Differential Regulation of Intracellular Signaling Systems by Sodium Fluoride in Rat Glioma Cells

Abstract: We investigated the rapid and slow effects of NaF on intracellular signaling systems such as Ca²⁺ homeostasis and cyclic GMP (cGMP) generation in rat glioma C6 cells, using the Ca²⁺-sensitive dye fura-2 and cGMP enzyme immunoassay. We found that the following: (a) NaF enhanced cGMP generation in a concentration-dependent manner. This enhancement was abolished by pretreatment with 100 µ M BAPTA tetraacetoxymethyl ester or in the presence of W-7 in a concentration-dependent manner. N ^G-Monomethyl- l -arginine (NMMA), a competitive inhibitor of nitric oxide synthase (NOS), also inhibited the NaF-induced generation of cGMP. These results suggest that NaF-induced cGMP generation occurs via a calcium/calmodulin- and NOS-dependent pathway. (b) The basal intracellular Ca²⁺ concentration ([Ca²⁺]_i) was transiently greater at 1 and 3 h after pretreatment with NaF. W-7 and W-13 antagonized the increase in [Ca²⁺]_i, whereas NMMA had little effect. This suggests that the NaF-induced change in basal [Ca²⁺]_i was mediated by a calmodulin-dependent pathway but was independent of a NOS-sensitive pathway. (c) The serotonin (5-HT)-induced intracellular mobilization of Ca²⁺ was reduced by pretreating the cells with NaF. The reduction in Ca²⁺ mobilization was antagonized by genistein, a tyrosine kinase inhibitor. W-7, W-5, and H-8 had no effect. Results suggest that NaF differentially regulates the cGMP generation, basal [Ca²⁺]_i, and 5-HT_2A receptor function in C6 glioma cells.     

SFA-1, a novel cellular gene induced by human T-cell leukemia virus type 1, is a member of the transmembrane 4 superfamily.

A novel cellular gene termed SFA-1 was isolated by differential hybridization of a cDNA library, using probes obtained from an adult T-cell leukemia cell line in comparison with probes obtained from normal CD4+ T cells and the MOLT-4 cell line. The mRNA of the SFA-1 gene is approximately 1.6 kb in size and encodes a protein of 253 amino acids, containing four putative transmembrane domains, a number of cysteine residues, and one potential N-glycosylation site in a major hydrophilic region between the third and fourth transmembrane domains. Expression of the SFA-1 gene was either absent or present at a low level in lymphoid cells but was up-regulated after transformation by human T-cell leukemia virus type 1 and transactivated by Tax. SFA-1 was broadly expressed in many human cell types and conserved in different species. Computer-aided comparison showed that SFA-1 had significant sequence homology and common structural features with members of the transmembrane 4 superfamily. SFA-1 antigen was detected as a 29-kDa membrane protein by immunoblotting, using anti-SFA-1 monoclonal antibody.     

Comparison of fish assemblages among an artificial reef, a natural reef and a sandy-mud bottom site on the shelf off Iwate, northern Japan

Synopsis Fish assemblages at an artificial reef site, a natural reef site and a sandy-mud bottom site, on the shelf (depth 130 m) off Iwate Prefecture, northern Japan, were surveyed by using a bottom trammel net from May 1987 to March 1993. A total of 12 173 fishes of 48 species were recorded. Physiculus maximowiczi was dominant and comprised 69% of the total numerical abundance. Total fish number was lowest in March at all the 3 sites when P. maximowiczi migrated to deeper and warmer waters. Assemblage equitability and species diversity also varied seasonally in accordance with the abundance fluctuation of P. maximowiczi. P. maximowiczi, Alcichthys alcicornis and Hexagrammos otakii were more abundant at the artificial reef and natural reef sites, while Dexistes rikuzenius and Hemitripterus villosus were more abundant at the sandy-mud bottom site; total fish abundance was largest at the artificial reef site mainly due to the large number of P. maximowiczi. Species richness was similar among sites, but equitability, and consequently species diversity, was lowest at the artificial reef site. The main effect of the artificial reef seemed the attraction of P. maximowiczi from nearby bottoms, especially from natural rocky reefs; its large abundance determined the structure of the artificial reef fish community.     

Hyperinduction of nitrile hydratase acting on indole-3-acetonitrile in Agrobacterium tumefaciens

 We investigated the optimum conditions for the formation of nitrile hydratase (NHase), which acts on indole-3-acetonitrile, in Agrobacterium tumefaciens. Good inducers for enzyme formation have been found to be roughly classified into three representative types of amides such as pivalamide, crotonamide and ɛ-caprolactam. When the strain was cultivated in the optimum culture medium containing ɛ-caprolactam as an inducer, in particular, the specific activity of NHase in the culture was 13 000 times higher than that without addition of amides, nitriles or acids. In this case, NHase formed accounted for 12% of the total cellular soluble protein. The purified NHase did not act on ɛ-caprolactam, and ɛ-caprolactam was not degraded during the cultivation by the strain, suggesting that ɛ-caprolactam seems to keep driving the NHase induction mechanism. Received: 3 March 1995/Received revision: 13 July 1995/Accepted: 7 September 1995     

A recombination outside the BB deletion refines the location of the X linked retinitis pigmentosa locus RP3.

Genetic loci for X-linked retinitis pigmentosa (XLRP) have been mapped between Xp11.22 and Xp22.13 (RP2, RP3, RP6, and RP15). The RP3 gene, which is responsible for the predominant form of XLRP in most Caucasian populations, has been localized to Xp21.1 by linkage analysis and the map positions of chromosomal deletions associated with the disease. Previous linkage studies have suggested that RP3 is flanked by the markers DXS1110 (distal) and OTC (proximal). Patient BB was thought to have RP because of a lesion at the RP3 locus, in addition to chronic granulomatous disease, Duchenne muscular dystrophy (DMD), mild mental retardation, and the McLeod phenotype. This patient carried a deletion extending approximately 3 Mb from DMD in Xp21.3 to Xp21.1, with the proximal breakpoint located approximately 40 kb centromeric to DXS1110. The RP3 gene, therefore, is believed to reside between DXS1110 and the proximal breakpoint of the BB deletion. In order to refine the location of RP3 and to ascertain patients with RP3, we have been analyzing several XLRP families for linkage to Xp markers. Linkage analysis in an American family of 27 individuals demonstrates segregation of XLRP with markers in Xp21.1, consistent with the RP3 subtype. One affected mate shows a recombination event proximal to DXS1110. Additional markers within the DXS1110-OTC interval show that the crossover is between two novel polymorphic markers, DXS8349 and M6, both of which are present in BB DNA and lie centromeric to the proximal breakpoint. This recombination places the XLRP mutation in this family outside the BB deletion and redefines the location of RP3.     

Phylogenetic relationship of medicinally importantCnidium officinale and Japanese Apiaceae based onrbcL sequences

Cnidium officinale Makino is important medicinally and economically, but its origin is uncertain. The phylogenetic relationship ofC. officinale is provided from the analyses based on the ribulose-1,5-bisphosphate carboxylase/oxgenase gene (rbcL) sequences of 41 species which represent the 34 genera of Aplaceae, the four genera of Araliaceae, and one genus each of Pittosporaceae, Cornaceae, and Caprifoliaceae. The strict consensus tree obtained supports a close relationship ofC. officinale to the Chinese members ofLigusticum, especially toL. chuanxiong. Additionally, the tree shows (1) polyphyly of the genusLigusticum and (2) monophyly of the subfamily Apioideae. Within Apioideae, we recognized some groups in our phylogenetic tree. The grouping is discordant in several respects with the traditional tribal divisions based mainly on fruit morphology.     

Immunocytochemical study of seminal γ-glutamyltransferase using monoclonal antibodies

We produced three monoclonal antibodies, SG1, SG2 and SG3, specific for human seminal -glutamyltransferase when characterized by enzyme-linked immunosorbent assay and immunoblotting. Seminal -glutamyltransferase was localized, by immunostaining, to the epithelial cells of the ductus epididymidis, seminal vesicle and prostate gland with SG1, those of the prostate gland with SG2, and those of the seminal vesicle with SG3. Rabbit polyclonal anti-seminal -glutamyltransferase serum reacted with the proximal convolution of the kidney and the bile capillaries of the liver, and with the epithelial cells of the reproductive organs. However, immunoreactivity was not observed in the kidney or liver with the monoclonal antibodies. Thus, these monoclonal antibodies are probably all specific to seminal -glutamyltransferase but recognize different epitopes.     

Visualization of mitotic chromosomes in filamentous fungi by fluorescence staining and fluorescence in situ hybridization

Mitotic chromosomes of the plant pathogenic filamentous fungi Botrytis cinerea and Alternaria alternata were observed. Chromosomes prepared by the germ tube burst method were stained with the fluorescent dye 4,6-diamidino-2-phenylindole (DAPI) to yield figures with good resolution. Using this method, component chromosomes were clearly distinguished and the chromosome number could be determined. Fluorescence in situ hybridization (FISH) was also successfully applied to the specimens, revealing one ribosomal RNA gene cluster, or nucleolus organizer region (NOR) in the genome of each fungus. A long attenuated chromatid thread expanding from a condensed metaphase chromosome, which had been called a thread-like structure in B. cinerea, was proved to be an NOR. This is the first report of the successful application of FISH to the chromosomes of filamentous fungi.     

Induction of G protein-coupled peptide receptor EBI 1 by human herpesvirus 6 and 7 infection in CD4+ T cells.

EBI 1, a putative lymphocyte-specific G protein-coupled peptide receptor, was induced by human herpesvirus 6 or 7 infection in CD4+ T cells, and its expression increased early after infection and reached a plateau at 48 h. The induction of the EBI 1 gene by human herpesvirus 6 or 7 infection was not mediated by soluble factors but by the virus itself. Deduced from comparisons of the amino acid sequences among members of the G protein-coupled receptor superfamily, these findings suggest that EBI 1 may be a member of the leukocyte chemotactic peptide receptor family.