Phenotypic changes in CD8+ peripheral blood lymphocytes in cats infected with feline immunodeficiency virus

It is well documented that several cell surface molecules of T lymphocytes are altered by immune activation. We previously reported that feline immunodeficiency virus (FIV) infection induces a reduction in CD8beta chain expression of peripheral blood lymphocytes (PBLs) in cats. In this study, we performed three-color flow-cytometric analysis for activation-associated cell surface molecules (CD2, CD11a, CD45RA-like and major histocompatibility complex antigen class II (MHC II)) and light scatters (cellular size and complexity) to examine whether phenotypic changes also occurred in CD4(+) PBLs in addition to CD8(+) PBLs, of five FIV-infected cats and one uninfected cat. It was shown that (i) CD8alpha(+) PBLs, but not CD4(+) PBLs, had a distinct subpopulation with increased CD11a expression accompanying a reduced CD8beta chain and increased intracellular granules (ii) CD8alpha(+) PBLs, but not CD4(+) PBLs, expressed CD45RA-like antigen with diverse expression levels and (iii) MHC II expression was greater in CD8alpha(+) PBLs than CD4(+) PBLs, and the CD8beta chain reduction was correlated with the MHC II decrease within CD8alpha(+) PBLs. These results suggest that FIV infection induces phenotypically heterogeneous subpopulations in CD8(+) PBLs, including activated phenotypes, rather than in CD4(+) PBLs.     

A novel DNA polymerase homologous to Escherichia coli DNA polymerase I from a higher plant, rice (Oryza sativa L.)

A novel DNA polymerase, designated as OsPolI-like, has been identified from the higher plant, rice (Oryza sativa L. cv. Nipponbare). The OsPolI-like cDNA was 3765 bp in length, and the open reading frame encoded a predicted product of 977 amino acid residues with a molecular weight of 100 kDa. The OsPolI-like gene has been mapped to chromosome 8 and contains 12 exons and 11 introns. The encoded protein showed a high degree of sequence and structural homology to Escherichia coli pol I protein, but differed from DNA polymerase γ and θ. The DNA polymerase domain of OsPolI-like showed DNA polymerase activity. Subcellular fractionation analysis suggested that the protein is localized in the plastid. Northern and western blotting, and in situ hybridization analyses demonstrated preferential expression of OsPolI-like in meristematic tissues such as shoot apical meristem, root apical meristem, leaf primordia and the marginal meristem. Interestingly, no expression was detected in mature leaves, although they have a high chloroplast content. These properties indicated that OsPolI-like is a novel plant DNA polymerase. The function of OsPolI-like is discussed in relation to plastid maturation.     

Empirical evaluation of a dynamic experiment design method for prediction of MHC class I-binding peptides

The ability to predict MHC-binding peptides remains limited despite ever expanding demands for specific immunotherapy against cancers, infectious diseases, and autoimmune disorders. Previous analyses revealed position-specific preference of amino acids but failed to detect sequence patterns. Efforts to use computational analysis to identify sequence patterns have been hampered by the insufficiency of the number/quality of the peptide binding data. We propose here a dynamic experiment design to search for sequence patterns that are common to the MHC class I-binding peptides. The method is based on a committee-based framework of query learning using hidden Markov models as its component algorithm. It enables a comprehensive search of a large variety (20(9)) of peptides with a small number of experiments. The learning was conducted in seven rounds of feedback loops, in which our computational method was used to determine the next set of peptides to be analyzed based on the results of the earlier iterations. After these training cycles, the algorithm enabled a real number prediction of MHC binding peptides with an accuracy surpassing that of the hitherto best performing positional scanning method.     

The expression of the rice (Oryza sativa L.) homologue of Snm1 is induced by DNA damages

We isolated and characterized the rice homologue of the DNA repair gene Snm1 (OsSnm1). The length of the cDNA was 1862bp; the open reading frame encoded a predicted product of 485 amino acid residues with a molecular mass of 53.2kDa. The OsSnm1 protein contained the conserved beta-lactamase domain in its internal region. OsSnm1 was expressed in all rice organs. The expression was induced by MMS, H(2)O(2), and mitomycin C, but not by UV. Transient expression of an OsSnm1/GFP fusion protein in onion epidermal cells revealed the localization of OsSnm1 to the nucleus. These results suggest that OsSnm1 is involved not only in the repair of DNA interstrand crosslinks, but also in various other DNA repair pathways.     

Myeloid leukemia factor 1 regulates p53 by suppressing COP1 via COP9 signalosome subunit 3

Myeloid leukemia factor 1 (MLF1) was first identified as the leukemic fusion protein NPM-MLF1 generated by the t(3;5)(q25.1;q34) chromosomal translocation. Although MLF1 expresses normally in a variety of tissues including hematopoietic stem cells and the overexpression of MLF1 correlates with malignant transformation in human cancer, little is known about how MLF1 is involved in the regulation of cell growth. Here we show that MLF1 is a negative regulator of cell cycle progression functioning upstream of the tumor suppressor p53. MLF1 induces p53-dependent cell cycle arrest in murine embryonic fibroblasts. This action requires a novel binding partner, subunit 3 of the COP9 signalosome (CSN3). A reduction in the level of CSN3 protein with small interfering RNA abrogated MLF1-induced G1 arrest and impaired the activation of p53 by genotoxic stress. Furthermore, ectopic MLF1 expression and CSN3 knockdown inversely affect the endogenous level of COP1, a ubiquitin ligase for p53. Exogenous expression of COP1 overcomes MLF1-induced growth arrest. These results indicate that MLF1 is a critical regulator of p53 and suggest its involvement in leukemogenesis through a novel CSN3-COP1 pathway.     

Effect of Hypochlorite-Based Disinfectants on Inactivation of Murine Norovirus and Attempt to Eliminate or Prevent Infection in Mice by Additionto Drinking Water

We evaluated the in vitro efficacy of weak acid hypochlorous solution (WAHS) against murine norovirus (MNV) by plaque assay and compared the efficacy with diluted NaOCl (Purelox) and 70% ethanol. WAHS was as effective as 70% ethanol and diluted Purelox for 0.5-min reactions. For 0.5-min reactions in the presence of mouse feces emulsion, the efficacy of WHAS and 1:600 diluted Purelox was decreased, reducing the virus titers by 2.3 and 2.6 log₁₀, respectively, while 70% ethanol reduced the titer by more than 5 log₁₀. However, WAHS showed more than 5 log₁₀ reductions for the 5-min reaction even in the presence of feces emulsion. Since WAHS showed enough efficacy in inactivating MNV in vitro, we tried to eliminate MNV from MNV-infected mice by substituting WAHS for their drinking water. However, MNV was found to be positive in feces of mice drinking WAHS by an RT-nested PCR and plaque assay. To investigate whether hypochlorite-based disinfectants could prevent infection of a mouse with MNV, WAHS or 1:6,000 diluted Purelox was substituted for the drinking water of mice for 2 or 4 weeks, and then the mice were placed in a cage with an MNV-infected mouse. The supply of disinfectants was continued after cohabitation, but MNV was detected in the feces of all the mice at 1 week after cohabitation. In this study, we tried to eliminate and prevent MNV infection from mice by supplying hypochlorite-based disinfectants as an easy and low-cost method. Unfortunately, drinking disinfectants was ineffective, so it is important to keep the facility environment clean by use of effective disinfectants. Also, animals introduced into facilities should be tested as MNV free by quarantine and periodically confirmed as MNV free by microbiological monitoring.     

Characterization of marine X-family DNA polymerases and comparative analysis of base excision repair proteins

While mammalian DNA polymerase β (Pol β), which is a member of the Pol X family, play important roles in base excision repair (BER) that efficiently removes DNA base lesions arising from both endogenous and exogenous agents, this protein has been found only a subset of animals. To understand natural evolution of this enzyme, we isolated and characterized Pol β from jellyfish Aurelia sp.1. (AsPol β). Despite of phylogenetic distance and environmental differences between jellyfish and mammals, in vitro assays showed biochemical characteristics of AsPol β were very similar to those of a mammalian counterpart. We also searched two other homologs of mammalian genes that were involved in short patch (sp) BER in the nucleotide sequence database, and found that both of these homologs were encoded in the genomes of a lineage from Cnidarians through mammals and Arthropods. This study suggests that a DNA repair mechanism resembling mammalian sp-BER may be largely limited to a subset of animals. On the basis of our findings and previous reports, we discuss possible evolutional model of Pol β and the other members of the Pol X family.     

Highly conserved configuration of catalytic amino acid residues among calicivirus-encoded proteases

A common feature of caliciviruses is the proteolytic processing of the viral polyprotein catalyzed by the viral 3C-like protease encoded in open reading frame 1 (ORF1). Here we report the identification and structural characterization of the protease domains and amino acid residues in sapovirus (SaV) and feline calicivirus (FCV). The in vitro expression and processing of a panel of truncated ORF1 polyproteins and corresponding mutant forms showed that the functional protease domain is 146 amino acids (aa) in SaV and 154 aa in FCV. Site-directed mutagenesis of the protease domains identified four amino acid residues essential to protease activities: H(31), E(52), C(116), and H(131) in SaV and H(39), E(60), C(122), and H(137) in FCV. A computer-assisted structural analysis showed that despite high levels of diversity in the primary structures of the protease domains in the family Caliciviridae, the configurations of the H, E, C, and H residues are highly conserved, with these residues positioned closely along the inner surface of the potential binding cleft for the substrate. These results strongly suggest that the H, E, C, and H residues are involved in the formation of a conserved catalytic surface of the SaV and FCV 3C-like proteases.