelle shape and function, is synthesized at several subcellular localizations via semiredundant pathways. Previously, we demonstrated in budding yeast that the PE synthase Psd1, which primarily operates on the mitochondrial inner membrane, is additionally targeted to the ER. While ER-localized Psd1 is required to support cellular growth in the absence of redundant pathways, its physiological function is unclear. We now demonstrate that ER-localized Psd1 sublocalizes on the ER to lipid droplet (LD) attachment sites and show it is specifically required for normal LD formation. We also find that the role of phosphatidylserine decarboxylase (PSD) enzymes in LD formation is conserved in other organisms. Thus we have identified PSD enzymes as novel regulators of LDs and demonstrate that both mitochondria and LDs in yeast are organized and shaped by the spatial positioning of a single PE synthesis enzyme.     

Topography, substratum and benthic macrofaunal relationships on a tropical mesophotic shelf margin, central Great Barrier Reef, Australia

Habitats and ecological communities occurring in the mesophotic region of the central Great Barrier Reef (GBR), Australia, were investigated using autonomous underwater vehicle (AUV) from 51 to 145 m. High-resolution multibeam bathymetry of the outer-shelf at Hydrographers Passage in the central GBR revealed submerged linear reefs with tops at 50, 55, 80, 90, 100 and 130 m separated by flat, sandy inter-reefal areas punctuated by limestone pinnacles. Cluster analysis of AUV images yielded five distinct site groups based on their benthic macrofauna, with rugosity and the presence of limestone reef identified as the most significant abiotic factors explaining the distribution of macrofaunal communities. Reef-associated macrofaunal communities occurred in three distinct depth zones: (1) a shallow (<60 m) community dominated by photosynthetic taxa, notably scleractinian corals, zooxanthellate octocorals and photosynthetic sponges; (2) a transitional community (60–75 m) comprising both zooxanthellate taxa and azooxanthellate taxa (notably gorgonians and antipatharians); and (3) an entirely azooxanthellate community (>75 m). The effects of depth and microhabitat topography on irradiance most likely play a critical role in controlling vertical zonation on reef substrates. The lower depth limits of zooxanthellate corals are significantly shallower than that observed in many other mesophotic coral ecosystems. This may be a result of resuspension of sediments from the sand sheets by strong currents and/or a consequence of cold water upwelling.     

Cell cycle control during the contact mediated growth inhibition of rat C6 glioma cells

Two separate control processes govern the cell cycle of rat C6 glioma cells. In subconfluent cultures growth inhibition is caused by cell contact interactions and the cell cycle is regulated primarily by changes in the duration of S phase. During advanced multilayering, medium depletion becomes the primary mechanism of growth inhibition and causes a pronounced G1 accumulation. Contact modulation acts by altering the velocity with which cells progress through the cell cycle, while depletion causes cycle arrest.     

The regulation of sterol metabolism by cell interactions

Total and free cholesterol levels in C6 glial cells are regulated by a cell interaction-dependent mechanism that operates independently of exogenous cholesterol and serum lipoproteins. This mechanism, which is activated by changes in culture density, coordinately regulates the activities of HMG-CoA reductase and acyl-CoA:cholesterol acyltransferase (ACAT). Both enzyme activities are low in sparse density cultures, rise as density increases from sparse to moderate, and decrease with further density increases. When culture density is abruptly elevated, both enzyme activities decay rapidly and with biphasic kinetics. Neither enzyme phosphorylation nor diffusible cytosolic factors appear to be directly involved in density suppression of HMG-CoA reductase. Studies with human fibroblasts that are defective in LDL receptor function demonstrate that density regulation does not require a functional LDL receptor. Extracellular matrix and soluble factors have also been ruled out as intercellular mediators. The specific growth rate of C6 cultures changes with density in the same manner as sterol metabolism. The possibility that growth and sterol metabolism are regulated by a common cell interaction-dependent mechanism is discussed.     

Effect of hypoxia on prostacyclin production in cultured pulmonary artery endothelium

Exposure of cultured bovine pulmonary artery endothelial cells to varying levels of hypoxia (10% or 0% O₂) for 4 hours resulted in a significant dose-dependent inhibition in endothelial prostacyclin synthesis (51% and 98%, at the 10% and 0% O₂ levels respectively, p <0.05, compared to 21% O₂ exposure values). Release of ³H-arachidonic acid from cellular pools was not altered by hypoxia. Some of the cells were incubated with arachidonic acid (20 μM for 5 min) or PGH₂ (4 μM for 2 min) immediately after exposure. Endothelium exposed to 0% O₂, but not to 10% O₂, produced significantly less prostacyclin after addition of either arachidonic acid (25 ± 5% of 21% O₂ exposure values, n=6, p <0.01) or PGH₂ (31 ± 3% of 21% O₂ exposure values, n=6, p <0.05). These results suggest that hypoxia inhibits cyclooxygenase at the 10% O₂ level and both cyclooxygenase and prostacyclin synthetase enzymes at the 0% O₂ exposure levels. Exposure of aortic endothelial cells resulted in a 44% inhibition of prostacyclin at the 0% exposure level. No significant alteration in prostacyclin production was found in pulmonary vascular smooth muscle cells exposed to hypoxia. These data suggest that the increased prostacyclin production reported in lungs exposed to hypoxia is not due to a direct effect of hypoxia on the main prostacyclin producing cells of the pulmonary circulation.