Immunoregulation in human Schistosomiasis by idiotypic interactions and lymphokine-mediated mechanisms

Anti-idiotypic (anti-Id) T cells from schistosomiasis patients or former patients proliferate upon exposure to polyclonal or monoclonal anti-soluble egg antigen (SEA) antibodies. Chloroquine does not inhibit, the response, which is induced by F(ab')2 (but not soluble Fab) fragments of these antibodies. Purified T cells from former patients require macrophages or exogenous IL-1 to respond to anti-SEA Ids and can respond to matrix-bound Fab fragments in the presence of IL-1. These anti-Id T cells recognize the Ids directly. Chronic schistosomiasis patients immunoregulate the production of a non-IL-2 lymphokine that stimulates IL-2 receptor expression on resting T cells. This regulation is reversed upon chemotherapeutic cure.

Genotype by environment interaction for seed yield per plant in rapeseed using AMMI model

The objective of this study was to assess genotype by environment interaction for seed yield per plant in rapeseed cultivars grown in Northern Serbia by the AMMI (additive main effects and multiplicative interaction) model. The study comprised 19 rapeseed genotypes, analyzed in seven years through field trials arranged in a randomized complete block design, with three replicates. Seed yield per plant of the tested cultivars varied from 1.82 to 19.47 g throughout the seven seasons, with an average of 7.41 g. In the variance analysis, 72.49% of the total yield variation was explained by environment, 7.71% by differences between genotypes, and 19.09% by genotype by environment interaction. On the biplot, cultivars with high yield genetic potential had positive correlation with the seasons with optimal growing conditions, while the cultivars with lower yield potential were correlated to the years with unfavorable conditions. Seed yield per plant is highly influenced by environmental factors, which indicates the adaptability of specific genotypes to specific seasons.

Control of the rat angiotensin I converting enzyme gene by CRE-like sequences

We characterized the role of potential cAMP-responsive elements (CRE) in basal and in induced angiotensin converting enzyme (ACE) gene promoter activity in order to shed light on the regulation of somatic ACE expression. We identified stimulators and repressors of basal expression between 122 and 288 bp and between 415 and 1303 bp upstream from the transcription start site, respectively, using a rabbit endothelial cell (REC) line. These regions also contained elements associated with the response to 8BrcAMP. When screening for CRE motifs we found pCRE, a proximal sequence between 209 and 222 bp. dCRE, a distal tandem of two CRE-like sequences conserved between rats, mice and humans, was detected between 834 and 846 bp. Gel retardation analysis of nuclear extracts of REC indicated that pCRE and dCRE bind to the same protein complexes as bound by a canonical CRE. Mutation of pCRE and dCRE in REC established the former as a positive element and the latter as a negative element. In 293 cells, a renal cell line, pCRE and dCRE are negative regulators. Co-transfection of ATF-2 or ATF-2 plus c-Jun repressed ACE promoter activity, suggesting that the ACE gene is controlled by cellular stress. Although mapping of cAMP responsiveness was consistent with roles for pCRE and dCRE, mutation analysis indicated that they were not required for cAMP responsiveness. We conclude that the basal activity of the somatic ACE promoter is controlled by proximal and distal CREs that can act as enhancers or repressors depending on the cell context.

Molecular analysis of the bovine coronavirus S1 gene by direct sequencing of diarrheic fecal specimens

Bovine coronavirus (BCoV) causes severe diarrhea in newborn calves, is associated with winter dysentery in adult cattle and respiratory infections in calves and feedlot cattle. The BCoV S protein plays a fundamental role in viral attachment and entry into the host cell, and is cleaved into two subunits termed S1 (amino terminal) and S2 (carboxy terminal). The present study describes a strategy for the sequencing of the BCoV S1 gene directly from fecal diarrheic specimens that were previously identified as BCoV positive by RT-PCR assay for N gene detection. A consensus sequence of 2681 nucleotides was obtained through direct sequencing of seven overlapping PCR fragments of the S gene. The samples did not undergo cell culture passage prior to PCR amplification and sequencing. The structural analysis was based on the genomic differences between Brazilian strains and other known BCoV from different geographical regions. The phylogenetic analysis of the entire S1 gene showed that the BCoV Brazilian strains were more distant from the Mebus strain (97.8% identity for nucleotides and 96.8% identity for amino acids) and more similar to the BCoV-ENT strain (98.7% for nucleotides and 98.7% for amino acids). Based on the phylogenetic analysis of the hypervariable region of the S1 subunit, these strains clustered with the American (BCoV-ENT, 182NS) and Canadian (BCQ20, BCQ2070, BCQ9, BCQ571, BCQ1523) calf diarrhea and the Canadian winter dysentery (BCQ7373, BCQ2590) strains, but clustered on a separate branch of the Korean and respiratory BCoV strains. The BCoV strains of the present study were not clustered in the same branch of previously published Brazilian strains (AY606193, AY606194). These data agree with the genealogical construction and suggest that at least two different BCoV strains are circulating in Brazil.

Polymorphism in the lymphotoxin-alpha gene, position +252 (rs909253), is not associated with preeclampsia development in Brazilian women

PURPOSE: To investigate the frequencies of polymorphic allele and genotypes for the LT-α gene, position +252 (rs909253), in Brazilian women with preeclampsia.METHODS: This is a case-control study, in which 30 women with preeclampsia, classified according to the criteria of the National High Blood Pressure Education Program, and 115 women in the control group, with at least two healthy pregnancies, were selected. Peripheral blood was collected, and DNA was extracted, followed by genotyping, using specific primers and restriction analysis. The genotypes obtained were AA, AG and GG. Statistical analysis was performed using the χ2association test. The Hardy-Weinberg Equilibrium was tested using the Haploview Program.RESULTS: The results showed no association between genotypes and preeclampsia development (χ2=2.0; p=0.4). When the AG and GG genotypes were grouped according to allele G presence or absence (genotype AA), the data showed that the presence of allele G was not significantly different between cases (women with preeclampsia) and controls (χ2=0.0; p=1.0). The LT-α gene polymorphism, position +252 (rs909253), seems not to be an important candidate for the development of preeclampsia. Other inflammatory genes should be researched, and studies involving gene-environment interactions should be performed, in order to reach a better understanding of the etiology of the preeclampsia.

Protein-DNA associations in a gender-specific gene of Schistosoma mansoni: characterization by UV cross-linking, DNase I footprinting and band shift assays

Protein extracts obtained from male and female shistosomes were incubated with a gender-specific gene, F-10, transcribed only in adult females and encoding a major egg-shell protein. The protein/DNA interaction was measured using the band shift, DNase-I-footprinting and UV cross-linking techniques. The results showed a clear band shift when a 302 bp restriction fragment containing the 3'end of the gene was incubated with either female or male proteins. This fragment also contained a putative steroid hormone regulatory element (HRE). In contrast, only the male proteins produced a shift with the 495 bp fragment corresponding to the middle region of the gene. DNase I footprinting showed that proteins from males and females interacted with the F-10 gene by binding to multiple adjacent sites along the DNA, thus generatingrelatively long protected fragments of approximately 100 bp. This result suggested that the adjacent binding of several moles of proteins occured at the 5'end of the gene. UV cross-linking between schistosome proteins and a 21 bp synthetic oligonucleotide the F-10 HRE, evidence proteins having MWS of 30,45 and 65 kDNA. These proteins are presumably involved in the regulation of transcription of the F-10 gene.

Self-assembly of supermolecular species directed by hydrogen bonding and aromatic π-π stacking interactions

A new Cu(II) trimers, [Cu3(dcp)2(H2O)8]. 4DMF, with the ligand 3,5-pyrazoledicarboxylic acid monohydrate (H3dcp) has been prepared by solvent method. Its solid-state structure has been characterized by elemental analysis, thermal analysis (TGA and DSC), and single crystal X-ray diffraction. X-ray crystallographic studies reveal that this complex has extended 1-D,2-D and 3-D supramolecular architectures directed by weak interactions (hydrogen bond and aromatic π-π stacking interaction) leading to a sandwich solid-state structure.

Epidemiological studies in the information and genomics era: experience of the Clinical Genome of Cancer Project in São Paulo, Brazil

Genomics is expanding the horizons of epidemiology, providing a new dimension for classical epidemiological studies and inspiring the development of large-scale multicenter studies with the statistical power necessary for the assessment of gene-gene and gene-environment interactions in cancer etiology and prognosis. This paper describes the methodology of the Clinical Genome of Cancer Project in São Paulo, Brazil (CGCP), which includes patients with nine types of tumors and controls. Three major epidemiological designs were used to reach specific objectives: cross-sectional studies to examine gene expression, case-control studies to evaluate etiological factors, and follow-up studies to analyze genetic profiles in prognosis. The clinical groups included patients' data in the electronic database through the Internet. Two approaches were used for data quality control: continuous data evaluation and data entry consistency. A total of 1749 cases and 1509 controls were entered into the CGCP database from the first trimester of 2002 to the end of 2004. Continuous evaluation showed that, for all tumors taken together, only 0.5% of the general form fields still included potential inconsistencies by the end of 2004. Regarding data entry consistency, the highest percentage of errors (11.8%) was observed for the follow-up form, followed by 6.7% for the clinical form, 4.0% for the general form, and only 1.1% for the pathology form. Good data quality is required for their transformation into useful information for clinical application and for preventive measures. The use of the Internet for communication among researchers and for data entry is perhaps the most innovative feature of the CGCP. The monitoring of patients' data guaranteed their quality.

Mutations in the rpoB gene of rifampicin-resistant Mycobacterium tuberculosis strains isolated in Brazil and France

We evaluated the mutations in a 193bp of the rpoB gene by automated sequencing of rifampicin (RMP)-resistant and susceptible Mycobacterium tuberculosis strains isolated from Brazil (25 strains) and France (37 strains). In RMP-resistant strains, mutations were identified in 100% (16/16) from France and 89% (16/18) from Brazil. No mutation was detected in the 28 RMP-susceptible strains. Among RMP-resistant or RMP-susceptible strains deletion was observed. A double point mutation which had not been reported before was detected in one strain from France. Among French resistant strains mutations were found in codons 531 (31.2%), 526, 513 and 533 (18.7% each). In Brazilian strains the most common mutations were in codons 531 (72.2%), 526 (11.1%) and 513 (5.5%). The heterogeneity found in French strains may be related to the fact that most of those strains were from African or Asian patients.

Genetic diversity of Colombian sylvatic Trypanosoma cruzi isolates revealed by the ribosomal DNA

American trypanosomiasis is a common zoonosis in Colombia and Trypanosoma cruzi presents a wide distribution throughout the country. Although some studies based on enzyme electrophoresis profiles have described the population structure of the parasite, very few molecular analyses of genotipic markers have been conducted using Colombian strains. In this study, we amplified the non-transcribed spacer of the mini-gene by PCR, typing the isolates as T. cruzi I, T. cruzi zymodeme 3 or T. rangeli. In addition, the internal transcribed spacers of the ribosomal gene concomitant with the 5.8S rDNA were amplified and submitted to restriction fragment polymorphism analysis. The profiles were analyzed by a numerical methodology generating a phenetic dendrogram that shows heterogeneity among the T. cruzi isolates. This finding suggests a relationship between the complexity of the sylvatic transmission cycle in Colombia and the diversity of the sylvan parasites.

Alternative genotyping method for the single nucleotide polymorphism A2959G (AF159246) of the bovine CAST gene

The objective of this work was to genotype the single nucleotide polymorphism (SNP) A2959G (AF159246) of bovine CAST gene by PCR-RFLP technique, and to report its use for the first time. For this, 147 Bos indicus and Bos taurus x Bos indicus animals were genotyped. The accuracy of the method was confirmed through the direct sequencing of PCR products of nine individuals. The lowest frequency of the meat tenderness favorable allele (A) in Bos indicus was confirmed. The use of PCR-RFLP for the genotyping of the bovine CAST gene SNP was shown to be robust and inexpensive, which will greatly facilitate its analysis by laboratories with basic structure.

Multivariate analyses of genotype x environment interaction of popcorn

The objectives of this work were to evaluate the genotype x environment (GxE) interaction for popcorn and to compare two multivariate analyses methods. Nine popcorn cultivars were sown on four dates one month apart during each of the agricultural years 1998/1999 and 1999/2000. The experiments were carried out using randomized block designs, with four replicates. The cv. Zélia contributed the least to the GxE interaction. The cv. Viçosa performed similarly to cv. Rosa-claro. Optimization of GxE was obtained for cv. CMS 42 for a favorable mega-environment, and for cv. CMS 43 for an unfavorable environment. Multivariate analysis supported the results from the method of Eberhart & Russell. The graphic analysis of the Additive Main effects and Multiplicative Interaction (AMMI) model was simple, allowing conclusions to be made about stability, genotypic performance, genetic divergence between cultivars, and the environments that optimize cultivar performance. The graphic analysis of the Genotype main effects and Genotype x Environment interaction (GGE) method added to AMMI information on environmental stratification, defining mega-environments and the cultivars that optimized performance in those mega-environments. Both methods are adequate to explain the genotype x environment interactions.

Modulation of intercellular communication by differential regulation and heteromeric mixing of co-expressed connexins

Intercellular communication may be regulated by the differential expression of subunit gap junction proteins (connexins) which form channels with differing gating and permeability properties. Endothelial cells express three different connexins (connexin37, connexin40, and connexin43) in vivo. To study the differential regulation of expression and synthesis of connexin37 and connexin43, we used cultured bovine aortic endothelial cells which contain these two connexins in vitro. RNA blots demonstrated discordant expression of these two connexins during growth to confluency. RNA blots and immunoblots showed that levels of these connexins were modulated by treatment of cultures with transforming growth factor-ß1. To examine the potential ability of these connexins to form heteromeric channels (containing different connexins within the same hemi-channel), we stably transfected connexin43-containing normal rat kidney (NRK) cells with connexin37 or connexin40. In the transfected cells, both connexin proteins were abundantly produced and localized in identical distributions as detected by immunofluorescence. Double whole-cell patch-clamp studies showed that co-expressing cells exhibited unitary channel conductances and gating characteristics that could not be explained by hemi-channels formed of either connexin alone. These observations suggest that these connexins can readily mix with connexin43 to form heteromeric channels and that the intercellular communication between cells is determined not only by the properties of individual connexins, but also by the interactions of those connexins to form heteromeric channels with novel properties. Furthermore, modulation of levels of the co-expressed connexins during cell proliferation or by cytokines may alter the relative abundance of different heteromeric combinations.

Low frequency of p53 mutations in cervical carcinomas among Brazilian women

Human papillomavirus (HPV) infections of the high-risk types are strongly linked to the development of cervical carcinoma. The HPV oncoproteins E6 and E7 are thought to play a crucial role in this process through their interactions with the p53 protein and the retinoblastoma susceptibility gene product pRb, respectively. E6 binds to p53 protein promoting its degradation. This is considered to contribute to the oncogenesis of HPV-associated anogenital cancer. On the other hand, in HPV-negative cervical carcinoma, p53 mutations are thought to have a role in the transformation process. A total of 122 HPV-positive cervical carcinoma tissue samples were evaluated for the presence of mutations in exons 5-8 of the p53 gene by single-stranded conformation polymorphism analysis and DNA sequencing. Only four missense point mutations were detected. These findings suggest that other mechanisms independent of p53 inactivation may play a role in the genesis of cervical carcinomas.

Excitotoxic median raphe lesions aggravate working memory storage performance deficits caused by scopolamine infusion into the dentate gyrus of the hippocampus in the inhibitory avoidance task in rats

The interactions between the median raphe nucleus (MRN) serotonergic system and the septohippocampal muscarinic cholinergic system in the modulation of immediate working memory storage performance were investigated. Rats with sham or ibotenic acid lesions of the MRN were bilaterally implanted with cannulae in the dentate gyrus of the hippocampus and tested in a light/dark step-through inhibitory avoidance task in which response latency to enter the dark compartment immediately after the shock served as a measure of immediate working memory storage. MRN lesion per se did not alter response latency. Post-training intrahippocampal scopolamine infusion (2 and 4 µg/side) produced a more marked reduction in response latencies in the lesioned animals compared to the sham-lesioned rats. Results suggest that the immediate working memory storage performance is modulated by synergistic interactions between serotonergic projections of the MRN and the muscarinic cholinergic system of the hippocampus.