Carbon monoxide protects against hyperoxia-induced endothelial cell apoptosis by inhibiting reactive oxygen species formation

Hyperoxia causes cell injury and death associated with reactive oxygen species formation and inflammatory responses. Recent studies show that hyperoxia-induced cell death involves apoptosis, necrosis, or mixed phenotypes depending on cell type, although the underlying mechanisms remain unclear. Using murine lung endothelial cells, we found that hyperoxia caused cell death by apoptosis involving both extrinsic (Fas-dependent) and intrinsic (mitochondria-dependent) pathways. Hyperoxia-dependent activation of the extrinsic apoptosis pathway and formation of the death-inducing signaling complex required NADPH oxidase-dependent reactive oxygen species production, because this process was attenuated by chemical inhibition, as well as by genetic deletion of the p47(phox) subunit, of the oxidase. Overexpression of heme oxygenase-1 prevented hyperoxia-induced cell death and cytochrome c release. Likewise, carbon monoxide, at low concentrations, markedly inhibited hyperoxia-induced endothelial cell death by inhibiting cytochrome c release and caspase-9/3 activation. Carbon monoxide, by attenuating hyperoxia-induced reactive oxygen species production, inhibited extrinsic apoptosis signaling initiated by death-inducing signal complex trafficking from the Golgi apparatus to the plasma membrane and downstream activation of caspase-8. We also found that carbon monoxide inhibited the hyperoxia-induced activation of Bcl-2-related proteins involved in both intrinsic and extrinsic apoptotic signaling. Carbon monoxide inhibited the activation of Bid and the expression and mitochondrial translocation of Bax, whereas promoted Bcl-X(L)/Bax interaction and increased Bad phosphorylation. We also show that carbon monoxide promoted an interaction of heme oxygenase-1 with Bax. These results define novel mechanisms underlying the antiapoptotic effects of carbon monoxide during hyperoxic stress.     

G1-G2 aggrecan product that can be generated by M-calpain on truncation at Ala709-Ala710 is present abundantly in human articular cartilage

To elucidate the specific function of m-calpain in the metabolism of aggrecan in human articular cartilage, the prevalence and localization of a large glycosaminoglycan-bearing aggrecan product generated by m-calpain in human osteoarthritis (OA) cartilage were investigated. Extracts of human OA articular cartilage were analysed by immunostaining using new polyclonal anti-VPGVA antiserum that detects the COOH terminal neoepitope IVTQVVPGVA(709) generated by m-calpain-related cleavage within the keratan sulphate rich region of human aggrecan. Immunoblotting analyses of aggrecan populations in guanidine hydrochloride-extracts showed that OA cartilages contained anti-VPGVA positive aggrecan products with the COOH terminal neoepitope ... VPGVA(709), resulting from truncation between the Ala(709)-Ala(710) m-calpain-related cleavage site. This aggrecan product consisted of two NH(2) terminal globular domain (G1 and G2) and KS side chains. Immunohistochemical staining showed that anti-VPGVA positive staining was localized within chondrocytes and spread to the surrounding interterritorial matrix. Confocal microscopic analysis showed subcellular colocalization of anti-VPGVA and anti m-calpain. These results indicate that the aggrecan product with the COOH terminal neoepitope VPGVA(709) is synthesized regularly by intracellular processing in chondrocytes, and is present abundantly as a limited form of aggrecan. M-calpain is the major candidate of the proteinase to generate this aggrecan product during the intracellular aggrecan processing.     

Platinum nanoparticle is a useful scavenger of superoxide anion and hydrogen peroxide

Bimetallic nanoparticles consisting of gold and platinum were prepared by a citrate reduction method and complementarily stabilized with pectin (CP-Au/Pt). The percent mole ratio of platinum was varied from 0 to 100%. The CP-Au/Pt were alloy-structured. They were well dispersed in water. The average diameter of platinum nanoparticles (CP-Pt) was 4.7 +/- 1.5 nm. Hydrogen peroxide (H(2)O(2)) was quenched by CP-Au/Pt consisting of more than 50% platinum whereas superoxide anion radical (O(2)(-)) was quenched by any CP-Au/Pt. The CP-Au/Pt quenched these two reactive oxygen species in dose-dependent manners. The CP-Pt is the strongest quencher. The CP-Pt decomposed H(2)O(2) and consequently generated O(2) like catalase. The CP-Pt actually quenched O(2)(-) which was verified by a superoxide dismutase (SOD) assay kit. This quenching activity against O(2)(-) persisted like SOD. Taken together, CP-Pt may be a SOD/catalase mimetic which is useful for medical treatment of oxidative stress diseases.     

A ROCK inhibitor permits survival of dissociated human embryonic stem cells

Poor survival of human embryonic stem (hES) cells after cell dissociation is an obstacle to research, hindering manipulations such as subcloning. Here we show that application of a selective Rho-associated kinase (ROCK) inhibitor, Y-27632, to hES cells markedly diminishes dissociation-induced apoptosis, increases cloning efficiency (from approximately 1% to approximately 27%) and facilitates subcloning after gene transfer. Furthermore, dissociated hES cells treated with Y-27632 are protected from apoptosis even in serum-free suspension (SFEB) culture and form floating aggregates. We demonstrate that the protective ability of Y-27632 enables SFEB-cultured hES cells to survive and differentiate into Bf1(+) cortical and basal telencephalic progenitors, as do SFEB-cultured mouse ES cells.     

The PDZ-LIM protein CLP36 is required for actin stress fiber formation and focal adhesion assembly in BeWo cells

CLP36 belongs to the ALP subfamily of PDZ-LIM proteins and has a PDZ domain at its N-terminal and a LIM domain at its C-terminal. It has been shown that CLP36 is localized to stress fibers through interaction with α-actinin, but its function is still unclear. To investigate the role of CLP36 in stress fibers, we suppressed CLP36 expression in BeWo cells by RNAi and examined the phenotypic changes. CLP36-knockdown resulted in cell spreading and the loss of stress fibers and focal adhesions. These changes were reversed by addition of exogenous CLP36, but not by addition of mutant forms of CLP36 that lacked the PDZ or LIM domain. These findings indicate that CLP36 plays a critical role in stress fiber formation and the assembly of focal adhesions in BeWo cells. In addition, the PDZ and LIM domains are both essential for CLP36 to function.     

Fibrillarin, a nucleolar protein, is required for normal nuclear morphology and cellular growth in HeLa cells

Fibrillarin is a key small nucleolar protein in eukaryotes, which has an important role in pre-rRNA processing during ribosomal biogenesis. Though several functions of fibrillarin are known, its function during the cell cycle is still unknown. In this study, we confirmed the dynamic localization of fibrillarin during the cell cycle of HeLa cells and also performed functional studies by using a combination of immunofluorescence microscopy and RNAi technique. We observed that depletion of fibrillarin has almost no effect on the nucleolar structure. However, fibrillarin-depleted cells showed abnormal nuclear morphology. Moreover, fibrillarin depletion resulted in the reduction of the cellular growth and modest accumulation of cells with 4n DNA content. Our data suggest that fibrillarin would play a critical role in the maintenance of nuclear shape and cellular growth.     

Purification and characterization of nitrate reductase from nodule cytosol of soybean plants

Nodulated soybean ( Glycine max [L.] Merr.) plants were grown in a nitrogen-free liquid culture medium prepared with distilled water. The cytosol fraction from root nodules showed a significant level of NADH-dependent nitrate reductase activity, even when the root did not show activity. This nitrate reductase was purified by column chromatography and native polyacrylamide gel electrophoresis (PAGE). The purified protein showed a main band at 100 kDa on sodium dodecyl sulfate (SDS)-PAGE. The K _m value for nitrate was 0.16 m M , and the highest activity was obtained at around pH 7.5. These characteristics are very similar to the inducible type of nitrate reductase, previously purified from soybean leaves. The developmental change in activity of this enzyme corresponded to that in nitrogenase activity.     

MKBP, a Novel Member of the Small Heat Shock Protein Family, Binds and Activates the Myotonic Dystrophy Protein Kinase

Muscle cells are frequently subjected to severe conditions caused by heat, oxidative, and mechanical stresses. The small heat shock proteins (sHSPs) such as αB-crystallin and HSP27, which are highly expressed in muscle cells, have been suggested to play roles in maintaining myofibrillar integrity against such stresses. Here, we identified a novel member of the sHSP family that associates specifically with myotonic dystrophy protein kinase (DMPK). This DMPK-binding protein, MKBP, shows a unique nature compared with other known sHSPs: (a) In muscle cytosol, MKBP exists as an oligomeric complex separate from the complex formed by αB-crystallin and HSP27. (b) The expression of MKBP is not induced by heat shock, although it shows the characteristic early response of redistribution to the insoluble fraction like other sHSPs. Immunohistochemical analysis of skeletal muscle cells shows that MKBP localizes to the cross sections of individual myofibrils at the Z-membrane as well as the neuromuscular junction, where DMPK has been suggested to be concentrated. In vitro, MKBP enhances the kinase activity of DMPK and protects it from heat-induced inactivation. These results suggest that MKBP constitutes a novel stress-responsive system independent of other known sHSPs in muscle cells and that DMPK may be involved in this system by being activated by MKBP. Importantly, since the amount of MKBP protein, but not that of other sHSP family member proteins, is selectively upregulated in skeletal muscle from DM patients, an interaction between DMPK and MKBP may be involved in the pathogenesis of DM.     

ANOMALOUS STIMULATION OF PROTEIN SYNTHESIS: EVIDENCE FOR TRANSLATIONAL CONTROL IN PRIMARY CELLS

Chick cells in primary culture synthesize protein at an accelerated rate following recovery from prolonged (2-12 hr) inhibition of protein synthesis. This 'overshoot' is followed by a return to the normal rate of synthesis.
Both actinomycin and fluorophenylalanine provided during the recovery period enhance the 'overshoot' and prevent the return to normal.
The results are consistent with the proposal that protein synthesis is restricted by an inhibitor or 'governor', itself a protein of relatively short half life.
Neither L nor HeLa cells demonstrate an 'overshoot' during the recovery phase.     

Coelastrella astaxanthina sp. nov. (Sphaeropleales,Chlorophyceae), a novel microalga isolated from an asphalt surface in midsummer in Japan

We taxonomically examined an algal strain (Ki‐4) isolated from a dry asphalt surface in mid‐summer of 2003. The vegetative cells were solitary, covered by a cell wall ornamented with meridional ribs, and contained hollow spherical chloroplasts and a pyrenoid. The cells exhibited a high tolerance to unfavorable photo‐oxidative stress conditions, and in culture developed a reddish coloration due to the accumulation of a water‐soluble astaxanthin‐binding protein, which was identified as a novel protein in organisms. 18S rDNA and ITS2 sequence analyses revealed that Ki‐4 belongs to Coelastrella sensu lato (Scenedesmaceae), but was taxonomically distinct from other members of the genus. On the basis of its phylogenetic position and morphological features, including the structures of cell wall ribs, we identify Ki‐4 as a new algal species in the genus Coelastrella, for which we propose the name Coelastrella astaxanthina sp. nov.