Subcellular localization of herpes simplex virus type 1 UL51 protein and role of palmitoylation in Golgi apparatus targeting

The herpes simplex virus type 1 (HSV-1) UL51 gene products are virion-associated phosphoproteins with apparent molecular masses of 27, 29, and 30 kDa in HSV-1-infected cells. In this study, we have investigated the intracellular localization and distribution of UL51 protein both in infected cells and in transfected cells expressing only UL51. We found that this protein colocalized closely with Golgi marker proteins such as the Golgi-58K protein and GM130 in transfected cells expressing only UL51. However, in infected cells, the UL51 protein localized to the juxtanuclear region but only partially colocalized with the Golgi maker proteins. Mutant protein analysis revealed that the N-terminal 15 amino acid residues of the UL51 protein sufficed for this Golgi localization property. The UL51 protein redistributed on addition of brefeldin A. This was prevented by pretreatment with 2-deoxyglucose and sodium azide, which results in ATP depletion, but not by pretreatment with NaF and AlCl(3), which activates heterotrimeric G proteins. Moreover, we found that palmitoylation of the UL51 protein through the N-terminal cysteine at position 9 was necessary for its Golgi localization. Protease digestion analysis suggested that the UL51 protein localized on the cytoplasmic face of the membrane in UL51-transfected cells, while in infected cells it localized mainly to the inside of cytoplasmic vesicles and/or the viral envelope. Transmission immunoelectron microscopy revealed an association of UL51 protein-specific labeling with cytoplasmic virions and also with some membranous structure. We infer from these observations that internalization of UL51 protein into the cytoplasmic vesicle and/or virion may occur in association with viral envelopment in HSV-infected cells.     

Role of immunoreactions and mast cells in pathogenesis of human endometriosis-morphologic study and gene expression analysis-

STUDY OBJECTIVES: To investigate the pathophysiology of human endometriosis, we examined by morphological and molecular biological methods. METHODS: Samples of ovarian endometriosis and normal ovarian tissues were obtained laparoscopically after informed consent. A morphological study by toluidine blue staining, immunohistochemistry of c-kit and electron microscopy demonstrated the localization of mast cells in the stromal lesions of endometriosis. Oligonucleotide microarrays were used for gene expression analysis. RESULTS: Infiltration of numerous mast cells and development of fibrosis was observed throughout the stromal lesions. Gene expression analysis by oligonucleotide microarrays indicated inflammatory immunoreactions in the lesions. Expressions of the FCER1G and PGDS, which are considered to be mast cell-specific genes, were upregulated in the ovarian endometriotic lesions as compared to the normal ovarian tissues. Furthermore, expressions of genes associated with immunological inflammation, such as IL-8, GRO1, GRO2, CXCR4, MCP1, and those related to tissue remodeling (MMP, COL4A2, and COL5A2) were also higher in endometriotic lesions than in the normal ovarian tissue. CONCLUSIONS: Thus it is likely that mast cells and their related inflammatory immunoreactions via chemokines play important roles in producing fibrosis and adhesions in endometriotic lesions.     

Effect of enzymatic modification of dietary wheat flour for reducing its allergenicity on oral sensitization to and intestinal absorption of ovalbumin

Increase in plasma immunoglobulin G specific to orally administered ovalbumin in Brown Norway rats was retarded by feeding enzyme-treated wheat flour when compared with untreated flour. Because plasma ovalbumin concentrations after feeding ovalbumin tended to be lower in mice fed enzyme-treated flour than in those fed untreated flour, suppression of ovalbumin absorption may be relevant to retarded sensitization observed in rats.     

Transcriptional pattern of a novel gene,expressed specifically after the point-of-no-return during sexualization,in planaria

We have investigated sexualization of asexual worms in the planarian Dugesia ryukyuensis. During sexualization there is a point from which an animal cannot return to the asexual state (point-of-no-return). To isolate the genes related to the point-of-no-return, we performed differential screening and isolated one novel gene that was expressed specifically in yolk glands of the worms after the point-of-no-return and named it Dryg. It encoded 655 amino acids with a predicted molecular mass of 79 kDa. We performed a series of experiments using Dryg as a molecular marker in the yolk gland. At first, we monitored how the yolk gland was formed during sexualization. The expression in sexualizing worms at stage 3 is limited to a single type of cell that has characteristics of neoblasts, the totipotent somatic cells; however, the expression is observed in the yolk gland in sexualized worms. Furthermore, we monitored yolk glands for expression during regeneration. The original yolk glands seem to disappear after ablation, then new yolk glands appeared along the ventral nerve cords. Because this expression pattern looks like that of sexualizing worms at stage 3, we speculate that yolk gland cells may differentiate from neoblasts during regeneration as observed during sexualization.     

Growth promotion of mycelia of the Matsutake mushroom Tricholoma matsutake by D-isoleucine

Mycelial growth of the Matsutake mushroom (Tricholama matsutake) was much slower than that of the other mushroom species. We found that the addition of D-isoleucine to the culture medium strikingly promoted mycelia growth. The other amino acids tested had no effect on this growth promotion.     

N-linked glycan structures of human lactoferrin produced by transgenic rice

Human lactoferrin was produced in genetically engineered rice. N-linked glycan structures of recombinant human lactoferrin were determined. The oligosaccharides liberated by hydrazinolysis were labeled with 2-aminopyridine (PA). The PA-labeled glycans were purified by reverse-phase and size-fractionation HPLCs. The structures of these glycans were identified by HPLC, exoglycosidase digestion, and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The glycan structures determined were ManFucXylGlcNAc(2) (3.4%), Man(2)FucGlcNAc(2) (2.1%), Man(3)FucGlcNAc(2) (2.5%), Man(3)FucXylGlcNAc(2) (42.5%), two isomers of Man(2)FucXylGlcNAc(2) (39.1%), Man(3)XylGlcNAc(2) (6.5%), and Man(2)XylGlcNAc(2) (3.9%).     

Distribution and phylogenetic analysis of family 19 chitinases in Actinobacteria

In organisms other than higher plants, family 19 chitinase was first discovered in Streptomyces griseus HUT6037, and later, the general occurrence of this enzyme in Streptomyces species was demonstrated. In the present study, the distribution of family 19 chitinases in the class Actinobacteria and the phylogenetic relationship of Actinobacteria family 19 chitinases with family 19 chitinases of other organisms were investigated. Forty-nine strains were chosen to cover almost all the suborders of the class Actinobacteria, and chitinase production was examined. Of the 49 strains, 22 formed cleared zones on agar plates containing colloidal chitin and thus appeared to produce chitinases. These 22 chitinase-positive strains were subjected to Southern hybridization analysis by using a labeled DNA fragment corresponding to the catalytic domain of ChiC, and the presence of genes similar to chiC of S. griseus HUT6037 in at least 13 strains was suggested by the results. PCR amplification and sequencing of the DNA fragments corresponding to the major part of the catalytic domains of the family 19 chitinase genes confirmed the presence of family 19 chitinase genes in these 13 strains. The strains possessing family 19 chitinase genes belong to 6 of the 10 suborders in the order Actinomycetales, which account for the greatest part of the Actinobacteria: Phylogenetic analysis suggested that there is a close evolutionary relationship between family 19 chitinases found in Actinobacteria and plant class IV chitinases. The general occurrence of family 19 chitinase genes in Streptomycineae and the high sequence similarity among the genes found in Actinobacteria suggest that the family 19 chitinase gene was first acquired by an ancestor of the Streptomycineae and spread among the Actinobacteria through horizontal gene transfer.