Identification of a 521-kilodalton protein (Gli521) involved in force generation or force transmission for Mycoplasma mobile gliding

Several mycoplasma species are known to glide on solid surfaces such as glass in the direction of the membrane protrusion, but the mechanism underlying this movement is unknown. To identify a novel protein involved in gliding, we raised monoclonal antibodies against a detergent-insoluble protein fraction of Mycoplasma mobile, the fastest glider, and screened the antibodies for inhibitory effects on gliding. Five monoclonal antibodies stopped the movement of gliding mycoplasmas, keeping them on the glass surface, and all of them recognized a large protein in immunoblotting. This protein, named Gli521, is composed of 4,738 amino acids, has a predicted molecular mass of 520,559 Da, and is coded downstream of a gene for another gliding protein, Gli349, which is known to be responsible for glass binding during gliding. Edman degradation analysis indicated that the N-terminal region is processed at the peptide bond between the amino acid residues at positions 43 and 44. Analysis of gliding mutants isolated previously revealed that the Gli521 protein is missing in a nonbinding mutant, m9, where the gli521 gene is truncated by a nonsense mutation at the codon for the amino acid at position 1170. Immunofluorescence and immunoelectron microscopy indicated that Gli521 localizes all around the base of the membrane protrusion, at the "neck," as previously observed for Gli349. Analysis of the inhibitory effects of the anti-Gli521 antibody on gliding motility revealed that this protein is responsible for force generation or force transmission, a role distinct from that of Gli349, and also suggested conformational changes of Gli349 and Gli521 during gliding.     

Toll-like receptor 3 and STAT-1 contribute to double-stranded RNA+ interferon-gamma-induced apoptosis in primary pancreatic beta-cells

Viral infections and local production of cytokines probably contribute to the pathogenesis of Type 1 diabetes. The viral replicative intermediate double-stranded RNA (dsRNA, tested in the form of polyinosinic-polycytidylic acid, PIC), in combination with the cytokine interferon-gamma (IFN-gamma), triggers beta-cell apoptosis. We have previously observed by microarray analysis that PIC induces expression of several mRNAs encoding for genes downstream of Toll-like receptor 3 (TLR3) signaling pathway. In this report, we show that exposure of beta-cells to dsRNA in combination with IFN-alpha, -beta, or -gamma significantly increases apoptosis. Moreover, dsRNA induces TLR3 mRNA expression and activates NF-kappaB and the IFN-beta promoter in a TRIF-dependent manner. dsRNA also induces an early (1 h) and sustained increase in IFN-beta mRNA expression, and blocking IFN-beta with a specific antibody partially prevents PIC plus IFN-gamma-induced beta-cell death. On the other hand, dsRNA plus IFN-gamma does not induce apoptosis in INS-1E cells, and expression of TLR3 and type I IFNs mRNAs is not detected in these cells. Of note, disruption of the STAT-1 signaling pathway protects beta-cells against dsRNA plus IFN-gamma-induced beta-cell apoptosis. This study suggests that dsRNA plus IFN-gamma triggers beta-cell apoptosis by two complementary pathways, namely TLR3-TRIF-NF-kappaB and STAT-1.     

Intracellular cAMP controls a physical association of V-1 with CapZ in cultured mammalian endocrine cells

V-1, an ankyrin repeat protein with the activity to control tyrosine hydroxylase (TH) gene expression and transmitter release in PC12D cells, associates with CapZ, an actin capping protein, and thereby regulates actin polymerization in vitro. In this study, immunoprecipitation and Western blot analysis showed that V-1 was physically associated with CapZ-beta in PC12D transfectants overexpressing V-1. These proteins were co-localized in the soma of Purkinje cells of rat cerebellum as assayed by immunohistochemistry. Furthermore, in the V-1 transfectants, the amount of CapZ which physically associated with V-1 was steeply reduced at 2h after treatment with forskolin, but was thereafter increased to reach its initial level at 12h after forskolin-treatment. These results suggest that the association of V-1 with CapZ is controlled by a cAMP-dependent signalling pathway probably to play a functional role in the regulatory mechanism of actin dynamics in the endocrine system and the central nervous system.     

Biochemistry and physiology of mitochondrial ion channels involved in cardioprotection

Over the past decades there has been considerable progress in understanding the multifunctional roles of mitochondrial ion channels in metabolism, energy transduction, ion transport, signaling, and cell death. Recent data have suggested that some of these channels function under physiological condition, and others may be activated in response to pathological insults and play a key role in cytoprotection. This review outlines our current understanding of the molecular identity and pathophysiological roles of the mitochondrial ion channels in the heart with particular emphasis on cardioprotection against ischemia/reperfusion injury, and future research on mitochondrial ion channels.     

Overexpression of glutathione peroxidase attenuates myocardial remodeling and preserves diastolic function in diabetic heart

Oxidative stress plays an important role in the structural and functional abnormalities of diabetic heart. Glutathione peroxidase (GSHPx) is a critical antioxidant enzyme that removes H(2)O(2) in both the cytosol and mitochondia. We hypothesized that the overexpression of GSHPx gene could attenuate left ventricular (LV) remodeling in diabetes mellitus (DM). We induced DM by injection of streptozotocin (160 mg/kg ip) in male GSHPx transgenic mice (TG+DM) and nontransgenic wildtype littermates (WT+DM). GSHPx activity was higher in the hearts of TG mice compared with WT mice, with no significant changes in other antioxidant enzymes. LV thiobarbituric acid-reactive substances measured in TG+DM at 8 wk were significantly lower than those in WT+DM (58 +/- 3 vs. 71 +/- 5 nmol/g, P < 0.05). Heart rate and aortic blood pressure were comparable between groups. Systolic function was preserved normal in WT+DM and TG+DM mice. In contrast, diastolic function was impaired in WT+DM and was improved in TG+DM as assessed by the deceleration time of peak velocity of transmitral diastolic flow and the time needed for relaxation of 50% maximal LV pressure to baseline value (tau; 13.5 +/- 1.2 vs. 8.9 +/- 0.7 ms, P < 0.01). The TG+DM values were comparable with those of WT+Control (tau; 7.8 +/- 0.2 ms). Improvement of LV diastolic function was accompanied by the attenuation of myocyte hypertrophy, interstitial fibrosis, and apoptosis. Overexpression of GSHPx gene ameliorated LV remodeling and diastolic dysfunction in DM. Therapies designed to interfere with oxidative stress might be beneficial to prevent cardiac abnormalities in DM.     

FIA functions as an early signal component of abscisic acid signal cascade in Vicia faba guard cells

An abscisic acid (ABA)-insensitive Vicia faba mutant, fia (fava bean impaired in ABA-induced stomatal closure) had previously been isolated. In this study, it was investigated how FIA functions in ABA signalling in guard cells of Vicia faba. Unlike ABA, methyl jasmonate (MeJA), H(2)O(2), and nitric oxide (NO) induced stomatal closure in the fia mutant. ABA did not induce production of either reactive oxygen species or NO in the mutant. Moreover, ABA did not suppress inward-rectifying K(+) (K(in)) currents or activate ABA-activated protein kinase (AAPK) in mutant guard cells. These results suggest that FIA functions as an early signal component upstream of AAPK activation in ABA signalling but does not function in MeJA signalling in guard cells of Vicia faba.