Two-dimensional QSAR studies on arylpiperazines as high-affinity 5-HT(1A) receptor ligands

5-HT(1A) receptor plays an important role in the delayed onset of antidepressant action of a class of selective serotonin reuptake inhibitors. Moreover, 5-HT(1A) receptor levels have been shown to be altered in patients suffering from major depression. In this work, hologram quantitative structure-activity relationship (HQSAR) studies were performed on a series of arylpiperazine compounds presenting affinity to the 5-HT(1A) receptor. The models were constructed with a training set of 70 compounds. The most significant HQSAR model (q(2) = 0.81, r(2) = 0.96) was generated using atoms, bonds, connections, chirality, and donor and acceptor as fragment distinction, with fragment size of 6-9. Predictions for an external test set containing 20 compounds are in good agreement with experimental results showing the robustness of the model. Additionally, useful information can be obtained from the 2D contribution maps.

Probing conformational changes in orphan nuclear receptor: The NGFI-B intermediate is a partially unfolded dimer

Human nerve growth factor-induced B (NGFI-B) is a member of the NR4A subfamily of orphan nuclear receptors (NRs). Lacking identified ligands, orphan NRs show particular co-regulator proteins binding properties, different from other NRs, and they might have a non-classical quaternary organization. A body of evidence suggests that NRs recognition of and binding to ligands, DNA, homo- and heterodimerization partners and co-regulator proteins involve significant conformational changes of the NR ligand-binding domains (LBDs). To shed light on largely unknown biophysical properties of NGFI-B, here we studied structural organization and unfolding properties of NGFI-B ligand (like)-binding domain induced by chemical perturbation. Our results show that NGFI-B LBD undergoes a two-state guanidine hydrochloride (GndHCl) induced denaturation, as judged by changes in the a-helical content of the protein monitored by circular dichroism spectroscopy (CD). In contrast, changes in the tertiary structure of NGFI-B LBD, reported by intrinsic fluorescence, reveal a clear intermediate state. Additionally, SAXS results demonstrate that the intermediate observed by intrinsic fluorescence is a partially folded homodimeric structure, which further unfolds without dissociation at higher GndHCl concentrations. This partially unfolded dimeric assembly of NGFI-B LBD might resemble an intermediate that this domain access momentarily in the native state upon interactions with functional partners. (C) 2008 Elsevier B.V. All rights reserved.

Expressão gênica de bcl2, receptor de estradiol e receptores de progesterona A e B em endométrio eutópico e ectópico de pacientes inférteis sem e com endometriose

A endometriose caracteriza-se pela presença de tecido endometriótico – glândulas e estroma – fora da cavidade uterina. A doença acomete superfícies peritoniais da pélvis, ovários e septo rectovaginal, sendo mais comum a endometriose pélvica. Embora os dados sobre a prevalência exata de endometriose sejam incertos, as evidências indicam uma prevalência em torno de 5 a 10% das mulheres em idade reprodutiva. Dor, dismenorréia, dispareunia e infertilidade são sintomas freqüentes da doença. Além de apresentar dependência aos hormônios sexuais, a endometriose está associada a um conjunto de desordens de diferentes etiologias, sendo considerada uma doença multifatorial de caráter genético e imune. Neste sentido, a carência de respostas imunológicas adequadas, aliada a um provável desequilíbrio entre os processos de proliferação e apoptose celular, pode ter um papel importante na instalação e manutenção das lesões endometrióticas. O objetivo deste trabalho foi caracterizar a expressão de genes relacionados aos processos de proliferação e diferenciação celular em implantes endometrióticos e endométrio eutópico de mulheres inférteis sem e com endometriose, através da análise semiquantitativa por RT-PCR. Foram estudadas 34 pacientes consultando por infertilidade e submetidas à laparoscopia diagnóstica. Quinze pacientes (31,7 ± 1,5 anos) apresentavam endometriose e 19 (32,9 ± 0,9 anos) foram consideradas controles inférteis sem endometriose. Os dois grupos apresentaram IMC e SHBG similares. As biópsias de endométrio foram realizadas, em fase folicular, nas pacientes do grupo controle (C) e nas pacientes com endometriose (endométrio eutópico (EUT) e ectópico (ECT)). A presença do mRNA para os receptores de estrogênio e progesterona detectada nos 3 grupos estudados, sustenta a existência de sensibilidade tecidual local aos hormônios sexuais. Nas condições experimentais do presente estudo não foi observada diferença estatística na expressão gênica de bcl2 entre os grupos. A expressão do gene do ER endometrial também foi similar entre os grupos controle, eutópico e ectópico. Contudo, o endométrio ectópico apresentou níveis de mRNA mais elevados para PR-A (0,84  0,02 UA) em relação aos grupos controle e eutópico (0,60  0,04 UA e 0,63  0,04 UA; p < 0,05). A expressão gênica do PR-B não foi estatisticamente diferente entre os grupos do presente trabalho. Porém, houve forte correlação negativa entre a expressão dos genes bcl2 e PR-B nas amostras de endométrio sem e com endometriose (r = - 0,795 e p = 0,018), sugerindo que um papel anti-proliferativo do PR-B possa ser operativo neste modelo de estudo.

Leukotriene B(4) is essential for selective eosinophil recruitment following allergen challenge of CD4(+) cells in a model of chronic eosinophilic inflammation

Subcutaneous heat-coagulated egg white implants (EWI) induce chronic, intense local eosinophilia in mice, followed by asthma-like responses to airway ovalbumin challenge. Our goal was to define the mechanisms of selective eosinophil accumulation in the EWI model. EWI carriers were challenged i.p. with ovalbumin and the contributions of cellular immunity and inflammatory mediators to the resulting leukocyte accumulation were defined through cell transfer and pharmacological inhibition protocols. Eosinophil recruitment required Major Histocompatibility Complex Class It expression, and was abolished by the leukotriene B4 (LTB4) receptor antagonist CP 105.696, the 5-lipoxygenase inhibitor BWA4C and the 5-lipoxygenase activating protein inhibitor MK886. Eosinophil recruitment in EWI carriers followed transfer of: a) CD4(+) (but not CD4(-)) cells, harvested from EWI donors and restimulated ex vivo; b) their cell-free supernatants, containing LTB4. Restimulation in the presence of MK886 was ineffective. CC chemokine receptor ligand (CCL)5 and CCL2 were induced by ovalbumin challenge in vivo. mRNA for CCL17 and CCL11 was induced in ovalbumin-restimulated CD4(+) cells ex vivo. MK886 blocked induction of CCL17 Pretreatment of EWI carriers with MK886 eliminated the effectiveness of exogenously administered CCL11, CCL2 and CCL5. In conclusion, chemokine-producing, ovalburnin-restimulated CD4(+) cells initiate eosinophil recruitment which is strictly dependent on LTB4 production. (C) 2008 Elsevier B.V. All rights reserved.

Regeneration of class III furcation defects with basic fibroblast growth factor (b-FGF) associated with GTR. A descriptive and histometric study in dogs

Background: the poor predictability of periodontal regenerative treatment of Class III furcation defects stimulates the study of alternatives to improve its results, such as the use of polypeptide growth factors. The objective of this study was to evaluate, both histologically and histometrically, the effects of topical application of basic fibroblast growth factor (b-FGF) associated with guided tissue regeneration (GTR) in the treatment of Class III defects surgically induced in dogs.Methods: All second and fourth premolars of 5 mongrel dogs were used and randomly assigned to one of three treatment groups: group 1 (control), treated with scaling and root planing, tetracycline hydrochloride (125 mg/ml) conditioning, and GTR with a collagen membrane; group 2, same treatment as group 1 plus 0.5 mg of b-FGF; group 3, same treatment as group 1 plus 1.0 mg of b-FGF. After a 90-day healing period, routine histologic processing and staining with hematoxylin and eosin and Masson trichrome were performed.Results: the descriptive analysis indicated better regenerative results in both groups treated with b-FGF while the histometric data, analyzed by means of analysis of variance (ANOVA), showed greater filling of the defects in group 2 in comparison to the defects in groups 3 and 1, respectively, which was represented by a smaller area of plaque-occupied space (P = 0.004) as well as a greater amount of newly formed cementum (P = 0.002).Conclusions: These results indicate that b-FGF, especially in smaller doses, may enhance the regenerative results in Class III furcation lesions, leading to greater filling of these defects with both mineralized and non-mineralized tissues.

A dominant function of p38 mitogen-activated protein kinase signaling in receptor activator of nuclear factor-kappa B ligand expression and osteoclastogenesis induction by Aggregatibacter actinomycetemcomitans and Escherichia coli lipopolysaccharide

Background and Objective: Lipopolysaccharide from gram-negative bacteria is one of the microbial-associated molecular patterns that initiate the immune/inflammatory response, leading to the tissue destruction observed in periodontitis. The aim of this study was to evaluate the role of the p38 mitogen-activated protein kinase (MAPK) signaling pathway in lipopolysaccharide-induced receptor activator of nuclear factor-kappa B ligand (RANKL) expression by murine periodontal ligament cells.Material and Methods: Expression of RANKL and osteoprotegerin mRNA was studied by reverse transcription-polymerase chain reaction upon stimulation with lipopolysaccharide from Escherichia coli and Aggregatibacter actinomycetemcomitans. The biochemical inhibitor SB203580 was used to evaluate the contribution of the p38 MAPK signaling pathway to lipopolysaccharide-induced RANKL and osteoprotegerin expression. Stable cell lines expressing dominant-negative forms of MAPK kinase (MKK)-3 and MKK6 were generated to confirm the role of the p38 MAPK pathway. An osteoclastogenesis assay using a coculture model of the murine monocytic cell line RAW 264.7 was used to determine if osteoclast differentiation induced by lipopolysaccharide-stimulated periodontal ligament was correlated with RANKL expression.Results: Inhibiting p38 MAPK prior to lipopolysaccharide stimulation resulted in a significant decrease of RANKL mRNA expression. Osteoprotegerin mRNA expression was not affected by lipopolysaccharide or p38 MAPK. Lipopolysaccharide-stimulated periodontal ligament cells increased osteoclast differentiation, an effect that was completely blocked by osteoprotegerin and significantly decreased by inhibition of MKK3 and MKK6, upstream activators of p38 MAPK. Conditioned medium from murine periodontal ligament cultures did not increase osteoclast differentiation, indicating that periodontal ligament cells produced membrane-bound RANKL.Conclusion: Lipopolysaccharide resulted in a significant increase of RANKL in periodontal ligament cells. The p38 MAPK pathway is required for lipopolysaccharide-induced membrane-bound RANKL expression in these cells.

Distinct localization of T cell Agrin during antigen presentation - evidence for the expression of Agrin receptor(s) in antigen-presenting cells

Agrin is over-expressed by activated and autoimmune T cells, and synergizes with the T cell receptor (TCR) to augment cell activation. In the present study, we show that Agrin accumulates to distinct areas of the plasma membrane and that cell activation causes its redistribution. During antigen presentation, Agrin primarily accumulates to the periphery of the mature immunological synapse, mostly in lamellipodia-like protrusions that wrap around the antigen-presenting cell and, conversely, anti-Agrin sera induced a significant redistribution of TCR at the plasma membrane. We also provide evidence for the expression of Agrin receptors in peripheral blood monocytes, dendritic cells and a fraction of B cells. Interestingly, interferon-a treatment, which induces the expression of Agrin in T cells, also augmented Agrin binding to monocytes. Stimulation of monocytes with recombinant Agrin induced the clustering of surface receptors, including major histocompatibility complex class II, activation of intracellular signalling cascades, as well as enhanced dsRNA-induced expression of pro-inflammatory cytokines interleukin-6 and tumour necrosis factor-a. Collectively, these results confirm the location of Agrin at the immunological synapse between T cells and antigen-presenting cells and justify further characterization of its receptors in the immune system.

Swimming training exacerbates pathological cardiac hypertrophy in kinin B(2) receptor-deficient mice

Kallikrein-kinin system exerts cardioprotective effects against pathological hypertrophy. These effects are modulated mainly via B(2) receptor activation. Chronic physical exercise can induce physiological cardiac hypertrophy characterized by normal organization of cardiac structure. Therefore, the aim of this work was to verify the influence of kinin B(2) receptor deletion on physiological hypertrophy to exercise stimulus. Animals were submitted to swimming practice for 5 min or for 60 min, 5 days a week, during 1 month and several cardiac parameters were evaluated. Results showed no significantly difference in heart weight between both groups, however an increased left ventricle weight and myocyte diameter were observed after the 60 min swimming protocol, which was more pronounced in B(2)(-/-) mice. In addition, sedentary B(2)(-/-) animals presented higher left ventricle mass when compared to wild-type (WT) mice. An increase in capillary density was observed in exercised animals, however the effect was less pronounced in B(2)(-/-) mice. Collagen, a marker of pathological hypertrophy, was increased in B(2)(-/-) mice submitted to swimming protocol, as well as left ventricular thickness, suggesting that these animals do not respond with physiological hypertrophy for this kind of exercise. In conclusion, our data suggest an important role for the kinin B(2) receptor in physiological cardiac hypertrophy. (c) 2007 Elsevier B.V. All rights reserved.

Marginal activity of progesterone receptor B (PR-B) in dogs but high incidence of mammary cancer

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Association of apolipoprotein B and adiponectin receptor 1 genes with carcass, bone integrity and performance traits in a paternal broiler line

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Porphyromonas gingivalis stimulates bone resorption by enhancing RANKL (Receptor Activator of NF-κB Ligand) through activation of toll-like receptor 2 in osteoblasts

Periodontitis has been associated with rheumatoid arthritis. In experimental arthritis, concomitant periodontitis caused by oral infection with Porphyromonas gingivalis enhances articular bone loss. The aim of this study was to investigate how lipopolysaccharide (LPS) from P. gingivalis stimulates bone resorption. The effects by LPS P. gingivalis and four other TLR2 ligands on bone resorption, osteoclast formation, and gene expression in wild type and Tlr2-deficient mice were assessed in ex vivo cultures of mouse parietal bones and in an in vivo model in which TLR2 agonists were injected subcutaneously over the skull bones. LPS P. gingivalis stimulated mineral release and matrix degradation in the parietal bone organ cultures by increasing differentiation and formation of mature osteoclasts, a response dependent on increased RANKL (receptor activator of NF-κB ligand). LPS P. gingivalis stimulated RANKL in parietal osteoblasts dependent on the presence of TLR2 and through a MyD88 and NF-κB-mediated mechanism. Similarly, the TLR2 agonists HKLM, FSL1, Pam2, and Pam3 stimulated RANKL in osteoblasts and parietal bone resorption. LPS P. gingivalis and Pam2 robustly enhanced osteoclast formation in periosteal/endosteal cell cultures by increasing RANKL. LPS P. gingivalis and Pam2 also up-regulated RANKL and osteoclastic genes in vivo, resulting in an increased number of periosteal osteoclasts and immense bone loss in wild type mice but not in Tlr2-deficient mice. These data demonstrate that LPS P. gingivalis stimulates periosteal osteoclast formation and bone resorption by stimulating RANKL in osteoblasts via TLR2. This effect might be important for periodontal bone loss and for the enhanced bone loss seen in rheumatoid arthritis patients with concomitant periodontal disease.

A transcriptional study in mice with different ethanol-drinking profiles: Possible involvement of the GABA(B) receptor

Previous studies have suggested that gamma-aminobutyric acid-B (GABA(B)) receptor agonists effectively reduce ethanol intake. The quantification using real-time polymerase chain reaction of Gabbr1 and Gabbr2 mRNA from the prefrontal cortex, hypothalamus, hippocampus, and striatum in mice exposed to an animal model of the addiction developed in our laboratory was performed to evaluate the involvement of the GABAB receptor in ethanol consumption. We used outbred, Swiss mice exposed to a three-bottle free-choice model (water, 5% v/v ethanol, and 10% v/v ethanol) that consisted of four phases: acquisition (AC), withdrawal (W), reexposure (RE), and quinine-adulteration (AD). Based on individual ethanol intake, the mice were classified into three groups: "addicted" (A group; preference for ethanol and persistent consumption during all phases), "heavy" (H group; preference for ethanol and a reduction in ethanol intake in the AD phase compared to AC phase), and "light" (L group; preference for water during all phases). In the prefrontal cortex in the A group, we found high Gabbr1 and Gabbr2 transcription levels, with significantly higher Gabbr1 transcription levels compared with the C (ethanol-naive control mice). L, and H groups. In the hippocampus in the A group, Gabbr2 mRNA levels were significantly lower compared with the C, L, and H groups. In the striatum, we found a significant increase in Gabbr1 transcription levels compared with the C, L, and H groups. No differences in Gabbr1 or Gabbr2 transcription levels were observed in the hypothalamus among groups. In summary, Gabbr1 and Gabbr2 transcription levels were altered in cerebral areas related to drug taking only in mice behaviorally classified as "addicted" drinkers, suggesting that these genes may contribute to high and persistent ethanol consumption. (C) 2012 Elsevier Inc. All rights reserved.

Inhibition of cannabinoid CB1 receptor upregulates Slc2a4 expression via nuclear factor-kappa B and sterol regulatory element-binding protein-1 in adipocytes

Evidences have suggested that the endocannabinoid system is overactive in obesity, resulting in enhanced endocannabinoid levels in both circulation and visceral adipose tissue. The blockade of cannabinoid receptor type 1 (CB1) has been proposed for the treatment of obesity. Besides loss of body weight, CB1 antagonism improves insulin sensitivity, in which the glucose transporter type 4 (GLUT4) plays a key role. The aim of this study was to investigate the modulation of GLUT4-encoded gene (Slc2a4 gene) expression by CB1 receptor. For this, 3T3-L1 adipocytes were incubated in the presence of a highly selective CB1 receptor agonist (1 mu M arachidonyl-2'-chloroethylamide) and/or a CB1 receptor antagonist/inverse agonist (0.1, 0.5, or 1 mu M AM251, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide). After acute (2 and 4 h) and chronic (24 h) treatments, cells were harvested to evaluate: i) Slc2a4, Cnr1 (CB1 receptor-encoded gene), and Srebf1 type a (SREBP-1a type-encoded gene) mRNAs (real-time PCR); ii) GLUT4 protein (western blotting); and iii) binding activity of nuclear factor (NF)-kappa B and sterol regulatory element-binding protein (SREBP)-1 specifically in the promoter of Slc2a4 gene (electrophoretic mobility shift assay). Results revealed that both acute and chronic CB1 receptor antagonism greatly increased (similar to 2.5-fold) Slc2a4 mRNA and protein content. Additionally, CB1-induced upregulation of Slc2a4 was accompanied by decreased binding activity of NF-kappa B at 2 and 24 h, and by increased binding activity of the SREBP-1 at 24 h. In conclusion, these findings reveal that the blockade of CB1 receptor markedly increases Slc2a4/GLUT4 expression in adipocytes, a feature that involves NF-kappa B and SREBP-1 transcriptional regulation. Journal of Molecular Endocrinology (2012) 49, 97-106