Angiotensin II (Ang II) plays an important role in several cardiovascular diseases associated with vascular smooth muscle cell (VSMC) growth and migration. Src activity is known to be required for the migration of a number of cell types. p130Cas was reported to be essential for cell migration and actin filament reorganization. Mitogen-activated protein (MAP) kinases were also reported to be critical regulatory factors for growth and migration of VSMC. However, precise intracellular mechanisms involving c-Src, p130Cas, and MAP kinases in Ang IIstimulated migration of VSMC have not been well elucidated. Here we demonstrated that Ang II rapidly and significantly stimulated tyrosine phosphorylation of Src and Cas and their association in rat aortic smooth muscle cells (RASMC). Ang II-stimulated tyrosine phosphorylation of Src and Cas and activation of ERK1/2 and JNK, but not p38, were potently inhibited by Src family tyrosine kinase inhibitors, herbimycin A (HA) and PP2. Ang II-stimulated Src and Cas association, tyrosine phosphorylation of Cas, and activation of ERK1/2 and JNK were suppressed in kinase-inactive Src (KI Src)-overexpressed RASMC. Ang II-stimulated JNK activation but not ERK1/2 activation was blocked in substrate domain-deleted Cas (⌬SD Cas)-overexpressed RASMC. In addition, HA, PP2, ERK1/2 inhibitor, 2Ј-amino-3Ј-methoxyflavone (PD98059) and JNK inhibitor, and anthra[1,9-cd]pyrazol-6(2H)-one (SP600125) significantly inhibited Ang II-stimulated migration of RASMC. Ang II-induced colocalization of Src and Cas and migration were inhibited in both KI Src-and ⌬SD Cas-overexpressed RASMC. These findings suggest that Src and Cas are essentially but differentially involved in Ang II-stimulated migration of VSMC through the activation of ERK1/2 and JNK.Angiotensin II (Ang II) plays an important role in several cardiovascular diseases associated with vascular smooth muscle cell (VSMC) growth (Berk et al., 1989) and migration (Griendling et al., 1996), such as atherosclerosis, restenosis after angioplasty, and hypertension. It was indicated that tyrosine phosphorylation is a critical episode in several Ang II-stimulated signaling events (Marrero et al., 1996). Ang II induces tyrosine phosphorylation of proteins with apparent molecular masses of 42, 44, 70 to 80, 110 to 130, and 190 kDa in VSMC (Tsuda et al., 1991). The 130-, 60-, 44-, and 42-kDa proteins that were tyrosine-phosphorylated in response to Ang II in VSMC were identified as p130Cas [a CT-10 -regulated kinase (Crk)-associated substrate] (Takahashi et al., 1998), 60-kDa c-Src, a nonreceptor tyrosine kinase (Ishida et al., 1995), and mitogen-activated protein (MAP) kinase isozymes (Tsuda et al., 1992), respectively.Cell migration requires cytoskeletal reorganization involving phosphorylation of cytoskeleton-associated tyrosine kinases and formation of focal-adhesion complexes (Ilic et al.,

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In this study we investigated the in vitro responses of peripheral blood mononuclear preparations and purified monocytes to Clostridium difficile toxin A. In contrast to the responses of T and B cells, exposure to toxin A led to a rapid loss of monocytes in a time-and dose-dependent fashion (the majority of cells were lost within 24 h of exposure to >100 ng of toxin per ml). Transmission electron microscopy, flow cytometry, and fluorescence microscopy after propidium iodide and Hoechst staining showed that cell death in purified preparations of monocytes following exposure to 100 and 1,000 ng of toxin A per ml occurred by apoptosis. Further studies showed that 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazole-carbocyanine iodide aggregates were retained within toxin A-exposed monocyte mitochondria, but cytochrome c was released, suggesting that the apoptotic cascade was triggered in the absence of mitochondrial permeability transition. There was also an increase in caspase-3 activity in toxin A-stimulated monocytes. Following exposure to very high concentrations of toxin A (30 g/ml), monocyte cell death was predominantly of the necrotic type, with rapid extracellular release of lactate dehydrogenase. These studies demonstrated that C. difficile toxin A has a cell-specific effect, in which monocytes exhibit greater susceptibility than lymphocytes and their death is induced in a concentration-dependent manner.

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OBJECTIVES To evaluate the relationship between prostate stem cell antigen (PSCA) expression level in transitional cell carcinoma (TCC) of the urinary bladder and various clinicopathological features, including stage and grade; and to determine whether PSCA mRNA expression predicts disease recurrence in superficial (not muscle‐invasive) TCC of the bladder. PATIENTS AND METHODS Real‐time reverse transcriptase‐polymerase chain reaction (RT‐PCR) was performed on 97 TCC tissue samples and in 36 samples of normal bladder urothelium; the findings were analysed in relation to clinicopathological factors. Immunohistochemical expression was examined using light and confocal immunofluorescence microscopy to validate the RT‐PCR data. RESULTS Twenty‐seven patients developed disease recurrence, while the remaining 22 had no evidence of recurrence of superficial TCC of the bladder. There was significantly higher PSCA mRNA expression in TCC than in normal urothelium samples (P = 0.008). Superficial (TaT1) tumours had significantly higher PSCA expression than muscle‐invasive (≥ pT2) tumours (P < 0.001). There was no significant difference between patients with G1–2 tumours and those with G3 tumours (P = 0.109). Immunohistochemical analysis showed markedly greater PSCA expression in superficial than invasive TCC. Notably, from a multivariate analysis, the expression level of PSCA was an independent predictor of disease recurrence in superficial TCC (P = 0.012). CONCLUSIONS These findings suggest that the PSCA expression level measured by real‐time RT‐PCR could be a valuable prognostic marker for tumour recurrence in superficial TCC of the bladder.