Reduction of the Fe(III)-tyrosyl radical center of Escherichia coli ribonucleotide reductase by dithiothreitol

The active form of protein B2, the small subunit of ribonucleotide reductase from Escherichia coli, contains a binuclear ferric center and a free radical localized to tyrosine 122 of the polypeptide chain. MetB2 is an inactive form that lacks the tyrosine radical but retains the Fe(III) center. We earlier reported (Fontecave, M., Eliasson, R., and Reichard, P. (1989) J. Biol. Chem. 264, 9164-9170) that enzymes from E. coli interconvert B2 and metB2, possibly as part of a regulatory mechanism. Introduction of the tyrosyl radical into metB2 occurred in two steps: first, the Fe(III) center was reduced to Fe(II), generating "reduced B2"; next oxygen regenerated non-enzymatically both Fe(III) and the tyrosyl radical. Here we demonstrate that dithiothreitol (DTT) between pH 8 and 9.5 also slowly converts metB2 to B2 in the presence of oxygen. Also in this case the reaction occurs stepwise with reduced B2 as an intermediate. DTT reduces Fe(III) of both metB2 and B2. In the latter case this reaction is accompanied by the immediate loss of the tyrosyl radical. Our results indicate that the tyrosyl radical can exist only in the presence of an intact Fe(III) center. In reduced B2 iron is loosely bound to the protein, dissociates on standing and is readily removed by chelating agents. Binding decreases at higher pH. Loss of iron from reduced B2 explains why ferrous iron stimulates and iron chelators inhibit reactivation of metB2. We propose that the reactivation of mammalian ribonucleotide reductase by DTT (Thelander, M., Gr?slund, A., and Thelander, L. (1983) Biochem. Biophys. Res. Commun. 110, 859-865) may proceed via a mechanism similar to the one found here for E. coli protein B2.     

Genetic diversity in oak populations under intensive management for fuelwood in the Sierra de Zongolica,Mexico

Firewood and charcoal are used on a daily basis both in rural areas and in cities. This type of energy is produced by one of the most ancient traditional methods, known as coppice, which harvest tree sprouts. There is controversy about its effects on forests: it preserves populations and tree cover of species used, but reduces density, inhibits sexual reproduction and generates genetic erosion. We inquired if it was possible to identify a loss of genetic diversity in oak populations traditionally used for charcoal by the Zongolica Nahuas in Veracruz state, Mexico. We studied populations of Quercus laurina, Quercus calophylla and Quercus rugosa in three different altitudes. Molecular analysis with eight nuclear codominant microsatellites was performed to determine the diversity, structure and gene flow of these species. Results for Q. laurina were Na = 8.458, I = 1.766, Ho = 0.679, polymorphism = 100%, Fis = 0.079, with intraindividual variation of 81.55%. For Q. calophylla: Na = 7.250, I = 1.563, Ho = 0.646, polymorphism = 91.67%, Fis = 0.083, with intraindividual variation of 83.80%. For Q. rugosa: Na = 6.958, I = 1.510, Ho = 0.574, polymorphism = 91.67%, Fis = 0.204, with intraindividual variation of 81.99%; this species shows signals of an early genetic isolation process. Our findings indicate that Quercus genetic diversity for the three species is high and comparable with oak species in Mexico and worldwide. We conclude that at the present, coppice is preserving a historical diversity in adult trees kept alive through sprouting. Nonetheless, problems with coppice systems elsewhere, unregulated harvesting and expansion of pine plantation in the region suggest that further studies, hand in hand with a landscape management approach that improve charcoal and firewood production, may be valuable for Sierra de Zongolica genetic biodiversity conservation.     

Trypanosoma cruzi: cruzipain and membrane-bound cysteine proteinase isoform(s) interacts with human alpha(2)-macroglobulin and pregnancy zone protein

Plasmatic levels of pregnancy zone protein (PZP) increase in children with acute Chagas disease. PZP, as well as alpha2-macroglobulin (alpha2-M), are able to interact with Trypanosoma cruzi proteinases. The interaction of alpha2-M and PZP with cruzipain, the major cysteine proteinase of T. cruzi, was investigated. Several molecular changes on both alpha-M inhibitors under reaction with cruzipain were found. PAGE analysis showed: (i) formation of complexes of intermediate mobility and tetramerization of native alpha2-M and PZP, respectively; (ii) limited proteolysis of bait region in alpha2-M and PZP, and (iii) covalent binding of cruzipain to PZP and alpha2-M. Conformational and structural changes experimented by alpha-Ms correlate with modifications of the enzyme electrophoretic mobility and activity. Cruzipain-alpha-M complexes were also detected by gelatin SDS-PAGE and immunoblotting using polyclonal anti-cruzipain antibodies. Concomitantly, alpha2-M and PZP impaired the activity of cruzipain towards Bz-Pro-Phe-Arg-pNA substrate. In addition, alpha-Ms were able to form covalent complexes with membrane isoforms of cysteine proteinases cross-reacting with cruzipain. The present study suggests that both human alpha-macroglobulin inhibitors could prevent or minimize harmful action of cruzipain on host's molecules and hypothetically regulate parasite functions controlled by cruzipain.     

On the promoting action of tamoxifen in a model of hepatocarcinogenesis induced by p-dimethylaminoazobenzene in CF1 mice

BACKGROUND AND AIMS: Tamoxifen (TMX) has proven to be an effective palliative treatment for advanced breast cancer with low reported incidence of side effects. TMX has been demonstrated to be an initiator and/or a promoter in the rat model of hepatocarcinogenesis. To document the long-term effect of TMX in mice treated with p-dimethylaminoazobenzene (DAB), we have investigated the time response action of these drugs on different biochemical parameters. METHODS: A group of animals was placed on dietary DAB (0.5%, w/w) during a period of 28 weeks. Control animals received a standard laboratory diet. Two other groups of non-treated and DAB-treated animals received TMX citrate (0.025%, w/w) in the diet since day 20. RESULTS: The activities of the enzymes involved in heme synthesis and degradation as evaluated in the DAB group was not further affected by TMX. DAB and/or TMX treatment significantly increased the content of total cytochrome P450 and also the activity of glutathione S-transferase indicating liver damage. In all treated groups oxidative stress and an adaptive response of the natural defense system (catalase and superoxide dismutase) were demonstrated. Histological and morphological studies revealed liver cell hyperplasia in DAB treated group; however, only in the DAB+TMX group solid, trabecular and acinar hepatocellular carcinoma was confirmed at the end of the experimental trial. CONCLUSION: We have demonstrated that TMX produced changes in hepatic enzyme activities which may be relevant for the metabolism and disposition of this and/or other drugs. Because liver tumors could be initiated and promoted by several agents which need to be activated, the possible hazard of TMX should be considered. This study reports that long-term treatment with TMX enhances hepatocarcinogenesis induced by DAB. The widespread use of TMX as an anticancer agent adds to the significance of this study.     

Survival of spray-dried <Emphasis Type="Italic">Lactobacillus kefir</Emphasis> is affected by different protectants and storage conditions

Survival of two Lactobacillus kefir strains after spray drying in reconstituted skim milk with or without the addition of 12.5 g monosodium glutamate/l, 20 g sucrose/l, or 20 g fructo-oligosaccharides (FOS)/l and during subsequent storage under different conditions of temperature (20 and 30°C) and relative humidity (RH) (0, 11 and 23%) was evaluated. After being dried, L. kefir 8321 and L. kefir 8348 had a decrease in viability of 0.29 and 0.70 log cfu/ml respectively, while the addition of different protectants improved the survival of both strains significantly. During storage, bacterial survival was significantly higher under lower conditions of RH (0–11%), and monosodium glutamate and FOS proved to be the best protectants.     

Inorganic polyphosphate from the immunobiotic Lactobacillus rhamnosus CRL1505 prevents inflammatory response in the respiratory tract

Lactobacillus (L.) rhamnosus CRL1505 accumulates inorganic polyphosphate (polyP) in its cytoplasm in response to environmental stress. The aim of this study was to evaluate the potential effects of polyP from the immunobiotic CRL1505 on an acute respiratory inflammation murine animal model induced by lipopolysaccharide (LPS).First, the presence of polyP granules in the cytoplasm of CRL1505 strain was evidenced by specific staining. Then, it was demonstrated in the intracellular extracts (ICE) of CRL1505 that polyP chain length is greater than 45 phosphate residues. In addition, the functionality of the genes involved in the polyP metabolism (ppk, ppx1 and ppx2) was corroborated by RT-PCR. Finally, the possible effect of the ICE of CRL1505 strain containing polyP and a synthetic polyP was evaluated in vivo using a murine model of acute lung inflammation. It was observed that the level of cytokines pro-inflammatory (IL-17, IL-6, IL-2, IL-4, INF-γ) in serum was normalized in mice treated with ICE, which would indicate that polyP prevents the local inflammatory response in the respiratory tract. The potential application of ICE from L. rhamnosus CRL1505 as a novel bioproduct for the treatment of respiratory diseases is one of the projections of this work.     

Characterization of components of the anaerobic ribonucleotide reductase system from Escherichia coli.

Anaerobic growth of Escherichia coli induces an oxygen-sensitive ribonucleoside triphosphate reductase system, different from the aerobic ribonucleoside diphosphate reductase (EC 1.17.4.1) of aerobic E. coli and higher organisms (Fontecave, M., Eliasson, R., and Reichard, P. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2147-2151). We have now purified and characterized two proteins from the anaerobic system, provisionally named dA1 and dA3. dA3 is the actual ribonucleoside triphosphate reductase; dA1 has an auxiliary function. From gel filtration, dA1 and dA3 have apparent molecular masses of 27 and 145 kDa, respectively. In denaturing gel electrophoresis, dA3 gives two bands of closely related polypeptides with apparent molecular masses of 77 (beta 1) and 74 (beta 2) kDa. Immunological and structural evidence suggests that beta 2 is a degradation product of beta 1 and that the active enzyme is a dimer of beta 1. dA1 activity coincides on denaturing gels with a band of 29 kDa and thus appears to be a monomer. The reaction requires, in addition, an extract from E. coli heated for 30 min at 100 degrees C. Potassium is one required component, but one or several others remain unidentified and are provisionally designated fraction RT. With dA3, dA1, RT, and potassium ions, CTP reduction shows absolute requirements for S-adenosylmethionine, NADPH (with NADH as a less active substitute), dithiothreitol, and magnesium ions, and is strongly stimulated by ATP, probably acting as an allosteric effector. Micromolar concentrations of several chelators inhibit CTP reduction completely, suggesting the involvement of (a) transition metal(s).     

The activating component of the anaerobic ribonucleotide reductase from Escherichia coli. An iron-sulfur center with only three cysteines

Class III anaerobic ribonucleotide reductase small component, named protein beta, contains a (4Fe-4S) center. Its function is to mediate electron transfer from reduced flavodoxin to S-adenosylmethionine, required for the introduction of a glycyl radical in the large component, named protein alpha, which then becomes active for the reduction of ribonucleotides. By site-directed mutagenesis we demonstrate that the three cysteines of the conserved CXXXCXXC sequence are involved in iron chelation. Such a sequence is also present in the activase of the pyruvate formate-lyase and in the biotin synthase, both carrying an iron-sulfur center involved in reductive activation of S-adenosylmethionine. Even though they are able to bind iron in the (4Fe-4S) form, as shown by M?ssbauer spectroscopy, the corresponding Cys to Ala mutants are catalytically inactive. Mutation of the two other cysteines of the protein did not result in inactivation. We thus conclude that the (4Fe-4S) cluster has, in the wild type protein, only three cysteine ligands and a fourth still unidentified ligand.     

In Silico Structural and Functional Characterization of the RSUME Splice Variants

RSUME (RWD-containing SUMO Enhancer) is a small protein that increases SUMO conjugation to proteins. To date, four splice variants that codify three RSUME isoforms have been described, which differ in their C-terminal end. Comparing the structure of the RSUME isoforms we found that, in addition to the previously described RWD domain in the N-terminal, all these RSUME variants also contain an intermediate domain. Only the longest RSUME isoform presents a C-terminal domain that is absent in the others. Given these differences, we used the shortest and longest RSUME variants for comparative studies. We found that the C-terminal domain is dispensable for the SUMO-conjugation enhancer properties of RSUME. We also demonstrate that these two RSUME variants are equally induced by hypoxia. The NF-κB signaling pathway is inhibited and the HIF-1 pathway is increased more efficiently by the longest RSUME, by means of a greater physical interaction of RSUME267 with the target proteins. In addition, the mRNA and protein levels of these isoforms differ in human glioma samples; while the shortest RSUME isoform is expressed in all the tumors analyzed, the longest variant is expressed in most but not all of them. The results presented here show a degree of redundancy of the RSUME variants on the SUMO pathway. However, the increased inhibition conferred by RSUME267 over the NF-κB signaling pathway, the increased activation over the HIF-1 pathway and the different expression of the RSUME isoforms suggest specific roles for each RSUME isoform which may be relevant in certain types of brain tumors that express RSUME, like human pituitary adenomas and gliomas.     

Exercise through a cardiac rehabilitation program attenuates oxidative stress in patients submitted to coronary artery bypass grafting*

Background: Cardiovascular disease is the main cause of morbidity and mortality in the world and oxidative stress has been implicated in the pathogenesis. Cardiac rehabilitation in patients with coronary artery disease submitted to coronary artery bypass grafting may prevent cardiovascular events probably through the attenuation of oxidative stress. The aim of this study was to evaluate the benefits of a cardiac rehabilitation program in the control of the systemic oxidative stress.

Methods: The studied population consisted of 40 patients, with chronic stable coronary artery disease submitted to coronary artery bypass grafting, who attended a cardiac rehabilitation program. Biomarkers of oxidative stress were evaluated in the blood of these patients at different moments.

Results: After the onset of cardiac rehabilitation, there was a significant and progressive decrease in thiobarbituric acid reactive substances levels and protein carbonyls, an initial increase and subsequent decrease in superoxide dismutase, catalase and glutathione peroxidase activities. Also, a progressive increase of uric acid, while ferric reducing antioxidant power levels increased only at the end of the cardiac rehabilitation and a tendency to increase of glutathione contents.

Conclusions: The results suggest that regular exercise through a cardiac rehabilitation program can attenuate oxidative stress in chronic coronary artery disease patients submitted to coronary artery bypass grafting.