Expression of brain-specific angiogenesis inhibitor 3 (BAI3) in normal brain and implications for BAI3 in ischemia-induced brain angiogenesis and malignant glioma

Murine brain-specific angiogenesis inhibitor 1 and 2 (mBAI1, mBAI2) are involved in angiogenesis after cerebral ischemia. In this study, mBAI3 was cloned and characterized. Northern and Western blot analyses demonstrated a unique developmental expression pattern in the brain. The level of mBAI3 in brain peaked 1 day after birth, unlike mBAI1 and mBAI2, which peaked 10 days after birth. In situ hybridization analyses of the brain showed the same localization of BAI3 as BAI1 and BAI2, which includes most neurons of cerebral cortex and hippocampus. In the in vivo focal cerebral ischemia model, BAI3 expression decreased from 0.5 h after hypoxia until 8 h, but returned to control level after 24 h. The expression of vascular endothelial growth factor following ischemia showed an inverse pattern. The decreased expressions of BAIs in high-grade gliomas were observed, but BAI3 expression was generally lower in malignant gliomas than in normal brain. Our results indicate that the expression and distribution of BAI3 in normal brain, but not its developmental expression, are very similar to those of BAI1 and BAI2, and that BAI3 may participate in the early phases of ischemia-induced brain angiogenesis and in brain tumor progression.     

Functional polymorphism in the carboxyl terminus of the alpha-subunit of the human epithelial sodium channel

A common human epithelial sodium channel (ENaC) polymorphism, alphaT663A, is present in the cytoplasmic C terminus of the alpha-subunit, although it is unclear whether this polymorphism segregates with blood pressure. We examined whether this polymorphism was associated with differences in functional Na(+) channel expression. Whole cell amiloride-sensitive currents in Xenopus oocytes expressing wild type channels (alphaT663betagamma) were significantly approximately 1.3-2.0-fold higher than currents measured in oocytes expressing channels with an Ala, Gly or Leu, or Lys at position alpha663. In contrast, differences in functional human ENaC expression were not observed with oocytes expressing channels having Thr (wild type), Ser, or Asp at this position. The surface expression of channels, measured using an epitope-tagged beta-subunit, was significantly reduced in oocytes expressing alphaT663Abetagamma when compared with oocytes expressing alphaT663betagamma. The corresponding polymorphism was generated in the mouse alpha-subunit (malphaA692T) and was not associated with differences in functional alphabetagamma-mouse ENaC expression. The polymorphism is present in a region that is not well conserved between human and mouse. We generated a mouse/human chimera by replacement of the distal C terminus of the mouse alpha-subunit with the distal C terminus of the human alpha-subunit. Co-expression of this m(1-678)/h(650-669)T663A chimera with mouse betagamma led to a significant reduction in whole cell Na(+) currents and surface expression when compared with m(1-678)/h(650-669)T663-mbetagamma. Our results suggest that halphaT663A is a functional polymorphism that affects human ENaC surface expression.     

Modulation of Thr phosphorylation of integrin beta1 during muscle differentiation

By using transient elevations of cytosolic free calcium levels triggered by integrin antibody or laminin (Kwon, M. S., Park, C. S., Choi, K., Park, C.-S., Ahnn, J., Kim, J. I., Eom, S. H., Kaufman, S. J., and Song, W. K. (2000) Mol. Biol. Cell 11, 1433-1443), we have demonstrated that protein phosphatase 2A (PP2A) is implicated in the regulation of reversible phosphorylation of integrin. In E63 skeletal myoblasts, the treatment of PP2A inhibitors such as okadaic acid and endothall induces an increase of phosphorylation of integrin beta1A and thereby inhibits integrin-induced elevation of cytosolic calcium level and formation of focal adhesions. None of these effects were in differentiated myotubes expressing the alternate beta1D isoform. In the presence of okadaic acid, PP2A in association with integrin beta1A was reduced on myoblasts, whereas beta1D on myotubes remained bound with PP2A. Both co-immunoprecipitation and in vitro phosphatase assays revealed that dephosphorylation of residues Thr788-Thr789 in the integrin beta1A cytoplasmic domain is dependent upon PP2A activity. Mutational analysis of the cytoplasmic domain and confocal microscopy experiments indicated that substitution of Thr788-Thr789 with Asn788-Asn789 is of critical importance for regulating the function of integrin beta1. These results suggest that PP2A may be a primary regulator of threonine phosphorylation of integrin beta1A and subsequent activation of downstream signaling molecules. Taken together, we propose that dephosphorylation of residues Thr788-Thr789 in the cytoplasmic domain of integrin beta1A may contribute to the linkage of integrins to focal adhesion sites and induce the association with cytoskeleton proteins. The switch of integrin beta1A to beta1D isoform in myotubes therefore may be a mechanism to escape from phospho-regulation by PP2A and promotes a more stable association of the cytoskeleton with the extracellular matrix.     

Human cytomegalovirus UL18 alleviated human NK-mediated swine endothelial cell lysis

Human cytomegalovirus UL18, a MHC class I homologue, is known to serve as a natural killer cell (NK) decoy and to ligate NK inhibitory receptors to prevent lysis of an infected target cell. To explore whether the cell surface expression of UL18 represents a potential immune suppressive approach to evade NK-mediated cytotoxicity in the prevention of xenograft rejection, we examined the effect of the UL18 expression in vitro upon human NK-mediated cytotoxicity against swine endothelial cells (SECs). UL18 expression on SECs by a retroviral vector (PLNCX2) significantly suppressed NK-mediated SEC lysis by approximately 25-100%. The protective effect of UL18 could be mediated through ILT-2 inhibitory receptor on NKs. Additionally, the interaction between UL18 and NKs resulted in the significant reduction of IFN-gamma production. This study demonstrates that UL18 can serve as an effective tool for the evasion of NK-mediated cytotoxicity and for the inhibition of IFN-gamma production during xenograft rejection.     

Epigallocatechin-3-gallate, constituent of green tea, suppresses the LPS-induced phenotypic and functional maturation of murine dendritic cells through inhibition of mitogen-activated protein kinases and NF-kappaB

The effects of epigallocatechin-3-gallate (EGCG) on dendritic cells (DC) maturation were investigated. EGCG, in a dose-dependent manner, profoundly inhibited CD80, CD86, and MHC class I and II expression on bone marrow-derived murine myeloid DC. EGCG restored the decreased dextran-FITC uptake and inhibited enhanced IL-12 production by LPS-treated DC. EGCG-treated DC were poor stimulators of nai;ve allogeneic T-cell proliferation and reduced levels of IL-2 production in responding T cells. EGCG-pretreated DC inhibited LPS-induced MAPKs, such as ERK1/2, p38, JNK, and NF-kappaB p65 translocation. Therefore, the molecular mechanisms by which EGCG antagonized LPS-induced DC maturation appeared to involve the inhibition of MAPK and NF-kappaB activation. These novel findings provide new insight into the immunopharmacological role of EGCG and suggest a novel approach to the manipulation of DC for therapeutic application of autoimmune and allergic diseases.     

Ginsenoside Rg3 antagonizes NMDA receptors through a glycine modulatory site in rat cultured hippocampal neurons

We previously reported that ginseng, a well-known herbal medicine, inhibited NMDA receptors in cultured hippocampal neurons. Here, we further examined the detailed mechanism of ginseng-mediated inhibition using its main active ingredient, ginsenoside Rg3. Co-application of ginsenoside Rg3 with increasing concentrations of NMDA did not change the EC50 of NMDA to the receptor, suggesting that ginsenoside Rg3 inhibits NMDA receptors without competing with the NMDA-binding site. Ginsenoside Rg3-mediated inhibition also occurred in a distinctive manner from the well-characterized NMDA receptor open channel blocker, MK-801. However, ginsenoside Rg3 produced its effect in a glycine concentration-dependent manner and shifted the glycine concentration-response curve to the right without changing the maximal response, suggesting the role of ginsenoside Rg3 as a competitive NMDA receptor antagonist. We also demonstrated that ginsenoside Rg3 significantly protected neurons against NMDA insults. Therefore, these results suggest that ginsenoside Rg3 protects NMDA-induced neuronal death via a competitive interaction with the glycine-binding site of NMDA receptors in cultured hippocampal neurons.