Molecular analysis of VCA1008: a putative phosphoporin of Vibrio cholerae

The PhoB/PhoR-dependent response to inorganic phosphate (Pi)-starvation in Vibrio cholerae O1 includes the expression of vc0719 for the response regulator PhoB, vca0033 for an alkaline phosphatase and vca1008 for an outer membrane protein (OMP). Sequences with high identity to these genes have been found in the genome of clinical and environmental strains, suggesting that the Pi-starvation response in V. cholerae is well conserved. VCA1008, an uncharacterized OMP involved in V. cholerae pathogenicity, presents sequence similarity to porins of Gram-negative bacteria such as phosphoporin PhoE from Escherichia coli . A three-dimensional model shows that VCA1008 is a 16-stranded pore-forming β-barrel protein that shares three of the four conserved lysine residues responsible for PhoE anionic specificity with PhoE. VCA1008 β-barrel apparently forms trimers that collapse into monomers by heating. Properties such as heat modifiability and resistance to denaturation by sodium dodecyl sulfate at lower temperatures permitted us to suggest that VCA1008 is a classical porin, more precisely, a phosphoporin due to its Pi starvation-induced PhoB-dependent expression, demonstrated by electrophoretic mobility shift assay and promoter fusion- lacZ assays.     

Alpha thalassemia and alpha-MRE haplotypes in Uruguayan patients with microcytosis and hypochromia without anemia

Alpha thalassemia is the most common genetic disorder across the world, being the α-^3.7 deletion the most frequent mutation. In order to analyze the spectrum and origin of alpha thalassemia mutations in Uruguay, we obtained a sample of 168 unrelated outpatients with normal hemoglobin levels with microcytosis and hypochromia from two cities: Montevideo and Salto. The presence of α-thalassemia mutations was investigated by gap-PCR, restriction endonucleases analysis and HBA2 and HBA1 genes sequencing, whereas the alpha-MRE haplotypes were investigated by sequencing. We found 55 individuals (32.7%) with α-thalassemia mutations, 51(30.4%) carrying the -α^3.7 deletion, one with the -α^4.2 deletion and three having the rare punctual mutation HBA2:c.-59C>T. Regarding alpha-MRE analysis, we observed a significant higher frequency of haplotype D, characteristic of African populations, in the sample with the -α^3.7 deletion. These results show that α-thalassemia mutations are an important determinant of microcytosis and hypochromia in Uruguayan patients with microcytosis and hypochromia without anemia, mainly due to the -α^3.7 deletion. The alpha-MRE haplotypes and the α-thalassemia mutations spectrum suggest a predominant, but not exclusive, African origin of these mutations in Uruguay.     

Arsenotoxicidad aguda experimental en ratones Balb/c: marcadores orgánicos y compromiso esplénico

IntroducciónEl arsénico es un tóxico ambiental ampliamente diseminado en todo el mundo. En hombres y animales, diversos órganos y tejidos son blancos de sus efectos deletéreos, entre ellos, el los del sistema inmunológico.ObjetivoDeterminar la intoxicación aguda por arsénico en tejidos y células diana de ratones Balb/c in vivo.Materiales y métodosSe aplicó una inyección intraperitoneal de 9,5 o 19 mg/kg de arsenito de sodio (NaAsO₂) o un volumen equivalente de solución fisiológica como control, en ratones Balb/c con 3 por cada grupo experimental. Tras media hora, los animales fueron sacrificados y se extrajeron bazos, timos, hígados, riñones y sangre. En cada muestra, se determinó la concentración de arsénico, polifenoles y hierro, y también, se evaluaron marcadores oxidativos, como peróxidos, productos avanzados de oxidación proteica y grupos sulfhidrilos libres. En los esplenocitos obtenidos del bazo, se determinaron la viabilidad celular y el potencial mitocondrial.ResultadosLa dosis aguda inyectada de NaAsO₂ redujo la función mitocondrial de los esplenocitos, lo que derivó en muerte celular. La presencia confirmada de arsénico en las muestras de bazo y la citotoxicidad resultante, produjeron disminución de los polifenoles y de los grupos sulfhidrilos libres, y alteraron el contenido y la distribución del hierro, pero no se aumentó la producción de peróxidos.ConclusiónEstos hallazgos aportan evidencia científica sobre los cambios en biomarcadores involucrados en la inmunotoxicidad del arsénico y ofrecen, además, una metodología para ensayar potenciales tratamientos frente a la acción deletérea de este compuesto en el sistema inmunológico.Palabras clave: arsénico, toxicidad aguda, estrés oxidativo, sistema inmunológico, ratones     

Expression of Human Cu-Zn Superoxide Dismutase Gene in Transgenic Mice: Model for Gene Dosage Effect in Down Syndrome

It was suggested that increased Cu-Zn superoxide dismutase (SOD-I) might be involved in the various biological abnormalities found in Down's syndrome (DS) such as premature aging and Alzheimer-type neurological lesions. As a model system for testing this hypothesis we have developed two strains of transgenic mice carrying only one copy of the human SOD-I gene. In the first strain (TGI). no expression has been found by northern blot analysis. The second strain (TG2) exhibited human SOD-I mRNA and increased SOD-l activity in the brain (1.93 fold), in the heart (I.69 fold), thymus (I.49 fold) and to a lesser extent in muscle (1.25 fold), liver (1.19 fold), kidney (1.18 fold), spleen (1.35 fold), lung (1.26 fold) and erythrocytes (1.09 fold). In this strain, increased SOD-1 activity in the brain did not induce modifications in the seleno-dependent glutathione peroxidase. glutathione reductase and glutathione S-transferase activities. In brain homogenates, we have focused our studies on Tau proteins which are known to be the major antigenic components of paired helical filaments (P11F). both in DS and Alzheimer's disease. Our results suggested that, in our experimental conditions, the overexpression of SOD-I did not induce the modifications of Tau proteins similar to those seen during neurofibrillary degeneration.     

Bacterial dynamics of sugarcane silage in the tropics

The objective of this study was to evaluate changes in the bacterial community in sugarcane silage, in distinct soil types along the storage period. We depicted the bacterial community associated with sugarcane, before and after ensiling, through a massive sequencing of the gene 16S rRNA using MiSeq platform. The ensilage process shifted the composition of the bacterial community from the heterofermentative lactic acid bacteria Leuconostoc to bacteria belonging to the genera Acinetobacter, Ralstonia and Novosphingobium. However, this shift did not convey statically significant differences in alfa diversity metrics. In addition, similarity percentage analysis showed that the bacterial Operational Taxonomic Units that were primarily responsible for the observed differences were Leuconostoc, Pseudomonas, Acinetobacter, Ralstonia, Fructobacillus, Novosphingobium, Lactobacillus, Burkholderia and Clostridium sensu stricto 1. The storage period was the most important factor responsible for changes in the bacterial community of silages. Results confirmed that the type of soil did not influence the dissimilarity found among samples.     

XRCC4 rs28360071 intronic variant is associated with increased risk for infant acute lymphoblastic leukemia with KMT2A rearrangements

Early age acute leukemia (EAL) shows a high frequency of KMT2A-rearrangements (KMT2A-r). Previous investigations highlighted double-strand breaks arising from maternal exposure to xenobiotics during pregnancy as a risk factor for EAL and KMT2A-r. In this case-control study, we investigated the relationship between EAL and genetic variants of the nonhomologous end-joining (XRCC6 rs5751129, XRCC4 rs6869366 and rs28360071), since they might affect DNA repair capacity, leading to KMT2A-r and leukemogenesis. Samples from 577 individuals (acute lymphoblastic leukemia-ALL, n=164; acute myeloid leukemia-AML, n=113; controls, n=300) were genotyped. No significant association was found for rs5751129 and rs6869366, whereas rs28360071 was associated with an increased risk for ALL with KMT2A-r (IIxID: OR - Odds ratio 2.23, CI 1.17-4.25, p=0.014). Bone marrow samples from ALL patients showed a higher expression of XRCC4 compared to AML patients (p=0.025). Human Splicing Finder 3.1 predicted that the deleted allele of rs28360071 is potentially associated with the activation of a 5’ cryptic splice site in intron 3 of XRCC4. The sequencing of cDNA did not show any differences on the splicing process for the rs28360071 genotypes. Our results suggest that the deleted allele for rs28360071 increases the risk for ALL with KMT2A-r, but not by modifying the XRCC4 expression levels or its structure.     

Biosynthesis of antimalarial lignans from Holostylis reniformis

Holostylis reniformis biosynthesizes 8-8′ linked lignans without 9,9′-oxygenation. To elucidate the biosynthetic pathways to these lignans, the reputed precursors [U-¹⁴C]phenylalanine, [9-³H₁]coniferyl alcohol, and [9-³H₁]isoeugenol were administered to roots of the plant, which led to the incorporation of ³H and ¹⁴C into ten 2,7′ linked-lignans (aryltetralone lignans) and two 7,7′-epoxylignans (furan lignans). These administration experiments demonstrated that the lignans were propenylphenol-derived and that H. reniformis can exhibit regioselective control over radical-radical coupling (via isoeugenol radicals). Regiospecific control over propenylphenol-derived lignan biosynthesis was observed, together with diastereoselective control of C2-C7′ bond formation for the aryltetralone lignans (7′R). These experiments provide evidence that isoeugenol is a biosynthetic intermediate to the aryltetralone and furan lignans.     

SPINT2 Deregulation in Prostate Carcinoma

SPINT2 is a tumor suppressor gene that inhibits proteases implicated in cancer progression, like HGFA, hepsin and matriptase. Loss of SPINT2 expression in tumors has been associated with gene promoter hypermethylation; however, little is known about the mechanisms of SPINT2 deregulation in prostate cancer (PCa). We aimed to analyze SPINT2 expression levels and understand the possible regulation by SPINT2 promoter hypermethylation in PCa. In a cohort of 57 cases including non-neoplastic and PCa tissues, SPINT2 expression and promoter methylation was analyzed by immunohistochemistry and methylation-specific PCR, respectively. Methylation status of the SPINT2 promoter was also evaluated by bisulfite sequencing and 5-aza-2’-deoxycytidine treatment. Oncomine and TCGA databases were used to perform in silico PCa analysis of SPINT2 mRNA and methylation levels. A reduction in SPINT2 expression levels from non-neoplastic to PCa tissues was observed; however, none of the cases exhibited SPINT2 promoter methylation. Both bisulfite sequencing and 5-aza demonstrated that SPINT2 promoter is not methylated in PCa cells. Bioinformatics approaches did not show downregulation of SPINT2 at the mRNA level and, in corroboration with our results, SPINT2 promoter region is reported to be unmethylated. Our study suggests an involvement of SPINT2 in PCa tumorigenesis, probably in association with a post-translational regulation of SPINT2.     

CYTOGENETIC STUDIES OF SOME HYPOCHOERIS SPECIES (COMPOSITAE) FROM BRAZIL

Karyotypes of nine Brazilian taxa of genus Hypochoeris were studied utilizing root-tip mitotic metaphases. Two distinct groups were found. One group includes six species that showed high asymmetric bimodal karyotypes, while the second group has two species that have a karyotype similar to those observed in European species. All the species have karyotypes with 2n = 8 that are very uniform within each group, with only small morphological differences. Nucleolar organizing region and C-band patterns are shown for H. brasiliensis.