No abstract available.
Mass Screening* ; src Homology Domains*

No abstract available.
Ethanol*

No abstract available.
Cell Cycle*

The cytolytic activity of CD4' T cells, both human and murine, has been clearly demonstrated in the immune response to mycobacterial infection and suggested to play a significant role in the protection and immunopathology. However, Uttle is known about the differentiation of CD4' CTL. In order to address this issue, we examined the influences of some factors on the generation of CD4' CTL specific to mycobacterial antigens. After 7 days' stimulation of PBMCs from healthy tuberculin reactors with mycobacterial antigens, the cytolytic activity of purised CD4' T cells toward autologous macrophages infected with mycobacteria was measured by Cr release assay. First, we found that both of live M. tubeiculosis and soluble antigens (ST-CF) induced the cytolytic activity of CD4' T cells, although the inducibility of the former was slightly greater than the latter. Second, the cytolytic activity was maximally induced at the relatively low antigen concentration (0.2:1 bacteria:monocyte ratio or 0.5 mg/ml of ST-CF). Finally, in the presence of increasing amounts of neutralizing anti-IL-12 or anti-IFN-r MoAb, the cytolytic activity of CD4+ T cells was decreased in a dose-dependent manner. These results suggest that low dose of antigen, its particulate type give mycobacteria), IL-12, and IFN-r give some positive signals for the generation of CD4+ CTL.
Humans ; Interleukin-12 ; Macrophages ; T-Lymphocytes* ; Tuberculin

TNF-alpha, a trimeric cytokine, was known to inhibit differention of preadipocytes to adipocytes. In the present study, we investigated signal mediators working downstream of TNF-alpha using murine 3T3-L1 cells. TNF-alpha induced activation of both c-jun NH2-terminal kinase (JNK) and nuclear transcription factor-kappaB (NF-kappaB) in 3T3-L1 cells. Blockage of these two mediators activities by specific inhibitors, SP600125 and Ad-IkappaBalpha-SR restored adipogenesis differentiation suggesting their involvement in the inhibited differentiation of 3T3-L1 cells by TNF-alpha. Consistent with previous studies, peroxisome proliferator-activated receptor gamma (PPARgamma) a key transcriptional regulator was remarkably reduced by TNF-alpha treatment. Compared with adipogenesis, however, SP600125, a chemical JNK inhibitor hardly relieved TNF-alpha effect on PPARgamma expression whereas S32A/S36A mutant of IkappaBalpha considerably recovered PPARgamma expression, indicating that two signal mediators exploit separable main routes to achieve reduced adipogenesis. These results suggest that inhibition of 3T3-L1 cells differentiation by TNF-alpha is partly implemented through NF-kappaB and one of its downstream effectors be PPARgamma.
3T3-L1 Cells ; Adipocytes/cytology/drug effects ; Animals ; Cell Differentiation/*drug effects ; Gene Expression Regulation/*drug effects ; Mice ; NF-kappa B/*metabolism ; Promoter Regions (Genetics)/genetics ; RNA, Messenger/genetics/metabolism ; Receptors, Cytoplasmic and Nuclear/*genetics ; Support, Non-U.S. Gov't ; Transcription Factors/*genetics ; Tumor Necrosis Factor/*pharmacology

To investigate the interaction of stimulatory GTP binding protein (G(s)) pathways with others, we overexpressed wild type alpha subunit of G(s) (G(s) alpha), constitutively activated R201E G(s) alpha, and dominant negative G226A G(s) alpha in COS-1 cells by transfection with DEAE-dextran, respectively, The expression of various G proteins in the transfected cells was analyzed after 72 h by quantitative Western blots, and cAMP production by stimulation with isoproterenol and forskolin was quantitated using cAMP binding proteins, The expression of Gs alpha increased about 5-fold in the transfected cells, with concomitant increase in the small forms. However, there was no significant alteration the in the level of the alpha subunit of inhibitory G protein (G(i)) and G(q), and the beta subunits of G proteins. The cAMP level without stimulation increased in the cells transfected with G(s) alpha regardless to the type of mutation, Treatment with either isoproterenol or forskolin resulted in comparable increase of the cAMP level in all the transfected cells, though the ratio to its respective basal level was smaller in the G(s) alpha-transfected cells, From this experiment, we found that the expression of the other G proteins and the signaling pathway producing cAMP did not change significantly by transiently expressing wild type, constitutively activated type, and dominant negative type of G(s) alpha. Analysis of the effects of long-term expression of Gs alpha would contribute to better understanding on how the G(s) alpha signaling system interacts with other signaling pathways and how it adapts to the changed environments.
Animals ; Blotting, Western ; Carrier Proteins ; Colforsin ; COS Cells* ; Cyclic AMP ; DEAE-Dextran ; GTP-Binding Proteins* ; Isoproterenol ; Protein Engineering ; Recombinant Proteins ; Transfection

Glucocorticoid receptor (GR) functions as a suppressor of inflammation by inhibiting the expression of many cytokine gene activated by NF-κB. The goal of this study is to investigate the mechanism by which GR repress NF-κB activation in lung epithelial cells. We used A549 and BEAS-2B lung epithelial cell lines. Using IgGκ-NF-κB luciferase reporter gene construct, we found that dexamethasone significantly suppressed TNF-α-induced NF-κB activation and the overexpression of GR showed dose-dependent reduction of TNF-α-induced NF-κB activity in both cell lines. However, DNA binding of NF-κB induced by TNF-α in electromobility shift assay was not inhibited by dexamethasone. Super shift assay with anti-p65 antibody demonstrated the existence of p65 in NF-κB complex induced by TNF-α Western blot showed that IκBα degradation induced by TNF-α was not affected by dexamethasone and IκBκ was not induced by dexamethasone, neither. To evaluate p65 specific transactivation, we adopted co-transfection study of Ga14-p65TA1 or TA2 fusion protein expression system together with 5xGa14-luciferase vector. Co-transfection of GR with Ga14-p65TA1 or TA2 repressed luciferase activity profoundly to the level of 10-20% of p65TA1- or TA2-induced transcriptional activity. And this transrepressional effect was abolished by co-transfection of CBP or SRC-1 expression vectors. These results suggest that Gr-mediated transrepression of NF-κB in lung epithelial cells is through competing for binding to limiting amount of transcriptional coactivators, CBP or SRC-1.
Blotting, Western ; Cell Line ; Dexamethasone* ; DNA ; Epithelial Cells* ; Genes, Reporter ; Inflammation ; Luciferases ; Lung ; NF-kappa B* ; Receptors, Glucocorticoid ; Transcriptional Activation*

Angiotensin II (Ang II) stimulates migration of vascular smooth muscle cell (VSMC) in addition to its contribution to contraction and hypertrophy. It is well established that Rho GTPases regulate cellular contractility and migration by reorganizing the actin cytoskeleton. Ang II activates Rac1 GTPase, but its upstream guanine nucleotide exchange factor (GEF) remains elusive. Here, we show that Ang II-induced VSMC migration occurs in a betaPIX GEF-dependent manner. betaPIX-specific siRNA treatment significantly inhibited Ang II-induced VSMC migration. Ang II activated the catalytic activity of betaPIX towards Rac1 in dose- and time-dependent manners. Activity reached a peak at 10 min and declined close to a basal level by 30 min following stimulation. Pharmacological inhibition with specific kinase inhibitors revealed the participation of protein kinase C, Src family kinase, and phosphatidylinositol 3-kinase (PI3-K) upstream of betaPIX. Both p21-activated kinase and reactive oxygen species played key roles in cytoskeletal reorganization downstream of betaPIX-Rac1. Taken together, our results suggest that betaPIX is involved in Ang II-induced VSMC migration.
1-Phosphatidylinositol 3-Kinase/metabolism ; Angiotensin II/*metabolism ; Animals ; *Cell Movement ; Cells, Cultured ; Guanine Nucleotide Exchange Factors/genetics/*metabolism ; Muscle, Smooth, Vascular/cytology ; Myocytes, Smooth Muscle/*cytology ; NADPH Oxidase/metabolism ; Protein Kinase C/metabolism ; RNA, Small Interfering/genetics ; Rats ; Rats, Sprague-Dawley ; p21-Activated Kinases/metabolism ; rac1 GTP-Binding Protein/metabolism ; src-Family Kinases/metabolism

Cyclooxygenase-2 (COX-2) has been reported to be associated with tumor development and progression as well as to protect cells from apoptosis induced by various cellular stresses. Through a tetracycline-regulated COX-2 overexpression system, we found that COX-2 inhibits detachment-induced apoptosis (anoikis) in a human bladder cancer cell line, EJ. We also found that the inhibition of anoikis by COX-2 results from activation of the PI-3K/Akt pathway as evidenced by suppression of the COX-2 effect on anoikis by a PI-3K inhibitor, LY294002. Furthermore, COX-2 enhanced Mcl-1 expression in the anoikis process, implying that Mcl-1 also may be involved in mediating the survival function of COX-2. Together, these results suggest that COX-2 inhibits anoikis by activation of the PI-3K/Akt pathway and probably by enhancement of Mcl-1 expression in human bladder cancer cells. This anti- anoikis effect of COX-2 may be a part of mechanisms to promote tumor development and progression.
1-Phosphatidylinositol 3-Kinase/*metabolism ; Anoikis/*physiology ; Bladder Neoplasms/*metabolism/pathology ; Enzyme Activation ; Humans ; Neoplasm Proteins/*metabolism ; Prostaglandin-Endoperoxide Synthase/*metabolism ; Protein-Serine-Threonine Kinases/*metabolism ; Proto-Oncogene Proteins/*metabolism ; Proto-Oncogene Proteins c-bcl-2/*metabolism ; Research Support, Non-U.S. Gov't ; Signal Transduction ; Transfection ; Tumor Cells, Cultured

Increased expression of a number of proinflammatory genes, including IL-8, is associated with inflammatory conditions such as asthma. Glucocorticoid receptor (GR)beta, one of the GR isoforms, has been suggested to be upregulated in asthma associated with glucocorticoid insensitivity and to work as a dominant negative inhibitor of wild type GRalpha. However, recent data suggest that GRbeta is not a dominant negative inhibitor of GRalpha in the transrepressive process and has its own functional role. We investigated the functional role of GRbeta expression in the suppressive effect of glucocorticoids on tumor necrosis factor (TNF)-alpha-induced IL-8 release in an airway epithelial cell line. GRbeta expression was induced by treatment of epithelial cells with either dexamethasone or TNF-alpha. GRbeta was able to inhibit glucocorticoid-induced transcriptional activation mediated by binding to glucocorticoid response elements (GREs). The suppressive effect of dexamethasone on TNF-alpha-induced IL-8 transcription was not affected by GRbeta overexpression, rather GRbeta had its own weak suppressive activity on TNF-alpha-induced IL-8 expression. Overall histone deacetylase activity and histone acetyltransferase activity were not changed by GRbeta overexpression, but TNF-alpha-induced histone H4 acetylation at the IL-8 promoter was decreased with GRbeta overexpression. This study suggests that GRbeta overexpression does not affect glucocorticoid-induced suppression of IL-8 expression in airway epithelial cells and GRbeta induces its own histone deacetylase activity around IL-8 promoter site.
Acetylation ; Cell Line, Tumor ; Dexamethasone/pharmacology ; Epithelial Cells/metabolism ; *Gene Expression Regulation ; Histones/*metabolism ; Humans ; Interleukin-8/*genetics/metabolism ; Receptors, Glucocorticoid/genetics/*metabolism ; Transcriptional Activation ; Transfection ; Tumor Necrosis Factor-alpha/*antagonists & inhibitors/pharmacology