919 resultados para THYROTROPIN-RELEASING-HORMONE


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A new family of compounds is presented as potential carbon monoxide releasing molecules (CORMs). These compounds, based on tetrachlorocarbonyliridate(III) derivatives, were synthesized and fully characterized by X-ray diffraction, electrospray mass spectrometry, IR. NMR, and density functional theory calculations. The rate of CO release was studied via the myoglobin assay. The results showed that the rate depends on the nature of the sixth ligand, trans to CO, and that a significant modulation on the release rate can be produced by changing the ligand. The reported compounds are soluble in aqueous media, and the rates of CO release are comparable with those for known CORMs, releasing CO at a rate of 0.03-0.58 mu M min(-1) in a 10 mu M solution of myoglobin and 10 mu M of the complexes.

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Thyroid hormone receptors (TRs) are ligand-gated transcription factors with critical roles in development and metabolism. Although x-ray structures of TR ligand-binding domains (LBDs) with agonists are available, comparable structures without ligand (apo-TR) or with antagonists are not. It remains important to understand apo-LBD conformation and the way that it rearranges with ligands to develop better TR pharmaceuticals. In this study, we conducted hydrogen/deuterium exchange on TR LBDs with or without agonist (T(3)) or antagonist (NH(3)). Both ligands reduce deuterium incorporation into LBD amide hydrogens, implying tighter overall folding of the domain. As predicted, mass spectroscopic analysis of individual proteolytic peptides after hydrogen/deuterium exchange reveals that ligand increases the degree of solvent protection of regions close to the buried ligand-binding pocket. However, there is also extensive ligand protection of other regions, including the dimer surface at H10-H11, providing evidence for allosteric communication between the ligand-binding pocket and distant interaction surfaces. Surprisingly, C-terminal activation helix H12, which is known to alter position with ligand, remains relatively protected from solvent in all conditions suggesting that it is packed against the LBD irrespective of the presence or type of ligand. T(3), but not NH(3), increases accessibility of the upper part of H3-H5 to solvent, and we propose that TR H12 interacts with this region in apo-TR and that this interaction is blocked by T(3) but not NH(3.) We present data from site-directed mutagenesis experiments and molecular dynamics simulations that lend support to this structural model of apo-TR and its ligand-dependent conformational changes. (Molecular Endocrinology 25: 15-31, 2011)

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To shed more light on the molecular requirements for recognition of thyroid response elements (TRES) by thyroid receptors (TRs), we compared the specific aspects of DNA TRE recognition by different TR constructs. Using fluorescence anisotropy, we performed a detailed and hierarchical study of TR-TRE binding. This wits done by comparing the binding affinities of three different TR constructs for four different TRE DNA elements, including palindromic sequences and direct repeats (F2, PAL, DR-1, and DR-4) as well as their interactions with nonspecific DNA sequences. The effect of MgCl(2) on suppressing of nonselective DNA binding to TR was also investigated. Furthermore, we determined the dissociation constants of the hTR beta DBD (DNA binding domain) and hTR beta DBD-LBD (DNA binding and ligand binding domains) for specific TRES. We found that a minimum DNA recognition peptide derived from DBD (H1TR) is sufficient for recognition and interaction with TREs, whereas scrambled DNA sequences were unrecognized. Additionally, we determined that the TR DBD binds to F2, PAL, and DR-4 with high affinity and similar K(d) values. The TR DBD-LBD recognizes all the tested TRES but binds preferentially to F2, with even higher affinity. Finally, our results demonstrate the important role played by LBDs in modulating TR-DNA binding.

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The ligand binding domain (LBD) of nuclear hormone receptors adopts a very compact, mostly alpha-helical structure that binds specific ligands with very high affinity. We use circular dichroism spectroscopy and high-temperature molecular dynamics Simulations to investigate unfolding of the LBDs of thyroid hormone receptors (TRs). A molecular description of the denaturation mechanisms is obtained by molecular dynamics Simulations of the TR alpha and TR beta LBDs in the absence and in the presence of the natural ligand Triac. The Simulations Show that the thermal unfolding of the LBD starts with the loss of native contacts and secondary Structure elements, while the Structure remains essentially compact, resembling a molten globule state. This differs From most protein denaturation simulations reported to date and suggests that the folding mechanism may start with the hydrophobic collapse of the TR LBDs. Our results reveal that the stabilities of the LBDs of the TR alpha and TR beta Subtypes are affected to different degrees by the binding of the isoform selective ligand Triac and that ligand binding confers protection against thermal denaturation and unfolding in a subtype specific manner. Our Simulations indicate two mechanisms by which the ligand stabilizes the LBD: (1) by enhancing the interactions between H8 and H 11, and the interaction of the region between H I and the Omega-loop with the core of the LBD, and (2) by shielding the hydrophobic H6 from hydration.

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Nuclear receptors are important targets for pharmaceuticals, but similarities between family members cause difficulties in obtaining highly selective compounds. Synthetic ligands that are selective for thyroid hormone (TH) receptor beta (TR beta) vs. TR alpha reduce cholesterol and fat without effects on heart rate; thus, it is important to understand TR beta-selective binding. Binding of 3 selective ligands (GC-1, KB141, and GC-24) is characterized at the atomic level; preferential binding depends on a nonconserved residue (Asn-331 beta) in the TR beta ligand-binding cavity (LBC), and GC-24 gains extra selectivity from insertion of a bulky side group into an extension of the LBC that only opens up with this ligand. Here we report that the natural TH 3,5,3`-triodothyroacetic acid (Triac) exhibits a previously unrecognized mechanism of TR beta selectivity. TR x-ray structures reveal better fit of ligand with the TR alpha LBC. The TR beta LBC, however, expands relative to TR alpha in the presence of Triac (549 angstrom(3) vs. 461 angstrom(3)), and molecular dynamics simulations reveal that water occupies the extra space. Increased solvation compensates for weaker interactions of ligand with TR beta and permits greater flexibility of the Triac carboxylate group in TR beta than in TR alpha. We propose that this effect results in lower entropic restraint and decreases free energy of interactions between Triac and TR beta, explaining subtype-selective binding. Similar effects could potentially be exploited in nuclear receptor drug design.

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Transplantation of pancreatic islets isolated from organ donors constitutes a promising alternative treatment for type 1 diabetes, however, it is severely limited by the shortage of organ donors. Ex vivo islet cell cultures appear as an attractive but still elusive approach for curing type 1 diabetes. It has recently been shown that, even in the absence of fibrotic over-growth, several factors, such as insufficient nutrition of the islet core, represent a major barrier for long-term survival of islets grafts. The use of immobilized dispersed cells may contribute to solve this problem due to conceivably easier nutritional and oxygen support to the cells. Therefore, we set out to establish an immobilization method for primary cultures of human pancreatic cells by adsorption onto microcarriers (MCs). Dispersed human islets cells were seeded onto Cytodex1 microcarriers and cultured in bioreactors for up to eight days. The cell number increased and islet cells maintained their insulin secretion levels throughout the time period studied. Moreover, the cells also presented a tendency to cluster upon five days culturing. Therefore, this procedure represents a useful tool for controlled studies on islet cells physiology and, also, for biotechnological applications.

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The study proposed to describe sexual development in pelagic stage loggerhead sea turtles Caretta caretta and compare this to hatchlings and adults. It is meant as an ontogenic approach, in order to understand reproductive development and population composition and their dynamics in the pelagic environment. The study focused on the pelagic loggerheads that are found in the waters offshore Madeira Island (Portugal) in the North-eastern Atlantic and use it as a developmental habitat. The innovating character of this work relied on the lack of any description regarding the gonad ontogenesis and reproductive development for the pelagic stage in any of the 7 existing sea turtle species, all of them in danger of extinction. Three methods were used to diagnose the sex of each juvenile individual and asses the level of reproductive development: (1) laparoscopy, (2) gonad biopsy and (3) the assessment of two sex steroids circulating levels, namely testosterone and estradiol. In order to cover all life stages and compare data obtained for the juvenile stage, hatchlings and nesting female adults were sampled at the nearest nesting rookery at Boa Vista Island in the Cape Verde Archipelago. Gonads from dead hatchlings were collected for gonad histology and blood was collected from nesting females for sex steroids assessment. Laparoscopies revealed to be a valid sexing method for the juvenile stage, since gonads are morphologically differentiated at these size classes. Moreover, laparoscopy was validated using gonad histology. Gonad histology of juveniles showed that gonads are already completely differentiated into ovaries or testes at the size classes examined, but development seems to be quiescent. Males present already developed seminiferous tubules with spermatogonia lining the interior of the seminiferous tubule. Female gonads present oocytes at different development stages, but only oocytes up to stage III were observed. The maximum oocyte diameter in each individual correlated with body size, suggesting that reproductive development is an on-going process in juvenile females. The circulating levels of both testosterone and estradiol in juveniles of both sexes were very low and consistently lower than the ones observed in the nesting females from Boa Vista Island. No bimodal distribution was found for any of the sex steroids analysed and thus circulating hormone levels were not a reliable tool for sexing juvenile individuals with a non-invasive technique. The ratio testosterone:estradiol did not show a bimodal distribution either. The levels of testosterone correlated with sea surface temperature. The fact that temperatures observed during this study were below 24ºC might have hindered a differential testosterone pattern between juvenile males and females. Sex ratios for this population were generated according to laparoscopy results and compared among years and size classes. An overall sex ratio of 2 females for each male was found, but they varied among size classes but not among years. Possible causes for the sex ratios observed are discussed. This study is a contribution to our knowledge on the pelagic stage of loggerhead turtles, namely on the population structure regarding sex ratio, which is a vital tool for implementing conservation strategies.

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This work was carried out to verify the effect of a glyphosate-based herbicide on Jundia hormones (cortisol, 17 beta-estradiol and testosterone), oocyte and swim-up fry production. Earthen ponds containing Jundia females were contaminated with glyphosate (3.6 mg/L); blood samples were collected from eight females from each treatment immediately before, or at 1, 10, 20 30 and 40 days following contamination. A typical post-stress rise in cortisol levels was observed at the 20th and 40th days following exposure to glyphosate. At the 40th day, 17 beta-estradiol was decreased in the exposed females. A similar number of oocytes were stripped out from females from both groups, however, a lower number of viable swim-up fry were obtained from the herbicide exposed females, which also had a higher liver-somatic index (LSI). The results indicate that the presence of glyphosate in water was deleterious to Rhamdia quelen reproduction, altering steroid profiles and egg viability. (c) 2006 Elsevier B.V. All rights reserved.

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The present study investigated how the timing of the administration of estradiol benzoate (EB) impacted the synchronization of ovulation in fixed-time artificial insemination protocols of cattle. To accomplish this, two experiments were conducted, with EB injection occurring at different times: at withdrawal of the progesterone-releasing (N) intravaginal device or 24 h later. The effectiveness of these times was compared by examining ovarian follicular dynamics (Experiment 1, n = 30) and conception rates (Experiment 2, n = 504). In Experiment 1, follicular dynamics was performed in 30 Nelore cows (Bos indicus) allocated into two groups. on a random day of the estrous cycle (Day 0), both groups received 2 mg of EB i.m. and a P4-releasing intravaginal device, which was removed on Day 8, when 400 IU of eCG and 150 mu g of PGF were administered. The control group (G-EB9; n = 15) received 1 mg of EB on Day 9, while Group EB8 (G-EB8; n = 15) received the same dose a day earlier. Ovarian ultrasonographic evaluations were performed every 8 h after device removal until ovulation. The timing of EB administration (Day 8 compared with Day 9) did affect the interval between P4 device removal to ovulation (59.4 +/- 2.0 h compared with 69.3 +/- 1.7 h) and maximum diameter of dominant (1.54 +/- 0.06 a cm compared with 1.71 +/- 0.05 b cm, P = 0.03) and ovulatory (1.46 +/- 0.05 a cm compared with 1.58 +/- 0.04 b cm, P < 0.01) follicles. In Experiment 2,504 suckling cows received the same treatment described in Experiment 1, but insemination was performed as follows: Group EB8-AI48h (G-EB8-AI48h; n = 119) and Group EB8-AI54h (G-EB8-AI54h; n = 134) received 1 mg of EB on Day 8 and FrAI was performed, respectively, 48 or 54 h after P4 device removal. Group EB9-AI48h (G-EB9-AI48h; n = 126) and Group EB9-AI54h (G-EB9-AI54h n = 125) received the same treatments and underwent the same FTAI protocols as G-EB8-AI48h and G-EB8-AI54h, respectively; however, EB was administered on Day 9. Conception rates were greater (P < 0.05) in G-EB9-AI54h 163.2% (79/125) a], G-EB9-AI48h [58.7% (74/126) a] and G-EB8-AI48h [58.8% (70/119) a] than in G-EB8-AI54h [34.3% (46/134) b]. We concluded that when EB administration occurred at device withdrawal (D8), the interval to ovulation shortened and dominant and ovulatory follicle diameters decreased. Furthermore, when EB treatment was performed 24 h after device removal, FTAI conducted at either 48 or 54 h resulted in similar conception rates. However, EB treatment on the same day as device withdrawal resulted in a lesser conception rate when FTAI was conducted 54 h after device removal. (C) 2007 Elsevier B.V. All rights reserved.

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Obesity is rampant in modern society and growth hormone (GH) could be useful as adjunct therapy to reduce the obesity-induced cardiovascular damage. To investigate GH effects on obesity, initially 32 male Wistar rats were divided into two groups (n = 16): control (C) was fed standard-chow and water and hyper-caloric (H) was fed hypercaloric chow and 30% sucrose in its drinking water. After 45 days, both C and H groups were divided into two subgroups (n = 8): C + PL was fed standard-chow, water and received saline subcutaneously; C + GH was fed standard-chow, water, and received 2 mg/kg/day GH subcutaneously; H + PL was fed hypercaloric diet, 30% sucrose in its drinking water, and received saline subcutaneously; and H + GH was fed hypercaloric diet, 30% sucrose in its drinking water, and received GH subcutaneously. After 75 days of total experimental period, H + PL rats were considered obese, having higher body weight, body mass index, Lee-index, and atherogenic index (AI) compared to C + PL. Obesity was accompanied by enhanced myocardial lipid hydroperoxide (LH) and lactate dehydrogenase (LDH), as well of depressed energy expenditure (RMR) and oxygen consumption(VO(2))/body weight. H + GH rats had higher fasting RMR, as well as lower AI and myocardial LH than H + PL. Comparing C + GH with C + PL, despite no effects on morphometric parameters, lipid profile, myocardial LH, and LDH activity, GH enhanced fed RMR and myocardial pyruvate dehydrogenase. In conclusion, the present study brought new insights into the GH effects on obesity related cardiovascular damage demonstrating, for the first time, that GH regulated cardiac metabolic pathways, enhanced energy expenditure and improved the lipid profile in obesity condition. Growth hormone in standard fed condition also offered promising therapeutic value enhancing pyruvate-dehydrogenase activity and glucose oxidation in cardiac tissue, thus optimizing myocardial energy metabolism.

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

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OBJETIVO: O presente estudo teve como objetivo avaliar os genes PROP1 e HESX1 em um grupo de pacientes com displasia septo-óptica (DSO) e deficiência hormonal hipofisária (combinada - DHHC; ou deficiência isolada de GH - DGH). Onze pacientes com apresentação clínica e bioquímica consistente com DHHC, DGH ou DSO foram avaliados. SUBJECTS and METHODS: em todos os pacientes, o gene HESX1 foi analisado pelo sequenciamento direto e, nos casos de DHHC, o gene PROP1 foi também sequenciado. RESULTADOS: Um polimorfismo no gene HESX1 (1772 A > G; N125S) foi identificado em um paciente com DSO. Foram encontrados três pacientes portadores da variação alélica 27 T > C; A9A e 59 A > G; N20S no éxon 1 do gene PROP1. Mutações no gene PROP1 e HESX1 não foram identificadas nesses pacientes com DGH, DHHC e DSO esporádicos. CONCLUSÃO: Alterações genéticas em um ou diversos outros genes ou mecanismos não genéticos devem estar implicados nesse processo patogênico.

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Purpose: to compare the efficacy of recombinant LH supplementation for controlled ovarian stimulation in recombinant FSH and GnRH-agonist protocol.Methods: Search strategies included on-line surveys of databases. The fixed effects model was used for odds ratio and effect size (weighted mean difference). Four trials fulfilled the inclusion criteria.Results: a fewer days of stimulation (p < 0.0001), a fewer total amount of r-FSH administered (p < 0.0001) and a higher serum estradiol levels on the day of hCG administration (p < 0.0001) were observed for the r-LH supplementation protocol. However, differences were not observed in number of oocyte retrieved, number of mature oocytes, clinical pregnancy per oocyte retrieval, implantation and miscarriage rates.Conclusions: more randomized controlled trials are necessary before evidence-based recommendations regarding exogenous LH supplementation in ovarian stimulation protocols with FSH and GnRH-agonist for assisted reproduction treatment can be provided.