Determinação sérica de haptoglobina, ceruloplasmina, alpha(1)-glicoproteína ácida, transferrina e alpha(1)-antitripsina, em equinos com cólica

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

Detecção de Histophilus somni (Haemophilus somnus) no sêmen bovino mediante reação em cadeia pela polimerase (PCR)

The present study evaluated the use of PCR for Histophilus somni detection in bovine semen. Semen samples were experimentally infected with H. somni at dilutions ranging from 107 to 101 bacteria/mL and subjected to DNA extraction by the phenol/chloroform method, followed by PCR amplification. The amplification products were analyzed by electrophoresis in 8% acrylamide gel. The oligonucleotide primers used yielded an amplification fragment of 400 base pairs from the bacterial DNA. Positive amplification was obtained even for the 101 bacteria/mL dilution. PCR proved to be an efficient method for the detection of H. somni. The results obtained in this study have brought relevant information for the diagnosis of H. somni, justifying the need for the diagnosis of this bacterium in bulls, especially in semen samples that should be free of contamination. The PCR method has shown to be a useful tool for the quality control of semen produced in artificial insemination centers.

EX-SITU BIOREMEDIATION OF BRAZILIAN SOIL CONTAMINATED WITH PLASTICIZERS PROCESS WASTES

The aim of this research was to evaluate the bioremediation of a soil contaminated with wastes from a plasticizers industry, located in Sao Paulo, Brazil. A 100-kg soil sample containing alcohols, adipates and phthalates was treated in an aerobic slurry-phase reactor using indigenous and acclimated microorganisms from the sludge of a wastewater treatment plant of the plasticizers industry (11gVSS kg(-1) dry soil), during 120 days. The soil pH and temperature were not corrected during bioremediation; soil humidity was corrected weekly to maintain 40%. The biodegradation of the pollutants followed first-order kinetics; the removal efficiencies were above 61% and, among the analyzed plasticizers, adipate was removed to below the detection limit. Biological molecular analysis during bioremediation revealed a significant change in the dominant populations initially present in the reactor.

Interaction between serotonin 5-HT2A receptor gene and dopamine transporter (DAT1) gene polymorphisms influences personality trait of persistence in Austrian Caucasians

We examined 89 normal volunteers using Cloninger's Temperament and Character Inventory (TCI). Genotyping the 102T/C polymorphism of the serotonin 5HT2A receptor gene and the ser9gly polymorphism in exon 1 of the dopamine D3 receptor (DRD3) gene was performed using PCR-RFLP, whereas the dopamine transporter (DAT1) gene variable number of tandem repeats (VNTR) polymorphism was investigated using PCR amplification followed by electrophoresis in an 8% acrylamide gel with a set of size markers. We found a nominally significant association between gender and harm avoidance (P=0.017; women showing higher scores). There was no association of either DAT1, DRD3 or 5HT2A alleles or genotypes with any dimension of the TCI applying Kruskal-Wallis rank-sum tests. Comparing homozygote and heterozygote DAT1 genotypes, we found higher novelty seeking scores in homozygotes (P=0.054). We further found a nominally significant interaction between DAT1 and 5HT2A homo-/heterozygous gene variants (P=0.0071; DAT1 and 5HT2A genotypes P value of 0.05), performing multivariate analysis of variance (MANOVA). Examining the temperamental TCI subscales, this interaction was associated with persistence (genotypes: P=0.004; homo-/heterozygous gene variants: P=0.0004). We conclude that an interaction between DAT1 and 5HT2A genes might influence the temperamental personality trait persistence.

Enhancing BOLD response in the auditory system by neurophysiologically tuned fMRI sequence

Auditory neuroscience has not tapped fMRI's full potential because of acoustic scanner noise emitted by the gradient switches of conventional echoplanar fMRI sequences. The scanner noise is pulsed, and auditory cortex is particularly sensitive to pulsed sounds. Current fMRI approaches to avoid stimulus-noise interactions are temporally inefficient. Since the sustained BOLD response to pulsed sounds decreases with repetition rate and becomes minimal with unpulsed sounds, we developed an fMRI sequence emitting continuous rather than pulsed gradient sound by implementing a novel quasi-continuous gradient switch pattern. Compared to conventional fMRI, continuous-sound fMRI reduced auditory cortex BOLD baseline and increased BOLD amplitude with graded sound stimuli, short sound events, and sounds as complex as orchestra music with preserved temporal resolution. Response in subcortical auditory nuclei was enhanced, but not the response to light in visual cortex. Finally, tonotopic mapping using continuous-sound fMRI demonstrates that enhanced functional signal-to-noise in BOLD response translates into improved spatial separability of specific sound representations.

Decline in Coccolithophore Diversity and Impact on Coccolith Morphogenesis Along a Natural CO2 Gradient

A natural pH gradient caused by marine CO2 seeps off Vulcano Island (Italy) was used to assess the effects of ocean acidification on coccolithophores, which are abundant planktonic unicellular calcifiers. Such seeps are used as natural laboratories to study the effects of ocean acidification on marine ecosystems, since they cause long-term changes in seawater carbonate chemistry and pH, exposing the organisms to elevated CO2 concentrations and therefore mimicking future scenarios. Previous work at CO2 seeps has focused exclusively on benthic organisms. Here we show progressive depletion of 27 coccolithophore species, in terms of cell concentrations and diversity, along a calcite saturation gradient from Omega calcite 6.4 to <1. Water collected close to the main CO2 seeps had the highest concentrations of malformed Emiliania huxleyi. These observations add to a growing body of evidence that ocean acidification may benefit some algae but will likely cause marine biodiversity loss, especially by impacting calcifying species, which are affected as carbonate saturation falls.

Phosphorylation of ubiquitin-activating enzyme in cultured cells.

Ubiquitin-activating enzyme, E1, is the first enzyme in the pathway leading to formation of ubiquitin-protein conjugates. E1 exists as two isoforms in human cells which are separable by electrophoresis. These isoforms migrate with apparent molecular sizes of 110 kDa and 117 kDa in SDS/polyacrylamide gels. Immunoprecipitation of E1 from lysates of HeLa cells metabolically labeled with [32P]phosphate indicated the presence of a phosphorylated form of E1 which migrates at 117 kDa. Phospho amino acid analysis identified serine as the phosphorylated residue in E1. Phosphorylated E1 was also detected in normal and transformed cells from another human cell line. Phosphatase-catalyzed dephosphorylation of E1 in vitro did not eliminate the 117-kDa E1 isoform detected by Coomassie staining after SDS/polyacrylamide gel electrophoresis, thereby demonstrating that phosphorylation is not the sole structural feature differentiating the isoforms of E1. These observations suggest new hypotheses concerning mechanisms of metabolic regulation of the ubiquitin conjugation pathway.

Avaliação do efeito do composto tipo heparina isolado do caranguejo Chaceon fenneri na hemostasia e na morte celular

Heparin is a pharmaceutical animal widely used in medicine due to its potent anticoagulant effect. Furthermore, it has the ability to inhibit the proliferation, invasion and adhesion of cancer cells to vascular endothelium. However, its clinical applicability can be compromised by side effects such as bleeding. Thus, the search for natural compounds with low bleeding risk and possible therapeutic applicability has been targeted by several research groups. From this perspective, this study aims to evaluate the hemorrhagic and anticoagulant activities and citotoxic effect for different tumor cell lines (HeLa, B16-F10, HepG2, HS-5,) and fibroblast cells (3T3) of the Heparin-like from the crab Chaceon fenneri (HEP-like). The HEP-like was purified after proteolysis, ion-exchange chromatography, fractionation with acetone and characterized by electrophoresis (agarose gel) and enzymatic degradation. Hep-like showed eletroforetic behavior similar to mammalian heparin, and high trisulfated /Nacetylated disaccharides ratio. In addition, HEP-like presented low in vitro anticoagulant activity using aPTT and a minor hemorrhagic effect when compared to mammalian heparin. Furthermore, the HEP-like showed significant cytotoxic effect (p<0.001) on HeLa, HepG2 and B16-F10 tumor cells with IC50 values of 1000 ug/mL, after incubation for 72 hours. To assess the influence of heparin-like on the cell cycle in HeLa cells, analysis was performed by flow cytometry. The results of this analysis showed that HEP-like influence on the cell cycle increasing S phase and decreasing phase G2. Thus, these properties of HEP-like make these compounds potential therapeutic agents

Investigation of H19/RsaI Polymorphism in Children With Low Birth Weight in Pernambuco, Brazil

Background: H19 is a strong candidate gene for influencing birth weight variation and is exclusively imprinted maternally. In an attempt to understand the relationship of this gene polymorphism with low birth weight children, we investigated association of H19/RsaI polymorphism with low birth weight and normal birth weight in children and their mothers. Objectives: The aim of our study was to establish the association between H19 gene polymorphism and LW in children born in Pernambuco, state of Brazil. Patients and Methods: It were selected 89 children, 40 low birth weight (LW) and 49 normal birth weight (NW) and 71 mothers (40 mothers of newborns NW and 31 mothers of newborns LW) attended at Dom Malan Hospital, Petrolina, Pernambuco - Brazil. Peripheral blood samples were collected from patients and genomic DNA was extracted and detected by electrophoresis agarose gel, stained by Blue Green Loading Dye. DNA PCR amplification was done using the primers H1 (sense) and H3 (antisense). PCR products were digested with RsaI and electrophoresed on agarose gel stained by ethidium bromide. Statistical analyses were performed using the program BioEstat version 5.0. Results: The RsaI polymorphism in the H19 gene showed that genotype frequencies did not differ statistically between low birth weight (AA = 12.5%, AB = 45%, BB = 42.5%) and control (AA = 8.6% AB = 36.73%, BB= 55.10% groups) and the allele frequencies were not significantly different (P = 0.2897). We also did not observe any association between maternal H19 allele polymorphism and low birth weight newborns (P =0.7799) or normal birth weight children (P = 0.8976). Conclusions: The small size of sample may be the explanation for these results; future studies with more patients are needed to confirm the effect of H19/RsaI polymorphism on birth weight of LW newborns.

Prevalência da infecção por HPV e perfil das mulheres frente ao exame de papanicolau no município de São José de Mipibú/RN.

The Human Papillomavirus (HPV) infection is the major sexually transmitted disease all over the world. There are many factors associated to infection and the virus persistency in the organism. This study aims to evaluate the women's knowledge, attitudes and practice about the Papanicolaou test (Pap), as well as analyze the HPV and Chlamydia trachomatis infections prevalences in sexually active women from the city of São José do Mipibu/RN/Brazil. This research was divided in two steps (step I and step II), using different methodologies and samples each. The samples collected in each step, even socio-demographic or from uterus cervix, are from different patients e were analyzed separated. In step I was evaluated 267 rural and urban zone women s knowledge, attitudes and practices about the Pap by home interview. In the step II were included 605 women with age ranged from 15 to 71 years old, with mean of 33,5 years old and from each one were collected two cervical samples, one for Pap and other for molecular biology, beside the epidemiological interview to investigate the correlation between prevalence of HPV infection and risk factors. To molecular analyses, the samples were processed using a mammal rapid DNA extraction technique protocol. For C. trachomatis DNA detection were used the CP24/27 primers, and GP5+/GP6+ to HPV. PCR products were analyzed by electrophoresis on 8% polyacrylamide gels, followed by silver staining. The results of the step I showed that, in spite of only 46,1% of the interviewed women they have demonstrated to possess appropriate knowledge on the Pap test, the attitude and practice proportions were significantly larger, 63,3% and 64,4% respectively. The largest education degree presented association with adaptation of the knowledge, attitudes and practice, while neglect, lack of solicitation of the exam for the doctor and shame, came as main barriers for the accomplishment of the exam. In the stage II the HPV general prevalence was 28,9%, being 26,7% in the women with normal cytology or benign alterations, 26,7% in the ones that had atypical squamous cells of undetermined significance (ASC-US) and 80% in those with Low grade squamous intraepithelial lesion (LSIL). the HPV infection prevalence was larger in the patients with up to 30 years of age and in the unmarried women, and those that had more than one sexual partner presented larger infection risk. The results show that the sexual relationship with multiple partners increased the infection risk for HPV and consequently the possibility of the occurrence of lesions uterine cervix

Laser Printer Attribution: Exploring New Features And Beyond.

With a huge amount of printed documents nowadays, identifying their source is useful for criminal investigations and also to authenticate digital copies of a document. In this paper, we propose novel techniques for laser printer attribution. Our solutions do not need very high resolution scanning of the investigated document and explore the multidirectional, multiscale and low-level gradient texture patterns yielded by printing devices. The main contributions of this work are: (1) the description of printed areas using multidirectional and multiscale co-occurring texture patterns; (2) description of texture on low-level gradient areas by a convolution texture gradient filter that emphasizes textures in specific transition areas and (3) the analysis of printer patterns in segments of interest, which we call frames, instead of whole documents or only printed letters. We show by experiments in a well documented dataset that the proposed methods outperform techniques described in the literature and present near-perfect classification accuracy being very promising for deployment in real-world forensic investigations.

Endothelial Nitric Oxide Genotypes and Haplotypes Are Not Associated with End-Stage Renal Disease

The identification of genetic markers associated with chronic kidney disease (CKD) may help to predict its development. Because reduced nitric oxide (NO) bioavailability and endothelial dysfunction are involved in CKD, genetic polymorphisms in the gene encoding the enzyme involved in NO synthesis (endothelial NO synthase [eNos]) may affect the susceptibility to CKD and the development of end-stage renal disease (ESRD). We compared genotype and haplotype distributions of three relevant eNOS polymorphisms (T(-786) C in the promoter region, Glu298Asp in exon 7, and 4b/4a in intron 4) in 110 healthy control subjects and 127 ESRD patients. Genotypes for the T(-786) C and Glu298Asp polymorphisms were determined by TaqMan (R) Allele Discrimination assay and real-time polymerase chain reaction. Genotypes for the intron 4 polymorphism were determined by polymerase chain reaction and fragment separation by electrophoresis. The software program PHASE 2.1 was used to estimate the haplotypes frequencies. We considered significant a probability value of p < 0.05/number of haplotypes (p < 0.05/8 = 0.0063). We found no significant differences between groups with respect to age, ethnicity, and gender. CKD patients had higher blood pressure, total cholesterol, and creatinine levels than healthy control subjects (all p < 0.05). Genotype and allele distributions for the three eNOS polymorphisms were similar in both groups (p > 0.05). We found no significant differences in haplotype distribution between groups (p > 0.05). The lack of significant associations between eNOS polymorphisms and ESRD suggests that eNOS polymorphisms may not be relevant to the genetic component of CKD that leads to ESRD.

Endothelial Nitric Oxide Synthase Haplotypes Associated with Aura in Patients with Migraine

There is strong evidence implicating nitric oxide (NO) in the pathophysiology of migraine and aura. Therefore, genetic polymorphisms in the endothelial NO synthase (eNOS) gene have been studied as candidate markers for migraine susceptibility. We compared for the first time the distribution of eNOS haplotypes including the three clinically relevant eNOS polymorphisms (T(-786)C in the promoter, rs2070744; Glu298Asp in exon 7, rs1799983; and a 27 bp variable number of tandem repeats in intron 4) and two additional tagging single-nucleotide polymorphisms (rs3918226 and rs743506) in 178 women with migraine (134 without aura and 44 with aura) and 117 healthy controls (control group). Genotypes were determined by TaqMan allele discrimination assay, real-time polymerase chain reaction, and polymerase chain reaction followed by fragment separation by electrophoresis. The GA (rs743506) genotype was more common in the control group than in women with migraine (odds ratio = 0.47, 95% confidence interval [CI] 0.29-0.78, p<0.01). No significant differences were found in allele distributions for the five eNOS polymorphisms. However, the haplotypes including the variants ""C C a Glu G"" and the variants ""C C b Glu G"" were more common in women with migraine with aura than in women with migraine without aura (odds ratio = 30.71, 95% CI = 1.61-586.4 and odds ratio = 17.26, 95% CI = 1.94-153.4, respectively; both p<0.0015625). These findings suggest that these two eNOS haplotypes affect the susceptibility to the presence of aura in patients with migraine.

Occurrence of phytoplasma associated with sugarcane yellow leaf syndrome in three regions of Sao Paulo State, Brazil

Sugarcane yellow leaf syndrome caused serious damage to crops in the Sao Paulo State, Brazil, in the 1990`s. The syndrome was also reported in other countries and investigations into the etiology revealed an association between the disease and virus and/or phytoplasma. The disease is potentially important and occurs endemically in that State, and for this reason the present study was conducted in order to demonstrate the occurrence of phytoplasma in three traditional sugarcane-producing areas, in Sao Paulo State, through molecular detection From naturally infected plants. Symptomatic plants belonging to varieties SP71-6163, SP71-6180 and SP89-1115 were sampled from Piracicaba, Jau and Ribeirao Preto, and total DNA was extracted from foliar tissues. Nested PCR was conducted with primer pairs P1/ Tint-16F2n/R2, and the amplified products were analyzed by electrophoresis on agarose gels. Amplified DNA fragments of 1.2 kb evidenced the presence of phytoplasma in 36% of symptomatic plants and revealed its Occurrence in all sampled regions. The results demonstrated that phytoplasma is associated with the disease and that it is important to keep a safe inspection of nurseries and monitoring plants in the field, as well as to Select Sugarcane genotypes with a good level of resistance in breeding programs.

Dehydromonocrotaline generates sequence-selective N-7 guanine alkylation and heat and alkali stable multiple fragment DNA crosslinks

Monocrotaline is a pyrrolizidine alkaloid known to cause toxicity in humans and animals. Its mechanism of biological action is still unclear although DNA crosslinking has been suggested to a play a role in its activity. In this study we found that an active metabolite of monocrotaline, dehydromonocrotaline (DHM), alkylates guanines at the N7 position of DNA with a preference for 5'-GG and 5'-GA sequences; In addition, it generates piperidine- and heat-resistant multiple DNA crosslinks, as confirmed by electrophoresis and electron microscopy. On the basis of these findings, we propose that DHM undergoes rapid polymerization to a structure which is able to crosslink several fragments of DNA.