Induction of DNA breaks and apoptosis in crosslink-hypersensitive V79 cells by the cytostatic drug β-D-glucosyl-ifosfamide mustard

To study molecular aspects of cytotoxicity of the anticancer drug β-D-glucose-ifosfamide mustard we investigated the potential of the agent to induce apoptosis and DNA breakage. Since β-D-glucose-ifosfamide mustard generates DNA interstrand crosslinks, we used as an in vitro model system a pair of isogenic Chinese hamster V79 cells differing in their sensitivity to crosslinking agents. CL-V5B cells are dramatically more sensitive (30-fold based on D10 values) to the cytotoxic effects of β-D-glucose-ifosfamide mustard as compared to parental V79B cells. After 48 h of pulse-treatment with the agent, sensitive cells but not the resistant parental line undergo apoptosis and necrosis, with apoptosis being the predominant form of cell death (70 and 20% of apoptosis and necrosis, respectively). Apoptosis increased as a function of dose and was accompanied by induction of DNA double-strand breaks in the hypersensitive cells. Furthermore, a strong decline in the level of Bcl-2 protein and activation of caspases-3, -8 and -9 were observed. The resistant parental cells were refractory to all these parameters. Bcl-2 decline in the sensitive cells preceded apoptosis, and transfection-mediated overexpression of Bcl-2 protected at least in part from apoptosis. From the data we hypothesize that non-repaired crosslinks induced by β-D-glucose-ifosfamide mustard are transformed into double-strand breaks which trigger apoptosis via a Bcl-2 dependent pathway

Na(+)-D-glucose cotransporter SGLT1 is pivotal for intestinal glucose absorption and glucose-dependent incretin secretion.

To clarify the physiological role of Na(+)-D-glucose cotransporter SGLT1 in small intestine and kidney, Sglt1(-/-) mice were generated and characterized phenotypically. After gavage of d-glucose, small intestinal glucose absorption across the brush-border membrane (BBM) via SGLT1 and GLUT2 were analyzed. Glucose-induced secretion of insulinotropic hormone (GIP) and glucagon-like peptide 1 (GLP-1) in wild-type and Sglt1(-/-) mice were compared. The impact of SGLT1 on renal glucose handling was investigated by micropuncture studies. It was observed that Sglt1(-/-) mice developed a glucose-galactose malabsorption syndrome but thrive normally when fed a glucose-galactose-free diet. In wild-type mice, passage of D-glucose across the intestinal BBM was predominantly mediated by SGLT1, independent the glucose load. High glucose concentrations increased the amounts of SGLT1 and GLUT2 in the BBM, and SGLT1 was required for upregulation of GLUT2. SGLT1 was located in luminal membranes of cells immunopositive for GIP and GLP-1, and Sglt1(-/-) mice exhibited reduced glucose-triggered GIP and GLP-1 levels. In the kidney, SGLT1 reabsorbed ∼3% of the filtered glucose under normoglycemic conditions. The data indicate that SGLT1 is 1) pivotal for intestinal mass absorption of d-glucose, 2) triggers the glucose-induced secretion of GIP and GLP-1, and 3) triggers the upregulation of GLUT2

Analysis of anisotropy and strain rate sensitivity of open-cell metal foam

This paper addresses numerical and experimental analysis of the m.pore® aluminium foam. Numerical models are based on computed tomography data in order to capture the complex material meso-structure. Uni-axial experimental tests were performed for quasi-static loading and an excellent agreement with numerical results has been obtained. Numerical analyses were extended for characteristic strain rates in order to analyse the strain rate sensitivity and anisotropy. Both, the micro-inertia and the base material strain rate sensitivity have an influence on the dynamic behaviour of the cellular meta

Cloning of a membrane-associated protein which modifies activity and properties of the Na⁺-D-glucose cotransporter

An expression library from porcine kidney cortex was screened with a monoclonal antibody (R4A6) which stimulates high-affinity phlorizin binding in kidney and intestine but does not react with the membrane protein (SGLT1) which mediates Na+-coupled transport of D-glucose (Hediger, M.A., Coady, M.J., Ikeda, T.S., and Wright, E.M. (1987) Nature 330, 379-381). A cDNA (RS1) was obtained which codes for a hydrophilic Mr 66,832 polypeptide and contains a predicted hydrophobic alpha-helix at the COOH terminus. After expression in Xenopus oocytes RS1 protein was found associated with the plasma membrane. RS1-homologous mRNAs were detected in renal outer cortex and outer medulla, small intestine, liver, and LLCPK1 cells, but not in skeletal muscle, heart muscle, Madin-Darby canine kidney (MDCK) cells, renal inner medulla, and Xenopus oocytes. After nondenaturing gel electrophoresis of renal brush-border membranes comigration of RS1- and SGLT1-homologous proteins as a high molecular weight complex was demonstrated. RS1 altered the expression of Na+-glucose cotransport by SGLT1 in Xenopus oocytes. There was no effect on the expression of the nonhomologous transporters for Na+-gamma-aminobutyric acid cotransport and for Na+-independent glucose transport. However, RS1 also changed the expression of the SGLT1-homologous Na+-myo-inositol cotransporter from MDCK cells. The Vmax of methyl-alpha-D-glucopyranoside (AMG) transport expressed after injection of a small amount of SGLT1-cRNA was increased 40-fold when a stoichiometric amount of RS1-cRNA was coinjected. In addition the voltage and glucose dependence of expressed AMG uptake and the concentration dependence of transport inhibition by phlorizin were changed when stoichiometric amounts of RS1-cRNA were coinjected with SGLT1-cRNA. Thus with SGLT1 one apparent transport site (K0.5 about 100 microM) and one apparent phlorizin inhibition site (Ki about 5 microM) was observed whereas with SGLT1 plus RS1 two apparent transport sites (K0.5(1) about 20 microM, K0.5(2) about 1 mM) and two apparent phlorizin inhibition sites (Ki(1) about 0.3 microM, Ki(2) about 30 microM) were found as has been described in brush-border membrane vesicles of kidney and intestine (see e.g. Koepsell, H., Fritzsch, G., Korn, K., and Madrala, A. (1990) J. Membr. Biol. 114, 113-132). The data suggest that the Na+-D-glucose cotransporter and possibly also other SGLT1-type Na+-cotransporters contain RS1-type regulatory subunits