Structural modeling and electrostatic properties of aspartate transcarbamylase from Saccharomyces cerevisiae

In Saccharomyces cerevisiae the first two reactions of the pyrimidine pathway are catalyzed by a multifunctional protein which possesses carbamylphosphate synthetase and aspartate transcarbamylase activities. Genetic and proteolysis studies suggested that the ATCase activity is carried out by an independently folded domain. In order to provide structural information for ongoing mutagenesis studies, a model of the three-dimensional structure of this domain was generated on the basis of the known X-ray structure of the related catalytic subunit from E. coli ATCase. First, a model of the catalytic monomer was built and refined by energy minimization. In this structure, the conserved residues between the two proteins were found to constitute the hydrophobic core whereas almost all the mutated residues are located at the surface. Then, a trimeric structure was generated in order to build the active site as it lies at the interface between adjacent chains in the E. coli catalytic trimer. After docking a bisubstrate analog into the active site, the whole structure was energy minimized to regularize the interactions at the contact areas between subunits. The resulting model is very similar to that obtained for the E. coli catalytic trimer by X-ray crystallography, with a remarkable conservation of the structure of the active site and its vicinity. Most of the interdomain and intersubunit interactions that are essential for the stability of the E. coli catalytic trimer are maintained in the yeast enzyme even though there is only 42% identity between the two sequences. Free energy calculations indicate that the trimeric assembly is more stable than the monomeric form. Moreover an insertion of four amino acids is localized in a loop which, in E. coli ATCase, is at the surface of the protein. This insertion exposes hydrophobic residues to the solvent. Interestingly, such an insertion is present in all the eukaryotic ATCase genes sequenced so far, suggesting that this region is interacting with another domain of the multifunctional protein. © 1994 Wiley-Liss, Inc.     

Impact du thé vert “Camellia sinensis” sur les effets du vanadium sur la croissance et l’appareil génital du ratWistar mâle

Some heavy metals are known to exert harmful effects by generating an oxidative stress which, in turn, can affect the sexual and reproductive functions of male animals. The addition of antioxidants to the diet could decrease the cytotoxic effect related to oxidative stress (in the presence of heavy metals as food or water contaminants). As a contribution to this problem, the protective effect ofCamellia sinensis green tea, which is know to be rich in antioxidant compounds (polyphenols, etc.), was studied in vanadium-treated adult male rats, with particular attention to growth and genital tract function. White male Wistar rats were given ammonium metavanadate in drinking water (0.46 g/L) for 90 days One group of animals received green tea supplement in drinking water and the control group did not. Chronic vanadium intoxication (without green tea supplement) induced a low growth rate and relative atrophy of the testes, epididymis, prostate and seminal vesicles. Motility and number of spermatozoa were also decreased. Histological examination of the testes revealed atrophy of the seminiferous tubules and defects of spermatogenesis leading to the absence of spermatozoa in 50% of seminiferous tubules. Blood testosterone levels, evaluated by radioimmunoassay, were also decreased from day 2 to day 20. In control animals, these levels were 0.717±0.107 ng/ml; 4.366±0.666 ng/ml and 1.979±0.42 ng/ml on day 2, day 10 and day 20, respectively. After vanadium treatment, they were reduced to 0.043±0.012 ng/ml, 2.494±0.17 ng/ml and 1.086±0.53 ng/ml, respectively, at the same periods. These morphological, histological and functional disorders mostly occured during the first phase of the intoxication period (day 2 to day 10) and were subsequently attenuated, indicating adaptation to the poisoning. In rats receiving green tea, vanadium ingestion did not modify growth rate compared to control animals. Very minor changes were observed in the genital tract. Testicular atrophy and absence of spermatozoa were observed in only some seminiferous tubules. Our results underscore the protective effect of green tea on vanadium poisoning. Polyphenols, which are abundant in green tea, are known to chelate iron. It is proposed that polyphenols may also form insoluble complexes with vanadium, allowing it to be eliminated in the feces. This could explain the decreased effects of vanadium poisoning under our experimental conditions.     

Selective pressure, putative recombination events and evolutionary relationships among members of the family Closteroviridae: A proposal for a new classification

Molecular evolution of viruses is driven by both positive selection and recombination. In an effort to determine putative recombination events in the entire genome of members of the family Closteroviridae comprising 54 accessions retrieved from the international databases, a detail study was carried out by using RDP algorithm version 3.31β. Only isolates of Citrus tristeza virus and Grapevine leafroll-associated virus 3 were potential recombinants. None of members of Crinivirus genus was possible recombinant, but in turn, they were under positive selection that might be correlated with vector and/or host interactions. In contrast, Citrus tristeza virus isolates were under purifying selection as well as one-third of isolates of Grapevine leafroll-associated virus 3. The evolutionary history of all members of the Closteroviridae showed that they split into three distinct clusters corresponding to the three genera constituting this family. Moreover, the inferred phylogeny reshuffled the existing classification actually adopted by the International Committee on Taxonomy of Viruses. In this regard, each genus should be constituted by two distinct subgroups.     

Putative Recombination Events and Evolutionary History of Five Economically Important Viruses of Fruit Trees Based on Coat Protein-Encoding Gene Sequence Analysis

To enhance the knowledge of recombination as an evolutionary process, 267 accessions retrieved from GenBank were investigated, all belonging to five economically important viruses infecting fruit crops (Plum pox, Apple chlorotic leaf spot, Apple mosaic, Prune dwarf, and Prunus necrotic ringspot viruses). Putative recombinational events were detected in the coat protein (CP)-encoding gene using RECCO and RDP version 3.31β algorithms. Based on RECCO results, all five viruses were shown to contain potential recombination signals in the CP gene. Reconstructed trees with modified topologies were proposed. Furthermore, RECCO performed better than the RDP package in detecting recombination events and exhibiting their evolution rate along the sequences of the five viruses. RDP, however, provided the possible major and minor parents of the recombinants. Thus, the two methods should be considered complementary.     

Evolution of Pseudomonas aeruginosa cells towards a filterable stage in seawater

In sterile nutrient-free seawater, the number of Pseudomonas aeruginosa culturable cells decreased progressively over time and the bacteria developed cells capable of passing through a 0.45 micron pore membrane. This development was more rapid in non-autoclaved, stirred seawater and the recovery of filterable cells varied depending on the membrane type used. Minicells were observed under an electron microscope. They yielded normal cells in bacteriological media with analogous colonies and an unchanged antibiotic resistance profile. Some biochemical characters, such as gelatinase or urease activity, were however modified in the filterable cells.     

IgG Western Blot for Confirmatory Diagnosis of Equivocal Cases of Toxoplasmosis by EIA-IgG and Fluorescent Antibody Test

The performance values of available techniques used in serodiagnosis of toxoplasmosis are satisfactory but they raise problems of equivocal and discordant results for very low IgG titers. Recently marketed, LDBio-Toxo II IgG Western blot (IB) showed an excellent correlation with the dye test. We estimated the proportion of equivocal and discordant results between the enzyme immunoassay Platelia Toxo IgG (EIA-IgG) and fluorescent antibody test (FAT) and assessed the usefulness of the IB as a confirmatory test. Out of 2,136 sera collected from pregnant women, 1,644 (77.0%) tested unequivocally positive and 407 (19.0%) were negative in both EIA-IgG and FAT. The remaining 85 (4%) sera showed equivocal or discordant results. Among them, 73 (85.9%) were positive and 12 (14.1%) were negative in IB. Forty-one (89.1%) equivocal sera in EIA-IgG and 46 (86.8%) equivocal sera in FAT were positive in IB. Reducing the cut-off values of both screening techniques improved significantly their sensitivity in detecting very low IgG titers at the expense of their specificity. In conclusion, equivocal results in routine-used techniques and their discordance in determination of the immune status in pregnancy women were not uncommon. IB test appeard to be highly useful in these situations as a confirmatory technique.