Adil Anwar

NAD(P)H:quinone oxidoreductase 1 (NQO1): chemoprotection, bioactivation, gene regulation and genetic polymorphisms

NAD(P)H:quinone oxidoreductase 1 (NQO1) is an obligate two-electron reductase that is involved in chemoprotection and can also bioactivate certain antitumor quinones. This review focuses on detoxification reactions catalyzed by NQO1 and its role in antioxidant defense via the generation of antioxidant forms of ubiquinone and vitamin E. Bioactivation reactions catalyzed by NQO1 are also summarized and the development of new antitumor agents for the therapy of solid tumors with marked NQO1 content is reviewed. NQO1 gene regulation and the role of the antioxidant response element and the xenobiotic response element in transcriptional regulation is summarized. An overview of genetic polymorphisms in NQO1 is presented and biological significance for chemoprotection, cancer susceptibility and antitumor drug action is discussed.

Demonstration of Functional Requirement of Polypyrimidine Tract-binding Protein by SELEX RNA during Hepatitis C Virus Internal Ribosome Entry Site-mediated Translation Initiation

Polypyrimidine tract-binding protein (PTB) has been previously shown to physically interact with the hepatitis C virus (HCV) RNA genome at its 5′- and 3′-noncoding regions. Using high affinity SELEX RNA molecules, we present evidence for the functional requirement of PTB during HCV internal ribosome entry site (IRES)-controlled translation initiation. This study was carried out in rabbit reticulocyte translation lysates in which the HCV IRES-driven reporter RNA was introduced along with the PTB-specific SELEX RNA molecules. The SELEX RNAs specifically inhibited the HCV IRES function in the context of mono- and dicistronic mRNAs. The cap-dependent translation of a reporter (chloramphenicol acetyltransferase) RNA or naturally capped brome mosaic virus RNA, however, was not affected by the presence of SELEX during in vitro translation assays. The SELEX-mediated inhibition of the HCV IRES is shown to be relieved by the addition of recombinant human PTB in an add-back experiment. Thein vivo requirement of PTB was further confirmed by cotransfection of Huh7 cells with reporter RNA and PTB-specific SELEX RNA. The HCV IRES activity was inhibited by the SELEX RNA in these cells, but not by an unrelated control RNA. Together, these results demonstrate the functional requirement of cellular PTB in HCV translation and further support the feasible use of SELEX RNA strategy in demonstrating the functional relevance of cellular protein(s) in complex biological processes.

Alkaline protease from Spilosoma obliqua: potential applications in bio‐formulations

Some properties of the purified alkaline protease from larvae of the insect Spilosoma obliqua (Lepidoptera) and its potential application as an additive in various bio‐formulations are reported. The novel feature of the present study is the use of insect protease. The protease was found to be compatible with some of the commercial detergents tested, and was also effective in cleaving various protein substrates tested, albeit to different extents, implying broader substrate specificity and effectiveness of the protease against a wide variety of stains. This property of the protease can also be exploited by using it as an active component in enzymic debriders in view of its ability to digest various protein substrates. The insect protease appears to be potentially useful as an additive in detergent, stain remover and other bio‐formulations.

Protein kinase C inhibitor Gö6976 augments caffeine-induced reversal of chemoresistance to cis-diamminedichloroplatinum-II (CDDP) in a human ovarian cancer model

OBJECTIVESnNovel strategies for the treatment of chemoresistant ovarian cancer are needed. Caffeine and related compounds have been shown to over-ride G2/M arrest in ovarian cancer cells, increasing toxicity to chemotherapy. Newer compounds have been developed which may have the same effect as and exhibit synergism with caffeine, allowing the use of lower doses.nnnMETHODSnWe investigated the effects of caffeine and Gö6976 in the presence of CDDP in the SKOV3 and A2780 cell lines using proliferation, cell-cycle analysis, apoptosis, and AKT expression.nnnRESULTSnProliferation of cancer cells was decreased in a dose-dependent manner with caffeine and CDDP, but doses of caffeine required for significant inhibition were higher than that achievable in patients. Gö6976, a global PKC inhibitor with G2/M over-ride capability similar to caffeine, when combined with caffeine and CDDP at doses below that required for cell-cycle over-ride produced the growth inhibitory effects of a ten-fold higher caffeine concentration in both cell lines. CDDP induced G2/M arrest was significantly abrogated by caffeine but not by Gö6976 alone and no additional effect was seen on G2/M over-ride by the addition of Gö6976 to caffeine. Addition of Gö6976 to caffeine and CDDP did increase apoptosis but without altering phospho-AKT.nnnCONCLUSIONSnGö6976, when added to caffeine at doses below that required for cell-cycle over-ride, augments caffeine in overcoming CDDP resistance in this experimental system. G2/M over-ride is not the mechanism underlying the inhibition of proliferation. An AKT-independent apoptotic mechanism may be responsible.

Photoprotective Effects of Bucillamine Against UV-induced Damage in an SKH-1 Hairless Mouse Model†

UVB exposure of skin results in various biologic responses either through direct or indirect damage to DNA and non‐DNA cellular targets via the formation of free radicals, reactive oxygen species (ROS) and inflammation. Bucillamine [N‐(2‐mercapto‐2‐methylpropionyl)‐l‐cysteine] is a cysteine‐derived compound that can replenish endogenous glutathione due to its two donatable thiol groups, and functions as an antioxidant. In this study, we investigated the effects of bucillamine on UVB‐induced photodamage using the SKH‐1 hairless mouse model. We have demonstrated that UVB exposure (two consecutive doses, 230u2003mJu2003cm−2) on the dorsal skin of SKH‐1 mice induced inflammatory responses (edema, erythema, dermal infiltration of leukocytes, dilated blood vessels) and p53 activation as early as 6u2003h after the last UVB exposure. Bucillamine pretreatment (20u2003mgu2003kg−1 of body weight, administered subcutaneously) markedly attenuated UVB‐mediated inflammatory responses and p53 activation. We have also demonstrated that the stabilization and upregulation of p53 by UVB correlated with phosphorylation of Ser‐15 and Ser‐20 residues of p53 protein and that bucillamine pretreatment attenuated this effect. We propose that bucillamine has potential to be effective as a photoprotective agent for the management of pathologic conditions elicited by UV exposure.

Role of naturally occuring autoantibodies in senescence of normal and ATP depleted goat erythrocytes

The possible role of autoantibodies in the senescence of goat erythrocytes has been investigated. For this purpose goat gammaglobulin was purified to homogenity and antigoat gammaglobulin was raised in rabbits. Using the peroxidase labelled antigoat gammaglobulin it was possible to detect the presence of auto anti band‐3 antibodies in goat sera. The goat erythrocytes were aged in vitro by ATP‐depletion, which resulted in appearence of a 52,000 mol. wt. polypeptide. The in vitro aged goat erythrocyte membrane bound substantial amounts of auto anti band‐3 antibodies as visualized by immumoblots.