Étude de l’effet des thiosemicarbazones sur Toxoplasma gondii

Toxoplasmosis is a neglected disease, with an estimated occurrence of one-third of the population worldwide. Research in medicinal chemistry has for some years been pursuing the development of new drugs against toxoplasmosis, because current treatments cause serious side effects in the patient. The use of thiosemicarbazones as an alternative option for the treatment of various diseases has been published in recent years, due to their, among others, anticancer, antimalarial, antitrypanosomal, antibacterial, and antitoxoplasmosis activities, the latter being the subject of this study, which is based upon biological analyses and tests of the response of Toxoplasma gondii in the presence of thiosemicarbazones.     

Inheritance of parthenocarpy in summer squash (Cucurbita pepo L.)

The inheritance of the tendency to set parthenocarpic fruit in the summer squash (Cucurbita pepo L.) line Whitaker was studied. Two parental lines, Whitaker (parthenocarpic) and Caserta (non-parthenocarpic), and the F1 and F2 generations and backcrosses to both parents were tested. The parthenocarpic tendency of individual plants was scored on a scale from 1 (non-parthenocarpic fruit) to 5 (parthenocarpic fruit). The Whitaker line produced parthenocarpic fruit and had a mean score of 4.2, whereas Caserta did not set parthenocarpic fruit and had a score of 1.55. The heritability estimates indicated that genetic gains from selection were feasible. The additive-dominant model showed a good fit, with epistasis being negligible or nonexistent. The hypothesis of monogenic inheritance with incomplete dominance was not rejected within the degree of dominance range from 0.2 to 0.5. These results indicate that parthenocarpy is controlled by a single locus, with incomplete dominance in the direction of parthenocarpic expression.     

Silencing the expression of mitochondrial acyl-CoA thioesterase I and acyl-CoA synthetase 4 inhibits hormone-induced steroidogenesis

Arachidonic acid and its lypoxygenated metabolites play a fundamental role in the hormonal regulation of steroidogenesis. Reduction in the expression of the mitochondrial acyl-CoA thioesterase (MTE-I) by antisense or small interfering RNA (siRNA) and of the arachidonic acid-preferring acyl-CoA synthetase (ACS4) by siRNA produced a marked reduction in steroid output of cAMP-stimulated Leydig cells. This effect was blunted by a permeable analog of cholesterol that bypasses the rate-limiting step in steroidogenesis, the transport of cholesterol from the outer to the inner mitochondrial membrane. The inhibition of steroidogenesis was overcome by addition of exogenous arachidonic acid, indicating that the enzymes are part of the mechanism responsible for arachidonic acid release involved in steroidogenesis. Knocking down the expression of MTE-I leads to a significant reduction in the expression of steroidogenic acute regulatory protein. This protein is induced by arachidonic acid and controls the rate-limiting step. Overexpression of MTE-I resulted in an increase in cAMP-induced steroidogenesis. In summary, our results demonstrate a critical role for ACS4 and MTE-I in the hormonal regulation of steroidogenesis as a new pathway of arachidonic acid release different from the classical phospholipase A2 cascade.     

Characterization of oligosaccharides from the chondroitin/dermatan sulfates. 1H-NMR and 13C-NMR studies of reduced trisaccharides and hexasaccharides

Chondroitin and dermatan sulfate (CS and DS) chains were isolated from bovine tracheal cartilage and pig intestinal mucosal preparations and fragmented by enzymatic methods. The oligosaccharides studied include a disaccharide and hexasaccharides from chondroitin ABC lyase digestion as well as trisaccharides already present in some commercial preparations. In addition, other trisaccharides were generated from tetrasaccharides by chemical removal of nonreducing terminal residues. Their structures were examined by high-field 1H and 13C NMR spectroscopy, after reduction using sodium borohydride. The main hexasaccharide isolated from pig intestinal mucosal DS was found to be fully 4-O-sulfated and have the structure: DeltaUA(beta1-3)GalNAc4S(beta1-4)L-IdoA(alpha1-3)GalNAc4S(beta1-4)L-IdoA(alpha1-3)GalNAc4S-ol, whereas one from bovine tracheal cartilage CS comprised only 6-O-sulfated residues and had the structure: DeltaUA(beta1-3)GalNAc6S(beta1-4)GlcA(beta1-3)GalNAc6S(beta1-4)GlcA(beta1-3)GalNAc6S-ol. No oligosaccharide showed any uronic acid 2-sulfation. One novel disaccharide was examined and found to have the structure: GalNAc6S(beta1-4)GlcA-ol. The trisaccharides isolated from the CS/DS chains were found to have the structures: DeltaUA(beta1-3)GalNAc4S(beta1-4)GlcA-ol and DeltaUA(beta1-3)GalNAc6S(beta1-4)GlcA-ol. Such oligosaccharides were found in commercial CS/DS preparations and may derive from endogenous glucuronidase and other enzymatic activity. Chemically generated trisaccharides were confirmed as models of the CS/DS chain caps and included: GalNAc6S(beta1-4)GlcA(beta1-3)GalNAc4S-ol and GalNAc6S(beta1-4)GlcA(beta1-3)GalNAc6S-ol. The full assignment of all signals in the NMR spectra are given, and these data permit the further characterization of CS/DS chains and their nonreducing capping structures.     

Hereditary hemochromatosis in a Brazilian university hospital in São Paulo State (1990-2000)

Hereditary hemochromatosis (HH) is the most common genetic disease among individuals of European descent. Two mutations (845G-->A, C282Y and 187C-->G, H63D) in the hemochromatosis gene (HFE gene) are associated with HH. About 85-90% of patients of northern European descent with HH are C282Y homozygous. The prevalence of HH in the Brazilian population, which has a very high level of racial admixture, is unknown. The aims of the present study were to identify individuals with diagnostic criteria for HH among patients with a body iron overload attended at the university hospital of the Faculty of Medicine of Ribeirao Preto from 1990 to 2000, and to evaluate the prevalence of HFE mutations. We screened first-degree relatives for HFE mutations. Four of 72 patients (three men and one woman, mean age 47 years) fulfilled the criteria for HH. HFE mutations were studied in three patients [two C282Y homozygotes (patients 1 and 2) and one H63D heterozygote]. Patient 1 had four children (all C282Y heterozygotes with no iron overload) and seven brothers and sisters: two sisters (66 and 76 years old) were C282Y homozygotes and both had an iron overload (a liver biopsy in one showed severe iron deposits), one sister (79 years old) was a compound heterozygote with no iron overload, one brother (78 years old) was a C282Y heterozygote with no iron overload, two individuals were H63D heterozygotes (one brother, 49 years old, obese, with a body iron overload and abnormal liver enzymes - a biopsy showed non-alcoholic steatohepatitis, and one 70-year-old sister with no iron overload). Patient 2 had two children (22 and 24 years old who were C282Y heterozygotes with no iron overload) but no brothers or sisters. These results showed that HH was uncommon among individuals attended at our hospital, although HFE mutations were found in all patients. Familial screening is valuable for the early diagnosis of individuals at risk since it allows treatment to be initiated before the onset of the clinical manifestations of organ damage associated with HH.     

Expression of heat shock protein in broiler embryo tissues after acute cold or heat stress

This study evaluated the expression of heat shock protein 70 kD (hsp70) in broiler chicken embryos subjected to cold (Experiment I) or high incubation temperature (Experiment II). In each experiment, fertile eggs were distributed in three incubators kept at 37.8 degrees C. At day 13 (D13), D16, and D19 of incubation, the embryos were subjected to acute cold (32 degrees C) or heat (40 degrees C) for 4-6 hr. Immediately after cold or heat exposure, samples from the liver, heart, breast muscle, brain, and lungs of 40 embryos were taken per age and treatment (control or stressed embryos). A tissue pool from 10 embryos was used as 1 replication. The levels of hsp70 in each tissue sample was quantified by Western blot analysis. The data were analyzed in a 3 x 2 factorial arrangement of treatments with four replications. hsp70 was detected in all embryo tissues, and the brain contained 2- to 5-times more hsp70 protein compared to the other tissues in either cold or heat stressed embryos. hsp70 increases were observed in the heart and breast muscle of cold stressed embryos at D16 and D19, respectively. Heat stressed embryos showed an increase of hsp70 in the heart at D13 and D19, and in the lung at D19 of incubation. Younger embryos had higher hsp70 synthesis than older embryos, irrespective of the type of thermal stressor. The results indicate that the expression of hsp70 in broiler chicken embryos is affected by cold and heat distress, and is tissue- and age-dependent.     

Cloning, expression, and structural analysis of recombinant BJcuL, a c-type lectin from the Bothrops jararacussu snake venom

The lactose-binding lectin from Bothrops jararacussu venom (BJcuL) is a homodimer belonging to group VII of the c-type animal lectins. BJcuL has also been shown to serve as an interesting tool for combating tumor progression by inhibiting cancer and endothelial cell growth. However, detailed structural studies of BJcuL and its biological mechanisms of cytotoxicity are yet to be reported, perhaps because of the non-availability of recombinant proteins in necessary quantities. Intending to increase the present information about structural and consequently the understating of biological studies, the cDNA coding for BJcuL from a venom gland has been cloned and sequenced. The mature protein-coding region was amplified by PCR with specific oligonucleotides, and subcloned into the pET-15b vector to express the recombinant BJcuL in Escherichia coli BL21 (DE3). The deduced amino acid sequence exhibits a high degree of sequence identity with c-type lectins (CTLs) and c-type lectin-like domains (CTLDs). An insoluble and inactive 18.5-kDa protein was overexpressed after 1.0mM IPTG induction. The recombinant BJcuL was recovered and denatured in a buffer with 6M urea and purified on a nickel-affinity column. Protein refolding was carried out on this column, during procedure purification, followed by dialysis against CTBS and then by gel filtration for separation of the active dimmer. The refolding process of rBJcuL and the analysis of its structure were confirmed by biological assay, circular dichroism, and MALDI-TOF.     

Effects of the parasite Probopyrus ringueleti (Isopoda) on glucose, glycogen and lipid concentration in starved Palaemonetes argentinus (Decapoda)

Effects of the branchial ectoparasite isopod Probopyrus ringueleti on the utilization of glucose, glycogen and total lipids on starved shrimp Palaemonetes argentinus were evaluated, as well as the isopod responses to host starvation. Shrimp were maintained for 12 d under laboratory conditions. Parasitized and unparasitized shrimp were starved for 15 d, and glucose, glycogen and total lipid concentrations were determined at 0, 24, 72, 168 and 360 h of starvation. During starvation, control animals (unparasitized) preferentially metabolized lipids and preserved their carbohydrate reserves. Parasitized shrimp had lower lipid concentrations than unparasitized shrimp, and preferentially metabolized carbohydrates. Bopyrids displayed a similar response, with glycogen depleted at the beginning of the starvation period followed by subsequent reestablishment. Our results demonstrated that bopyrids affect host energy allocation. The lower initial lipid concentration of parasitized shrimp suggested that the host is disadvantaged from both food deprivation and isopod nutritional demands. A possible dependency of P. ringueleti on the mechanisms that control host metabolic processes was also suggested.