High frequency of BRAF mutations in nevi

To evaluate the timing of mutations in BRAF (v-raf murine sarcoma viral oncogene homolog B1) during melanocytic neoplasia, we carried out mutation analysis on microdissected melanoma and nevi samples. We observed mutations resulting in the V599E amino-acid substitution in 41 of 60 (68%) melanoma metastases, 4 of 5 (80%) primary melanomas and, unexpectedly, in 63 of 77 (82%) nevi. These data suggest that mutational activation of the RAS/RAF/MAPK pathway in nevi is a critical step in the initiation of melanocytic neoplasia but alone is insufficient for melanoma tumorigenesis.

Ataxia-telangiectasia-mutated (ATM) and NBS1-dependent phosphorylation of Chk1 on Ser-317 in response to ionizing radiation

In mammals, the ATM (ataxia-telangiectasia-mutated) and ATR (ATM and Rad3-related) protein kinases function as critical regulators of the cellular DNA damage response. The checkpoint functions of ATR and ATM are mediated, in part, by a pair of checkpoint effector kinases termed Chk1 and Chk2. In mammalian cells, evidence has been presented that Chk1 is devoted to the ATR signaling pathway and is modified by ATR in response to replication inhibition and UV-induced damage, whereas Chk2 functions primarily through ATM in response to ionizing radiation (IR), suggesting that Chk2 and Chk1 might have evolved to channel the DNA damage signal from ATM and ATR, respectively. We demonstrate here that the ATR-Chk1 and ATM-Chk2 pathways are not parallel branches of the DNA damage response pathway but instead show a high degree of cross-talk and connectivity. ATM does in fact signal to Chk1 in response to IR. Phosphorylation of Chk1 on Ser-317 in response to IR is ATM-dependent. We also show that functional NBS1 is required for phosphorylation of Chk1, indicating that NES1 might facilitate the access of Chk1 to ATM at the sites of DNA damage. Abrogation of Chk1 expression by RNA interference resulted in defects in IR-induced S and G2/M phase checkpoints; however, the overexpression of phosphorylation site mutant (S317A, S345A or S317A/S345A double mutant) Chk1 failed to interfere with these checkpoints. Surprisingly, the kinase-dead Chk1 (D130A) also failed to abrogate the S and G2 checkpoint through any obvious dominant negative effect toward endogenous Chk1. Therefore, further studies will be required to assess the contribution made by phosphorylation events to Chk1 regulation. Overall, the data presented in the study challenge the model in which Chk1 only functions downstream from ATR and indicate that ATM does signal to Chk1. In addition, this study also demonstrates that Chk1 is essential for IR-induced inhibition of DNA synthesis and the G2/M checkpoint.

Escalation of Drug Use in Early-Onset Cannabis Users vs Co-twin Controls

Context Previous studies have reported that early initiation of cannabis (marijuana) use is a significant risk factor for other drug use and drug-related problems. Objective To examine whether the association between early cannabis use and subsequent progression to use of other drugs and drug abuse/dependence persists after controlling for genetic and shared environmental influences. Design Cross-sectional survey conducted in 1996-2000 among an Australian national volunteer sample of 311 young adult (median age, 30 years) monozygotic and dizygotic same-sex twin pairs discordant for early cannabis use (before age 17 years). Main Outcome Measures Self-reported subsequent nonmedical use of prescription sedatives, hallucinogens, cocaine/other stimulants, and opioids; abuse or dependence on these drugs (including cannabis abuse/dependence); and alcohol dependence. Results Individuals who used cannabis by age 17 years had odds of other drug use, alcohol dependence, and drug abuse/dependence that were 2.1 to 5.2 times higher than those of their co-twin, who did not use cannabis before age 17 years. Controlling for known risk factors (early-onset alcohol or tobacco use, parental conflict/separation, childhood sexual abuse, conduct disorder, major depression, and social anxiety) had only negligible effects on these results. These associations did not differ significantly between monozygotic and dizygotic twins. Conclusions Associations between early cannabis use and later drug use and abuse/dependence cannot solely be explained by common predisposing genetic or shared environmental factors. The association may arise from the effects of the peer and social context within which cannabis is used and obtained. In particular, early access to and use of cannabis may reduce perceived barriers against the use of other illegal drugs and provide access to these drugs.

The highly rearranged mitochondrial genome of the plague thrips, Thrips imaginis (Insecta : thysanoptera): Convergence of two novel gene boundaries and an extraordinary arrangement of rRNA genes

To help understand the mechanisms of gene rearrangement in the mitochondrial (mt) genomes of hemipteroid insects, we sequenced the mt genome of the plague thrips, Thrips imaginis (Thysanoptera). This genome is circular, 15,407 by long, and has many unusual features, including (1) rRNA genes inverted and distant from one another, (2) an extra gene for tRNA-Ser, (3) a tRNA-Val lacking a D-arm, (4) two pseudo-tRNA genes, (5) duplicate control regions, and (6) translocations and/or inversions of 24 of the 37 genes. The mechanism of rRNA gene transcription in T. imaginis may be different from that of other arthropods since the two rRNA genes have inverted and are distant from one another. Further, the rRNA genes are not adjacent or even close to either of the two control regions. Tandem duplication and deletion is a plausible model for the evolution of duplicate control regions and for the gene translocations, but intramitochondrial recombination may account for the gene inversions in T. imaginis. All the 18 genes between control regions #1 and #2 have translocated and/or inverted, whereas only six of the 20 genes outside this region have translocated and/or inverted. Moreover, the extra tRNA gene and the two pseudo-tRNA genes are either in this region or immediately adjacent to one of the control regions. These observations suggest that tandem duplication and deletion may be facilitated by the duplicate control regions and may have occurred a number of times in the lineage leading to T. imaginis. T. imaginis shares two novel gene boundaries with a lepidopsocid species from another order of hemipteroid insects, the Psocoptera. The evidence available suggests that these shared gene boundaries evolved by convergence and thus are not informative for the interordinal phylogeny of hemipteroid insects. We discuss the potential of hemipteroid insects as a model system for studies of the evolution of animal rut genomes and outline some fundamental questions that may be addressed with this system.

Effects of additional sequences directly downstream from the AUG on the expression of GFP gene

We have studied the expression of the green fluorescent protein (GFP) gene to gain more understanding of the effects of additional nucleotide triplets (codons) downstream from the initiation codon on the translation of the GFP mRNA in CHO and Cos1 cells. A leader sequence of six consecutive identical codons (GUG, CUC, AGU or UCA) was introduced into a humanized GFP (hm gfp) gene downstream from the AUG to produce four GFP gene variants. Northern blot and RT-PCR analysis indicated that mRNA transcription from the GFP gene was not significantly affected by any of the additional sequences. However, immunoblotting and FACS analysis revealed that AGU and UCA GFP variants produced GFP at a mean level per cell 3.5-fold higher than the other two GFP variants and the hm gfp gene. [35S]-Methionine labeling and immunoprecipitation demonstrate that GFP synthesis was very active in UCA variant transfected-cells, but not in GUG variant and hm gfp transfected-cells. Moreover, proteasome inhibitor MG-132 treatment indicated that the GFPs encoded by each of the GFP variants and the hm gfp were equally stable, and this together with the comparable mRNA levels observed for each construct suggested that the different steady-state GFP concentrations observed reflected different translation efficiencies of the various GFP genes. In addition, the CUC GFP variant, when transiently transfected into CHO or COS-1 cells, did not produce any GFP expressing cells (fully green cells), and the GUG variant produced GFP expressing cells less than 10%, while AGU and UCA GFP variants up to 30–35% in a time course study from 8 to 36 h posttransfection. Analysis of the potential secondary structure of the GFP variant mRNAs especially in the translation initiation region suggested that the secondary structure of the GFP mRNAs was unlikely to explain the different translation efficiencies of the GFP variants. The present findings indicate that a change of the initiation context of the GFP gene by addition of extra coding sequence can alter the translation efficiency of GFP mRNA, providing a means of more efficient expression of GFP in eukaryotic cells.

ATP activates ataxia-telangiectasia mutated (ATM) in vitro - Importance of autophosphorylation

Ataxia-telangiectasia Mutated (ATM), mutated in the human disorder ataxia-telangiectasia, is rapidly activated by DNA double strand breaks. The mechanism of activation remains unresolved, and it is uncertain whether autophosphorylation contributes to activation. We describe an in vitro immunoprecipitation system demonstrating activation of ATM kinase from unirradiated extracts by preincubation with ATP. Activation is both time- and ATP concentration-dependent, other nucleotides fail to activate ATM, and DNA is not required. ATP activation is specific for ATM since it is not observed with kinase-dead ATM, it requires Mn2+, and it is inhibited by wortmannin. Exposure of activated ATM to phosphatase abrogates activity, and repeat cycles of ATP and phosphatase treatment reveal a requirement for autophosphorylation in the activation process. Phosphopeptide mapping revealed similarities between the patterns of autophosphorylation for irradiated and ATP-treated ATM. Caffeine inhibited ATM kinase activity for substrates but did not interfere with ATM autophosphorylation. ATP failed to activate either A-T and rad3-related protein (ATR) or DNA-dependent protein kinase under these conditions, supporting the specificity for ATM. These data demonstrate that ATP can specifically induce activation of ATM by a mechanism involving autophosphorylation. The relationship of this activation to DNA damage activation remains unclear but represents a useful model for understanding in vivo activation.

Epitope map for a growth hormone receptor agonist monoclonal antibody, MAb 263

Monoclonal antibody (MAb) 263 is a widely used monoclonal antibody that recognizes the extracellular domain (ECD) of the GH receptor. It has been shown to act as a GH agonist both in vitro and in vivo, and we report here that it must be divalent to exert its effect on the full-length receptor. To understand the mechanism of its agonist action, we have determined the precise epitope for this antibody using a novel random PCR mutagenesis approach together with expression screening in yeast. A library of 5200 clones of rabbit GH receptor ECD mutants were screened both with MAb 263 and with an anticarboxy-tag antibody to verify complete ECD expression. Sequencing for clones that expressed complete ECD but were not MAb 263 positive identified 20 epitope residues distributed in a discontinuous manner throughout the ECD. The major part of the epitope, as revealed after mapping onto the crystal structure model of the ECD molecule, was located on the side and upper portion of domain 1, particularly within the D - E strand disulfide loop 79 - 96. Molecular dynamics docking of an antibody of the same isotype as MAb 263 was used to dock the bivalent antibody to the 1528-Angstrom(2) epitope and to visualize the likely consequences of MAb binding. The minimized model enables the antibody to grasp two receptors in a pincer-like movement from opposite sides, facilitating alignment of the receptor dimerization domains in a manner similar to, but not identical with, GH.

Solution structure of CnErg1 (Ergtoxin), a HERG specific scorpion toxin

The three-dimensional structure of chemically synthesized CnErg1 (Ergtoxin), which specifically blocks HERG (human ether-a-go-go-related gene) K+ channels, was determined by nuclear magnetic resonance spectroscopy. CnErg1 consists of a triple-stranded beta-sheet and an a-helix, as is typical of K+ channel scorpion toxins. The peptide structure differs from the canonical structures in that the first beta-strand is shorter and is nearer to the second beta-strand rather than to the third beta-strand on the C-terminus. There is also a large hydrophobic patch on the surface of the toxin, surrounding a central lysine residue, Lys13. We postulate that this hydrophobic patch is likely to form part of the binding surface of the toxin. (C) 2003 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Cytotoxic iron chelators: Characterization of the structure, solution chemistry and redox activity of ligands and iron complexes of the di-2-pyridyl ketone isonicotinoyl hydrazone (HPKIH) analogues

Di-2-pyridyl ketone isonicotinoyl hydrazone (HPKIH) and a range of its analogues comprise a series of monobasic acids that are capable of binding iron (Fe) as tridentate (N,N,O) ligands. Recently, we have shown that these chelators are highly cytotoxic, but show selective activity against cancer cells. Particularly interesting was the fact that cytotoxicity of the HPKIH analogues is maintained even after complexation with Fe. To understand the potent anti-tumor activity of these compounds, we have fully characterized their chemical properties. This included examination of the solution chemistry and X-ray crystal structures of both the ligands and Fe complexes from this class and the ability of these complexes to mediate redox reactions. Potentiometric titrations demonstrated that all chelators are present predominantly in their charge-neutral form at physiological pH (7.4), allowing access across biological membranes. Keto-enol tautomerism of the ligands was identified, with the tautomers exhibiting distinctly different protonation constants. Interestingly, the chelators form low-spin (diamagnetic) divalent Fe complexes in solution. The chelators form distorted octahedral complexes with Fe-II, with two tridentate ligands arranged in a meridional fashion. Electrochemistry of the Fe complexes in both aqueous and non-aqueous solutions revealed that the complexes are oxidized to their ferric form at relatively high potentials, but this oxidation is coupled to a rapid reaction with water to form a hydrated (carbinolamine) derivative, leading to irreversible electrochemistry. The Fe complexes of the HPKIH analogues caused marked DNA degradation in the presence of hydrogen peroxide. This observation confirms that Fe complexes from the HPKIH series mediate Fenton chemistry and do not repel DNA. Collectively, studies on the solution chemistry and structure of these HPKIH analogues indicate that they can bind cellular Fe and enhance its redox activity, resulting in oxidative damage to vital biomolecules.

Angola, jeje e ketu : memórias e identidades em casas e nações de candomblé na Região Metropolitana da Grande Vitória (ES)

Angola, jeje e ketu: Memórias e identidades em casas e nações de candomblé na Região Metropolitana da Grande Vitória (ES), que é o tema desta dissertação, requer adentrar em análises de categorias nativas do povo de santo, e, em seguida, passar a questões teóricas sobre esses temas. Na cidade de Serra encontram-se as quatro casas de santo onde a pesquisa de campo foi realizada com três iyalorixás e um babalorixá, que dividem suas memórias e experiências religiosas compondo um exercício teórico sobre a história e a formação do candomblé no Espírito Santo. Nesse estado, que não é referência dessa religião, a mesma encontra-se em ascensão. A preocupação dos integrantes das comunidades de terreiros é transformar parte das tradições orais em produção escrita. Tendo em vista os processos políticos de reconhecimento legal da diversidade cultural, o debate se deu em torno de hibridização e múltiplas formas de identidade. O universo encantador e mágico do candomblé é composto pelos toques dos atabaques, danças rituais e fé em forças da natureza. Os “filhos da diáspora africana” trazidos para o Brasil eram de várias regiões da África, o que nos permite entender a diversidade cultural que marca esses grupos. Em função do sincretismo entre as próprias religiões de matriz africana e delas com o catolicismo e as doutrinas espiritualistas, essas religiões encontram-se de norte a sul do país. Este encontro de crenças e rituais é tão evidente que já não dizemos religiões “africanas” e sim religiões “afro-brasileiras”. O candomblé, desde o seu surgimento, vem sendo criado e recriado pela transmissão de suas tradições e ritos. A tradição oral nas comunidades de terreiro é um dos elementos demarcadores da construção da sua identidade, a partir de uma organização interna e do aprendizado hierarquicamente transmitido pelos depositários do saber, seguindo uma ordem de senioridade de iniciação, os antigos são detentores dos saberes e segredos. Por ser uma religião iniciática, o aprendizado ocorre permanentemente, em especial o da língua ritual, onde o exercício e o contato levam a transmissão cultural. O povo de santo reconstrói uma ligação com uma comunidade imaginada que remonta a África e desenvolve relações de parentesco ficcional entre os membros das comunidades de terreiro e forma uma família de santo e de axé.

Avaliação do teste Quantiferon TB Gold in tube no diagnóstico de infecção latente pelo Mycobacterium tuberculosis em profissionais de saúde da atenção básica

Introdução: Os ensaios de liberação do interferon- γ (ELIG) surgiram como uma alternativa para o diagnóstico de infecção latente pelo Mycobacterium tuberculosis (ILTB). Neste estudo, nós comparamos o desempenho de um dos ELIG, teste Quantiferon TB Gold in tube – QFT, com a prova tuberculínica (PT) em dois pontos de corte (≥ 5 mm e ≥ 10 mm), em profissionais de saúde da atenção básica à saúde (ABS). Métodos: Estudo transversal realizado em profissionais de saúde da ABS de quatro capitais nacionais com alta incidência de TB. O resultado do teste QFT foi comparado com o resultado da PT nos pontos de corte ≥ 5mm e ≥ 10 mm. Resultados: Foram incluídos 632 profissionais de saúde. Ao considerar o ponto de corte ≥ 10 mm para a PT, a concordância entre QFT e a PT foi de 69% (k = 0,31) e para o ponto de corte ≥ 5 mm, a concordância entre os testes foi de 57% (k = 0,22). Devido a baixa concordância entre a PT e o QFT, nós avaliamos os possíveis fatores associados com a discordância entre eles. Ao comparar o grupo PT- / QFT- com o grupo PT+ / QFT-, no ponto de corte ≥ 5 mm, a idade entre 41-45 [OR = 2,70, IC 95%: 1,32-5,51] e 46-64 [OR = 2,04, IC 95%: 1,05-3,93], presença de cicatriz vacinal do BCG [OR = 2,72, IC 95%: 1,40-5,25] e trabalhar apenas na ABS [OR = 2,30, IC 95 %: 1,09-4,86] apresentaram associação estatística significativa. Para o ponto de corte ≥ 10 mm, a presença de cicatriz vacinal do BCG [OR = 2,26, IC 95%: 1,03-4,91], ter tido contato domiciliar com paciente portador de tuberculose ativa [OR = 1,72, IC 95%: 1,01-2,92] e ter feito a PT anteriormente [OR = 1,66, IC 95%: 1,05-2,62] revelaram associação estatística significativa. Curiosamente, a discordância observada no grupo PT- / QFT + não apresentou associação estatistica com nenhuma das variáveis consideradas, independentemente do ponto de corte da PT. Conclusões: Apesar de termos identificado que a vacina BCG contribuiu para a discordância entre os testes, as recomendações brasileiras para o início do tratamento da ILTB não devem ser alteradas devido as limitações do QFT.

Experiências formativas no início da docência mediadas pelo PIBID educação física da Universidade Estadual de Feira de Santana, Bahia

O presente trabalho objetiva compreender como o Coordenador, os Supervisores e os Bolsistas do PIBID Educação Física da Universidade Estadual de Feira de Santana (UEFS) no município de Feira de Santana – Bahia percebem os processos de iniciação à docência na formação inicial de professores de Educação Física no PIBID Educação Física – UEFS. Por meio da Pesquisa Narrativa, numa perspectiva de pesquisa e de formação, oportunizamos aos sujeitos da pesquisa entrevistas narrativas compostas pela reflexividade crítica necessária na constituição da docência. Analisamos a produção acadêmica sobre a temática da iniciação à docência no tempo da formação inicial nos bancos de dados da Associação Nacional de Pós-Graduação e Pesquisa em Educação (ANPED) e do Congresso Brasileiro de Ciências do Esporte (CONBRACE) e discutimos com Antonio Nóvoa, Carlos Marcelo e Molina Neto sobre as concepções de formação e as experiências vividas no início da docência e buscamos em Walter Benjamin e Jorge Larrosa ancoragens para o entendimento do conceito de experiência. Os resultados apontam para o reconhecimento do PIBID como um programa que aproxima do campo de atuação interferindo na escolha pela docência. Sinalizam que o programa possibilita a articulação entre a universidade e a escola fortalecendo a perspectiva de formação que acontece coletivamente. Confirmam que o PIBID permite a constatação dos desafios presentes na escola e da necessidade de transformação desta realidade com a articulação dos saberes acadêmicos e os escolares. Neste sentido, constatamos a afirmação da entrada na carreira, como uma experiência formativa que intensifica o desenvolvimento profissional docente e, portanto, deve ser concebida como política nacional institucionalizada de iniciação à docência.

Survival Analysis Employed in Predicting Corporate Failure: A Forecasting Model Proposal

In face of the current economic and financial environment, predicting corporate bankruptcy is arguably a phenomenon of increasing interest to investors, creditors, borrowing firms, and governments alike. Within the strand of literature focused on bankruptcy forecasting we can find diverse types of research employing a wide variety of techniques, but only a few researchers have used survival analysis for the examination of this issue. We propose a model for the prediction of corporate bankruptcy based on survival analysis, a technique which stands on its own merits. In this research, the hazard rate is the probability of ‘‘bankruptcy’’ as of time t, conditional upon having survived until time t. Many hazard models are applied in a context where the running of time naturally affects the hazard rate. The model employed in this paper uses the time of survival or the hazard risk as dependent variable, considering the unsuccessful companies as censured observations.

XRCC3241 Polymorphism influence on genotoxicity biomarkers frequency in workers occupationaly exposed to formaldehyde

Formaldehyde (FA) is ubiquitous in the environment and is a chemical agent that possesses high reactivity. Occupational exposure to FA has been shown to induce nasopharyngeal cancer and has been classified as carcinogenic to humans (group 1) on the basis of sufficient evidence in humans and sufficient evidence in experimental animals. The exposure to this substance is epidemiologically linked to cancer and nuclear changes detected by the cytokinesis-block micronucleus test (CBMN). This method is extensively used in molecular epidemiology, since it determines several biomarkers of genotoxicity, such as micronucleus (biomarkers of chromosomes breakage or loss), nucleoplasmic bridges (biomarker of chromosome rearrangement, poor repair and / or telomeres fusion) and nuclear buds (biomarker of elimination of amplified DNA). The gene X-ray repair cross-complementing group 3 (XRCC3) is involved in homologous recombination repair of cross-links and chromosomal double-strand breaks and at least one polymorphism has been reported in codon 241, a substitution of a methionine for a threonine.

Interaction of formaldehyde and tobacco smoking in the frequency of micronucleus and the XRCC3 Thr241Met polymorphism

Formaldehyde is classified by IARC as carcinogenic to humans (nasopharyngeal cancer). Tobacco smoke has been epidemiologically associated to a higher risk of development of cancer, especially in the oral cavity, larynx and lungs, as these are places of direct contact with many carcinogenic tobacco’s compounds. XRCC3 is involved in homologous recombination repair of cross-links and chromosomal double-strand breaks (Thr241Met polymorphism). The aim of the study is to determine whether there is an in vivo association between genetic polymorphism of the gene XRCC3 and the frequency of genotoxicity biomarkers in subjects exposed or not to formaldehyde and with or without tobacco consumption.