Dithiothreitol induces the sacrificial antioxidant property of human serum albumin in a metal-catalyzed oxidation and gamma-irradiation system

The species *OH or H2O2 are produced by both metal-catalyzed oxidation (MCO) of reducing equivalents and gamma-irradiation. Intact or Cys-34-modified human serum albumin (HSA) was significantly degraded in the MCO system containing dithiothreitol (DTT) as electron donor, but as long as it lasted, HSA prohibited *OH or H2O2 from initiating molecular damage of DNA. However, in the GSH and ascorbate (nonthiol) MCO system, HSA was not sacrificially degraded, and indeed accelerated the formation of DNA strand breaks. In the y-irradiation system producing *OH from H2O, only DTT attenuated the generation of DNA strand breaks by HSA. It did not degrade more H2O2 in the presence of reduced GSH (thiol-linked peroxidase) than in its absence. Therefore it would seem that in an MCO system, the antioxidant activity of HSA depends on the effectiveness of reducing equivalents to induce exposure of a functional group scavenging the *OH or H2O2 species, by reduction of its disulfide-bonds. In the presence of DTT, disulfide bonds in HSA were quantitatively reduced to cysteinyl residues but not significantly reduced by ascorbate or GSH. In conclusion, the antioxidant activity of HSA in the D     

Rkp1/Cpc2, a fission yeast RACK1 homolog, is involved in actin cytoskeleton organization through protein kinase C, Pck2, signaling

The Rkp1/Cpc2, a fission yeast RACK1 homolog, interacted with Pck2, one of the known PKC homologs, in vivo and in vitro. The rkp1-deletion mutants (Deltarkp1) are elongated and the pck2-deletion mutant (Deltapck2) showed abnormal morphology. The double-deletion mutant (Deltarkp1Deltapck2) showed more aberrant cell shapes and was sensitive to high salt concentration. Both Deltarkp1 and Deltapck2 cells were sensitive to latrunculin B (Lat B) which inhibits actin polymerization. The cells expressing the human RACK1 homolog complemented the latrunculin B sensitivity of Deltarkp1 indicating that human RACK1 is a functional homolog of Rkp1/Cpc2. We propose that Rkp1/Cpc2 may function as a receptor for Pck2 in the regulation of actin cytoskeleton organization during cell wall synthesis and morphogenesis of Schizosaccharomyces pombe.     

The PUB domain: a putative protein-protein interaction domain implicated in the ubiquitin-proteasome pathway

Cytoplasmic peptide:N-glycanase (PNGase) is a de-N-glycosylating enzyme which may be involved in the proteasome-dependent pathway for degradation of misfolded glycoproteins formed in the endoplasmic reticulum (ER) that are exported into the cytoplasm. A cytoplasmic PNGase found in Saccharomyces cerevisiae, Png1p, is widely distributed in higher eukaryotes as well as in yeast (Suzuki, T., et al. J. Cell Biol. 149, 1039-1051, 2000). The recently uncovered complete genome sequence of Arabidopsis thaliana prompted us to search for the protein homologue of Png1p in this organism. Interestingly, when the mouse Png1p homologue sequence was used as a query, not only a Png1p homologue containing a transglutaminase-like domain that is believed to contain a catalytic triad for PNGase activity, but also four proteins which had a domain of 46 amino acids in length that exhibited significant similarity to the N-terminus of mouse Png1p were identified. Moreover, three of these homologous proteins were also found to possess a UBA or UBX domain, which are found in various proteins involved in the ubiquitin-related pathway. We name this newly found homologous region the PUB (Peptide:N-glycanase/UBA or UBX-containing proteins) domain and propose that this domain may mediate protein-protein interactions.     

Gene profile of replicative senescence is different from progeria or elderly donor

In vitro cellular senescence of human diploid fibroblast has been a good model for aging research, which shows similar phenotypes to in vivo aging. Gene expression profiling would provide an insight to understand the mechanism of senescence. Using cDNA microarray containing 384 known genes, we compared the expression profiles of three different types of aging models: replicative senescence, fibroblasts from progeria or from elderly donor. Although all of them showed senescence phenotypes, distinct sets of genes were altered in each group. Pairwise plots or cluster analysis of activation fold of gene expression revealed closer relationships between fibroblasts from progeria or from old individual, but not between replicative senescence fibroblasts and either models. Differential expression pattern of several genes were confirmed by RT-PCR. We suggest that the replicative senescence model might behave differently to other types of aging models due to the distinct gene expression.     

Arsenic trioxide induces G2/M growth arrest and apoptosis after caspase-3 activation and bcl-2 phosphorylation in promonocytic U937 cells

Arsenic trioxide has recently been shown to inhibit growth and induce apoptosis in acute promyelocytic leukemia (APL), but little is known about the molecular mechanisms mediating these effects. Here we demonstrate that treatment of promonocytic U937 cells with arsenic trioxide leads to G2/M arrest which was associated with a dramatic increase in the levels of cyclin B and cyclin B-dependent kinase and apoptosis. We further show that apoptosis occurs after bcl-2 phosphorylation and caspase-3 activation followed by cleavage of PARP and PLC-gamma1 degradation and DNA fragmentation. The arsenic trioxide-induced apoptosis could be blocked by the protein synthesis inhibitor cycloheximide. In addition, pretreatment of U937 cells with the DNA polymerase inhibitor aphidicolin also blocked apoptosis, but did not cause the arrest of cells in the G2/M phase. The findings suggest that arsenic trioxide exerts its growth-inhibitory effects by modulating expression and/or activity of several key G2/M regulatory proteins. Furthermore, arsenic trioxide-mediated G2/M arrest correlates with the onset of apoptosis.     

Mechanistic studies on class I polyhydroxybutyrate (PHB) synthase from Ralstonia eutropha: class I and III synthases share a similar catalytic mechanism

The Class I and III polyhydroxybutyrate (PHB) synthases from Ralstonia eutropha and Chromatium vinosum, respectively, catalyze the polymerization of beta-hydroxybutyryl-coenzyme A (HBCoA) to generate PHB. These synthases have different molecular weights, subunit composition, and kinetic properties. Recent studies with the C. vinosum synthase suggested that it is structurally homologous to bacterial lipases and allowed identification of active site residues important for catalysis [Jia, Y., Kappock, T. J., Frick, T., Sinskey, A. J., and Stubbe, J. (2000) Biochemistry 39, 3927-3936]. Sequence alignments between the Class I and III synthases revealed similar residues in the R. eutropha synthase. Site-directed mutants of these residues were prepared and examined using HBCoA and a terminally saturated trimer of HBCoA (sT-CoA) as probes. These studies reveal that the R. eutropha synthase possesses an essential catalytic dyad (C319-H508) in which the C319 is involved in covalent catalysis. A conserved Asp, D480, was shown not to be required for acylation of C319 by sT-CoA and is proposed to function as a general base catalyst to activate the hydroxyl of HBCoA for ester formation. Studies of the [(3)H]sT-CoA with wild-type and mutant synthases reveal that 0.5 equiv of radiolabel is covalently bound per monomer of synthase, suggesting that a dimeric form of the enzyme is involved in elongation. These studies, in conjunction with search algorithms for secondary structure, suggest that the Class I and III synthases are mechanistically similar and structurally homologous, despite their physical and kinetic differences.     

Localization of phospholipase C-gamma1 signaling in caveolae: importance in EGF-induced phosphoinositide hydrolysis but not in tyrosine phosphorylation

Upon epidermal growth factor treatment, phospholipase C-gamma1 (PLC-gamma1) translocates from cytosol to membrane where it is phosphorylated at tyrosine residues. Caveolae are small plasma membrane invaginations whose structural protein is caveolin. In this study, we show that the translocation of PLC-gamma1 and its tyrosine phosphorylation are localized in caveolae by caveolin-enriched low-density membrane (CM) preparation and immunostaining of cells. Pretreatment of cells with methyl-beta-cyclodextrin (MbetaCD), a chemical disrupting caveolae structure, inhibits the translocation of PLC-gamma1 to CM as well as phosphatidylinositol (PtdIns) turnover. However, MbetaCD shows no effect on tyrosine phosphorylation level of PLC-gamma1. Our findings suggest that, for proper signaling, PLC-gamma1 phosphorylation has to occur at PtdInsP(2)-enriched sites.     

Inhibition of cytokine-induced vascular cell adhesion molecule-1 expression; possible mechanism for anti-atherogenic effect of Agastache rugosa

Adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1) play an important role during the early stages of atherogenesis. Agastache rugosa has an anti-atherogenic effect in low density lipoprotein receptor -/- mice. Moreover, A. rugosa reduced macrophage infiltration and VCAM-1 expression has been localized in aortic endothelium that overlies early foam cell lesions. This study ascertained that tilianin (100 microM), a major component of A. rugosa, inhibits the tumor necrotic factor-alpha (TNF-alpha)-induced expression of VCAM-1 by 74% in cultured human umbilical vein endothelial cells (HUVECs). Also, tilianin (100 microM) reduced TNF-alpha-induced activation of nuclear factor-kappaB in HUVECs.