Discovery of two novel laccase-like multicopper oxidases from Pleurotus citrinopileatus and their application in phenolic oligomer synthesis.

BACKGROUND: Laccases and laccase-like multicopper oxidases (LMCOs) oxidize a vast array of phenolic compounds and amines, releasing water as a byproduct. Their low substrate specificity is responsible for their tremendous biotechnological interest, since they have been used for numerous applications. However, the laccases characterized so far correspond to only a small fraction of the laccase genes identified in fungal genomes. Therefore, the knowledge regarding the biochemistry and physiological role of minor laccase-like isoforms is still limited. RESULTS: In the present work, we describe the isolation, purification and characterization of two novel LMCOs, PcLac1 and PcLac2, from Pleurotus citrinopileatus. Both LMCOs were purified with ion-exchange chromatographic methods. PcLac2 was found to oxidize a broader substrate range than PcLac1, but both LMCOs showed similar formal potentials, lower than those reported previously for laccases from white-rot fungi. Proteomic analysis of both proteins revealed their similarity with other well-characterized laccases from Pleurotus strains. Both LMCOs were applied to the oxidation of ferulic and sinapic acid, yielding oligomers with possible antioxidant activity. CONCLUSIONS: Overall, the findings of the present work can offer new insights regarding the biochemistry and variability of low-redox potential laccases of fungal origin. Low-redox potential biocatalysts could offer higher substrate selectivity than their high-redox counterparts, and thus, they could be of applied value in the field of biocatalysis.

FTacV study of electroactive immobilized enzyme/free substrate reactions: Enzymatic catalysis of epinephrine by a multicopper oxidase from Thermothelomyces thermophila.

In the present work, a kinetic analysis is made concerning the reaction of an electroactive immobilized enzyme with a free substrate, based on a Michaelis-Menten scheme. The proposed kinetic equations are investigated numerically for conditions describing large amplitude fast Fourier transform alternating current voltammetry (FTacV), under different reaction states (transient or steady state for the reaction intermediate as well as quasi or complete reversibility of the electrochemical step). The dependence of two chief observables that occur from the analysis of the results of the method, that is, the maximum of the harmonics and the potential shift of the corresponding dominant peaks, on substrate concentration is presented. The FTacV method is applied experimentally for an immobilized laccase-like multicopper oxidase from Thermothelomyces thermophila, TtLMCO1, and its reaction with epinephrine. From the experimental findings it is shown that the intrinsic characteristics of the system do not lead to the extraction of the desired kinetic data although indications on the relation between the kinetic constants is revealed. Finally, the response of the third harmonic for the first additions of epinephrine at subnanomolarity range can be exploited for the detection of epinephrine at rather low concentrations.

Kinetic and amperometric study of the MtPerII peroxidase isolated from the ascomycete fungus Myceliophthora thermophila.

The enzyme MtPerII is a new peroxidase which has been isolated only recently from fungus Myceliophthora thermophila and has significant thermostability and stability at high H2O2 concentrations. In the present work, an electrochemical kinetic study, based on cyclic voltammetry, is performed for the first time for the catalytic decomposition of H2O2 by MtPerII, at 18°C. Leuco methylene blue (LMB) is used as a mediator and the catalytic and Michaelis constants are determined, assuming a Michaelis-Menten mechanism. Experimental evidence suggest the absence of inhibition by H2O2, for concentrations up to 16mM, and increasing catalytic activity for temperatures up to 50°C. Moreover, a modified electrode is constructed, by attempting the entrapment of MtPerII on a dodecanothiol self-assembled monolayer on gold. The modified electrode is studied chronoamperometrically in solutions containing methylene blue mediator and different concentrations of H2O2. It is shown that adsorbed MtPerII retains its activity and the modified electrode exhibits a considerably high linear region for the detection of H2O2. The experimental findings indicate that MtPerII is a new candidate for analytical and industrial applications.

Pretreatment with sevoflurane attenuates direct lung injury.

BACKGROUND: Sevoflurane exerts effects on pulmonary cells that could protect against lung injury. We evaluated the potential of pretreatment with sevoflurane to attenuate lipopolysaccharide (LPS)-induced lung injury. METHODS: LPS was administered intratracheally in Wistar rats to induce lung injury. Sevoflurane was administered for 30 min at 0.25, 0.5 or 1.0 MAC 15 min before LPS or for 30 min at 0.5 MAC 24 hours before LPS. After initial analysis of bronchoalveolar lavage fluid (BALF) cells and total protein, the group of 0.5 MAC 15 min before LPS was further analyzed for surfactant aggregates subfractions, plasma malondialdehyde levels and lung histology. RESULTS: LPS instillation resulted in neutrophils sequestration in the lungs, loss of alveolar macrophages, increased BALF total protein and decreased large surfactant aggregates. Only inhalation of sevoflurane for 30 min at 0.5 MAC 15 min before LPS installation effectively reduced neutrophil accumulation, preserved alveolar epithelial cells and reduced total protein content in BALF. This regimen also reduced plasma malondialdehyde levels and increased large surfactant aggregates, despite the application of mechanical ventilation. This effect was preserved after LPS instillation and the favorable composition of surfactant was maintained. CONCLUSION: Pretreatment with sevoflurane effectively attenuates direct severe lung injury, possibly by inhibition of neutrophil accumulation and alteration of the surfactant composition.

"Menstrual irregularities in PCOS. Does it matter when it starts?".

BACKGROUND: PCOS is presented by a broad spectrum of menstrual irregularities appearing often at puberty or later on during the reproductive years in women suffering from this multifaceted syndrome. To our knowledge, there is no evidence to suggest whether the time of onset of menstrual irregularities (peri or post pubertal) indicates a differential metabolic and/or hormonal profile as well as ovarian ultrasonographic findings, in adulthood in women with PCOS. AIM OF THE STUDY: To compare anthropometric, hormonal-metabolic profile and ultrasound findings in PCOS women with peripubertal onset of menstrual disorders with the corresponding data obtained from PCOS patients with post pubertal onset of menstrual irregularities, matched for BMI and age. PATIENTS-METHODS: 89 PCOS women were evaluated cross-sectionally at the age of 25 years. In 49 subjects menstrual irregularities were present from menarche, whereas in 40 women the irregularities appeared at least 3 years post menarche. RESULTS: Anthropometric parameters were comparable between the 2 groups. The 2 groups did not differ on metabolic and hormonal profile as well as ovarian ultrasound findings. CONCLUSIONS: These data indicate that the timing of menstrual irregularities, do not appear to have an impact, on hormonal/metabolic profile and ovarian ultrasound morphology in patients diagnosed with PCOS, later in life.

Effect of eating frequency on body composition in 9-11-year-old children.

The aim of this study was twofold: a) to examine the association between eating frequency and body composition in children, and b) to identify possible factors that may explain this relationship. Body composition (anthropometry) and dietary intake (3-day food records) were assessed in a cohort of 151 children. After excluding the underreporters (n = 20), data from 131 children (66 boys and 65 girls) aged 9.9 +/- 0.1 yr with a BMI of 19.6 +/- 0.4 kg/m (2) (means +/- se) were used for further analysis. Children were categorized in tertiles based on the daily number of eating episodes. Physical activity was assessed in a subgroup of 48 volunteers with 4-day accelerometry (RT3, Stayhealthy Inc., Monrovia, CA, USA). The number of eating episodes was inversely associated (p < 0.05) with the sum of skinfolds (r = - 0.17) and % body fat (r = - 0.18) after controlling for age and sex. Frequent eaters presented lower total (p < 0.05) and central adiposity (p < 0.01) compared with the infrequent ones. This was despite the fact that energy intake was higher for the frequent eaters (2077.0 +/- 64.3 vs. 1813.0 +/- 37.8 kcals/day for the frequent and the infrequent eaters, respectively, p < 0.05). Actually, frequent eaters devoted more time to physical activity than infrequent ones (624.7 +/- 13.5 vs. 559.2 +/- 23.1 min/day, p < 0.05). In conclusion, high eating frequency was associated with more favorable body composition in this cohort of school children. Increased energy expenditure due to physical activity may, at least in part, explain the favorable body composition of children who eat frequently.