QGP-1 cells release 5-HT via TRPA1 activation; a model of human enterochromaffin cells

Molecular and Cellular Biochemistry - Recently, we discovered that transient receptor potential ankyrin1 channel (TRPA1) is highly expressed in human and rat enterochromaffin (EC) cells, and those...     

Hydrogen peroxide as an effecter on the inactivation of particulate methane monooxygenase under aerobic conditions

Particulate methane monooxygenase (pMMO), a copper-containing membrane protein, catalyzes methane hydroxylation under aerobic conditions. We found that the activity of pMMO was increased by catalase, implying that hydrogen peroxide (H₂O₂) is generated by pMMO with duroquinol, an electron donor for pMMO, and that the generated H₂O₂ inhibits pMMO activity. In addition, reversible inhibition of pMMO with H₂O₂ was observed upon treatment of pMMO with H₂O₂ followed by the addition of catalase, and H₂O₂ formation by pMMO with duroquinol was detected using a fluorescence probe. The redox behavior of type 2 copper in pMMO measured by the electron paramagnetic resonance revealed that H₂O₂ re-oxidizes the type 2 copper in pMMO reduced with duroquinol.     

Association of the Ser326Cys polymorphism in the OGG1 gene with type 2 DM

The association of the Ser326Cys polymorphism of the 8-oxoguanine glycosylase 1 (OGG1) gene with type 2 diabetes was examined using a Japanese population (n (M/W): 4585 (2085/2500); age: 62.6 ± 10.9 years). HbA1c levels and frequency of diabetic subjects were significantly higher in subjects with genotypes with Cys allele than in those without (p = 0.032 and 0.037, respectively). Multiple logistic regression analysis showed that genotypes with Cys allele were significantly associated with diabetes (OR: 1.32, p = 0.0289). In subjects whose glucose tolerance was classified by FPG and 2-h PG (n = 1.634), the association was more substantial (genotypes with Cys allele vs. without, OR: 1.70, p = 0.0059; genotypes Cys/Cys vs. Ser/Ser, OR: 2.19, p = 0.0008). In subjects with genotype Ser/Ser, the insulin secretion index, HOMA-β, increased in the subjects with glucose intolerance and decreased in the subjects with diabetes, while, in subjects with genotypes Ser/Cys + Cys/Cys, HOMA-β decreased as the glucose tolerance progressed (p for trend = 0.010).     

Repulsive guidance molecule b inhibits neurite growth and is increased after spinal cord injury

Neuronal axons are guided by attractive and repulsive cues in their local environment. Since the identification of the repulsive guidance molecule (RGM) a (RGMa) as an axon repellent in the visual system, diverse functions, as part of the developing and adult central nervous system (CNS), have been ascribed to it. The binding of RGMa to its receptor neogenin has been shown to induce RhoA activation, leading to inhibitory/repulsive behavior and the collapse of the neuronal growth cone. In this paper, we provide evidence to suggest the involvement of RGMb, another member of the RGM family, in the rat CNS. RGMb inhibits neurite outgrowth in postnatal cerebellar granule neurons (CGNs) in vitro. RGMb is expressed by oligodendrocytes and neurons in the adult rat CNS, and the expression of this molecule is upregulated around the site of spinal cord injury. RGMb is present in myelin isolated from an adult rat brain. RGMb and neogenin are coexpressed in CGNs and entorhinal cortex neurons. These findings suggest that RGMb is a myelin-derived inhibitor of axon growth in the CNS. Inhibition of RGMb may provide an alternative approach for the treatment of spinal injuries.     

Unc5B associates with LARG to mediate the action of repulsive guidance molecule

Neuronal axons are guided by attractive and repulsive cues in their local environment. Because the repulsive guidance molecule A (RGMa) was originally identified as an axon repellent in the visual system, diverse functions in the developing and adult central nervous system have been ascribed to it. RGMa binding to its receptor neogenin induces RhoA activation, leading to inhibitory/repulsive behavior and collapse of the neuronal growth cone. However, the precise mechanisms that regulate RhoA activation are poorly understood. In this study, we show that Unc5B, a member of the netrin receptor family, interacts with neogenin as a coreceptor for RGMa. Moreover, leukemia-associated guanine nucleotide exchange factor (LARG) associates with Unc5B to transduce the RhoA signal. Focal adhesion kinase (FAK) is involved in RGMa-induced tyrosine phosphorylation of LARG as well as RhoA activation. These findings uncover the molecular basis for diverse functions mediated by RGMa.     

Glucose-induced production of hydrogen sulfide may protect the pancreatic beta-cells from apoptotic cell death by high glucose

We examined the expression of the major H₂S-producing enzymes, cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). CBS was ubiquitously distributed in the mouse pancreas, but CSE was found only in the exocrine. Freshly isolated islets expressed CBS, while CSE was faint. However, high glucose increased the CSE expression in the beta-cells. l-Cysteine or NaHS suppressed islet cell apoptosis with high glucose, and increased glutathione content in MIN6 beta-cells. Pretreatment with l-cysteine improved the secretory responsiveness following stimulation with glucose. The CSE inhibitor dl-propargylglycine antagonized these l-cysteine effects. We suggest H₂ S may function as an ‘intrinsic brake’ which protects beta-cells from glucotoxicity.     

Gangliosides and β1‐Integrin Are Required for Caveolae and Membrane Domains

Caveolae are plasma membrane domains involved in the uptake of certain pathogens and toxins. Internalization of some cell surface integrins occurs via caveolae suggesting caveolae may play a crucial role in modulating integrin‐mediated adhesion and cell migration. Here we demonstrate a critical role for gangliosides (sialo‐glycosphingolipids) in regulating caveolar endocytosis in human skin fibroblasts. Pretreatment of cells with endoglycoceramidase (cleaves glycosphingolipids) or sialidase (modifies cell surface gangliosides and glycoproteins) selectively inhibited caveolar endocytosis by >70%, inhibited the formation of plasma membrane domains enriched in sphingolipids and cholesterol (‘lipid rafts'), reduced caveolae and caveolin‐1 at the plasma membrane by approximately 80%, and blunted activation of β1‐integrin, a protein required for caveolar endocytosis in these cells. These effects could be reversed by a brief incubation with gangliosides (but not with asialo‐gangliosides or other sphingolipids) at 10°C, suggesting that sialo‐lipids are critical in supporting caveolar endocytosis. Endoglycoceramidase treatment also caused a redistribution of focal adhesion kinase, paxillin, talin, and PIP Kinase Iγ away from focal adhesions. The effects of sialidase or endoglycoceramidase on membrane domains and the distribution of caveolin‐1 could be recapitulated by β1‐integrin knockdown. These results suggest that both gangliosides and β1‐integrin are required for maintenance of caveolae and plasma membrane domains.     

Myelin-associated glycoprotein inhibits microtubule assembly by a Rho-kinase-dependent mechanism

Myelin-associated glycoprotein (MAG) and Nogo are potent inhibitors of neurite outgrowth from a variety of neurons, and they have been identified as possible components of the central nervous system myelin that prevents axonal regeneration in the adult vertebrate central nervous system. The activation of RhoA and Rho-kinase is reported to be an essential part of the signaling mechanism of these proteins. Here, we report that the collapsing response mediator protein-2 (CRMP-2) is phosphorylated by a Rho-kinase-dependent mechanism downstream of MAG or Nogo-66. The overexpression of the nonphosphorylated form of CRMP-2 at threonine 555, which is the phosphorylation site for Rho-kinase, counteracts the inhibitory effect of MAG on the postnatal cerebellar neurons. Additionally, the expression of the dominant negative form of CRMP-2 or knockdown of the gene using small interference RNA (siRNA) mimics the effect of MAG in vitro. Consistent with the function of CRMP-2, which promotes microtubule assembly, microtubule levels are down-regulated in the cerebellar neurons that are stimulated with MAG in vitro. Reduction in the density of microtubules is also observed in the injured axons following the spinal cord injury, and this effect depends on the Rho-kinase activity. Our data suggest the important roles of CRMP-2 and microtubules in the inhibition of the axon regeneration by the myelin-derived inhibitors.     

Proteomic analysis of laser-microdissected paraffin-embedded tissues: (2) MRM assay for stage-related proteins upon non-metastatic lung adenocarcinoma

A preceding paper suggested 81 candidates of stage-specifically expressed proteins for either stage IA or IIIA by global shotgun proteomics and spectral counting. Six proteins, a subset of these proteins, were chosen for a further verification study since they are potentially soluble and/or secretory, which nature is convenient for detecting them in blood in clinical practice. The multiple-reaction monitoring (MRM) quantitative analysis suggested that napsin-A and anterior gradient protein 2 homolog (hAG-2) out of the 6 candidates would be useful for determining stage IA or IIIA and are related to metastasis. In the study we noted that stage IIIA patients with better outcome showed napsin-A profiles similar to that of stage IA patients. We therefore examined 14 additional patients for analysis, which contained the IA-stage patients of poorer outcome and the IIIA-stage patients of better outcome. The MRM analysis of napsin-A for all patients suggests that napsin-A contents correlate with better outcome in stage IA. This and discovery studies demonstrate that direct isolation of tumor cells alone by laser microdissection (LMD) greatly reduces complexity on comprehensive analyses, and that MRM mass spectrometry using the endogenous internal standard is a feasible technology for quantitative verification of target proteins in formalin-fixed paraffin embedded (FFPE) tissues.     

Interleukin-17A regulates ependymal cell proliferation and functional recovery after spinal cord injury in mice

Ependymal cells have been suggested to act as neural stem cells and exert beneficial effects after spinal cord injury (SCI). However, the molecular mechanism underlying ependymal cell regulation after SCI remains unknown. To examine the possible effect of IL-17A on ependymal cell proliferation after SCI, we locally administrated IL-17A neutralizing antibody to the injured spinal cord of a contusion SCI mouse model, and revealed that IL-17A neutralization promoted ependymal cell proliferation, which was paralleled by functional recovery and axonal reorganization of both the corticospinal tract and the raphespinal tract. Further, to test whether ependymal cell-specific manipulation of IL-17A signaling is enough to affect the outcomes of SCI, we generated ependymal cell-specific conditional IL-17RA-knockout mice and analyzed their anatomical and functional response to SCI. As a result, conditional knockout of IL-17RA in ependymal cells enhanced both axonal growth and functional recovery, accompanied by an increase in mRNA expression of neurotrophic factors. Thus, Ependymal cells may enhance the regenerative process partially by secreting neurotrophic factors, and IL-17A stimulation negatively regulates this beneficial effect. Molecular manipulation of ependymal cells might be a viable strategy for improving functional recovery.Subject terms: Neuroimmunology, Spinal cord injury