Blockage of autophagic flux is associated with lymphocytosis and higher percentage of tumoral cells in chronic lymphocytic leukemia of B cells.

PURPOSE: Autophagy has lately emerged as an important biological process with implications in several hematological pathologies. Recently, a growing body of evidence supports a putative role of autophagy in chronic lymphocytic leukemia; however, no definitive clue has been established so far. To elucidate this issue, we have developed a pilot study to measure autophagic flux in peripheral blood mononuclear cells from chronic lymphocytic leukemia patients, and explored its correlation with classical clinical/analytical parameters. METHODS/PATIENTS: Thirty-three chronic lymphocytic leukemia patients participated in the study. Autophagic flux in peripheral blood mononuclear cells was determined by western blot measuring the levels of the proteins p62 and lipidated LC3. Moreover, p62 mRNA levels were analyzed by RT-qPCR. RESULTS: Lymphocytosis and the percentage of tumoral lymphocytes in chronic lymphocytic leukemia patients statistically correlate with a blocked autophagic flux. CONCLUSION: Alterations in autophagic flux could play an important role in the physiopathology of chronic lymphocytic leukemia.

Protective effect of cyclodextrins on the quality parameters of roast preserved pepper.

Total phenolics (TP), vitamin C, antioxidant activity and colour of preserved peppers were evaluated at 4, 25 and 50 ℃ storage during 30-day intervals. Except for 4 ℃, TP decreased during storage at 25 ℃ and 50 ℃, being softer for fortified samples with ß-CDs. The protective effect was evident, since 50 ℃ samples containing ß-CDs exhibited lower TP loss (19%) than control samples (38%) for 5 months storage. A decrease in the vitamin C content was observed for both samples as time and temperature progressed. In samples stored at 50 ℃ the protective effect of ß-CD only was evident at the first month, since fortified samples showed lower vitamin C loss (10%) than control samples. The fortified samples with ß-CDs exhibited lowest antioxidant activity loss (40%) during 90-day storage at 50 ℃, than control samples (64%). The colour changes were in line with those observed for total phenolics and at the end of study, the presence of 1% ß-CDs delayed the darkening of samples at both (25 and 50 ℃) storage conditions.

Fluorimetric determination of sulphathiazole in honey by means the formation of CDs inclusion complexes.

The inclusion complex of sulphathiazole in ß-cyclodextrin has been investigated. A 1:2 stoichiometry of the complex was established and formation constants K2 (42.83 ± 3.27 M(-1)) and K1 (4.98 ± 0.36 M(-1)) were calculated by using the changes produced on the native fluorescence of the drug, when included on the hydrophobic cyclodextrin cavity. An enhancement in the fluorescence emission of sulphathiazole and protection of the drug against photochemical reactions has been attained upon inclusion. In solutions of ß-CD dual emission (458 nm) was noticed in STZ. Formation of the inclusion complex of STZ should result in dual emission, which is due to a twisted intramolecular charge transfer band (TICT). A fluorimetric method for the determination of sulphathiazole has been proposed and applied in honey without sample treatment. The optimized fluorimetric method showed detection and quantitation limits of 9.74 ng/g and 32.48 ng/g, respectively. Selectivity is high, showing no cross-reactivity to other chemically related antibiotics. The results obtained for blind honey samples (mean recovery 97%), were in good agreement with those obtained by liquid chromatography separation and mass spectrometry detection (LC-MS) (mean recovery 102%), showing that the proposed method might be used for the determination of sulphathiazole residues without expensive equipment.

Red cabbage ( Brassica oleracea ) as a new source of high-thermostable peroxidase.

Soluble and membrane-bound peroxidases (PODs) were extracted from red cabbage using Triton X-114. Optimum activity was obtained at pH 4.0 for both enzymes, and both were inactivated by sodium dodecyl sulfate (SDS). The K(M) and V(m) values for H(2)O(2) were found to be 0.98 mM and 8.1 µM/min, respectively, for soluble POD and 0.82 mM and 6.1 µM/min, respectively, for membrane-bound POD. When the 2,2'-azinobis(3-ethylbenzothiazolinesulfonic acid (ABTS) concentration was increased, maintaining a steady concentration of H(2)O(2), the activity was inhibited at the highest ABTS concentrations in soluble POD. Ascorbic acid was found to be the most active modulator of POD activity. The effect of cyclodextrins was also studied, and the complexation constant between ABTS and hydroxypropyl-ß-cyclodextrins (HP-ß-CDs) was calculated (K(c) = 312 M(-1)). Membrane-bound POD is more thermostable than soluble POD, losing >90% of relative activity after 5 min of incubation at 76.6 and 30.2 °C, respectively.

Effect of ripening on protein content and enzymatic activity of Crimson Seedless table grape.

The evolution of °Brix, protein content, polyphenoloxidase activity and peroxidase activity during the ripening of Crimson Seedless table grape was studied in three consecutive years (2006, 2007 and 2008). The total protein content was determined according to Bradford's dye binding method, and polyphenoloxidase (PPO) and peroxidase (POD) were extracted using Triton X-114 and characterised using spectrophotometric methods. The year had a statistically significant effect on all the studied parameters and there was an interannual correlation in the evolution of protein, PPO, POD and °Brix. All the studied parameters were statistically correlated, except POD activity with protein content. Weather conditions during the ripening period had a greater effect on protein content than PPO and POD activity.

Kinetic characterisation and thermal inactivation study of red alga (Mastocarpus stellatus) peroxidase.

Peroxidase (POD) was extracted from red alga (Mastocarpus stellatus) using Triton X-114 and characterised by UV-spectrophotometry. Optimum activity using 2,2´-azinobis(3-ethylbenzothiazolinesulphonic acid) (ABTS) as the H-donor was obtained at pH 5.0. In the presence of the anionic detergent, sodium dodecyl sulphate (SDS), however, POD was inactivated at all the pH values studied and totally inactivated at 1mM SDS. When the enzyme was kinetically characterised, the KM and Vm values for ABTS were found to be 13mM and 40µM/min, respectively. In addition, when the H2O2 concentration was increased, at a fixed concentration of ABTS, the activity was inhibited at the highest H2O2 concentrations. In a study of the effect of several reducing agents, l-cysteine was found to be the most active. A thermal inactivation study showed a first-order inactivation kinetic, and the Arrhenius plot yielded a straight line with a slope equivalent to an activation energy of 121.6kJ/mol. Significant inactivation occurred at temperatures of>35°C, with>90% of the relative activity being lost after only 5min of incubation at 48.4°C.