TNF-alpha polymorphisms are associated with obsessive-compulsive disorder

Introduction: Several lines of evidence support an immunologic involvement in obsessive-compulsive disorder (OCD): the increased prevalence of OCD in patients with rheumatic fever (RF), and the aggregation of obsessive-compulsive spectrum disorders among relatives of RF probands. Tumor necrosis factor alpha is a proinflammatory cytokine involved in RF and other autoimmune diseases. Polymorphisms in the promoter region of the TNFA gene have been associated with RE Given the association between OCD and RF, the goal of the present study was to investigate a possible association between polymorphisms within the promoter region of TNFA and OCD. Materials and methods: Two polymorphisms were investigated: -308 G/A and -238 G/A. The allelic and genotypic frequencies of these polymorphisms were examined in 111 patients who fulfilled DSM-IV criteria for OCD and compared with the frequencies in 250 controls. Results: Significant associations were observed between both polymorphisms and OCD. For -238 G/A, an association between the A allele and OCD was observed (X-2 = 12.05, p = 0.0005). A significant association was also observed between the A allele of the -308 G/A polymorphism and OCD (X-2 = 7.09, p = 0.007). Finally, a haplotype consisting of genotypes of these two markers was also examined. Significant association was observed for the A-A haplotype (p = 0.0099 after correcting for multiple testing). Discussion: There is association between the -308 G/A and -238 G/A TNFA polymorphisms and OCD in our Brazilian sample. However, these results need to be replicated in larger samples collected from different populations. (c) 2008 Elsevier Ireland Ltd. All rights reserved.

Overexpression and Purification of the Oat (Avena Fatua) Protein AFN1 and Construction of a pMAL/AFN1 Expression Plasmid

Abscisic acid (ABA) is an important phytohormone with regulatory roles in many physiological processes. ABA expression is induced by environmental stresses such as drought and it is known to be an inhibitor of seed germination. A wild oat (Avena fatua) called AFN1 has been hypothesized to initiate the early stages of germination as its mRNA accumulates in nondormant seed embryos during imbibition. The polypeptide sequence of AFN1 suggests that it is an ABA glucosyl transferase. Glucosylation by AFN1 and thereby inactivation of ABA could lead to seed germination. In order to understand the role of AFN1 in germination, an ample quantity of AFN1 polypeptide is needed to test for enzymatic ABA glucosylase activity. My work has been to overexpress recombinant AFN1containing a (His)6 tag using a pRSETC E.coli expression system followed by Purification of the AFN1 protein by means of a nickel-affinity column that bind to the (His)6 tag. Due to the insufficient yield of AFN1 fusion protein obtained with this procedure, another method using a pMAL-c2x vector is now being employed. The pMAL expression system provides a method for expressing and purifying protein by tagging proteins with maltose-binding protein (MBP). It is anticipated that MBP tag will be advantageous as it can make the fusion protein more soluble and thereby yield a larger quantity of protein. Currently, work is underway on the construction of pMAL/AFN1 plasmid.

Characterization of AFN1, a Gene Associated with Cereal Grain Germination

The AFN1 gene is transiently expressed in germinating oat grains. As AFN1 is not expressed in dormant oat grains during imbibition, we hypothesize that AFN1 may be involved in stimulating the germination process. Sequence analysis of an AFN1 cDNA clone indicates that the AFN1 polypeptide is similar to a previously identified abscisic acid (ABA) glucosyl transferase. This suggests that AFN1 may be acting to glucosylate ABA, thereby inactivating it. As the hormone ABA is known to inhibit germination, ABA glucosylation/inactivation could lead to germination in grains expressing AFN1. To test this hypothesis, we have constructed an expression plasmid that encodes an MBP::AFN1 (maltose binding protein) fusion protein. E. coli cells carrying the expression plasmid were found to produce the MBP::AFN1 fusion protein as a substantial fraction of total protein. We are currently in the process of purifying the MBP::AFN1 fusion protein by affinity chromatography, so that it can be assayed for ABA glucosyl transferase activity. We also wish to test the effect of AFN1 gene expression during grain imbibition on the germination behavior of the grains. To this end, we have constructed plasmids for the overexpression and RNAi-based suppression of AFN1 in transgenic plants. These plasmids have been introduced into oat cells by particle bombardment and we are in the process of regenerating transgenic plants for study.

Characterization of the Wheat Grain PKABA1-Interacting Protein TaWD40

Abscisic acid (ABA)-mediated gene expression is a critical component of plant responses to this important hormone, which affects plant growth, development, and responses to environmental stresses. Plant responses to ABA are mediated by a number of factors including PKABA1, an ABA induced protein kinase involved in ABA-suppressed gene expression in cereal grains, and TaWD40, which has previously been shown to physically interact with PKABA1. A full-length 1.9 kb TaWD40 cDNA, CK210682, was sequenced as part of this project. Based on the deduced protein sequence, it is thought that TaWD40 may belong to the family of E3 ubiquitin ligases, possibly targeting PKABA1 for destruction. Construction of expression plasmids for overproduction of the TaWD40 polypeptide in E. coli is currently underway. The TaWD40 cDNA has been successfully amplified from the source plasmid and inserted into an intermediate plasmid, pCR2.1. The TaWD40 cDNA is currently being cloned from the pCR2.1 intermediate plasmid into two different expression vectors, pRSET-A and pMAL-c2x, for future protein production and purification.

Genetic variability among the wild boars (Sus scrofa scrofa), crossbred animals and pigs using microsatellite markers (STRs)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Non invasive methods for genetic analysis applied to ecological and behavioral studies in Latino-America

Documenting the presence and abundance of the neotropical mammals is the first step for understanding their population ecology, behavior and genetic dynamics in designing conservation plans. The combination of field research with molecular genetics techniques are new tools that provide valuable biological information avoiding the disturbance in the ecosystems, trying to minimize the human impact in the process to gather biological information. The objective of this paper is to review the available non invasive sampling techniques that have been used in Neotropical mammal studies to apply to determine the presence and abundance, population structure, sex ratio, taxonomic diagnostic using mitochondrial markers, and assessing genetic variability using nuclear markers. There are a wide range of non invasive sampling techniques used to determine the species identification that inhabit an area such as searching for tracks, feces, and carcasses. Other useful equipment is the camera traps that can generate an image bank that can be valuable to assess species presence and abundance by morphology. With recent advances in molecular biology, it is now possible to use the trace amounts of DNA in feces and amplify it to analyze the species diversity in an area, and the genetic variability at intraspecific level. This is particularly helpful in cases of sympatric and cryptic species in which morphology failed to diagnose the taxonomic status of several species of brocket deer of the genus Mazama.

Synthetic lethality between eIF5A and Ypt1 reveals a connection between translation and the secretory pathway in yeast

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Loss of nuclear poly(A)-binding protein 1 causes defects in myogenesis and mRNA biogenesis

The nuclear poly(A)-binding protein 1 (PABPN1) is a ubiquitously expressed protein that plays a critical role in polyadenylation. Short expansions of the polyalanine tract in the N-terminus of PABPN1 lead to oculopharyngeal muscular dystrophy (OPMD), which is an adult onset disease characterized by eyelid drooping, difficulty in swallowing and weakness in the proximal limb muscles. Although significant data from in vitro biochemical assays define the function of PABPN1 in control of poly(A) tail length, little is known about the role of PABPN1 in mammalian cells. To assess the function of PABPN1 in mammalian cells and specifically in cells affected in OPMD, we examined the effects of PABPN1 depletion using siRNA in primary mouse myoblasts from extraocular, pharyngeal and limb muscles. PABPN1 knockdown significantly decreased cell proliferation and myoblast differentiation during myogenesis in vitro. At the molecular level, PABPN1 depletion in myoblasts led to a shortening of mRNA poly(A) tails, demonstrating the cellular function of PABPN1 in polyadenylation control in a mammalian cell. In addition, PABPN1 depletion caused nuclear accumulation of poly(A) RNA, revealing that PABPN1 is required for proper poly(A) RNA export from the nucleus. Together, these experiments demonstrate that PABPN1 plays an essential role in myoblast proliferation and differentiation, suggesting that it is required for muscle regeneration and maintenance in vivo.

Primary non-gestational choriocarcinoma of the uterine cervix: A case report

Background. Primary non-gestational choriocarcinoma of the female genital tract has been described in the ovaries and is very unusual in other genital sites.Case. Primary non-gestational uterine cervical choriocarcinoma was diagnosed in a patient, 32, single, without previous sexual contact nor antecedent pregnancy, admitted to the hospital with irregular vaginal hemorrhaging. Pelvic examination realized under anesthetic revealed a tumor mass occupying the uterine cervix. Metastases investigation was realized and the patient was accepted as FIGO IV: risk factor of 13. She was submitted to intensive chemotherapy and hysterectomy, showing general recovery, but died from drug-resistant disease 12 months later. Histological, immunohistochemical, and molecular genetics studies confirmed non-gestational choriocarcinoma.Conclusion. Primary non-gestational uterine cervical choriocarcinoma may arise from germ cell tumor or epithelial tissue. (c) 2005 Elsevier B.V. All rights reserved.

Novel insights into the genomic basis of citrus canker based on the genome sequences of two strains of Xanthomonas fuscans subsp aurantifolii

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Identification of a microRNA signature associated with progression of leukoplakia to oral carcinoma

MicroRNAs (miRs) are non-coding RNA molecules involved in cancer initiation and progression. Deregulated miR expression has been implicated in cancer; however, there are no studies implicating an miR signature associated with progression in oral squamous cell carcinoma (OSCC). Although OSCC may develop from oral leukoplakia, clinical and histological assessments have limited prognostic value in predicting which leukoplakic lesions will progress. Our aim was to quantify miR expression changes in leukoplakia and same-site OSCC and to identify an miR signature associated with progression. We examined miR expression changes in 43 sequential progressive samples from 12 patients and four non-progressive leukoplakias from four different patients, using TaqMan Low Density Arrays. The findings were validated using quantitative RT-PCR in an independent cohort of 52 progressive dysplasias and OSCCs, and five non-progressive dysplasias. Global miR expression profiles distinguished progressive leukoplakia/OSCC from non-progressive leukoplakias/normal tissues. One hundred and nine miRs were highly expressed exclusively in progressive leukoplakia and invasive OSCC. miR-21, miR-181b and miR-345 expressions were consistently increased and associated with increases in lesion severity during progression. Over-expression of miR-21, miR-181b and miR-345 may play an important role in malignant transformation. Our study provides the first evidence of an miR signature potentially useful for identifying leukoplakias at risk of malignant transformation.

A Streamlined DNA Tool for Global Identification of Heavily Exploited Coastal Shark Species (Genus Rhizoprionodon)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evaluating the karyotypic diversity in species of Hyla (Anura; Hylidae) with 2n=30 chromosomes based on the analysis of ten species

Ten species of Hyla with 2n = 30 from Brazilian fauna were analysed cytogenetically. Hyla minuta is the unique presenting all bi-armed metacentric or submetacentric chromosomes in the karyotype, therefore, with the highest FN = 60. The remaining species have a variable number of uni-armed telocentric or subtelo-centric chromosomes: H. cruzi, H. elianeae, and H. rubicundula with three pairs (FN = 54), H. berthalutzae, H. elegans, H. microps, and H. nana with four pairs (FN = 52), and H. nahdereri and H. sanborni with five pairs (FN = 50). The uni-armed elements are among pairs 5, 6, 7, 11, 14, and 15, which also appeared with metacentric or submetacentric morphology. The remaining chromosome pairs 1, 2, 3, 4, 8, 9,10, 12, and 13 were never found to be telocentric or subtelocentric. AgNOR patterns are species-specific, the majority of the species exhibiting a single pair with AgNORs, with the exception of H. elegans and H. nana with more than one chromosome pair bearing this cytological marker. C banding was obtained in H. berthalutzae, H. cruzi, H. elegans, H. elianeae, H. microps, H. minuta, H. nahdereri, and H. nana, which showed positively stained centromeric heterochromatin. Our analysis confirms the great karyotypic diversity in the species of Hyla with 2n = 30, with no species sharing identical karyotypes.

Fatores genéticos e ambientais envolvidos na carciogênese gástrica

RACIONAL: O câncer de estômago é o segundo tipo mais comum de neoplasia no mundo. A carcinogênese de estômago é processo de múltiplos passos, podendo manifestar-se em várias etapas como gastrite superficial, gastrite atrófica crônica, metaplasia intestinal, displasia e, finalmente, como um carcinoma. Essas condições costumam ser seqüenciais e ocorrer num período de muitos anos como resultado da exposição a uma variedade de fatores endógenos e exógenos, que causam alterações genéticas. Os recentes avanços da genética molecular têm mostrado que o acúmulo dessas várias anormalidades, incluindo a ativação de oncogenes e a inativação de genes supressores de tumores, resultam no desenvolvimento do câncer. Alterações genéticas descritas em carcinomas gástricos incluem amplificações e mutações dos genes c-ERBB2, K-RAS, c-MET e TP53. O ganho de cromossomos também foi encontrado em várias combinações com perda de outros cromossomos e pode estar associado com a expressão elevada de oncogenes, que contribuem com a progressão tumoral. CONCLUSÃO: Essas mudanças genéticas em carcinomas evidenciam o processo de múltiplas etapas da carcinogênese gástrica, por meio do acúmulo de uma série de alterações.

Allelic imbalance studies of chromosome 9 suggest major differences in chromosomal instability among nonmelanoma skin carcinomas

CONTEXTO: Vários estudos de perda de heterozigozidade na região 9p21-p22, que abriga os genes supressores tumorais CDKN2a/p16INK4a, p19ARF e p15INK4b, têm sido realizados em uma ampla série de tumores humanos, incluindo os melanomas familiares. Perdas e ganhos em outras regiões do cromossomo 9 também têm sido observados com freqüência e podem indicar mecanismos adicionais no processo de tumorigênese dos carcinomas basocelulares da pele. OBJETIVO: Investigar o equilíbrio alélico existente na região 9p21-p22 em carcinomas basocelulares. TIPO DE ESTUDO: Análise molecular de marcadores de microssatélites em tumores e controles. LOCAL: Dois serviços de dermatologia de atendimento terciário em universidades públicas de São Paulo e o Laboratório de Genética Molecular do Câncer da Universidade Estadual de Campinas (UNICAMP), Brasil. PARTICIPANTES: Examinamos 13 casos benignos, incluindo 4 queratoses solares, 3 queratoacantomas, 3 nevos melanocíticos, 2 doenças de Bowen e 1 neurofibroma cutâneo, além de 58 tumores malignos da pele: 14 de células escamosas, 40 carcinomas basocelulares e 4 melanomas; em pacientes consecutivamente encaminhados à clínica de Dermatologia da Unicamp e que concordaram em participar do estudo. VARIÁVEIS ESTUDADAS: O tumor principal e uma porção normal de pele não-adjacente foram removidos cirurgicamente de pacientes que consecutivamente procuraram os ambulatórios de dermatologia da Universidade Estadual de Campinas (UNICAMP) e da Universidade Estadual de São Paulo (Unesp), São Paulo, por causa de lesões cutâneas. Extraímos DNA tanto de tecido tumoral como do correspondente tecido normal de cada paciente. Para amplificar regiões de repetição polimórfica de microssatélites do cromossomo 9, foram utilizados quatro pares de primers, sendo dois deles destinados à região 9p21-p22. RESULTADOS: Identificamos oito casos (20%) de desequilíbrio alélico entre os carcinomas basocelulares, sendo dois casos de perda de heterozigozidade e seis casos de instabilidade de microssatélite na região 9p21-p22. Outros marcadores também mostravam anormalidades em três destes tumores, enquanto nenhuma alteração foi detectada entre os casos benignos e nos outros tumores malignos. CONCLUSÃO: Esta dependência fenotípica sugere que existem diferenças importantes no comportamento das formas mais comuns de tumores cutâneos não-melanocíticos em relação à sua tendência para instabilidade de microssatélite no cromossomo 9. Considerando-se que os genes CDKN2a/p16INK4a, p19ARF e p15INK4b não parecem responsáveis pelas anormalidades observadas, outros genes em 9p21-p22 podem estar envolvidos na etiopatogenia e na progressão dos carcinomas basocelulares.