ABSTRACT
To determine the roles of nitric oxide (NO) and its metabolite, peroxynitrite (ONOO(-)), on osteoblastic activation, we investigated the effects of a NO donor [ethanamine, 2, 2'-(hydroxynitrosohydrazono)bis- (dNO)], an O(-2) donor (pyrogallol), and an ONOO(-) scavenger (urate) on alkaline phosphatase (ALPase) activity and osteocalcin gene expression, which are indexes of osteoblastic differentiation. dNO elevated ALPase activity in the osteogenic MC3T3-E1 cell line. The combination of dNO and pyrogallol reduced both ALPase activity and osteocalcin gene expression. Because both indexes were recovered by urate, ONOO(-), unlike NO itself, inhibited the osteoblastic differentiation. Furthermore, treatment with a combination of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) was found to yield ONOO(-) as well as NO and O(-2). The reductions in ALPase activity and osteocalcin gene expression were also restored by urate. We conclude that ONOO(-) produced by TNF-alpha and IL-1beta, but not NO per se, would overcome the stimulatory effect of NO on osteoblastic activity and inhibit osteoblastic differentiation.
Subject(s)
Interleukin-1/pharmacology , Nitrates/metabolism , Osteoblasts/physiology , Tumor Necrosis Factor-alpha/pharmacology , Alkaline Phosphatase/metabolism , Animals , Cell Line , Drug Synergism , Free Radical Scavengers , Gene Expression , Mice , Nitrates/analysis , Nitric Oxide Donors/pharmacology , Osteocalcin/genetics , Pyrogallol/pharmacology , RNA, Messenger/metabolism , Superoxide Dismutase/pharmacology , Superoxides/metabolismABSTRACT
The delta-sarcoglycan (SG) gene is deleted in hamsters with hereditary cardiomyopathies. Immunological analyses of heart before, but not after, the progression of cardiomyopathy (CM) revealed that the BIO 14.6 strain, a model of hypertrophic CM, heterogeneously preserved alpha- and gamma-SG with loss of beta- and delta-SG. In contrast, the TO-2 strain, a model of dilated CM, did not show either SG. Furthermore, in vivo transfer of the full length delta-SG gene to TO-2 hearts expressed all four SGs. Thus, this age- and strain-dependent features suggest a more feasible setting for TO-2 than BIO 14.6 to verify both CM progression and the efficacy of gene therapy.
Subject(s)
Cardiomyopathies/genetics , Cytoskeletal Proteins/genetics , Gene Transfer Techniques , Membrane Glycoproteins/genetics , Age Factors , Animals , Cardiomyopathies/therapy , Cricetinae , Cytoskeletal Proteins/metabolism , Disease Models, Animal , Dystroglycans , Dystrophin/analysis , Gene Expression , Genetic Therapy , Immunoblotting , Immunohistochemistry , Male , Membrane Glycoproteins/metabolism , Mesocricetus , Myocardium/cytology , Myocardium/metabolism , Sarcoglycans , TransfectionABSTRACT
Both efficient gene transfer and the exact identification of gene product are required for gene therapy. Gene transfection of green fluorescence protein (GFP) might be useful for the reporter. After in vivo cotransfection of GFP and beta-galactosidase (beta-Gal) genes in Sendai virus-coated proteoliposomes to rat hearts, we compared the sensitivity and specificity of three methods: GFP detection, histochemical staining (HC) of beta-Gal activity, and immunostaining (IS) of the beta-Gal protein. Fluorescence microscopy and double staining of HC and IS revealed that both GFP and IS were equally sensitive and fourfold superior to HC at the peak of gene expression. However, different from skeletal muscle, the GFP of transfected cardiomyocytes showed two demerits: the fluorescence quenching due to the intense staining of beta-Gal activity, and nonspecific autofluorescence from myocardium. Thus, specific IS would be so far the most reliable to identify the gene product in heart.
Subject(s)
Heart/virology , Proteolipids/genetics , Respirovirus/genetics , Transfection/methods , Animals , Genes, Reporter , Green Fluorescent Proteins , Histocytochemistry , Luminescent Proteins/analysis , Luminescent Proteins/genetics , Male , Microscopy, Fluorescence , Myocardium/metabolism , Rats , Rats, Wistar , beta-Galactosidase/analysis , beta-Galactosidase/geneticsABSTRACT
Previous studies demonstrated two myosin heavy chain isoforms in vascular smooth muscles with SDS-polyacrylamide gel electrophoresis; MHC1 (204 kDa) and MHC2 (200 kDa). We report the existence of a novel myosin heavy chain isoform, MHC3 (196 kDa), which was exclusively contained in inferior vena cava. Equal amount of MHC1 and MHC2 was observed in aorta and pulmonary artery, respectively. However, inferior vena cava contained only MHC3. Proteolytic artifact was refuted by immunoblotting of tissue homogenates without purification, or SDS-polyacrylamide gel electrophoresis of myosin bands isolated by pyrophosphate gel electrophoresis. Furthermore, alpha-chymotryptic cleavage of MHC1, MHC2, and MHC3 displayed different peptide maps, indicating the primary structural difference among all three isoforms.
