Oxidant,antioxidant and physical exercise

Generation of reactive oxygen species (ROS) is a normal process in the life of aerobic organisms. Under physiological conditions, these deleterious species are mostly removed by the cellular antioxidant systems, which include antioxidant vitamins, protein and non-protein thiols, and antioxidant enzymes. Since the antioxidant reserve capacity in most tissues is rather marginal, strenuous physical exercise characterized by a remarkable increase in oxygen consumption with concomitant production of ROS presents a challenge to the antioxidant systems.An acute bout of exercise at sufficient intensity has been shown to stimulate activities of antioxidant enzymes. This could be considered as a defensive mechanism of the cell under oxidative stress. However, prolonged heavy exercise may cause a transient reduction of tissue vitamin E content and a change of glutathione redox status in various body tissues. Deficiency of antioxidant nutrients appears to hamper antioxidant systems and augment exercise-induced oxidative stress and tissue damage. Chronic exercise training seems to induce activities of antioxidant enzymes and perhaps stimulate GSH levels in body fluids. Recent research suggest that supplementation of certain antioxidant nutrients are necessary for physically active individuals.     

Interaction of porphyrins with heme proteins – a brief review

In the past years, in our laboratory, several cell lines have been generated starting from a human liver (H7). Some of them have been used successfully in studies of the infection with and propagation of Hepatitis B and Hepatitis C viruses. Recently, several lines of evidence indicated that the origin of these cell lines was uncertain. Therefore, we now have determined the genetic characteristics of these cell lines in comparison to HepG2 cells received from ATCC and to HepG2 isolates grown at other laboratories. Quadruplex fluorescent short tandem repeat (STR) typing and karyotyping were performed. In addition, some biochemical characteristics of selected clones were studied. Genetically, all H7-derived cell lines were identical to HepG2 cells. However, some liver-specific functions varied between the different sub-cloned lines. The H7-derived cell lines that were generated proved to be sub-cloned lines of HepG2. The problem of cross-contamination during cloning of cell lines appears to be not uncommon. We found that two out of six HepG2 isolates obtained from other laboratories were not derived from the same individual as the original HepG2 cells. Therefore, STR typing should be applied as a rapid and sensitive technique to determine and monitor the origin of cell lines and to safeguard against contamination.     

Identification,purification and partial characterization of tissue inhibitor of matrix metalloproteinase-2 in bovine pulmonary artery smooth muscle

Bovine pulmonary artery smooth muscle possesses the tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) as revealed by Western immunoblot study of its cytosol fraction with bovine polyclonal TIMP-2 antibody. This potent polypeptide inhibitor of matrix metalloproteinases (MMPs) was purified to homogeneity from cytosol fraction of bovine pulmonary artery smooth muscle. This inhibitor was purified by ammonium sulfate precipitation followed by gelatin sepharose and lentil lectin sepharose affinity chromatography and continuous elution electrophoresis by Prep Cell Model 491 (Bio-Rad, USA). SDS-PAGE revealed that the inhibitor has an apparent molecular mass of 21 kDa and was confirmed as TIMP-2 by (i) Western immunoblot assay using bovine polyclonal TIMP-2 antibody; and also by (ii) amino terminal amino acid sequence analysis of the purified inhibitor is found to be identical with TIMP-2 obtained from other sources. The purified 21 kDa inhibitor was found to be active against matrix metalloproteinase-2 (MMP-2, 72 kDa gelatinase) and matrix metalloproteinase-9 (MMP-9, 92 kDa gelatinase), the ambient MMPs in the pulmonary artery smooth muscle. The inhibitor was also found to be sensitive to the activated 72 kDa gelatinase-TIMP-2 complex and also active human interstitial collagenase. By contrast, it was found to be insensitive to the serine proteases: trypsin and plasmin. The inhibitor was heat and acid resistant and it had the sensitivity to trypsin degradation and reduction-alkylation. Treatment of the inhibitor with hydrogen peroxide, superoxide generating system (hypoxanthine plus xanthine oxidase) and peroxynitrite inactivated the inhibitor.     

Identification,purification and partial characterization of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in bovine pulmonary artery smooth muscle

Bovine pulmonary artery smooth muscle tissue possesses the tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) as revealed by immunoblot studies of the cytosolic fraction with polyclonal TIMP-1 antibody. In this report, we described the purification and partial characterization of the inhibitor from the cytosolic fraction of the smooth muscle. This inhibitor was purified by a series of anion-exchange, gel filtration and affinity chromatographic procedure. The purified inhibitor showed an apparent molecular mass of 30 kDa in SDS-PAGE. Amino terminal sequence analysis for the first 22 amino acids of the purified inhibitor was also found to be identical to bovine TIMP-1. This glycosylated inhibitor was found to be active against matrix metallorpoteinase-9 (MMP-9, gelatinase B), the ambient matrix metalloproteinase in the pulmonary smooth muscle. The purified TIMP-1 was also found to be sensitive to pure rabbit and human fibroblast collagenase and type IV collagenase. In contrast, it had minimum inhibitory activity against bacterial collagenase. It was also found to be inactive against the serine proteases trypsin and plasmin. The inhibitor was heat and acid resistant and it had the sensitivity to trypsin degradation and reduction-alkylation.     

Proteolytic activation of protein kinase Calpha by peroxynitrite in stimulating cytosolic phospholipase A2 in pulmonary endothelium: involvement of a pertussis toxin sensitive protein

We sought to determine the roles of PKCalpha and G(i)alpha in regulating cPLA(2) activity in bovine pulmonary artery endothelial cell membrane under peroxynitrite (ONOO(-)) stimulation. Treatment of bovine pulmonary artery endothelial cells with ONOO(-) markedly stimulates the cell membrane associated protease activity, protein kinase C (PKC) activity, phospholipase A(2) (PLA(2)) activity, and arachidonic acid (AA) release from the cells. ONOO(-) significantly increases (Ca(2+))(i) in the cells, and pretreatment with the intracellular Ca(2+) chelator BAPTA-AM prevents the increase in (Ca(2+))(i), protease activity, PKC activity, and cPLA(2) activity in the cell membrane and AA release from the cells. Pretreatment of the cells with arachidonyl trifluoromethyl ketone (AACOCF(3)) (a cPLA(2) inhibitor) prevents ONOO(-)-stimulated cPLA(2) activity and AA release without producing a significant alteration of the protease activity. Pretreatment with vitamin E and aprotinin prevents ONOO(-)-induced increase in the protease activity, PKC activity, and cPLA(2) activity in the cell membrane and AA release from the cells. Pretreatment with the PKC inhibitor calphostin C prevents ONOO(-)-caused increase in PKC activity and cPLA(2) activity in the cell membrane and AA release from the cells. An immunoblot study of the cell membrane isolated from the ONOO(-)-treated cells with polyclonal PKCalpha antibody elicited an increase in the 80 kDa immunoreactive protein band along with an additional 47 kDa immunoreactive fragment. An immunoblot study with anti-nitrotyrosine antibody revealed that ONOO(-) induces nitration of tyrosine residues in PKCalpha. Pretreatment of the cells with aprotinin abolished the 47 kDa immunoreactive fragment in the immunoblot. An immunoblot study of the endothelial cell membrane with polyclonal cPLA(2) antibody revealed that treatment of the cells with ONOO(-) markedly increases the cPLA(2) immunoreactive protein profile in the membrane. Pretreatment of the endothelial cells with Go6976, a PKCalpha inhibitor, prevents the increase in PKC activity and cPLA(2) activity in the cell membrane under ONOO(-)-triggered condition. It, therefore, appears from the present study that treatment of the cells with ONOO(-) causes an increase in the protease activity, and that plays an important role in activating PKCalpha, which subsequently stimulates cPLA(2) activity in the cell membrane and AA release from the cells. An immunoblot assay with polyclonal G(i)alpha antibody elicited an immunoreactive band having a molecular mass of 41 kDa. Pretreatment of the cells with pertussis toxin markedly inhibits ONOO(-)-induced increase in cPLA(2) activity and AA release without significantly altering (Ca(2+))(i), protease activity, and PKC activity in the cell membrane. Treatment of the cells with ONOO(-) causes phosphorylation of G(i)alpha in the cell membrane, and pretreatment with Go6976 prevents its phosphorylation. We suggest the existence of a pertusssis toxin sensitive G protein-mediated mechanism for activation of cPLA(2) by ONOO(-) in bovine pulmonary artery endothelial cell membrane, which is regulated by PKCalpha-dependent phosphorylation and sensitive to aprotinin for its inhibition.     

Isolation of MMP-2 from MMP-2/TIMP-2 complex: characterization of the complex and the free enzyme in pulmonary vascular smooth muscle plasma membrane

We have previously indicated that bovine pulmonary artery smooth muscle plasma membrane possesses a complex of 72-kDa gelatinase and TIMP-2 (MMP-2/TIMP-2 complex) [Mol. Cell. Biochem. 258 (2004) 73]. In this paper, we described isolation of MMP-2 from the MMP-2/TIMP-2 complex, characterizations of the isolated MMP-2 and also the complex. MMP-2/TIMP-2 complex was purified from bovine pulmonary vascular smooth muscle plasma membrane using a combination of purification steps. Heparin-sepharose (100 mM NaCl eluate)-purified preparation contained the MMP-2/TIMP-2 complex. The MMP-2/TIMP-2 complex, which was electrophoresed under reducing condition on the SDS-PAGE and immunobloted with a mixture of polyclonal MMP-2 and TIMP-2 antibodies, revealed two separate immunoreactive bands at their respective electrophoretic migration. Continuous elution electrophoresis of the complex resulted to MMP-2 free of any detectable TIMP-2. The homogeneity of the isolated MMP-2 and the complex was demonstrated by SDS-PAGE under nonreducing condition and also by nondenaturing native-PAGE. The purified TIMP-2 free enzyme electrophoresed as a single band of 72-kDa, which could be activated rapidly and fully by aminophenylmercuric acetate (APMA) with the formation of 62-kDa and 45-kDa active species like native MMP-2 purified from the same source (bovine pulmonary artery smooth muscle). Identical treatment of the MMP-2/TIMP-2 complex with APMA resulted to significantly slower and partial conversion of the active species. Addition of pure TIMP-2 to the TIMP-2 free MMP-2 formed a complex with the progelatinase and prevented the rapid autolytic conversion induced by APMA. Immunoblot study with polyclonal MMP-2 antibody suggested that the isolated 72-kDa gelatinase is the MMP-2. We have also presented additional data indicating that the isolated preparation of 72-kDa gelatinase exhibited properties that are identical with MMP-2 obtained from different sources.     

Indenone derivatives as inhibitor of human DNA dealkylation repair enzyme AlkBH3

The mammalian AlkB homologue-3 (AlkBH3) is a member of the dioxygenase family of enzymes that in humans is involved in DNA dealkylation repair. Because of its role in promoting tumor cell proliferation and metastasis of cancer, extensive efforts are being directed in developing selective inhibitors for AlkBH3. Here we report synthesis, screening and evaluation of panel of arylated indenone derivatives as new class of inhibitors of AlkBH3 DNA repair activity. An efficient synthesis of 2,3-diaryl indenones from 2,3-dibromo indenones was achieved via Suzuki-Miyaura cross-coupling. Using a robust quantitative assay, we have obtained an AlkBH3 inhibitor that display specific binding and competitive mode of inhibition against DNA substrate. Finally, we established that this compound could prevent the proliferation of lung cancer cell line and enhance sensitivity to DNA damaging alkylating agent.     

The 5′-Nor Aristeromycin Analogues of 5′-Deoxy-5′-Methylthioadenosine and 5′-Deoxy-5′-Thiophenyladenosine

To extend the potential of 5′-noraristeromycin (and its enantiomer) as potential antiviral candidates, the enantiomers of the carbocyclic 5′-nor derivatives of 5′-methylthio-5′-deoxyadenosine and 5′-phenylthio-5′-deoxyadenosine have been synthesized and evaluated. None of the compounds showed meaningful antiviral activity.