The induction of microsomal NADPH:cytochrome P450 and NADH:cytochrome b(5) reductases by long-term salt treatment of cotton (Gossypium hirsutum L.) and bean (Phaseolus vulgaris L.) plants.

We studied the effect of salinity on the activity of microsomal NADPH:cytochrome P450 reductase (CPR, EC 1.6.2.4) and NADH:ferricytochrome b(5) oxidoreductase (B5R, EC 1.6.2.2) in two dicotyledonous plant species differing in their sensitivity to salt, cotton (Gossypium hirsutum L. cv Ogosta) and common bean (Phaseolus vulgaris L. cv Dobrujanski 7). A significant inhibition of fresh weight of salt-treated bean plants was observed, while cotton was affected to a much lesser degree. NaCl application resulted in a significant increase in the activity of both reductases, but was more pronounced in salt-tolerant cotton. We suppose that alterations in B5R and CPR activities may be targeted to the maintenance of membrane lipids. Most probably, plants use both enzymes (B5R and CPR) and their respective electron donors (NADH and NADPH) to reduce cytochrome b(5), which can donate reducing equivalents to a series of lipid-modification reactions such as desaturation and hydroxylation.     

Thermodynamic and kinetic properties of the outer membrane cytochrome OmcF,a key protein for extracellular electron transfer in Geobacter sulfurreducens

Gene knock-out studies on Geobacter sulfurreducens have shown that the monoheme c-type cytochrome OmcF is essential for the extracellular electron transfer pathways involved in the reduction of iron and uranium oxy-hydroxides, as well as, on electricity production in microbial fuel cells. A detailed electrochemical characterization of OmcF was performed for the first time, allowing attaining kinetics and thermodynamic data. The heterogeneous electron transfer rate constant was determined at pH?7 (0.16?±?0.01?cm?s^?1) indicating that the protein displays high electron transfer efficiency compared to other monoheme cytochromes. The pH dependence of the redox potential indicates that the protein has an important redox-Bohr effect in the physiological pH range for G. sulfurreducens growth. The analysis of the structures of OmcF allowed us to assign the redox-Bohr centre to the side chain of His⁴⁷ residue and its pK_a values in the reduced and oxidized states were determined (pK_ox?=?6.73; pK_red?=?7.55). The enthalpy, entropy and Gibbs free energy associated with the redox transaction were calculated, pointing the reduced form of the cytochrome as the most favourable. The data obtained indicate that G. sulfurreducens cells evolved to warrant a down-hill electron transfer from the periplasm to the outer-membrane associated cytochrome OmcF.     

Effect of Retreatment with Interferon Alone or Interferon plus Ribavirin on Hepatitis C Virus Quasispecies Diversification in Nonresponder Patients with Chronic Hepatitis C

Alpha interferon (IFN-alpha) treatment is effective on a long-term basis in only 15 to 25% of patients with chronic hepatitis C. The results of recent trials indicate that response rates can be significantly increased when IFN-alpha is given in combination with ribavirin. However, a large number of patients do not respond even to combination therapy. Nonresponsiveness to IFN is characterized by evolution of the hepatitis C virus (HCV) quasispecies. Little is known about the changes occurring within the HCV genomes when nonresponder patients are retreated with IFN or with IFN plus ribavirin. In the present study we have examined the genetic divergence of HCV quasispecies during unsuccessful retreatment with IFN or IFN plus ribavirin. Fifteen nonresponder patients with HCV-1 (4 patients with HCV-1a and 11 patients with HCV-1b) infection were studied while being retreated for 2 months (phase 1) with IFN-alpha (6 MU given three times a week), followed by IFN plus ribavirin or IFN alone for an additional 6 months (phase 2). HCV quasispecies diversification in the E2 hypervariable region-1 (HVR1) and in the putative NS5A IFN sensitivity determining region (ISDR) were analyzed for phase 1 and phase 2 by using the heteroduplex tracking assay and clonal frequency analysis techniques. A major finding of this study was the relatively rapid evolution of the HCV quasispecies observed in both treatment groups during the early phase 1 compared to the late phase 2 of treatment. The rate of quasispecies diversification in HVR1 was significantly higher during phase 1 versus phase 2 both in patients who received IFN plus ribavirin (P = 0.017) and in patients who received IFN alone (P = 0. 05). A trend toward higher rates of quasispecies evolution in the ISDR was also observed during phase 1 in both groups, although the results did not reach statistical significance. However, the NS5A quasispecies appeared to be rather homogeneous and stable in most nonresponder patients, suggesting the presence of a single well-fit major variant, resistant to antiviral treatment, in agreement with published data which have identified an IFN sensitivity determinant region within the NS5A. During the entire 8 months of retreatment, there was no difference in the rate of fixation of mutation between patients who received combination therapy and patients who were treated with IFN alone, suggesting that ribavirin had no major effects on the evolution of the HCV quasispecies after the initial 2 months of IFN therapy.