Determinants of Slow Walking Speed in Ambulatory Patients Undergoing Maintenance Hemodialysis

Walking ability is significantly lower in hemodialysis patients compared to healthy people. Decreased walking ability characterized by slow walking speed is associated with adverse clinical events, but determinants of decreased walking speed in hemodialysis patients are unknown. The purpose of this study was to identify factors associated with slow walking speed in ambulatory hemodialysis patients. Subjects were 122 outpatients (64 men, 58 women; mean age, 68 years) undergoing hemodialysis. Clinical characteristics including comorbidities, motor function (strength, flexibility, and balance), and maximum walking speed (MWS) were measured and compared across sex-specific tertiles of MWS. Univariate and multivariate logistic regression analyses were performed to examine whether clinical characteristics and motor function could discriminate between the lowest, middle, and highest tertiles of MWS. Significant and common factors that discriminated the lowest and highest tertiles of MWS from other categories were presence of cardiac disease (lowest: odds ratio [OR] = 3.33, 95% confidence interval [CI] = 1.26–8.83, P<0.05; highest: OR = 2.84, 95% CI = 1.18–6.84, P<0.05), leg strength (OR = 0.62, 95% CI = 0.40–0.95, P<0.05; OR = 0.57, 95% CI = 0.39–0.82, P<0.01), and standing balance (OR = 0.76, 95% CI = 0.63–0.92, P<0.01; OR = 0.81, 95% CI = 0.68–0.97, P<0.05). History of fracture (OR = 3.35, 95% CI = 1.08–10.38; P<0.05) was a significant factor only in the lowest tertile. Cardiac disease, history of fracture, decreased leg strength, and poor standing balance were independently associated with slow walking speed in ambulatory hemodialysis patients. These findings provide useful data for planning effective therapeutic regimens to prevent decreases in walking ability in ambulatory hemodialysis patients.     

MiR-376c Down-Regulation Accelerates EGF-Dependent Migration by Targeting GRB2 in the HuCCT1 Human Intrahepatic Cholangiocarcinoma Cell Line

MicroRNA miR-376c was expressed in normal intrahepatic biliary epithelial cells (HIBEpiC), but was significantly suppressed in the HuCCT1 intrahepatic cholangiocarcinoma (ICC) cell line. The biological significance of the down-regulation of miR-376c in HuCCT1 cells is unknown. We hypothesized that miR-376c could function as a tumor suppressor in these cells. To test this hypothesis, we sought the targets of miR-376c, and characterized the effect of its down-regulation on HuCCT1 cells. We performed proteomic analysis of miR-376c-overexpressing HuCCT1 cells to identify candidate targets of miR-376c, and validated these targets by 3′-UTR reporter assay. Transwell migration assays were performed to study the migratory response of HuCCT1 cells to miR-376c overexpression. Furthermore, microarrays were used to identify the signaling that were potentially involved in the miR-376c-modulated migration of HuCCT1. Finally, we assessed epigenetic changes within the potential promoter region of the miR-376c gene in these cells. Proteomic analysis and subsequent validation assays showed that growth factor receptor-bound protein 2 (GRB2) was a direct target of miR-376c. The transwell migration assay revealed that miR-376c significantly reduced epidermal growth factor (EGF)-dependent cell migration in HuCCT1 cells. DNA microarray and subsequent pathway analysis showed that interleukin 1 beta and matrix metallopeptidase 9 were possible participants in EGF-dependent migration of HuCCT1 cells. Bisulfite sequencing showed higher methylation levels of CpG sites upstream of the miR-376c gene in HuCCT1 relative to HIBEpiC cells. Combined treatment with the DNA-demethylating agent 5-aza-2′-deoxycytidine and the histone deacetylase inhibitor trichostatin A significantly upregulated the expression of miR-376c in HuCCT1 cells. We revealed that epigenetic repression of miR-376c accelerated EGF-dependent cell migration through its target GRB2 in HuCCT1 cells. These findings suggest that miR-376c functions as a tumor suppressor. Since metastasis is the major cause of death in ICC, microRNA manipulation could lead to the development of novel anti-cancer therapy strategies for ICC.     

Requirement of protein co-factor for the DNA-binding function of the human ski proto-oncogene product.

We identified the human c-ski gene product (c-Ski) as a protein with the apparent molecular weight of 100,000, p100c-ski, by using a c-Ski-specific polyclonal antibody. p100c-ski was a nuclear protein and p100c-ski in nuclear extracts of Molt4 cells bound to calf thymus DNA cellulose, but the bacterially synthesized c-Ski did not, suggesting that Ski was associated with another protein(s) and that the Ski complex had DNA-binding activity. This hypothesis was supported by the finding that the bacterially synthesized Ski bounds to DNA cellulose after being mixed with a nuclear extract of Molt4 cells. By use of a series of deletion mutants of Ski synthesized in an in vitro translation system, two portions in Ski were found to be necessary for the DNA binding of the Ski complex: the N-proximal portion containing a cystein/histidine-rich domain and the C-terminal portion including a region rich in basic amino acids.     

Phorbol myristate acetate inhibits increases in membrane fluidity induced by anti-IgM in B cells

Anti-IgM or anti-IgD stimulates B cells to induce increases in inositol phospholipid metabolism and intracellular free calcium concentration [( Ca2+]i). Anti-IgM also causes increases in membrane fluidity that occur more promptly than those in [Ca2+]i in resting B cells as well as BAL17 B lymphoma cells. However, other B cell activators such as LPS or PMA did not induce the membrane fluidity changes. Furthermore, sodium fluoride, which is considered to be an activator of the guanine nucleotide-binding protein, caused increases in membrane fluidity as well as increased [Ca2+]i or inositol phospholipid metabolism. Anti-IgM- or sodium fluoride-induced increases in membrane fluidity were inhibited by 20-min pretreatment of cells with PMA, but not by 24-h pretreatment. These results indicate that membrane fluidity changes are closely associated with increased [Ca2+]i after cross-linkage of membrane Ig and are regulated by protein kinase C in B cells.     

Association of Lps gene with natural resistance of mouse macrophages against Legionella pneumophila

We have cloned a 1.6-kb region of chromosomal DNA from Thermoplasma acidophilum into Escherichia coli using as a probe part of the Methanococcus vannielii fus-gene. The sequence of the clone was highly homologous to part of the corresponding Methanococcus vannielii gene. By chromosome walking, a 4.7-kb EcoRI fragment containing the complete gene was isolated. Nucleotide sequencing revealed an open reading frame of 2196 nucleotides. The deduced amino acid sequence contains the known peptide sequence around the ADP-ribosylation site of T. acidophilum elongation factor 2, which unequivocally confirms that the fus-gene has been cloned. The amino acid sequence was compared to that of hamster and E. coli, as well as to known archaebacterial EF-2 sequences.     

Multiplication of Legionella pneumophila Philadelphia-1 in cultured peritoneal macrophages and its correlation to susceptibility of animals

Intracellular growth of Legionella pneumophila Philadelphia-1 strain in peritoneal macrophages (PMP) from various rodents was measured and its correlation to the level of susceptibility of the animal was examined. In guinea pig PMP, the organism grew well and the guinea pig was very susceptible to it (50% lethal dose, LD50 = 7.6 X 10(4)). On the other hand, the bacteria hardly multiplied in mouse PMP and the animal was resistant to infection (LD50 = 6.7 X 10(7)). Intracellular growth rate correlated well with susceptibility in these animals. In golden hamsters, a discrepancy between intracellular growth and susceptibility was found. The organism grew intracellularly as rapid as in guinea pig PMP, but the golden hamster was very resistant to infection (LD50 = 2.2 X 10(8)). In rat PMP, the organism did not grow intracellularly during a 24-h period of infection, but started to grow after that and the growth rate thereafter was as rapid as in guinea pig PMP. WKA rats were resistant and the LD50 in the animal was 1.9 X 10(7). In vivo natural resistance of rats and golden hamsters to the organism was considered to be a result of other factors than macrophages.     

Regulation of B-lymphocyte activation, proliferation, and immunoglobulin secretion

Lymphocyte growth and differentiation are controlled by signals resulting from the interaction of antigen and cellular products, such as lymphokines, with specific cell membrane receptors. Resting B lymphocytes can be activated by low concentrations (1-5 micrograms/ml) of antibodies to membrane IgM, which is the B-lymphocyte receptor for antigen. The binding of anti-IgM to B cells causes a rapid increase in intracellular free calcium concentration ([Ca2+]i), in inositol phosphate concentration, and in protein kinase activity. Moreover, the effects of anti-IgM on B cells are mimicked by the combined use of calcium ionophores and phorbol esters. Since phorbol esters activate protein kinase c, this suggests that the increase in [Ca2+]i and in phosphatidylinositol metabolism stimulated by anti-IgM are critical events in B-cell activation. The entry into S phase of B cells stimulated with anti-IgM depends on the action of a T-cell-derived factor designated B-cell stimulatory factor (BSF)-1. This is a 20,000-Da protein which is a powerful inducer of class II major histocompatibility complex molecules. Although an important cofactor for B-cell proliferative responses to anti-IgM, its major locus of action is on resting B cells. B cells stimulated with anti-IgM and BSF-1 do not synthesize secretory IgM. However, if two additional T-cell-derived factors, B151-TRF and interleukin-2, are added to cultures, a substantial proportion of stimulated B cells produce secretory IgM. BSF-1 has also been shown to participate in the "switch" in Ig class expression. Resting B cells cultured with lipopolysaccharide will switch to IgG1 secretion in the presence of purified BSF-1.     

BSF-1 action on resting B cells does not require elevation of inositol phospholipid metabolism or increased [Ca2+]i

B cell stimulatory factor-1 (BSF-1) acts on resting B cells to increase expression of class II major histocompatibility complex (MHC) molecules and to prepare for more prompt entry into S phase in response to anti-IgM and lipopolysaccharide. It also acts as a costimulant, with low concentrations of anti-IgM, to cause resting B cells to synthesize DNA. Unlike anti-IgM, BSF-1 does not cause elevation in inositol phospholipid metabolism or in concentration of intracellular free calcium, nor does it enhance such biochemical responses to anti-IgM. Furthermore, increased expression of class II MHC molecules to BSF-1 is observed when essentially all extracellular calcium is chelated by EGTA, whereas lower concentrations of EGTA completely inhibit increases in class II molecules in response to anti-IgM. These results indicate that BSF-1 effects on resting B cells are not mediated by the inositol phospholipid metabolic pathway.