Suriender Kumar

Identification and evaluation of anti Hepatitis C Virus phytochemicals from Eclipta alba

ETHNOPHARMACOLOGICAL RELEVANCE Eclipta alba, traditionally known as bhringraj, has been used in Ayurvedic medicine for more than 1000 years in India. It is used for the treatment of infective hepatitis, liver cirrhosis, liver enlargement and other ailments of liver and gall bladder in India. The aim of this study was to evaluate anti-hepatitis C virus activity present in the Eclipta alba extract, perform bioassay based fractionation and identify anti-HCV phytochemicals from the active fractions. MATERIALS AND METHODS Identification of active compounds was performed by bio-activity guided fractionation approach. Active isolates were separated by the combination of silica gel chromatography and preparative scale reverse phase HPLC. Eclipta alba extract and its isolates were examined for their ability to inhibit HCV replicase (HCV NS5B) activity in vitro and HCV replication in a cell culture system carrying replicating HCV subgenomic RNA replicon. The purified isolates were also examined for their binding affinity to HCV replicase by fluorescence quenching and their cytotoxicity by MTT assay. RESULTS Eclipta alba extract strongly inhibited RNA dependent RNA polymerase (RdRp) activity of HCV replicase in vitro. In cell culture system, it effectively inhibited HCV replication which resulted in reduced HCV RNA titer and translation level of viral proteins. Bioassay-based fractionations of the extracts and purification of anti-HCV phytochemicals present in the active fractions have identified three compounds, wedelolactone, luteolin, and apigenin. These compounds exhibited dose dependent inhibition of HCV replicase in vitro, and anti-HCV replication activity in the cell culture system CONCLUSION Eclipta alba extract and phytochemicals isolated from active fractions display anti-HCV activity in vitro and in cell culture system. The standardized Eclipta alba extract or its isolates can be used as an effective alternative and complementary treatment against HCV.

Modulation of proteases and their inhibitors in immortal human osteoblast-like cells by tumor necrosis factor-alpha in vitro

Inflammatory cytokines like interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-α) are linked to abnormal cartilage and bone loss in a variety of pathological conditions. We have investigated the effect of TNF-α on the synthesis and/ or steady-state mRNA levels of collagen, alkaline phosphatase (ALP), plasminogen activators (PAs) and their inhibitor PAI-1, and collagenases (MMPs) and their inhibitor TIMP-1 by human osteoblastic, HOS TE85, cells in monolayer cultures. HOS TE85 cells possess ∼2000 TNF-α receptors per cell with aKd value of 0.67 nM and receptor of ∼ 60 kDa. TNF-α enhances urokinase-plasminogen activator (u-PA) activity and steady-state mRNA levels twofold without affecting tissue-plasminogen activator (t-PA) or PAI-1. The increase in u-PA mRNA is due to enhanced transcription of this gene. mRNA levels or activities of collagenase 1 (MMP-1), 72- and 92-kDa gelatinases (MMP-2 and MMP-9) are also nearly doubled with little change in the level of expression of TIMP-1. TNF-α does not significantly affect the activity or mRNA levels of ALP. TNF-α decreases collagen as well as general protein synthesis. However, the steady-state mRNA for the α2 chain of collagen type I is increased three- to fourfold. These results show that TNF-α may increase pathological bone turnover by enhancing the rate of transcription of proteases capable of degrading the nonmineralized osteoid layer and decelerating the maturation of the extracellular matrix formed by osteoblasts.

Stimulation of the synthesis of hepatic fatty acid synthesizing enzymes of hypophysectomized rats by 3,5,3′-l-triiodothyronine

Abstract The levels of hepatic fatty acid synthesizing enzymes, acetyl-CoA carboxylase and fatty acid synthetase, are lowered to about one-tenth of the controls in hypophysectomized animals, whereas the lung enzymes decrease by only 25–30%. Administration of 3,5,3′- l -triiodothyronine to the hypophysectomized animals returns the hepatic and lung enzyme activities to the control values. Optimum levels are achieved at a dose of about 150 μg/100 g body wt and 3–4 days after triiodothyronine administration. The triiodothyronine response can be reduced by 80% with actinomycin-D or cycloheximide but not with hydrocortisone hemisuccinate. Antibody-antigen titrations and measurements of the rate of synthesis of fatty acid synthetase are indicative of increased synthesis of fatty acid synthetase and not of activation of the preexisting inactive species. These measurements provide evidence for the involvement of hormones other than insulin in the control of synthesis of fatty acid synthesizing enzymes.

Nutritional and hormonal control of lung and liver fatty acid synthesis

Abstract During starvation and in streptozotocin-induced diabetes, the total activities of rat lung acetyl CoA carboxylase and fatty acid synthetase are reduced to one-third of the normal values. Refeeding of the starved animals or administration of insulin to diabetic animals restores the levels to the original values. The insulin effect is dose and time dependent. These data contrast with those in the liver, where a 30- to 50-fold depression of these enzymes is observed in the diabetic state and administration of insulin is actually followed by doubling of the activity over normal controls. Fat-free high-fructose diet (containing 60% fructose by weight) enhances the activities of liver enzymes 3- to 6-fold over the values of controls on laboratory diet but has no effect on the lung enzymes. Long-term feeding of fructose diet also increases the activities of liver enzymes from diabetic animals to twice the value of normal controls on laboratory diet. Insulin administration to fructose-fed diabetic animals restores the enzyme activities to those obtained with fructose-fed normal controls. However, the stimulation of lung enzymes of diabetic animals can be effected either by fructose or by insulin. Antigen-antibody titrations and measurements of the rate of protein synthesis show that the increased activity of the lung and liver fatty acid synthetase is due to enhanced content rather than increased specific activity. These data suggest that insulin or fructose effects on fatty acid-synthesizing enzymes are mediated through intermediate(s) whose concentration is affected in the experimental diabetes. Furthermore, all tissues may not have stringent insulin requirements since the lung enzymes can be stimulated by fructose alone.

Fatty acid synthetase complex: Selective inactivation by phenylmethylsulphonyl fluoride

Abstract Reaction of pigeon and rat liver fatty acid synthetases with phenylmethylsulphonyl fluoride at pH 7.0 results in rapid and complete loss of activity for fatty acid synthesis. Acetyl and malonyl transacylation, two reductions, dehydration and condensation-CO 2 exchange reactions are not appreciably altered in the modified enzyme. However, the deacylation of palmityl CoA is completely inhibited. Complete inactivation results in the incorporation of about 1.9 moles of 14 C-phenylmethylsulphonyl groups/mole of the enzyme complex. These results suggest that either two moles of a fatty acyl deacylase or two deacylases with different fatty acyl chain length specificities may be functional in the enzyme complex.

Rat liver fatty acid synthetase: Inactivation of the component enzyme catalysing condensation-CO2 exchange reaction by chloroacetyl CoA

Summary Rat liver fatty acid synthetase is inhibited by chloroacetyl CoA. The inhibition is rapid and results from the covalent transfer of chloroacetyl group to the enzyme. A stoichiometric relationship exists between the moles of inhibitor bound and the loss of enzyme activity for fatty acid synthesis. Two moles of inhibitor are bound for complete inactivation and this inhibition results from the loss of activity for the critical condensation-CO2 exchange component reaction. These and other data are consistent with the presence of two sites on the enzyme for condensation between enzyme bound acetyl-and malonyl groups.

Ultrastructural analysis of mineralized matrix from human osteoblastic cells: Effect of tumor necrosis factor-alpha

Recent work by a number of investigators has demonstrated that the process of bone matrix formation and mineralization is under the influence of growth factors and cytokines present in the local environment. Utilizing primary and established osteoblast cell culture systems, these studies have examined the regulation of bone matrix protein synthesis and deposition into the extracellular matrix (ECM) and subsequent mineralization. In previous studies, we have utilized the human osteoblastic cell line, HOS TE85, to study the effects of Tumor Necrosis Factor - alpha (TNF-α) on the regulation of matrix proteins and proteolytic function in monolayer cultures as well as during the development and calcification of ECM formed by HOS TE85 cells during extended culture. Our studies demonstrate that TNF-α inhibited formation and mineralization of nodules. In the study reported here, we evaluated the ultrastructural morphology of the cell-matrix complex formed by HOS TE85 cells in the presence and absence of TNF-α at selected time points during the matrix development process utilizing both transmission electron microscopy and light microscopy. In the presence of TNF-α, the cell-matrix complex does not develop normally, with a lack of organization and mineralization, when compared to untreated cells. The lack of mineralization appears to result from the lack of normal collagen fibril deposition and formation of an appropriate ECM essential for the mineralization process. These results support our previous observations that TNF-α inhibits HOS TE85 cells from forming a mineralizing ECM by inhibiting incorporation of collagen into the ECM and inducing the synthesis of proteolytic enzymes capable of degrading collagen in the ECM.

Immunological and catalytic cross reactivity studies of fatty acid synthetase complexes from avian and mammalian livers

Abstract Fatty acid-synthetase complexes from avian and mammalian livers catalyse similar series of enzymatic reactions. We have studied the nature of immunological similarities and differences among these antigens by double diffusion analysis. Anti-chicken and anti-pigeon liver fatty acid-synthetase-γ-globulins give precipitin reactions with the antigens from avian livers, but not from rat liver, and the antibody prepared against the mammalian enzyme does not cross react with the avian enzymes. Minor antigenic differences seem to exist between the enzymes from avian livers as shown by the formation of spurs in immunodiffusion analysis. Since homlogous antibodies inhibit the activity for fatty acid-synthesis of the respective antigens, we considered the possibility of inhibition by nonhomologous antibodies. The antibodies against the avian enzymes inhibit the activity of avian enzymes though the titre values are considerably different. However, the rat liver enzyme is inhibited only to a small extent. Correspondingly, the antibody against the rat enzyme only partially inhibits the avian enzymes. Even though the degree of cross precipitation among avian and mammalian enzymes is absent, catalytic cross inhibition is exhibited by nonhomologous antibodies to a small extent. We also measured the activity for model reactions of fatty acid-synthesis in antigen · antibody complex to characterize the nature of inhibition by the antibody. Condensation-CO 2 exchange is the only reaction completely inhibited in the complex. The activities for other partial reactions show little or no change. To further characterize the nature of antigenic fragment, we have determined immunological and catalytic efficiency of the chicken enzyme modified with iodoacetic acid, iodoacetamide, palmitoyl CoA, trypsin and urea. The first four modifying agents lead to complete inhibition of activity for fatty acidsynthesis, but to only partial inhibition of activity for the reduction of acetoacetyl- N -acetyl cysteamine. However, the immunological reactivity is completely retained. The enzyme treated with 2 M urea gives results similar to other modifying agents but treatment with 4 and 6 M urea leads to complete loss of catalytic and immunological reactivity. These data imply that the major antigenic fragment encompasses the condensing site of the enzyme complex but that complete loss of immunological and catalytic reactivity requires complete disruption of overall enzyme conformation.

Irreversible inactivation of chicken liver fatty acid synthetase by its substrates acetyl and malonyl CoA: Effect of temperature and NADP+ on fatty acid and triacetic acid lactone synthesis

Abstract Chicken liver fatty acid synthetase is irreversibly inactivated by malonyl CoA and by acetyl and malonyl CoA. Two active forms of the enzyme existing above and below 11.5° are inactivated at different rates. Activities for fatty acid and triacetic acid lactone synthesis are lost at about the same rate and NADP + protects the enzyme against inactivation. Inactivation results from the enhanced covalent binding of malonyl groups in addition to those required for fatty acid synthesis.

Inactivation of chicken liver fatty acid synthetase by malonyl CoA

Abstract Chicken liver fatty acid synthetase is inactivated by malonyl CoA. The rate and extent of inactivation depends upon the ratio of concentration of malonyl CoA to enzyme rather than the absolute concentrations. The rate of inactivation is enhanced by acetyl CoA but is slowed down by NADP+. Inactivation does not result in the dissociation of enzyme complex and is complete when 6–7 moles of malonyl group are covalently bound per mole of the enzyme.