Molecular epidemiology of norovirus from patients with acute gastroenteritis in northwestern Spain.

The high incidence of norovirus (NoV) infections seems to be related to the emergence of new variants that evolved by genetic drift of the capsid gene. In this work, that represents a first effort to describe the molecular epidemiology of NoV in the northwest of Spain, a total of eight different NoV genotypes (GII.1, GII.3, GII.4, GII.6, GII.7, GII.12, GII.13, GII.14) were detected. The major genotypes observed were GII.4 (45·42%) and GII.14 (34·9%), being detected in all age groups. In addition, and although most of GII.4 sequences belonged to 2006b (7·2%) and 2010 (50·35%) variants, the presence of new NoV variants was observed. Phylogenetic analysis revealed that a high number of GII.4 sequences (35·24%) could be assigned to the newly emerging Sydney 2012 variant, even during late 2010. The high prevalence of NoV GII.14 observed in this study may indicate the emergence of this genotype in Spain.

Genotyping of hepatitis A virus detected in bivalve shellfish in Galicia (NW Spain).

Hepatitis A virus (HAV) represents a significant public health problem due to its high persistence in the environment and its transmission through contaminated water and food. Bivalve shellfish are filter feeders that can bioaccumulate human pathogens found in contaminated waters, their consumption being a potential cause of hepatitis A outbreaks. In this work, cultured and wild bivalve shellfish from the Ría de Vigo (Galicia, NW Spain) were analysed for the presence and genotyping of HAV. A total of 160 shellfish samples were collected between March 2004 and December 2006, including 68 samples from cultured mussels (Mytilus galloprovincialis), 30 from wild clams (Rupitapes decussatus), 31 from wild cockles (Cerastoderma edule) and 31 from wild mussel. HAV detection, carried out by quantitative RT-PCR, was positive for 29 (42.6%) cultured and 40 (43.5%) wild samples, with levels ranging from 3.1 x 10(2) and 1.4 x 10(10) RNA copies/g of shellfish digestive tissue. The phylogenetic analysis of VP1-P2A and VP3-VP1 regions, separately or as concatenated sequences, revealed that all HAV strains analysed belong to subgenotype IB. These results indicate a high prevalence of this subgenotype in the area studied.

Measurement of relative antioxidant activity of compounds: a methodological note.

The relative activities of some hydrogen-donating antioxidants were assessed by comparing their activities with that of Trolox (Trolox equivalent antioxidant capacity, TEAC) for scavenging the ABTS radical cation (ABTS.+) generated in the aqueous phase. We have verified, however, that TEAC values may change with the concentration of compounds and with the measuring times used. Not withstanding, TEAC values do not differ significantly if the compounds have kinetic curves of ABTS.+ formation similar to that of Trolox. This is the case with ascorbic acid, whose TEAC values, determined by using five concentrations at three different measuring times, are very close. For the flavonoids studied (catechin, rutin, naringenin and silibinin) which have kinetic curves of ABTS.+ formation different from that of Trolox, the TEAC values decrease with increasing concentrations of the compounds for each measuring time, and increase with increasing measuring times for each concentration. In the present study, we conclude that, in order to evaluate relative antioxidant activities of compounds by the ABTS assay, it is essential to perform kinetic studies to assess scavenging of ABTS.+ by these compounds. Therefore, when the TEAC values of compounds are determined for more than one measuring time, we may be sure that all the antioxidant potential of compounds is being considered and whether or not it is possible to establish a hierarchy for their antioxidant activities.

[Antioxidant effect of drugs used in cardiovascular therapy]. / Efeito antioxidante de Fármacos usados em terapêutica cardiovascular.

Oxidative stress is implicated in the pathogenesis of various cardiovascular disorders. The knowledge, characterisation and comparison of the different antioxidant properties of some cardiovascular drugs might lead to new therapeutic approaches. Blood constituents are biological products easier to obtain. Their oxidative damage is accepted to be involved in several pathogenic pathways such as atherogenesis. Blood products can be utilised as in vitro models of macromolecular and cellular oxidative damage. It is well known that transition metals catalyse the generation of more damaging reactive oxygen species. We studied the antioxidant effect of four beta-adrenergic blocking agents (pindolol, propranolol, atenolol and metoprolol) and of one calcium channel antagonist (nifedipine), on the plasma oxidative damage induced by copper. Lipid peroxidation was evaluated by measuring fluorescent substances. We added the drugs to the assay system, before and after the induction of peroxidation by copper, to elucidate their ability to prevent and/or block lipid peroxidation. We observed that pindolol, propranolol and nifedipine have antioxidant properties, in the assay system, when added 15 min before the addition of copper. This effect is demonstrated by the delay in initiation and decrease in formation of lipid peroxidation products. Nifedipine also has a remarkable chain breaking effect. We compared the drugs with ascorbic acid in their relative antioxidant effect: nifedipine > ascorbate > pindolol > propranolol.

[Stress]. / Stress.

The general adaptation syndrome is discussed on the light of recent discoveries on hypothalamic peptides and of their possible influence in survival and in induction of diseases. The problem of stress in alcoholism is reviewed. The author ends with a short souvenir of Hans Selye.

[Effect of silibinin on oxidative damage of blood constituents]. / Effet de la silibinine sur la lésion oxydante des constituants du sang.

Silibinin (SDH) is a flavonoid with ascertained hepatoprotective effects, which have been partially attributed to its antioxidant properties. Oxidation of blood constituents could have a role in atherogenesis and interfere with the rheologic properties of the blood. In this study we investigated, whether SDH could protect some blood constituents against oxidative modification. In human plasma we measured TBARS and fluorescence generation as indicators of copper or azobis amidinopropane hydrochloride (AAPH) at 760 mm Hg PO2-induced lipid peroxidation. SDH at 50 microM inhibited copper-induced TBARS formation by 25% and fluorescence by 47%. SDH also inhibited AAPH-induced lipid peroxidation, but at 175 microM concentration only. Oxidative modification of albumine was evaluated by fluorescence generation. SDH at 50 microM inhibited copper/hydrogen peroxide fluorescence generation by 54% and at 2.5 microM it inhibited EDTA-Fe (II)/hydrogen peroxide fluorescence generation by 31%. The protection of albumin by SDH was confirmed by SDS-PAGE electrophoresis. Copper-induced red-cell lipid peroxidation was evaluated by TBARS formation. SDH at 250 microM inhibited copper-induced lipid peroxidation and hemolysis by 45% and 94%, respectively. SDH also inhibited hemolysis in red-cell suspensions exposed to hydrogen peroxide, but not lipid peroxidation. Our results show that SDH may protect blood constituents from oxidative damage.

[Alcohol and free radicals. Various consequences: protein synthesis, endocrine disorders, immunity. Role of stress]. / Alcool e radicais livres. Algumas consequências: síntese proteica, disendocrinias, imunidade. Importância do stress.

Ethanol is a powerful generator of oxygen free radicals, when metabolized in the liver or in other organs. Isoenzyme 2E1 of cytochrome P450 and aldehyde oxidase are the main mechanisms for the generation of these radicals. A consequence of free radical generation is a decrease in protein synthesis. As a result we have endocrine and immunity alterations. The paper ords with a brief discussion of stress associated to alcoholism.

Superoxide anion radical generation during the oxidation of various amines by diamine oxidase.

Diamine oxidase (DAO) or histaminase is an enzyme which deaminates histamine and several aliphatic amines to their corresponding aldehydes. Hydrogen peroxide and ammonia are side products of this reaction. The purpose of the present work was to evaluate if determination of produced hydrogen peroxide reflects DAO activity or if intermediate formation of the superoxide radical could be a reason for lack of correspondence between oxygen uptake and hydrogen peroxide production at different pH. Superoxide radical formation was determined by cytochrome c reduction in the presence and absence of superoxide dismutase (SOD). Oxygen uptake was measured with an oxygen electrode and hydrogen peroxide production by a spectrophotometric method. At pH 6.6 there was no superoxide production, but at pH 7.4 there was some, and it increased markedly at pH 9.5. Oxygen uptake also increased with increasing pH, especially with histamine as substrate. These results lead us to suggest that the mechanism of action of DAO involves the intermediate generation of superoxide radicals.

Different effects of thiol and nonthiol ace inhibitors on copper-induced lipid and protein oxidative modification.

Differences among angiotensin-converting enzyme inhibitors (ACEI) in scavenging reactive oxygen species were described and mainly attributed to the presence or absence of a thiol group. Plasma constituents and red cells are known targets for oxidative damage. Transition metals, like copper, are well known catalizers of free radical generation. In the present study we compared the abilities of captopril (a thiol ACEI), enalaprilat, and lisinopril (two nonthiol ACEI) for inhibiting copper-induced thiobarbituric acid reactive substances (TBARS) formation and fluorescence generation in whole human plasma and low-density lipoprotein. The effects of those ACEI on copper/hydrogen peroxide-induced fluorescence development and electrophoretic mobility modification in albumin and on copper-induced TBARS formation and hemolysis in human red cells were also compared. Captopril was more effective than the two nonthiol ACEI in inhibiting plasma and LDL lipid peroxidation, but it was ineffective in inhibiting the albumin oxidative modification that was moderately inhibited by enalaprilat and lisinopril. On the contrary, the inhibitory effects of the three ACEI on copper-induced lipid peroxidation and hemolysis in red cell suspensions were more uniform. This as yet unreported red cell protective effect may deserve pharmacological evaluation. Our results show that captopril is a more effective antioxidant than the nonthiol ACEI in some systems. However, the nonthiol ACEI also have the ability to partially protect some targets against oxidative damage. These observations suggest that the presence of a thiol group in the ACEI structure is not the only determinant for the antioxidant properties.

Hyaluronic acid in progressive systemic sclerosis.

BACKGROUND: The glycosaminoglycans metabolism is disturbed in progressive systemic sclerosis (PSS). Serum hyaluronic acid (HA) is elevated in this disease. OBJECTIVE: This study was conducted to determine the HA plasma concentrations of patients with PSS according to the different stages of the disease. METHODS: We studied 48 patients divided into three subgroups: subgroup 1 (n = 10), with skin compromise without evidence of other organ involvement; subgroup 2 (n = 21), with skin and esophagus involvement; subgroup 3 (n = 17), with skin, lung and other internal organ involvement. A radiometric assay was performed for quantification of HA. RESULTS: Our results confirm the increase in plasma HA in patients with PSS. They also suggest that lung involvement is the main feature responsible for high plasma concentrations of HA. The plasma HA levels were elevated in patients compared to normals (p <0.001). Significant differences were observed between subgroups 1 and 3 (p <0.01) and between subgroups 2 and 3 (p <0.01). A positive correlation between disease severity scores and plasma HA values was observed (p <0.01). CONCLUSION: An important elevation of HA plasma levels could be a serologic marker of disease severity, progression and degree of visceral involvement.

Kinetics of the inhibition of xanthine dehydrogenase and of the reversible and irreversible forms of xanthine oxidase by silibinin and bendazac.

Xanthine oxidase exists in vivo predominantly as a NAD(+)-dependent dehydrogenase form (xanthine dehydrogenase) which can be transformed into oxygen-dependent oxidase forms as a result of sulfhydryl oxidation (reversible xanthine oxidase) or proteolysis (irreversible xanthine oxidase). Xanthine oxidase has been hypothesized to be a potential source of oxygen-derived free radicals during reperfusion of ischemic tissues. Xanthine dehydrogenase was purified from rat liver and converted into reversible xanthine oxidase by heating at 37 °C and into irreversible xanthine oxidase by proteolysis with trypsin. Silibinin and bendazac are compounds used in therapeutics and to which free radical scavenging properties were ascribed. The effects of the compounds silibinin and bendazac on the different forms of the enzyme were studied. Silibinin inhibited all the forms of the enzyme but bendazac inhibited only reversible and irreversible xanthine oxidase. The inhibitions seem to be mixed non-competitive-competitive. The authors discuss the hypothesis that selective inhibitors of xanthine oxidase, preventing the interruption of uric acid formation, may have some advantage over the inhibitors of both xanthine dehydrogenase and xanthine oxidase in the treatment and prevention of situations such as ischemia and reperfusion syndromes.

[Pathologic states related to alcoholism]. / Patologias relacionadas com o alcoolismo.

The Author reviews the pathological consequences of chronic alcoholism with particular emphasis to the consequences of malnutrition, especially vitamin B-1 deficiency, immune deficiencies, adaptation of cytochrome 2E1, testicular atrophy, alterations of the skin, bones, liver, muscles, cardiac and neuropsychiatric.

[An analysis of plasma histaminase in schizophrenic patients and its relationship to clinical parameters]. / Análise da histaminase plasmática em doentes esquizofrénicos e sua relação com paraâmetros clínicos.

Previous studies have investigated putative alterations in histamine and histamine receptors in schizophrenia, and evidence in favour of the role of this amine as a neurotransmitter or as a neuromodulator was found. In the present study the activity of plasmatic histaminase was analysed, with histamine and with cadaverine as substrates, in a group of 23 schizophrenic patients and compared with that of healthy controls (n = 32). Plasma histaminase activity was determined using Gordon and Peters spectrophotometric method, and the results were expressed in mumoles of H2O2 transformed/hour/litre of plasma at 25 degrees C. Plasmatic histaminase, using histamine as substrate, was significantly increased in schizophrenic patients as a whole compared with the healthy controls. On the other hand, when cadaverine was used as substrate plasma histaminase was significantly reduced in female schizophrenics but not in males. When patients were divided according to 17 clinical characteristics it was found that the following subgroups were significantly associated to high levels of plasma histaminase (using histamine as substrate): the non mentally deteriorated schizophrenic patients compared with mentally deteriorated schizophrenic patients and those with thymic symptoms as opposed to those without thymic symptoms. An extension of this series and a prospective analysis are required to further define the clinical and biological significance of the alteration of this biochemical parameter in schizophrenia, and particularly in relation with a subgroup of schizophrenia with a more favourable course.

Evidence for free radical generation due to NADH oxidation by aldehyde oxidase during ethanol metabolism.

Several studies associate ethanol hepatic toxicity to the generation of reactive oxygen species. Ethanol metabolism by alcohol dehydrogenase (ADH) originates acetaldehyde and NADH, with the subsequent increase of the NADH/NAD+ ratio. Some authors have suggested that the oxidation of acetaldehyde by aldehyde oxidase (AO) may be responsible for oxyradical generation during ethanol metabolism. In this study we demonstrated that AO acts not only upon acetaldehyde but also upon NADH, with superoxide anion radical (O2.-) formation. The apparent Km of NADH for AO was approximately 28 microM, a much smaller value than the one reported for acetaldehyde (1 mM). The NADH oxidation by AO promoted the O2.- generation and the ADP-Fe(3+)-dependent microsomal lipid peroxidation in a NADH and AO concentration-dependent manner. If in these experiments NADH is substituted by ethanol, NAD+, and ADH, a higher level of lipid peroxidation will be obtained. To explain this observation a vicious cycle which increases the oxyradical production is suggested: ADH reduces NAD+ to NADH, which is oxidized by AO, generating reactive oxidative species plus NAD+ available again for reduction by ADH. From the studies which were done in the presence of some antioxidants it was observed that the addition of SOD and/or catalase did not inhibit lipid peroxidation, but these results do not exclude the participation of reactive oxygen species. Our studies indicate that the NADH oxidation by AO may play a role in ethanol-induced generation of reactive oxygen species, contributing to its hepatotoxicity.

Effects of zinc on copper-induced and spontaneous lipid peroxidation.

Zinc (Zn) is an essential nonredox metal that has been regarded as having antioxidant properties. Some epidemiological indications and therapeutic results point to a role of Zn in restricting the development and the progression of some diseases. Redox-active metals like iron and copper are involved in oxidative injury mechanisms, and a decrease in the Zn:Cu ratio may be associated with certain pathologies. We studied the effect of Zn on the copper-induced lipid peroxidation in diluted human plasma. Lipid peroxidation was evaluated by measuring the formation of conjugated dienes and of thiobarbituric acid reactive products. We found that 20 microM Zn reduced the 125-microM copper-dependent formation of conjugated dienes by 27% and of thiobarbituric acid reactive products by 49%, during a 3-h incubation period. The inhibition of lipid peroxidation by 125 microM Zn is almost total in the same conditions. The time-course study of the inhibitory effect of 125 microM Zn showed that it lasted for 7 h, which was the maximum incubation period tested. We also found that Zn had an inhibitory effect on the spontaneous lipid peroxidation in rat brain whole homogenates. Our results support the antioxidant properties of Zn, which may be potentially relevant to the protection of human plasma constituents, competing with the transition metals for redox reactions.

pH dependence of lipid peroxidation and albumin oxidative modification: possible implications to the pH paradox.

The hydrogen ion concentration may have an important influence in biological free radical reactions. We studied the effect of an acidic pH on two models of free radical-mediated damage: copper-induced lipid peroxidation in plasma and copper/hydrogen peroxide-induced oxidative modification of albumin. A reduction of pH from 7·4 to 6·6 decreased diene conjugation by 32%, thiobarbituric acid-reactive substances formation by 25% and fluorescence generation by 53% in plasma exposed to cupric chloride. At pH values lower than 6·6 an even greater inhibition of lipid peroxidation in plasma was obtained. Visible fluorescence development in albumin by exposure to site-specific generation of free radicals was also increasingly reduced by decreasing pH values. From pH 7·4 to 6·6 there was a 50% fluorescence generation inhibition. The observed partial protection of lipids and proteins against oxidative damage by an acidic pH alerts to the need for rigorously controlling the pH values when assaying compounds for antioxidant properties in vitro. It may also contribute to the explanation for the protective effect of an acidic pH against anoxic cell injury and for cell death that is precipitated by a rapid return to a normal pH following reperfusion (the 'pH paradox').

[Effect of aluminum on the non-enzymatic oxidation of dopamine]. / Efeito do alumínio na oxidação não enzimática da dopamina.

The effect of different concentrations of aluminum sulphate on the nonenzymatic oxidation of dopamine was studied in order to evaluate the action of this metal on neuromelanin synthesis. Data shows that under the studied conditions, aluminum partially inhibits dopamine self-oxidation, decreasing the formation of some intermediate compounds, namely dopaminequinone and dopaminochrome. If neuromelanins have a cytoprotective function in the central nervous system, possibly acting as intracellular scavengers of free radicals and redox metal ions, their decrease due to aluminum could be responsible for serious damage to neuronal tissues.

Scavenging of reactive oxygen species by silibinin dihemisuccinate.

Silibinin dihemisuccinate (SDH) is a flavonoid of plant origin with hepatoprotective effects which have been partially attributed to its ability to scavenge oxygen free radicals. In the present paper the antioxidant properties of SDH were evaluated by studying the ability of this drug to react with relevant biological oxidants such as superoxide anion radical (O2-), hydrogen peroxide (H2O2), hydroxyl radical (HO.) and hypochlorous acid (HOCl). In addition, its effect on lipid peroxidation was investigated. SDH is not a good scavenger of O2- and no reaction with H2O2 was detected within the sensitivity limit of our assay. However, it reacts rapidly with HO. radicals in free solution at approximately diffusion-controlled rate (K = (1.0-1.2) x 10(10)/M/sec) and appears to be a weak iron ion chelator. SDH at concentrations in the micromolar range protected alpha 1-antiproteinase against inactivation by HOCl, showing that it is a potent scavenger of this oxidizing species. Luminol-dependent chemiluminescence induced by HOCl was also inhibited by SDH. The reaction of SDH with HOCl was monitored by the modification of the UV-visible spectrum of SDH. The studies on rat liver microsome lipid peroxidation induced by Fe(III)/ascorbate showed that SDH has an inhibitory effect, which is dependent on its concentration and the magnitude of lipid peroxidation. This work supports the reactive oxygen species scavenger action ascribed to SDH.