Two mouse piwi-related genes: miwi and mili

Genes belonging to the piwi family are required for stem cell self-renewal in diverse organisms. We cloned mouse homologues of piwi by RT-PCR using degenerative primers. The deduced amino acid sequences of mouse homologues MIWI and MILI showed that each contains a well-conserved C-terminal PIWI domain and that each shares significant homology with PIWI and their human counterparts HIWI. Both miwi and mili were found in germ cells of adult testis by in situ hybridization, suggesting that these genes may function in spermatogenesis. Furthermore, mili was expressed in primordial germ cells (PGCs) of developing mouse embryos and may therefore play a role during germ cell formation. MIWI may be involved in RNA processing or translational regulation, since MIWI was found to possess RNA binding activity. Our data suggest that miwi and mili regulate spermatogenesis and primordial germ cell production.     

Cytokine expression in rat molar gingival periodontal tissues after topical application of lipopolysaccharide

It is well known that proinflammatory cytokines produced by host cells play an important role in periodontal tissue destruction. However, the localization of the cytokines in in vivo periodontal tissues during development of periodontal disease has not been determined. Immunohistochemical expression of proinflammatory cytokines including IL-1!, IL-1#, and TNF-! was examined at 1 and 3 h, and 1, 2, 3, and 7 days after topical application of lipopolysaccharide (LPS; 5 mg/ml in physiological saline) from E. coli into the rat molar gingival sulcus. In the normal periodontal tissues, a small number of cytokine-positive epithelial cells were seen in the junctional epithelium (JE), oral sulcular and oral gingival epithelium, in addition to macrophages infiltrating in the subjunctional epithelial area and osteoblasts lining the alveolar bone surface. Epithelial remnants of Malassez existing throughout periodontal ligament were intensely positive for IL-1# but negative for the other two cytokines. At 3 h after the LPS treatment, almost all cells in the JE were strongly positive for the cytokines examined. In addition, several cytokine-positive cells, including neutrophils, macrophages, and fibroblasts, were seen in the subjunctional epithelial connective tissue. At day 2, expression of the cytokines in the JE gradually decreased, while cytokine-positive cells in the connective tissue increased in number. Positive staining of the cytokines was seen in osteoclasts and preosteoclasts which appeared along the alveolar bone margin in this period. The number of cytokine-positive cells decreased by day 7. These findings indicate that, in addition to macrophages, neutrophils, and fibroblasts, the JE cells are a potent source of TNF-!, IL-1!, and IL-1# reacting to LPS application, and suggest that JE cells may play an important role in the first line of defense against LPS challenge, and the proinflammatory cytokines transiently produced by various host cells may be involved in the initiation of inflammation and subsequent periodontal tissue destruction.     

Cloning of a gene encoding acetate kinase from Methanosarcina mazei 2-P isolated from a Japanese paddy field soil

The ack gene encoding acetate kinase from the mesophilic Methanosarcina mazei 2-P, isolated from a paddy field soil in Japan, was cloned, sequenced, and functionally expressed in Escherichia coli. The terminal region of the putative pta gene, probably encoding phosphotransacetylase, was found upstream of the ack gene. The deduced amino acid sequence of the acetate kinase is 86.5% identical to that of the Methanosarcina thermophila acetate kinase. The activity of the His(6)-tagged acetate kinase purified from E. coli JM109 was optimal at 35 degrees C.     

Attenuated fever in pregnant rats is associated with blunted syntheses of brain cyclooxygenase-2 and PGE2

Attenuation of fever occurs in pregnant animals. This study examined a hypothesis that brain production of PGE(2), the final mediator of fever, is suppressed in pregnant animals. Near-term pregnant rats and age-matched nonpregnant female rats were injected with lipopolysaccharide (100 microg/kg) intraperitoneally. Four hours later, colonic temperature was measured, their cerebrospinal fluid (CSF) was sampled for PGE(2) assay, and their brains were processed for immunohistochemistry of cyclooxygenase-2, an enzyme involved in PGE(2) biosynthesis. In the pregnant rats, lipopolysaccharide injection resulted in significantly smaller elevations in both colonic temperature and CSF-PGE(2) level than in nonpregnant rats. In the pregnant rats, lipopolysaccharide-induced cyclooxygenase-2 expression was blunted in terms of the number of positive cells. There was a significant correlation between PGE(2) level in CSF and the number of cyclooxygenase-2-positive endothelial cells. These results suggest that suppressed PGE(2) production in the brain is one cause for the attenuated fever response at near-term pregnancy and that this suppressed PGE(2) production is due to the suppressed induction of cyclooxygenase-2 in brain endothelial cells.     

ecNOS gene polymorphism is associated with endothelium-dependent vasodilation in Type 2 diabetes

The association between endothelial constitutive nitric oxide synthase (ecNOS) gene polymorphism and vascular endothelial function has not been clarified. We investigated the impact of ecNOS gene polymorphism on endothelial function in 95 patients with Type 2 diabetes (ecNOS genotype: 4b/b, n = 62; 4b/a, n = 30; 4a/a, n = 3). Flow-mediated (endothelium dependent, FMD) and nitroglycerin-induced (endothelium independent, NTG) vasodilations of the right brachial artery were studied using a phase-locked echotracking system. There were no significant differences in clinical characteristics among the ecNOS genotypes. The FMD was significantly lower in the patients with ecNOS4a allele than in those without ecNOS4a allele (P < 0.05). Multiple regression analysis showed that ecNOS4a allele and mean blood pressure were significant independent determinants for reduced FMD in all patients (R(2) = 0.122, P = 0.0025). The ecNOS4a allele was an independent determinant for reduced FMD in smokers but not in nonsmokers. These results suggest that ecNOS4a allele is a genetic risk factor for endothelial dysfunction in diabetic patients, especially in smokers.     

Occurrence of an alpha-galacturonosyl-ceramide in the dioxin-degrading bacterium Sphingomonas wittichii

The chemical structure of two glycosphingolipids (GSLs) found in the dioxin-degrading bacterium Sphingomonas wittichii strain RW1 was investigated by means of mass spectrometry and (1)H-nuclear magnetic resonance spectroscopy. One of the GSLs was alpha-D-glucuronosyl-ceramide, commonly present in Sphingomonas spp., and the other was proved to be alpha-D-galacturonosyl-ceramide, whose sugar configuration has not been reported before. In both GSLs the ceramide portion was composed of myristic acid or 2-hydroxy-myristic acid as the fatty acid, and 2-amino-1,3-octadecanediol or 2-amino-cis-13,14-methylene-1,3-eicosanediol as the dihydrosphingosine.     

Synthesis of an ether-linked alkyl 5a-carba-beta-D-glucoside,a 5a-carba-beta-D-galactoside,a 2-acetamido-2-deoxy-5a-carba-beta-D-glucoside,and an alkyl 5a'-carba-beta-lactoside

For the purpose of providing biologically stable building blocks for the biocombinatorial synthesis using a living cell, some ether-linked alkyl 5a-carba-beta-D-glycoside primers were prepared. The key step of the synthesis was coupling of 1-bromo-n-alkanes with the 1-OH unprotected derivatives of 5a-carba-sugar analogues of D-glucose, D-galactose, and 2-acetamido-2-deoxy-D-glucose (N-acetyl-D-glucosamine), in DMF in the presence of sodium hydride. Alternatively, alkyl carba-lactoside was synthesized by incorporation of a 5a-carba-beta-D-galactose residue into the 4-position of dodecyl beta-D-glucopyranoside. A strong and specific inhibition of beta-galactosidase (K(i) 0.67 microM, bovine liver) was found for dodecyl 5a-carba-beta-D-galactopyranoside.     

Characterization of nuclear gangliosides in rat brain: concentration, composition, and developmental changes

Nuclear gangliosides were characterized using two distinct fractions of large (N1) and small (N2) nuclear populations from rat brain. The ganglioside concentration of N1 nuclei from adult rat brain was 0.92 microg sialic acid/mg protein, which was about 3.8 times higher than that of N2 nuclei. N1 and N2 nuclear gangliosides showed similar compositional profiles; they contained major gangliosides of GM1, GD1a, GD1b, and GT1b, with GM3 in lesser amounts. c-Series gangliosides such as GT3, GQ1c, and GP1c were also detected in both nuclear preparations. Nuclear localization of gangliosides was confirmed by immunofluorescence with anti-GM1 antibody, cholera toxin B subunit, and c-series ganglioside-specific monoclonal antibody A2B5. Developmental changes of nuclear gangliosides were examined using rats of different ages ranging from embryonic day 14 (E14) to postnatal 7 weeks. The concentration of N1 nuclear gangliosides changed only slightly during development and did not correlate with that of whole-brain gangliosides. The developmental pattern of ganglioside composition of N1 nuclei was also distinguished from that of microsomal membranes; the ganglioside changes in N1 nuclei included reduced expression of di- and polysialogangliosides at E16 and higher proportions of GM3 at early and late stages of the period. These findings suggest that gangliosides in nuclear membranes are developmentally regulated in a distinct manner in brain cells.     

Specific recognition and cleavage of galectin-3 by Leishmania major through species-specific polygalactose epitope

Lipophosphoglycan is a major surface molecule of Leishmania, protozoa parasites, which are the causative agents of leishmaniasis, a disease that annually afflicts millions of people worldwide. The oligosaccharide structures of lipophosphoglycan varies among species, and epitopes of these species-specific oligosaccharides are suggested to be implicated in the interaction of Leishmania with macrophages as well as species-specific tissue tropism observed in leishmaniasis. The recognition of the species-specific variation of oligosaccharides is likely to be mediated by host carbohydrate-binding proteins, lectins, but the identities of the lectins remain elusive. Galectin-3 is a mammalian soluble beta-galactoside-binding lectin and is expressed in macrophages, dendritic cells, and keratinocytes, as well as fibroblasts, all of which are present in the site of Leishmania infection. In this paper, we found that galectin-3 binds to lipophosphoglycan of Leishmania major but not to those of Leishmania donovani through L. major-specific polygalactose epitopes. Association of galectin-3 with L. major led to the cleavage of galectin-3, resulting in truncated galectin-3 containing the C-terminal lectin domain but lacking the N-terminal domain implicated in lectin oligomerization. This cleavage was inhibited by the galectin-3 antagonist lactose, as well as 1,10-ortho-phenanthroline, suggesting that galectin-3 is cleaved by zinc metalloproteases after its binding to lipophosphoglycans. The modulation of various innate immunity reactions by galectin-3 is affected by its oligomerization; therefore, we propose the L. major-specific truncation of galectin-3 may contribute to the species-specific immune responses induced by Leishmania.     

Neuronal differentiation-dependent expression of the disialic acid epitope on CD166 and its involvement in neurite formation in Neuro2A cells

We previously demonstrated that alpha2,8-linked disialic acid (diSia) residues occur in several glycoproteins of mammalian brains (Sato, C., Fukuoka, H., Ohta, K., Matsuda, T., Koshino, R., Kobayashi, K., Troy, F. A., II, and Kitajima, K. (2000) J. Biol. Chem. 275, 15422-15431). The role of the diSia epitope on these glycoproteins is not known, whereas the importance of the diSia epitope on glycolipids is well documented in neurite formation. In this study, we demonstrated that the diSia epitope (Neu5Acalpha2 --> 8Neu5Acalpha2 --> 3Gal) on glycoproteins, but not on glycolipids, is involved in neurite formation in a mouse neuroblastoma cell line, Neuro2A, based on the following lines of evidence. First, the amount of diSia epitope on glycoproteins increased during retinoic acid-induced neurite formation. Second, retinoic acid treatment primarily increased the diSia epitope on a 100-kDa glycoprotein. We identified this protein as CD166 (SC1), an immunoglobulin superfamily cell adhesion molecule involved in neurite extension. Third, a monoclonal antibody against the diSia epitope specifically inhibited neurite formation. We also demonstrated that alpha2,8-sialyltransferase III mRNA expression increased 1.7-fold after the induction of neurite formation, suggesting that alpha2,8-sialyltransferase III is responsible for formation of the diSia epitope on CD166.