Differentiation of restriction sites in ribosomal DNA in the genusApodemus

Southern blot analysis of ribosomal DNA (rDNA) from seven species ofApodemus was carried out in order to examine the genetic relationships between the species. Analysis of heterogeneity in rDNA spacers inA. sylvaticus, A. flavicollis, A. semotus, A. agrarius, A. argenteus, A. speciosus, andA. peninsulae, using 13 different restriction enzymes and cloned mouse rDNA probes, revealed that the families of rDNA in these species can be characterized by restriction maps which show the major constituents of rDNA repeating units (repetypes). Based on differences in the arrangement of restriction sites, sequence divergence among the different major repetypes was estimated. Among the seven species ofApodemus examined, the major repetypes ofA. flavicollis andA. sylvaticus were the most closely related, having only 1.0% sequence divergence. These repetypes and those of the remaining five species differ substantially from one another, with 4.3–8.5% divergence.This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.     

In vitro tumor cell killing by peritoneal macrophages from mitomycin C-treated rats

Summary The local cellular response induced by intraperitoneal injection of mitomycin C was examined in terms of cell-mediated cytotoxicity for tumor cells. An in vitro cytolysis assay involving ¹²⁵I-iododeoxyuridine-labeled tumor target cells revealed that treatment of normal ACI/N rats (200 g) with a single intraperitoneal injection of mitomycin C (50, 100, or 200 g) induced tumoricidal macrophages in the peritoneal cavity. The tumoricidal activity was dependent on the dose of mitomycin C injected and it was detectable as early as 1 day after the intraperitoneal injection of mitomycin C. In addition to the increased tumoricidal activity, the functional activities of the peritoneal macrophages were found to be increased with respect both to uptake of 2-deoxy-d-glucose and to phagocytosis of latex beads. Additional experiments excluded the possibility that the tumor cell cytolysis was the result of direct cytotoxicity by mitomycin C that might have been incorporated in the peritoneal macrophages or of nutrient depletion in the medium during the cytolysis assay. Furthermore, endotoxin contamination of the mitomycin C, which might have produced the activated macrophages, was not detected. The mechanism by which mitomycin C injected intraperitoneally induced the tumoricidal macrophages locally remains uncertain; however, it is possible also in clinical situations.     

An antagonistic effect between aziridine and diaziridine on their cytotoxic activities against L-1210 mouse leukemia cells

The effect of the combination of 1-methyl-2-$\text{p}$-chlorophenylaziridine and 1,2-dimethyl-3-$\text{p}$-chlorophenyldiaziridine on cytotoxic activities against L-1210 mouse leukemia cells was studied. Both compounds clearly showed an antagonistic interaction. The results supported our hypothesis that nitrosomethane formed by the fragmentation of aziridine via the N-oxide in a cellular system causes the cytotoxic behavior of the N-methyl aziridines.     

Involvement of phospholipase D activation in endothelin-1-induced release of arachidonic acid in osteoblast-like cells

In a previous study, we have shown that endothelin-1 (ET-1) activates phospholipase D independently from protein kinase C in osteoblast-like MC3T3-E1 cells. It is well recognized that phosphatidylycholine hydrolysis by phospholipase D generates phosphatidic acid, which can be further degraded by phosphatidic acid phosphohydrolase to diacylglycerol. In the present study, we investigated the role of phospholipase D activation in ET-1-induced arachidonic acid release and prostaglandin E₂ (PGE₂) synthesis in osteoblast-like MC3T3-E1 cells. ET-1 stimulated arachidonic acid release dose-dependently in the range between 0.1 nM and 0.1 μM. Propranolol, an inhibitor of phosphatidic acid phosphohydrolase, significantly inhibited the ET-1-induced arachidonic acid release in a dose-dependent manner as well as the ET-1-induced diacylglycerol formation. 1,6-bis-(cyclohexyloxyminocarbonylamino)-hexane (RHC-80267), an inhibitor of diacylglycerol lipase, significantly suppressed the ET-1-induced arachidonic acid release. The pretreatment with propranolol and RHC-80267 also inhibited the ET-1-induced PGE₂ synthesis. These results strongly suggest that phosphatidylcholine hydrolysis by phospholipase D is involved in the arachidonic acid release induced by ET-1 in osteoblast-like cells. J. Cell. Biochem. 64:376–381. © 1997 Wiley-Liss, Inc.     

Polymorphic microsatellite loci and low-invasive DNA sampling inMacaca cyclopis

Polymorphic genetic markers and methods for DNA sampling in the field are the basic requirements for studies on population and conservation genetics of wildMacaca cyclopis. In this paper we screened microsatellites for their polymorphism and accessed the validity in paternity identification and gene typing of DNA samples from various sources. Among the 36 primer sets tested, 21 are polymorphic with an average observed heterozygosity 0.56. All theeight loci examined for a parent-offspring triad followed Medelian inheritance. Application of the two most polymorphic loci in paternity identification of a daptive group showed that the top-ranking male sired all the juveniles. DNA samples from wound and menstrual bleeding, or from ejaculates and hairs produced concordant microsatellite banding patterns for specific individuals. The success in DNA extraction from samples collected low-invasively and the polymorphic loci screened in this study can be applied in future studies on population and conservation genetics of natural primate populations.     

Cellobiose transport by mixed ruminal bacteria from a Cow.

The transport of cellobiose in mixed ruminal bacteria harvested from a holstein cow fed an Italian ryegrass hay was determined in the presence of nojirimycin-1-sulfate, which almost inhibited cellobiase activity. The kinetic parameters of cellobiose uptake were 14 microM for the Km and 10 nmol/min/mg of protein for the Vmax. Extracellular and cell-associated cellobiases were detected in the rumen, with both showing higher Vmax values and lower affinities than those determined for cellobiose transport. The proportion of cellobiose that was directly transported before it was extracellularly degraded into glucose increased as the cellobiose concentration decreased, reaching more than 20% at the actually observed levels of cellobiose in the rumen, which were less than 0.02 mM. The inhibitor experiment showed that cellobiose was incorporated into the cells mainly by the phosphoenolpyruvate phosphotransferase system and partially by an ATP-dependent and proton-motive-force-independent active transport system. This finding was also supported by determinations of phosphoenolpyruvate phosphotransferase-dependent NADH oxidation with cellobiose and the effects of artificial potentials on cellobiose transport. Cellobiose uptake was sensitive to a decrease in pH (especially below 6.0), and it was weakly but significantly inhibited in the presence of glucose.     

Chemiluminescence of iron-chlorophyllin.

The iron-chlorophyllin complex was found to be chemiluminescent (CL) in an acetonitrile (22%)/water mixed solvent. In the presence of hydrogen peroxide, when iron-chlorophyllin was added to the mixed solvent, a sharp CL signal immediately appeared. Also, analysis of the absorption spectra revealed decomposition of iron-chlorophyllin (based on decrease in absorbance at 396 nm), hence iron-chlorophyllin is the CL substance. Moreover, the CL intensity decreased in the presence of potassium thiocyanate (KSCN), indicating that the axial coordinative position of iron-chlorophyllin acts as a point of catalytic activation. In addition, when fluorophores were present with iron-chlorophyllin CL, their CL intensity values were similar to or greater than that of the well-known trichlorophenylperoxalate ester (TCPO) CL. Thus, during the decomposition reaction of iron-chlorophyllin, the latter transfers its energy to the coexisting fluorophores. Moreover, since the decomposed compound in this CL reaction had a fluorescence, it was found that the iron-chlorophyllin also functions as an energy donor. Therefore, the iron-chlorophyllin complex acts not only as a CL substance, but also as a catalyst and energy donor in the reaction.     

Relationship between scapular position and structural strength of rib cage in quadruped animals

Determining scapular position is a major issue in reconstructing the skeletal systems of extinct quadruped archosaurs and mammals, because the proximal portion of the scapulae has no direct skeletal joint with the vertebrae or ribs. When quadrupeds stand or walk, their trunk is suspended between the forelimbs by the serratus muscles, which arises from the lateral sides of the “thoracic” ribs and inserts into the proximal portion of the costal surface of the scapula. Therefore, the “thoracic” ribs are subjected to a static or dynamic vertical compression between the lifting force from the muscle and the gravitational force from the vertebral column. To investigate the body support function of the ribs, we analyzed the mechanical strength of the ribs of extant tetrapods by the two‐dimensional finite element method, and compared the degree of strength through their craniocaudal scapular positions. The result of this simulation showed that the “thoracic” ribs of quadrupeds, to which the serratus muscles attach, have a relatively higher strength against compaction than the other ribs. In bipeds, however, we did not find a similar correlation between the strength of ribs and the serratus muscle. This implies that the location of robust ribs is associated with the arrangement of the serratus muscle, and provides a probable candidate for determination of the scapular position for extinct quadruped archosaurs and mammals. J. Morphol. 2009. © 2009 Wiley‐Liss, Inc.     

Hypoxia-Inducible Factors Activate CD133 Promoter through ETS Family Transcription Factors

CD133 is a cellular surface protein that has been reported to be a cancer stem cell marker, and thus it is considered to be a potential target for cancer treatment. However, the mechanism regulating CD133 expression is not yet understood. In this study, we analyzed the activity of five putative promoters (P1–P5) of CD133 in human embryonic kidney (HEK) 293 cells and colon cancer cell line WiDr, and found that the activity of promoters, particularly of P5, is elevated by overexpression of hypoxia-inducible factors (HIF-1α and HIF-2α). Deletion and mutation analysis identified one of the two E-twenty six (ETS) binding sites (EBSs) in the P5 region as being essential for its promoter activity induced by HIF-1α and HIF-2α. In addition, a chromatin imunoprecipitation assay demonstrated that HIF-1α and HIF-2α bind to the proximal P5 promoter at the EBSs. The immunoprecipitation assay showed that HIF-1α physically interacts with Elk1; however, HIF-2α did not bind to Elk1 or ETS1. Furthermore, knockdown of both HIF-1α and HIF-2α resulted in a reduction of CD133 expression in WiDr. Taken together, our results revealed that HIF-1α and HIF-2α activate CD133 promoter through ETS proteins.     

Production of anti-Candida antibodies in mice with gut colonization of Candida albicans

BACKGROUND: Production of antibodies that are specific for allergens is an important pathological process in inflammatory allergic diseases. These contain the antibodies against antigens of Candida albicans, one of the normal microbial flora in an intestinal tract. We studied the effects of the prednisolone administration on the production of anti-Candida antibodies in the gastrointestinally C. albicans-colonized mice. METHODS AND MATERIALS: BALB/c mice, treated with antibacterial antibiotics to decontaminate indigenous intestinal bacterial flora, were inoculated intragastrically with C. albicans. The mice, in which C. albicans grows intestinally, were administered prednisolone to induce temporary immunosuppression. The Candida growth in their intestinal tract and their antibody response to Candida were examined. RESULTS: Antibiotic treatment allowed establishment of C. albicans gastrointestinal colonization, but did not cause subsequent systemic dissemination of C. albicans in all the animals. When these animals received an additional treatment with prednisolone, they showed a significantly higher population of C. albicans in their feces than those of animals treated with antibiotics alone, and the organisms were recovered even from their kidney. This systemic dissemination by C. albicans appeared to be temporal, because all the mice survived without any symptoms for more than 2 months. Examination of the serum titers of total immunoglobulin (Ig)E antibodies and specific IgE and IgG antibodies against Candida antigens demonstrated that titers of total IgE increased, partially by day 14 and clearly at day 27, in prednisolone-treated Candida-colonized mice. Without prednisolone treatment, an increment of the serum titer was scarcely observed. By day 27, corresponding to the increase of total IgE, the anti-Candida IgE and IgG titer increased in mice of the prednisolone-treated group. CONCLUSION: Administration of prednisolone to Candida-colonized mice can induce production of the IgG, IgE antibodies against Candida antigens, perhaps through temporal systemic dissemination of Candida from the intestinal tract.