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.     

Functional changes and motility characteristics of Japanese Black bull spermatozoa separated by Percoll

Spermatozoa from two Japanese Black bulls (Bull-ATF and Bull-KTG) were separated by centrifugation at 700 x g for 15min in modified TALP with or without 45-90% Percoll. Control washed spermatozoa and those collected from the bottom of 45 and 90% Percoll fractions were examined for viability and membrane integrity (using Hoechst bis-benzimide 33258 or propidium iodide and 6-carboxyfluorescein diacetate (PI-CFDA)), acrosomal status (using fluorescence isothiocyanate (FITC) conjugated Pisum Sativum agglutinin (PSA) and Peanut agglutinin (PNA), Naphthol Yellow S and Erythrosin B (NE) or triple staining (TS)), capacitation status (using chlortetracycline (CTC)), motility characteristics (using a computer-assisted sperm motion analysis system (CASA)) and for in vitro fertility. Percoll-separated spermatozoa showed greater viability and membrane integrity than controls, as determined by supravital staining. Differences were observed in the results regarding viability and acrosomal status of spermatozoa among sperm staining methods. Bull-ATF, which showed significantly greater in vitro fertility than Bull-KTG (P<0.05), showed a significantly higher rate of CTC-B-pattern (capacitated) spermatozoa (P<0.01) than Bull-KTG. The motility characteristics of control washed spermatozoa and those separated by 45-90% Percoll were analyzed by CASA. More motile and progressively motile spermatozoa were observed in the fraction at the bottom of the 90% Percoll solution than in the 45% Percoll fraction or in controls (P<0.01). Moreover, the spermatozoa of Bull-KTG, which showed lower in vitro fertility than Bull-ATF, did not show significant differences in motility from those of Bull-ATF. These results provided basic information about Japanese Black bull spermatozoa, and suggested that spermatozoa with greater motility and viability can be obtained by Percoll separation than without separation. However, Percoll separation did not enhance their in vitro fertility.     

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.     

Dietary fructooligosaccharides induce immunoregulation of intestinal IgA secretion by murine Peyer's patch cells

Probiotic supplements induce immunological responses in the host, and dietary fructooligosaccharides (FOS) stimulate the growth of selected intestinal microflora. In this study we investigated the immunological influences of orally administrated FOS. BALB/c mice were orally administered 0-7.5% FOS for 6 weeks, and the intestinal mucosal immune responses were measured. In the 2.5%-FOS group, fecal IgA was significantly increased. IgA secretion by Peyer's patch (PP) cells was upregulated in a dose-dependent way in response to FOS and CD4+ T cells from PP showed a dose-dependent increase in production of interferon-gamma and interleukin (IL) 10, and a high response in production of IL-5 and IL-6. In contrast, FOS suppressed serum IgG1. Our findings suggest that FOS supplementation changes the intestinal environment of microflora, and leads to upregulation of IgA secretion in CD4+ PP cells in intestinal mucosa, and to suppression of the systemic immune response to type 2 helper T (Th2) dominant.     

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.     

A novel ER-derived compartment,the ER body,selectively accumulates a beta-glucosidase with an ER-retention signal in Arabidopsis

The ER body is a novel compartment that is derived from endoplasmic reticulum (ER) in Arabidopsis. In contrast to whole seedlings which have a wide distribution of the ER bodies, rosette leaves have no ER bodies. Recently, we reported that wound stress induces the formation of many ER bodies in rosette leaves, suggesting that the ER body plays a role in the defense system of plants. ER bodies were visualized in transgenic plants (GFP-h) expressing green fluorescent protein (GFP) with an ER-retention signal, HDEL. These were concentrated in a 1000-g pellet (P1) of GFP-h plants. We isolated an Arabidopsis mutant, nai1, in which fluorescent ER bodies were hardly detected in whole plants. We found that a 65-kDa protein was specifically accumulated in the P1 fraction of GFP-h plants, but not in the P1 fraction of nai1 plants. N-terminal peptide sequencing revealed that the 65-kDa protein was a beta-glucosidase, PYK10, with an ER-retention signal, KDEL. Immunocytochemistry showed that PYK10 was localized in the ER bodies. Compared with the accumulation of GFP-HDEL, which was associated with both cisternal ER and ER bodies, the accumulation of PYK10 was much more specific to ER bodies. PYK10 was one of the major proteins in cotyledons, hypocotyls and roots of Arabidopsis seedlings, while PYK10 was not detected in rosette leaves that have no ER bodies. These findings indicated that PYK10 is the main component of ER bodies. It is possible that PYK10 produces defense compounds when plants are damaged by insects or wounding.     

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%).     

Total degradation of pentachloroethane by an engineered Alcaligenes strain expressing a modified camphor monooxygenase and a hybrid dioxygenase

We engineered biphenyl-degrading Alcaligenes sp. strain KF711 for total degradation of pentachloroethane (PCA), which expresses a modified camphor monooxygenase and a hybrid dioxygenase consisting of TodC1 (a large subunit of toluene dioxygenase of Pseudomonas putida F1) and BphA2-BphA3-pbhA4 (a small subunit, ferredoxin and ferredoxin reductase of biphenyl dioxygenase, respectively, in strain KF707). Modified camphor monooxygenase genes (camCAB) were supplied as a plasmid and the todC1 gene was integrated within the chromosomal bph gene cluster by a single crossover recombination. The resultant strain KF711S-3cam dechlorinated PCA to trichloroethene by the action of the modified camphor monooxygenase under anaerobic conditions. The same strain subsequently degraded trichloroethene formed oxidatively by the action of the Tol-Bph hybrid dioxygenase under aerobic conditions. Thus sequential anaerobic and aerobic treatments of the KF711S-3cam resting cells resulted in efficient and total degradation of PCA.