Cloning and functional studies of splice variants of the alpha -subunit of the amiloride-sensitive Na+ channel

The -subunit of the amiloride-sensitive epithelialNa⁺ channel (ENaC) is criticalin forming an ion conductive pore in the membrane. We have identifiedthe wild-type and three splice variants of the human ENaC (hENaC)from the human lung cell line H441, using RT-PCR. These splice variantscontain various structures in the extracellular domain, resultingin premature truncation (hENaCx), 19-amino acid deletion(hENaC19), and 22-amino acid insertion (hENaC+22).Wild-type hENaC and splice variants were functionally characterizedin Xenopus oocytes by coexpression with hENaC - and -subunits. Unlike wild-type hENaC,undetectable or substantially reduced amiloride-sensitive currents wereobserved in oocytes expressing these splice variants. Wild-typehENaC was the most abundantly expressed hENaC mRNA species in alltissues in which its expression was detected. These findings indicate that the extracellular domain is important to generate structural andfunctional diversity of hENaC and that alternative splicing may playa role in regulating hENaC activity.

     

Primary structure and properties of Mn-superoxide dismutase from scallop adductor muscle

Manganese-superoxide dismutase was purified to homogeneity from scallop adductor muscle using DEAE-Sephacel, Buthyl-Cellulofine and Superdex 200 pg column chromatographies. The molecular weights of the purified enzyme were calculated to be 22,321.4 according to time-of-flight mass spectrometry, and to be approximately 95,000 and 93,000 on Superdex 200 pg column chromatography and non-denatured PAGE, respectively, and were calculated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 24,000 and 25,000 in the absence and 25,000 in the presence of beta-mercaptoethanol. These findings suggested that the native enzyme is composed of four identical subunits. Other properties of scallop adductor muscle manganese-superoxide dismutase, including pH stability and heat stability, were also determined. We determined the partial amino acid sequences of purified manganese-superoxide dismutase using digestions by bromocyan and lysyl endopeptidase and also determined the manganese-superoxide dismutase cDNA structure. The amino acid sequence of the enzyme obtained using both methods showed homology to those of vertebrates such as human, bovine, chicken, Xenopus and zebrafish manganese-superoxide dismutases (64.91, 65.35, 64.47, 63.27 and 64.60%, respectively). We also predicted the 3D structure of scallop adductor muscle manganese-superoxide dismutase using molecular operating environment and compared its structure with those of other manganese-superoxide dismutases. The overall structure of scallop adductor muscle manganese-superoxide dismutase was very similar to those of other species, including human and Aspergillus.     

Thrombopoietin and interleukin-2 induce association of CRK with STAT5

Crk (Crk I and II) proteins and closely related CrkL are adapters which are commonly involved in various signaling processes in various cells, and these proteins share many ligands. Whether they have redundant or distinct physiologic roles is unclear. By coprecipitation and far Western blotting analysis, we demonstrate that Crk (I/II) binds to tyrosine phosphorylated STAT5 in cells stimulated by cytokines such as thrombopoietin (TPO) and interleukin-2 (IL-2). The association did not require nuclear elements and can be observed in primary cells as this was also demonstrated in TPO-stimulated platelets. Using a beta-casein promoter STAT5 binding site as a probe, we have also demonstrated that CrkL (a close relative of Crk) antiserum, but not Crk antiserum, supershifted the STAT5-DNA complex by an electrophoretic mobility shift assay, suggesting that CrkL, but not Crk, is the major component of the complex. Thus, Crk and CrkL may have distinct roles in the regulation of STAT5.     

Phosphoglucomutase-1 subtypes: polymorphic occurrence of PGM1*7+ and geographical variation in Japan

The distribution of gene frequencies in the phosphoglucomutase-1 (PGM1) system was investigated in two Japanese populations from Yamaguchi (Western Japan) and Okinawa (Southern Japan) using an improved isoelectric focusing method permitting the successful detection of the most anodal variant PGM1 3+. PGM1*7+ occurred with a polymorphic frequency of 0.012-0.021. A difference in the gene frequency was observed between the two populations. In comparison with neighboring populations, the Yamaguchi population was similar to Mongolians and Koreans in North China, and Okinawa to Zhuang in South China.     

Multiple, small exposures of far-ultraviolet or mid-ultraviolet light change the sensitivity to acute ultraviolet exposures measured by cell lethality and mutagenesis in V79 Chinese hamster cells

Chinese hamster V79 cells (subline MI2G) were exposed repeatedly to fractionated doses of germicidal 254 nm light (far-uv) at 6 J.m-2/fraction/day or sunlight-simulating 290-330 nm (mid-uv) at 150 J.m-2/fraction/day and sensitivities to cell killing action and mutation of far-uv and mid-uv were examined. As the number of exposure fractions increased, the cell cultures became resistant to cell killing induced by both far-uv and mid-uv. Increases in both Do and Dq were observed. Treatment with exposures of 6 J.m-2 far-uv is more efficient in yielding cell cultures that are resistant than exposures of 150 J.m-2 mid-uv. In contrast to the cells exposed to repeated far-uv, the cells exposed to repeated mid-uv were relatively more resistant to cell killing effects of mid-uv than far-uv, suggesting a possible role of photolesions other than pyrimidine dimers. When mutants resistant to 6-thioguanine were assayed during repeated exposure to far- or mid-uv light, the yield was initially linear with accumulating dose. At high total accumulated doses, the frequency decreased gradually (6 J.m-2 mid-uv) or reached a plateau (150 J.m-2 mid-uv). The sensitivity of N80 cells (exposed to 80 fractions of mid-uv) to mutation induction by uv light is higher than that of the original MI2G cells, whereas U81 cells (exposed to 81 fractions of far-uv) have a sensitivity similar to that of the original cells. Although an initial decrease in resistance to cell killing was observed, resistant cells retained their characteristics after 100 days in culture without further exposure. Cross-resistance to X rays was not shown. The data in this paper suggest that the capacity for repair of photolesions in DNA by repair processes was enhanced in cell cultures by repeated exposure to far-uv or mid-uv and that this altered the cells' ability to cope with lethal and mutagenic lesions. It remains to be seen if these changes in cell sensitivity were brought about by selective or inductive processes or a combination of both.     

Landform effects on fire behavior and post-fire regeneration in the mixed forests of northern Japan

This study was conducted to clarify the characteristics of landform effects on fire behavior and post-fire regeneration and to examine regional differences in the effects of fire within a mixed-forest area with various topographic conditions in Hokkaido, northern Japan. Fire spread was controlled by topographic barriers such as ridges and valleys, although this was not clear in the areas with serpentinite. Fire frequency was higher on windward slopes of the prevailing wind, and the size of the burned area varied with the underlying geology, which controlled the areal extent of hillside slopes. In areas with terrace deposits, Wakkanai Formation or serpentinized rock, burned areas were large and included few unburned stands. The type of vegetation that grew following the fires varied with slope aspect and topographic position.Sasa grasslands often formed at wind-exposed sites. The grasslands were well developed in the three areas with the previously mentioned geology, where large-scale burning had occurred. Structure of the re-established forest stands was also affected by landforms. Canopy height and the maximum diameter at breast height varied with slope aspect, topographic position and elevation, as well as with stand age. Tree size was found to decrease at wind-exposed sites within high-elevation zones.     

A single cell observation of staurosporine effect on the Ca signals in rat basophilic leukemia cells

A digital imaging fluorescence microscope was used to study the effect of a protein kinase inhibitor staurosporine on the antigen-dependent calcium signals in an individual rat basophilic leukemia cell (RBL-2H3). Although dose dependency of staurosporine was different from one cell to another, staurosporine inhibited, at low concentration, the calcium influx from the external medium into RBL-2H3 cells. At high concentration, however, it inhibited both the removal of calcium ion from internal stores and the calcium influx from the external medium. These results indicated that staurosporine is necessary for the inhibition of the calcium influx from the external medium and that a protein kinase (possibly protein kinase C) is involved in the calcium influx from the external medium into the cytoplasm.     

Antigenic determinants on rat metallothionein: fine epitope mapping for a murine monoclonal antibody and rabbit polyclonal antisera.

In order to determine the epitope of metallothionein (MT) to a murine monoclonal antibody (MT 189-14-7) which had been produced by immunization with rat MT 2 (Kikuchi et al. (1988) Mol. Immunol. 25, 1033-1036), various lengths of synthetic oligopeptides were tested for their inhibitory activities in competitive radioimmunoassay (RIA). The amino-terminal acetylated pentapeptide, AcMDPNC, exhibited an inhibitory activity comparable to that of native MTs, whereas the acetylated tetrapeptide, AcMDPN, and the deacetylated heptapeptide, MDPNCSC, were much less inhibitory. The results suggest that the major part of the epitope structure of MT to the MT 189-14-7 monoclonal antibody is located within the amino-terminal acetylated pentapeptide, AcMDPNC. The specificities of polyclonal rabbit anti-MT antisera raised against the same immunogen were also determined by using various animal MTs and synthetic peptides as inhibitors in the RIA. Among three antisera tested, two reacted with several amino-terminal oligopeptides similarly to the MT 189-14-7 antibody. The major epitope structures to these polyclonal antibodies were shown to be located within the acetylated tetrapeptide, AcMDPN. Another antiserum contained at least two different populations of antibodies: one consisted of antibodies reactive with the amino-terminal synthetic peptides, while the other was not reactive with them. These results suggest that, in the rabbit also, the amino-terminal region common to various animal MTs can be an epitope to antibodies raised against rat MT, as shown in the mouse. Moreover, the results indicate that the synthetic amino-terminal peptides are useful for determination of the specificity of polyclonal rabbit anti-MT antibodies, which have been widely used for the quantification of MTs.     

Production and characterization of monoclonal antibodies to nivalenol tetraacetate and their application to enzyme-linked immunoassay of nivalenol

Three monoclonal antibodies were obtained by the fusion of mouse myeloma cells with splenocytes isolated from BALB/c mice that had been immunized with 8-hydroxy-3,4,7,15-tetraacetyl-nivalenol hemiglutarate covalently bound to bovine serum albumin. These anti-nivalenol tetraacetate monoclonal antibodies were of the IgG type and highly specific to nivalenol tetraacetate, with an apparent association constant of about 10(8)M-1. The relative cross-reactivities of one monoclonal antibody with nivalenol tetraacetate, acetyl T-2 toxin, and scirpenol triacetate were found to be 1.0, 0.02 and 0.03, respectively. Other derivatives showed no cross-reactivity at all. An indirect enzyme-linked immunosorbent assay (ELISA) based on the competitive binding principle was developed using the antibody from clone D18.102.59. The sensitivity of the system was about 0.1 ng of nivalenol tetraacetate per assay. Comparison of nivalenol levels detected in naturally contaminated barley samples by competitive indirect ELISA and gas chromatography (GC) showed good agreement, indicating that the antibody is useful for the measurement of nivalenol in naturally contaminated cereals and grains.