CLIC-5A functions as a chloride channel in vitro and associates with the cortical actin cytoskeleton in vitro and in vivo

CLIC-5A is a member of the chloride intracellular channel protein family, which is comprised of six related human genes encoding putative chloride channels. In this study, we found that reconstitution of purified recombinant CLIC-5A into artificial liposomes resulted in a dose-dependent chloride efflux that was sensitive to the chloride channel blocker IAA-94. CLIC-5A was originally isolated as a component of an ezrin-containing cytoskeletal complex from human placental microvilli. Here we show that similar protein complexes can be isolated using either immobilized CLIC-5A or the C-terminal F-actin-binding domain of ezrin and that actin polymerization is required for de novo assembly of these complexes. To investigate the behavior of CLIC-5A in vivo, JEG-3 placental choriocarcinoma cells were stably transfected with epitope-tagged CLIC-5A. In fixed cells, CLIC-5A displayed a polarized distribution and colocalized with ezrin in apical microvilli. Microvillar localization of CLIC-5A was retained after Triton X-100 extraction and was disrupted by treatment with latrunculin B. In transient transfections assays, we mapped a region between residues 20 and 54 of CLIC-5A that is required for targeting of CLIC-5A to microvilli in JEG-3 cells. Interestingly, expression of CLIC-5A in JEG-3 cells did not enhance the rate of iodide efflux in intact cells, suggesting that if CLIC-5A is a chloride channel, its channel activity may be restricted to intracellular membrane compartments in these cells. Regardless of its role in ion transport, CLIC-5A, like ezrin, may play an important role in the assembly or maintenance of F-actin-based structures at the cell cortex.     

CLIC4 is enriched at cell-cell junctions and colocalizes with AKAP350 at the centrosome and midbody of cultured mammalian cells

CLIC4 is a member of the chloride intracellular channel (CLIC) protein family whose principal cellular functions are poorly understood. Recently, we demonstrated that several CLIC proteins, including CLIC4, interact with AKAP350. AKAP350 is concentrated at the Golgi apparatus, centrosome, and midbody and acts as a scaffolding protein for several protein kinases and phosphatases. In this report, we show that endogenous CLIC4 and AKAP350 colocalize at the centrosome and midbody of cultured cells by immunofluorescence microscopy. Unlike AKAP350, CLIC4 is not enriched in the Golgi apparatus but is enriched in mitochondria, actin-based structures at the cell cortex, and the nuclear matrix, indicating that CLIC4-AKAP350 interactions are regulated at specific subcellular sites in vivo. In addition to the centrosome and midbody, CLIC4 colocalizes with AKAP350 and the tight junction protein ZO-1 in the apical region of polarized epithelial cells, suggesting that CLIC4 may play a role in maintaining apical-basolateral membrane polarity during mitosis and cytokinesis. Biochemical studies show that CLIC4 behaves mainly as a soluble cytosolic protein and can associate with proteins of the microtubule cytoskeleton. The localization of CLIC4 to the cortical actin cytoskeleton and its association with AKAP350 at the centrosome and midbody suggests that CLIC4 may be important for regulating cytoskeletal organization during the cell cycle. These findings lead to the conclusion that CLIC4 and possibly other CLIC proteins have alternate cellular functions that are distinct from their proposed roles as chloride channels.     

STUDY ON THE PROLIFERATIVE STATE OF HAEMOPOIETIC STEM CELLS (CFU)

Hydroxyurea was used to study the proliferation rate of haemopoietic stem cells (CFUJ in normal mice, after irradiation or transplantation into irradiated recipients. It was demonstrated that the proliferation rate of endogenous CFU_S (endo-CFU,) and exogenous CFU_S (exo-CFU_s) are identical. After irradiation (650 R) the surviving endo-CFU_s begin to proliferate immediately. By contrast exo-CFU, transplanted into the irradiated recipient mouse (850 R), begin to proliferate only after about 30 hr. However, injection of isoproterenol (which stimulates adenyl cyclase) or dibutyryl cyclic adenosine 3′,5′-monophosphate shortly after marrow cell graft, triggers the transplanted CFU_S into cell cycle as shown by an almost immediately increased sensitivity to hydroxyurea. Isoproterenol is capable of inducing DNA synthesis also in stem cells of normal mice but it takes about 20 hr before CFU, become to be increasingly sensitive to hydroxyurea.     

An ordinary differential equation model for the multistep transformation to cancer

Cancer is viewed as a multistep process whereby a normal cell is transformed into a cancer cell through the acquisition of mutations. We reduce the complexities of cancer progression to a simple set of underlying rules that govern the transformation of normal cells to malignant cells. In doing so, we derive an ordinary differential equation model that explores how the balance of angiogenesis, cell death rates, genetic instability, and replication rates give rise to different kinetics in the development of cancer. The key predictions of the model are that cancer develops fastest through a particular ordering of mutations and that mutations in genes that maintain genomic integrity would be the most deleterious type of mutations to inherit. In addition, we perform a sensitivity analysis on the parameters included in the model to determine the probable contribution of each. This paper presents a novel approach to viewing the genetic basis of cancer from a systems biology perspective and provides the groundwork for other models that can be directly tied to clinical and molecular data.     

Integrin alphavbeta8 functions as a receptor for foot-and-mouth disease virus: role of the beta-chain cytodomain in integrin-mediated infection

Field isolates of foot-and-mouth disease virus (FMDV) have been shown to use three alphav integrins, alphavbeta1, alphavbeta3, and alphavbeta6, as cellular receptors. Binding to the integrin is mediated by a highly conserved RGD motif located on a surface-exposed loop of VP1. The RGD tripeptide is recognized by several other members of the integrin family, which therefore have the potential to act as receptors for FMDV. Here we show that SW480 cells are made susceptible to FMDV following transfection with human beta8 cDNA and expression of alphavbeta8 at the cell surface. The involvement of alphavbeta8 in infection was confirmed by showing that virus binding and infection of the transfected cells are inhibited by RGD-containing peptides and by function-blocking monoclonal antibodies specific for either the alphavbeta8 heterodimer or the alphav chain. Similar results were obtained with a chimeric alphavbeta8 including the beta6 cytodomain (alphavbeta8/6), showing that the beta6 cytodomain can substitute efficiently for the corresponding region of beta8. In contrast, virus binding to alphavbeta6 including the beta8 cytodomain (alphavbeta6/8) was lower than that of the wild-type integrin, and this binding did not lead to infection. Further, the alphavbeta6 chimera was recognized poorly by antibodies specific for the ectodomain of alphavbeta6 and displayed a relaxed sequence-binding specificity relative to that of wild-type integrin. These data suggest that the beta6 cytodomain is important for maintaining alphavbeta6 in a conformation required for productive infection by FMDV.     

Early events in integrin alphavbeta6-mediated cell entry of foot-and-mouth disease virus

We have shown that foot-and-mouth disease virus (FMDV) infection mediated by the integrin alphavbeta6 takes place through clathrin-dependent endocytosis but not caveolae or other endocytic pathways that depend on lipid rafts. Inhibition of clathrin-dependent endocytosis by sucrose treatment or expression of a dominant-negative version of AP180 inhibited virus entry and infection. Similarly, inhibition of endosomal acidification inhibited an early step in infection. Blocking endosomal acidification did not interfere with surface expression of alphavbeta6, virus binding to the cells, uptake of the virus into endosomes, or cytoplasmic virus replication, suggesting that the low pH within endosomes is a prerequisite for delivery of viral RNA into the cytosol. Using immunofluorescence confocal microscopy, FMDV colocalized with alphavbeta6 at the cell surface but not with the B subunit of cholera toxin, a marker for lipid rafts. At 37 degrees C, virus was rapidly taken up into the cells and colocalized with markers for early and recycling endosomes but not with a marker for lysosomes, suggesting that infection occurs from within the early or recycling endosomal compartments. This conclusion was supported by the observation that FMDV infection is not inhibited by nocodazole, a reagent that inhibits vesicular trafficking between early and late endosomes (and hence trafficking to lysosomes). The integrin alphavbeta6 was also seen to accumulate in early and recycling endosomes on virus entry, suggesting that the integrin serves not only as an attachment receptor but also to deliver the virus to the acidic endosomes. These findings are all consistent with FMDV infection proceeding via clathrin-dependent endocytosis.     

Influence of treatment of diabetic rats with combinations of pycnogenol, beta-carotene, and alpha-lipoic acid on parameters of oxidative stress

Treatment with antioxidants may act more effectively to alter markers of free radical damage in combinations than singly. This study has determined whether treatment with combinations of pycnogenol, beta-carotene, and alpha-lipoic acid was more effective at reducing oxidative stress in diabetic rats than treatment with these antioxidants alone. It is not feasible, based on this study, to assume that there are interactive effects that make combinations of these antioxidants more effective than any one alone to combat oxidative stress. Female Sprague-Dawley rats, normal and streptozotocin-induced diabetic, were treated (10 mg/kg/day ip for 14 days) with pycnogenol, beta-carotene, pycnogenol + beta-carotene, or pycnogenol + beta-carotene + alpha-lipoic acid; controls were untreated. Concentrations of thiobarbituric acid reactive substances, glutathione and glutathione disulfide, and activities of glutathione reductase, glutathione peroxidase, superoxide dismutase, and catalase were measured in liver, kidney, and heart. Four types of effects were observed: (1) treatment with beta-carotene alone either reversed (cardiac glutathione disulfide) or elevated (cardiac glutathione, hepatic glutathione peroxidase activity) levels seen in diabetic animals; (2) beta-carotene alone produced no effect, but pycnogenol both alone and in combinations elevated (renal glutathione peroxidase and glutathione reductase activities, hepatic glutathione reductase activity and glutathione disulfide) or depressed (cardiac glutathione disulfide) levels seen in untreated diabetic animals; (3) all treatments with antioxidants, either alone or in combination, either normalized (lipid peroxidation in all tissues), elevated (hepatic GSH, cardiac glutathione peroxidase activity), or had no effect on (activities of hepatic catalase and superoxide dismutase in all tissues) levels seen in diabetic animals; (4) in only one case (cardiac glutathione reductase activity) levels in diabetic animals treated with combinations of antioxidants were normal, but elevated in animals treated with either antioxidant alone. Antioxidant effects seem to be dependent on the nature of the antioxidant used and not on combination effects.     

Relationship between histology,development and tumorigenesis of mammary gland in female rat

The mammary gland is a dynamic organ that undergoes structural and functional changes associated with growth, reproduction, and post-menopausal regression. The postnatal transformations of the epithelium and stromal cells of the mammary gland may contribute to its susceptibility to carcinogenesis. The increased cancer incidence in mammary glands of humans and similarly of rodents in association with their development is believed to be partly explained by proliferative activity together with lesser degree of differentiation, but it is not completely understood how the virgin gland retains its higher susceptibility to carcinogenesis. During its developmental cycle, the mammary gland displays many of the properties associated with breast cancer. An early first full-term pregnancy may have a protective effect. Rodent models are useful for investigating potential breast carcinogens. The purpose of this review is to help recognizing histological appearance of the epithelium and the stroma of the normal mammary gland in rats, and throughout its development in relation to tumorigenic potential.