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The effect of nickel (Ni2+) in the alpha2-macroglobulin-methylamine (alpha2M*) and low-density lipoprotein receptor (LRP/alpha2MR) interaction was investigated through ligand binding assays featuring thioglycolate-elicited macrophages of mice. Results have shown that Ni(2+) abrogated the binding of alpha2M* with LRP/alpha2MR through blocking the carboxyl terminal receptor recognition domain.

Angiostatin blocks tumor angiogenesis in vivo, almost certainly through its demonstrated ability to block endothelial cell migration and proliferation. Although the mechanism of angiostatin action remains unknown, identification of [F.sub.1]-[F.sub.0] ATP synthase as the major angiostatin-binding site on the endothelial cell surface suggests that ATP metabolism may play a role in the angiostatin response. Previous studies noting the presence of [F.sub.1] ATP synthase subunits on endothelial cells and certain cancer cells did not determine whether this enzyme was functional in ATP synthesis. We now demonstrate that all components of the [F.sub.1] ATP synthase catalytic core are present on the endothelial cell surface, where they colocalize into discrete punctate structures. The surfaceassociated enzyme is active in ATP synthesis as shown by dual-label TLC and bioluminescence assays. Both ATP synthase and ATPase activities of the enzyme are inhibited by angiostatin as well as by antibodies directed against the [Alpha]- and [Beta]-subunits of ATP synthase in cell-based and biochemical assays. Our data suggest that angiostatin inhibits vascularization by suppression of endothelialsurface ATP metabolism, which, in turn, may regulate vascular physiology by established mechanisms. We now have shown that antibodies directed against subunits of ATP synthase exhibit endothelial cell-inhibitory activities comparable to that of angiostatin, indicating that these antibodies function as angiostatin mimetics.

Objective: Tetrameric α2-macroglobulin (α2M), a plasma panproteinase inhibitor, is activated upon interaction with a proteinase, and undergoes a major conformational change exposing a receptor recognition site in each of its subunits. Activated α2M (α2M") binds to cancer cell surface GRP78 and triggers proliferative and antiapoptotic signaling. We have studied the role of α2M" in the regulation of mTORC1 and TORC2 signaling in the growth of human prostate cancer cells. Methods: Employing immunoprecipitation techniques and Western blotting as well as kinase assays, activation of the mTORC1 and mTORC2 complexes, as well as down stream targets were studied. RNAi was also employed to silence expression of Raptor, Rictor, or GRP78 in parallel studies. Results: Stimulation of cells with α2M" promotes phosphorylation of mTOR, TSC2, S6-Kinase, 4EBP, AktT308, and AktS473 in a concentration and time-dependent manner. Rheb, Raptor, and Rictor also increased. α2M" treatment of cells elevated mTORC1 kinase activity as determined by kinase assays of mTOR or Raptor immunoprecipitates. mTORC1 activity was sensitive to LY294002 and rapamycin or transfection of cells with GRP78 dsRNA. Down regulation of Raptor expression by RNAi significantly reduced α2M"-induced S6-Kinase phosphorylation at T389 and kinase activity in Raptor immunoprecipitates. α2M"-treated cells demonstrate about a twofold increase in mTORC2 kinase activity as determined by kinase assay of S473 phosphorylation and levels of p-S473 in mTOR and Rictor immunoprecipitates. mTORC2 activity was sensitive to LY294002 and transfection of cells with GRP78 dsRNA, but insensitive to rapamycin. Down regulation of Rictor expression by RNAi significantly reduces α2M"-induced phosphorylation of S473 phosphorylation in Rictor immunoprecipitates. Conclusion: Binding of α2M" to prostate cancer cell surface GRP78 upregulates mTORC1 and mTORC2 activation and promotes protein synthesis in the prostate cancer cells. [ABSTRACT FROM AUTHOR]

Examines the role of endothelial cell surface F[sub 1]-F[sub 0] ATP synthase in ATP synthesis. Inhibition of ATP synthase by angiotensin; Role of ATP metabolism in angiostatin response; Fusion of angiostatin to bovine F[sub 1] ATP synthase.

Chronic treatment with ethanol damages pericentral regions of the liver selectively, and reactive oxygen species such as H2O2 may be involved in the mechanism of hepatotoxicity. To test this idea, the effect of chronic treatment with ethanol on rates of H2O2 production was measured in tissue cylinders isolated from periportal and pericentral regions of livers from ethanol-treated rats. Rates of hydrogen peroxide production, assessed from the oxidation of methanol to formaldehyde by catalase-H2O2, were similar in tissue cylinders isolated from periportal regions in control and ethanol-treated rats. In contrast, rates of H2O2 production were over 4-fold higher in tissue isolated from pericentral regions of livers from ethanol-treated than control animals (1.7±0.5 vs. 0.4 ±0.3 nmol/min/mg protein, respectively). Rates of H2O2-generating acyl CoA oxidase activity were equivalent in tissue cylinders from periportal regions of livers from both groups (approximately 2 nmol/min/mg protein), but were over 2-fold higher in tissue cylinders from pericentral regions of livers from ethanol-treated rats than from controls. In contrast, catalase activity was increased nearly 2-fold in homogenates from both periportal and pericentral regions by ethanol treatment while glutathione peroxidase activity was decreased significantly in both regions. These data demonstrate that ethanol increases H2O2 generation in pericentral regions of the liver lobule in part by elevating rates of peroxisomal β-oxidation of acyl CoA compounds and are consistent with the hypothesis that local increases in H2O2 production may be involved in the mechanism of ethanol-induced hepatotoxicity. [ABSTRACT FROM AUTHOR]