An active site-tyrosine-containing heptapeptide from D-amino acid oxidase

The flavoenzyme D-amino acid oxidase (Eo) is rapidly chlorinated by N-chloro-D-leucine (Rudie, N.G., Porter, D.J.T., and Bright, H.J. (1980) J. Biol. Chem. 255, 498-508). We have carried out chymotryptic digestion of E0-36Cl2 and find that all of the radiolabel is located in a heptapeptide having [3.5-36Cl2]chlorotyrosine as the COOH-terminal residue. This heptapeptide, having the sequence -Asp-Leu-Glu-Arg-Gly-Ile-Tyr-, is located within a larger fragment obtained previously from cyanogen bromide cleavage of E0. These results demonstrate that the target for chlorination in E0 must be a single tyrosine residue and provide, when taken together with previous findings, the first clear evidence for the identity and location of an active site residue in the polypeptide chain of D-amino oxidase.     

Carvedilol protects against cisplatin-induced oxidative stress, redox state unbalance and apoptosis in rat kidney mitochondria

Cisplatin is a highly effective chemotherapeutic agent which causes severe nephrotoxicity. Studies have suggested that reactive oxygen species, mainly generated in mitochondria, play a central role in cisplatin-induced renal damage. A wide range of antioxidants have been evaluated as possible protective agents against cisplatin-induced nephrotoxicity; however a safe and efficacious compound has not yet been found. The present study is the first to evaluate the protective potential of carvedilol, a beta-blocker with strong antioxidant properties, against the mitochondrial oxidative stress and apoptosis in kidney of rats treated with cisplatin. The following cisplatin-induced toxic effects were prevented by carvedilol: increased plasmatic levels of creatinine and blood urea nitrogen (BUN); lipid peroxidation, oxidation of cardiolipin; oxidation of protein sulfhydryls; depletion of the non-enzymatic antioxidant defense and increased activity of caspase-3. Carvedilol per se did not present any effect on renal mitochondria. It was concluded that carvedilol prevents mitochondrial dysfunction and renal cell death through the protection against the oxidative stress and redox state unbalance induced by cisplatin. The association of carvedilol to cisplatin chemotherapy was suggested as a possible strategy to minimize the nephrotoxicity induced by this antitumor agent.     

Prevalence and follow-up of occult HCV infection in an italian population free of clinically detectable infectious liver disease

Background

Occult hepatitis C virus infection (OCI) is a recently described phenomenon characterized by undetectable levels of HCV-RNA in serum/plasma by current laboratory assays, with identifiable levels in peripheral blood mononuclear cells (PBMCs) and/or liver tissue by molecular tests with enhanced sensitivity. Previous results from our group showed an OCI prevalence of 3.3% in a population unselected for hepatic disease. The present study aimed to evaluate OCI prevalence in a larger cohort of infectious liver disease-free (ILDF) subjects. Clinical follow-up of OCI subjects was performed to investigate the natural history of the infection.

Methods and Findings

439 subjects referred to a Turin Blood Bank for phlebotomy therapy were recruited. They included 314 ILDF subjects, 40 HCV-positive subjects and 85 HBV-positive subjects, of whom 7 were active HBV carriers. Six subjects (4/314 ILDF subjects [1.27%] and 2/7 active HBV carriers [28%]) were positive for HCV-RNA in PBMCs, but negative for serological and virological markers of HCV, indicating OCI. HCV genotypes were determined in the PBMCs of 3/6 OCI subjects two had type 1b; the other had type 2a/2c. OCI subjects were followed up for at least 2 years. After 12 months only one OCI persisted, showing a low HCV viral load (3.73×10¹ UI/ml). By the end of follow-up all OCI subjects were negative for HCV. No seroconversion, alteration of liver enzyme levels, or reduction of liver synthesis occurred during follow-up.

Conclusions

This study demonstrated the existence of OCI in ILDF subjects, and suggested a high OCI prevalence among active HBV carriers. Follow-up suggested that OCI could be transient, with a trend toward the decrease of HCV viral load to levels undetectable by conventional methods after 12–18 months. Confirmation studies with a longer follow-up period are needed for identification of the OCI clearance or recurrence rates, and to characterize the viruses involved.     

Pulsatile Flow Inside Moderately Elastic Arteries,Its Modelling and Effects of Elasticity

Pulsatile flow inside a moderately elastic circular conduit with a smooth expansion is studied as a model to understand the influence of wall elasticity in artery flow. The solution of the simultaneous fluid-wall evolution is evaluated by a perturbative method, where the zeroth order solution is represented by the flow in a rigid vessel; the first order correction gives the wall motion and induced flow modification without the need to solve the difficult coupled problem. Such an approach essentially assumes a locally infinite celerity, therefore it represent a good approximation for the fluid-wall interaction in sites of limited extent (branches, stenosis, aneurism, etc.), which include typical situations associated with vascular diseases. The problem is solved numerically in the axisymmetric approximation; the influence of wall elasticity on the flow and on the unsteady wall shear stress is studied in correspondence of parameters taken from realistic artery flow. Attention is posed to the role of phase difference between the incoming pressure and flow pulses.     

Antioxidant activity of isocoumarins isolated from Paepalanthus bromelioides on mitochondria

The isocoumarins (1-50 microM) paepalantine (9,10-dihydroxy-5,7-dimethoxy-1H-naptho(2,3c)pyran-1-one), 8,8'-paepalantine dimer, and vioxanthin isolated from Paepalanthus bromelioides, were assessed for antioxidant activity using isolated rat liver mitochondria and non-mitochondrial systems, and compared with the flavonoid quercetin. The paepalantine and paepalantine dimers, but not vioxanthin, were effective at scavenging both 1,1-diphenyl-2-picrylhydrazyl (DPPH(*)) and superoxide (O(2)(-)) radicals in non-mitochondrial systems, and protected mitochondria from tert-butylhydroperoxide-induced H(2)O(2) accumulation and Fe(2+)-citrate-mediated mitochondrial membrane lipid peroxidation, with almost the same potency as quercetin. These results point towards paepalantine, followed by paepalantine dimer, as being a powerful agent affording protection, apparently via O(2)(-) scavenging, from oxidative stress conditions imposed on mitochondria, the main intracellular source and target of those reactive oxygen species. This strong antioxidant action of paepalantine was reproduced in HepG2 cells exposed to oxidative stress condition induced by H(2)O(2).     

DNA polymerase switching: effects on spontaneous mutagenesis in Escherichia coli

Escherichia coli possesses five known DNA polymerases (pols). Pol III holoenzyme is the cell's main replicase, while pol I is responsible for the maturation of Okazaki fragments and filling gaps generated during nucleotide excision repair. Pols II, IV and V are significantly upregulated as part of the cell's global SOS response to DNA damage and under these conditions, may alter the fidelity of DNA replication by potentially interfering with the ability of pols I and III to complete their cellular functions. To test this hypothesis, we determined the spectrum of rpoB mutations arising in an isogenic set of mutL strains differentially expressing the chromosomally encoded pols. Interestingly, mutagenic hot spots in rpoB were identified that are susceptible to the actions of pols I–V. For example, in a recA730 lexA (Def) mutL background most transversions were dependent upon pols IV and V. In contrast, transitions were largely dependent upon pol I and to a lesser extent, pol III. Furthermore, the extent of pol I-dependent mutagenesis at one particular site was modulated by pols II and IV. Our observations suggest that there is considerable interplay among all five E. coli polymerases that either reduces or enhances the mutagenic load on the E. coli chromosome.     

Phenobarbital and 6-aminonicotinamide effect on cerebral enzymatic activities related to energy metabolism in different rat brain areas

The effect of phenobarbital (100 mg/kg i.p.) and 6-aminonicotinamide (6AN) (35 mg/kg i.p.) on enzyme activities related to energy transduction was investigated on the homogenate in toto, non-synaptic mitochondrial fraction and synaptosomal fraction isolated from different rat brain areas (cerebral cortex, hippocampus, hypothalamus, striatum, and medulla oblongata). 6AN treatment decreased: (a) phosphofructokinase in all the areas tested; (b) lactate dehydrogenase on the homogenate in toto in striatum and hypothalamus, and on the synaptosomal fraction in cerebral cortex and corpus striatum; (c) succinate dehydrogenase on non-synaptic mitochondrial fraction in hippocampus and striatum. Finally, aspartate aminotransferase was increased on non-synaptic mitochondrial fraction in striatum and medulla oblongata. Phenobarbital treatment induced an increase of total NADH cytochrome c reductase on mitochondrial fraction in hippocampus and hypothalamus, and a decrease of cytochrome oxidase activity on non-synaptic mitochondrial fraction in hypothalamus and medulla oblongata.