Mutational analysis of genes p14ARF, p15INK4b, p16INK4a, and PTEN in human nervous system tumors

The cancer is one of the most common and severe problems in clinical medicine, and nervous system tumors represent about 2% of the types of cancer. The central role of the nervous system in the maintenance of vital activities and the functional consequences of the loss of neurons can explain how severe brain cancers are. The cell cycle is a highly complex process, with a wide number of regulatory proteins involved, and such proteins can suffer alterations that transform normal cells into malignant ones. The INK4 family members (CDK inhibitors) are the cell cycle regulators that block the progression of the cycle through the R point, causing an arrest in G1 stage. The p14ARF (alternative reading frame) gene is a tumor suppressor that inhibits p53 degradation during the progression of the cell cycle. The PTEN gene is related to the induction of growth suppression through cell cycle arrest, to apoptosis and to the inhibition of cell adhesion and migration. The purpose of the present study was to assess the mutational state of the genes p14ARF, p15INK4b, p16INK4a, and PTEN in 64 human nervous system tumor samples. Homozygous deletions were found in exon 2 of the p15INK4b gene and exon 3 of the p16INK4a gene in two schwannomas. Three samples showed a guanine deletion (63 codon) which led to a loss of heterozygosity in the p15 gene, and no alterations could be seen in the PTEN gene. Although the group of patients was heterogeneous, our results are in accordance with other different studies that indicate that homozygous deletion and loss of heterozygosity in the INK4 family members are frequently observed in nervous system tumors.

Land planarians (Platyhelminthes) as a model organism for fine-scale phylogeographic studies: understanding patterns of biodiversity in the Brazilian Atlantic Forest hotspot

The Brazilian Atlantic Forest is one of the richest biodiversity hotspots of the world. Paleoclimatic models have predicted two large stability regions in its northern and central parts, whereas southern regions might have suffered strong instability during Pleistocene glaciations. Molecular phylogeographic and endemism studies show, nevertheless, contradictory results: although some results validate these predictions, other data suggest that paleoclimatic models fail to predict stable rainforest areas in the south. Most studies, however, have surveyed species with relatively high dispersal rates whereas taxa with lower dispersion capabilities should be better predictors of habitat stability. Here, we have used two land planarian species as model organisms to analyse the patterns and levels of nucleotide diversity on a locality within the Southern Atlantic Forest. We find that both species harbour high levels of genetic variability without exhibiting the molecular footprint of recent colonization or population expansions, suggesting a long-term stability scenario. The results reflect, therefore, that paleoclimatic models may fail to detect refugia in the Southern Atlantic Forest, and that model organisms with low dispersal capability can improve the resolution of these models.

Gene expression profiling of papillary thyroid carcinoma identifies transcripts correlated with BRAF mutational status and lymph node metastasis

Purpose: To identify papillary thyroid carcinoma (PTC)-associated transcripts, we compared the gene expression profiles of three Serial Analysis of Gene Expression libraries generated from thyroid tumors and a normal thyroid tissue. Experimental Design: Selected transcripts were validated in a panel of 57 thyroid tumors using quantitative PCR (qPCR). An independent set of 71 paraffin-embedded sections was used for validation using immunohistochemical analysis. To determine if PTC-associated gene expression could predict lymph node involvement, a separate cohort of 130 primary PTC (54 metastatic and 76 nonmetastatic) was investigated. The BRAF(V600E) mutational status was compared with qPCR data to identify genes that might be regulated by abnormal BRAF/MEK/extracellular signal-regulated kinase signaling. Results: We identified and validated new PTC-associated transcripts. Three genes (CST6, CXCL14, and DHRS3) are strongly associated with PTC. Immunohistochemical analysis of CXCL14 confirmed the qPCR data and showed protein expression in PTC epithelial cells. We also observed that CST6, CXCL14, DHRS3, and SPP1 were associated with PTC lymph node metastasis, with CST6, CXCL14, and SPP1 being positively correlated with metastasis and DHRS3 being negatively correlated. Finally, we found a strong correlation between CST6 and CXCL14 expression and BRAF(V600E) mutational status, suggesting that these genes may be induced subsequently to BRAF activation and therefore may be downstream in the BRAF/MEK/extracellular signal-regulated kinase signaling pathway. Conclusion: CST6, CXCL14, DHRS3, and SPP1 may play a role in PTC pathogenesis and progression and are possible molecular targets for FTC therapy.

Mutational analysis of the necdin gene in patients with congenital isolated hypogonadotropic hypogonadism

Context: Necdin activates GNRH gene expression and is fundamental for the development, migration, and axonal extension of murine GNRH neurons. In humans, necdin plays a potential role in the hypogonadotropic hypogonadism phenotype in patients with Prader-Willi syndrome. Aim: To investigate necdin gene (NDN) variants in patients with isolated hypogonadotropic hypogonadism (IHH). Patients and methods: We studied 160 Brazilian patients with IHH, which includes 92 with Kallmann syndrome and 68 with normosmic IHH. Genomic DNA was extracted and the single NDN exon was amplified and sequenced. To measure GNRH transcriptional activity, luciferase reporter plasmids containing GNRH regulatory regions were transiently transfected into GT1-7 cells in the presence and absence of overexpressed wild-type or mutant necdin. Results: A heterozygous variant of necdin, p.V318A, was identified in a 23-year-old male with Kallmann syndrome. The p.V318A was also present in affected aunt and his father and was absent in 100 Brazilian control subjects. Previous FGFR1 gene analysis revealed a missense mutation (p.P366L) in this family. Functional studies revealed a minor difference in the activation of GNRH transcription by mutant protein compared with wild type in that a significant impairment of the necdin protein activity threshold was observed. Conclusion: A rare variant of necdin (p.V318A) was described in a family with Kallmann syndrome associated with a FGFR1 mutation. Familial segregation and in vitro analysis suggested that this non-synonymous variant did not have a direct causative role in the hypogonadism phenotype. NDN mutations are not a frequent cause of congenital IHH.

Agribusiness Opportunity Costs and Environmental Legal Protection: Investigating Trade-Off on Hotspot Preservation in the State of So Paulo, Brazil

Prior to deforestation, So Paulo State had 79,000 km(2) covered by Cerrado (Brazilian savanna) physiognomies, but today less than 8.5% of this biodiversity hotspot remains, mostly in private lands. The global demand for agricultural goods has imposed strong pressure on natural areas, and the economic decisions of agribusiness managers are crucial to the fate of Cerrado domain remaining areas (CDRA) in Brazil. Our aim was to investigate the effectiveness of Brazilian private protected areas policy, and to propose a feasible alternative to promote CDRA protection. This article assessed the main agribusiness opportunity costs for natural areas preservation: the land use profitability and the arable land price. The CDRA percentage and the opportunity costs were estimated for 349 municipal districts of So Paulo State through secondary spatial data and profitability values of 38 main agricultural products. We found that Brazilian private protected areas policy fails to preserve CDRA, although the values of non-compliance fines were higher than average opportunity costs. The scenario with very restrictive laws on private protected areas and historical high interest rates allowed us to conceive a feasible cross compliance proposal to improve environmental and agricultural policies.

Community perceptions of four protected areas in the Northern portion of the Cerrado hotspot, Brazil

Establishing effective networks of protected areas (PAS) is one of the major goals of conservation strategies worldwide. However, the success of PAS in promoting biodiversity conservation depends on their integration to local and regional contexts, reducing and mitigating human impacts originating from buffer zones. Community perceptions affect interactions between residents and PAS, and thereby conservation effectiveness. Research at Tocantins state (northern Brazilian Cerrado), aimed to analyse local community perceptions of four PAs, discussing how different factors may influence these. Perceptions were assessed through standardized interviews applied to PA employees and 275 local inhabitants. There was modest community participation in PA establishment and management. Residents were aware of the PAS` existence, but were unfamiliar with their goals. Length of residency and occupation of inhabitants influenced their PA perceptions, shaping different people-park relations in each of the four studied PAs. Involvement of local residents in PA planning and management represents a central strategy to strengthen local support for PAS over the long term. In those areas that still have settlements inside their boundaries, community relocation should follow a careful participatory process to avoid significant changes in local perceptions and attitudes towards these PAS, crucial for conserving Brazilian biodiversity.

Structural modeling and mutational analysis of yeast eukaryotic translation initiation factor 5A reveal new critical residues and reinforce its involvement in protein synthesis

Eukaryotic translation initiation factor 5A (eIF5A) is a protein that is highly conserved and essential for cell viability. This factor is the only protein known to contain the unique and essential amino acid residue hypusine. This work focused on the structural and functional characterization of Saccharomyces cerevisiae eIF5A. The tertiary structure of yeast eIF5A was modeled based on the structure of its Leishmania mexicana homologue and this model was used to predict the structural localization of new site-directed and randomly generated mutations. Most of the 40 new mutants exhibited phenotypes that resulted from eIF-5A protein-folding defects. Our data provided evidence that the C-terminal alpha-helix present in yeast eIF5A is an essential structural element, whereas the eIF5A N-terminal 10 amino acid extension not present in archaeal eIF5A homologs, is not. Moreover, the mutants containing substitutions at or in the vicinity of the hypusine modification site displayed nonviable or temperature-sensitive phenotypes and were defective in hypusine modification. Interestingly, two of the temperature-sensitive strains produced stable mutant eIF5A proteins - eIF5A(K56A) and eIF5A(Q22H,L93F)- and showed defects in protein synthesis at the restrictive temperature. Our data revealed important structural features of eIF5A that are required for its vital role in cell viability and underscored an essential function of eIF5A in the translation step of gene expression.

Répteis da Estação Ecológica Serra Geral do Tocantins, Brasil Central

Os Cerrados sul-americanos abrigam alta diversidade de répteis, incluindo elevado número de endemismos. No entanto, o conhecimento desta diversidade é ainda incompleto frente à acelerada transformação das paisagens naturais no Brasil central. Constituem, portanto, uma das regiões prioritárias para estudo e conservação da biodiversidade mundial. Estudos intensivos sobre a fauna de répteis do Cerrado são necessários e urgentes para melhor compreensão dos processos que levaram à sua origem e distribuição e para subsidiar ações de conservação. Por meio de métodos padronizados, amostramos duas regiões ainda inexploradas da Estação Ecológica Serra Geral do Tocantins, situada na região do Jalapão. Registramos 45 espécies de répteis para a EESGT e entorno, o que representa uma riqueza alta e comparável à de outras regiões bem amostradas do Cerrado. Curvas de acumulação e estimadores indicam que a riqueza local de lagartos e anfisbenídeos aproxima-se da riqueza real enquanto a de serpentes é subestimada. A distribuição não-aleatória das espécies na paisagem concorda com evidências anteriores sugerindo utilização diferencial dos hábitats pelos répteis. Reunindo os resultados do presente estudo com os de levantamentos prévios realizados na região, registramos 88 espécies de répteis para o Jalapão sendo oito registros novos que incluem Bachia oxyrhina uma espécie recém descrita da região. As espécies da área apresentam três padrões gerais de distribuição: (1) espécies endêmicas do Cerrado, (2) espécies compartilhadas com domínios da diagonal de formações abertas sul-americanas, e (3) espécies de ampla ocorrência, compartilhadas também com ecossistemas florestais. Prevalecem espécies de ampla distribuição, porém é grande o número de espécies típicas do Cerrado, incluindo cinco possivelmente endêmicas do Jalapão, e há contribuição importante da fauna da Caatinga. A distribuição dos répteis em escala local e regional demonstra a necessidade de considerar a heterogeneidade paisagística para o planejamento de diretrizes visando à conservação em regiões do Cerrado. Por sua grande extensão, posição biogeográfica e complexidade de relevo e tipos de hábitat, a EESGT tem papel fundamental para a preservação e conhecimento da diversidade de répteis do Cerrado.

Birds from cerradão woodland, an overlooked forest of the Cerrado region, Brazil

The Cerrado region still receives relatively little ornithological attention, although it is regarded as the only tropical savanna in the world considered to be a biodiversity hotspot. Cerradão is one of the least known and most deforested Cerrado physiognomies and few recent bird surveys have been conducted in these forests. In order to rescue bird records and complement the few existing inventories of this under-studied forest type in the state of São Paulo, we looked for published papers on birds of cerradão. Additionally we surveyed birds at a 314-ha cerradão remnant located in central São Paulo, Brazil, from September 2005-December 2006 using unlimited distance transect counts. Out of 95 investigations involving cerradão bird studies, only 17 (18%) investigations teased apart bird species recorded inside cerradão from those recorded in other physiognomies of Cerrado. Except for one study, no research found more than 64 species in this type of forest, a result shared within many regions from Brazil and Bolivia. Differences in species richness do not seem be related with levels of disturbance of landscape or fragment size. Considering all species recorded in cerradão in Brazil and Bolivia, a compilation of data accumulated 250 species in 36 families and 15 orders. In recent surveys at central São Paulo, we recorded 48 species in 20 families, including the Pale-bellied Tyrant-Manakin Neopelma pallescens, threatened in São Paulo, and the Helmeted Manakin Antilophia galeata, near threatened in the state and endemic to the Cerrado region. Among the most abundant species inside this fragment, none was considered to be neither threatened nor endemic.

Vertebrados da Estação Ecológica Serra Geral do Tocantins: faunística, biodiversidade e conservação no Cerrado brasileiro

Inventários e estudos faunísticos detalhados sobre vertebrados são uma das fontes mais relevantes de dados para interpretações de padrões detalhados de diversidade biológica. Dados básicos e de boa qualidade sobre faunística são ainda mais urgentes em regiões pouco estudadas e sob intensa ameaça antrópica, tais como a região do Cerrado, um dos 34 hotspots globais para a conservação da biodiversidade. Apresentamos aqui uma síntese dos resultados dos inventários de vertebrados na Estação Ecológica Serra Geral do Tocantins (~716.000 ha), a segunda maior unidade de conservação em todo o Cerrado. Foram registradas 450 espécies de vertebrados na EESGT e entorno imediato, incluindo 17 espécies ameaçadas, 50 espécies endêmicas do Cerrado e 11 espécies com distribuição potencialmente restrita. Do total de espécies amostradas, 180 são novos registros para a região do Jalapão. Ao menos 12 espécies amostradas foram consideradas potenciais espécies novas, das quais quatro foram descritas recentemente, a partir do material obtido no inventário. Os resultados evidenciam que a EESGT é uma das mais importantes áreas protegidas no Brasil central, contribuindo para a persistência de espécies ameaçadas, dependentes dos últimos grandes blocos contínuos de vegetação nativa de Cerrado. Nossos resultados indicam ainda que a conservação da EESGT e suas principais subunidades é crucial para a representatividade do sistema de áreas protegidas do Cerrado, protegendo potenciais endemismos restritos que aliam alta vulnerabilidade intrínseca e valor como indicadores de padrões e processos biogeográficos formadores da rica e cada vez mais ameaçada fauna Neotropical.

Reduced transcription of TCOF1 in adult cells of Treacher Collins syndrome patients

Background: Treacher Collins syndrome (TCS) is an autosomal dominant craniofacial disorder caused by frameshift deletions or duplications in the TCOF1 gene. These mutations cause premature termination codons, which are predicted to lead to mRNA degradation by nonsense mediated mRNA decay (NMD). Haploinsufficiency of the gene product (treacle) during embryonic development is the proposed molecular mechanism underlying TCS. However, it is still unknown if TCOF1 expression levels are decreased in postembryonic human cells. Methods: We have estimated TCOF1 transcript levels through real time PCR in mRNA obtained from leucocytes and mesenchymal cells of TCS patients (n = 23) and controls (n = 18). Mutational screening and analysis of NMD were performed by direct sequencing of gDNA and cDNA, respectively. Results: All the 23 patients had typical clinical features of the syndrome and pathogenic mutations were detected in 19 of them. We demonstrated that the expression level of TCOF1 is 18-31% lower in patients than in controls (p < 0.05), even if we exclude the patients in whom we did not detect the pathogenic mutation. We also observed that the mutant allele is usually less abundant than the wild type one in mesenchymal cells. Conclusions: This is the first study to report decreased expression levels of TCOF1 in TCS adult human cells, but it is still unknown if this finding is associated to any phenotype in adulthood. In addition, as we demonstrated that alleles harboring the pathogenic mutations have lower expression, we herein corroborate the current hypothesis of NMD of the mutant transcript as the explanation for diminished levels of TCOF1 expression. Further, considering that TCOF1 deficiency in adult cells could be associated to pathologic clinical findings, it will be important to verify if TCS patients have an impairment in adult stem cell properties, as this can reduce the efficiency of plastic surgery results during rehabilitation of these patients.

Real-time PCR investigation on the expression of sboA and ituD genes in Bacillus spp

Aims: To investigate the expression of sboA and ituD genes among strains of Bacillus spp. at different pH and temperature. Methods and Results: Different Bacillus strains from the Amazon basin and Bacillus subtilis ATCC 19659 were investigated for the production of subtilosin A and iturin A by qRT-PCR, analysing sboA and ituD gene expression under different culture conditions. Amazonian strains presented a general gene expression level lower than B. subtilis ATCC 19659 for sboA. In contrast, when analysing the expression of ituD gene, the strains from the Amazon, particularly P40 and P45B, exhibited higher levels of expression. Changes in pH (6 and 8) and temperature (37 and 42 degrees C) caused a decrease in sboA expression, but increased ituD expression among strains from Amazonian environment. Conclusions: Temperature and pH have an important influence on the expression of genes sboA (subtilosin A) and ituD (iturin A) among Bacillus spp. The strains P40 and P45B can be useful for the production of antimicrobial peptide iturin A. Significance and Impact of the Study: Monitoring the expression of essential biosynthetic genes by qRT-PCR is a valuable tool for optimization of the production of antimicrobial peptides.

Rare canopy species in communities within the Atlantic Coastal Forest in Rio de Janeiro State, Brazil

Rare species are one of the principal components of the species richness and diversity encountered in Dense Ombrophilous Tropical Forests. This study sought to analyze the rare canopy species within the Atlantic Coastal Forest in Rio de Janeiro State, Brazil. Six different communities were examined: Dense Ombrophilous alluvial Forest; Dense sub-montane Ombrophilous Forest; Dense Montane Ombrophilous in Serra do Mar and Serra da Mantiqueira. In each area the vegetation was sampled within forty 10 x 25 m plots alternately distributed along a linear transect. All trees with DBH (1.3 m above ground level) a parts per thousand yen5 cm were sampled. The canopy was characterized using the allometric relationship between diameter and height, and included all trees with BDH a parts per thousand yen10 cm and height a parts per thousand yen10 m. A total of 64 families, 206 genera, and 542 species were sampled, of which 297 (54.8%) represented rare species (less than one individual per hectare). The percentage of rare species varied from 34 to 50% in each of the different communities sampled. A majority of these rare trees belonged to the Rosidae, and a smaller proportion to the Dilleniidae. It was concluded that there was no apparent pattern to rarity among families, that rarity was probably derived from a number of processes (such as gap formation), and that a great majority of the rare species sampled were consistently rare. This indicates that the restricted geographic distribution and high degree of endemism of many arboreal taxa justifies the conservation of even small fragments of Atlantic Forest.

Identification of novel L2HGDH gene mutations and update of the pathological spectrum

L-2-hydroxyglutaric aciduria (L-2-HGA, MIM 236792) is a neurometabolic disorder caused by the toxic accumulation of high concentration of L-2-hydroxyglutaric acid in plasma and cerebrospinal fluid. Distinct mutations on the L2HGDH gene have been associated with the clinical and biochemical phenotype. Here we present three novel mutations (Gln197X, Gly211Val and c.540+1 G>A), which increase the present deleterious collection of L2HGDH gene up to 35 mutations that we have compiled in this study. In addition, we used the haplotypic information based on polymorphic markers to demonstrate the common origin of Gly57Arg harboring chromosomes. Journal of Human Genetics (2010) 55, 55-58; doi: 10.1038/jhg.2009.110; published online 13 November 2009

NKX2.5 mutations in patients with non-syndromic congenital heart disease

Background: Cardiac development is a complex and multifactorial biological process. Heterozygous mutations in the transcription factor NKX2.5 are between the first evidence of a genetic cause for congenital heart defects in human beings. In this study, we evaluated the presence and frequency of mutations in the NKX2.5 gene on 159 unrelated patients with a diverse range of non-syndromic congenital heart defects (conotruncal anomalies, septal defects, left-sided lesions, right-sided lesions, patent ductus arteriosus and Ebstein`s anomaly). Methods: The coding region of the NKX2.5 locus was amplified by polymerase chain reaction and mutational analysis was performed using denaturing high performance liquid chromatography (DHPLC) and DNA sequencing. Results: We identified two distinct mutations in the NKX2.5 coding region among the 159 (1.26%) individuals evaluated. An Arg25Cys mutation was identified in a patient with Tetralogy of Fallot. The second mutation found was an Ala42Pro in a patient with Ebstein`s anomaly. Conclusions: The association of NKX2.5 mutations is present in a small percentage of patients with non-syndromic congenital heart defects and may explain only a few cases of the disease. Screening strategies considering the identification of germ-line molecular defects in congenital heart disease are still unwarranted and should consider other genes besides NKX2.5. (C) 2008 Elsevier Ireland Ltd. All rights reserved.