Novel checkpoint response to genotoxic stress mediated by nucleolin-replication protein a complex formation

Human replication protein A (RPA), the primary single-stranded DNA-binding protein, was previously found to be inhibited after heat shock by complex formation with nucleolin. Here we show that nucleolin-RPA complex formation is stimulated after genotoxic stresses such as treatment with camptothecin or exposure to ionizing radiation. Complex formation in vitro and in vivo requires a 63-residue glycine-arginine-rich (GAR) domain located at the extreme C terminus of nucleolin, with this domain sufficient to inhibit DNA replication in vitro. Fluorescence resonance energy transfer studies demonstrate that the nucleolin-RPA interaction after stress occurs both in the nucleoplasm and in the nucleolus. Expression of the GAR domain or a nucleolin mutant (TM) with a constitutive interaction with RPA is sufficient to inhibit entry into S phase. Increasing cellular RPA levels by overexpression of the RPA2 subunit minimizes the inhibitory effects of nucleolin GAR or TM expression on chromosomal DNA replication. The arrest is independent of p53 activation by ATM or ATR and does not involve heightened expression of p21. Our data reveal a novel cellular mechanism that represses genomic replication in response to genotoxic stress by inhibition of an essential DNA replication factor.     

Role of ceramide in Ca2+-sensing receptor-induced apoptosis

Increased extracellular Ca(2+) ([Ca(2+)](o)) can damage tissues, but the molecular mechanisms by which this occurs are poorly defined. Using HEK 293 cell lines that stably overexpress the Ca(2+)-sensing receptor (CaR), a G protein-coupled receptor, we demonstrate that activation of the CaR leads to apoptosis, which was determined by nuclear condensation, DNA fragmentation, caspase-3 activation, and increased cytosolic cytochrome c. This CaR-induced apoptotic pathway is initiated by CaR-induced accumulation of ceramide which plays an important role in inducing cell death signals by distinct G protein-independent signaling pathways. Pretreatment of wild-type CaR-expressing cells with pertussis toxin inhibited CaR-induced [(3)H]ceramide formation, c-Jun phosphorylation, and caspase-3 activation. The ceramide accumulation, c-Jun phosphorylation, and caspase-3 activation by the CaR can be abolished by sphingomyelinase and ceramide synthase inhibitors in different time frames. Cells that express a nonfunctional mutant CaR that were exposed to the same levels of [Ca(2+)](o) showed no evidence of activation of the apoptotic pathway. In conclusion, we report the involvement of the CaR in stimulating programmed cell death via a pathway involving GTP binding protein alpha subunit (Galpha(i))-dependent ceramide accumulation, activation of stress-activated protein kinase/c-Jun N-terminal kinase, c-Jun phosphorylation, caspase-3 activation, and DNA cleavage.     

A replication competent adenovirus 5 host range mutant-simian immunodeficiency virus (SIV) recombinant priming/subunit protein boosting vaccine regimen induces broad,persistent SIV-specific cellular immunity to dominant and subdominant epitopes in Mamu-A*01 rhesus macaques

CTL are important in controlling HIV and SIV infection. To quantify cellular immune responses induced by immunization, CD8(+) T cells specific for the subdominant Env p15m and p54m epitopes and/or the dominant Gag p11C epitope were evaluated by tetramer staining in nine macaques immunized with an adenovirus (Ad) 5 host range mutant (Ad5hr)-SIVenv/rev recombinant and in four of nine which also received an Ad5hr-SIVgag recombinant. Two Ad5hr-SIV recombinant priming immunizations were followed by two boosts with gp120 protein or an envelope polypeptide representing the CD4 binding domain. Two mock-immunized macaques served as controls. IFN-gamma-secreting cells were also assessed by ELISPOT assay using p11C, p15m, and p54m peptide stimuli and overlapping pooled Gag and Env peptides. As shown by tetramer staining, Ad-recombinant priming elicited a high frequency of persistent CD8(+) T cells able to recognize p11C, p15m, and p54m epitopes. The presence of memory cells 38 wk postinitial immunization was confirmed by expansion of tetramer-positive CD8(+) T cells following in vitro stimulation. The SIV-specific CD8(+) T cells elicited were functional and secreted IFN-gamma in response to SIV peptide stimuli. Although the level and frequency of response of peripheral blood CD8(+) T cells to the subdominant Env epitopes were not as great as those to the dominant p11C epitope, elevated responses were observed when lymph node CD8(+) T cells were evaluated. Our data confirm the potency and persistence of functional cellular immune responses elicited by replication competent Ad-recombinant priming. The cellular immunity elicited is broad and extends to subdominant epitopes.     

Human tryptophanyl-tRNA synthetase can efficiently complement the Saccharoymces cerevisiae homologue,Wrs1P

A denitrifying bacterium, strain YG1, capable of degrading pyrrolidine under denitrifying conditions, was isolated. On the basis of phenotypic and phylogenetic characteristics, it was identified as a member of the genus Pseudomonas. During the anaerobic degradation of pyrrolidine, YG1 reduced a stoichiometric amount of nitrate to nitrogen gas, demonstrating that the degradation of pyrrolidine is coupled with respiratory nitrate reduction. YG1 also degraded pyrrolidine with a higher degradation rate under aerobic conditions than under denitrifying conditions.     

Frequent activation of AKT2 kinase in human pancreatic carcinomas

Activation of AKT/protein kinase B promotes a variety of biological activities important in tumorigenesis, such as cell survival and cell cycle progression. We previously demonstrated amplification and overexpression of the AKT2 gene in a subset of human pancreatic carcinomas. In this investigation, we assessed AKT2 catalytic activity in 50 frozen pancreatic tissues (37 carcinomas, four benign tumors and nine normal pancreata) by in vitro kinase assay. Twelve of 37 (32%) pancreatic carcinomas showed markedly elevated levels of AKT2 activity compared to normal pancreata and begin pancreatic tumors. To delineate mechanisms contributing to AKT2 activation in malignant pancreatic tumors, we examined the status of upstream components of the phosphatilydlinositol 3-kinase (PI3K)/AKT pathway. Western blot analysis revealed loss of PTEN protein expression in two of the 12 pancreatic carcinomas with activated AKT2. In vitro PI3K assays demonstrated high levels of PI3K activity in seven carcinoma specimens that showed AKT2 activation. Immunohistochemical staining confirmed high levels of phosphorylated (active) AKT in malignant pancreatic tumors compared to normal pancreata. Overall, these data suggest that upstream perturbations of the PI3K/AKT pathway contribute to frequent activation of AKT2 in pancreatic cancer, which may contribute to the pathogenesis of this highly aggressive form of human malignancy.     

Micronucleus induction in mice exposed to diazoaminobenzene or its metabolites,benzene and aniline: implications for diazoaminobenzene carcinogenicity

Diazoaminobenzene (DAAB), a manufacturing intermediate metabolized primarily to the known carcinogens benzene and aniline, has been identified as an impurity in a number of dyes and coloring agents that are components of cosmetics, food products, and pharmaceuticals. Several structural analogs of DAAB are carcinogenic as well. DAAB was selected for metabolism and toxicity studies by the National Toxicology Program (NTP) based on the potential for human exposure, positive Salmonella data, and lack of adequate toxicological data. In the toxicology studies in mice, DAAB exhibited properties similar to benzene and aniline. Because both these metabolites induce micronuclei (MN) in rodent bone marrow erythrocytes, DAAB was tested for induction of micronuclei in male B6C3F(1) mice. DAAB was administered twice by corn oil gavage at 24 h intervals, at doses of 25, 50, and 100 mg/kg per day. In addition, comparative micronucleus tests were conducted with benzene, aniline, and a mixture of benzene plus aniline; doses were based on the respective molar equivalents of each metabolite to DAAB. It was hypothesized that any observed increase in micronuclei seen in DAAB-treated mice would be due primarily to the effects of the benzene metabolite, as benzene is a more potent inducer of chromosomal damage than aniline. Results of this study showed that DAAB and benzene were effective inducers of micronuclei, with stronger responses noted for DAAB at higher doses. Positive results were also obtained with the mixture of benzene and aniline, although the magnitude of the response was lower than for DAAB. Aniline gave a weak positive response at doses exceeding its molar equivalent to 100 mg/kg DAAB. Overall, the data indicated that DAAB is a potent inducer of micronuclei in mice, and its activity appears to be closely related to the activity of benzene, one of its primary metabolites. The results are consistent with a prediction of carcinogenicity for DAAB.     

Mouse ribonuclease III. cDNA structure, expression analysis, and chromosomal location

Background  

Members of the ribonuclease III superfamily of double-stranded(ds)-RNA-specific endoribonucleases participate in diverse RNA maturation and decay pathways in eukaryotic and prokaryotic cells. A human RNase III orthologue has been implicated in ribosomal RNA maturation. To better understand the structure and mechanism of mammalian RNase III and its involvement in RNA metabolism we determined the cDNA structure, chromosomal location, and expression patterns of mouse RNase III.