Human recombinant erythropoietin (rHuEPO) induces cytosolic free calcium ([Ca2+]i) mobilization, an activation of mitogen-activated protein (MAP) kinase and DNA synthesis in several tissues. We explored the mechanism of rHuEPO-induced [Ca2+]i mobilization and its role in the activation of MAP kinase and DNA synthesis in vascular smooth muscle cells (VSMC).[Ca2+]i concentrations were measured by fura-2. MAP kinase activation was analyzed using an immunocomplex kinase assay and Western blotting. DNA synthesis was measured as an incorporation of 5-bromo-2'-deoxyuridine.Although addition of rHuEPO significantly increased [Ca2+]i, either in the presence or absence of extracellular Ca2+, the peak level and sustained elevation of [Ca2+]i were significantly reduced in the absence of extracellular Ca2+. Pretreatment with genistein completely blocked the elevation of [Ca2+]i in both conditions. Calphostin C and staurosporine did not completely block the elevation of [Ca2+]i. Staurosporine reduced its peak level in a dose-dependent manner, whereas calphostin C reduced its peak level at concentrations over 1 nmol/l in the presence of extracellular Ca2+. Similar results to those with staurosporine were observed with nifedipine. In the absence of extracellular Ca2+, their dose-dependent effects disappeared even though rHuEPO increased [Ca2+]i. rHuEPO activated MAP kinase and DNA synthesis, both of which were significantly suppressed by the chelation of intracellular Ca2+.These findings suggest that rHuEPO increases [Ca2+]i by both Ca2+ influx and Ca2+ release from intracellular stores. Tyrosine phosphorylation is critical in the regulation of [Ca2+]i, but protein kinase C activation is important only in the regulation of Ca2+ influx. Dihydropyridine-sensitive L-type Ca2+ channels seem to be involved in rHuEPO-induced Ca2+ influx. In addition, increase of [Ca2+]i by rHuEPO stimulates MAP kinase activation and DNA synthesis in VSMC.
Abstract A CD8+ Ts clone 13G2 was established from lymph node cells of bovine alpha s1-casein-primed C57BL/6 mice by in vitro antigenic stimulation followed by maintenance with IL-2-containing medium. The clone suppressed the Ag-induced proliferative responses of CD4+ Th cell clones without detectable cytotoxicity for both APC and responding T cells. The clone was able to suppress the in vitro proliferative response and antibody formation of Ag-primed lymph node cells. The suppression was Ag-nonspecific and not restricted to the MHC. The clone was able to suppress the proliferation of Th clones induced by an immobilized anti-TCR antibody in which APC was absent. The clone was, however, unable to suppress the proliferation of Th clones induced by anti-CD3 or IL-2. Thus, the mechanism of suppression by 13G2 was found to be due to a direct action on Th by inhibiting a consequence of signal transduction initiated through the TCR.
The spectrum of classical and non-classical HLA genes related to the risk of multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD) in the Japanese population has not been studied in detail. We conducted a case-control analysis of classical and non-classical HLA genes. We used next-generation sequencing (NGS)-based HLA genotyping methods for mapping risk for 45 MS patients, 31 NMOSD patients, and 429 healthy controls. We evaluated the association of the HLA variants with the risk of MS and NMOSD using logistic regression analysis and Fisher's exact test. We confirmed that HLA-DRB1*15:01 showed the strongest association with MS (P = 2.1 × 10−5; odds ratio [OR] = 3.44, 95% confidence interval [95% CI] = 1.95–6.07). Stepwise conditional analysis identified HLA-DRB1*04:05, HLA-B*39:01, and HLA-B*15:01 as being associated with independent MS susceptibility (PConditional < 8.3 × 10−4). With respect to amino acid polymorphisms in HLA genes, we found that phenylalanine at HLA-DQβ1 position 9 had the strongest effect on MS susceptibility (P = 3.7 × 10−8, OR = 3.48, 95% CI = 2.23–5.43). MS risk at HLA-DQβ1 Phe9 was independent of HLA-DRB1*15:01 (PConditional = 1.5 × 10−5, OR = 2.91, 95% CI = 1.79–4.72), while HLA-DRB1*15:01 was just significant when conditioned on HLA-DQβ1 Phe9 (PConditional = 0.037). Regarding a case-control analysis for NMOSD, HLA-DQA1*05:03 had a significant association with NMOSD (P = 1.5 × 10−4, OR = 6.96, 95% CI = 2.55–19.0). We identified HLA variants associated with the risk of MS and NMOSD. Our study contributes to the understanding of the genetic architecture of MS and NMOSD in the Japanese population.
To further characterize the alpha- and beta-intercalated cells (alpha-IC, beta-IC) in the isolated and perfused connecting tubule (CNT), cortical collecting duct (CCD) and outer medullary collecting duct in the inner stripe (OMCDi) of rabbit kidneys, we studied the effects of various transport inhibitors on electrical parameters. They included inhibitors of Cl-/HCO3- exchanger (4-acetamino-4'-isothiocyanostilbene-2,2'-disulfonic acid, SITS), carbonic anhydrase (acetazolamide) and Na(+)-K(+)-ATPase (ouabain). Upon addition of 10(-3) M SITS to the bath, the basolateral membrane voltage (VB) of alpha-IC in the OMCDi and CCD was significantly hyperpolarized by 20.8 +/- 4.6 (n = 5) and 29.8 +/- 5.6 mV (n = 11), respectively. On the other hand, luminal addition of SITS had no effects on VB of alpha-IC in the OMCDi and CCD. Neither bath nor lumen SITS affected VB of beta-IC in the CCD and CNT. When 10(-4) M acetazolamide was added to the bath, VB of alpha-IC in the OMCDi and CCD was significantly hyperpolarized by 20.0 +/- 4.1 (n = 4) and 18.6 +/- 1.7 mV (n = 3), respectively. Similarly, 10(-4) M acetazolamide in the bath caused the basolateral membrane of beta-IC in the CCD and CNT to hyperpolarize significantly by 34.3 +/- 7.9 (n = 6) and 21.6 +/- 2.9 mV (n = 3), respectively. Luminal addition of acetazolamide had no effect on VB of alpha-IC in the CCD and OMCDi and beta-IC in the CCD and CNT.(ABSTRACT TRUNCATED AT 250 WORDS)
The genetic cause of cardiac conduction system disease (CCSD) has not been fully elucidated. Whole-exome sequencing (WES) can detect various genetic variants; however, the identification of pathogenic variants remains a challenge. We aimed to identify pathogenic or likely pathogenic variants in CCSD patients by using WES and 2015 American College of Medical Genetics and Genomics (ACMG) standards and guidelines as well as evaluating the usefulness of functional studies for determining them.We performed WES of 23 probands diagnosed with early-onset (<65 years) CCSD and analysed 117 genes linked to arrhythmogenic diseases or cardiomyopathies. We focused on rare variants (minor allele frequency < 0.1%) that were absent from population databases. Five probands had protein truncating variants in EMD and LMNA which were classified as 'pathogenic' by 2015 ACMG standards and guidelines. To evaluate the functional changes brought about by these variants, we generated a knock-out zebrafish with CRISPR-mediated insertions or deletions of the EMD or LMNA homologs in zebrafish. The mean heart rate and conduction velocities in the CRISPR/Cas9-injected embryos and F2 generation embryos with homozygous deletions were significantly decreased. Twenty-one variants of uncertain significance were identified in 11 probands. Cellular electrophysiological study and in vivo zebrafish cardiac assay showed that two variants in KCNH2 and SCN5A, four variants in SCN10A, and one variant in MYH6 damaged each gene, which resulted in the change of the clinical significance of them from 'Uncertain significance' to 'Likely pathogenic' in six probands.Of 23 CCSD probands, we successfully identified pathogenic or likely pathogenic variants in 11 probands (48%). Functional analyses of a cellular electrophysiological study and in vivo zebrafish cardiac assay might be useful for determining the pathogenicity of rare variants in patients with CCSD. SCN10A may be one of the major genes responsible for CCSD.
The beamlines of SPring-8 are classified by synchrotron radiation source into undulator, wiggler and bending-magnet beamlines for safety analysis. The shielding design calculations for synchrotron radiation are performed with a new shielding design code, STAC8, taking into consideration the linear-polarization effect, the slant length of the shield hutch wall and the build-up effect as functions of scattering angle and azimuthal angle.
Microorganisms growing in natural habitats are permanently confronted with a wide variety of external stresses. In fungi, including Aspergillus nidulans, one of the general and ultimate strategies to survive in harsh habitats is the development of stress-tolerant conidia. In this study, we found that one of the basic-region leucine zipper transcription factors, named AtfA, plays a crucial role in the acquisition of tolerance against oxidative and heat stress on conidia, but not on mycelia, although this factor is not involved in asexual development per se.
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