Identification and characterization of HLA-B*5401-restricted HIV-1-Nef and Pol-specific CTL epitopes

The identification of HIV-1 cytotoxic T lymphocyte (CTL) epitopes presented by each HLA allele and the characterization of their CTL responses are important for the study of pathogenesis of AIDS and the development of a vaccine against it. In the present study, we focused on identification and characterization of HIV-1 epitopes presented by HLA-B*5401, which is frequently found in the Asian population, because these epitopes have not yet been reported. We identified these epitopes by using 17-mer overlapping peptides derived from HIV-1 Gag, Pol, and Nef. Seven of these 17-mer peptides induced HLA-B*5401-restricted CD8+ T cell responses. Only five HLA-B*5401-restricted Pol- or Nef-specific CD8+ T cell responses were detected in the analysis using 11-mer overlapping peptides. Three Pol and two Nef optimal peptides were identified by further analysis using truncated peptides. These epitope-specific CTLs effectively killed HLA-B*5401-expressing target cells infected with HIV-1 recombinant vaccinia virus, indicating that these peptides were naturally processed by HLA-B*5401 in HIV-1-infected cells. These epitope-specific CD8+ T cells were elicited in more than 25% of chronically HIV-1-infected individuals carrying HLA-B*5401. Therefore, these epitopes should prove useful for studying the pathogenesis of AIDS in Asia and developing a vaccine against HIV-1.     

The role of CD98 in astrocytic neoplasms

The high expression of CD98 was reported in some normal tissues, including blood brain barrier, activated lymphocytes, the basal layer of skin, proximal tubles of kidney, placenta, testis and a wide variety of tumors. The CD98 complex consists of an 80-85kD heavy chain (4F2hc/FRP-1) and a 40-45kD light chain. CD98hc, 4F2hc, and FRP-1 are the same glycosylated protein each other and define antigenicity of CD98. LAT1, the sodium-independent L-type amino acid transporter 1, has been identified as a light chain of the CD98 heterodimer from C6 glioma cells. LAT1 also corresponds to TA1, an oncofetal antigen that is expressed primarily in fetal tissues and cancer cells such as glioma cells. Increased LAT1 expression was found in various malignancies including human gliomas. Several studies implicated the important role of LAT1 and 4F2hc in malignant transformation and carcinogenesis. The LAT1-CD98 pathway may represent a unique therapeutic target for cancer intervention.     

A subpopulation of bone marrow cells depleted by a novel antibody, anti-Liv8, is useful for cell therapy to repair damaged liver

We previously reported a new in vivo model named as "GFP/CCl(4) model" for monitoring the transdifferentiation of green fluorescent protein (GFP) positive bone marrow cell (BMC) into albumin-positive hepatocyte under the specific "niche" made by CCl(4) induced persistent liver damage, but the subpopulation which BMCs transdifferentiate into hepatocytes remains unknown. Here we developed a new monoclonal antibody, anti-Liv8, using mouse E 11.5 fetal liver as an antigen. Anti-Liv8 recognized both hematopoietic progenitor cells in fetal liver at E 11.5 and CD45-positive hematopoietic cells in adult bone marrow. We separated Liv8-positive and Liv8-negative cells and then transplanted these cells into a continuous liver damaged model. At 4 weeks after BMC transplantation, more efficient repopulation and transdifferentiation of BMC into hepatocytes were seen with Liv8-negative cells. These findings suggest that the subpopulation of Liv8-negative cells includes useful cells to perform cell therapy on repair damaged liver.     

Okadaic Acid as a Probe to Analyse the Cell Cycle Progression in Plant Cells

Previously we have demonstrated the dynamic change of microtubules (MTs) during cell cycle progression using highly synchronized tobacco BY-2 cells and characterized the specific transition points of MT organization (Hasezawa and Nagata, 1991). In this study the effect of okadaic acid (OA), a specific inhibitor of protein phosphatase 1 and 2A, on such changes of MTs during cell cycle was examined. These experiments revealed that cell cycle was arrested before the formation of the preprophase band (PPB), at anaphase and at the border of M/G₁. Although the block at the anaphase seemed to be analogous to that observed in animal cells (Yamashita et al., 1990), the other two blocks were specific to plant cells. It is interesting that these two blocks coincided with the transition points of MT organization, as revealed in the previous study (Hasezawa and Nagata, 1991). Thus it is proposed that phosphorylation is involved in MT organization, although the effect of OA has been shown mainly to be the activation of cdc-2/histone H1 kinase in animal cells. Another inhibitor of protein phosphatase 1 and 2A, calyculin A (CLA), showed very similar effects on the cell cycle progression. The use of such inhibitors to dissect the cell cycle progression of plant cells is discussed.     

Specific enzyme complex of beta-1,4-galactosyltransferase-II and glucuronyltransferase-P facilitates biosynthesis of N-linked human natural killer-1 (HNK-1) carbohydrate

Human natural killer-1 (HNK-1) carbohydrate is highly expressed in the nervous system and is involved in synaptic plasticity and dendritic spine maturation. This unique carbohydrate, consisting of a sulfated trisaccharide (HSO(3)-3GlcAβ1-3Galβ1-4GlcNAc-), is biosynthesized by the successive actions of β-1,4-galactosyltransferase (β4GalT), glucuronyltransferase (GlcAT-P and GlcAT-S), and sulfotransferase (HNK-1ST). A previous study showed that mice lacking β4GalT-II, one of seven β4GalTs, exhibited a dramatic loss of HNK-1 expression in the brain, although β4GalT-I-deficient mice did not. Here, we investigated the underlying molecular mechanism of the regulation of HNK-1 expression. First, focusing on a major HNK-1 carrier, neural cell adhesion molecule, we found that reduced expression of an N-linked HNK-1 carbohydrate caused by a deficiency of β4GalT-II is not likely due to a general loss of the β1,4-galactose residue as an acceptor for GlcAT-P. Instead, we demonstrated by co-immunoprecipitation and endoplasmic reticulum-retention analyses using Neuro2a (N2a) cells that β4GalT-II physically and specifically associates with GlcAT-P. In addition, we revealed by pulldown assay that Golgi luminal domains of β4GalT-II and GlcAT-P are sufficient for the complex to form. With an in vitro assay system, we produced the evidence that the kinetic efficiency k(cat)/K(m) of GlcAT-P in the presence of β4GalT-II was increased about 2.5-fold compared with that in the absence of β4GalT-II. Finally, we showed that co-expression of β4GalT-II and GlcAT-P increased HNK-1 expression on various glycoproteins in N2a cells, including neural cell adhesion molecule. These results indicate that the specific enzyme complex of β4GalT-II with GlcAT-P plays an important role in the biosynthesis of HNK-1 carbohydrate.     

The mechanism of fibril formation of a non-inhibitory serpin ovalbumin revealed by the identification of amyloidogenic core regions

Ovalbumin (OVA), a non-inhibitory member of the serpin superfamily, forms fibrillar aggregates upon heat-induced denaturation. Recent studies suggested that OVA fibrils are generated by a mechanism similar to that of amyloid fibril formation, which is distinct from polymerization mechanisms proposed for other serpins. In this study, we provide new insights into the mechanism of OVA fibril formation through identification of amyloidogenic core regions using synthetic peptide fragments, site-directed mutagenesis, and limited proteolysis. OVA possesses a single disulfide bond between Cys(73) and Cys(120) in the N-terminal helical region of the protein. Heat treatment of disulfide-reduced OVA resulted in the formation of long straight fibrils that are distinct from the semiflexible fibrils formed from OVA with an intact disulfide. Computer predictions suggest that helix B (hB) of the N-terminal region, strand 3A, and strands 4-5B are highly β-aggregation-prone regions. These predictions were confirmed by the fact that synthetic peptides corresponding to these regions formed amyloid fibrils. Site-directed mutagenesis of OVA indicated that V41A substitution in hB interfered with the formation of fibrils. Co-incubation of a soluble peptide fragment of hB with the disulfide-intact full-length OVA consistently promoted formation of long straight fibrils. In addition, the N-terminal helical region of the heat-induced fibril of OVA was protected from limited proteolysis. These results indicate that the heat-induced fibril formation of OVA occurs by a mechanism involving transformation of the N-terminal helical region of the protein to β-strands, thereby forming sequential intermolecular linkages.     

Sensitive determination of aspirin and its metabolites in plasma by LC-UV using on-line solid-phase extraction with methylcellulose-immobilized anion-exchange restricted access media

We describe a sensitive determination of aspirin (ASA) and its three metabolites (salicylic acid [SA], 2,3-dihydroxybenzoic acid [2,3-DHBA], and 2,5-dihydroxybenzoic acid [gentisic acid (GA)]) in rat plasma. Analysis was carried out by on-line solid-phase extraction (SPE) using a methylcellulose-immobilized-strong anion-exchanger (MC-SAX), followed by liquid chromatography (LC) coupled with UV detection. The lower limits of quantitation for ASA and SA were 60 ng/mL in 100 microL of plasma, respectively. This method was validated with respect to intra- and inter-day precision, accuracy, and linearity up to concentrations of 20,000 ng/mL for ASA, SA, 2,3-DHBA and gentisic acid, respectively. The method was successfully applied to an analysis of the pharmacokinetics of ASA and SA in rats.     

Dietary adaptations of temperate primates: comparisons of Japanese and Barbary macaques

Habitat, diet and leaf chemistry are compared between Japanese and Barbary macaques to reveal the similarities and differences in dietary adaptations of temperate primates living at the eastern and western extremes of the genus Macaca. Tree species diversity and proportion of fleshy-fruited species are much higher in Japan than in North Africa. Both species spend considerable annual feeding time on leaves. Japanese macaques prefer fruits and seeds over leaves, and Barbary macaques prefer seeds. These characteristics are adaptive in temperate regions where fruit availability varies considerably with season, since animals can survive during the lean period by relying on leaf and other vegetative foods. The two species are different with respect to the higher consumption of herbs by Barbary macaques, and the leaves consumed contain high condensed and hydrolysable tannin for Barbary but not for Japanese macaques. Barbary macaques supplement less diverse tree foods with herbs. Because of the low species diversity and high tannin content of the dominant tree species, Barbary macaques may have developed the capacity to cope with tannin. This supports the idea that digestion of leaves is indispensable to survive in temperate regions where fruit and seed foods are not available for a prolonged period during each year.     

Probabilistic protein function prediction from heterogeneous genome-wide data

Dramatic improvements in high throughput sequencing technologies have led to a staggering growth in the number of predicted genes. However, a large fraction of these newly discovered genes do not have a functional assignment. Fortunately, a variety of novel high-throughput genome-wide functional screening technologies provide important clues that shed light on gene function. The integration of heterogeneous data to predict protein function has been shown to improve the accuracy of automated gene annotation systems. In this paper, we propose and evaluate a probabilistic approach for protein function prediction that integrates protein-protein interaction (PPI) data, gene expression data, protein motif information, mutant phenotype data, and protein localization data. First, functional linkage graphs are constructed from PPI data and gene expression data, in which an edge between nodes (proteins) represents evidence for functional similarity. The assumption here is that graph neighbors are more likely to share protein function, compared to proteins that are not neighbors. The functional linkage graph model is then used in concert with protein domain, mutant phenotype and protein localization data to produce a functional prediction. Our method is applied to the functional prediction of Saccharomyces cerevisiae genes, using Gene Ontology (GO) terms as the basis of our annotation. In a cross validation study we show that the integrated model increases recall by 18%, compared to using PPI data alone at the 50% precision. We also show that the integrated predictor is significantly better than each individual predictor. However, the observed improvement vs. PPI depends on both the new source of data and the functional category to be predicted. Surprisingly, in some contexts integration hurts overall prediction accuracy. Lastly, we provide a comprehensive assignment of putative GO terms to 463 proteins that currently have no assigned function.