Gender difference in the Oatp1-mediated tubular reabsorption of estradiol 17beta-D-glucuronide in rats

The gender difference in the urinary excretion of estradiol-17beta-glucuronide (E(2)-17betaG) was examined in rats. The urinary clearance of E(2)-17betaG was >250 times lower in male than in female rats. No such major gender difference was observed in its biliary excretion or metabolism in kidney homogenate. Both plasma protein binding and inulin clearance were comparable in male and female rats, suggesting that this gender difference cannot be explained by glomerular filtration. The urinary clearance with respect to the plasma unbound E(2)-17betaG in male rats was <1% of the glomerular filtration rate, indicating its potential reabsorption by the kidney, and this increased to a level comparable with that found in female rats when dibromosulfophthalein was coinfused. A marked increase in E(2)-17betaG urinary excretion was also observed in male rats that had undergone orchidectomy. Testosterone injections given to female rats reduced the urinary excretion to a level comparable with that of control male rats. The concomitant change in the expression of the gene product for organic anion-transporting polypeptide Oatp1, of which E(2)-17betaG is a typical substrate, was found in the kidney membrane fractions after these treatments. These results suggest that urinary E(2)-17betaG excretion is subject to hormonal regulation and that the large gender difference can be explained by regulation in Oatp1-mediated reabsorption.     

Contribution of IL-18 to Th1 response and host defense against infection by Mycobacterium tuberculosis: a comparative study with IL-12p40

The present study was conducted to critically determine the protective role of IL-18 in host response to Mycobacterium tuberculosis infection. IL-18-deficient (knockout (KO)) mice were slightly more prone to this infection than wild-type (WT) mice. Sensitivity of IL-12p40KO mice was lower than that of IL-12p40/IL-18 double KO mice. IFN-gamma production caused by the infection was significantly attenuated in IL-18KO mice compared with WT mice, as indicated by reduction in the levels of this cytokine in sera, spleen, lung, and liver, and its synthesis by spleen cells restimulated with purified protein derivatives. Serum IL-12p40 level postinfection and its production by peritoneal exudate cells stimulated with live bacilli were also significantly lower in IL-18KO mice than WT mice, suggesting that attenuated production of IFN-gamma was secondary to reduction of IL-12 synthesis. However, this was not likely the case, because administration of excess IL-12 did not restore the reduced IFN-gamma production in IL-18KO mice. In further studies, IL-18 transgenic mice were more resistant to the infection than control littermate mice, and serum IFN-gamma level and its production by restimulated spleen cells were increased in the former mice. Taken together, our results indicate that IL-18 plays an important role in Th1 response and host defense against M. tuberculosis infection although the contribution was not as profound as that of IL-12p40.     

Molecular cloning and characterization of CALP/KChIP4, a novel EF-hand protein interacting with presenilin 2 and voltage-gated potassium channel subunit Kv4

Presenilin (PS) genes linked to early-onset familial Alzheimer's disease encode polytopic membrane proteins that are presumed to constitute the catalytic subunit of gamma-secretase, forming a high molecular weight complex with other proteins. During our attempts to identify binding partners of PS2, we cloned CALP (calsenilin-like protein)/KChIP4, a novel member of calsenilin/KChIP protein family that interacts with the C-terminal region of PS. Upon co-expression in cultured cells, CALP was directly bound to and co-localized with PS2 in endoplasmic reticulum. Overexpression of CALP did not affect the metabolism or stability of PS complex, and gamma-cleavage of betaAPP or Notch site 3 cleavage was not altered. However, co-expression of CALP and a voltage-gated potassium channel subunit Kv4.2 reconstituted the features of A-type K(+) currents and CALP directly bound Kv4.2, indicating that CALP functions as KChIPs that are known as components of native Kv4 channel complex. Taken together, CALP/KChIP4 is a novel EF-hand protein interacting with PS as well as with Kv4 that may modulate functions of a subset of membrane proteins in brain.     

Moritella japonica sp. nov., a novel barophilic bacterium isolated from a Japan Trench sediment

Strain DSK1 is a novel moderately barophilic bacterium isolated from the Japan Trench at a depth of 6,356 m. Phylogenetic analysis based on 16S ribosomal DNA sequences showed that strain DSK1 represents a separate lineage with the Shewanella barophiles branch and is closely related to Moritella marina. Comparisons of the temperature and pressure range for growth and some biochemical characteristics indicate that strain DSK1 differs from M. marina and Shewanella barophilic species. Furthermore, strain DSK1 displays a low level of DNA similarity to the Moritella and Shewanella type strains; however this isolate characteristically produces DHA (22:6) as a membrane fatty acids, and the fatty acid profile of this strain is similar to that of M. marina. Because of these differences, strain DSK1 appears to represent a novel deep-sea Moritella species. The name Moritella japonica is proposed. The type strain is JCM 10249.     

Graviresponse and its regulation from the aspect of molecular levels in higher plants: growth and development, and auxin polar transport in etiolated pea seedlings under microgravity.

In STS-95 space experiments we have demonstrated that microgravity conditions resulted in automorphosis in etiolated pea (Pisum sativum L. cv. Alaska) seedlings (Ueda et al. 1999). Automorphosis-like growth and development in etiolated pea seedlings were also induced under simulated microgravity conditions on a 3-dimensional (3-D) clinostat, epicotyls being the most oriented toward the direction far from the cotyledons. Detail analysis of epicotyl bending revealed that within 36 h after watering, no significant difference in growth direction of epicotyls was observed in between seedlings grown on the 3-D clinostat and under 1 g conditions, differential growth near the cotyledonary node resulting in epicotyl bending of ca. 45 degrees toward the direction far from the cotyledons. Thereafter epicotyls continued to grow almost straightly keeping this orientation on the 3-D clinostat. On the other hand, the growth direction in etiolated seedlings changed to antigravity direction by negative gravitropic response under 1 g conditions. Automorphological epicotyl bending was also phenocopied by the application of auxin polar transport inhibitors such as 9-hydroxyfluorene-9-carboxylic acid, N-(1-naphtyl)phthalamic acid and 2,3,5-triiodobenzoic acid. These results together with the fact that auxin polar transport activity in etiolated pea epicotyls was substantially reduced in space suggested that reduced auxin polar transport is closely related to automorphosis. Strenuous efforts to learn how gravity contributes to the auxin polar transport in etiolated pea epicotyls in molecular bases resulted in successful identification of PsPIN2 and PsAUX1 encoding putative auxin-efflux and influx carrier proteins, respectively. Based on the results of these gene expression under simulated microgravity conditions, a possible role of PsPIN2 and PsAUX1 genes for auxin polar transport in etiolated pea seedlings will be discussed.     

Larval growth and development in the caddisfly Cheumatopsyche brevilineata under natural thermal regimes

Cheumatopsyche brevilineata (Iwata) is a filter‐feeding caddisfly without distinct or distinguishable cohorts. In a semi‐natural channel, we reared fourth and fifth instar larvae of C. brevilineata in individual cages with hourly recording of water temperature. We calculated the individual growth rate from the wet‐weight gain of each larva, and the development rate from the ratio of larvae that progressed to the next instar or pupal stage during each rearing experiment. We analyzed the linear regressions of growth (increase in size) and development (physiological and morphological progression toward maturity) rates against the statistical parameters of water temperatures during each rearing period, i.e. mean and given percentiles of water temperatures. We presumed that the most appropriate parameter of water temperature to explain larval growth and development would show a peak value of the determination coefficients (r²) in the linear regressions. There were highly significant regressions in the growth rates for fourth and fifth instar larvae and in development rates for fourth instar larvae against every statistical parameter of water temperature, but not in the development rates for fifth instar larvae. For the growth of fourth and fifth instar larvae, we could not specify the most appropriate parameters of water temperatures, because we observed no clear peaks in the determination coefficients. For the development of fourth instar larvae, this parameter could be the 65th percentile value, where the development zero temperature and effective degree‐days were 11.1°C and 56 degree‐days, respectively.     

Ozone exposure generates free radicals in the blood samples in vitro. Detection by the ESR spin-trapping technique

Generation of free radicals in the reaction of ozone with blood samples and related salt solutions was investigated in vitro by using ESR spin-trapping technique with DMPO. In the reactions of low levels of ozone, a carbon-centered radical was spin-trapped with DMPO, giving rise to the 6-line ESR signal in both whole blood and blood plasma. In the blood plasma, DMPO-spin adduct of hydroxyl radical (DMPO-OH) was detected together with the spin adduct of carbon-centered radical. The present spin-trapping study demonstrates that, when exposed to ozone, 0.9% NaCl solution in the presence of DMPO gives rise to the formation of DMPO-OH. The addition effects of ethanol, which is a ^·OH scavenger, into the NaCl solution reveal that DMPO-OH is produced by the reaction of DMPO with both ^·OH and unidentified oxidants originated from the reaction of Cl^- and ozone. Based on these observations, we consider that ^·OH is generated similarly in the blood plasma exposed to ozone. The ESR study of DMPO-spin adducts in the ozone-exposed aqueous solution of NaOCl indicates that Cl^- reacts with ozone to give ClO^-. Presumably, further oxidation of ClO^- by ozone leads to the formation of ^·OH and the unidentified oxidants.     

Differentiation ofMelampsora rust species on willows in Japan using PCR-RFLP analysis of ITS regions of ribosomal DNA

To develop a reliable method for identifyingMelampsora species parasitic on willows in Japan, we differentiated 10Melampsora species by PCR-RFLP analysis. Internal transcribed, spacer (ITS) regions, including 5.8S ribosomal DNA, of 63 collections of 10Melampsora species and 4 collections of unidentified species were amplified by PCR. The fragments from the 67 collections varied in size (approximately 880 bp, 860 bp and 840 bp). The restriction sites in the amplified DNA fragments were mapped after the RFLP analysis using four restriction enzymes,Dra I,EcoRI,SspI andTaqI. All the collections were divided into 11 RFLP types. In the 6 species,M. caprearum, M. epiphylla, M. kamikotica, M. larici-urbaniana, M. microsora andM. yezoensis, the RFLP type was species-specific. The RFLP type ofM. chelidonii-pierotii andM. coleosporioides was identical. The collections ofM. epitea were separated into three RFLP types. One of these three types was identical with the type ofM. humilis. It is suggested that the PCR-RFLP analysis of ITS regions is a useful and reliable method for species identification ofMelampsora. Contribution No. 131, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba.