A monoclonal antibody against horse kidney (Na+ + K+)-ATPase inhibits sodium pump and E2K to E1 conversion of (Na+ + K+)-ATPase from outside of the cell membrane

Monoclonal antibodies against horse kidney outer medulla (Na+ + K+)-ATPase were prepared. One of these antibodies (M45-80), was identified as an IgM, recognized the alpha subunit of the enzyme. M45-80 had the following effects on horse kidney (Na+ + K+)-ATPase: (1) it inhibited the enzyme activity by 50% in 140 mM Na+ and by 80% in 8.3 mM Na+; (2) it increased the Na+ concentration necessary for half-maximal activation (K0.5 for Na+) from 12.0 to 57.6 mM, but did not affect K0.5 for K+; (3) it slightly increased the K+-dependent p-nitrophenylphosphatase (K-pNPPase) activity; (4) it inhibited phosphorylation of the enzyme with ATP by 30%, but did not affect the step of dephosphorylation; and (5) it enhanced the ouabain binding rate. These data are compatible with a stabilizing effect on the E2 form of (Na+ + K+)-ATPase. M45-80 was concluded to bind to the extracellular surface of the plasmamembrane, based on the following evidence: (1) M45-80 inhibited by 50% the ouabain-sensitive 86Rb+ uptake in human intact erythrocytes from outside of the cells; (2) the inhibition of (Na+ + K+)-ATPase activity in right-side-out vesicles of human erythrocytes was greater than that in inside-out vesicles; and (3) the fluorescence intensity due to FITC-labeled rabbit anti-mouse IgM that reacted with M45-80 bound to the right-side-out vesicles was much greater than that in the case of the inside-out vesicles.     

Isolation and characterization of dominant dwarf mutants, <Emphasis Type="Italic">Slr1-d</Emphasis>, in rice

sd1 is known as the ‘green revolution’ gene in rice because its application in rice breeding has dramatically increased rice yield. Since the ‘green revolution,’ sd1 has been extensively used to produce modern semi-dwarf varieties. The extensive use of limited dwarfing sources may, however, cause a bottleneck effect in the genetic background of rice varieties. To circumvent this problem, novel and useful sources of dwarf genes must be identified. In this study, we identified three semi-dominant dwarf mutants. These mutants were categorized as dn-type dwarf mutants according to the elongation pattern of internodes. Gibberellin (GA) response tests showed that the mutants were still responsive to GA, although at a reduced rate. Map-based cloning revealed that the dwarf phenotype in these mutants was caused by gain-of-function mutations in the N-terminal region of SLR1. Degradation of the SLR1 protein in these mutants occurred later than in the wild type. Reduced interaction abilities of the SLR1 protein in these mutants with GID1 were also observed using the yeast two-hybrid system. Crossing experiments indicated that with the use of an appropriate genetic background, the semi-dominant dwarf alleles identified in this study could be used to alleviate the deficiency of dwarfing genes for breeding applications.     

Effect of carbamylcholine on Harderian gland morphology in rats

Summary To determine the effect of cholinergic secretagogue on the Harderian gland of rats, several light- and electron-microscopic parameters were morphometrically assessed at different time intervals after carbamylcholine injection. In controls, two types of glandular cells (type A cells having 40–55 large vacuoles per cell profile and type B cells containing 30–38 smaller vacuoles per cell profile) and myoepithelial cells were recognized. At 5 min after injection of carbamylcholine, when rats secreted bloody tears, many alveoli showing narrower lumina and exocytotic figures in both types of cells were observed. Some vacuoles, which were covered by thin cytoplasmic sheets, protruded into the alveolar lumina. However, there was no evidence of apocrine or holocrine secretion. At 30 min and 120 min after injection, most of the alveolar lumina were dilated, and a pronounced decrease in the number of vacuoles in the glandular cells was observed. At 300 min after injection, the secretory vacuoles in both cell types reaccumulated. Transitional forms between the two cell types were not observed. The two types of Harderian gland cells can therefore be considered independent populations rather than different secretory stages of the same cell. It appears that the secretory process of the Harderian gland of rat is affected by cholinergic stimulation of the two types of glandular cells and of myoepithelial cells.     

Dendritic cell-derived TNF-α is responsible for development of IL-10-producing CD4 T cells

Immature dendritic cells (DCs) appear to be involved in peripheral immune tolerance via induction of IL-10-producing CD4⁺ T cells. We examined the role of TNF-α in generation of the IL-10-producing CD4⁺ T cells by immature DCs. Immature bone marrow-derived DCs from wild type (WT) or TNF-α^−/− mice were cocultured with CD4⁺ T cells from OVA specific TCR transgenic mice (OT-II) in the presence of OVA_323-339 peptide. The WT DCs efficiently induced the antigen-specific IL-10-producing CD4⁺ T cells, while the ability of the TNF-α^−/− DCs to induce these CD4⁺ T cells was considerably depressed. Addition of exogenous TNF-α recovered the impaired ability of the TNF-α^−/− DCs to induce IL-10-producing T cells. However, no difference in this ability was observed between TNF-α^−/− and WT DCs after their maturation by LPS. Thus, TNF-α appears to be critical for the generation of IL-10-producing CD4⁺ T cells during the antigen presentation by immature DCs.     

The inhibitory effects of a RANKL-binding peptide on articular and periarticular bone loss in a murine model of collagen-induced arthritis: a bone histomorphometric study

IntroductionWe designed OP3-4 (YCEIEFCYLIR), a cyclic peptide, to mimic the soluble osteoprotegerin (OPG), and was proven to bind to RANKL (receptor activator of NF-κB ligand), thereby inhibiting osteoclastogenesis. We recently found that another RANKL binding peptide, W9, could accelerate bone formation by affecting RANKL signaling in osteoblasts. We herein demonstrate the effects of OP3-4 on bone formation and bone loss in a murine model of rheumatoid arthritis.MethodsTwenty-four seven-week-old male DBA/1J mice were used to generate a murine model of collagen-induced arthritis (CIA). Then, vehicle or OP3-4 (9 mg/kg/day or 18 mg/kg/day) was subcutaneously infused using infusion pumps for three weeks beginning seven days after the second immunization. The arthritis score was assessed, and the mice were sacrificed on day 49. Thereafter, radiographic, histological and biochemical analyses were performed.ResultsThe OP3-4 treatment did not significantly inhibit the CIA-induced arthritis, but limited bone loss. Micro-CT images and quantitative measurements of the bone mineral density revealed that 18 mg/kg/day OP3-4 prevented the CIA-induced bone loss at both articular and periarticular sites of tibiae. As expected, OP3-4 significantly reduced the CIA-induced serum CTX levels, a marker of bone resorption. Interestingly, the bone histomorphometric analyses using undecalcified sections showed that OP3-4 prevented the CIA-induced reduction of bone formation-related parameters at the periarticular sites.ConclusionThe peptide that mimicked OPG prevented inflammatory bone loss by inhibiting bone resorption and stimulating bone formation. It could therefore be a useful template for the development of small molecule drugs for inflammatory bone loss.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0753-8) contains supplementary material, which is available to authorized users.     

Cloning and mapping of cat (Felis catus) immunoglobulin and T-cell receptor genes

 Molecular cloning and chromosomal mapping of the cat immunoglobulin (Ig) and T-cell receptor (TcR) genes were carried out to provide basic information for genetic analysis of immunologic diseases including leukemias and lymphomas in cats. We cloned two Ig constant genes, IGHM and IGHG and three TcR constant genes, TRAC, TRGC, and TRDC, by polymerase chain reaction (PCR) amplification of cDNA from cat peripheral blood mononuclear cells. For chromosomal mapping of the Ig and TcR loci including the IGK, IGL, and TRB on the cat genome, we performed PCR screening of DNAs from 37 cat × rodent somatic cell hybrids by using specific primers for the given genes. Consequently, three loci for IGH, TRA, and TRD, and two loci for TRB and TRG were found to be syntenic and assigned to cat chromosomes (FCA) B3 and A2, respectively. Further, IGK and IGL loci were mapped on FCA A3 and D3, respectively. These findings support the notion that the genetic linkages between the Ig and TcR genes are extensively conserved between humans and cats. Received: 18 June 1997 / Revised: 12 August 1997     

Pioglitazone ameliorates endothelial dysfunction in obese rats with nephropathy

Endothelial dysfunction is a key event in the development of renovascular complications in the metabolic syndrome. The aim of this study was to elucidate the pathogenetic mechanisms involved in renovascular injuries in the Zucker obese rat, a model of the metabolic syndrome, and to examine the therapeutic effects of pioglitazone, a thiazolidinedione. Obese rats fed high-protein diet (OHP) for 12 weeks exhibited nephropathy and endothelial dysfunction, which were improved by pioglitazone. Accumulation of nitrotyrosine, a tracer of nitrative stress, was increased in aorta of the OHP group. The mRNA expressions of NADPH oxidase components and inducible nitric oxide synthase in the aorta were enhanced in the OHP group. Pioglitazone reduced nitrotyrosine in the aorta of the OHP group, inhibiting the augmented expression levels of both. These results suggest that nitrative stress could cause endothelial dysfunction in the rat model of metabolic syndrome with nephropathy, and that pioglitazone ameliorates these injuries, presumably by reducing nitrative stress.     

Effect of selenium on determination of mercury in animal tissues.

Selenium in animal tissues was found to influence the reactivity of mercury in the tissues with stannous chloride or with stannous chloride plus cadmium chloride added as reducing agents for the determination of mercury by the method developed by L. Magos (1971, Analyst, 96, 847–853) and L. Magos and T. W. Clarkson (1972, J. Assoc. Offic. Anal. Chem., 55, 966–971). The recovery of mercury in the tissues of animals to which inorganic mercury and selenite were simultaneously administered was low compared to the case in which inorganic mercury alone was administered. Of the in vitro interactions of inorganic mercury and selenite examined in tissue homogenates and blood samples, only those interactions in blood samples caused the difficulty in mercury analysis mentioned above, i.e., there was a marked decrease in recovery of mercury when an equimolar amount of each compound was added to the blood. These facts suggest that selenium and inorganic mercury in the animal tissues are likely to interact with each other and might form a chemically stable state of inorganic mercury which resists reduction with stannous chloride in the procedure for mercury determination.