Cdc42 regulates arsenic-induced NADPH oxidase activation and cell migration through actin filament reorganization

Although arsenic is a human carcinogen, the molecular mechanisms of its action remain to be understood. The present study reports that exposure to arsenic induced actin filament reorganization, resulting in lamellipodia and filopodia structures through the activation of Cdc42 in SVEC4-10 endothelial cells. It was also found that arsenic induced the formation of the superoxide anion (O2*) in SVEC4-10 cells. Immunoprecipitation and Western blotting analysis demonstrated that arsenic stimulation induced serine phosphorylation of p47phox, a key component of NADPH oxidase, indicating that arsenic induces O2* formation through NADPH oxidase activation. Inhibition of arsenic-induced actin filament reorganization by either overexpression of a dominant negative Cdc42 or pretreatment of an actin filament stabilizing regent, jasplakinolide, abrogated arsenic-induced NADPH oxidase activation, showing that the activation of NADPH oxidase was regulated by Cdc42-mediated actin filament reorganization. This study also showed that overexpression of a dominant negative Rac1 was sufficient to abolish arsenic-induced O2*- production, implying that Rac1 activities are required for Cdc42-mediated NADPH oxidase activation in response to arsenic stimulation. Furthermore, arsenic stimulation induced cell migration, which can be inhibited by the inactivation of either Cdc42 or NADPH oxidase. Taken together, the results indicate that arsenic is able to activate NADPH oxidase through Cdc42-mediated actin filament reorganization, leading to the induction of an increase in cell migration in SVEC4-10 endothelial cells.     

Theoretical analysis of twist/bend ratio and mechanical moduli of bacterial flagellar hook and filament

Certain motile bacteria employ rotating flagella for propulsion. The relative flexibility of two key components of the flagellum, filament and hook, is partially responsible for the mechanistic workings of this motor. A new computational method, the quantized elastic deformational model, was employed in this article to calculate the dimensionless twist/bend ratio (EI/GJ) of the filament and hook, providing a quantitative means to compare their relative stiffness. Both ratios were much <1.0, an average of 0.0440 for the filament and 0.0512 for the hook, indicating that within each structure bending is favored over twisting. These two ratios, along with previous experimental measurements, allowed us to propose a theoretical Young's modulus (E) between 10(6) and 10(7) dyn/cm(2) for the hook. This value is orders of magnitude smaller than experimentally determined Young's moduli of the filament, hence in agreement with empirical evidence linking compliance in the flagellum mainly to the hook.     

Pathogenic human thyroglobulin peptides in HLA-DR3 transgenic mouse model of autoimmune thyroiditis

To identify pathogenic epitopes on human thyroglobulin (hTg), a homodimer of 660kDa, we have applied a computer-based algorithm to predict potential HLA-DR3-binding peptides and have tested them in DR3-transgenic mice. Of the 39 peptides selected, four stimulated a proliferative response from hTg-primed cells of DR3+ mice, but not DQ8+ mice. Of the four peptides, one, hTg2079, was consistently pathogenic. Thyroiditis was not only produced by adoptive transfer of hTg-primed, hTg2079-activated cells but also by direct immunization with the peptide. These results demonstrate the utility of using this computer-based algorithm with synthetic peptides to help identify pathogenic T cell epitopes on hTg.     

Versatile modes of peptide recognition by the AAA+ adaptor protein SspB

Energy-dependent proteases often rely on adaptor proteins to modulate substrate recognition. The SspB adaptor binds peptide sequences in the stress-response regulator RseA and in ssrA-tagged proteins and delivers these molecules to the AAA+ ClpXP protease for degradation. The structure of SspB bound to an ssrA peptide is known. Here, we report the crystal structure of a complex between SspB and its recognition peptide in RseA. Notably, the RseA sequence is positioned in the peptide-binding groove of SspB in a direction opposite to the ssrA peptide, the two peptides share only one common interaction with the adaptor, and the RseA interaction site is substantially larger than the overlapping ssrA site. This marked diversity in SspB recognition of different target proteins indicates that it is capable of highly flexible and dynamic substrate delivery.     

Nonrandom dimerization of murine leukemia virus genomic RNAs

Retroviral genomes consist of two unspliced RNAs linked noncovalently in a dimer. Although these two RNAs are generally identical, two different RNAs can be copackaged when virions are produced by coinfected cells. It has been assumed, but not tested, that copackaging results from random RNA associations in the cytoplasm to yield encapsidated RNA homodimers and heterodimers in Hardy-Weinberg proportions. Here, virion RNA homo- and heterodimerization were examined for Moloney murine leukemia virus (MLV) using nondenaturing Northern blotting and a novel RNA dimer capture assay. The results demonstrated that coexpressed MLV RNAs preferentially self-associated, even when RNAs were identical in known packaging and dimerization sequences or when they differed overall by less than 0.1%. In contrast, HIV-1 RNAs formed homo- and heterodimers in random proportions. We speculate that these species-specific differences in RNA dimer partner selection may at least partially explain the higher frequency of genetic recombination observed for human immunodeficiency virus type 1 than for MLV.     

Cr(VI) increases tyrosine phosphorylation through reactive oxygen species-mediated reactions

While Cr (VI)containing compounds are well established carcinogens, the mechanisms of their action remain to be investigated. In this study we show that Cr (VI) causes increased tyrosine phosphorylation in human lung epithelial A549 cells in a timedependent manner. Nacetylcysteine (NAC), a general antioxidant, inhibited Cr (VI)induced tyrosine phosphorylation. Catalase, a scavenger of H₂O₂, sodium formate and aspirin, scavengers of hydroxyl radical (OH), also inhibited the increased tyrosine phosphorylation induced by Cr (VI). SOD, an inhibitor of superoxide radical (O₂ ^–), caused less inhibition. ESR study shows that incubation of Cr (VI) with the A549 cells generates OH radical. The generation of radical was decreased by addition of catalase and sodium formate, while SOD did not have any inhibitory effect. Oxygen consumption measurements show that addition of f Cr (VI) to A549 cells resulted in enhanced molecular oxygen consumption. These results indicate that Cr (VI) can induce an increase in tyrosine phosphorylation. H₂O₂ and OH radicals generated during the process are responsible for the increased tyrosine phosphorylation induced by Cr (VI).     

Role of CD8(+) lymphocytes in control of simian immunodeficiency virus infection and resistance to rechallenge after transient early antiretroviral treatment

Transient antiretroviral treatment with tenofovir, (R)-9-(2-phosphonylmethoxypropyl)adenine, begun shortly after inoculation of rhesus macaques with the highly pathogenic simian immunodeficiency virus (SIV) isolate SIVsmE660, facilitated the development of SIV-specific lymphoproliferative responses and sustained effective control of the infection following drug discontinuation. Animals that controlled plasma viremia following transient postinoculation treatment showed substantial resistance to subsequent intravenous rechallenge with homologous (SIVsmE660) and highly heterologous (SIVmac239) SIV isolates, up to more than 1 year later, despite the absence of measurable neutralizing antibody. In some instances, resistance to rechallenge was observed despite the absence of detectable SIV-specific binding antibody and in the face of SIV lymphoproliferative responses that were low or undetectable at the time of challenge. In vivo monoclonal antibody depletion experiments demonstrated a critical role for CD8(+) lymphocytes in the control of viral replication; plasma viremia rose by as much as five log units after depletion of CD8(+) cells and returned to predepletion levels (as low as <100 copy Eq/ml) as circulating CD8(+) cells were restored. The extent of host control of replication of highly pathogenic SIV strains and the level of resistance to heterologous rechallenge achieved following transient postinoculation treatment compared favorably to the results seen after SIVsmE660 and SIVmac239 challenge with many vaccine strategies. This impressive control of viral replication was observed despite comparatively modest measured immune responses, less than those often achieved with vaccination regimens. The results help establish the underlying feasibility of efforts to develop vaccines for the prevention of AIDS, although the exact nature of the protective host responses involved remains to be elucidated.