Mitochondrial gene divergence of Colombian Drosophila pseudoobscura

Isolated populations of drosophila pseudoobscura, separated from North American populations by about 2,400 km, were found in Colombia in 1960. We compared for sequences of the small ribosomal RNA (srRNA) gene on the mitochondria between North American and Colombian D. pseudoobscura in order to clarify the age of the Colombian isolates. The North American populations were not genetically different from each other but were genetically different from the Colombian populations. The Mexican strains represent the area from which the Colombian founders might have come. The estimated net nucleotide divergence between Mexican and Colombian D. pseudoobscura indicates that the Colombian population is not an ancient lineage. Phylogenies using both distance and parsimony methodologies reinforced this conclusion. The Colombian samples group together with both methods but, according to the bootstrap analysis, not significantly. It appears that the populations have not been separated long enough for their DNA sequences to show much divergence.      

Constitutive expression of CD69 in interspecies T-cell hybrids and locus assignment to human chromosome 12

In this study we describe the generation and characterization of interspecies somatic cell hybrids between human activated mature T cells and mouse BW5147 thymoma cells. A preferential segregation of human chromosomes was observed in the hybrids. Phenotypic analysis of two hybrids and their clones demonstrated coexpression of CD4 and CD69 antigens in the same cells. Segregation analysis of an informative family of hybrids followed by molecular and karyotype studies clearly demonstrated that the locus encoding CD69 antigen mapped to human chromosome 12. Although the expression of CD69 antigen is an early event after T-lymphocyte activation and rapidly declines in absence of exogenous stimuli, in the hybrids described in this study the expression was constitutive, similarly to what was previously found in early thymocyte precursors and mature thymocytes. In this respect it was important to note that the behavior of the hybrids in culture strongly suggested a dominant influence of the thymus-derived mouse tumor cell genome in controlling the constitutive expression of human CD69. These hybrids may thus provide a system to study the genetic and molecular mechanisms controlling the expression and function of this activation antigen. Address correspondence and offprint requests to: R. S. Accolla, Istituto di Scienze Immunologiche, Facolta'di Medicina e Chirurgia, Universita'di Verona, Policlinico Borgo Roma, 37100 Verona, Italy.     

Evidence for the presence of both D-1 and D-2 dopamine receptors in human esophagus.

Clinical and pharmacological evidence suggested that dopamine is involved in the control of esophageal motility. The present study was designed to determine whether or not dopamine receptors are present in human esophagus. With this aim we measured adenylate cyclase activity as a biochemical index of dopamine receptor function in esophageal specimens taken from five patients during surgery for upper esophageal carcinoma. The selective D-1 agonist fenoldopam stimulated cAMP formation in the lower esophageal sphincter, but not in the esophageal body; this effect was prevented by the selective D-1 antagonist SCH 23390 and by d-butaclamol. Bromocriptine, a selective D-2 stimulator, inhibited adenylate cyclase activity in the lower esophageal sphincter, an effect blocked by the D-2 antagonist (-)sulpiride. No effects of bromocriptine were found in the esophageal body. These data indicate that both D-1 and D-2 receptors are present in the lower esophageal sphincter, but not in esophageal body and emphasize the role of dopamine in the regulation of esophageal function.     

Benzo(a)pyrene Exposure Causes Genotoxic and Biochemical Changes in the Midge Larvae of <Emphasis Type="Italic">Chironomus sancticaroli</Emphasis> Strixino &amp; Strixino (Diptera: Chironomidae)

Benzo(a)pyrene (BaP) is a carcinogenic polycyclic aromatic hydrocarbon, also found in nature due to human activities. BaP adheres to sediments showing toxic effects on benthic organisms, including midge larvae of the family Chironomidae. We tested for toxic effects of benzo(a)pyrene on Chironomus sancticaroli Strixino & Strixino 1981 using biochemical and genotoxic biomarkers, to identify changes in metabolic and antioxidant pathways, besides neurotoxic and DNA damage. Enzyme activity was compared by exposing larvae to four nominal concentrations (0.47, 2.13, 3.41, and 4.73 μg l^?1) and DNA damage to two concentrations (0.47 and 4.73 μg l^?1), after exposure at 24, 48, 72, and 96 h. BaP caused neurotoxic effect, showing acetylcholinesterase alterations at different treatments. Changes in the biotransformation pathway were detected, with an increased activity of alpha and beta esterase in 48 h and reduction of glutathione-S-transferase activity in all periods at the highest concentrations. Damage to the antioxidant system was observed by the increase of the superoxide dismutase and reduction of the catalase, in 48 h. Genotoxicity was detected by an increased DNA damage at 48 and 72 h. The lowest concentration (0.47 μg l^?1), even presenting low mortality, also altered the biochemical parameters of the larvae. Thus, these results indicate that BaP causes metabolic, neurotoxic, and genotoxic effects on C. sancticaroli, even at low concentrations and short-term exposure. BaP can cause damage of immature invertebrates, and the ecological dynamics can be affected, since these organisms have trophic importance in the aquatic environment.     

Molecular Detection of HpmA and HlyA Hemolysin of Uropathogenic Proteus mirabilis

Urinary tract infection (UTI) is one of the bacterial infections frequently documented in humans. Proteus mirabilis is associated with UTI mainly in individuals with urinary tract abnormality or related with vesicular catheterism and it can be difficult to treat because of the formation of stones in the bladder and kidneys. These stones are formed due to the presence of urease synthesized by the bacteria. Another important factor is that P. mirabilis produces hemolysin HpmA, used by the bacteria to damage the kidney tissues. Proteus spp. samples can also express HlyA hemolysin, similar to that found in Escherichia coli. A total of 211 uropathogenic P. mirabilis isolates were analyzed to detect the presence of the hpmA and hpmB genes by the techniques of polymerase chain reaction (PCR) and dot blot and hlyA by PCR. The hpmA and hpmB genes were expressed by the RT-PCR technique and two P. mirabilis isolates were sequenced for the hpmA and hpmB genes. The presence of the hpmA and hpmB genes was confirmed by PCR in 205 (97.15 %) of the 211 isolates. The dot blot confirmed the presence of the hpmA and hpmB genes in the isolates that did not amplify in the PCR. None of the isolates studied presented the hlyA gene. The hpmA and hpmB genes that were sequenced presented 98 % identity with the same genes of the HI4320 P. mirabilis sample. This study showed that the PCR technique has good sensitivity for detecting the hpmA and hpmB genes of P. mirabilis.     

Genome Size Evaluation in Tetraodontiform Fishes from the Neotropical Region

Smooth pufferfish of the family Tetraodontidae had become pure genomic models because of the remarkable compaction of their genome. This trait seems to be the result of DNA loss following its divergence from the sister family Diodontidae, which possess larger genomes. In this study, flow cytometry was used for estimate the genome size of four pufferfish species from the Neotropical region. Cytogenetic data and confocal microscopy were also used attempting to confirm relationships between DNA content and cytological parameters. The haploid genome size was 0.71?±?0.03 pg for Sphoeroides greeleyi, 0.34?±?0.01 pg for Sphoeroides spengleri, 0.82?±?0.03 pg for Sphoeroides testudineus (all Tetraodontidae), and 1.00?±?0.03 pg for Chilomycterus spinosus (Diodontidae). These differences are not related with ploidy level, because 46 chromosomes are considered basal for both families. The value for S. spengleri represents the smallest vertebrate genome reported to date. Since erythrocyte cell and nuclear sizes are strongly correlated with genome size, the variation in this last is considered under both adaptive and evolutionary perspectives.     

Development of a cost-effective high-throughput process of microsatellite analysis involving miniaturized multiplexed PCR amplification and automated allele identification

Background

Microsatellites are nucleotide sequences of tandem repeats occurring throughout the genome, which have been widely used in genetic linkage analysis, studies of loss of heterozygosity, determination of lineage and clonality, and the measurement of genome instability or the emergence of drug resistance reflective of mismatch repair deficiency. Such analyses may involve the parallel evaluation of many microsatellite loci, which are often limited by sample DNA, are labor intensive, and require large data processing.

Results

To overcome these challenges, we developed a cost-effective high-throughput approach of microsatellite analysis, in which the amplifications of microsatellites are performed in miniaturized, multiplexed polymerase chain reaction (PCR) adaptable to 96 or 384 well plates, and accurate automated allele identification has been optimized with a collective reference dataset of 5,508 alleles using the GeneMapper software.

Conclusions

In this investigation, we have documented our experience with the optimization of multiplex PCR conditions and automated allele identification, and have generated a unique body of data that provide a starting point for a cost-effective, high-throughput process of microsatellite analysis using the studied markers.

     

A Multiple Aminoacyl-tRNA Synthetase Complex That Enhances tRNA-Aminoacylation in African Trypanosomes

The genes for all cytoplasmic and potentially all mitochondrial aminoacyl-tRNA synthetases (aaRSs) were identified, and all those tested by RNA interference were found to be essential for the growth of Trypanosoma brucei. Some of these enzymes were localized to the cytoplasm or mitochondrion, but most were dually localized to both cellular compartments. Cytoplasmic T. brucei aaRSs were organized in a multiprotein complex in both bloodstream and procyclic forms. The multiple aminoacyl-tRNA synthetase (MARS) complex contained at least six aaRS enzymes and three additional non-aaRS proteins. Steady-state kinetic studies showed that association in the MARS complex enhances tRNA-aminoacylation efficiency, which is in part dependent on a MARS complex-associated protein (MCP), named MCP2, that binds tRNAs and increases their aminoacylation by the complex. Conditional repression of MCP2 in T. brucei bloodstream forms resulted in reduced parasite growth and infectivity in mice. Thus, association in a MARS complex enhances tRNA-aminoacylation and contributes to parasite fitness. The MARS complex may be part of a cellular regulatory system and a target for drug development.     

Evidence for a trans-acting activator function regulating the expression of the human CD5 antigen

Interspecies somatic cell hybrids were generated by fusing the mouse T-lymphoma cell line, BW5147, with normal human T lymphocytes at different stages of differentiation. Thymocytes, activated peripheral T lymphocytes, or an activated T-cell clone were used as human partners, respectively, in three independent fusions. Irrespective of the human cell partner used for fusion, a certain number of hybrids lost CD5 surface expression over a period of time in culture. Analysis at the phenotype and genetic level showed that lack of CD5 expression was due neither to segregation of human autosome 11, on which the CD5 gene has been mapped, nor to deletion of the CD5 structural gene. Furthermore, loss of CD5 surface expression correlated with the absence of specific mRNA. Since these hybrids preferentially segregate human chromosomes, these results indicate the existence of a non-syntenic trans-active locus, or loci, positively controlling the expression of the human CD5 gene.