Replication Region Fragments Cloned from Flac+ Are Identical to EcoRI Fragment f5 of F

The replication region fragments from Flac⁺ cloned in plasmids pSC138 and pML31 are identical with each other and with EcoRI fragment 5 of plasmid F.     

Cloning and characterization of EcoRI and HindIII restriction endonuclease-generated fragments of antibiotic resistance plasmids R6-5 and R6

Summary DNA fragments generated by the EcoRI or HindIII endonucleases from the low copy number antibiotic resistance plasmids R6 and R6-5 were separately cloned using the high copy number ColEl or pML21 plasmid vectors and the insertional inactivation procedure. The hybrid plasmids that were obtained were used to determine the location of the EcoRI and HindIII cleavage sites on the parent plasmid genomes by means of electron microscope heteroduplex analysis and agarose gel electrophoresis. Ultracentrifugation of the cloned fragments in caesium chloride gradients localized the high buoyant density regions of R6-5 to fragments that carry the genes for resistance to streptomycin-spectinomycin, sulfonamide, and mercury and a low buoyant density region to fragments that carry the tetracycline resistance determinant. Functional analysis of hybrid plasmids localized a number of plasmid properties such as resistances to antibiotics and mercury and several replication functions to specific regions of the R6-5 genome. Precise localisation of the genes for resistance to chloramphenicol, kanamycin, fusidic acid and tetracycline was possible due to the presence of identified restriction endonuclease cleavage sites within these determinants.Only one region competent for autonomous replication was identified on the R6-5 plasmid genome and this was localized to EcoRI fragment 2 and HindIII fragment 1. However, two additional regions of replication activity designated RepB and RepC, themselves incapable of autonomous replication but capable of supporting replication of a linked ColE1 plasmid in polA^– bacteria, were also identified.     

Evaluation of DNA vaccination of spotted sand bass (Paralabrax maculatofasciatus) with two major outer-membrane protein-encoding genes from Aeromonas veronii

Genes encoding two major outer membrane proteins (OMPs) of the bacterial pathogen Aeromonas veronii, Omp38 and Omp48, were used to construct DNA vaccines. The protective effect of such vaccines against motile aeromonad septicaemia was evaluated in spotted sand bass (Paralabrax maculatofasciatus), an endemic species of the Mexican Northwest Pacific coast and a potential resource for the aquaculture industry. Weak protein expression, as determined by immunoblotting, was observed after transfection of eukaryotic cells with the DNA vaccines. Fish immunized with a single intramuscular injection of 20 microg of the omp38 and omp48 DNA vaccines showed slightly, but significantly elevated serum antibody levels 4 and 6 weeks after vaccination, compared to fish vaccinated with the control plasmid pcDNA3.1. Spotted sand bass vaccinated with the omp38 and omp48 DNA vaccines and challenged with A. veronii by intraperitoneal route recorded a relative percent survival (RPS) between 50 and 60%. Histopathological signs of motile aeromonad septicaemia were observed in around 40% of omp38 and omp48-vaccinated fish and 80% of pcDNA3.1-vaccinated control fish. The results indicate that P. maculatofasciatus vaccinated with a single dose of DNA plasmids encoding the major OMPs from A. veronii shows partial protection against infection and mortality by A. veronii experimental infection.     

Effects of follicle-stimulating hormone and vitamin A upon purinergic secretion by rat Sertoli cells

Follicle-stimulating hormone (FSH) and vitamin A (retinol) are two of the main regulators of the male reproductive system. Recently, it has been described that extracellular purines can affect some important reproductive-related functions in Sertoli cells and germinative cells, by activating specific purinergic receptors. In this work, we report that both FSH and retinol are able to induce changes in the levels of extracellular purines of cultured rat Sertoli cells. FSH induced an increase in adenosine, mainly caused by enhanced ecto-ATPase activity, while retinol increased xanthine and hypoxanthine levels, and decreased uric acid concentration by an unknown mechanism. These data indicate that purinergic signaling may be involved in the control and/or regulation of some of the reproductive-related actions of these hormones. (Mol Cell Biochem 278: 185–194, 2005)     

Lessons from a national survey of Chagas disease transmission risk in Colombia

In recent years, there has been a revitalization of large-scale programmes to control parasitic disease in developing countries. In 1997, the Governments of Colombia, Venezuela, Ecuador and Peru committed themselves to replicate the cost-effective elimination of Trypanosoma cruzi transmission achieved in the Southern Cone by using insecticides against the domestic triatomine vectors (in combination with blood-bank screening). Central American Governments launched a complementary initiative. All plan to interrupt vectorial transmission throughout the region by 2010 but specific targets are decided nationally. In this article, we highlight the novel approach taken by the Colombian Government for determining the geographic distribution of Chagas disease risk to select where to intervene first.     

Clinical and pathological importance of vacA allele heterogeneity and cagA status in peptic ulcer disease in patients from North Brazil

We have examined the prevalence of gene cagA and vacA alleles in 129 patients, 69 with gastritis and 60 with peptic ulcer diseases from North Brazil and their relation with histopathological data. vacA and cagA genotype were determined by polymerase chain reaction. Hematoxylin-eosin staining was used for histological diagnosis. 96.6% of the patients were colonized by Helicobacter pylori strains harboring single vacA genotype (nont-mixed infection). Among them, 11.8% had subtype s1a, 67.8% had subtype s1b, and 17% subtype s2. In regard to the middle region analysis, m1 alleles were found in 75.4% and m2 in 21.2% of patients. The cagA gene was detected in 78% patients infected with H. pylori and was associated with the s1-m1 vacA genotype. The H. pylori strains, vacA s1b m1/cagA-positive, were associated with increased risk of peptic ulcer disease and higher amounts of lymphocytic and neutrophilic infiltrates and the presence of intestinal metaplasia. These findings show that cagA and vacA genotyping may have clinical relevance in Brazil.     

Post mortem time and season alter subpopulation characteristics of Iberian red deer epididymal sperm

We have studied the effect of post mortem time and season on sperm subpopulation pattern and characteristics. We used epididymal samples from free-ranging Iberian red deers harvested during the hunting season. We studied samples at different moments of the year (rut, transition period and post-rut), and at different times post mortem (up to 4 days). Sperm were extracted from the cauda epididymis and their motility was evaluated by means of a CASA system. A principal component and clustering analysis were carried out to identify subpopulations. Post mortem time caused a significant decrease in motility quality, and a general deterioration in subpopulation characteristics. We found three subpopulations the first day, and the one indicating good sperm quality decreased with post mortem time until it disappeared on the fourth day. This may indicate considerable impairment of the samples after 72 h post mortem, which could compromise their use in AI programs. With regard to season, subpopulation pattern and characteristics were better in the transition and post-rut periods. Moreover, we found one subpopulation formed by mature spermatozoa, which increased from rut to post-rut. This might be a negative fact, because samples collected after the rut may undergo hypermaturation, which possibly impairs fertility. Our results are of interest for the management of wildlife germplasm banks based on post mortem sperm recovery.