Estudo da diversidade genética de Podosphaera xanthii através de marcadores AFLP e seqüências ITS

O meloeiro (Cucumis melo L.) é uma frutífera largamente cultivada no Brasil, principalmente no nordeste brasileiro, onde é produzida principalmente para a exportação. Plantas da família do meloeiro, como pepino e abóbora, podem ser severamente afetadas pelo oídio, causado por Podosphaera xanthii.. Este fungo apresenta diversas raças fisiológicas cuja correta identificação é importante para o manejo da doença, já que o uso de variedades resistentes é o método mais eficaz de seu controle. No entanto, a identificação destas raças por meio da prática tradicional de inoculações em uma série diferenciadora de variedades de meloeiro é laboriosa e passível de erros. Devido a isso, um método alternativo seria o uso de marcadores moleculares para determinar de forma rápida a identidade das raças. O objetivo deste estudo foi o de analisar a variabilidade entre isolados de P. xanthii previamente classificados em raças através da técnica de AFLP e do seqüenciamento da região ITS 5.8S do rDNA. A partir dos marcadores AFLP obteve-se um dendrograma no qual não houve separação dos isolados quanto às suas raças, origem geográfica ou hospedeiro de origem. Com esta técnica verificou-se alta variabilidade entre isolados, com similaridade genética máxima de 69% e similaridade mínima de 23%. Ao contrário da informação gerada por AFLP, não foi observada variação na sequência da região ITS 5.8S entre isolados. Desta forma, a análise por AFLP indicou que os isolados tem composição genética heterogênea muito embora este fato não tenha sido evidenciado pelo sequenciamento da região ITS.

Estimativas de polinização cruzada em população de Spondias tuberosa Arruda (Anacardiaceae) usando marcador AFLP

O umbuzeiro é uma das espécies mais importantes do semiárido brasileiro, devido à sua capacidade de produzir frutos em ambiente de estresse hídrico. O objetivo deste estudo foi estimar taxas de polinização cruzada em umbuzeiro (), considerando-se a frequência de heterozigotos observada e esperada nas populações maternal e de descendentes (M1), respectivamente, e a estimativa de multilocos (M2), de forma a orientar programas de preservação e melhoramento genético da espécie. Amostras de DNA extraído de uma população maternal de 96 plantas, estabelecidas no campo em 1991, em Petrolina, PE, bem como DNA de um indivíduo descendente de cada planta-mãe, foram analisadas para 16 bandas polimórficas das combinações de primer de AFLPAAA_CTG e AAA_CTC. No M1, considerou-se a frequência de heterozigotos estimada pela raiz quadrada de 1 menos a frequência de ausência de fragmentos AFLP, tanto para a população maternal (frequência observada) quanto para a população de descendentes (frequência esperada), enquanto no M2 a estimativa foi obtida por estimativa multilocos (m). As estimativas de no M1 variaram de 1,74 a 0,50, com média de 1,063. Como valores de acima de 1,0 são biologicamente inaceitáveis, ficou demonstrado que M1 não foi apropriado para estimar . As frequências de óvulos e pólen para 15 locos de AFLP foram iguais, indicando boa adequação do modelo de acasalamento misto no M2. A taxa m obtida pelo M2 foi de 0,719, próxima de estimativas prévias com isoenzimas em outras populações da espécie. Os resultados indicam que o umbuzeiro é predominantemente de polinização cruzada e há necessidade de amostras amplas para preservar a variabilidade genética da espécie.

Genetic diversity of teak (Tectona grandis L.F.) from different provenances using microsatellite markers

Teak (Tectona grandis) is one of the main timber species in the world with high economic value, famous for its beauty, strength and durability. The objective of this work was to characterize the genetic diversity of teak genotypes used in Brazilian plantations. Nine microsatellite primers were used to assess 60 teak genotypes, including 33 genotypes from seeds of plantations and 14 clones from Cáceres municipality, Mato Grosso State, Brazil, and 13 clones from Honduras, Malaysia, India, Indonesia, Ivory Coast and Solomon Islands. Two groups of genotypes were detected using the Bayesian Structure analysis: 80% were placed in group 1, represented by genotypes from Cáceres and one from Malaysia, and 20% allocated in group 2, composed of clones from India, Solomon Islands, Malaysia and Honduras and the clones from the Ivory Coast. Most of the genetic variability (73%) was concentrated within groups according to AMOVA analysis. Genetic parameters were estimated for the two groups obtained in the analysis of Structure. Moderate genetic diversity was found, with 4.1 alleles per locus, on average, and an average heterozygosity of 0.329, which was lower than the expected heterozygosity (He = 0.492). Group 1 showed the lowest values for these parameters. Suggestions were made concerning the identification of contrasting genotypes to be used as parents in breeding programs.

Prevalence of radiographic markers of femoroacetabular impingement in asymptomatic adults

OBJECTIVE: to determine the prevalence of radiographic signs of femoroacetabular impingement (FAI) in asymptomatic adults and correlate them with data from physical examinations. METHODS: We conducted a cross-sectional study with 82 asymptomatic volunteers, 164 hips, between 40 and 60 years of age, selected by convenience. They were submitted to anamnesis and clinical examination of the hip, anteroposterior (AP) pelvis radiographs with three incidences, Dunn 45° and Lequesne false profile of each hip, to measure the variables. We measured the alpha angle, anterior offset of the femoral neck, cervical diaphyseal angle, CE angle of Wiberg, acetabular index, Sharp angle, and the crossing, ischial spine and posterior wall signs. RESULTS: our sample consisted of 66% women, mean age of 50.4 years. The average alpha angle was 45.10°, SD=8.6. One quarter of the hips showed alpha angle greater than or equal to 50°; among men the prevalence was 34%, and among women, 11%. We found indicative radiographic signs of femoroacetabular impingement in 42.6% of hips, whether femoral or acetabular, and the increased alpha angle was related to the decrease in hip internal rotation (p<0.001). CONCLUSION: the radiographic findings of femoroacetabular impingement in asymptomatic patients were frequent in the studied sample. The increase in alpha angle was associated with decreased internal rotation.

Increased oxidative stress markers may be a promising indicator of risk for primary ovarian insufficiency: a cross-sectional case control study

PURPOSE: The aim of this study was to evaluate serum levels of inducible nitric oxide synthase (INOS), myeloperoxidase (MPO), total antioxidant status (TAS), and total oxidative status (TOS) in women with primary ovarian insufficiency (POI) and to compare them with healthy fertile women. We also examined the possible risk factors associated with POI.METHODS: This cross-sectional case control study was conducted in Zekai Tahir Burak Women's Health Education and Research Hospital. The study population consisted of 44 women with POI (study group) and 36 healthy fertile women (control group). In all patients, serum levels of INOS, MPO, TAS, and TOS were determined. INOS and MPO levels were measured by enzyme-linked immunosorbent assay whereas colorimetric method was used for evaluating TAS and TOS levels. Age, body mass index (BMI), obstetric history, smoking status, family history, comorbidities, sonographic findings, complete blood count values, C-reactive protein and baseline hormone levels were also analyzed. Student's t-test or Mann-Whitney U test was used to compare continuous variables between the groups; categorical data were evaluated by using Pearson χ2 or Fisher exact test, when appropriate. Binary logistic regression method was used to identify risk factors for POI.RESULTS: We found significantly elevated levels of INOS (234.1±749.5 versus133.8±143.0; p=0.005), MPO (3,438.7±1,228.6 versus 2,481.9±1,230.1; p=0.001), and TOS (4.3±1.4 versus 3.6±1.4; p=0.02) in the sera of the study group when compared to the BMI-age matched control group. However, difference in serum levels of TAS were not significant between the 2 groups (1.7±0.2 versus 1.6±0.2; p=0.15). Logistic regression method demonstrated that BMI <25 kg/m2, nulliparity, family history of POI, smoking, and elevated serum levels of INOS, MPO, and TOS were independent risk factors for POI.CONCLUSION: We found an increase in INOS, MPO, and TOS in women with POI. These serum markers may be promising in early diagnosis of POI. Further large-scale studies are required to determine whether oxidative stress markers have a role in diagnosing POI.

Expression of the Immunohistochemical Markers p16 and Ki-67 and Their Usefulness in the Diagnosis of Cervical Intraepithelial Neoplasms

Objective The aim of this study was to determine the expression of the immunohistochemical markers p16 and Ki-67 in cervical intraepithelial neoplasms and their influence on the level of agreement among different observers and for the same observer. Methods The study included 184 patients with cervical intraepithelial neoplasms previously confirmed through biopsies performed between 2005 and 2006. Three pathologists reviewed the biopsies by using hematoxylin-eosin staining to reach a consensus on the diagnosis. Subsequently, an immunohistochemical study analyzed the expression of p16 and Ki-67 in such cases. Results The comparison among the reviewing pathologists revealed only moderate agreement (kappa = 0.44). The agreement improved when the differentiation of highgrade lesions (cervical intraepithelial neoplasm - CIN - 3) was analyzed (kappa = 0.59). p16 staining exhibited a high negative predictive value and sensitivity; however, the specificity was low. Overall, both qualitative and quantitative analyses of p16 and a quantitative analysis Ki-67 exhibited low accuracy. The agreement among diagnoses before immunohistochemistry was 0.47. The use of immunohistochemistry increased the agreement to 0.68. Conclusion Our study showed that the agreement among observers using traditional diagnostic criteria of cervical intraepithelial lesions can improve with the use of immunohistochemistry.

Detection of illegal estrogen administration through immunohistochemical markers in the bovine prostate

The immunodetection of diverse cell markers was evaluated in prostatic samples from bullocks, and bullocks showing epithelial hyperplasia-metaplasia, with oestrogen-induced changes, and in experimental samples from bullocks inoculated with dietylstilbestrol (DES). Antigen-retrieval procedures allowed the use of tissues that had been fixed in formalin for long periods. Three tissue markers were chosen for the study: cytokeratins 13 and 16, vimentin and desmin. Monoclonal antibody K8.12 (specific for cytokeratins 13 and 16) stained basal cells and hyperplastic-metaplastic epithelium; monoclonal antivimentin, and desmin, allowed the definition of fibromuscular changes.

Nuclear and mitochondrial DNA markers in traceability of retail beef samples

Several characteristics are important in a traceability system of animal products, such as age at slaughter, breed composition, besides information of the productive chain. In general, the certification agent records information about the animals and the system which it came from, although cannot guarantee that the slaughtering, meat processing and distribution are error proof. Besides, there is a differential price, at least at the international market, based on sex and breed composition of the animals. Genetic markers allow identification of characteristics controlled in the beef cattle traceability program, as sex and breed composition, in order to correctly identify and appraise the final product for the consumer. The hypothesis of this study was that the majority beef samples retailed in the local market originate from female with a great participation of zebu breeds. Therefore, the objective of this work was to characterize retail beef samples with DNA markers that identify cattle sex and breed composition. Within 10 beef shops localized in Pirassununga, SP, Brazil, 61 samples were collected, all were genotyped as harboring Bos taurus mitochondrial DNA and 18 were positive for the Y chromosome amplification (male). For the marker sat1711b-Msp I the frequency of the allele A was 0.278 and for the marker Lhr-Hha I the frequency of the allele T was 0.417. The results of sat1711b-Msp I and Lhr-Hha I allelic frequencies are suggestive that the proportion of indicus genome compared with the taurine genome in the market meat is smaller than the observed in the Nellore breed. The procedure described in this study identified sex and subspecies characteristics of beef meat samples, with potential application in meat products certification in special as an auxiliary tool in beef cattle traceability programs.

Evidence of natural hybridization and introgression in Bulbophyllum involutum Borba, Semir & F. Barros and B. weddellii (Lindl.) Rchb. f. (Orchidaceae) in the Chapada Diamantina, Brazil, by using allozyme markers

Hybridization between B. involutum and B. weddellii (Orchidaceae) has been first observed in the Serra do Cipó, Minas Gerais State, Brazil, the hybrid being described as B. ×cipoense Borba & Semir. In this study, allozime electrophoresis was used to test the hypothesis of occurrence of hybridization between these two species, as suggested by morphological characters, in the Chapada Diamantina, Bahia State, Brazil. The lack of a diagnostic locus does not allow definite confirmation of the natural hybridization, although this hypotheses is reinforced by the absence of exclusive alleles in the putative hybrid individuals. The existence of several different genotypes points out to either population derived from multiple hybridization events or the hybrids produced offspring. Homozigosity in some morphologically intermediate individuals of alelles which are exclusive to B. involutum and high genetic similarity between them reinforce the hypotheses of introgression in B. involutum, but not in B. weddellii. Genetic variability observed in B. weddellii (He = 0.21) and B. involutum (He = 0.35) is high. Bulbophyllum weddellii and B. involutum presented very high genetic similarity values (0.94). These species, although vegetatively similar, have been placed in different sections based on floral morphology. The results suggest that these species may be more related than previously supposed.

Reading the human Y chromosome: the emerging DNA markers and human genetic history

Reading the human Y chromosome: the emerging DNA markers and human genetic history.

Isozyme markers and genetic variability in three species of Centrosema (Leguminosae)

Isozyme patterns and their genetic control in three Centrosema species are described. Seven isozymatic systems (aspartate aminotransferase, glucose-6-phosphate isomerase, phosphoglucomutase, anodal peroxidase, malate dehydrogenase, 6-phosphogluconate dehydrogenase, and isocitrate dehydrogenase) were studied in 18 populations and several breeding lines of C. acutifolium, C. brasilianum and C. pubescens, using starch gel electrophoresis techniques. All systems, except glucose-6-phosphate isomerase, are described for the first time in these species. A total of 17 isozyme loci were scored; this represents the largest set of Mendelian loci known up to now in Centrosema species. Isozyme polymorphism and variability within and between populations and species were relatively high and allowed discrimination among species

A search for RFLP markers to identify genes for aluminum tolerance in maize

The objective of this study was to identify restriction fragment length polymorphism (RFLP) markers linked to QTLs that control aluminum (Al) tolerance in maize. The strategy used was bulked segregant analysis (BSA) and the genetic material utilized was an F2 population derived from a cross between the Al-susceptible inbred line L53 and Al-tolerant inbred line L1327. Both lines were developed at the National Maize and Sorghum Research Center - CNPMS/EMBRAPA. The F2 population of 1554 individuals was evaluated in a nutrient solution containing a toxic concentration of Al and relative seminal root length (RSRL) was used as a phenotypic measure of tolerance. The RSRL frequency distribution was continuous, but skewed towards Al-susceptible individuals. Seedlings of the F2 population which scored the highest and the lowest RSRL values were transplanted to the field and subsequently selfed to obtain F3 families. Thirty F3 families (15 Al-susceptible and 15 Al-tolerant) were evaluated in nutrient solution, using an incomplete block design, to identify those with the smallest variances for aluminum tolerance and susceptibility. Six Al-susceptible and five Al-tolerant F3 families were chosen to construct one pool of Al-susceptible individuals, and another of Al-tolerant, herein referred as "bulks", based on average values of RSRL and genetic variance. One hundred and thirteen probes were selected, with an average interval of 30 cM, covering the 10 maize chromosomes. These were tested for their ability to discriminate the parental lines. Fifty-four of these probes were polymorphic, with 46 showing codominance. These probes were hybridized with DNA from the two contrasting bulks. Three RFLPs on chromosome 8 distinguished the bulks on the basis of band intensity. DNA of individuals from the bulks was hybridized with these probes and showed the presence of heterozygous individuals in each bulk. These results suggest that in maize there is a region related to aluminum tolerance on chromosome 8

Low levels of sex hormone-binding globulin and hyperproinsulinemia as markers of increased pancreatic ß-cell demand in men

Low levels of sex hormone-binding globulin (SHBG) are considered to be an indirect index of hyperinsulinemia, predicting the later onset of diabetes mellitus type 2. In the insulin resistance state and in the presence of an increased pancreatic ß-cell demand (e.g. obesity) both absolute and relative increases in proinsulin secretion occur. In the present study we investigated the correlation between SHBG and pancreatic ß-cell secretion in men with different body compositions. Eighteen young men (30.0 ± 2.4 years) with normal glucose tolerance and body mass indexes (BMI) ranging from 22.6 to 43.2 kg/m2 were submitted to an oral glucose tolerance test (75 g) and baseline and 120-min blood samples were used to determine insulin, proinsulin and C-peptide by specific immunoassays. Baseline SHBG values were significantly correlated with baseline insulin (r = -0.58, P<0.05), proinsulin (r = -0.47, P<0.05), C-peptide (r = -0.55, P<0.05) and also with proinsulin at 120 min after glucose load (r = -0.58, P<0.05). Stepwise regression analysis revealed that proinsulin values at 120 min were the strongest predictor of SHBG (r = -0.58, P<0.05). When subjects were divided into obese (BMI >28 kg/m2, N = 8) and nonobese (BMI £25 kg/m2, N = 10) groups, significantly lower levels of SHBG were found in the obese subjects. The obese group had significantly higher baseline proinsulin, C-peptide and 120-min proinsulin and insulin levels. For the first time using a specific assay for insulin determination, a strong inverse correlation between insulinemia and SHBG levels was confirmed. The finding of a strong negative correlation between SHBG levels and pancreatic ß-cell secretion, mainly for the 120-min post-glucose load proinsulin levels, reinforces the concept that low SHBG levels are a suitable marker of increased pancreatic ß-cell demand.

Metabolic markers following beta-adrenoceptor agonist infusion in footshock-stressed rats

Stress hormones can alter metabolic functions in adipose tissue and liver, as well as the sensitivity of rat white adipocytes and rat atrial responses to ß-adrenergic agonists. In this study, we examined the effects of three daily footshock stress sessions on the plasma corticosterone, glucose, glycerol and triacylglycerol levels of fed, conscious male rats, and on the plasma glucose, glycerol and triacylglycerol levels of the same rats following iv infusions of ß-adrenergic agonists (isoproterenol: 0.4 nmol kg-1 min-1, noradrenaline: 5.0 µg kg-1 day-1, and BRL 37344 ([±]-[4-(2-[(2-[3-chlorophenyl]-2-hydroxyethyl)amino]propyl)phenoxy]acetic acid), a selective ß3-adrenoceptor agonist: 0.4 nmol kg-1 min-1). Plasma corticosterone levels increased significantly after each stress session, while triacylglycerol levels increased after the first session and glucose increased after the second and third sessions. Glycerol levels were unaltered after stress. These results suggest that repeated footshock stress may induce a metabolic shift from triacylglycerol biosynthesis to glucose release by hepatic tissue, with glycerol serving as one of the substrates in both pathways. Stressed rats were more sensitive to infusion of noradrenaline plus prazosin and to infusion of isoproterenol, with elevated plasma glucose, glycerol and triacylglycerol levels. The higher sensitivity of stressed rats to isoproterenol and noradrenaline was probably related to the permissive effect of plasma corticosterone. Only BRL 37344 increased plasma glycerol levels in stressed rats, probably because ß3-adrenoceptors are not involved in hepatic triacylglycerol synthesis, thus allowing glycerol to accumulate in plasma.

Allelic frequencies of six polymorphic markers for risk of prostate cancer

The aim of the present study was to evaluate the distribution of polymorphisms for the androgen receptor (AR) (CAG, StuI, GGN), SRD5A2 (Ala49Thr, Val89Leu) and CYP17 (MspA1) genes that are considered to be relevant for risk of prostate cancer. We studied 200 individuals from two cities in the State of São Paulo, by PCR, PCR-RFLP and ASOH techniques. The allelic frequencies of the autosomal markers and the StuI polymorphism of the AR gene were very similar to those described in most North American and European populations. In relation to the CAG and GGN number of repeats, the study subjects had smaller repeat lengths (mean of 20.65 and 22.38, respectively) than those described in North American, European and Chinese populations. In the present study, 30.5% of the individuals had less than 22 CAG repeats and 45.5% had less than 23 GGN repeats. When both repeat lengths are considered jointly, this Brazilian population is remarkably different from the others. Further studies on prostate cancer patients need to be conducted to assess the significance of these markers in the Brazilian population.