RESUMO
BRCA2 tumor suppressor protein ensures genome integrity by mediating DNA repair via homologous recombination (HR). This function is executed in part by its canonical DNA binding domain located at the C-terminus (BRCA2CTD), the only folded domain of the protein. Most germline pathogenic missense variants are located in this highly conserved region which binds to single-stranded DNA (ssDNA) and to the acidic protein DSS1. These interactions are essential for the HR function of BRCA2. Here, we report that the variant R2645G, identified in breast cancer and located at the DSS1 interface, unexpectedly increases the ssDNA binding activity of BRCA2CTDin vitro. Human cells expressing this variant display a hyper-recombination phenotype, chromosomal instability in the form of chromatid gaps when exposed to DNA damage, and increased PARP inhibitor sensitivity. In mouse embryonic stem cells (mES), this variant alters viability and confers sensitivity to cisplatin and Mitomycin C. These results suggest that BRCA2 interaction with ssDNA needs to be tightly regulated to limit HR and prevent chromosomal instability and we propose that this control mechanism involves DSS1. Given that several missense variants located within this region have been identified in breast cancer patients, these findings might have clinical implications for carriers.
Assuntos
Proteína BRCA2 , DNA de Cadeia Simples , Ligação Proteica , Humanos , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Animais , Camundongos , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , Instabilidade Cromossômica , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Cisplatino/farmacologia , Dano ao DNA , Mutação de Sentido Incorreto , Feminino , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Células-Tronco Embrionárias Murinas/metabolismo , Linhagem Celular Tumoral , Mitomicina/farmacologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Complexo de Endopeptidases do ProteassomaRESUMO
Germline inactivating mutations in the BRCA1 and BRCA2 genes are responsible for hereditary breast and ovarian cancer syndrome (HBOCS). Genetic testing of these genes identifies a significant proportion of variants of uncertain significance (VUS). Elucidation of the clinical impact of these variants is an important challenge in genetic diagnostics and counseling. In this study, we assess the RNA effect of 28 BRCA1 and BRCA2 VUS identified in our set of HBOCS families with the aim of gaining insight into their clinical relevance. mRNA was extracted from VUS carriers and controls lymphocytes cultured for 5-6 days and treated with puromycin. RNA was reverse transcribed to perform transcriptional analysis for the study of splicing aberrations. In silico prediction tools were used to select those variants most likely to affect the RNA splicing process. Six out of the 28 variants analyzed showed an aberrant splicing pattern and could therefore be classified as probably pathogenic mutations. Reclassification of VUS improves the genetic counseling and clinical surveillance of carriers of these mutations and highlights the importance of RNA studies in routine diagnostic laboratories.
Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Mutação em Linhagem Germinativa/genética , Síndrome Hereditária de Câncer de Mama e Ovário/genética , RNA Mensageiro/genética , Feminino , Humanos , Splicing de RNA/genética , Transcrição Gênica/genéticaRESUMO
BACKGROUND: Accurate evaluation of unclassified sequence variants in cancer predisposition genes is essential for clinical management and depends on a multifactorial analysis of clinical, genetic, pathologic, and bioinformatic variables and assays of transcript length and abundance. The integrity of assay data in turn relies on appropriate assay design, interpretation, and reporting. METHODS: We conducted a multicenter investigation to compare mRNA splicing assay protocols used by members of the ENIGMA (Evidence-Based Network for the Interpretation of Germline Mutant Alleles) consortium. We compared similarities and differences in results derived from analysis of a panel of breast cancer 1, early onset (BRCA1) and breast cancer 2, early onset (BRCA2) gene variants known to alter splicing (BRCA1: c.135-1G>T, c.591C>T, c.594-2A>C, c.671-2A>G, and c.5467+5G>C and BRCA2: c.426-12_8delGTTTT, c.7988A>T, c.8632+1G>A, and c.9501+3A>T). Differences in protocols were then assessed to determine which elements were critical in reliable assay design. RESULTS: PCR primer design strategies, PCR conditions, and product detection methods, combined with a prior knowledge of expected alternative transcripts, were the key factors for accurate splicing assay results. For example, because of the position of primers and PCR extension times, several isoforms associated with BRCA1, c.594-2A>C and c.671-2A>G, were not detected by many sites. Variation was most evident for the detection of low-abundance transcripts (e.g., BRCA2 c.8632+1G>A Δ19,20 and BRCA1 c.135-1G>T Δ5q and Δ3). Detection of low-abundance transcripts was sometimes addressed by using more analytically sensitive detection methods (e.g., BRCA2 c.426-12_8delGTTTT ins18bp). CONCLUSIONS: We provide recommendations for best practice and raise key issues to consider when designing mRNA assays for evaluation of unclassified sequence variants.
Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Testes Genéticos/métodos , Testes Genéticos/normas , Laboratórios/normas , Splicing de RNA , Predisposição Genética para Doença , Humanos , Análise Multivariada , Guias de Prática Clínica como Assunto , Sítios de Splice de RNA , Sensibilidade e EspecificidadeRESUMO
Allantoate degradation is an essential step for recycling purine-ring nitrogen in all plants, but especially in tropical legumes where the ureides allantoate and allantoin are the main compounds used to store and transport the nitrogen fixed in nodules. Two enzymes, allantoate amidohydrolase (AAH) and allantoate amidinohydrolase (allantoicase), could catalyze allantoate breakdown, although only AAH-coding sequences have been found in plant genomes, whereas allantoicase-related sequences are restricted to animals and some microorganisms. A cDNA for AAH was cloned from Phaseolus vulgaris leaves. PvAAH is a single-copy gene encoding a polypeptide of 483 amino acids that conserves all putative AAH active-site domains. Expression and purification of the cDNA in Nicotiana benthamiana showed that the cloned sequence is a true AAH protein that yields ureidoglycine and ammonia, with a Km of 0.46 mM for allantoate. Optimized in vitro assay, quantitative RT-PCR and antibodies raised to the PvAAH protein were used to study AAH under physiological conditions. PvAAH is ubiquitously expressed in common bean tissues, although the highest transcript levels were found in leaves. In accordance with the mRNA expression levels, the highest PvAAH activity and allantoate concentration also occurred in the leaves. Comparison of transcript levels, protein amounts and enzymatic activity in plants grown with different nitrogen sources and upon drought stress conditions showed that PvAAH is regulated at posttranscriptional level. Moreover, RNAi silencing of AAH expression increases allantoate levels in the transgenic hairy roots, indicating that AAH should be the main enzyme involved in allantoate degradation in common bean.
Assuntos
Regulação da Expressão Gênica de Plantas , Nitrogênio/metabolismo , Phaseolus/enzimologia , Ureo-Hidrolases/genética , Animais , Anticorpos , Sequência de Bases , DNA Complementar/genética , Secas , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Inativação Gênica , Dados de Sequência Molecular , Especificidade de Órgãos , Phaseolus/genética , Folhas de Planta/enzimologia , Folhas de Planta/genética , Plantas Geneticamente Modificadas , RNA de Plantas/genética , Coelhos , Análise de Sequência de DNA , Nicotiana/genética , Nicotiana/metabolismo , Ureia/análogos & derivados , Ureia/metabolismo , Ureo-Hidrolases/metabolismoRESUMO
Recently, it has been reported that biallelic mutations in the ERCC4 (FANCQ) gene cause Fanconi anemia (FA) subtype FA-Q. To investigate the possible role of ERCC4 in breast and ovarian cancer susceptibility, as occurs with other FA genes, we screened the 11 coding exons and exon-intron boundaries of ERCC4 in 1573 index cases from high-risk Spanish familial breast and ovarian cancer pedigrees that had been tested negative for BRCA1 and BRCA2 mutations and 854 controls. The frequency of ERCC4 mutation carriers does not differ between cases and controls, suggesting that ERCC4 is not a cancer susceptibility gene. Interestingly, the prevalence of ERCC4 mutation carriers (one in 288) is similar to that reported for FANCA, whereas there are approximately 100-fold more FA-A than FA-Q patients, indicating that most biallelic combinations of ERCC4 mutations are embryo lethal. Finally, we identified additional bone-fide FA ERCC4 mutations specifically disrupting interstrand cross-link repair.
Assuntos
Alelos , Neoplasias da Mama/congênito , Proteínas de Ligação a DNA/genética , Predisposição Genética para Doença , Variação Genética , Neoplasias Ovarianas/genética , Substituição de Aminoácidos , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Estudos de Casos e Controles , Proteínas de Ligação a DNA/metabolismo , Éxons , Feminino , Síndrome Hereditária de Câncer de Mama e Ovário , Heterozigoto , Humanos , Mutação , Neoplasias Ovarianas/metabolismo , Fenótipo , EspanhaRESUMO
Comprehensive genetic testing of the breast cancer susceptibility genes BRCA1 and BRCA2 identified approximately 16% of variants of unknown significance (VUS), a significant proportion of which could affect the correct splicing of the genes. Our aim is to establish a workflow for classifying VUS in these complex genes, the first stage of which is splicing analysis. We used a combined approach consisting of five in silico splicing prediction programs and RT-PCR analysis for a set of 26 variants not previously studied at the mRNA level and six variants that had already been studied, four of which were used as positive controls as they were found to affect the splicing of these genes and the other two were used as negative controls. We identified a splicing defect in 8 of the 26 newly studied variants and ruled out splicing alteration in the remaining 18 variants. The results for the four positive and the two negative control variants were consistent with results presented in the literature. Our results strongly suggest that the combination of RNA analysis and in silico programs is an important step towards the classification of VUS. The results revealed a very high correlation between experimental data and in silico programs when using tools for predicting acceptor/donor sites but a lower correlation in the case of tools for identifying ESE elements.
Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Síndrome Hereditária de Câncer de Mama e Ovário/genética , RNA Mensageiro/genética , Processamento Alternativo , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Sequência de Bases , Simulação por Computador , Feminino , Predisposição Genética para Doença , Humanos , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Análise de Sequência de RNARESUMO
BACKGROUND: Genes that, when mutated, cause Fanconi anemia or greatly increase breast cancer risk encode for proteins that converge on a homology-directed DNA damage repair process. Mutations in the SLX4 gene, which encodes for a scaffold protein involved in the repair of interstrand cross-links, have recently been identified in unclassified Fanconi anemia patients. A mutation analysis of SLX4 in German or Byelorussian familial cases of breast cancer without detected mutations in BRCA1 or BRCA2 has been completed, with globally negative results. METHODS: The genomic region of SLX4, comprising all exons and exon-intron boundaries, was sequenced in 94 Spanish familial breast cancer cases that match a criterion indicating the potential presence of a highly-penetrant germline mutation, following exclusion of BRCA1 or BRCA2 mutations. RESULTS: This mutational analysis revealed extensive genetic variation of SLX4, with 21 novel single nucleotide variants; however, none could be linked to a clear alteration of the protein function. Nonetheless, genotyping 10 variants (nine novel, all missense amino acid changes) in a set of controls (138 women and 146 men) did not detect seven of them. CONCLUSIONS: Overall, while the results of this study do not identify clearly pathogenic mutations of SLX4 contributing to breast cancer risk, further genetic analysis, combined with functional assays of the identified rare variants, may be warranted to conclusively assess the potential link with the disease.
Assuntos
Neoplasias da Mama/genética , Genes BRCA1 , Genes BRCA2 , Mutação de Sentido Incorreto/genética , Recombinases/genética , Análise Mutacional de DNA , Éxons/genética , Família , Feminino , Predisposição Genética para Doença , Mutação em Linhagem Germinativa/genética , Humanos , Íntrons/genética , MasculinoRESUMO
BACKGROUND: Blood transfusion centres should understand the epidemiology of emerging diseases that are transmissible through the transfusion of blood components. The risk of transmission of arboviruses through this route has become apparent in recent years. The aim of our study is to summarise the reported prevalence (viraemic rate, seroprevalence and/or antigen detection) of Chikungunya (CHIKV), Dengue (DENV) and Zika (ZIKV) viruses in blood donors according to screening test used and world region. MATERIALS AND METHODS: We conducted a systematic literature review and meta-analysis having searched for information in the main bibliographic databases (MEDLINE, Embase, and Scopus). The prevalence for each of the viruses was calculated according to the screening test used and geographic location. RESULTS: We included 18 records on CHIKV, 71 on DENV, and 27 on ZIKV. The highest prevalences of RNA for CHIKV were 1.9% in Puerto Rico (2014), 1.0% in Thailand (2009), and 1.0% in French Polynesia (2014-15). The highest prevalences of RNA for DENV were 5.5% in Saudi Arabia (2015-16), 2.3% in Madeira, Portugal (2012-13), and 0.6% in Brazil (2012). The highest prevalences of RNA for ZIKV were 2.8% in French Polynesia (2013-14), 2.7% in Brazil (2015-16), and 1.8% in Martinique (2016). Overall seroprevalence, as assessed by IgG antibodies, was 21.6% for CHIKV, 24.0% for DENV, and 5.1% for ZIKV. DISCUSSION: Our study shows a high proportion of donors who are viraemic and asymptomatic, especially during outbreaks, with prevalences surpassing 5% for DENV, 1% for CHIKV, and 2% for ZIKV. These data confirm a clear threat to blood transfusion safety. The elevated seroprevalence for these three arboviruses is also indicative of their wide circulation in populations, correlating with an increased risk of infected but asymptomatic donors. Health centres and institutions must address this threat, especially in tropical regions where the biggest outbreaks occur.
Assuntos
Febre de Chikungunya , Dengue , Infecção por Zika virus , Zika virus , Doadores de Sangue , Febre de Chikungunya/epidemiologia , Dengue/diagnóstico , Humanos , Prevalência , RNA , Estudos Soroepidemiológicos , Viremia , Zika virus/genética , Infecção por Zika virus/epidemiologiaRESUMO
Nucleotides are molecules of great importance in plant physiology. In addition to being elementary units of the genetic material, nucleotides are involved in bio-energetic processes, play a role as cofactors, and are also components of secondary metabolites and the hormone cytokinin. The common bean (Phaseolus vulgaris) is a legume that transports the nitrogen fixed in nodules as ureides, compounds synthetized from purine nucleotides. The first step in this pathway is the removal of the 5'-phosphate group by a phosphatase. In this study, a gene that codes for a putative nucleotidase (PvNTD2) has been identified in P. vulgaris. The predicted peptide contains the conserved domains for haloacid dehalogenase-like hydrolase superfamily. The protein has been overexpressed in Escherichia coli, and the purified protein showed molybdate-resistant phosphatase activity with nucleoside monophosphates as substrates, confirming that the identified gene codes for a nucleotidase. The optimum pH for the activity was 7-7.5. The recombinant enzyme did not show special affinity for any particular nucleotide, although the behaviour with AMP was different from that with the other nucleotides. The activity was inhibited by adenosine, and a regulatory role for this nucleoside was proposed. The expression pattern of PvNTD2 shows that it is ubiquitously expressed in all the tissues analysed, with higher expression in nodules of adult plants. The expression was maintained during leaf ontogeny, and it was induced during seedling development. Unlike PvNTD1, another NTD previously described in common bean, the high expression of PvNTD2 was maintained during nodule development, and its possible role in this organ is discussed.
RESUMO
French bean (Phaseolus vulgaris) is a legume that transports most of the atmospheric nitrogen fixed in its nodules to the aerial parts of the plant as ureides. Changes in ureide content and in enzymatic activities involved in their metabolism were identified in the cotyledons and embryonic axes during germination and early seedling development. Accumulation of ureides (ca. 1300 nmol per pair of cotyledons) was observed in the cotyledons of dry seeds. Throughout germination, the total amount of ureides slightly decreased to about 1200 nmol, but increased both in cotyledons and in embryonic axes after radicle emergence. In the axes, the ureides were almost equally distributed in roots, hypocotyls and epicotyls. The pattern of ureide distribution was not affected by the presence of nitrate or sucrose in the media up to 6 days after imbibition. Ureides are synthesized from purines because allopurinol (a xanthine dehydrogenase inhibitor) blocks the increase of ureides. Allantoin and allantoate-degrading activities were detected in French bean dried seeds, whereas no ureidoglycolate-degrading activity was detected. During germination, the levels of the three activities remain unchanged in cotyledons. After radicle emergence, the levels of activities in cotyledons changed. Allantoin-degrading activity increased, allantoate-degrading activity decreased and ureidoglycolate-degrading activity remained undetectable in cotyledons. In developing embryonic axes, the three activities were detected throughout germination and early seedling development. The embryonic axes are able to synthesize ureides, because those compounds accumulated in axes without cotyledons.
Assuntos
Cotilédone/metabolismo , Phaseolus/metabolismo , Plântula/metabolismo , Ureia/análogos & derivados , Ureia/metabolismo , Alantoína/metabolismo , Cotilédone/crescimento & desenvolvimento , Germinação , Nitrogênio/metabolismo , Phaseolus/embriologia , Phaseolus/crescimento & desenvolvimento , Plântula/crescimento & desenvolvimentoRESUMO
Ureides are nitrogenous compounds with a special function in some legume under nitrogen fixing conditions, the ureidic legumes. In this group, ureides are the predominant nitrogen transport molecule from nodules to the upper part, whereas amidic legumes use amides as nitrogen transport compounds. In this study, the ureide levels have been analysed in seedlings from four ureidic and four amidic legume plants. It has been found that the differentiation among ureide and amide plants already exists in seedlings during early seedling development, with high levels of ureide and allantoinase activity in cotyledons and embryonic axes from ureide plants. Since ureides have been implicated in the response of plant to several stress, total hydrosoluble antioxidant capacity and the levels of several antioxidant activities have been determined and compared among these two legume groups. The total antioxidant capacity did not follow any differential pattern in cotyledons or embryonic axes for the analysed plants. The levels of superoxide dismutase, guaiacol peroxidase and ascorbate peroxidase in both embryonic axes and cotyledons are statistical different between amide and ureide seedlings, whereas the catalase activity was similar among these groups of plants. We discuss than amides and ureides could follow different strategies to protect against oxidation.
Assuntos
Antioxidantes/metabolismo , Cotilédone/metabolismo , Fabaceae/metabolismo , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Plântula/metabolismo , Fixação de Nitrogênio/fisiologiaRESUMO
Honey bee plays the leading role in the pollination of many wild plants and crops, but it currently faces serious threats. Climate change is pointed out as one of the causes of the colony collapse disorder. Understanding the response of bees to the new climate change scenario is essential to face this challenge. Especially in the most sensitive bioclimatic zones, such as the Mediterranean areas. In this work, we remotely monitored the weight of the hives with an electronic device during a flowering period in the beekeeping seasons of 2016 and 2017, marked by extreme episodes of drought and high temperatures. We assessed bee colonies at the beginning, middle and at the end of the flowering as well, considering the adult bee population, bee brood, and pollen and honey reserves. The results showed that the flowering was reduced in three weeks in 2017 in comparison to 2016. In those years weight gain was 7.67â¯kg and 18.92â¯kg, respectively. The adverse conditions affected the evolution of the populations of bees and the reserves of honey and pollen in a meaningful way, increasing food stress for bees. It also affected the pollen spectrum and commercial characteristics of honey. Our results provide objective data about the effect of climate change on bees, but it also proved the relevant role of bees in the study of changes in the environment.
Assuntos
Abelhas/fisiologia , Mudança Climática , Estresse Fisiológico , Animais , Clima , Colapso da Colônia , Região do MediterrâneoRESUMO
The three-dimensional wavelet transform (3D-WT) has been proposed for volumetric data coding, since it can provide lossless coding and top-quality reconstruction: two key features highly relevant to medical imaging applications. In this paper, we present experimental results for four new algorithms based on the Classic 3D-WT. The proposed algorithms are capable of obtaining the wavelet coefficients after the spatial and, mainly, the temporal decomposition processes, reducing most redundancies in the video sequence and getting lower entropy values than the Classic algorithm. The new algorithms are based on the Temporal-Window method for carrying out the temporal decomposition. We have conducted a set of experimental evaluations for a representative data set of a modality of intrinsically volumetric medical imaging: angiography sequences.
Assuntos
Angiografia/métodos , Interpretação de Imagem Assistida por Computador/métodos , Algoritmos , Entropia , HumanosRESUMO
During leaf senescence resources are managed, with nutrients mobilized from older leaves to new sink tissues. The latter implies a dilemma in terms of resource utilization, the leaf senescence should increase seed quality whereas delay in senescence should improve the seed yield. Increased knowledge about nutrient recycling during leaf senescence could lead to advances in agriculture and improved seed quality. Macromolecules mobilized during leaf senescence include proteins and nucleic acids. Although nucleic acids have been less well studied than protein degradation, they are possible reservoirs of nitrogen and phosphorous. The present study investigated nuclease activities and gene expression patterns of five members of the S1/P1 family in French bean (Phaseolus vulgaris L. cv.)Page: 2 during leaf senescence. An in-gel assay was used to detect nuclease activity during natural and dark-induced senescence, with single-stranded DNA (ssDNA) used as a substrate. The results revealed two nucleases (glycoproteins), with molecular masses of 34 and 39kDa in the senescent leaves. The nuclease activities were higher at a neutral than at an acidic pH. EDTA treatment inhibited the activities of the nucleases, and the addition of zinc resulted in the recovery of these activities. Both the 34 and 39kDa nucleases were able to use RNA and double-stranded DNA (dsDNA) as substrates, although their activities were low when dsDNA was used as a substrate. In addition, two ribonucleases with molecular masses of 14 and 16kDa, both of which could only utilize RNA as a substrate, were detected in the senescent leaves. Two members of the S1/P1 family, PVN2 and PVN5, were expressed under the experimental conditions, suggesting that these two genes were involved in senescence. The nuclease activity of the glycoproteins and gene expression were similar under both natural senescence and dark-induced senescence conditions.
Assuntos
Expressão Gênica , Glicoproteínas/genética , Phaseolus/fisiologia , Proteínas de Plantas/genética , Glicoproteínas/metabolismo , Phaseolus/genética , Fotoperíodo , Folhas de Planta/metabolismo , Folhas de Planta/fisiologia , Proteínas de Plantas/metabolismoRESUMO
Nucleotidases are phosphatases that catalyze the removal of phosphate from nucleotides, compounds with an important role in plant metabolism. A phosphatase enzyme, with high affinity for nucleotides monophosphate previously identified and purified in embryonic axes from French bean, has been analyzed by MALDI TOF/TOF and two internal peptides have been obtained. The information of these peptide sequences has been used to search in the genome database and only a candidate gene that encodes for the phosphatase was identified (PvNTD1). The putative protein contains the conserved domains (motif I-IV) for haloacid dehalogenase-like hydrolases superfamily. The residues involved in the catalytic activity are also conserved. A recombinant protein overexpressed in Escherichia coli has shown molybdate resistant phosphatase activity with nucleosides monophosphate as substrate, confirming that the identified gene encodes for the phosphatase with high affinity for nucleotides purified in French bean embryonic axes. The activity of the purified protein was inhibited by adenosine. The expression of PvNTD1 gene was induced at the specific moment of radicle protrusion in embryonic axes. The gene was also highly expressed in young leaves whereas the level of expression in mature tissues was minimal.
Assuntos
Nucleotidases/genética , Phaseolus/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Escherichia coli/genética , Expressão Gênica , Nucleotidases/química , Nucleotidases/metabolismo , Phaseolus/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em TandemRESUMO
Plant nucleases are involved in nucleic acid degradation associated to programmed cell death processes as well as in DNA restriction, repair and recombination processes. However, the knowledge about the function of plant nucleases is limited. A major nuclease activity was detected by in-gel assay with whole embryonic axes of common bean by using ssDNA or RNA as substrate, whereas this activity was minimal in cotyledons. The enzyme has been purified to electrophoretic homogeneity from embryonic axes. The main biochemical properties of the purified enzyme indicate that it belongs to the S1/P1 family of nucleases. This was corroborated when this protein, after SDS-electrophoresis, was excised from the gel and further analysis by MALDI TOF/TOF allowed identification of the gene (PVN1) that codes this protein. The gene that codes the purified protein was identified. The expression of PVN1 gene was induced at the specific moment of radicle protrusion. The inclusion of inorganic phosphate to the imbibition media reduced the level of expression of this gene and the nuclease activity suggesting a relationship with the phosphorous status in French bean seedlings.
Assuntos
Desoxirribonucleases/genética , Genes de Plantas , Phaseolus/genética , Ribonucleases/genética , Plântula/metabolismo , Sementes/metabolismo , Sequência de Aminoácidos , Desoxirribonucleases/metabolismo , Expressão Gênica , Germinação , Dados de Sequência Molecular , Phaseolus/enzimologia , Phaseolus/metabolismo , Fósforo/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Ribonucleases/metabolismoRESUMO
Germline inactivating mutations in BRCA1 and BRCA2 genes are responsible for Hereditary Breast and Ovarian Cancer Syndrome (HBOCS). Genetic testing of these genes is available, although approximately 15% of tests identify variants of uncertain significance (VUS). Classification of these variants into pathogenic or non-pathogenic type is an important challenge in genetic diagnosis and counseling. The aim of the present study is to functionally assess a set of 7 missense VUS (Q1409L, S1473P, E1586G, R1589H, Y1703S, W1718L and G1770V) located in the C-terminal region of BRCA1 by combining in silico prediction tools and structural analysis with a transcription activation (TA) assay. The in silico prediction programs gave discrepant results making its interpretation difficult. Structural analysis of the three variants located in the BRCT domains (Y1703S, W1718L and G1770V) reveals significant alterations of BRCT structure. The TA assay shows that variants Y1703S, W1718L and G1770V dramatically compromise the transcriptional activity of BRCA1, while variants Q1409L, S1473P, E1586G and R1589H behave like wild-type BRCA1. In conclusion, our results suggest that variants Y1703S, W1718L and G1770V can be classified as likely pathogenic BRCA1 mutations.
Assuntos
Proteína BRCA1/química , Proteína BRCA1/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto/genética , Bioensaio , Simulação por Computador , Feminino , Humanos , Modelos Moleculares , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Ativação Transcricional/genéticaRESUMO
Common bean (Phaseolus vulgaris) seedlings accumulate ureides derived from purines after germination. The first step in the conversion of purines to ureides is the removal of the 5'-phosphate group by a phosphatase that has not been established yet. Two main phosphatase activities were detected in the embryonic axes of common bean using inosine monophosphate as substrate in an in-gel assay. Both activities differed in their sensitive to the common phosphatase inhibitor molybdate, with the molybdate-resistant as the first enzyme induced after radicle protrusion. The molybdate-resistant phosphatase has been purified to electrophoretic homogeneity and this is the first enzyme which shows this resistance purified and characterized from plant tissues. The native enzyme was a monomer of 55 kDa and it showed highest activity with nucleotides as substrates, with the K(m) values in the micromolar range. Among nucleotides, the highest specific constant (V(max)/K(m)) was observed for adenosine monophosphate. Furthermore, the enzyme was inhibited by nucleosides, the products of the enzymatic reaction, with maximum effect for adenosine. Common bean seedlings imbibed in the presence of adenosine monophosphate in vivo showed the highest molybdate-resistant phosphatase activity in the axes in addition to increased ureide content. The data presented suggests that purified phosphatase is involved in nucleotide metabolism in embryonic axes from common bean.
Assuntos
Monofosfato de Adenosina/metabolismo , Inosina Monofosfato/metabolismo , Phaseolus/enzimologia , Fosfatos/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas de Plantas/metabolismo , Purinas/metabolismo , Germinação/fisiologia , Cinética , Peso Molecular , Molibdênio/farmacologia , Nucleosídeos/metabolismo , Phaseolus/metabolismo , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/isolamento & purificação , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Plântula/metabolismo , Sementes/metabolismo , Especificidade por Substrato , Oligoelementos/farmacologiaRESUMO
OBJECTIVE: We wanted to assess the capability of MRI to quantitatively evaluate the therapeutic response to Crohn's disease (CD) relapse. SUBJECTS AND METHODS: Twenty patients with histologically proven CD were prospectively evaluated with MRI and ileocolonoscopy over a 2-year period. The MRI protocol included axial and coronal T2-weighted and contrast-enhanced T1-weighted sequences. MRI examinations were performed twice, once during an acute relapse of CD and the other at clinical remission. The terminal ileum and colon were divided into six segments/patient, and the endoscopy and histology findings were considered the standard of reference. These were compared on a segmental basis with the quantitative MRI findings regarding wall thickness and contrast enhancement. The results obtained in active and remission CD phases were likewise compared with the findings in 10 control subjects who underwent complete ileocolonoscopy for other reasons and had no pathological findings on ileocolonoscopy. RESULTS: Fifty three of 120 (44.2%) bowel segments showed pathologic changes on endoscopy and histology consistent with CD in active phase. On changing from the active disease phase to clinical remission, a significant decrease was observed in the wall thickness and contrast enhancement of the affected bowel wall. In the active phase of CD, the pathologic bowel segments presented with significantly greater contrast enhancement and wall thickness values compared with the healthy segments of CD and controls. On converting clinically into remission, contrast enhancement tended to normalize, whereas bowel wall thickness remained increased compared with the controls. CONCLUSION: MRI is able to detect pathologic bowel segments in CD, as it allows the measurement of significant variations in wall thickness and contrast enhancement on changing from the active phase of the disease to remission.