Multiple Regulatory Molecules Research Articles

Review: Enteroendocrine cells: Neglected players in gastrointestinal disorders?

Enteroendocrine cells (EEC) form the basis of the largest endocrine system in the body. They secrete multiple regulatory molecules which control physiological and homeostatic functions, particularly postprandial secretion and motility. Their key purpose is to act as sensors of luminal contents, either in a classical endocrine fashion, or by a paracrine effect on proximate cells, notably vagal afferent fibres. They also play a pivotal role in the control of food intake, and emerging data add roles in mucosal immunity and repair. We propose that EEC are fundamental in several gastrointestinal pathologies, notably Post-infectious Irritable Bowel Syndrome, infectious enteritis, and possibly inflammatory bowel disease. Further work is needed to fully illustrate the importance, detailed biology and therapeutic potential of these frequently overlooked cells.

Determinants of RASistance to Anti–Epidermal Growth Factor Receptor Agents

Determinants of <i>RAS</i>istance to Anti–Epidermal Growth Factor Receptor Agents

Preparation and Characterization of Monoclonal Antibodies Against Mouse Jab1/CSN5 Protein

Jab1, also known as the fifth component of the COP9 signalosome complex (CSN5), directly interacts with and regulates the activity and stability of multiple intracellular regulatory molecules, such as c-Jun, p27, p53, Cullin, Smad4, and HIF1alpha. In addition, a high level of Jab1 is observed in a variety of human cancers and is sometimes correlated with a poor prognosis, suggesting that Jab1 contributes to cancer cell proliferation and survival and could be a novel target of cancer therapy. In this report, we generated five mouse monoclonal antibodies to a bacterially produced recombinant mouse Jab1 protein and examined their capabilities and limitations in commonly used assays, including enzyme linked immunosorbent assay (ELISA), Western blotting with denatured and native polyacrylamide gel electrophoresis (PAGE), immunoprecipitation, and immunofluorescence microscopy, finding the most suitable antibody for each application. Because these antibodies proved useful for immunohistochemical staining for Jab1 in fixed sections of human cancer samples, they should be useful in determining the expression and subcellular distribution of Jab1 in human tumors.

Intracellular proteolytic activity of cathepsin B is associated with capillary‐like tube formation by endothelial cells in vitro

The lysosomal cysteine protease cathepsin B is implicated in degradation of extracellular matrix (ECM), a crucial step in a variety of physiological and pathological processes, including tumor dissemination and angiogenesis. In this study, we analyzed the contribution of extracellular and intracellular cathepsin B activity on the formation of capillary-like tubular structures by human umbilical vein endothelial cells (HUVECs) grown on Matrigel matrix, using general and specific cysteine protease inhibitors. We demonstrated, by confocal assay using quenched fluorescent protein substrate DQ-collagen IV, that endothelial cells degrade ECM both intracellularly and pericellularly. Intracellular cathepsin B activity detected by degradation of Z-Arg-Arg cresyl violet substrate was co-localized with the products of DQ-collagen IV degradation in the perinuclear region and in the capillary-like tubular structures. Treatment of cells with membrane-permeable CA-074 Me effectively abolished intracellular cathepsin B activity, and resulted in reduced tube length (32.3+/-9.4% at 10 microM), total tubule area (49.6+/-12.4% at 10 microM), and the number of branch points of tubules (47.5+/-7.7% at 10 microM) in a dose-dependent manner. In contrast, CA-074 (0.1-10 microM), a membrane-impermeable cathepsin B specific inhibitor, general cysteine protease inhibitors chicken cystatin (5 microM) and E-64 (10 microM), and the metalloprotease inhibitor Minocycline (10 microM) showed no significant inhibitory effect in our angiogenesis model. These results show that, besides multiple regulatory molecules, intracellular cathepsin B also contributes to the neovascularization process and should be considered as a potential therapeutic target.

An innate cell-mediated, murine ulcerative colitis-like syndrome in the absence of nuclear factor of activated T cells

Background & Aims: Nuclear factor of activated T cells transcription factors plays a central role in immunity by regulating the expression of multiple cytokines and other regulatory molecules, many of which have been heavily implicated in the pathogenesis of inflammatory bowel disease. However, few studies have directly investigated the nuclear factor of activated T cells proteins in inflammatory bowel disease. We describe here a specific role for nuclear factor of activated T cells c2 in the pathogenesis of murine inflammatory bowel disease. Methods: Mice deficient for nuclear factor of activated T cells c2, recombinase activating gene-2, or both and transgenic or nontransgenic for an anti-ovalbumin T-cell receptor or an anti-hen egg lysozyme B-cell receptor were studied. Adoptive transfers were performed of T or B cells or both from nuclear factor of activated T cells c2-deficient mice into nuclear factor of activated T cells c2-deficient recombinase activating gene-deficient animals, in the presence or absence of antibodies that neutralize interleukin-10 activity. Results: Nuclear factor of activated T cells c2-deficient, recombinase activating gene-deficient animals spontaneously developed a severe inflammatory bowel syndrome that resembled ulcerative colitis but was composed entirely of nonlymphocytes. The disease was suppressed by the adoptive transfer of polyclonal B-cell populations, even on neutralization of interleukin-10, but not by the presence of monoclonal T or B cells. Conclusions: Nuclear factor of activated T cells plays a critical role in the regulation of bowel inflammation by nonlymphoid immune cells, and B cells suppress bowel inflammation by innate immune cells. Such findings indicate a novel, interleukin-10-independent role for nuclear factor of activated T cells in the regulation of innate immunity and in intestinal immune tolerance.

The use of cyclin-dependent kinase inhibitors alone or in combination with established cytotoxic drugs in cancer chemotherapy.

Cyclin-dependent kinase (CDK) inhibitors are small molecule inhibitors of the kinases required for the orderly progression of cells, both normal and neoplastic, through the cell cycle. Because cell cycle dysregulation is such a common occurrence in neoplasia, the search for agents that might block cell cycle traverse has been the focus of intense interest. These efforts have led to the identification of a broad array of compounds that interfere directly with the function of CDKs. Two of these agents (flavopiridol and UCN-01) have now entered the clinical arena, and others are scheduled to do so in the near future. In preclinical studies, CDK inhibitors have shown the ability not only to block neoplastic cell proliferation, but also to induce, through a variety of mechanisms, programmed cell death. The latter capacity may stem from the diverse effects that CDK inhibitors exert on multiple kinases and apoptotic regulatory molecules. In addition, there is abundant preclinical evidence that CDK inhibitors can potentiate, generally in a dose- and sequence-dependent manner, the anti-tumor effects of many established cytotoxic agents. In clinical studies in humans, flavopiridol and UCN-01 have been shown to be tolerable, although clear evidence of single agent activity or enhancement of the efficacy of established agents has not yet emerged. This may reflect a failure to optimize drug schedules/pharmacokinetics, or to identify the critical molecular targets of these agents. Finally, in recent years, a rationale has emerged for combining CDK inhibitors with other molecularly targeted agents (i.e. differentiation-inducers and signal transduction modulators). Current research has basically two goals: (a). to identify CDK inhibitor concentrations and schedules that inhibit the growth of and induce apoptosis in specific tumor cell types; and (b). to establish a rational basis for combining CDK inhibitors with more conventional cytotoxic agents to enhance antitumor efficacy. This review gives a brief summary of such efforts, with an emphasis on agents and combinations that are in or near clinical development.

Oncoprotein v-Myb and Retinoic Acid Receptor α Are Mutual Antagonists

ABSTRACT: The process of hematopoiesis is critically dependent on correct interactions of multiple regulatory molecules and transcription factors. We have studied the interactions of the v-Myb and retinoic acid receptor proteins which have opposing effects on hematopoiesis. While v-mybacts as a transforming oncogene preventing differentiation of monoblasts to macrophages, RARα functions as an anti-oncogene arresting the growth of v-myb-transformed cells and allowing their final myeloid differentiation steps to occur. We found that the retinoic acid receptor α inhibits v-Myb transformation by suppressing the expression of v-Myb target genes typified by themim-1 gene. Conversely, v-Myb protein interferes with RARα transactivation function as well as with retinoic acid-induced apoptosis of HL-60 cells. These results demonstrate that retinoic acid receptor and v-Myb proteins act in antagonistic ways and reciprocally modify each other's functions.

Chemical kinetics of induced gene expression: activation of transcription by noncooperative binding of multiple regulatory molecules.

A chemical kinetics model is described for the regulation of gene expression by the progressive binding of regulatory molecules to specific binding sites on DNA. Chemical rate equations are formulated and solved for the accumulation of regulatory molecules on DNA, the change in the level of induced mRNA, and the change in the level of the encoded protein in the activated tissue. Some special cases are examined, including that of an activation threshold created by a requirement for the binding of a minimum number of regulatory molecules prior to gene activation. Experimental data for several hormone-activated genetic systems are analyzed in the frame of the proposed model, and kinetic parameters are predicted. The model accounts for a number of experimental characteristics of hormone-inducible genetic systems, including the existence of a lag in the time course of mRNA accumulation, the sigmoidal curve of induced mRNA kinetics, the effect of hormone on mRNA stabilization, and the induction parameters observed when hormone analogues are used. The model also provides an explanation for the phenotypes of genetic variants with altered inducibility as changes in the molecular kinetic parameters of gene activity.

Selective expression of rat pancreatic genes during embryonic development.

We present the developmental profiles of the mRNAs of 10 selectively expressed pancreatic exocrine genes and of insulin. The mRNA profiles fall into three related classes, but each profile is in some respect unique. The data on gene expression suggest there are four developmental states of the exocrine pancreas: early morphogenesis and low-level gene expression (the protodifferentiated state), the embryonic differentiated state, a modulated state in neonatal animals, and the adult differentiated state. Each state is characterized by distinct ratios of the exocrine mRNAs and presumably involves a distinct regulatory transition. This complex differentiative program must involve multiple regulatory molecules.