Occludin is an integral membrane protein of the epithelial cell tight junction (TJ). Its potential role in coordinating structural and functional events of TJ formation has been suggested recently. Using a rat salivary gland epithelial cell line (Pa-4) as a model system, we have demonstrated that occludin not only is a critical component of functional TJs but also controls the phenotypic changes associated with epithelium oncogenesis. Transfection of an oncogenic Raf-1 into Pa-4 cells resulted in a complete loss of TJ function and the acquisition of a stratified phenotype that lacked cell–cell contact growth control. The expression of occludin and claudin-1 was downregulated, and the distribution patterns of ZO-1 and E-cadherin were altered. Introduction of the human occludin gene into Raf-1–activated Pa-4 cells resulted in reacquisition of a monolayer phenotype and the formation of functionally intact TJs. In addition, the presence of exogenous occludin protein led to a recovery in claudin-1 protein level, relocation of the zonula occludens 1 protein (ZO-1) to the TJ, and redistribution of E-cadherin to the lateral membrane. Furthermore, the expression of occludin inhibited anchorage-independent growth of Raf-1–activated Pa-4 cells in soft agarose. Thus, occludin may act as a pivotal signaling molecule in oncogenic Raf- 1–induced disruption of TJs, and regulates phenotypic changes associated with epithelial cell transformation.

The enzyme activity of mitogen-activated protein kinase (MAP kinase) increases in response to agents acting on a variety of cell surface receptors, including receptors linked to heterotrimeric G proteins. In this report, we demonstrated that Raf-1 protein kinase activity in the mouse parotid glands was induced by chronic isoproterenol administration in whole animals. To investigate the molecular nature underlying cellular responses to Raf-1 activation, we have stably transfected rat salivary epithelial Pa-4 cells with human Raf-1-estrogen receptor fusion gene (⌬Raf-1:ER) and used mRNA differential display in search of messages induced by ⌬Raf-1:ER activation. Through this approach, the gene encoding non-histone chromosomal protein HMGI-C was identified as one of the target genes activated by oncogenic Raf-1 kinase. Activation of Raf-1 kinase resulted in a delayed and sustained increase of The Ras/Raf/MEK 1 /MAP kinase signaling pathway mediates diverse responses to extracellular stimuli and initiates cellular proliferation and oncogenic transformation in mammalian cells (1-5). Results from biochemical and genetic studies have emphasized that members of the Raf family of serine/threonine protein kinases (Raf-1, A-Raf, and B-Raf) are the pivotal components of intracellular signal transduction pathways that relay signals from a plethora of activated cell surface receptors to a variety of intracellular effectors (4, 5). Once activated, the membrane-associated Raf-1 phosphorylates and activates a set of dual specificity protein kinases MEK1 and MEK2 (6 -8), which in turn phosphorylate and activate the ubiquitous p42/ p44 MAP kinases (ERK2/ERK1 for extracellular signal-regulated kinase) (9, 10). Activation of ERK1/2 induces pleiotropic effects ranging from phosphorylation of membrane-bound proteins, such as cytosolic phospholipase A 2 , to the activation of transcription factors, such as Elk-1 (11-14).Although ERK1 and ERK2 are likely to be the essential mediators of Raf-1 action, there are situations in which Raf-1 exerts biological effects in the absence of detectable ERK1/2 activation (15-18). Recent observations that Raf-1 activates cdc25 protein phosphatase in HeLa cells (19) and p70 S6 kinase in Chinese hamster ovary cells (20) support the idea that there are branch points in the Raf/MEK/ERK signaling cascade. Moreover, it is also demonstrated that Raf-1 activates JNK (c-Jun terminal kinase) in 3T3 cells through an autocrine manner (21). Hence, it is possible that these different pathways are not mutually exclusive and could be activated in a cell typespecific manner to mediate intracellular signaling events.The pathway linking G protein-coupled receptors to Raf-1 activation is still poorly understood. Recent reports indicate that the Raf-1 signaling pathway can be activated by a number of receptors linked to G i , G q , or G s . For example, triggering of ␣ 2 -adrenergic receptor (22), m1 muscarinic receptor (23), and ␣ 1 -and ␤-adrenergic receptors (24, 25) can all potently stimulate ERK activities. Moreover, ...

Mesoporous activated carbons were prepared from coconut shells by the method of chemical activation with H 3 PO 4 . Effects of main influence factors on the yield and adsorption properties of activated carbon were studied via orthogonal experiments. Experimental results under the optimum conditions were as follows: the yield of the activated carbon was 36.90%; methylene blue adsorption was 21.5 mL/0.1 g; and the iodine number was 889.36 mg/g. The surface area of the activated carbon prepared was 891 m 2 /g, as determined by the BET method. HorvathKawazoe equations (H-K) and density functional theory (DFT) were introduced to analyze the porous structures of the activated carbon. It was shown that the activated carbon was mesoporous, with a total pore volume of 0.7233 mL/g, a micropore volume of 37.06%, a mesopore volume of 62.85%, and a macropore volume of 0.07%. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) studies demonstrated the results of the pore structure analysis.

Flavin-containing monooxygenases (FMO) are metabolic enzymes mediating the oxygenation of nucleophilic atoms such as nitrogen, sulfur, phosphorus, and selenium. These enzymes share similar properties to the cytochrome P450 system but can be differentiated through heat inactivation and selective substrate inhibition by methimazole. This study investigated 10 compounds with varying degrees of FMO involvement to determine the nature of the correlation between human in vitro and in vivo unbound intrinsic clearance. To confirm and quantify the extent of FMO involvement six of the compounds were investigated in human liver microsomal (HLM) in vitro assays using heat inactivation and methimazole substrate inhibition. Under these conditions FMO contribution varied from 21% (imipramine) to 96% (itopride). Human hepatocyte and HLM intrinsic clearance (CL) data were scaled using standard methods to determine the predicted unbound intrinsic clearance (predicted CL) for each compound. This was compared with observed unbound intrinsic clearance (observed CL) values back calculated from human pharmacokinetic studies. A good correlation was observed between the predicted and observed CL using hepatocytes ( = 0.69), with 8 of the 10 compounds investigated within or close to a factor of 2. For HLM the in vitro-in vivo correlation was maintained ( = 0.84) but the accuracy was reduced with only 3 out of 10 compounds falling within, or close to, twofold. This study demonstrates that human hepatocytes and HLM can be used with standard scaling approaches to predict the human in vivo clearance for FMO substrates.

An experimental study was carried out on the tensile and bending behavior of multi-axial warp-knitted fabric composites. Five specimens reinforced with multi-axial warp-knitted fabric/epoxy were manufactured by a vacuum-assisted resin transfer molding method. Quasi-static tensile and three-point bending tests were carried out in a number of orientations relative to the stitching direction: quadriaxial, triaxial, biaxial (±45° and 0/90°) and unidirectional. The results of the tests revealed that the quadriaxial and biaxial (±45°) samples showed quasi-isotropic behavior, whereas the other laminates showed anisotropic behavior. The influence of fiber volume fraction and the orientation distribution of the constituent material on the tensile and bending behavior were also analyzed. The relationships between the stress and strain and the tensile and bending behavior of different multi-axial warp-knitted fabric composite were obtained by polynomial fitting.