Rye (Secale cereale) supernumerary (B) chromosomes associated with heat tolerance during early stages of male sporogenesis.

BACKGROUND AND AIMS: Rye supernumerary (B) chromosomes have an accumulation mechanism involving the B subtelomeric domain highly enriched in D1100- and E3900-related sequences. In this work, the effects of heat stress during the early stages of male meiosis in 0B and +B plants were studied. METHODS: In-depth cytological analyses of chromatin structure and behaviour were performed on staged rye meiocytes utilizing DAPI, fluorescence in situ hybridization and 5-methylcytosine immune labelling. Quantitative real-time PCR was used to measure heat effects on the expression of the Hsp101 gene as well as the 3·9- and 2·7-kb E3900 forms in various tissues and meiotic stages. KEY RESULTS AND CONCLUSIONS: Quantitative real-time PCR established that heat induced equal up-regulation of the Hsp101 gene in 0B and 2B plants, with a marked peak in anthers with meiocytes staged at pachytene. Heat also resulted in significant up-regulation of E3900-related transcripts, especially at pachytene and for the truncated 2·7-kb form of E3900. Cytological heat-induced anomalies in prophase I, measured as the frequency of anomalous meiocytes, were significantly greater in 0B plants. Whereas telomeric sequences were widely distributed in a manner close to normal in the majority of 2B pachytene cells, most 0B meiocytes displayed abnormally clustered telomeres after chromosome pairing had occurred. Relevantly, bioinformatic analysis revealed a significantly high-density heat responsive cis regulatory sequence on E3900, clearly supporting stress-induced response of transcription for the truncated variant. Taken together, these results are the first indication that rye B chromosomes have implications on heat tolerance and may protect meiocytes against heat stress-induced damage.

Bisphenol A alters transcript levels of biomarker genes for Major Depressive Disorder in vascular endothelial cells and colon cancer cells.

Bisphenol A (BPA) is capable of mimicking endogenous hormones with potential consequences for human health and BPA exposure has been associated with several human diseases including neuropsychiatric disorders. Here, quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) results show that BPA at low concentrations (10 ng/mL and 1 µg/mL) induces differential transcript levels of four biomarker genes for Major Depressive Disorder (MDD) in HT29 human colon adenocarcinona cell line and Human Umbilical Vein Endothelial Cells (HUVEC). These results substantiate increasing concerns of BPA exposure in levels currently detected in humans.

Bisphenol A at the reference level counteracts doxorubicin transcriptional effects on cancer related genes in HT29 cells.

Human exposure to Bisphenol A (BPA) results mainly from ingestion of food and beverages. Information regarding BPA effects on colon cancer, one of the major causes of death in developed countries, is still scarce. Likewise, little is known about BPA drug interactions although its potential role in doxorubicin (DOX) chemoresistance has been suggested. This study aims to assess potential interactions between BPA and DOX on HT29 colon cancer cells. HT29 cell response was evaluated after exposure to BPA, DOX, or co-exposure to both chemicals. Transcriptional analysis of several cancer-associated genes (c-fos, AURKA, p21, bcl-xl and CLU) shows that BPA exposure induces slight up-regulation exclusively of bcl-xl without affecting cell viability. On the other hand, a sub-therapeutic DOX concentration (40 nM) results in highly altered c-fos, bcl-xl, and CLU transcript levels, and this is not affected by co-exposure with BPA. Conversely, DOX at a therapeutic concentration (4 µM) results in distinct and very severe transcriptional alterations of c-fos, AURKA, p21 and CLU that are counteracted by co-exposure with BPA resulting in transcript levels similar to those of control. Co-exposure with BPA slightly decreases apoptosis in relation to DOX 4 µM alone without affecting DOX-induced loss of cell viability. These results suggest that BPA exposure can influence chemotherapy outcomes and therefore emphasize the necessity of a better understanding of BPA interactions with chemotherapeutic agents in the context of risk assessment.

Bisphenol A disrupts transcription and decreases viability in aging vascular endothelial cells.

Bisphenol A (BPA) is a widely utilized endocrine disruptor capable of mimicking endogenous hormones, employed in the manufacture of numerous consumer products, thereby interfering with physiological cellular functions. Recent research has shown that BPA alters epigenetic cellular mechanisms in mammals and may be correlated to enhanced cellular senescence. Here, the effects of BPA at 10 ng/mL and 1 µg/mL, concentrations found in human samples, were analyzed on HT29 human colon adenocarcinona cell line and Human Umbilical Vein Endothelial Cells (HUVEC). Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) transcriptional analysis of the Long Interspersed Element-1 (LINE-1) retroelement showed that BPA induces global transcription deregulation in both cell lines, although with more pronounced effects in HUVEC cells. Whereas there was an increase in global transcription in HT29 exclusively after 24 h of exposure, this chemical had prolonged effects on HUVEC. Immunoblotting revealed that this was not accompanied by alterations in the overall content of H3K9me2 and H3K4me3 epigenetic marks. Importantly, cell viability assays and transcriptional analysis indicated that prolonged BPA exposure affects aging processes in senescent HUVEC. To our knowledge this is the first report that BPA interferes with senescence in primary vascular endothelial cells, therefore, suggesting its association to the etiology of age-related human pathologies, such as atherosclerosis.

Cytotoxicity of Eupatorium cannabinum L. ethanolic extract against colon cancer cells and interactions with Bisphenol A and Doxorubicin.

BACKGROUND: Eupatorium cannabinum L. has long been utilized in traditional medicine, however no information is available regarding cellular effects of full extracts. Here we assessed the effects of E. cannabinum ethanolic extract (EcEE) on the colon cancer line HT29. Potential interactions with bisphenol A (BPA) a synthetic phenolic compound to which humans are generally exposed and a commonly used chemotherapeutic agent, doxorubicin (DOX) were also evaluated. METHODS: HT29 cells were exposed to different concentrations (0.5 to 50 µg/ml) of EcEE alone or in combination with BPA or DOX. Cell viability was analyzed through resazurin assay. Gene transcription levels for NCL, FOS, p21, AURKA and bcl-xl were determined through qRT-PCR. Cytological analysis included evaluation of nuclear and mitotic anomalies after DAPI staining, immunodetection of histone H3 lysine 9 acetylation (H3K9ac) and assessment of DNA damage by TUNEL assay. RESULTS: Severe loss of HT29 cell viability was detected for 50 µg/ml EcEE immediately after 24 h exposure whereas the lower concentrations assayed (0.5, 5 and 25 µg/ml) resulted in significant viability decreases after 96 h. Exposure to 25 µg/ml EcEE for 48 h resulted in irreversible cell damage leading to a drastic decrease in cell viability after 72 h recovery in EcEE-free medium. 48 h 25 µg/ml EcEE treatment also induced alteration of colony morphology, H3K9 hyperacetylation, transcriptional up regulation of p21 and down regulation of NCL, FOS and AURKA, indicating reduced proliferation capacity. This treatment also resulted in drastic mitotic and nuclear disruption accompanied by up-regulation of bcl-xl, limited TUNEL labeling and nuclear size increase, suggestive of a non-apoptocic cell death pathway. EcEE/BPA co-exposure increased mitotic anomalies particularly for the lowest EcEE concentration, although without major effects on viability. Conversely, EcEE/DOX co-exposure decreased cell viability in relation to DOX for all EcEE concentrations, without affecting the DOX-induced cell cycle arrest. CONCLUSIONS: EcEE has cytotoxic activity on HT29 cancer cells leading to mitotic disruption and non-apoptotic cell death without severe induction of DNA damage. Interaction experiments showed that EcEE can increase BPA aneugenic effects and EcEE synergistic effects with DOX supporting a potential use as adjuvant in chemotherapeutic approaches.

[231 laparoscopic cholecystectomy in ambulatory: what results?]. / 231 colecistectomias laparoscópicas em ambulatório: Que resultados?

INTRODUCTION: Laparoscopic cholecystectomy is the gold standard procedure for the treatment of lithiasic gallblader pathology and acute cholecystitis. Whether or not it should be done as ambulatory surgery is still being discussed. The present study aims to analyze the quality and safety of laparoscopic cholecystectomy conducted by the Ambulatory Unit of Hospital de Braga and compare the results with those from other European surgical centers performing LC as ambulatory surgery. MATERIAL AND METHODS: Observational prospective study of patients submitted to laparoscopic cholecystectomy in ambulatory surgery during a period of 26 months. Data regarding patients' demography, peri- and postoperative complications, surgical time, time in recovery room, and readmission rates was collected. RESULTS: A total of 231 patients were subjected to Laparoscopic cholecystectomy in the ambulatory unit with overnight stay (time to discharge less than 24 hours). Three patients presented with intra-operative complications, which needed conversion to laparotomy, and four patients were admitted after surgery. The mean time for the procedure was 58 minutes and the mean time for recovery was 19h19 minutes. Postoperative morbidity was 7.8% with 2 nonscheduled admission having occurred. DISCUSSION: One of the controversies regarding laparoscopic cholecystectomy as an outpatient procedure is the need for overnight hospital surveillance. In our unit, we have started LC on an outpatient basis with an overnight stay. Using this protocol, over the past two years, we have operated 231 patients and our results show that this is a completely safe technique. CONCLUSIONS: Data from the study suggests that LC is a safe technique when performed in ambulatory practice, having similar results to other european surgical centers.

Introdução: A colecistectomia laparoscópica é actualmente o gold standard no tratamento da patologia litiásica vesicular e colecistite aguda. A sua realização em regime de cirurgia de ambulatório permanece em discussão. O presente estudo tem por objectivo analisar a qualidade e segurança das colecistectomias laparoscópicas realizadas pela Unidade de Cirurgia de Ambulatório do Hospital de Braga e comparar os resultados com outros centros europeus que realizam colecistectomia laparoscópica em regime de ambulatório.Material e Métodos: Estudo observacional prospectivo de doentes submetidos a colecistectomia laparoscópica em regime de ambulatório durante 26 meses. Foram recolhidos dados referentes à demografia do doente, complicações peri e pós-operatórias, tempo cirúrgico e tempo de permanência no recobro e internamentos não programados.Resultados: Foram submetidos a colecistectomia laparoscópica em regime de ambulatório com pernoita (alta < 24h) 231 doentes. Três doentes tiveram intercorrências intra-operatórias com necessidade de conversão para laparotomia e quatro doentes foram internados após a cirurgia. O tempo médio de cirurgia foi de 58 minutos e o tempo médio de recobro de 19h19m. A morbilidade pósoperatória foi de 7,8% tendo, ocorrido dois internamentos não programados.Discussão: Uma das discussões que existe em torno da colecistectomia laparoscópica em regime de ambulatório é a necessidade de vigilância hospitalar durante a primeira noite após a cirurgia. Na nossa unidade, iniciamos a realização da colecistectomia laparoscópica em regime de ambulatório com pernoita hospitalar. Os resultados de mais de dois anos e 231 doentes operados a colecistectomia laparoscópica mostram que é uma técnica perfeitamente segura em regime de ambulatório.Conclusões: Os dados do estudo mostram que a colecistectomia laparoscópica é uma técnica segura em regime de ambulatório, com resultados semelhantes comparativamente com outros centros europeus.

Bisphenol A at concentrations found in human serum induces aneugenic effects in endothelial cells.

Bisphenol A (BPA) is an endocrine disrupting chemical to which humans are exposed. Continuous environmental exposure to BPA leads to its detection in the majority of individuals from developed countries, with serum concentrations ranging from 0.5 to 10ng/ml in the general population and much higher when associated with occupational exposure. In this work, human umbilical vascular endothelial cells (HUVEC) and human colon adenocarcinona (HT29) cell lines were used to represent endothelial and digestive-tract tissues, which are in direct contact to BPA in vivo. Our results demonstrate that BPA has cell-type differential effects. Notably, BPA concentrations commonly found in humans induce micronucleus formation and interfere with cell-division processes in endothelial cells, resulting in mitotic abnormalities. We also found that BPA induces up-regulation of two genes encoding proteins associated with chromosome segregation, namely CDCA8 (borealin/cell division cycle A8) and SGOL2 (shugoshin-like2). Taken together, the aneugenic effects observed in endothelial cells (HUVECs) substantiate increasing concerns about BPA exposure at levels currently detected in humans.

Chromosome and DNA methylation dynamics during meiosis in the autotetraploid Arabidopsis arenosa.

Variation in chromosome number due to polyploidy can seriously compromise meiotic stability. In autopolyploids, the presence of more than two homologous chromosomes may result in complex pairing patterns and subsequent anomalous chromosome segregation. In this context, chromocenter, centromeric, telomeric and ribosomal DNA locus topology and DNA methylation patterns were investigated in the natural autotetraploid, Arabidopsis arenosa. The data show that homologous chromosome recognition and association initiates at telomeric domains in premeiotic interphase, followed by quadrivalent pairing of ribosomal 45S RNA gene loci (known as NORs) at leptotene. On the other hand, centromeric regions at early leptotene show pairwise associations rather than associations in fours. These pairwise associations are maintained throughout prophase I, and therefore likely to be related to the diploid-like behavior of A. arenosa chromosomes at metaphase I, where only bivalents are observed. In anthers, both cells at somatic interphase as well as at premeiotic interphase show 5-methylcytosine (5-mC) dispersed throughout the nucleus, contrasting with a preferential co-localization with chromocenters observed in vegetative nuclei. These results show for the first time that nuclear distribution patterns of 5-mC are simultaneously reshuffled in meiocytes and anther somatic cells. During prophase I, 5-mC is detected in extended chromatin fibers and chromocenters but interestingly is excluded from the NORs what correlates with the pairing pattern.

Rye Bs disclose ancestral sequences in cereal genomes with a potential role in gametophyte chromatid segregation.

Two sequence families, E3900 and D1100, are amplified on the subtelomeric domain of the long arm of rye B chromosomes, the region that controls its drive mechanism. In this work, polymerase chain reaction (PCR) with a number of primers spanning E3900 shows that the organization and nucleotide sequence of E3900-related portions are present and highly conserved on rye A chromosomes as well as in other cereals. Quantitative Real-Time PCR estimates two E3900 sequences to be represented in 100-150 copies on Bs and at least as single copies on As. A novel E3900-related sequence, with a deletion that results in a frameshift and subsequently an open reading frame with putative DNA binding motifs, is identified. Expression analysis of E3900 indicates identical transcription levels in leaves from plants with and without Bs, showing that the expression of these sequences must be silenced on Bs and tightly regulated on As in leaves. In contrast, E3900 transcription is upregulated during meiosis exclusively in plants with Bs, maintaining a high level of transcription in the gametophyte. Interestingly, Bs not only influence their own chromatid segregation but also that of the regular chromosome complement of both rye and wheat. There is a drastic increase in frequency of disrupted metaphase and anaphase cells in the first mitosis of pollen grains carrying Bs, which appears to be due to anomalous adherences between sister chromatids. Taken together, this work provides insight into how E3900 sequences are potentially associated with important evolutionary mechanisms involved in basic cellular processes.

Interplay of ribosomal DNA loci in nucleolar dominance: dominant NORs are up-regulated by chromatin dynamics in the wheat-rye system.

BACKGROUND: Chromatin organizational and topological plasticity, and its functions in gene expression regulation, have been strongly revealed by the analysis of nucleolar dominance in hybrids and polyploids where one parental set of ribosomal RNA (rDNA) genes that are clustered in nucleolar organizing regions (NORs), is rendered silent by epigenetic pathways and heterochromatization. However, information on the behaviour of dominant NORs is very sparse and needed for an integrative knowledge of differential gene transcription levels and chromatin specific domain interactions. METHODOLOGY/PRINCIPAL FINDINGS: Using molecular and cytological approaches in a wheat-rye addition line (wheat genome plus the rye nucleolar chromosome pair 1R), we investigated transcriptional activity and chromatin topology of the wheat dominant NORs in a nucleolar dominance situation. Herein we report dominant NORs up-regulation in the addition line through quantitative real-time PCR and silver-staining technique. Accompanying this modification in wheat rDNA trascription level, we also disclose that perinucleolar knobs of ribosomal chromatin are almost transcriptionally silent due to the residual detection of BrUTP incorporation in these domains, contrary to the marked labelling of intranucleolar condensed rDNA. Further, by comparative confocal analysis of nuclei probed to wheat and rye NORs, we found that in the wheat-rye addition line there is a significant decrease in the number of wheat-origin perinucleolar rDNA knobs, corresponding to a diminution of the rDNA heterochromatic fraction of the dominant (wheat) NORs. CONCLUSIONS/SIGNIFICANCE: We demonstrate that inter-specific interactions leading to wheat-origin NOR dominance results not only on the silencing of rye origin NOR loci, but dominant NORs are also modified in their transcriptional activity and interphase organization. The results show a cross-talk between wheat and rye NORs, mediated by ribosomal chromatin dynamics, revealing a conceptual shift from differential amphiplasty to 'mutual amphiplasty' in the nucleolar dominance process.

Transcriptionally active heterochromatin in rye B chromosomes.

B chromosomes (Bs) are dispensable components of the genomes of numerous species. Thus far, there is a lack of evidence for any transcripts of Bs in plants, with the exception of some rDNA sequences. Here, we show that the Giemsa banding-positive heterochromatic subterminal domain of rye (Secale cereale) Bs undergoes decondensation during interphase. Contrary to the heterochromatic regions of A chromosomes, this domain is simultaneously marked by trimethylated H3K4 and by trimethylated H3K27, an unusual combination of apparently conflicting histone modifications. Notably, both types of B-specific high copy repeat families (E3900 and D1100) of the subterminal domain are transcriptionally active, although with different tissue type-dependent activity. No small RNAs were detected specifically for the presence of Bs. The lack of any significant open reading frame and the highly heterogeneous size of mainly polyadenylated transcripts indicate that the noncoding RNA may function as structural or catalytic RNA.

Genomic analysis of Grapevine Retrotransposon 1 (Gret 1) in Vitis vinifera.

The complete sequence of the first retrotransposon isolated in Vitis vinifera, Gret 1, was used to design primers that permitted its analysis in the genome of grapevine cultivars. This retroelement was found to be dispersed throughout the genome with sites of repeated insertions. Fluorescent in situ hybridization indicated multiple Gret 1 loci distributed throughout euchromatic portions of chromosomes. REMAP and IRAP proved to be useful as molecular markers in grapevine. Both of these techniques showed polymorphisms between cultivars but not between clones of the same cultivar, indicating differences in Gret 1 distribution between cultivars. The combined cytological and molecular results suggest that Gret 1 may have a role in gene regulation and in explaining the enormous phenotypic variability that exists between cultivars.

Molecular and cytological characterization of genomic variability in hexaploid wheat 'Lindström'.

'Lindström' wheat (AABBDD+rye B chromosomes) was used to study the effects of alien chromatin introgressed into a wheat genetic background, subjecting the wheat genome to a new and transient allopolyploidisation episode. Using this experimental material, we have previously demonstrated that no large-scale chromosomal translocations occurred as a result of the genomic constitution of the addition line. However, we have shown that the presence of a number of rye B chromosomes is associated with changes in the interphase organization and expression patterns of wheat rDNA loci. We have now extended our studies to focus on a further characterization of 'Lindström' 5S rDNA loci and also on high molecular weight glutenin subunit (HMW-GS) patterns. In the process, we have uncovered an unusually large variant of the 5S rDNA locus on wheat chromosome 1B (not to be confused with rye B chromosomes) and 2 novel HMW glutenin y-type alleles. These changes are not directly related to variation in rye B chromosome number in the present material, but the fact that a new, and still segregating, 1Dy HMW-GS gene was identified indicates a recent timescale for its origin. Strikingly, the 'Lindström' 5S rDNA 1B locus integrates a unit sharing 94% homology with a rye 5S rDNA sequence, suggesting the possibility that the wheat locus was colonized by highly homologous rye sequences during the breeding of 'Lindström', when the rye and wheat genomes were together, albeit briefly, in the same nucleus.

[Evaluation of hospital resources for perinatal assistance in Brazil]. / Avaliação dos recursos hospitalares para assistência perinatal em Belo Horizonte, Minas Gerais.

OBJECTIVE: To verify the adequacy of hospital resources for perinatal care in Belo Horizonte, MG, Brazil in 1996. METHODS: This is a cross-sectional study conducted encompassing all the 28 maternity-hospitals in the city of Belo Horizonte, capital of the State of Minas Gerais, Brazil. A model of evaluation denominated "Níveis de Complexidade e Segurança em Potencial de Unidades Perinatais de Hospitais-Maternidade" [Levels of Complexity and Potential Safety in Maternity-Hospitals]. This model is based on the evaluation of three major areas in the hospital: general structure, perinatal clinical infra-structure and resources available in perinatal units. Two scores involving these three areas were created. When evaluating low risk perinatal care, the score may sum to a total of 1,000 points. On the other hand, when high to medium perinatal risk care is considered, the score may sum to 2,000 points. Hosptals that scored more than 500 points were classified in two levels: I-A and I-B (for low risk); and six levels I-A through III-B (for medium/high risk). RESULTS: Five hospitals were classified as level 0 according to the low risk score, that is, they were not considered adequate to assist any delivery. When the medium/high risk score was employed, a total of seven hospitals were considered inadequate to assist any delivery. CONCLUSIONS: This evaluation model was capable of classifying hospitals according to available infra-structure for perinatal care.

Avaliação dos recursos hospitalares para assistência perinatal em Belo Horizonte, Minas Gerais / Evaluation of hospital resources for perinatal assistance in Brazil

OBJETIVO: Verificar a adequação de recursos hospitalares no atendimento às gestantes/neonatos, no ano de 1996. MÉTODOS: Trata-se de um estudo transversal abrangendo 28 hospitais-maternidade em funcionamento no município de Belo Horizonte, MG. Utilizou-se um modelo de avaliação denominado "níveis de complexidade e segurança em potencial de unidades perinatais de hospitais-maternidade", elaborado a partir da avaliação de três grandes áreas hospitalares: infra-estrutura geral, infra-estrutura clínica-perinatal e recursos das unidades perinatais. Foram desenvolvidos dois escores de pontos envolvendo essas áreas hospitalares, totalizando 1.000 pontos quando considerada a assistência perinatal de risco habitual e 2.000 pontos para assistência perinatal de médio/alto risco. A partir de 500 pontos, os hospitais foram classificados em dois níveis: I-A e I-B (para risco habitual) e seis níveis de I-A a III-B (para médio/alto risco). RESULTADOS: Na avaliação com o escore proposto para risco habitual, cinco hospitais foram considerados inadequados para realizarem atendimento a qualquer tipo de parto, classificados como nível zero; esse número aumentou para sete, quando se utilizou o escore para parto de médio/alto risco. CONCLUSAO: O modelo de avaliação utilizado mostrou-se adequado para classificar os hospitais de acordo com a infra-estrutura disponível para assistência perinatal.

Evidence for 'cross-talk' between A and B chromosomes of rye.

Spontaneous DNA insertions from supernumerary B chromosomes (Bs) into the standard A chromosome complement were detected in rye (Secale cereale L.), using fluorescent in situ hybridization (FISH) analysis with the D1100 B-specific sequence probe. The insertions were seen in individuals derived from plants possessing deleted Bs, characterized in this study by not having the B-specific sequences that are normally found at the distal part of the long arm of the standard rye B. This result supports the case for the spontaneous introgression of B-specific DNA into the A chromosome genome, and it indicates that 'cross-talking' between A and B chromosomes may occur in wild populations.

Diabetes mellitus: conceito, fisiopatologia, diagnóstico e tratamento / Diabetes mellitus: concept, physiopathology, diagnosis and treatment

Este Informe Técnico tem a intençäo de promover o repasse de informaçöes sobre diabetes para os profissionais de saúde de todo o Estado de Minas Gerais, sensibilizando-os quanto a importância desta patologia em nosso meio e provendo-os das informaçöes necessárias para que possam atuar junto ao paciente diabético, seus familiares e a populaçäo em geral...