Impaired dendritic cell differentiation and maturation in the absence of C3

Human monocytes can be differentiated into immature dendritic cells (DCs) in the presence of serum and cytokines. One of the main functions of immature DCs is to capture and process antigens. Following maturation, they differentiate into antigen presenting cells. The role of complement in the differentiation process from monocytes towards immature DCs remains elusive. Here we demonstrate that complement 3 (C3) has a regulatory impact on the expression of specific DC surface molecules and DC-derived cytokine production during DC differentiation. We isolated human adherent peripheral blood mononuclear cells, which were cultured in the presence of GM-CSF plus IL-4 in medium supplemented with normal human serum or C3 deficient serum. The lack of C3 during DC differentiation negatively impacted the expression of C-type lectin receptor DC-SIGN, the antigen presenting molecules HLA-DR and CD1a, and the costimulatory molecules CD80 and CD86. Further, the spontaneous production of IL-6 and IL-12 was reduced in the absence of C3. Moreover, the maturation of immature DCs in response to LPS challenge was impaired in the absence of C3 as evidenced by reduced MHC-II, co-stimulatory molecule expression as well as modulated IL-12 and TNF-alpha production. Collectively, our results provide evidence for a novel role of C3 as a critical cofactor in human DC differentiation and maturation. (C) 2007 Elsevier Ltd. All rights reserved.

Characterization of Caulobacter crescentus response to low temperature and identification of genes involved in freezing resistance

Free-living bacteria must respond to a wide range of temperature changes, and have developed specific mechanisms to survive in extreme environments. In this work we describe a remarkable resistance of mesophilic bacterium Caulobacter crescentus to several cycles of freezing at -80 degrees C, which was able to grow at low temperatures. Exponentially growing cells and late stationary-phase cells presented higher freezing resistance at both -20 and -80 degrees C than early stationary-phase cells. Cryotolerance was observed when log-phase cultures grown at 30 degrees C were preincubated at 5, 15 or 20 degrees C before freezing at -20 degrees C. A transposon library was screened to identify mutants sensitive to freezing at -80 degrees C and three strains presenting < 10% survival were isolated. Identification of genes disrupted in each mutant showed that they encoded an AddA family DNA helicase, a DEAD/DEAH box RNA helicase and a putative RND (resistance, nodulation, cell division) efflux system component. These strains showed longer generation times than wild-type cells when growing at 15 degrees C, with the RNA helicase mutant presenting a severe growth defect. These analyses suggest that the singular intrinsic resistance to freezing of C. crescentus is in fact a consequence of several independent traits, especially the maintenance of a proper degree of supercoiling of nucleic acids.

Cathepsin L-like genes of Trypanosoma vivax from Africa and South America - characterization, relationships and diagnostic implications

We characterized sequences from genes encoding cathepsin L-like (CatL-like) cysteine proteases from African and South American isolates of Trypanosoma vivax and T. vivax-like organisms, and evaluated their suitability as genetic markers for population structure analysis and diagnosis. Phylogenetic analysis of sequences corresponding to CatL-like catalytic domains revealed substantial polymorphism, and clades of sequences (TviCatL1-9) were separated by large genetic distances. TviCatL1-4 sequences were from cattle isolates from West Africa (Nigeria and Burkina Faso) and South America (Brazil and Venezuela), which belonged to the same T. vivax genotype. T. vivax-like genotypes from East Africa showed divergent sequences, including TviCatL5-7 for isolates from Mozambique and TviCatL8-9 for an isolate from Kenya. Phylogenetic analysis of CatL-like gene data supported the relationships among trypanosome species reflected in the phylogenies based on the analysis of small subunit (SSU) of ribosomal RNA gene sequence data. The discovery of different CatL-like sequences for each genotype, defined previously by ribosomal DNA data, indicate that these sequences provide useful targets for epidemiological and population genetic studies. Regions in CatL-like sequences shared by all T. vivax genotypes but not by other trypanosomes allowed the establishment of a specific and sensitive diagnostic PCR for epidemiological studies in South America and Africa. (C) 2008 Elsevier Ltd. All rights reserved.

Sequence-specific reconstruction from fragmentary databases using seed sequences: implementation and validation on SAGE, proteome and generic sequencing data

Motivation: DNA assembly programs classically perform an all-against-all comparison of reads to identify overlaps, followed by a multiple sequence alignment and generation of a consensus sequence. If the aim is to assemble a particular segment, instead of a whole genome or transcriptome, a target-specific assembly is a more sensible approach. GenSeed is a Perl program that implements a seed-driven recursive assembly consisting of cycles comprising a similarity search, read selection and assembly. The iterative process results in a progressive extension of the original seed sequence. GenSeed was tested and validated on many applications, including the reconstruction of nuclear genes or segments, full-length transcripts, and extrachromosomal genomes. The robustness of the method was confirmed through the use of a variety of DNA and protein seeds, including short sequences derived from SAGE and proteome projects.

Analysis of genotypic variation in genes associated with virulence in Aggregatibacter actinomycetemcomitans clinical isolates

Background and Objective: Although certain serotypes of Aggregatibacter actinomycetemcomitans are associated more with aggressive periodontitis than are other serotypes, the correlation between distinct lineages and virulence traits in this species is poorly understood. This study aimed to evaluate the polymorphism of genes encoding putative virulence factors of clinical isolates, and to correlate these findings with A. actinomycetemcomitans serotypes, genotypes and periodontal status of the hosts. Material and Methods: Twenty-six clinical isolates from diverse geographic populations with different periodontal conditions were evaluated. Genotyping was performed using pulse-field gel electrophoresis. Polymorphisms in the genes encoding leukotoxin, Aae, ApaH and determinants for serotype-specific O polysaccharide were investigated. Results: The isolates were classified into serotypes a-f, and exhibited three apaH genotypes, five aae alleles and 25 macrorestriction profiles. Two serotype b isolates (7.7%), obtained from Brazilian patients with aggressive periodontitis, were associated with the highly leukotoxic genotype; these isolates showed identical fingerprint patterns and aae and apaH genotypes. Serotype c, obtained from various periodontal conditions, was the most prevalent among Brazilian isolates, and isolates were distributed in two aae alleles, but formed a genetically distinct group based on apaH analysis. Cluster analysis showed a close relationship between fingerprinting genotypes and serotypes/apaH genotypes, but not with aae genotypes. Conclusion: Apart from the deletion in the ltx promoter region, no disease-associated markers were identified. Non-JP2-like strains recovered from individuals with periodontal disease exhibited considerable genetic variation regarding aae/apaH genotypes, serotypes and XhoI DNA fingerprints.

The Thyroid Hormone Receptor (TR) beta-Selective Agonist GC-1 Inhibits Proliferation But Induces Differentiation and TR beta mRNA Expression in Mouse and Rat Osteoblast-Like Cells

Previous studies showed anabolic effects of GC-1, a triiodothyronine (T3) analogue that is selective for both binding and activation functions of thyroid hormone receptor (TR) beta 1 over TR alpha 1, on bone tissue in vivo. The aim of this study was to investigate the responsiveness of rat (ROS17/2.8) and mouse (MC3T3-E1) osteoblast-like cells to GC-1. As expected, T3 inhibited cellular proliferation and stimulated mRNA expression of osteocalcin or alkaline phosphatase in both cell lineages. Whereas equimolar doses of T3 and GC-1 equally affected these parameters in ROS17/2.8 cells, the effects of GC-1 were more modest compared to those of T3 in MC3T3-E1 cells. Interestingly, we showed that there is higher expression of TR alpha 1 than TR beta 1 mRNA in rat (similar to 20-90%) and mouse (similar to 90-98%) cell lineages and that this difference is even higher in mouse cells, which highlights the importance of TR alpha 1 to bone physiology and may partially explain the modest effects of GC-1 in comparison with T3 in MC3T3-E1 cells. Nevertheless, we showed that TR beta 1 mRNA expression increases (similar to 2.8- to 4.3-fold) as osteoblastic cells undergo maturation, suggesting a key role of TR beta 1 in mediating T3 effects in the bone forming cells, especially in mature osteoblasts. It is noteworthy that T3 and GC-1 induced TR beta 1 mRNA expression to a similar extent in both cell lineages (similar to 2- to 4-fold), indicating that both ligands may modulate the responsiveness of osteoblasts to T3. Taken together, these data show that TR beta selective T3 analogues have the potential to directly induce the differentiation and activity of osteoblasts.

Screening the Schistosoma mansoni transcriptome for genes differentially expressed in the schistosomulum stage in search for vaccine candidates

Schistosomiasis affects more than 200 million people worldwide; another 600 million are at risk of infection. The schistosomulum stage is believed to be the target of protective immunity in the attenuated cercaria vaccine model. In an attempt to identify genes up-regulated in the schistosomulum stage in relation to cercaria, we explored the Schistosoma mansoni transcriptome by looking at the relative frequency of reads in EST libraries from both stages. The 400 genes potentially up-regulated in schistosomula were analyzed as to their Gene Ontology categorization, and we have focused on those encoding-predicted proteins with no similarity to proteins of other organisms, assuming they could be parasite-specific proteins important for survival in the host. Up-regulation in schistosomulum relative to cercaria was validated with real-time reverse transcription polymerase chain reaction (RT-PCR) for five out of nine selected genes (56%). We tested their protective potential in mice through immunization with DNA vaccines followed by a parasite challenge. Worm burden reductions of 16-17% were observed for one of them, indicating its protective potential. Our results demonstrate the value and caveats of using stage-associated frequency of ESTs as an indication of differential expression coupled to DNA vaccine screening in the identification of novel proteins to be further investigated as potential vaccine candidates.

The differential roles of D-Pax2 variants in regulating Drosophila eye and bristle development

The ability to appropriately interact with the environment is crucial to an organism’s survival. The establishment of functional sensory systems, such as the bristles and eyes in Drosophila, is a critical event during the development of the organism. The transcription factor D Pax2 is involved in the differentiation of the shaft and glial cells in the developing bristle (Kavaler et al., Dev, 126:2261-2272, 1999) and of the cone and primary pigment cells in the developing eye (Fu and Noll, Genes Dev, 11:389-405, 1997). How D-Pax2 contributes to distinct differentiative pathways in different cell types is not known. Recent work by Anna Czechowski and Katherine Harmon (personal communication) identified a mutation in the D-Pax2 gene that introduced a stop codon at the end of exon 9, effectively truncating the protein. This mutation affects bristle, but not eye, development. We thus suspected regions after exon 9 are required for D-Pax2 function only in the bristles and may also be associated with alternative splicing of the D Pax2 transcript. We plan to assess the role of the carboxy terminal region of the protein by establishing transgenic lines bearing rescue constructs of D-Pax2 with either the complete coding sequence or with deletions of specific exons. To date, we have generated the first rescue construct bearing the complete coding region of the gene driven by a 3 KB upstream regulatory region of D-Pax2 and are currently generating transgenic fly lines with this construct.

Estudo da influência da variabilidade em genes de apolipoproteínas sobre níveis lipídicos e parâmetros de massa e gordura corporal na população de Porto Alegre

Apolipoproteínas constituem a parte proteica das lipoproteínas e de uma maneira geral desempenham papéis como proporcionar estabilidade estrutural, solubilizar lipídeos altamente hidrofóbicos, servir como ligantes a receptores ou agir como co-fatores para enzimas envolvidas no metabolismo. Diversos estudos têm mostrado que a variabilidade dos genes que codificam estas proteínas podem influenciar os níveis lipídicos em diversas populações. A variabilidade da apo A-IV também foi associada com variáveis antropométricas. Nesta investigação foram analisados 8 RFLPs nos genes das apolipoproteínas C-I (HpaI), C-II (AvaII), C-III (SacI, FokI e MspI) e A-IV (XbaI, HinfI e PvuII). A amostra foi composta por 391 indivíduos caucasóides de Porto Alegre (RS) e dados sobre hábitos de vida, dosagens lipídicas e medidas antropométricas foram obtidas para cada indivíduo. Os fragmentos de interesse de cada gene foram amplificados por PCR e os genótipos foram identificados por eletroforese em géis de agarose ou poliacrilamida corados com brometo de etídio.

A expressão do FOXP2 : uma introdução ao estudo das relações entre genes e linguagem

A aquisição da linguagem é um processo invariavelmente dependente da exposição ao ambiente. Contudo, a mais famosa e controversa hipótese desenvolvida por Chomsky sustenta que a criança traz para o processo de aprendizagem da língua um conjunto de recursos específicos – a Gramática Universal – para superar a pobreza de estímulos do ambiente e construir uma gramática final adequada, composta por princípios universais e parâmetros fixados de acordo com a experiência na comunidade lingüística. A Gramática Universal é, assim, entendida como uma dotação genética, necessária, mas não suficiente, para a aquisição da linguagem. Para além da hipótese do inatismo – central à tradição gerativista – a relação entre genes e linguagem vem sendo investigada há muitos anos, com evidências significativas de transmissão hereditária e etiologia genética para diversos tipos de distúrbios de linguagem. Mais recentemente, em 2001, foi descoberto o primeiro caso de um gene, o FOXP2, implicado na habilidade de adquirir e desenvolver a fala e a linguagem. Este trabalho parte de uma introdução ao processo de aquisição da linguagem em uma perspectiva inatista (Capítulo I) para enfocar os aspectos centrais à organização biológica da faculdade da linguagem (Capítulo II), propondo, a partir da análise dos efeitos da mutação do gene FOXP2, uma discussão introdutória sobre como o estudo dos distúrbios do desenvolvimento da linguagem, avaliados a partir de suas características fenotípica e genotípica, pode contribuir para a exploração de questões fundamentais acerca do desenvolvimento lingüístico e cognitivo (Capítulo III).

Mapeamento cromossômico de genes ribossomais em espécies estuarinas da família Gerreidae Identificação de uniformidade cariotípica e sua empregabilidade com fins biotecnológicos - REPROGEN

Perciformes are dominant in the marine environment, characterized as the largest and most diverse fish group. Some families, as Gerreidae, popularly known as silver jennies, carapebas, or mojarras have a high economic potential to marine fish farming, natural explotation and game fishing. Genetic information of these species are of fundamental importance for their management and production. Despite exist over 13,000 marine fish species described, only 2% were cytogenetically analyzed and less than 1% have some reproductive characteristics known. Induced breeding, cytogenetic characterization and cryopreservation of gametes, represent important areas in applied fish studies. In this project cytogenetic analyzes were performed to acess genetic aspects of Gerreidae species, distributed in coastal and estuarine regions of Northeast Brazil. Different methods for identifying chromosomal regions were employed using conventional techniques (Ag-NORs, C-banding), staining with base-specific fluorochromes (DAPI-CMA3), and physical mapping of ribosomal genes 18S and 5S rDNA, through hybridization in situ with fluorescent probes (FISH). The six species analyzed showed remarkable chromosome conservatism. The 18S and 5S ribosomal genes when analyzed in phylogenetic perspective demonstrate varied evolutionary dynamics, suggesting ocurrence of stasis process in some groups and greater dynamism in others. Double FISH with 18S and 5S probes showed both how efficient cytotaxonomic markers in the homogeneous karyotypes of this group of species. The karyotypic pattern identified in addition to the evolutionary aspects of karyotype, are suggestive of existence of low potential of post-zygotic barrier, prompting further research to prospect for artificial interspecific hybridization of these species of commercial importance

Identificação e caracterização de genes vip e cry coleóptero-específicos em isolados de Bacillus thuringiensis

O objetivo deste trabalho foi identificar e caracterizar os genes cry3, vip1, vip2 e vip1/vip2 em uma coleção de 1.078 isolados de Bacillus thuringiensis potencialmente tóxicos para larvas de coleópteros. Foram utilizados pares de oligonucleotídeos iniciadores gerais obtidos a partir de regiões conservadas dos genes e do alinhamento de sequências consenso. Posteriormente, os isolados positivos foram caracterizados por meio da técnica de PCR‑RFLP, tendo-se utilizado enzimas de restrição específicas, para identificar novas subclasses de genes nos isolados. Cento e cinquenta e um isolados foram positivos para os genes avaliados, com maior frequência para o gene vip1/vip2 (139 isolados). Pela técnica de PCR‑RFLP, foram observados 14 perfis polimórficos, o que indica a presença de diferentes alelos e, consequentemente, de distintas subclasses desses genes.

Estudo dos genes do complexo do antígeno leucocitário humano (hla) associados à susceptibilidade ao diabetes mellitus tipo 1

Of all of the genes associated with the development of Diabetes mellitus type 1 (T1D), the largest contribution comes from the genes in the Human Leukocyte Antigen (HLA) region, mostly the class II DR e DQ genes. Specific combinations of alleles DRB1, DQA1 and DQB1 constituting haplotypes, and further, a combination of more than one haplotype, providing multilocus genotypes are associated with susceptibility, protection and neutrality to DM1. Thus, the aim of present study was to verified the association of polymorphisms of HLA genes class II with susceptibility to type 1 diabetes mellitus (T1D). Ninety-two patients with T1D and 100 individuals normoglycemics (NG) aged between 6 and 20 years were studied. Genomic DNA was obtained from peripheral whole blood, collected in EDTA tube, using the extraction kit Illustra Triple Prep®, GE Healthcare. For HLA typing was used DNA LABType system by One Lambda kit applying Luminex® technology to the method of PCRSSO typing reverse. The alleles DRB1*03:01, *04:05, *04:01, *04:02, DQA1*03:01g, *05:01g, DQB1*02:01g, *03:02, the haplotypes DRB1*03:01-DQA1*05:01-DQB1*02:01, DRB1*04:05-DQA1*03:01g-DQB1*03:02, DRB1*04:02-DQA1*03:01g-DQB1*03:02, DRB1*04:01-DQA1*03:01g-DQB1*03:02 and DR3-DQ2/DR4-DQ8 genotype were significantly associated with the chance of developing T1D. The alleles DRB1*11:01, *15:03, *15:01, *13:01, DQA1*01:02, *04:01g, *01:03, DQB1*06:02, *03:01g, *06:03, *04:02, the haplotypes DRB1*11:01-DQA1*05:01-DQB1*03:01, DRB1*13:01-DQA1*01:03-DQB1*06:03 and DRX-DQX/DRX-DQX genotype, formed by other than the DR3-DQ2 or DR4-DQ8 haplotypes, were significantly associated with T1D protection Despite the major racial Brazilian, even at the regional level, these results are similar to the majority of alleles, genotypes and haplotypes of HLA class II-related susceptibility or resistance to T1D, extensively described in the literature for Caucasian population. Children with age at diagnosis less than 5 years of age had significantly higher frequency of the heterozygous genotype DR3-DQ2/DR4-DQ8 compared to children with age at diagnosis than 5 years old. These results also demonstrate strong association of the genetic profile of the class II HLA for this age group, possibly associated with the severity and rapid progression to the onset of T1D. The knowledge of HLA class II genes may be useful in genetic screens that allow the prediction of T1D

Padrões cromossômicos e mapeamento de genes ribossomais 18S e 5S em peixes pelágicos Atlânticos

Cytogenetic studies in fish have been contributed significantly to a better understanding of the marine biodiversity, presenting information related to characterization, evolution and conservation of species e fisheries stocks. Among the marine species which cytogenetic data are less well known pelagic forms are detached, that despite the economic importance and conservation efforts have been suffering great pressure from the artisanal and industrial fisheries. The present work characterized cytogenetically six species of large pelagic fish in the Atlantic, belonging to the Order Perciformes, among them, four species of Scombridae, Thunnus albacares, T. obesus, Scomberomorus brasiliensis and Acanthocybium solandri and two Coryphaenidae, Coryphaena equiselis and C. hippurus using Classical cytogenetic methods as conventional staining, C-banding and Ag-NORs and molecular through staining fluorochromes AT and GC-specific and mapping of ribosomal multigene families, 18S and 5S. The identification of phylogenetic patterns and cytotaxonomic markers between the species and the presence of sex chromosomes in at least one species of Coryphaenidae, are particularly useful in the formulating of phylogenetic hypotheses, as well as comparisons between groups and populations

Infectious bronchitis virus: detection and vaccine Strain differentiation by semi-nested RT-PCR

A semi-nested reverse transcription-polymerase chain reaction (Semi-N-RT-PCR) was developed and used to detect the S glycoprotein gene of infectious bronchitis virus (IBV) strains and to discriminate H120 vaccine strain from other strains. Viral RNA was extracted from the allantoic fluid of chicken embryos and from tissues of chickens experimentally infected with different strains of IBV. Amplification and identification of the viral RNA was performed using two sets of primers complementary to a region of the S glycoprotein gene in the Semi-N-RT-PCR assay. The pair of primers used in the first PCR consisted of universal oligonucleotides flanking a more variable region of S1-S2 gene. The second primer pair was used in the Semi-N-RT-PCR and was comprised of one of the primers from the first universal pair together with either another universal internal oligolucleotide or a oligonucleotide sequence specific for the H120 strain of IBV. The universal primers detected all reference IBV strains and field isolates tested herein. The Semi-N-RT-PCR had high sensitivity and specificity, and was able to differentiate the H120 vaccine strain from other reference IBV strains; including M41 strain. All tissue samples collected from chickens experimentally infected with H120 or M41 strains were positive in the semi-nested RT-PCR using universal primers, while only the H120-infected tissue samples were amplified by the set of primers containing the H120-oligonucleotide. In conclusion, the ability of Semi-N-RT-PCR to detect distinct IBV strains and preliminarily discriminate the vaccine strain (H120) closes a diagnostic gap and offers the opportunity to use comprehensive PCR procedures for the IBV diagnosis.