Micronuclei, genetic polymorphisms and cardiovascular disease mortality in a nested case-control study in Italy

AIM: To validate the predictive value of micronuclei (MN) in peripheral blood lymphocytes (PBL) and glutathione-S-transferases (GSTs) polymorphisms (GSTM1 and GSTT1) for mortality risk (MR) of cardiovascular diseases (CVD). METHODS: Blood samples from 1650 healthy subjects selected from the general population were collected between June 1991 and November 1993, and slides were immediately prepared for MN assessment. The vital status, or the cause of death, was monitored for all subjects until January 2005. At the end of the follow-up, 111 deaths were recorded and 39 CVD cases were observed (age range=42-88 years). Two thousand binucleated (BN) cells/subject were scored for the MN assay and GSTs genotypes were assessed on the DNA extracted from the blood or serum samples. RESULTS: A significantly higher MN frequency was recorded for the case group in comparison with the control group (n=67, Kruskall-Wallis test, p=0.006) and GSTT1 null genotype was significantly less frequent in CVD patients (chi(2)-test, p=0.036). The influence of other factors were evaluated using a unconditional logistic regression that confirmed a significant association of GSTT1 positive genotype with an increased OR for CVD (OR=6.29, 95% CI 1.32-29.95) beside a significant effect of age (OR=1.13, 95% CI 1.03-1.26 year(-1)). Finally, subjects with an higher MN frequency showed a higher MR for CVD (Log-rank test, p=0.001). CONCLUSIONS: MN confirmed to be a suitable cytogenetic biomarker for early prediction of CVD death. The GSTT1 positive genotype is associated with an increased MR for CVD.     

Stability and activity of a thermostable malic enzyme in denaturants and water-miscible organic solvents

A study was made of the effects of common protein denaturants and water-miscible organic solvents on both the stability and activity of the malic enzyme [(S)-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating); EC 1.1.1.40] from the extreme thermoacidophilic archaebacterium Sulfolobus solfataricus. At 25 degrees C, the enzyme was not inactivated in 4 M urea or 0.05% SDS over 24 h, while the half-life was 30 min in 6 M guanidine hydrochloride and 5 h in 0.075% SDS. The enzyme stability in water-miscible organic solvents at 25 degrees C is somewhat surprising: after a 24-h incubation, the enzyme was completely active in 50% dimethylformamide; it lost 15% of its initial activity in 50% methanol or 15% ethanol. However, the resistance to organic solvents was greatly reduced at higher temperatures. The enzyme was able to catalyze the malate conversion even in the presence of 1.5% Triton X-100 or sodium deoxycholate. A number of solvents were found to stimulate the malic activity independent of time. Studies with 50% methanol revealed that the activation was reversible and inversely related to the temperature; moreover, the solvent was demonstrated to exclusively affect the maximal velocity of catalysis, the Km values for both substrates being unchanged. Investigation was made to find out whether there was a correlation between enzyme stability, as well as activation, and hydrophobicity of the organic medium. The residual malic activity after incubation in the water/organic medium correlated inversely with the logarithm of the partition coefficient in octanol/H2O of the mixture used as a hydrophobicity index. On the other hand, the extent of activation depended directly on the logarithm of the molar concentration of the organic solvent required for maximal enzymatic activation. Because of its remarkable resistance to organic solvents required for maximal enzymatic activation. Because of its remarkable resistance to organic solvents and protein denaturants in general, the malic enzyme from Sulfolobus solfataricus can be considered suitable for biotechnological applications.     

Solution structure and backbone dynamics of the K18G/R82E Alicyclobacillus acidocaldarius thioredoxin mutant: a molecular analysis of its reduced thermal stability

No general strategy for thermostability has been yet established, because the extra stability of thermophiles appears to be the sum of different cumulative stabilizing interactions. In addition, the increase of conformational rigidity observed in many thermophilic proteins, which in some cases disappears when mesophilic and thermophilic proteins are compared at their respective physiological temperatures, suggests that evolutionary adaptation tends to maintain corresponding states with respect to conformational flexibility. In this study, we accomplished a structural analysis of the K18G/R82E Alicyclobacillus acidocaldarius thioredoxin (BacTrx) mutant, which has reduced heat resistance with respect to the thermostable wild-type. Furthermore, we have also achieved a detailed study, carried out at 25, 45, and 65 degrees C, of the backbone dynamics of both the BacTrx and its K18G/R82E mutant. Our findings clearly indicate that the insertion of the two mutations causes a loss of energetically favorable long-range interactions and renders the secondary structure elements of the double mutants more similar to those of the mesophilic Escherichia coli thioredoxin. Moreover, protein dynamics analysis shows that at room temperature the BacTrx, as well as the double mutant, are globally as rigid as the mesophilic thioredoxins; differently, at 65 degrees C, which is in the optimal growth temperature range of A. acidocaldarius, the wild-type retains its rigidity while the double mutant is characterized by a large increase of the amplitude of the internal motions. Finally, our research interestingly shows that fast motions on the pico- to nanosecond time scale are not detrimental to protein stability and provide an entropic stabilization of the native state. This study further confirms that protein thermostability is reached through diverse stabilizing interactions, which have the key role to maintain the structural folding stable and functional at the working temperature.     

Mitochondrial respiratory chain dysfunction, a non-receptor-mediated effect of synthetic PPAR-ligands: biochemical and pharmacological implications

Peroxisome proliferator activated receptors (PPARs) are a class of nuclear receptors involved in lipid and glucidic metabolism, immune regulation, and cell differentiation. Many of their biological activities have been studied by using selective synthetic activators (mainly fibrates and thiazolidinediones) which have been already employed in therapeutic protocols. Both kinds of drugs, however, showed pharmacotoxicological profiles, which cannot be ascribed by any means to receptor activation. To better understand these non-receptorial or extrareceptorial aspects, the effect of different PPAR-ligands on the metabolic status of human HL-60 cell line has been investigated. At this regard, NMR analysis of cell culture supernatants was accomplished in order to monitor modifications at the level of cell metabolism. Cell growth and chemiluminescence assays were employed to verify cell differentiation. Results showed that all the considered PPAR-ligands, although with different potencies and independently from their PPAR binding specificity, induced a significant derangement of the mitochondrial respiratory chain consisting in a strong inhibition of NADH-cytochrome c reductase activity. This derangement has been shown to be strictly correlated to the adaptive metabolic modifications, as evidenced by the increased formation of lactate and acetate, due to the stimulation of anaerobic glycolysis and fatty acid beta-oxidation. It is worthy noting that the mitochondrial dysfunction appeared also linked to the capacity of any given PPAR-ligand to induce cell differentiation. These data could afford an explanation of biochemical and toxicological aspects related to the therapeutic use of synthetic PPAR-ligands and suggest a revision of PPAR pathophysiologic mechanisms.     

Phenols removal in musts: Strategy for wine stabilization by laccase

The potential of laccase from Trametes versicolor for phenolic removal in must for wine stabilization was evaluated through a combination of an analytical methodology (capillary zone electrophoresis) and kinetics of phenols removal as the total antioxidant potential variation. Total phenolic content, total antioxidant potential and polyphenols were monitored from 0 to 3 h of must treatment. The results indicated that the treatment of a red must with laccase affect mainly the phenolic compounds responsible for the must antioxidant properties. The treatment of white musts with laccase showed higher reduction in total phenol than in the total antioxidant potential. Phenol degradation by laccase was very fast for catechins, and slowly for stilbenes (cis- and trans-resveratrol) and derivatives of cinnamic (ferulic and caffeic) and benzoic (syringic, vanillic, and gallic) acids. It is possible to conclude in this case that the use of laccase in white wines is perfectly feasible. This would allow softer and ecologically correct treatments, which would diminish the cost of processing and avoid deterioration of wines for long storage times.     

CD45 Isoform Profile Identifies Natural Killer (NK) Subsets with Differential Activity

The leucocyte-specific phosphatase CD45 is present in two main isoforms: the large CD45RA and the short CD45RO. We have recently shown that distinctive expression of these isoforms distinguishes natural killer (NK) populations. For example, co-expression of both isoforms identifies in vivo the anti tumor NK cells in hematological cancer patients. Here we show that low CD45 expression associates with less mature, CD56^bright, NK cells. Most NK cells in healthy human donors are CD45RA⁺CD45RO^-. The CD45RA^-RO⁺ phenotype, CD45RO cells, is extremely uncommon in B or NK cells, in contrast to T cells. However, healthy donors possess CD45RA^dimRO^- (CD45RA^dim cells), which show immature markers and are largely expanded in hematopoietic stem cell transplant patients. Blood borne cancer patients also have more CD45RA^dim cells that carry several features of immature NK cells. However, and in opposition to their association to NK cell progenitors, they do not proliferate and show low expression of the transferrin receptor protein 1/CD71, suggesting low metabolic activity. Moreover, CD45RA^dim cells properly respond to in vitro encounter with target cells by degranulating or gaining CD69 expression. In summary, they are quiescent NK cells, with low metabolic status that can, however, respond after encounter with target cells.     

Increased formation of short-chain organic acids after chronic ethanol administration and its interaction with the carnitine pool in rat

Organic acidurias are genetic disorders of mitochondrial metabolism that lead to the accumulation of organic acids in tissues and biological fluids. It has been demonstrated that interaction of carnitine with the cellular coenzyme A (CoA) pool, through the production of acyl-carnitines, is potentially critical for maintaining normal cellular metabolism under condition of impaired acyl-CoA use and that exposure of humans and other mammals to ethanol leads to impairment of mitochondrial function. The aim of the present study was to evaluate the role of chronic administration of ethanol on urinary excretion of short-chain organic acids and endogenous carnitines in rats. The data reported show that chronic administration of ethanol significantly increases urinary excretion of propionate, methylmalonate, as well as free acetate, butyrate, pyruvate, lactate, and beta-hydroxybutyrate. Chronic administration of propranolol abolished ethanol-dependent accumulation of propionate, suggesting involvement of beta-adrenergic mechanisms. Increased formation of propionate and methylmalonate was associated with decreased plasma carnitine levels and with increased excretion of specific acyl-carnitines, corresponding to the accumulating acyl groups. Our data indicate that chronic alcohol ingestion induces increased excretion of selected organic acids and that the endogenous carnitine pool might exert a protective role against the deleterious effects of accumulating short-chain organic acids.     

Spread of herbicide‐resistant weedy rice (red rice,Oryza sativa L.) after 5 years of Clearfield rice cultivation in Italy

The weedy relative of cultivated rice, red rice, can invade and severely infest rice fields, as reported by rice farmers throughout the world. Because of its close genetic relationship to commercial rice, red rice has proven difficult to control. Clearfield (Cl) varieties, which are resistant to the inhibiting herbicides in the chemical group AHAS (acetohydroxyacid synthase), provide a highly efficient opportunity to control red rice infestations. In order to reduce the risk of herbicide resistance spreading from cultivated rice to red rice, stewardship guidelines are regularly released. In Italy, the cultivation of Cl cultivars started in 2006. In 2010, surveillance of the possible escape of herbicide resistance was carried out; 168 red rice plants were sampled in 16 fields from six locations containing Cl and traditional cultivars. A first subsample of 119 plants was analysed after herbicide treatment and the resistance was found in 62 plants. Of these 119 plants, 78 plants were randomly selected and analysed at the level of the AHAS gene to search for the Cl mutation determining the resistant genotype: the Cl mutation was present in all the resistant plants. Nuclear and chloroplast microsatellite markers revealed a high correlation between genetic similarity and herbicide resistance. The results clearly show that Cl herbicide‐resistant red rice plants are present in the field, having genetic relationships with the Cl variety. Finding plants homozygous for the mutation suggests that the crossing event occurred relatively recently and that these plants are in the F₂ or later generations. These observations raise the possibility that Cl red rice is already within the cultivated rice seed supply.