Monitoring the efficacy of specific treatment in chronic Chagas disease by polymerase chain reaction and flow cytometry analysis

PCR and FC-ALTA were used to monitor parasite clearance in 54 chronic chagasic patients who had completed therapy with allopurinol (ALLO, n = 31) or itraconazole (ITRA, n = 23) ten years earlier. All patients maintained positive conventional serology. 25 of them showed positive XD (ALLO, n = 11 and ITRA, n = 14) and 29 negative XD (ALLO, n = 20 and ITRA, n = 9). 43 patients were positive by both techniques (ALLO, n = 23 and ITRA, n = 20). Seven of 54 patients were negative by PCR and positive by FC-ALTA and three of 54 were positive by PCR and negative by FC-ALTA. Only one case with both tests negative should be considered cured. Of 29 patients with negative XD, 14 treated ALLO (70 %) and nine with ITRA (77.8 %) showed positive PCR and FC-ALTA. These results do not show differences of efficacy among the drugs, and reinforce the relevance of using sensitive tools such as PCR and FC-ALTA for the follow-up of patients with chronic Chagas disease.     

Calcium-dependent conformational changes of membrane-bound Ebola fusion peptide drive vesicle fusion

The fusogenic subdomain of the Ebola virus envelope glycoprotein is an internal sequence located ca. 20 residues downstream the N-terminus of the glycoprotein transmembrane subunit. Partitioning of the Ebola fusion peptide into membranes containing phosphatidylinositol in the absence of Ca2+ stabilizes an alpha-helical conformation, and gives rise to vesicle efflux but not vesicle fusion. In the presence of millimolar Ca2+ the membrane-bound peptide adopts an extended beta-structure, and induces inter-vesicle mixing of lipids. The peptide conformational polymorphism may be related to the flexibility of the virus-cell intermembrane fusogenic complex.     

Direct calcium binding results in activation of brain serine racemase

Serine racemase (SR) is a brain enzyme present in glial cells, where it isomerizes L-serine into D-serine that, in turn, diffuses and coactivates the N-methyl-D-aspartate receptor through the binding to the so-called "glycine site." We have developed a method for the slow expression of SR in a eukaryotic vector that permits the correct insertion of the prosthetic group into the active site, rendering functional SR with a K(m) toward L-serine of 4.8 mm. Divalent cations such as calcium or manganese were necessary for complete enzyme activity, whereas the presence of chelators such as EDTA completely inhibited the enzyme. Moreover, direct binding of calcium to SR was evidenced using (45)Ca(2+). Gel filtration of the recombinant SR revealed the protein to be in a dimer-tetramer equilibrium. The addition of EDTA to a calcium-saturated serine racemase evokes a profound conformational change, as monitored by both fluorescence and circular dichroism techniques. Fluorescence titration allowed us to calculate a binding constant for calcium of 6.2 microm. Reagents that react with sulfhydryl groups, such as cystamine, were potent inhibitors of SR, in a clear reflection that one or more cysteine residues are important for enzyme activity. Additionally, 16 serine analogues were tested as a putative SR substrate or inhibitors. Significant inhibition was only observed for L-Ser-O-sulfate, L-cycloserine, and L-cysteine. Finally, activation of brain SR as a result of the changes in calcium concentration was studied in primary astrocytes. Treatment of astrocytes with the calcium ionophore, as well as with compounds that augment the intracellular calcium levels such as glutamate or kainate led to an increase in the amount of d-serine present in the extracellular medium. These results suggest that there might be a glutamatergic-mediated regulation of SR activity by intracellular calcium concentration.     

The losses of nutrients by fire in a savanna vegetation of Trachypogon

The losses of nutrients by fire were assessed in a Trachypogon savanna located in Calabozo, Venezuela. About 95% of the biomass, 97% of N, 61% of P, 76% of K and 65% of Ca and Mg were transferred to the atmosphere. Ash deposition returned between 21-34% of Mg, Ca, K and P and 0.2% of N. Previous data indicated that precipitation and atmospheric fixation (in the case of N) could replace the rest of N, Ca, K and Mg transferred to the atmosphere. As a consequence of frequent burning (annual or biannual), the soil of the savanna showed lower organic matter and available P and K content when it is compared with a 32 year protected savanna.     

Reproductive toxicity and infertility effect of Ferula hornonis extracts in mice

The anti-fertility, anti-implantation, and ovarian histological alterations of the ethanolic extract of Ferula hormonis have been investigated in female mice. The intragastric application of 3 mg/kg per day of such extract for 6 weeks resulted in a significant reduction in female mice fertility. Furthermore, it caused a decrease in the number of mated females, the total number of implantations, and the number of viable fetuses. These changes were also associated with ovarian atrophy and a concomitant increase in the connective tissue. The ova showed degeneration while most of the ovarian follicles suffered follicular atresia.     

Coupling reaction and properties of poly(ethylene glycol)-linked phospholipases A2

Secretory phospholipases A2 (PLA2) from Naja naja naja (cobra snake) venom, from Bothrops neuwiedii (crotalid snake) venom (two isoforms) and from bee venom were modified with tresylated monomethoxy poly(ethylene glycol) (TMPEG). The kinetic and inflammatory properties of the adducts (PEG-PLA2) were measured. As found by gel permeation chromatography, 95-100% of P-1 PLA2 from B. neuwiedii and PLA2 from N. naja naja venom change their chromatographic mobility after TMPEG treatment. By contrast, only 50-60% of both P-3-PLA2 from B. neuwiedii and PLA2 from bee venom modify their elution profile from Superdex 75. All the modified proteins preserved the enzymatic activity toward phospholipid monolayers, but with a reduced specific activity and greater lag times than the unmodified controls. These results suggest that the PEG-PLA2 complexes would have an altered interaction with lipid membranes. The PEG-linked proteins preserve their edema-inducing activity evaluated by the rat hind-paw edema test except for N. naja naja PEG-PLA2 in which inflammatory activity was significatively decreased. Altogether, the results show a partial dissociation of catalytic and inflammatory activities of Group II and III secretory PLA2s after their modification with PEG.     

Evaluation of ion exchange resins for removal of inhibitory compounds from corn stover hydrolyzate for xylitol fermentation

The use of dilute acids to catalyze the hydrolysis of hemicellulose to its sugar constituents is well-known and effective. However, a major problem associated with this pretreatment is the poor fermentability of the produced hydrolyzate as a result of the presence of the microorganism's inhibitory compounds. In the present work, seven ion-exchange resins were tested in order to detoxify corn stover hydrolyzate. Regarding xylose recovery, it was observed that more than 92% recovery was feasible. Furfural removal varied from 53.% to 99.%, and hydroxymethylfurfural (HMF) removal was effective between 37% and 100%. Acetic acid was totally removed by Purolite A 103 S resin. Corn stover hydrolyzate (CSH) treated with Purolite A 103 S, and Finex CS 14 GC resins, was tested as substrate for xylitol production using a yeast, Candida mogii. Product yields, Yp/s, of 0.41 and 0.37 g/g and cellular yields, Yx/s, of 0.24 and 0.13 g/g, respectively, were obtained using the two types of resin-treated hydrolyzates.