Genome-wide assessment of oxidatively generated DNA damage

In the tide of science nouveau after the completion of genome projects of various species, there appeared a movement to understand an organism as a system rather than the sum of cells directed for certain functions. With the advent and spread of microarray techniques, systematic and comprehensive genome-wide approaches have become reasonably possible and more required on the investigation of DNA damage and the subsequent repair. The immunoprecipitation-based technique combined with high-density microarrays or next-generation sequencing is one of the promising methods to provide access to such novel research strategies. Oxygen is necessary for most of the life on earth for electron transport. However, reactive oxygen species are inevitably generated, giving rise to steady-state levels of DNA damage in the genome, that may cause mutations leading to cancer, ageing and degenerative diseases. Previously, we showed that there are many factors involved in the genomic distribution of oxidatively generated DNA damage including chromosome territory, and proposed this sort of research area as oxygenomics. Recently, RNA is also recognized as a target of this kind of modification.     

The three genes lipB, lipC, and lipD involved in the extracellular secretion of the Serratia marcescens lipase which lacks an N-terminal signal peptide.

The extracellular lipase of Serratia marcescens Sr41, lacking a typical N-terminal signal sequence, is secreted via a signal peptide-independent pathway. The 20-kb SacI DNA fragment which allowed the extracellular lipase secretion was cloned from S. marcescens by selection of a phenotype conferring the extracellular lipase activity on the Escherichia coli cells. The subcloned 6.5-kb EcoRV fragment was revealed to contain three open reading frames which are composed of 588, 443, and 437 amino acid residues constituting an operon (lipBCD). Comparisons of the deduced amino acid sequences of the lipB, lipC, and lipD genes with those of the Erwinia chrysanthemi prtDEC, prtEEC, and prtFEC genes encoding the secretion apparatus of the E. chrysanthemi protease showed 55, 46, and 42% identity, respectively. The products of the lipB and lipC genes were 54 and 45% identical to the S. marcescens hasD and hasE gene products, respectively, which were secretory components for the S. marcescens heme-binding protein and metalloprotease. In the E. coli DH5 cells, all three lipBCD genes were essential for the extracellular secretion of both S. marcescens lipase and metalloprotease proteins, both of which lack an N-terminal signal sequence and are secreted via a signal-independent pathway. Although the function of the lipD gene seemed to be analogous to those of the prtFEC and tolC genes encoding third secretory components of ABC transporters, the E. coli TolC protein, which was functional for the S. marcescens Has system, could not replace LipD in the LipB-LipC-LipD transporter reconstituted in E. coli. These results indicated that these three proteins are components of the device which allows extracellular secretion of the extracellular proteins of S. marcescens and that their style is similar to that of the PrtDEF(EC) system.     

The ABC-exporter genes involved in the lipase secretion are clustered with the genes for lipase, alkaline protease, and serine protease homologues in Pseudomonas fluorescens no. 33

In Pseudomonas fluorescens no. 33, the lipase gene is clustered with the genes for alkaline protease, AprDEF exporter, and two homologue proteins of Serratia serine proteases (pspA and pspB). Secretion of the lipase and alkaline protease through AprDEF was shown in the Escherichia coli cells. Interestingly, the E. coli cells carrying the pspA gene secreted PspA to the media AprDEF-independently.     

The Human Cytomegalovirus UL76 Gene Regulates the Level of Expression of the UL77 Gene

Background

Human cytomegalovirus (HCMV) can be reactivated under immunosuppressive conditions causing several fatal pneumonitis, hepatitis, retinitis, and gastrointestinal diseases. HCMV also causes deafness and mental retardation in neonates when primary infection has occurred during pregnancy. In the genome of HCMV at least 194 known open reading frames (ORFs) have been predicted, and approximately one-quarter, or 41 ORFs, are required for viral replication in cell culture. In contrast, the majority of the predicted ORFs are nonessential for viral replication in cell culture. However, it is also possible that these ORFs are required for the efficient viral replication in the host. The UL77 gene of HCMV is essential for viral replication and has a role in viral DNA packaging. The function of the upstream UL76 gene in the HCMV-infected cells is not understood. UL76 and UL77 are cistons on the same viral mRNA and a conventional 5′ mRNA for UL77 has not been detected. The vast majority of eukaryotic mRNAs are monocistronic, i.e., they encode only a single protein.

Methodology/Principal Findings

To determine whether the UL76 ORF affects UL77 gene expression, we mutated UL76 by ORF frame-shifts, stop codons or deletion of the viral gene. The effect on UL77 protein expression was determined by either transfection of expression plasmids or infection with recombinant viruses. Mutation of UL76 ORF significantly increased the level of UL77 protein expression. However, deletion of UL76 upstream of the UL77 ORF had only marginal effects on viral growth.

Conclusions/Significance

While UL76 is not essential for viral replication, the UL76 ORF is involved in regulation of the level of UL77 protein expression in a manner dependent on the translation re-initiation. UL76 may fine-tune the UL77 expression for the efficient viral replication in the HCMV- infected cells.     

Phosphorylation of rat liver glycogen synthase bound to the glycogen particle

Rat liver glycogen synthase bound to the glycogen particle was partially purified by repeated high-speed centrifugation. This synthase preparation was labeled with ³²P by incubations with cAMP-dependent protein kinase and cAMP-independent synthase (casein) kinase-1 in the presence of [γ-³²P]ATP. The phosphorylated synthase was separated from other proteins in the glycogen pellet by immunoprecipitation with rabbit anti-rat liver glycogen synthase serum. Analysis of the immunoprecipitates by sodium dodecyl sulfate-gel electrophoresis showed that synthase subunits of M_r 85,000 and 80,000 were present in varying proportions. The ³²P-labeled synthase in the immunoprecipitate was digested with trypsin, and the resulting peptides were analyzed by isoelectric focusing. Synthase bound to the glycogen particle was phosphorylated by cAMP-dependent protein kinase at more sites and by cAMP-independent synthase (casein) kinase-1 at less sites than when the homogeneous synthase was incubated with these kinases. Phosphorylation of synthase in the glycogen pellet by either cAMP-dependent protein kinase or cAMP-independent synthase (casein) kinase-1 did not cause a significant inactivation as has been observed when the homogeneous synthase was incubated with these kinases. Inactivation of synthase in the glycogen pellet, however, can be achieved by the combination of both kinases. This inactivation appears to result from the phosphorylation of a new site by cAMP-independent synthase (casein) kinase-1 neighboring a site previously phosphorylated by cAMP-dependent protein kinase.     

Enteroviral aseptic meningitis in Japan, 1981-1991. A report of the National Epidemiological Surveillance of Infectious Agents in Japan.

In Japan, aseptic meningitis cases due to enterovirus infections increase every summer in various degrees with an incidence peak usually in July. During the past 11 years from 1981 through 1991, a total of 8,595 enterovirus isolations from aseptic meningitis cases were reported from 54 participating laboratories. Eight enterovirus types caused large epidemics; more than 100 isolations of each type from aseptic meningitis cases were reported for every epidemic year of the respective type. They were coxsackievirus (C) types B3 and B5, echovirus (E) types 4, 6, 7, 9, 18 and 30. Among these, the highest meningitis-associating frequency was reported for E30, representing 82.6% of the total isolations reported for the type during this period, followed by E4, 71.1%. The frequencies of E9, E7, E6 and CB5 were in a range from 54.5% to 44.4%, while that of E18 was 37.7% and that of CB3 21.0%. During the epidemics, enterovirus-associated meningitis was most frequently reported among children of 4-7 years of age. High frequencies were also shown in infants less than 1-year of age in some types. A total of 4,240 enteroviruses were isolated from cerebrospinal fluid of aseptic meningitis cases, representing 49.3% of the cases with enterovirus isolation.     

Synovial Mononuclear Cells Consist with T Cells Which Produce High Levels of Tumor Necrosis Factor α

To determine whether synovial mononuclear cells include a population of tumor necrosis factor α-produeing T cells, we measured tumor necrosis α levels in culture supernatants of synovial mononuclear cells by ELISA and analyzed tumor necrosis α mRNA-positive cell frequencies. There were no significant differences in the spontaneous levels of TNF α between synovial mononuclear cells and peripheral mononuclear cells. The frequency of tumor necrosis factor α mRNA-positive cells in synovial mononuclear cells was higher than that of peripheral mononuclear cells. When stimulated with a superantigen, mononuclear cells from the synovial fluid of rheumatoid arthritis patients showed higher levels of tumor necrosis factor α production (1,035 ± 817 pg/ml) than did mononuclear cells from their peripheral blood (236 ± 180 pg/ml). In addition, we observed that a few T cell clones were resistant to superantigenic restimulation in vitro. We conclude that when these types of T cells persist in the synovium, they play a role in the development of rheumatoid arthritis via a mechanism involving tumor necrosis factor α production.     

Vascular smooth muscle relaxation by endothelium-dependent beta 1-adrenergic action

1. Norepinephrine (NE) (10(-5) M) in rabbit aorta relaxed ring segments with endothelium precontracted with 10(-6) M NE, but not segments without endothelium. 2. The relaxation was inhibited with metoprolol and methylene blue, but not inhibited with yohimbine and indomethacin. 3. NE (10(-5) M) significantly elevated tissue c-GMP levels in segments with endothelium. 4. These studies suggest that the vascular relaxation by high doses of NE is mediated by the release of endothelium-derived relaxing factor (EDRF) induced by the stimulation of beta 1-adrenoceptor.