PSA and androgen-related gene (AR, CYP17, and CYP19) polymorphisms and the risk of adenocarcinoma at prostate biopsy

The aim of the present study was to examine the impact of polymorphisms in prostate-specific antigen (PSA) and androgen-related genes (AR, CYP17, and CYP19) on prostate cancer (PCa) risk in selected high-risk patients who underwent prostate biopsy. Blood samples and prostate tissues were obtained for DNA analysis. Single-nucleotide polymorphisms in the 50-untranslated regions (UTRs) of the PSA (substitution A > G at position -158) and CYP17 (substitution T > C at 50-UTR) genes were detected by polymerase chain reaction (PCR)-restriction fragment length polymorphism assays. The CAG and TTTA repeats in the AR and CYP19 genes, respectively, were genotyped by PCR-based GeneScan analysis. Patients with the GG genotype of the PSA gene had a higher risk of PCa than those with the AG or AA genotype (OR = 3.79, p = 0.00138). The AA genotype was associated with lower PSA levels (6.44 +/- 1.64 ng/mL) compared with genotypes having at least one G allele (10.44 +/- 10.06 ng/mL) (p = 0.0687, 95% CI - 0.3146 to 8.315, unpaired t-test). The multivariate analysis confirmed the association between PSA levels and PSA genotypes (AA vs. AG+GG; chi(2) = 0.0482) and CYP19 (short alleles homozygous vs. at least one long allele; chi(2) = 0.0110) genotypes. Genetic instability at the AR locus leading to somatic mosaicism was detected in one PCa patient by comparing the length of AR CAG repeats in matched peripheral blood and prostate biopsy cores. Taken together, these findings suggest that the PSA genotype should be a clinically relevant biomarker to predict the PCa risk.

Recoleta uterina como estratégia para aumentar a taxa de embriões em fêmeas bovinas de corte e leite

Embryo recovery rate in superovulated cows after uterine flushing is lower than the ovulation rate, which contributes to the relative inefficiency of the conventional cervical recovery procedure. A simple and easy alternative to improve embryo recovery rate is the uterine re-flushing procedure. To evaluate the effect of re-flushing and the influence of breed and operator on bovine embryo recovery rate, 38 Nelore and 19 Jersey females were stimulated using FSH, with embryos being collected by one of two trained operators. At the end of flushing, the catheter was sealed and maintained into the uterine body, filled with flushing medium, while females were released to a paddock for 30 to 50 min, to be submitted to the re-flushing procedure by the same operator. A total of 599 structures were recovered out of 57 procedures, from which 423 (70.6%) were obtained in the first flushing and 176 (29.4%) after re-flushing. Mean recovery rates of 7.4 and 3.1 structures were obtained after the first and second flushing, respectively, for a total of 10.5 structures per cow. Structures were obtained in 73.6% ( 42 out of 57) of the re-flushing procedures. No breed effect was observed on total ova or embryo recovery, with 10.9 total ova collected from Jersey and 10.3 from Nelore females. Likewise, the embryo recovery rate obtained following uterine flushing or re-flushing did not differ either between Jersey (8.1 and 2.7) and Nelore (7.0 and 3.2) animals, or between operators A (7.5 and 3.3) and B (7.3 and 2.9), respectively. In conclusion, the uterine re-flushing procedure significantly increased the rate of embryo recovery in Jersey and Nelore females, with no operator influence being observed.

PRACTICAL USE OF MOLECULAR MARKERS IN BEEF AND DAIRY ZEBU CATTLE IN BRAZIL

Selection of zebu (Bos indicus) beef and dairy cattle in Brazil and the validation process of genetic markers for growth, carcass and meat quality traits and also for milk production, fat and protein milk content are discussed as concerned to the concepts and details of their use as auxiliary tools in selection processes. It is highlighted, also, the importance of right selection of ova donor cows for production of embryos to be transferred.

Random X Inactivation and Extensive Mosaicism in Human Placenta Revealed by Analysis of Allele-Specific Gene Expression along the X Chromosome

Imprinted inactivation of the paternal X chromosome in marsupials is the primordial mechanism of dosage compensation for X-linked genes between females and males in Therians. In Eutherian mammals, X chromosome inactivation (XCI) evolved into a random process in cells from the embryo proper, where either the maternal or paternal X can be inactivated. However, species like mouse and bovine maintained imprinted XCI exclusively in extraembryonic tissues. The existence of imprinted XCI in humans remains controversial, with studies based on the analyses of only one or two X-linked genes in different extraembryonic tissues. Here we readdress this issue in human term placenta by performing a robust analysis of allele-specific expression of 22 X-linked genes, including XIST, using 27 SNPs in transcribed regions. We show that XCI is random in human placenta, and that this organ is arranged in relatively large patches of cells with either maternal or paternal inactive X. In addition, this analysis indicated heterogeneous maintenance of gene silencing along the inactive X, which combined with the extensive mosaicism found in placenta, can explain the lack of agreement among previous studies. Our results illustrate the differences of XCI mechanism between humans and mice, and highlight the importance of addressing the issue of imprinted XCI in other species in order to understand the evolution of dosage compensation in placental mammals.

The gene transformer-2 of Anastrepha fruit flies (Diptera, Tephritidae) and its evolution in insects

Background: In the tephritids Ceratitis, Bactrocera and Anastrepha, the gene transformer provides the memory device for sex determination via its auto-regulation; only in females is functional Tra protein produced. To date, the isolation and characterisation of the gene transformer-2 in the tephritids has only been undertaken in Ceratitis, and it has been shown that its function is required for the female-specific splicing of doublesex and transformer pre-mRNA. It therefore participates in transformer auto-regulatory function. In this work, the characterisation of this gene in eleven tephritid species belonging to the less extensively analysed genus Anastrepha was undertaken in order to throw light on the evolution of transformer-2. Results: The gene transformer-2 produces a protein of 249 amino acids in both sexes, which shows the features of the SR protein family. No significant partially spliced mRNA isoform specific to the male germ line was detected, unlike in Drosophila. It is transcribed in both sexes during development and in adult life, in both the soma and germ line. The injection of Anastrepha transformer-2 dsRNA into Anastrepha embryos caused a change in the splicing pattern of the endogenous transformer and doublesex pre-mRNA of XX females from the female to the male mode. Consequently, these XX females were transformed into pseudomales. The comparison of the eleven Anastrepha Transformer-2 proteins among themselves, and with the Transformer-2 proteins of other insects, suggests the existence of negative selection acting at the protein level to maintain Transformer-2 structural features. Conclusions: These results indicate that transformer-2 is required for sex determination in Anastrepha through its participation in the female-specific splicing of transformer and doublesex pre-mRNAs. It is therefore needed for the auto-regulation of the gene transformer. Thus, the transformer/transfomer-2 > doublesex elements at the bottom of the cascade, and their relationships, probably represent the ancestral state ( which still exists in the Tephritidae, Calliphoridae and Muscidae lineages) of the extant cascade found in the Drosophilidae lineage ( in which tra is just another component of the sex determination gene cascade regulated by Sex-lethal). In the phylogenetic lineage that gave rise to the drosophilids, evolution co-opted for Sex-lethal, modified it, and converted it into the key gene controlling sex determination.

Gene Expression Noise in Spatial Patterning: hunchback Promoter Structure Affects Noise Amplitude and Distribution in Drosophila Segmentation

Positional information in developing embryos is specified by spatial gradients of transcriptional regulators. One of the classic systems for studying this is the activation of the hunchback (hb) gene in early fruit fly (Drosophila) segmentation by the maternally-derived gradient of the Bicoid (Bcd) protein. Gene regulation is subject to intrinsic noise which can produce variable expression. This variability must be constrained in the highly reproducible and coordinated events of development. We identify means by which noise is controlled during gene expression by characterizing the dependence of hb mRNA and protein output noise on hb promoter structure and transcriptional dynamics. We use a stochastic model of the hb promoter in which the number and strength of Bcd and Hb (self-regulatory) binding sites can be varied. Model parameters are fit to data from WT embryos, the self-regulation mutant hb(14F), and lacZ reporter constructs using different portions of the hb promoter. We have corroborated model noise predictions experimentally. The results indicate that WT (self-regulatory) Hb output noise is predominantly dependent on the transcription and translation dynamics of its own expression, rather than on Bcd fluctuations. The constructs and mutant, which lack self-regulation, indicate that the multiple Bcd binding sites in the hb promoter (and their strengths) also play a role in buffering noise. The model is robust to the variation in Bcd binding site number across a number of fly species. This study identifies particular ways in which promoter structure and regulatory dynamics reduce hb output noise. Insofar as many of these are common features of genes (e. g. multiple regulatory sites, cooperativity, self-feedback), the current results contribute to the general understanding of the reproducibility and determinacy of spatial patterning in early development.

Retinoic Acid-Treated Pluripotent Stem Cells Undergoing Neurogenesis Present Increased Aneuploidy and Micronuclei Formation

The existence of loss and gain of chromosomes, known as aneuploidy, has been previously described within the central nervous system. During development, at least one-third of neural progenitor cells (NPCs) are aneuploid. Notably, aneuploid NPCs may survive and functionally integrate into the mature neural circuitry. Given the unanswered significance of this phenomenon, we tested the hypothesis that neural differentiation induced by all-trans retinoic acid (RA) in pluripotent stem cells is accompanied by increased levels of aneuploidy, as previously described for cortical NPCs in vivo. In this work we used embryonal carcinoma (EC) cells, embryonic stem (ES) cells and induced pluripotent stem (iPS) cells undergoing differentiation into NPCs. Ploidy analysis revealed a 2-fold increase in the rate of aneuploidy, with the prevalence of chromosome loss in RA primed stem cells when compared to naive cells. In an attempt to understand the basis of neurogenic aneuploidy, micronuclei formation and survivin expression was assessed in pluripotent stem cells exposed to RA. RA increased micronuclei occurrence by almost 2-fold while decreased survivin expression by 50%, indicating possible mechanisms by which stem cells lose their chromosomes during neural differentiation. DNA fragmentation analysis demonstrated no increase in apoptosis on embryoid bodies treated with RA, indicating that cell death is not the mandatory fate of aneuploid NPCs derived from pluripotent cells. In order to exclude that the increase in aneuploidy was a spurious consequence of RA treatment, not related to neurogenesis, mouse embryonic fibroblasts were treated with RA under the same conditions and no alterations in chromosome gain or loss were observed. These findings indicate a correlation amongst neural differentiation, aneuploidy, micronuclei formation and survivin downregulation in pluripotent stem cells exposed to RA, providing evidence that somatically generated chromosomal variation accompanies neurogenesis in vitro.

Morphology and biology of a new Pseudopolydora (Annelida: Spionidae) species from Brazil

Adults of Pseudopolydora rosebelae sp. nov. inhabit silty tubes on muddy bottoms in shallow water in southern Brazil, states of Sao Paulo and Rio de Janeiro. They are rare and extremely delicate, attaining 20 mm long for 55 chaetigers. The worms are distinctive by their colourful yellow and black pigmentation on the anterior part of body and palps, prominent transverse hood on the dorsal anterior edge of chaetiger 3, and lack of coloured respiratory pigment in blood. Of 12 examined individuals, all were females. Oogenesis is intraovarian; oocytes develop from chaetigers 14-15 to chaetigers 24-36. Recently laid oocytes were about 150 mu m in diameter, with embryos and developing larvae found in capsules inside female tubes in March-June. Broods comprised up to 23 capsules with 400 propagules. Capsules were joined to each other in a string and each attached by a single thin stalk to the inner wall of the tube. Larvae hatched at the 4-chaetiger stage and fed on plankton. Pelagic larvae are unique among Pseudopolydora in having large ramified mid-dorsal melanophores from chaetiger 3 onwards. Competent larvae are able to settle and metamorphose at the 15-chaetiger stage, but can remain planktonic up to 18 chaetigers. They have one pair of unpigmented ocelli and three pairs of black eyes in the prostomium, unpaired ramified mid-dorsal melanophores on chaetiger 1 and on the pygidium, ramified lateral melanophores on chaetigers 5-10, prominent yellow chromatophores in the prostomium, peristomium, on dorsal and ventral sides of chaetigers and in the pygidium. Branchiae are present on chaetigers 7-10, and gastrotrochs are arranged on chaetigers 3, 5, 7 and 12. Provisional serrated bristles are present in all notopodia, and hooks are present in neuropodia from chaetiger 8 onwards. Two pairs of provisional protonephridia are present in chaetigers 1 and 2, and adult metanephridia are present from chaetiger 4.

Investigating Giant (Gt) Repression in the Formation of Partially Overlapping Pair-Rule Stripes

Drosophila pair-rule genes are expressed in striped patterns with a precise order of overlap between stripes of different genes. We investigated the role of Giant (Gt) in the regulation of even-skipped, hairy, runt, and fushi tarazu stripes formed in the vicinity of Gt expression domains. In gt null embryos, specific stripes of eve, h, run, and ftz are disrupted. With an ectopic expression system, we verified that stripes affected in the mutant are also repressed. Simultaneously hybridizing gt misxpressing embryos with two pair-rule gene probes, we were able to distinguish differences in the repression of pairs of stripes that overlap extensively. Together, our results showed Gt repression roles in the regulation of two groups of partially overlapping stripes and that Gt morphogen activity is part of the mechanism responsible for the differential positioning of these stripes borders. We discuss the possibility that other factors regulate Gt stripe targets as well. Developmental Dynamics 239:2989-2999, 2010. (C) 2010 Wiley-Liss, Inc.

Psychometric properties of the DASS-Depression scale among a Brazilian population with chronic pain

Background: Depression is a common contributor to suffering and disability in people with chronic pain. However, the assessment of depression in this population has been hampered by the presence of a number of somatic symptoms that are shared between chronic pain, treatment side-effects and traditional concepts of depression. As a result, the use of depression measures that do not contain somatic items has been encouraged. Objective: This study examined the psychometric properties of the Depression sub-scale of the Depression Anxiety and Stress Scales (DASS) in a Brazilian chronic pain patient population. Method: Data on a number of measures were collected from 348 participants attending pain facilities. Results: Principal components and exploratory factor analyses indicated the presence of only one factor. Item analyses indicated adequate item-scale correlations. The Cronbach alpha was .96, which suggests an excellent internal consistency. Conclusion: The DASS-Depression scale has adequate psychometric properties and its further use with Brazilian chronic pain populations can now be supported. (c) 2008 Elsevier Inc. All rights reserved.

Testing motor fitness in karate

Background and Study Aim: Evaluation of sport skills test can be very useful tool for coach practice. The aim of the present paper was: (a) to evaluate the reliability and accuracy of the Specific Physical Fitness Tests (SPFT) (b) to review the results of karate athletes who represent different weight categories, and who are at different stages of schooling; (c) to establish grading criteria of physical fitness preparation. Material/Methods: The reseach was conducted among 219 Kyokushin karate players, whose profiles were presented as (chi) over bar +/- SD and their main characteristics were the following: age 26.8 +/- 4.67 (19-39) years, body mass 75.2 +/- 8.35 (50-97) kg and body height 176.4 +/- 5.67 (160-196) cm. The value of the BMI amounted to 24.1 +/- 2.17 (17.9-29.4) kg/m(2). All the subjects of the research had training experience of 10.5 +/- 3.71 (4-20) years and their degree of proficiency ranged from 4(th) kyu to 3(rd) dan. The physical fitness trials proposed by Story (1989) included: hip turning speed, speed punches, flexibility, rapid kicks, agility, and evasion actions. It was supplemented by a test of local strength endurance, composing a battery of the SPFT, which was implemented by first of the authors between 1991 and 2006. Results: SPFT is characterized by high reliability and it can be used to diagnose the physical fitness preparation and monitor the individual results of training. It discriminates accurately competitors with different sports level and it is characterized by very high accuracy, it is correlated with the test results of motor general physical fitness abilities and coordination abilities as well as it is connected with the somatic build of the athlete. The performance classification table was developed on the basis of our research. Discussion: Results obtained in SPFT were shortly discussed. Conclusions: The collected results of our research allowed us to come to, the conclusion: The table can be applied not only to assess karate fighters, but also adepts in taekwondo, kick-boxing, ju-jitsu, hapkido or other mixed martial arts.

Direct regeneration of protocorm-like bodies (PLBs) from leaf apices of Oncidium flexuosum Sims (Orchidaceae)

The present study describes the direct regeneration of protocorm-like bodies (PLBs) in leaf explants of the tropical species Oncidium flexuosum. The explants were inoculated in a solid, modified Murashige and Skoog (MS) medium with different concentrations of the growth regulator thidiazuron (TDZ) and with or without 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene acetic acid (NAA), and kept away from light or in a 16-h photoperiod. The presence of auxins, 2,4-D, and NAA inhibited the formation of PLBs. The highest frequency of explants that regenerated PLBs (80%) was obtained when they were maintained in a culture medium containing 1.5 mu M TDZ under dark conditions. In the same culture medium but under a 16-h photoperiod, 95% of the leaf explants presented necrosis. Therefore, darkness was crucial for the regeneration of PLBs in O. flexuosum leaf explants, which is in disagreement with the literature. PLBs developed from the division of epidermal and subepidermal cells mainly on the adaxial side of the apex region of the explant. Plants with well-developed leaves and roots grew after the PLBs were transferred to growth regulator-free medium under a 16-h photoperiod.

Transgenic Sweet Orange (Citrus sinensis L. Osbeck) Expressing the attacin A Gene for Resistance to Xanthomonas citri subsp citri

Genetic transformation with genes that code for antimicrobial peptides has been an important strategy used to control bacterial diseases in fruit crops, including apples, pears, and citrus. Asian citrus canker (ACC) caused by Xanthomonas citri subsp. citri Schaad et al. (Xcc) is a very destructive disease, which affects the citrus industry in most citrus-producing areas of the world. Here, we report the production of genetically transformed Natal, Pera, and Valencia sweet orange cultivars (Citrus sinensis L. Osbeck) with the insect-derived attacin A (attA) gene and the evaluation of the transgenic plants for resistance to Xcc. Agrobacterium tumefaciens Smith and Towns-mediated genetic transformation experiments involving these cultivars led to the regeneration of 23 different lines. Genetically transformed plants were identified by polymerase chain reaction, and transgene integration was confirmed by Southern blot analyses. Transcription of attA gene was detected by Northern blot analysis in all plants, except for one Natal sweet orange transformation event. Transgenic lines were multiplied by grafting onto Rangpur lime rootstock plants (Citrus limonia Osbeck) and spray-inoculated with an Xcc suspension (10(6) cfu mL(-1)). Experiments were repeated three times in a completely randomized design with seven to ten replicates. Disease severity was determined in all transgenic lines and in the control (non-transgenic) plants 30 days after inoculation. Four transgenic lines of Valencia sweet orange showed a significant reduction in disease severity caused by Xcc. These reductions ranged from 58.3% to 77.8%, corresponding to only 0.16-0.30% of leaf diseased area as opposed to 0.72% on control plants. One transgenic line of Natal sweet orange was significantly more resistant to Xcc, with a reduction of 45.2% comparing to the control plants, with only 0.14% of leaf diseased area. Genetically transformed Pera sweet orange plants expressing attA gene did not show a significant enhanced resistance to Xcc, probably due to its genetic background, which is naturally more resistant to this pathogen. The potential effect of attacin A antimicrobial peptide to control ACC may be related to the genetic background of each sweet orange cultivar regarding their natural resistance to the pathogen.

Effect of follicular wave synchronization on in vitro embryo production in heifers

Aiming to achieve the ideal time of ovum pick-up (OPU) for in vitro embryo production (IVP) in crossbred heifers, two Latin square design studies investigated the effect of ovarian follicular wave synchronization with estradiol benzoate (EB) and progestins. For each experiment, crossbred heifers stage of estrous cycle was synchronized either with a norgestomet ear implant (Experiment 1) or a progesterone intravaginal device (Experiment 2) for 7d, followed by the administration of 150 mu g D-cloprostenol. On Day 7, all follicles >3 mm in diameter were aspirated and implants/devices were replaced by new ones. Afterwards, implant/device replacement was conducted every 14 d. Each experiment had three treatment groups. In Experiment I (n = 12), heifers in Group 2X had their follicles aspirated twice a week and those in Groups 1X and 1X-EB were submitted to OPU once a week for a period of 28 d. Heifers from Group 1X-EB also received 2 mg EB i.m. immediately after each OPU session. In Experiment 2 (n = 11), animals from Group 0EB did not receive EB while heifers in Groups 2EB and 5EB received 2 and 5 mg of EB respectively, immediately after OPU. The OPU sessions were performed once weekly for 28 d. Therefore, in both experiments, four OPU sessions were performed in heifers aspirated once a week and in Experiment 1, eight OPU sessions were done in heifers aspirated twice a week. Additionally, during the 7-d period following follicular aspiration, ovarian ultrasonography examinations were conducted to measure diameter of the largest follicle and blood samples were collected for FSH quantification by RIA. In Experiment 1, all viable oocytes recovered were in vitro matured and fertilized. Results indicated that while progestin and EB altered follicular wave patterns, this treatment did not prevent establishment of follicular dominance on the ovaries of heifers during OPU at 7-d intervals. Furthermore, the proposed stage of follicular wave synchronization strategies did not improve the number and quality of the recovered oocytes, or the number of in vitro produced embryos. (C) 2009 Elsevier B.V. All rights reserved.

Factors affecting fertilisation and early embryo quality in single- and superovulated dairy cattle

Data on fertilisation and embryo quality in dairy cattle are presented and the main factors responsible for the low fertility of single-ovulating lactating cows and embryo yield in superovulated dairy cattle are highlighted. During the past 50 years, the fertility in high-producing lactating dairy cattle has decreased as milk production increased. Recent data show conception rates to first service to be approximately 32% in lactating cows, whereas in heifers it has remained above 50%. Fertilisation does not seem to be the principal factor responsible for the low fertility in single-ovulating cows, because it has remained above 80%. Conversely, early embryonic development is impaired in high-producing dairy cows, as observed by most embryonic losses occurring during the first week after fertilisation. However, in superovulated dairy cattle, although fertilisation failure is more pronounced, averaging approximately 45%, the percentage of fertilised embryos viable at 1 week is quite high (>70%). Among the multifactorial causes of low fertility in lactating dairy cows, high feed intake associated with low concentrations of circulating steroids may contribute substantially to reduced embryo quality. Fertilisation failure in superovulated cattle may be a consequence of inappropriate gamete transport due to hormonal imbalances.