TECNETIUM-99m AS ALTERNATIVE TO PRODUCE SOMATOSTATIN-LABELED DERIVATIVES: COMPARATIVE BIODISTRIBUTION EVALUATION WITH (111)In-DTPA-OCTREOTIDE

Synthetic somatostatin (SST) analogues have been used in the preparation of receptor-specific radiopharmaceuticals for diagnostic and therapy of neuroendocrine tumors. This work studied the labeling conditions with (99m)Tc and biological distribution in Swiss mice of two SST analogs (HYNIC-Tyr(3)-Octreotide and HYNIC-Tyr(3)-Octreotate) and compared the biodistribution pattern with (111)In-DTPA-Octreotide. Biological distribution studies were performed after injection of radiopharmaceuticals on Swiss mice. Labeling procedures resulted on high radiochemical yield for all three preparations and the labeled products presented high in vitro stability. Biological distribution studies evidenced similar general biodistribution of (99m)Tc-labeled peptides when compared with indium-labeled peptide with fast blood clearance and elimination by urinary tract. Kidneys uptake of (99m)Tc-HYNIC-TATE are similar to (111)In-DTPA-Octreotide, and both are significantly higher than (99m)Tc-HYNIC-OCT. All labeled peptides presented similar uptake on liver, but the retention in time at intestines, particularly at large intestine, was more expressive for (111)In-labeled peptide. The %ID of (99m)Tc-HYNIC-OCT and (99m)Tc-HYNIC-TATE in organs with high density of SST receptors like pancreas and adrenals were significant and similar to obtained for (111)In-DTPA-Octreotide, confirming the affinity of these radiopharmaceuticals for the receptors.

Coordinated expression of ER, PR and HER2 define different prognostic subtypes among poorly differentiated breast carcinomas

Aims: Histological grade is one of the most important prognostic factors in breast carcinomas, but poorly differentiated neoplasms still have quite heterogeneous biological behaviour, since they can be genetically classified as basal-like, HER2+ or even luminal. The aim was to analyse the frequency of oestrogen receptor (ER), progesterone receptor (PR) and HER2 expression profiles among breast carcinomas with < 10% tubular formation, and their correlation with classic prognostic factors. Methods and results: One hundred and thirty-four samples of paraffin-embedded tumours were studied retrospectively. The tumours were classified in to four groups by their ER/PR/HER2 profile: (i) ER+ and/or PR+ but HER2-; (ii) ER+ and/or PR+ and HER2+; (iii) ER- and/or PR- but HER2+; and (iv) ER-, PR- and HER2- (triple-negative). The histological features of triple-negative and HER2+ carcinomas overlap. The only difference was the expression of basal cytokeratins (basal CK), which was more frequent among triple-negative carcinomas. Basal-CK expression defined a more aggressive group of tumours, according to the pathological features, regardless of the immunohistochemical profile. Conclusions: Group 1 and 2 tumours (ER+ and/or PR+ tumours with or without HER2 expression) were not statistically different, suggesting that poorly differentiated carcinomas with hormone receptors correspond to the luminal B type of tumour. Among poorly differentiated breast carcinomas, the classic profile associated with basal-CK identifies distinct subtypes equivalent to those seen by genetic classification.

Estrogen receptor-positive breast carcinomas in younger women are different from those of older women: A pathological and immunohistochemical study

The higher frequency of triple-negative and HER-2-positive tumors detected in younger patients has been suggested as an explanation for the more aggressive tumor types observed in this age group. However, estrogen receptor (ER)-positive tumors are the most frequent subtype of breast carcinomas identified, even in younger patients. In this retrospective study, the morphological and immunohistochemical profiles of ER-positive breast carcinomas from women 35 yrs and younger that were diagnosed between 1997 and 2007 were evaluated. From these cases, 213 were selected based on the availability of pathology reports and paraffin blocks. For comparison, 117 consecutive cases of breast carcinomas diagnosed in patients >60 yrs from 2006 were included. Paraffin-embedded tumors were stained for expression of ER, progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2). Ki-67 antigen, epidermal growth factor receptor (EGFR), cytokeratin 5/6, p53, vimentin, CD117, and p63 using tissue microarrays. ER-positive carcinomas were diagnosed in 120 (56.1%) samples of the younger patient group and in 92 (78.6%) samples of the older patient group. Of these ER-positive carcinomas, 48 (40%) from the younger patient group presented the subtype luminal A, compared with 53 (57.6%) from the older patient group (p=0.01). Tumors from the younger patient group were also associated with increased vascular involvement, co-expression of HER-2, and decreased expression of CD117. These results highlight differences in expression markers and the pathology of ER-positive tumors detected in younger women, with a notable characteristic being co-expression of HER-2. (C) 2010 Elsevier Ltd. All rights reserved.

The Snake Venom Peptide Bj-PRO-7a Is a M1 Muscarinic Acetylcholine Receptor Agonist

Proline-rich peptides from Bothrops jararaca venom (Bj-PRO) were characterized based on the capability to inhibit the somatic angiotensin-converting enzyme. The pharmacological action of these peptides resulted in the development of Captopril, one of the best examples of a target-driven drug discovery for treatment of hypertension. However, biochemical and biological properties of Bj-PROs were not completely elucidated yet, and many recent studies have suggested that their activity relies on angiotensin-converting enzyme-independent mechanisms. Here, we show that Bj-PRO-7a (receptors were also responsive to Bj-PRO-7a application, whereas no such response was observed in undifferentiated P19 cells not expressing muscarinic receptors. As further support for its specific action on M1 receptors, the peptide did not activate M3 subtypes in transfected CHO cells. Our findings provide a novel M1 muscarinic receptor agonist that could be used for basic research and even for pharmacological applications. (C) 2010 International Society for Advancement of Cytometry

Ligand induced interaction of thyroid hormone receptor beta with its coregulators

Thyroid hormones exert most of their physiological effects through two thyroid hormone receptor (TR) subtypes, TR alpha and TR beta, which associate with many transcriptional coregulators to mediate activation or repression of target genes. The search for selective TR beta ligands has been stimulated by the finding that several pharmacological actions mediated by TR beta might be beneficial in medical conditions such as obesity, hypercholesterolemia and diabetes. Here, we present a new methodology which employs surface plasmon resonance to investigate the interactions between TR beta ligand binding domain (LBD) complexes and peptides derived from the nuclear receptor interaction motifs of two of its coregulators, SRC2 and DAX1. The effect of several TR beta ligands, including the TR beta selective agonist GC-I and the TR beta selective antagonist NH-3, were investigated. We also determined the kinetic rate constants for the interaction of TR beta-T3 with both coregulators, and accessed the thermodynamic parameters for the interaction with DAX1. Our findings Suggest that flexibility plays an important role in the interaction between the receptor and its coregulators. and point out important aspects of experimental design that should be addressed when using TR beta LBD and its agonists. Furthermore, the methodology described here may be useful for the identification of new TR beta ligands. (C) 2008 Elsevier Ltd. All rights reserved.

Structure-Based Approach for the Study of Estrogen Receptor Binding Affinity and Subtype Selectivity

Estrogens exert important physiological effects through the modulation of two human estrogen receptor (hER) subtypes, alpa (hER alpha) and beta (hER beta). Because the levels and relative proportion of hER alpha and hER beta differ significantly in different target cells, selective hER ligands could target specific tissues or pathways regulated by one receptor subtype without affecting the other. To understand the structural and chemical basis by which small molecule modulators are able to discriminate between the two subtypes, we have applied three-dimensional target-based approaches employing a series of potent hER-ligands. Comparative molecular field analysis (CoMFA) studies were applied to a data set of 81 hER modulators, for which binding affinity values were collected for both hER alpha and hER beta. Significant statistical coefficients were obtained (hER alpha, q(2) = 0.76; hER beta, q(2) = 0.70), indicating the internal consistency of the models. The generated models were validated using external test sets, and the predicted values were in good agreement with the experimental results. Five hER crystal structures were used in GRID/PCA investigations to generate molecular interaction fields (MIF) maps. hER alpha and hER beta were separated using one factor. The resulting 3D information was integrated with the aim of revealing the most relevant structural features involved in hER subtype selectivity. The final QSAR and GRID/PCA models and the information gathered from 3D contour maps should be useful for the design or novel hER modulators with improved selectivity.

Alpha7 Nicotinic Acetylcholine Receptor Expression and Activity During Neuronal Differentiation of PC12 Pheochromocytoma Cells

Nicotinic acetylcholine receptors (nAChR) exert pivotal roles in synaptic transmission, neuroprotection and differentiation. Particularly, homomeric alpha 7 receptors participate in neurite outgrowth, presynaptic control of neurotransmitter release and Ca(2+) influx. However, the study of recombinant alpha 7 nAChRs in transfected cell lines is difficult due to low expression of functional receptor channels. We show that PC12 pheochromocytoma cells induced to differentiation into neurons are an adequate model for studying differential nAChR gene expression and receptor activity. Whole-cell current recording indicated that receptor responses increased during the course of differentiation. Transcription of mRNAs coding for alpha 3, alpha 5, alpha 7, beta 2 and beta 4 subunits was present during the course of differentiation, while mRNAs coding for alpha 2, alpha 4 and beta 3 subunits were not expressed in PC12 cells. alpha 7 subunit expression was highest following 1 day of induction to differentiation. Activity of alpha 7 nAChRs, however, was most elevated on day 2 as revealed by inhibition experiments in the presence of 10 nM methyllycaconitine, rapid current decay and receptor responsiveness to the alpha 7 agonist choline. Increased alpha 7 receptor activity was noted when PC12 were induced to differentiation in the presence of choline, confirming that chronic agonist treatment augments nAChR activity. In summary, PC12 cells are an adequate model to study the role and pharmacological properties of this receptor during neuronal differentiation.

Estrogen receptor: Structural differences and potential implications on selectivity examined by the GRID/CPCA approach

Selective Estrogen Receptor Modulators ( SERMs) have been developed, but the selectivity towards the subtypes ( ER or ER is not well understood. Based on three-dimensional structural properties of ligand binding domains, a model that takes into account this aspect was developed via molecular interaction fields and consensus principal component analysis (GRID/CPCA).

Influência do bloqueador de receptor de angiotensina (Losartana potássica) na função renal e pressão arterial em cães GRMD

A distrofia muscular de Duchenne (DMD) é uma alteração neuromuscular caracterizada por contínua necrose muscular e degeneração, com eventual fibrose e infiltração por tecido adiposo. O aumento progressivo da fibrose intersticial no músculo impede a migração das células miogênicas, necessárias para a formação muscular. O modelo canino constitui-se nas melhores fenocópias da doença em humanos, quando comparados com outros modelos animais com distrofia. O tratamento antifibrose de pacientes DMD, tendo como alvo os mediadores da citocina, TGF-beta, e o tratamento com antiinflamatórios, podem limitar a degeneração muscular e contribuir para a melhora do curso da doença. O presente estudo teve como objetivo observar os possíveis efeitos adversos na fisiologia renal, por meio de avaliação bioquímica sanguínea e da pressão arterial, verificando a viabilidade do uso do Losartan (um inibidor de TGF-beta) nos cães afetados pela distrofia muscular. Foram utilizados quatro cães adultos, sendo dois machos e duas fêmeas. Utilizou-se a dose de 50mg de Losartan, administrada via oral, uma vez ao dia. Os exames clínicos, bem como alterações na função renal, o nível do potássio sérico e a pressão arterial não evidenciaram reação adversa durante todo o período do experimento. O uso de Losartan, por um período de 9 semanas, mostrou-se como uma terapia segura para o tratamento antifibrótico em cães adultos, não afetando a função renal ou pressão arterial dos animais.

AT1-receptor mediated vascular damage in myocardium, kidneys and liver in rats

The systemic aspect of vascular damage induced by angiotensin II (ANG II) has been poorly explored in the literature. Considering the presence of ANG II and its specific receptor AT1, in several organs, all tissues might be potentially affected by its effects. The aims of this study were: To evaluate the early histological changes in the heart, liver and kidneys, produced by ANG II infusion, to evaluate the protective effect of losartan. Wistar rats were distributed into three groups: control (no treatment), treated with ANG II, and treated with ANG II + losartan. ANG II was continuously infused over 72 hours by subcutaneous osmotic pumps. Histological sections of the myocardium, kidneys and liver were stained and observed for the presence of necrosis. There were ANG II-induced perivascular inflammation and necrosis of the arteriolar wall in the myocardium, kidney, and liver by, which were partially prevented by losartan. There was no significant correlation between heart and kidney damage. Tissue lesion severity was lower than that of vascular lesions, without statistical difference between groups. ANG II causes vascular injury in the heart, kidneys and liver, indicating a systemic vasculotoxic effect; the mechanisms of damage/protection vary depending on the target organ; perivascular lesions may occur even when anti-hypertensive doses of losartan are used.

Toll-like receptor 4 (TLR4) expression in human and murine pancreatic beta-cells affects cell viability and insulin homeostasis

Background: Toll-like receptor 4 (TLR4) is widely recognized as an essential element in the triggering of innate immunity, binding pathogen-associated molecules such as Lipopolysaccharide (LPS), and in initiating a cascade of pro-inflammatory events. Evidence for TLR4 expression in non-immune cells, including pancreatic beta-cells, has been shown, but, the functional role of TLR4 in the physiology of human pancreatic beta-cells is still to be clearly established. We investigated whether TLR4 is present in beta-cells purified from freshly isolated human islets and confirmed the results using MIN6 mouse insulinoma cells, by analyzing the effects of TLR4 expression on cell viability and insulin homeostasis. Results: CD11b positive macrophages were practically absent from isolated human islets obtained from nondiabetic brain-dead donors, and TLR4 mRNA and cell surface expression were restricted to beta-cells. A significant loss of cell viability was observed in these beta-cells indicating a possible relationship with TLR4 expression. Monitoring gene expression in beta-cells exposed for 48h to the prototypical TLR4 ligand LPS showed a concentration-dependent increase in TLR4 and CD14 transcripts and decreased insulin content and secretion. TLR4-positive MIN6 cells were also LPS-responsive, increasing TLR4 and CD14 mRNA levels and decreasing cell viability and insulin content. Conclusions: Taken together, our data indicate a novel function for TLR4 as a molecule capable of altering homeostasis of pancreatic beta-cells.

Interleukin-22 forms dimers that are recognized by two interleukin-22R1 receptor chains

Interleukin-22 (IL-22) is a class 2 cytokine whose primary structure is similar to that of interleukin 10 (IL-10) and interferon-gamma (IFN-gamma). IL-22 induction during acute phase immune response indicates its involvement in mechanisms of inflammation. Structurally different from IL-10 and a number of other members of IL-10 family, which form intertwined inseparable V-shaped dimers of two identical polypeptide chains, a single polypeptide chain of IL-22 folds on itself in a relatively globular structure. Here we present evidence, based on native gel electrophoresis, glutaraldehyde cross-linking, dynamic light scattering, and small angle x-ray scattering experiments, that human IL-22 forms dimers and tetramers in solution under protein concentrations assessable by these experiments. Unexpectedly, low-resolution molecular shape of IL-22 dimers is strikingly similar to that of IL-10 and other intertwined cytokine dimeric forms. Furthermore, we determine an ab initio molecular shape of the IL-22/IL-22R1 complex which reveals the V-shaped IL-22 dimer interacting with two cognate IL-22R1 molecules. Based on this collective evidence, we argue that dimerization might be a common mechanism of all class 2 cytokines for the molecular recognition with their respective membrane receptor. We also speculate that the IL-22 tetramer formation could represent a way to store the cytokine in nonactive form at high concentrations that could be readily converted into functionally active monomers and dimers upon interaction with the cognate cellular receptors.

Structural basis of GC-1 selectivity for thyroid hormone receptor isoforms

Background: Thyroid receptors, TRa and TR beta, are involved in important physiological functions such as metabolism, cholesterol level and heart activities. Whereas metabolism increase and cholesterol level lowering could be achieved by TR beta isoform activation, TRa activation affects heart rates. Therefore, beta-selective thyromimetics have been developed as promising drug-candidates for treatment of obesity and elevated cholesterol level. GC-1 [ 3,5-dimethyl-4-(4'-hydroxy- 3'-isopropylbenzyl)-phenoxy acetic acid] has ability to lower LDL cholesterol with 600-to 1400-fold more potency and approximately two-to threefold more efficacy than atorvastatin (Lipitor(C)) in studies in rats, mice and monkeys. Results: To investigate GC-1 specificity, we solved crystal structures and performed molecular dynamics simulations of both isoforms complexed with GC-1. Crystal structures reveal that, in TRa Arg228 is observed in multiple conformations, an effect triggered by the differences in the interactions between GC-1 and Ser277 or the corresponding asparagine (Asn331) of TR beta. The corresponding Arg282 of TR beta is observed in only one single stable conformation, interacting effectively with the ligand. Molecular dynamics support this model: our simulations show that the multiple conformations can be observed for the Arg228 in TR alpha, in which the ligand interacts either strongly with the ligand or with the Ser277 residue. In contrast, a single stable Arg282 conformation is observed for TR beta, in which it strongly interacts with both GC-1 and the Asn331. Conclusion: Our analysis suggests that the key factors for GC-1 selectivity are the presence of an oxyacetic acid ester oxygen and the absence of the amino group relative to T(3). These results shed light into the beta-selectivity of GC-1 and may assist the development of new compounds with potential as drug candidates to the treatment of hypercholesterolemia and obesity.

Association of alpha1a-adrenergic receptor polymorphism and blood pressure phenotypes in the Brazilian population

Background: The alpha1A-adrenergic receptor (alpha(1A)-AR) regulates the cardiac and peripheral vascular system through sympathetic activation. Due to its important role in the regulation of vascular tone and blood pressure, we aimed to investigate the association between the Arg347Cys polymorphism in the alpha(1A)-AR gene and blood pressure phenotypes, in a large sample of Brazilians from an urban population. Methods: A total of 1568 individuals were randomly selected from the general population of the Vitoria City metropolitan area. Genetic analysis of the Arg347Cys polymorphism was conducted by polymerase chain reaction/restriction fragment length polymorphism. We have compared cardiovascular risk variables and genotypes using ANOVA, and Chi-square test for univariate comparisons and logistic regression for multivariate comparisons. Results: Association analysis indicated a significant difference between genotype groups with respect to diastolic blood pressure (p = 0.04), but not systolic blood pressure (p = 0.12). In addition, presence of the Cys/Cys genotype was marginally associated with hypertension in our population (p = 0.06). Significant interaction effects were observed between the studied genetic variant, age and physical activity. Presence of the Cys/Cys genotype was associated with hypertension only in individuals with regular physical activity (odds ratio = 1.86; p = 0.03) or younger than 45 years (odds ratio = 1.27; p = 0.04). Conclusion: Physical activity and age may potentially play a role by disclosing the effects of the Cys allele on blood pressure. According to our data it is possible that the Arg347Cys polymorphism can be used as a biomarker to disease risk in a selected group of individuals.