Overexpression of plant uncoupling mitochondrial protein in transgenic tobacco increases tolerance to oxidative stress

An Arabidopsis thaliana cDNA clone encoding a plant uncoupling mitochondrial protein (AtPUMP1) was overexpressed in transgenic tobacco plants. Analysis of the AtPUMP1 mRNA content in the transgenic lines, determined by Northernblot, revealed variable levels of transgene expression. Antibody probing ofWestern blots of mitochondrial proteins from three independent transgenic lines showed significant accumulation of AtPUMP1 in this organelle. Overproduction of AtPUMP1 in transgenic tobacco plants led to a significantincrease in tolerance to oxidative stress promoted by exogenous hydrogen peroxide as compared to wild-type control plants. These results provide thefirst biological evidence for a role of PUMP in protection of plant cells against oxidative stress damage.     

Characterization and expression of a novel member (JBURE-II) of the urease gene family from jackbean [Canavalia ensiformis (L.) DC

Canavalia ensiformis (jackbean) seeds contain the proteins urease and canatoxin, a variant form of the jackbean urease. Here we have cloned a cDNA encoding another isoform of urease, called JBURE-II. This cDNA was obtained by RT-PCR using as template total RNA extracted from C. ensiformis tissues. Nucleotide sequence analysis showed that JBURE-II clones share 86% similarity with known jackbean urease. The presence in C. ensiformis of a family of urease-related genes with at least three members was demonstrated by Southern blot analysis. In order to understand the pattern of expression of the JBURE-II gene, we collected tissue samples from different stages of flower and embryo development. The results of RT-PCR show that JBURE-II is expressed from flower buds throughout seed maturation. Semi-quantitative RT-PCR indicates that expression of urease and JBURE-II genes is induced in seedlings and in leaves treated with abscisic acid, a phytohormone involved in seed maturation and wound response. This work constitutes the first report on the presence of a family of urease genes in jackbean, and provides characterization of a cDNA encoding a new member of this gene family.     

Docking and small angle X-ray scattering studies of purine nucleoside phosphorylase

Docking simulations have been used to assess protein complexes with some success. Small angle X-ray scattering (SAXS) is a well-established technique to investigate protein spatial configuration. This work describes the integration of geometric docking with SAXS to investigate the quaternary structure of recombinant human purine nucleoside phosphorylase (PNP). This enzyme catalyzes the reversible phosphorolysis of N-ribosidic bonds of purine nucleosides and deoxynucleosides. A genetic deficiency due to mutations in the gene encoding for PNP causes gradual decrease in T-cell immunity. Inappropriate activation of T-cells has been implicated in several clinically relevant human conditions such as transplant rejection, rheumatoid arthritis, lupus, and T-cell lymphomas. PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation and has been submitted to extensive structure-based drug design. The present analysis confirms the trimeric structure observed in the crystal. The potential application of the present procedure to other systems is discussed.     

Temperature and meal size effects on the postprandial metabolism and energetics in a boid snake

We investigated the combined effect of meal size and temperature on the aerobic metabolism and energetics of digestion in Boa constrictor amarali. Oxygen uptake rates (Vd2;o2) and the duration of the digestion were determined in snakes fed with meals equaling to 5%, 10%, 20%, and 40% of the snake's body mass at 25 degrees and 30 degrees C. The maximum Vd2;o2 values attained during digestion were greater at 30 degrees C than at 25 degrees C. Both maximal Vd2;o2 values and the duration of the specific dynamic action (SDA) were attained sooner at 30 degrees C than at 25 degrees C. Therefore, the temperature effect on digestion in Boa is characterized by the shortening of the SDA duration at the expense of increased Vd2;o2. Energy allocated to SDA was not affected by meal size but was greater at 25 degrees C compared to 30 degrees C. This indicates that a postprandial thermophilic response can be advantageous not only by decreasing the duration of digestion but also by improving digestive efficiency. Maximal Vd2;o2 and SDA duration increased with meal size at both temperatures.     

Molecular cytogenetic assignment of genes to bovine chromosome 5

Seven genes were assigned by molecular cytogenetic methods to bovine chromosome 5. To accomplish this, specific primers were either publicly available or were designed from highly conserved regions of the publicly available mammalian gene sequences. The identity of the amplified segments was verified by sequencing and alignment with the published sequences. The optimized primers that amplified the desired bovine genes were used for screening a bovine bacterial artificial chromosome library. The positive clones were localized to a specific band of bovine chromosome 5 by fluorescence in situ hybridization. The genes HOXC4, SP1 and IGFBP6 were localized to band q21, COL2A1 was localized to bands q21-q23, IGF1 was localized to band q26, MB to band q31 and the gene CYP2D6 was localized to band q35. The cytogenetic assignment of SP1, IGFBP6, COL2A1, IGF1, MB and CYP2D6 is first reported here and the assignment of HOXC4 refines the previous assignment of this gene. The identification and localization of these genes further support the development of the human to bovine comparative map through characterizing the homologous segments conserved in the evolution of these species. This information will be useful for the future localization of genes that affect economically important traits in bovines.     

Brazilian distribution of Amblyomma varium Koch, 1844 (Acari: Ixodidae), a common parasite of sloths (Mammalia: Xenarthra)

Amblyomma varium, commonly known in Brazil as the "carrapato-gigante-da-pregui a" (sloth's giant tick) is found from southern Central America to Argentina. The present study adds information on the geographical distribution of A. varium, as well as on their hosts, based on material deposited in the main Brazilian collections and on the available literature. Eighty-two vials, containing 191 adult specimens, deposited in five Acari collections between 1930 and 2001, were examined. These vials included data on the host and collection localities. The biology of A. varium is unknown. However it is known that, during the adult stage, the tick presents a high host specificity and is found almost exclusively on the sloths Bradypus tridactylus, B. variegatus, B.torquatus (Bradypodidae), Choloepus hoffmanni and C. didactylus (Megalonychidae). Based on the material examined, the states of Rond nia, Amazonas, Bahia and Alagoas are newly assigned to geographic distribution of A. varium in Brazil.     

Factors affecting the uptake of DMSO by the eggs and embryos of medaka,Oryzias latipes

High performance liquid chromatography (HPLC) was used to assess the uptake dynamics of the cryoprotectant DMSO by intact unfertilized eggs (stage 0), 8-cell (stage 5) and eyed embryos (stage 30) of medaka, Oryzias latipes, the relation of the internal concentration (Cin) of DMSO with fertilization and survival rates, and the effects of several factors on these processes. The factors examined were: cryoprotectant concentration (0.6, 1.2, 1.9 and 2.5 M), impregnation time (1, 3, 5, 10, 15 and 20 min), temperature (0, 5 and 20 degrees C), hydrostatic pressure (0 and 50 atm), and the osmotic conditions of the materials (normal or partially dehydrated). Cryoprotectant permeation, estimated from the initial rates of DMSO uptake, was higher in embryos than in eggs and increased with embryonic development; however, the DMSO Cin in eyed embryos reached a plateau at 1-5 min and could not be increased by prolonging impregnation. The highest fertilization and survival rates for any given DMSO Cin were obtained with high concentrations and short times of impregnation rather than low concentrations and long impregnation times. Application of hydrostatic pressure (50 atm) and exposure for 3 min to a 1 M trehalose solution prior to impregnation induced a substantial increase in the DMSO Cin of 8-cell embryos in comparison to untreated controls with no significant effect on survival. Hydrostatic pressure also promoted DMSO uptake in unfertilized eggs, but with rapid loss of viability, and was ineffective in eyed embryos. The uptake of DMSO and its toxicity to 8-cell embryos were directly proportional to the temperature of impregnation. The results of this study reveal important interactions between cryoprotectant concentration, impregnation time and the developmental stage (or type) of the materials and provide evidence that hydrostatic pressure, temperature of impregnation and the osmotic conditions of the materials can be manipulated to increase the uptake of cryoprotectant by fish eggs and embryos.     

Control of Chagas disease in blood banks in Colombia

The screening programs for the Chagas disease agent, Trypanosoma cruzi, were examined in Colombian blood banks and, as a consequence, several procedural improvements in the blood bank network were recommended. Screening strategies and techniques were examined, as well as the action taken when seropositive donors were discovered. From a total of 180 blood banks in 33 departments, 103 banks in 20 departments answered the survey. The 103 banks collected 291, 105 units of blood, corresponding to 66.6% of all units collected in the country in 1997. Of these blood units, 99.6% were screened for Chagas trypanosomes; 3,321 (1.2%) of 287,048 were found positive for anti-T. cruzi. The data were grouped by department; geographical differences for seroprevalence rates varied markedly between 0% and 12.6%. The most commonly used serological technique was ELISA, but only 33.2% of the positive samples for anti-T. cruzi underwent further confirmatory testing, mainly through indirect immunofluorescent test. Most (95.1%) of the blood banks used basic, internal quality control procedures, and 73.8% sent positive samples to other laboratories for external quality control.     

In vitro tests for haematotoxicity: prediction of drug-induced myelosuppression by the CFU-GM assay

In a prevalidation study, a standard operating procedure (SOP) for human and mouse in vitro tests was developed, for evaluating the potential haematotoxicity of xenobiotics in terms of their direct, adverse effects on the myeloid colony-forming unit (CFU-GM). Based on the adjustment of the mouse-derived maximum tolerated dose (MTD), a prediction model was set up to calculate the human MTD, and an international blind trial was designed to apply this model to the clinical neutropenia of 23 drugs including 17 antineoplastics. The model correctly predicted the human MTD for 20 drugs out of the 23 (87%). This high percentage of predictivity, and the reproducibility of the SOP testing, confirmed the scientific validation of this model, and suggest promising applications for developing and validating other in vitro methods for use in haematotoxicology.