A large scale preparative gel electrophoresis separation of α1 and α2 subunits of the voltage‐gated Ca2+ channel from rabbit skeletal muscle

Nyormoi, Okot; Schneider, Toni; Smith, Robert G.

doi:10.1002/elps.11501501178

Cited by 4 publications

(1 citation statement)

References 28 publications

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“…All extraction buffers contained a cocktail of protease inhibitors including 1 mg/ml of aprotinin, 1 mg/ml of leupeptin, 10 mM iodoacetamide, 1 mM benzamidine, and 2 mM PMSF as previously described. 72 Briefly, cells were grown to 70-80% confluence, scraped off the plates, washed once with PBS and once with hypotonic buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl 2 , 10 mM KCl, 2 mM PMSF, and 0.5 mM DTT), and chilled in hypotonic buffer in ice for 10 min. Cells were lysed in a 2-ml glass homogenizer with a loose-fitting pestle (type B).…”

Section: Protein Extractionmentioning

confidence: 99%

An MMP-2/MMP-9 inhibitor, 5a, enhances apoptosis induced by ligands of the TNF receptor superfamily in cancer cells

2003

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Several studies have shown that matrix metalloproteases (MMPs) promote tumor growth, invasion, and metastasis. Consequently, MMP inhibitors have been developed as a new class of anticancer drugs, many of which are in clinical trials. The exact mechanism of the antineoplastic activity of MMP antagonists is unknown. To investigate the mechanism, we hypothesized that MMP inhibitors enhance the actions of apoptosis-inducing agents. To test this hypothesis, we treated breast, melanoma, leukemia, osteosarcoma, and normal breast epithelial cells with (2R)-2-[(4-biphenylsulfonyl)amino]-3-phenylproprionic acid (compound 5a), an organic inhibitor of MMP-2/MMP-9, alone or in combination with TNFa or other apoptotic agents. FACS analysis showed that 5a interacted synergistically with ligands of the TNF receptor superfamily, including TNFa and TNF receptor-like apoptosisinducing ligand (TRAIL), and with a Fas-cross-linking antibody (CH11), UV, paclitaxel, thapsigargin, and staurosporin, to induce apoptosis in a cell-type-specific manner. Other MMP inhibitors did not synergize with TNFa. Compound 5a did not act directly on the mitochondrion or via changes in protein synthesis. Instead, the mechanism requires ligandreceptor interaction and caspase 8 activation. Investigation of the effect of 5a on tumor growth in vivo revealed that continuous treatment of subcutaneous melanoma with a combination of 5a plus TRAIL reduced tumor growth and angiogenesis in nude mice. Our data demonstrate that 5a possesses a novel proapoptotic function, thus providing an alternative mechanism for its antineoplastic action. These observations have important implications for combination cancer therapy.

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Section: Protein Extractionmentioning

confidence: 99%

An MMP-2/MMP-9 inhibitor, 5a, enhances apoptosis induced by ligands of the TNF receptor superfamily in cancer cells

2003

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Proteolytic activity in amyotrophic lateral sclerosis IgG preparations

Nyormoi

1996

Annals of Neurology

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Sporadic amyotrophic lateral sclerosis is a motor neuron disease of unknown origin. Autoimmunity against voltage-gated calcium channels is one mechanism hypothesized to be the cause of the disease. In support of this hypothesis, it was previously reported that amyotrophic lateral sclerosis IgG specifically blocked the binding of 8B7 monoclonal antibody to the alpha1 subunit of voltage-gated calcium channels, suggesting overlapping epitopes of the two antibodies. It is, however, possible that the 8B7 epitope was destroyed by proteases. Data presented here show that the blocking of 8B7 binding to the alpha1 subunit by diethylaminoethyl cellulose (DEAE)-purified amyotrophic lateral sclerosis IgG was not observed with Fab fragments of amyotrophic lateral sclerosis IgG. The blocking was prevented by serine protease inhibitors. Moreover, it was reproduced by plasminogen and urokinase. These observations suggest that raised proteolytic activity in amyotrophic lateral sclerosis IgG preparations may be responsible for the blockade of 8B7 monoclonal antibody demonstrated previously. They also indicate the need to be particularly cautious when interpreting the results of incubation in amyotrophic lateral sclerosis sera or IgG preparations. Furthermore, they suggest that proteases may be partly responsible for some of the effects previously described for amyotrophic lateral sclerosis IgG. However, the proteolytic activity needs to be better defined and its possible role in amyotrophic lateral sclerosis investigated.

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Calcium Channel Autoantibodies and Amyotrophic Lateral Sclerosis

Smith

Appel

1996

Autoantibodies

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A large scale preparative gel electrophoresis separation of α₁ and α₂ subunits of the voltage‐gated Ca²⁺ channel from rabbit skeletal muscle

Cited by 4 publications

References 28 publications

An MMP-2/MMP-9 inhibitor, 5a, enhances apoptosis induced by ligands of the TNF receptor superfamily in cancer cells

An MMP-2/MMP-9 inhibitor, 5a, enhances apoptosis induced by ligands of the TNF receptor superfamily in cancer cells

Proteolytic activity in amyotrophic lateral sclerosis IgG preparations

Calcium Channel Autoantibodies and Amyotrophic Lateral Sclerosis

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