Interleukin-4 enhances in vitro mineralization in human osteoblast-like cells.

The effect of human interleukin-4 (hIL-4) on mineralization in human osteoblast-like cells was investigated. Confluent cells were incubated with hIL-4 for 16 or 30 days in the presence or absence, respectively, of alpha-glycerophosphate (alpha-GP), which accelerates the mineralization process. hIL-4 (0.3 ng/ml) induced mineralization with 1.9-, 26- and 37-fold increases of hydroxyproline, calcium, and osteocalcin content, respectively, in the presence of alpha-GP. Mineralization was not induced with other cytokines, hIL-1, hIL-2, hIL-6, or mIL-4. hIL-4 also induced mineralization in the absence of alpha-GP in a manner different from that of 1 alpha, 25(OH)2 vitaminD3 (1,25(OH)2VD3). These findings suggest that IL-4 may play an important role in bone formation.     

Cloning and characterization of two forms of C-type natriuretic peptide receptor in rat brain.

Two similar membrane bound guanylate cyclases (GC-A and GC-B) are known as natriuretic peptide receptors, but have not been well characterized yet. In this study, we have isolated two forms of GC-B cDNA clones along with GC-A cDNA clones from rat brain. The two forms of rat GC-B differ from each other only by 75bp deletion at 3'-flanking region of the putative transmembrane domain, the shorter form lacking the nucleotide binding site by the deletion. Expression of these cDNAs on mammalian cells revealed that (1) GC-B is a specific receptor for CNP whereas GC-A is stimulated effectively both by ANP and BNP, and (2) the two forms of GC-B possess practically the same high binding affinity for CNP while the shorter form could not induce cGMP production by the binding of CNP. These data indicate that in rat brain is present the non-functional receptor for CNP caused by the short deletion.     

The evidence for post-meiotic expression of a testis-specific isoform of a regulatory subunit of calcineurin using a monoclonal antibody.

The expression of a regulatory subunit of calcineurin (CaN beta) during rat spermatogenesis was examined in rat testes using a monoclonal antibody Va1. Results showed that a testis-specific isoform of CaN beta was expressed only 3 weeks after birth, when meiosis begins, and increased in amount depending on the maturation of spermatogenesis. The matured sperm, which consists of only post-meiotic cells, is most likely to have only the testis-specific isoform of CaN beta. The brain type isoform of CaN beta was not detected in rat sperm. Immunoblot analysis of testes from different rodent species by a monoclonal antibody Va1 showed that all rodent species examined had their own homologues corresponding to a testis-specific isoform of CaN beta in rats, although they showed distinctively different molecular weights on SDS-PAGE compared to the testis-specific isoform in rats. Each homologue was shown to be specifically expressed in post-meiotic phase of spermatogenesis, as was seen in rats.     

Developmental changes in intestinal glucose transporter mRNA levels.

Developmental changes in glucose transporter mRNA levels in the jejunum of rats of different ages were examined by using slot blot RNA analysis. The level of SGLT1 mRNA did not change significantly through life. The GLUT5 mRNA level was highest in 10-day-old rats and then decreased reaching the adult level by day 20 after birth. The GLUT2 mRNA level was low in rats of 5 and 10 days old, but then increased progressively reaching the adult value by day 25 after birth. These results indicate that the expressions of intestinal facilitative glucose transporter genes change markedly in the third week after birth.     

Cloning and chromosomal mapping of the mouse DNA-dependent protein kinase gene

 Severe combined immune deficiency (scid) mice are assumed to have two types of abnormalities: one is high radiosensitivity and the other is abnormal recombination in immunoglobulin and T-cell receptor genes. The human chromosome 8 q1.1 region has an ability to complement the scid aberrations. Moreover, the localization of the subunit DNA-dependent protein kinase [DNA-PK_cs] participating in DNA double-strand break repair in the same locus was clarified. In scid mouse cells, the number of DNA-PK_cs products and extent of DNA-PK activity remarkably decrease. These observations gave rise to the assumption that DNA-PK_cs is the scid factor itself. In order to determine whether the DNA-PK _cs gene is the scid gene, we isolated the mouse DNA-PK _cs gene and investigated its chromosomal locus by fluorescence in situ hybridization (FISH). Consequently, it became clear that the mouse DNA-PK _cs gene existed in the centromeric region of mouse chromosome 16, determined by cross-genetic study, as a scid locus. This finding strongly suggests that mouse DNA-PK _cs is the scid gene. Received: 22 March 1996     

Characterization of spirochetes isolated from ticks (Ixodes tanuki, Ixodes turdus, and Ixodes columnae) and comparison of the sequences with those of Borrelia burgdorferi sensu lato strains.

Ixodes persulcatus serves as a tick vector for Borrelia garinii and Borrelia afzelii in Japan; however, unidentified spirochetes have been isolated from other species of ticks. In this study, 13 isolates from ticks (6 from Ixodes tanuki, 6 from Ixodes turdus, and 1 from Ixodes columnae) and 3 isolates from voles (Clethrionomys rufocanus) were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, rRNA gene restriction fragment length polymorphism, partial sequencing of the outer surface protein C (OspC) gene, whole DNA-DNA hybridization, and 16S rRNA gene sequence comparison. All of the results revealed that these Borrelia strains clearly represent at least two new species. A third is also likely, although additional strains have to be isolated and characterized before a separate species is designated. We designated all isolates of I. tanuki and C. rufocanus as group Hk501 and all isolates of I. turdus as group Ya501. Phylogenetic analysis based on 16S rRNA gene sequences distinguished these Borrelia strains from those belonging to hitherto known Borrelia species. Furthermore, the genomic groups, each with its own tick vectors with enzootic cycles, were quite different from each other and also from those of Lyme disease Borrelia species known to occur in Japan. The results of 16S rRNA gene sequence comparison suggest that the strain Am501 from I. columnae is related to group Hk501, although its level of DNA relatedness is less than 70%.     

RELATIONSHIPS AMONG MORPHOLOGICAL STATUS, STEROID HORMONES, AND POST-THAWING VIABILITY OF FROZEN SPERMATOZOA OF MALE MINKE WHALES (BALAENOPTERA ACUTOROSTRATA)

Spermatozoa from 21 mature minke whales ( Balaenoptera acutorostrata ) taken in the Antarctic Ocean for Japanese research were recovered from vasa deferentia, diluted 1:9 in a Tris-based diluent, and frozen at - 80°C on board the vessel. After a period ranging from 45 to 125 d, the samples were transferred to liquid nitrogen and transported to the laboratory. After thawing at 37°C the motility (percentage of motile spermatozoa), vitality (proportion of live spermatozoa), and sperm concentration were determined for each sample. These values were tested for correlations with morphological measurements (body size, body weight, testis weight) and serum concentrations of progesterone (Pd), estradiol-17β (E2), and testosterone (T). Ten of 21 samples had motile spermatozoa (2%-40%). Although no motile spermatozoa were observed in 1.1 samples, all sperm samples were examined by eosinnigrosin staining and showed vitality levels of 3%44%. It was found that the motility (Y = 0.54) and vitality (r = 0.53) of the spermatozoa were significantly (P < 0.01) correlated with the E₂ levels (8.50 ± 1.80 pg/ml). Serum T levels (0.07 ± 0.02 ngml) were significantly correlated with the E₂ levels (r = 0.58, P < 0.01>, but sperm concentrations were not correlated with either Ea or T levels. The present study demonstrates that spermatozoa of minke whales can be successfully cryopreserved.     

Organic-Solvent-Tolerant Bacterium Which Secretes Organic-Solvent-Stable Lipolytic Enzyme

A bacterial strain which could be grown in a medium containing organic solvents and which could secrete lipolytic enzyme was isolated. The stability of the lipolytic activity of the supernatant of the culture increased significantly in the presence of organic solvents such as toluene, cyclohexane, ethanol, and acetone.     

Characterization of spinach leaf α-l-arabinofuranosidases and β-galactosidases and their synergistic action on an endogenous arabinogalactan-protein

Two β-galaclosidases (β-Galase-I and -II, EC 3.2.1.23) and two α-l -arabinofuranosidases (α-l -Arafase-I and -II. EC 3.2.1.55). were purified from mesophyll tissues of spinach (Spinacia oleracea L.), using chromatography on DEAE-cellulose, lactose-conjugated Sepharose CL-4B, and Sephadex G-100, or on hydroxylapatite and Sephadex G-150. The apparent molecular mass (M_r) of β-Galase-I and -II, respectively, were estimated to be 38 000 and 58 000 on SDS-PAGE and 64 000 and 60 000 on gel-permeation chromatography, indicating that the former was a dimeric protein. The isoelectric points of β-Galase-I and -II were 6.9 and 5.2, respectively. Both enzymes hydrolyzed maximally p-nitrophenyl (PNP) β-galactoside at pH 4.3, and were activated about 2-fold in the presence of BSA (100 μg ml^?1). The activity of both enzymes was inhibited strongly by heavy metal ions and p-chloromercuriberszoate (p-CMB). d -Galactono-(1→4)-lactone and d -galactal served as potent competitive inhibitors for the enzymes. β-Galase-I and -II could be distinguished from each other in their relative rates and kinetic properties in the hydrolysis of aryl β-galactosides as well as of lactose and galacto-oligosaccharides. In particular. β-Galase-I exhibited a preferential exowise cleavage of β-1,6-galactotriose and β-1.3-galactan. α-l -Arafase-l (M_r 118000) and -II (M, 68 000) were optimally active on PNP α-l -arabinofuranoside at pH 4.8 and gave K_m values of 1.2 and 2.2 mM. respectively. l -Arabino-(1 → 4)-lactone. Ag⁺, and SDS acted as inhibitors for the isozymes. α-l Arafase-I was characterized by its activity to hydrolyze PNP β-d -xylopyranoside besides PNP α-l -arabinofuranoside. inhibition by d -xylose and d -glucono-(1 → 5)-lactone. and less sensitivity to Hg²⁺. Cu²⁺, and p-CMB. Sugar beet arabinan was hydrolyzed rapidly by α-l Arafase-II at one-half the rate for PNP α-l arabinofuranoside, while the polysaccharide was less susceptible to α-l Arafase-I. A spinach leaf arabinogalactan-protein was practically resistant to the action of β-Galases, but its susceptibility to the enzymes increased remarkably after prior hydrolysis with α-l Arafase-Il.