A novel seminal plasma glycoprotein of a teleost, the Nile tilapia (Oreochromis niloticus), contains a partial von Willebrand factor type D domain and a zona pellucida-like domain

Our previous study shows that seminal plasma of a teleost, the Nile tilapia, contains a glycoprotein Mr = 120,000 named as SPP (Seminal plasma glycoprotein)120 which forms a homopolymer that has sperm immobilizing activity. In order to elucidate the mechanisms of the formation of the homopolymer and the immobilization of sperm, molecular cloning of SPP120 was conducted. The cDNA for SPP120 contains a complete open reading frame encoding 797 amino acid residues with 14 potential N-glycosylation sites. The predicted amino acid sequence of SPP120 contains a partial von Willebrand factor type D domain and a zona pellucida domain, that are involved in protein-protein adhesion that form filamentous structures in various kinds of cells. This result suggests that SPP120 forms a homopolymer via these domains in seminal plasma and probably interacts with spermatozoa. Northern blotting reveals that the gene is also expressed in ovary, even in ovulated eggs. The results of in situ hybridization indicate that in testis the gene is expressed in Sertoli cells and epithelial cells of sperm ducts, and the localization corresponds to that of the protein analyzed by immunohistochemistry. In the ovary, the gene is expressed at the perinucleolus stage of oocytes; however, the protein is not detected in any cells other than oocytes.     

Effects of germinated barley foodstuff on microflora and short chain fatty acid production in dextran sulfate sodium-induced colitis in rats

Germinated barley foodstuff (GBF) administration has been previously reported to suppress dextran sulfate sodium (DSS)-induced experimental colitis. In this study, we investigated the roles of the intestinal microflora and short chain fatty acids (SCFAs) following administration of GBF in DSS-induced rat colitis. Sprague-Dawley rats were fed 3% (w/w of diet) DSS in GBF-diets for 5 days. The control rats were fed 3% DSS in cellulose-diets for 5 days. The administration of GBF effectively prevented bloody diarrhea and mucosal damage as compared to control rats. GBF significantly elevated fecal acetic acid and n-butyric acid levels. GBF tended to increase the number of eubacteria and that of bifidobacteria as compared to control rats. In addition, the number of enterobacteriaceae, the total number of aerobes and bacteroidaseae, were significantly lower in rats fed GBF than in the control group. It is suggested that the therapeutic effects of GBF for DSS-induced colitis depend mainly on increased SCFAs, which are accompanied by changes of composition of intestinal bacteria.     

Host-related variability in arbuscular mycorrhizal fungal structures in roots of <Emphasis Type="Italic">Hedera rhombea</Emphasis>, <Emphasis Type="Italic">Rubus parvifolius</Emphasis>, and <Emphasis Type="Italic">Rosa multiflora</Emphasis> under controlled conditions

The arbuscular mycorrhizal (AM) morphology of three host plant species inoculated with single and mixed fungal culture and the distribution of AM fungal species in roots of the hosts treated with a mixed culture of AM fungi were determined. The aim was to investigate the effect of host plants and AM fungi on AM morphology of coexisting plant species. Noncolonized rooted cuttings of Hedera rhombea (Miq) Bean, Rubus parvifolius L., and Rosa multiflora Thunb. were inoculated with five fungal species as single and mixed culture inocula. The fungal species used were Gigaspora rosea and Scutellospora erythropa, previously isolated from H. rhombea; Acaulospora longula and Glomus etunicatum from R. parvifolius; and Glomus claroideum from both plant species. A few hyphal and arbusculate coils were seen in the mixed culture-inoculated roots of R. parvifolius; all fungal treatments produced this Paris-type AM in H. rhombea and Arum-type AM in R. parvifolius, and R. multiflora indicates that AM morphology is strongly controlled by the identity of the host plants used in this study. AM fungal rDNA was extracted separately from roots of each replicate plant species inoculated with the mixed fungal culture, amplified, cloned, sequenced, and analyzed to determine the AM fungal species and their respective proportions in roots of each plant species. Glomus etunicatum and G. claroideum of the family Glomaceae generally occurred more frequently in R. parvifolius and R. multiflora, which form Arum-types, whereas S. erythropa, of the family Gigasporaceae, was the most frequently detected species in H. rhombea, which produced Paris-type AM. Although the genotype of the plant species used appears to determine the AM morphologies formed, there was preferential association between the hosts and AM fungal inoculants.     

Tick Surveillance for Relapsing Fever Spirochete Borrelia miyamotoi in Hokkaido,Japan

During 2012–2013, a total of 4325 host-seeking adult ticks belonging to the genus Ixodes were collected from various localities of Hokkaido, the northernmost island of Japan. Tick lysates were subjected to real-time PCR assay to detect borrelial infection. The assay was designed for specific detection of the Relapsing fever spirochete Borrelia miyamotoi and for unspecific detection of Lyme disease-related spirochetes. Overall prevalence of B. miyamotoi was 2% (71/3532) in Ixodes persulcatus, 4.3% (5/117) in Ixodes pavlovskyi and 0.1% (1/676) in Ixodes ovatus. The prevalence in I. persulcatus and I. pavlovskyi ticks were significantly higher than in I. ovatus. Co-infections with Lyme disease-related spirochetes were found in all of the tick species. During this investigation, we obtained 6 isolates of B. miyamotoi from I. persulcatus and I. pavlovskyi by culture in BSK-M medium. Phylogenetic trees of B. miyamotoi inferred from each of 3 housekeeping genes (glpQ, 16S rDNA, and flaB) demonstrated that the Hokkaido isolates were clustered with Russian B. miyamotoi, but were distinguishable from North American and European B. miyamotoi. A multilocus sequence analysis using 8 genes (clpA, clpX, nifS, pepX, pyrG, recG, rplB, and uvrA) suggested that all Japanese B. miyamotoi isolates, including past isolates, were genetically clonal, although these were isolated from different tick and vertebrate sources. From these results, B. miyamotoi-infected ticks are widely distributed throughout Hokkaido. Female I. persulcatus are responsible for most human tick-bites, thereby I. persulcatus is likely the most important vector of indigenous relapsing fever from tick bites in Hokkaido.     

Mutation of the PI3' kinase gene in a human colon carcinoma cell line, HCC2998

HCC2998 is a highly differentiated human colon carcinoma cell line, which has been shown to be converted to a poorly differentiated one after expression of a constitutively active phosphatidylinositol 3-kinase (PI3' kinase). These cells express aberrant sizes of a regulatory subunit of PI3' kinase, p85alpha, with molecular weights of 50 and 76 kDa at a very low level. To elucidate how these cells express these proteins, we analyzed mutations within the p85alpha gene. DNA sequencing analysis revealed that these mutant proteins were generated by independent point mutations in the two alleles of the p85alpha gene: one in the coding sequence, and the other in the acceptor sequence for splicing. Introduction of wild-type p85alpha into HCC2998 cells induced slight rounding of the cells and enhancement of mucin secretion. At the same time, a membrane receptor, ErbB3, was phosphorylated on tyrosine, which in turn, binds to PI3' kinase. Since ErbB3 is upstream of PI3' kinase, it is likely that there is an autocrine loop in which PI3' kinase is activated by ErbB3, which may contribute to dedifferentiation of the cells.     

Persistence and expression of circular DNAs encoding Drosophila amylase, bacterial chloramphenicol acetyltransferase, and others in Xenopus laevis embryos

We injected circular forms of several different DNAs into fertilized eggs of Xenopus laevis, and studied the persistence and expression of the injected DNAs during early embryonic development. When we injected plasmids which contained Drosophila amylase genes, the copy number of the injected DNA increased only slightly during cleavage, started to decrease at the blastula stage, then became very small at the tadpole stage. In such embryos, Drosophila amylase activity was detected at and after the gastrula stage. When we injected other kinds of circular DNAs (pX1r101A, cDm2055, pII25.1, pBR322, and pSP-64-L14), their copy number did not increase throughout the early stages. When circular plasmids that contained bacterial chloramphenicol acetyltransferase (CAT) genes were injected, their copy number usually did not increase, but sometimes, for unknown reasons, it increased extensively throughout the blastula to gastrula stages. In both cases, CAT enzyme activity started to be expressed during the blastula to gastrula stages and disappeared at the 2 day-old tadpole stage. The level of CAT enzyme activity was roughly proportional to the amount of CAT mRNA formed, and also to the copy number of injected genes. From these results, we concluded that in Xenopus embryos, exogenously-injected circular DNAs are preserved for the most part as circular DNAs, and that the increase in their copy number within the embryos is not prerequisite for the expression of their genetic information.     

Relationship between luminous fish and symbiosis. I. Comparative studies of lipopolysaccharides isolated from symbiotic luminous bacteria of the luminous marine fish, Physiculus japonicus

In order to investigate the relationship between host and symbiosis in the luminous marine fish, Physiculus japonicus, the bacterial lipopolysaccharides (LPS) of symbiotic luminous bacteria were compared serologically and electrophoretically. Five symbiotic luminous bacteria (PJ strains) were separately isolated from five individuals of this fish species caught at three points, off the coasts of Chiba, Nakaminato, and Oharai. LPS preparations were made from these bacteria by Westphal's phenol-water method and highly purified by repeated ultracentrifugation. These LPSs contained little or no 2-keto-3-deoxyoctonate and had powerful mitogenic activity. In sodium dodecylsulfate polyacrylamide gel electrophoresis, these PJ-1 to -5 LPSs were separated by their electrophoretic patterns into three groups; the first group included PJ-1 and PJ-4, the second group PJ-2 and PJ-3, and the third group PJ-5 alone. The results agreed with those of the double immunodiffusion test; precipitin lines completely coalesced within each group but not with other groups. In immunoelectrophoresis, one precipitin line was observed between anti PJ-2 LPS serum and PJ-5 LPS but the electrophoretic mobility of PJ-5 LPS was clearly different from that of the PJ-2 LPS group. Furthermore, in a 50% inhibition test with PJ-2 LPS by the passive hemolysis system, the doses of PJ-2 LPS, PJ-3 LPS, and PJ-5 LPS required for 50% inhibition (ID50) in this system were 0.25, 0.25, and 21.6 micrograms/ml for each alkali-treated LPS, respectively, and the ID50's of both PJ-1 LPS and PJ-4 LPS were above 1,000 micrograms/ml. These results indicate that PJ-5 LPS has an antigenic determinant partially in common with LPS from the PJ-2 group but not with LPS from the PJ-1 group and that the symbiotic luminous bacterium PJ-5 is more closely related to the PJ-2 group than to the PJ-1 group. These results show that the species Physiculus japonicus is symbiotically associated with at least three immunologically different strains of luminous marine bacteria in its specialized light organ.