Localization of phospholipase C-gamma1 signaling in caveolae: importance in EGF-induced phosphoinositide hydrolysis but not in tyrosine phosphorylation

Upon epidermal growth factor treatment, phospholipase C-gamma1 (PLC-gamma1) translocates from cytosol to membrane where it is phosphorylated at tyrosine residues. Caveolae are small plasma membrane invaginations whose structural protein is caveolin. In this study, we show that the translocation of PLC-gamma1 and its tyrosine phosphorylation are localized in caveolae by caveolin-enriched low-density membrane (CM) preparation and immunostaining of cells. Pretreatment of cells with methyl-beta-cyclodextrin (MbetaCD), a chemical disrupting caveolae structure, inhibits the translocation of PLC-gamma1 to CM as well as phosphatidylinositol (PtdIns) turnover. However, MbetaCD shows no effect on tyrosine phosphorylation level of PLC-gamma1. Our findings suggest that, for proper signaling, PLC-gamma1 phosphorylation has to occur at PtdInsP(2)-enriched sites.     

Inhibition of cytokine-induced vascular cell adhesion molecule-1 expression; possible mechanism for anti-atherogenic effect of Agastache rugosa

Adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1) play an important role during the early stages of atherogenesis. Agastache rugosa has an anti-atherogenic effect in low density lipoprotein receptor -/- mice. Moreover, A. rugosa reduced macrophage infiltration and VCAM-1 expression has been localized in aortic endothelium that overlies early foam cell lesions. This study ascertained that tilianin (100 microM), a major component of A. rugosa, inhibits the tumor necrotic factor-alpha (TNF-alpha)-induced expression of VCAM-1 by 74% in cultured human umbilical vein endothelial cells (HUVECs). Also, tilianin (100 microM) reduced TNF-alpha-induced activation of nuclear factor-kappaB in HUVECs.     

Fine tuning of TCR signaling by CD5

Current data indicate that CD5 functions as an inhibitor of TCR signal transduction. Consistent with this role, thymocyte selection in TCR transgenic/CD5(-/-) mice is altered in a manner suggestive of enhanced TCR signaling. However, the impact of CD5 deletion on thymocyte selection varies depending on the transgenic TCR analyzed, ranging from a slight to a marked shift from positive toward negative selection. An explanation for the variable effect of CD5 on selection is suggested by the observation that CD5 surface expression is regulated by TCR signal intensity during development and CD5 surface levels on mature thymocytes and T cells parallel the avidity of the positively selecting TCR/MHC/ligand interaction. In this study, we generated mice that overexpress CD5 during thymocyte development (CD5-tg), and then examined the effect of CD5 overexpression or CD5 deletion (CD5(-/-)) on selection of thymocytes that express the same TCR transgenes. The results demonstrate that the effect on thymocyte selection of altering CD5 expression depends on the avidity of the selecting interaction and, consequently, the level of basal (endogenous) CD5 surface expression. Substitution of endogenous CD5 with a transgene encoding a truncated form of the protein failed to rescue the CD5(-/-) phenotype, demonstrating that the cytoplasmic domain of CD5 is required for its inhibitory function. Together, these results indicate that inducible regulation of CD5 surface expression during thymocyte selection functions to fine tune the TCR signaling response.     

Susceptibility of avian hosts to experimental Gymnophalloides seoi infection

To determine whether avian species are susceptible to infection with Gymnophalloides seoi (a human-infecting intestinal trematode), we exposed 7 species of birds with metacercariae obtained from oysters. The birds were necropsied at days 2, 4, and 6 postinfection (PI). The highest worm recovery at day 6 PI was obtained from the Kentish plover (Charadrius alexandrinus; mean = 56.0%), followed by the Mongolian plover (C. mongolus; 49.3%), and the grey plover (Pluvialis squatarola; 32.3%). In contrast, no mature worms were recovered from the great knot (Calidris tenuirostris), dunlin (C. alpina), black-tailed gull (Larus crassirostris), and mallard (Anas platyrhynchos). Among the plovers, the worms attained the greatest size at day 6 PI (254.1 x 190.4 microm) in the Kentish plover, with a significantly higher number of eggs in the uterus. The 3 species of plovers are highly susceptible to experimental G. seoi infection, suggesting that they could play a role as definitive hosts for these worms in nature.     

Role of Hck in the pathogenesis of encephalomyocarditis virus-induced diabetes in mice

Soluble mediators such as interleukin-1beta, tumor necrosis factor alpha (TNF-alpha), and inducible nitric oxide synthase (iNOS) produced from activated macrophages play an important role in the destruction of pancreatic beta cells in mice infected with a low dose of the D variant of encephalomyocarditis (EMC-D) virus. The tyrosine kinase signaling pathway was shown to be involved in EMC-D virus-induced activation of macrophages. This investigation was initiated to determine whether the Src family of kinases plays a role in the activation of macrophages, subsequently resulting in the destruction of beta cells, in mice infected with a low dose of EMC-D virus. We examined the activation of p59/p56(Hck), p55(Fgr), and p56/p53(Lyn) in macrophages from DBA/2 mice infected with the virus. We found that p59/p56(Hck) showed a marked increase in both autophosphorylation and kinase activity at 48 h after infection, whereas p55(Fgr) and p56/p53(Lyn) did not. The p59/p56(Hck) activity was closely correlated with the tyrosine phosphorylation level of Vav. Treatment of EMC-D virus-infected mice with the Src kinase inhibitor, PP2, resulted in the inhibition of p59/p56(Hck) activity and almost complete inhibition of the production of TNF-alpha and iNOS in macrophages and the subsequent prevention of diabetes in mice. On the basis of these observations, we conclude that the Src kinase, p59/p56(Hck), plays an important role in the activation of macrophages and the subsequent production of TNF-alpha and nitric oxide, leading to the destruction of pancreatic beta cells, which results in the development of diabetes in mice infected with a low dose of EMC-D virus.     

Type D retrovirus Gag polyprotein interacts with the cytosolic chaperonin TRiC

The carboxy terminus-encoding portion of the gag gene of Mason-Pfizer monkey virus (M-PMV), the prototype immunosuppressive primate type D retrovirus, encodes a 36-amino-acid, proline-rich protein domain that, in the mature virion, becomes the p4 capsid protein. The p4 domain has no known role in M-PMV replication. We found that two mutants with premature termination codons that remove half or all of the p4 domain produced lower levels of stable Gag protein and of self-assembled capsids. Interestingly, yeast two-hybrid screening revealed that p4 specifically interacted with TCP-1gamma, a subunit of the chaperonin TRiC (TCP-1 ring complex). TRiC is a cytosolic chaperonin that is known to be involved in both folding and subunit assembly of a variety of cellular proteins. TCP-1gamma also associated with high specificity with the M-PMV pp24/16-p12 domain and human immunodeficiency virus p6. Moreover, in cells, Gag polyprotein associated with the TRiC chaperonin complex and this association depended on ATP hydrolysis. In the p4 truncation mutants, the Gag-TRiC association was significantly reduced. These results strongly suggest that cytosolic chaperonin TRiC is involved in Gag folding and/or capsid assembly. We propose that TRiC associates transiently with nascent M-PMV Gag molecules to assist in their folding. Consequently, properly folded Gag molecules carry out the intermolecular interactions involved in self-assembly of the immature capsid.     

Antioxidative and antitumor promoting effects of [6]-paradol and its homologs

Benzo[a]pyrene diol epoxide, a metabolite of benzo[a]pyrene (BaP), and chlorohydrin, the reaction product of chloride and the epoxide, form in vitro the same trans- and cis-stereoisomeric DNA adducts, but in different proportions. In this study, we asked whether the DNA adduct concentration can be kept the same by applying the appropriate dose of (+/-)-7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE)and (+/-)-7r,8t,9t-trihydroxy-10c-chloro-7,8,9,10-tetrahydrobenzo[a]pyrene (trans-BPDCH) to rodent skin and whether the DNA adducts formed differ only in their trans- and cis-stereoisomerism. Skin from C57Bl6 mice, spontaneous hypertension rats (SHR) and Sprague-Dawley (SD) rats was treated ex vivo immediately after the death of the animals with anti-BPDE and its corresponding bay region chlorohydrin trans-BPDCH and the epidermis was analyzed for DNA adducts 1h after the application. We found that adduct formation at the exocyclic amino groups of deoxyguanosine and deoxyadenosine in epidermal DNA followed a linear dose-response within 6--100 nmol/cm(2) with both chemicals. In order to achieve the same adduct concentration in mouse, spontaneous hypertension rat (SHR), and Sprague-Dawley (SD) rat skin, respectively, a 37-, 23- and 10-fold lower dose of anti-BPDE than of trans-BPDCH had to be applied. The order of 2'-deoxyguanosine (dGuo) adduct concentration with anti-BPDE was similar to what has been reported, but the order with trans-BPDCH was (+)-cis-BPDE-N(2)-dGuo adduct>(+)-trans-BPDE-N(2)-dGuo=(-)-trans-BPDE-N(2)-dGuo>(-)-cis-BPDE-N(2)-dGuo in mouse skin. Irrespective of species or strain, a significantly higher proportion of cis-adducts was obtained after treatment with trans-BPDCH than after treatment with anti-BPDE. Therefore, DNA adduct concentration can be kept the same by applying the appropriate dose of anti-BPDE and trans-BPDCH to rodent skin and the DNA adducts formed differ only in their trans- and cis-stereoisomerism.