Determinação da estrutura e estudo da função da metalotioneína de Synechococcus com ferramentas da bioinformática.

Este trabalho abordou a análise estrutural das MTs a fim de se modelar esta proteína e com isso compreender melhor o funcionamento da mesma. O conhecimento da cianobactéria e uma completa análise da MT poderia ser muito útil em um estudo de utilização desta na técnica da biorremediação para remover metais pesados do solo e da água decorrentes das praticas agrícolas atuais.bitstream/CNPTIA/9902/1/comuntec43.pdfAcesso em: 30 maio 2008

Prostaglandin receptors and role of G protein-activated pathways on corpora lutea of pseudopregnant rabbit in vitro

Studies were conducted to characterize receptors for prostaglandin (PG) F(2alpha) (PGF(2alpha)) and PGE(2), and the signalling pathways regulating total nitric oxide synthase activity and progesterone production in rabbit corpora lutea (CL) of different luteal stages. CL were obtained at days 4, 9 and 13 of pseudopregnancy and cultured in vitro for 2 h with PGF(2alpha) or PGE(2) and with activators and inhibitors of G protein (Gp), phospholipase C (PLC), protein kinase C (PKC), adenylate cyclase (AC) and protein kinase A (PKA). High affinity PGF(2alpha) receptor (K(d)=1.9+/-0.6 nM mean+/-s.e.m. ) concentrations increased (P< or =0.01) four- to five-fold from early to mid- and late-luteal phases (50.6+/-8.5, 188.3+/-36.1 and 231.4+/-38.8 fmol/mg protein respectively). By contrast, PGE(2) receptor (K(d)=1.6+/-0.5 nM) concentrations decreased (P< or =0.01) from day 4 to day 9 and 13 (27.5+/-7.7, 12.4+/-2.4 and 16.5+/-3.0 fmol/mg protein respectively). The Gp-dependent AC/PKA pathway was triggered only on day 4 CL, mimicking the PGE(2) treatment and increasing progesterone production. In both day 9 and day 13 CL, the Gp-activated PLC/PKC pathway evoked a luteolytic effect similar to that induced by PGF(2alpha). The time-dependent selective resistance to PGF(2alpha) and PGE(2) by rabbit CL is mediated by factors other than a lack of luteal receptor-ligand interactions

Expression patterns of cytokines, p53 and nitric oxide synthase enzymes in corpora lutea of pseudopregnant rabbits during spontaneous luteolysis

The gene expressions for macrophage chemoattractant protein-1 (MCP-1), interleukin (IL)-1 beta, IL-2 and p53 were examined by semi-quantitative RT-PCR in corpora lutea (CL) of rabbits during spontaneous luteolysis at days 13, 15, 18 and 22 of pseudopregnancy. In the same luteal tissue, total activity of nitric oxide (NO) synthase (NOS) and genes for both endothelial (eNOS) and inducible (iNOS) isoforms were also analysed. From day 13 to 15, MCP-1 and IL-1 beta mRNA levels rose (P &lt; or = 0.01) almost 2-fold, and the transcript for p53 almost 8-fold, but then all dropped (P &lt; or = 0.05) from day 18 onward. IL-2 mRNA abundance was higher (P &lt; or = 0.01) on day 13 and then gradually declined. During luteolysis, eNOS mRNA decreased 40% (P &lt; or = 0.05) by day 15, but thereafter remained unchanged, while iNOS mRNA was barely detectable and did not show any clear age-related pattern throughout the late luteal stages. Total NOS activity progressively increased (P &lt; or = 0.01) from day 13 to 18 of pseudopregnancy and then dropped to the lowest (P &lt; or = 0.01) levels on day 22. Luteal progesterone content also declined during CL regression from 411 to 17 pg/mg found on days 13 and 22 respectively, in parallel with the decrease in blood progesterone concentrations. These data further support a physiological role of NO as modulator of luteal demise in rabbits. Locally, luteal cytokines may be involved in the up-regulation of NOS activity, while downstream NO may inhibit steroroidogenesis and induce expression of p53 gene after removal of the protective action of progesterone

The physiological dilemma of the high progesterone syndrome in rabbit does

This work focused on the mechanisms that may cause multiple asynchronous ovulations and alter normal ovarian function in order to characterize the high progesterone (P+) syndrome in rabbit does, that, having abnormally high plasma progesterone concentration at the time of insemination, fail to become pregnant. At different luteal stages, at either days 4, 9, or 13 of pseudopregnancy, induced by GnRH injection (d-0), two groups of rabbits (n=5/group) were treated with saline or 0.8 \ub5g GnRH. Blood samples were collected from d-0 to d-26 of pseudopregnancy. At d-4, GnRH injection prolonged (P&lt;0.05) the functional CL life span by 3 to 4 d over that of controls. At d-9, GnRH caused a transient decline (P&lt;0.01) of progesterone for the following 3 d but, thereafter, increased again and remained higher (P&lt;0.01) than controls up to d-26. At d-13, progesterone fell to 1 ng/ml within one day following GnRH, but then gradually increased. Based of these progesterone profiles, it can be argued that, at both mid- and late-luteal phase, GnRH triggered luteolysis and induced ovulation followed by the formation of a new generation of CL. For the in vitro study, CL, collected at days 4, 9, and 13 of pseudopregnancy, were incubated with GnRH, GnRH-antagonist, PLA2 inhibitor, and PLC inhibitor. GnRH decreased (P&lt;0.01) progesterone secretion by d-9 and d-13 CL cultured in vitro; by converse, GnRH antagonist, increased (P&lt;0.01) progesterone release from d-4 CL. Co-incubation of GnRH with GnRH antagonist increased (P&lt;0.01) progesterone release in d-4 CL, but had an opposite effect (P&lt;0.01) on d-9 and d-13 CL. PLC inhibitor reversed the GnRH effects in both d-9 and d-13 CL, while PLA2 inhibitor did not change progesterone release. These data suggest that rabbit CL express a functional receptor for GnRH, likely of type II, that utilizes the PLC post transductional cascade. Luteal FSH-R and LH-R mRNA relative abundances did not differ between d-4 and d-9 CL, but were two- to three-fold (P 640.01) higher, respectively, at d-13. StAR mRNA was highly expressed at d-4 of pseudopregnancy, but then markedly declined (P 640.01) at d-9 and d-13. Taken together, our results show that GnRH triggers i) functional regression when CL acquire luteolytic capacity from d 9 of pseudopregnancy onward, and ii) multiple asynchronous ovulations, thus partly explaining the P+ syndrome associated with the simultaneous coexistence of two population of \u201cfresh\u201d and \u201cold\u201d CL, although not yet the underlying causes

Plasma CXCL10, sCD163 and sCD14 Levels Have Distinct Associations with Antiretroviral Treatment and Cardiovascular Disease Risk Factors

We investigate the associations of three established plasma biomarkers in the context of HIV and treatment-related variables including a comprehensive cardiovascular disease risk assessment, within a large ambulatory HIV cohort. Patients were recruited in 2010 to form the Royal Perth Hospital HIV/CVD risk cohort. Plasma sCD14, sCD163 and CXCL10 levels were measured in 475 consecutive patients with documented CVD risk (age, ethnicity, gender, smoking, blood pressure, BMI, fasting metabolic profile) and HIV treatment history including immunological/virological outcomes. The biomarkers assessed showed distinct associations with virological response: CXCL10 strongly correlated with HIV-1 RNA (p0.2). Associations between higher sCD163 and protease inhibitor therapy (p = 0.05) and lower sCD14 with integrase inhibitor therapy (p = 0.02) were observed. Levels of sCD163 were also associated with CVD risk factors (age, ethnicity, HDL, BMI), with a favourable influence of Framingham score <10% (p = 0.04). Soluble CD14 levels were higher among smokers (p = 0.002), with no effect of other CVD risk factors, except age (p = 0.045). Our findings confirm CXCL10, sCD163 and sCD14 have distinct associations with different aspects of HIV infection and treatment. Levels of CXCL10 correlated with routinely monitored variables, sCD163 levels reflect a deeper level of virological suppression and influence of CVD risk factors, while sCD14 levels were not associated with routinely monitored variables, with evidence of specific effects of smoking and integrase inhibitor therapy warranting further investigation

Multi-Operator Qualitative Behavioural Assessment for dogs entering the shelter

This study aimed to develop and validate a fast and straightforward welfare assessment system to help shelter staff in decision-making processes. For newly captured dogs entering the shelter, the animal control officer (at capture time) and the veterinary officer (at entrance examination) compiled a form attributing a qualitative score for the overall Stress level and for 5 Descriptors of dog behavioural traits. Furthermore, a tester filled out the same form by subjecting the dog to a test battery in his pen, besides performing behavioural observations. The veterinary officer and the tester repeated the evaluation protocol after four weeks of acclimatisation in the shelter. The analysis evaluated inter-observer and test–retest reliability, internal consistency, and construct validity of qualitative scores. Overall, we collected 258 forms regarding 189 dogs. Principal component (PC) extracted by Descriptors showed a good correlation with the Stress level score for veterinary and tester confirming the internal consistency of these scales while it was low for animal control officer form. Moreover, qualitative evaluations of the veterinarian and the tester showed congruent correlations with behavioural observations supporting their construct validity. Conversely, the scores expressed by the animal control officer were not consistent with quantitative observations. Then, the veterinary officer and tester forms could be validated and further simplified including only Stress level score while the control officer form requires a revision as it does not seem reliable. We did not find agreement between the Stress level scores expressed by animal control officer, veterinary and tester suggesting that the three contexts represent different stress stimuli to which the same dog reacts differently. The point of view of the three evaluators can increase the reliability of the assessment. Static but vigilant behaviours prevailed in newly sheltered dogs but activity, interactions and behavioural diversity increased in the second behavioural observation when the dogs were kept in multiple cages and four weeks of adaptation had passed. Stress level reduced and PCs tended towards the pole of sociability suggesting a reduction of stress after the period of adaptation to the socio-environmental conditions of the shelter. On the other hand, the considerable inter-individual variability in behaviours reflects differences in coping strategies and or in the manifestation of stress. Our simple tool can not replace a multidisciplinary approach to welfare assessment but could help shelter staff for individual management of dogs complying with their different adaptation skills

Ruthenium(II) Tris-Pyrazolylmethane Complexes in Transfer Hydrogenation Reactions

While ruthenium(II) arene complexes have been widely investigated for their potential in catalytic transfer hydrogenation, studies on homologous compounds replacing the arene ligand with the six-electron donor tris(1-pyrazolyl)methane (tpm) are almost absent in the literature. The reactions of [RuCl(κ3-tpm)(PPh3)2]Cl, 1, with a series of nitrogen ligands (L) proceeded with selective PPh3 mono-substitution, affording the novel complexes [RuCl(κ3-tpm)(PPh3)(L)]Cl (L=NCMe, 2; NCPh, 3; imidazole, 4) in almost quantitative yields. Products 2–4 were fully characterized by IR and multinuclear NMR spectroscopy, moreover the molecular structure of 4 was ascertained by single crystal X-ray diffraction. Compounds 2–4 were evaluated as catalytic precursors in the transfer hydrogenation of a series of ketones with isopropanol as the hydrogen source, and 2 exhibited the highest activity. Extensive NMR experiments and DFT calculations allowed to elucidate the mechanism of the transfer hydrogenation process, suggesting the crucial role played by the tpm ligand, reversibly switching from tri- to bidentate coordination during the catalytic cycle

Gold nanoparticles approach to detect chondroitin sulphate and hyaluronic acid urothelial coating

This study investigated the location of hyaluronic acid (HA)-and chondroitin sulphate (CS)-coated gold nanoparticles in rabbit bladder and evaluated gene expression of CD44, RHAMM and ICAM-1 receptors involved in HA and CS transport into the cell. Gold nanoparticles were synthesised by reduction of gold salts with HA or CS to form HA-AuNPs and CS-AuNPs. Bladder samples were incubated with CS-AuNPs and HA-AuNPs or without glycosaminoglycans. Transmission electron microscopy, optic microscopy and scanning electron microscopy were used to determine the location of the synthesised AuNPs. Real-time PCR was used to analyse expression of urothelial cell receptors CD44, RHAMM, ICAM-1, after ex vivo administration of CS-AuNPs and HA-AuNPs. We showed that HA-AuNPs and CS-AuNPs were located in the cytoplasm and tight junctions of urothelial umbrella cells; this appearance was absent in untreated bladders. There were no significant differences in gene expression levels for CD44, RHAMM and ICAM-1 receptors in treated versus control bladder tissues. In conclusion, we clearly showed the presence of exogenous GAGs in the bladder surface and the tight junctions between umbrella cells, which is important in the regeneration pathway of the urothelium. The GAGs-AuNPs offer a promising approach to understanding the biophysical properties and imaging of urothelial tissue

Leptin receptor expression and in vitro leptin actions on prostaglandin release and nitric oxide synthase activity in the rabbit oviduct

In this study, we have examined the presence and the distribution of receptors for leptin (Ob-R) in the oviduct of rabbits, and the effects of leptin on the release of prostaglandin (PG) F2alpha and PGE2 and on the activity of nitric oxide (NO) synthase (NOS) by oviducts cultured in vitro. Rabbits were killed during the follicular phase and the oviducts were incubated in vitro with leptin, PGF2alpha, PGE2, NO donor and inhibitors of NOS and cyclo-oxigenase (COX). Using immunohistochemistry, Ob-R-like positive reaction was observed only in the cytoplasm of secretory cells, having stronger intensity in the infundibulum and ampulla tracts than in the isthmus. Both leptin and NO donor inhibited PGE2 release, whereas they enhanced PGF2alpha release; NOS inhibitor alone or with leptin increased PGE2 and decreased PGF2alpha production; NOS activity was enhanced by leptin, while PGs did not affect this enzyme. This study suggests that the oviduct could be a potential target for endocrine regulation by leptin, whose circulating levels may act as a metabolic signal modulating oviductal PG release through mediation of the NOS/NO system