Calixarene and Resorcarene Based Receptors: From Structural and Thermodynamic Studies to the Synthesis of a New Mercury(II) Selective Material

Materials used in current technological approaches for the removal of mercury lack selectivity. Given that this is one of the main features of supramolecular chemistry, receptors based on calix[4]arene and calix[4]resorcarene containing functional groups able to interact selectively with polluting ions while discriminating against biologically essential ones were designed. Thus two receptors, a partially functionalized calix[4]arene derivative, namely, 5,11,17,23-tetra-tert-butyl [25-27-bis(diethyl thiophosphate amino)dihydroxy] calix[4]arene (1) and a fully functionalized calix[4]resorcarene, 4,6,10,12,16,18,22,24-diethyl thiophosphate calix[4]resorcarene (2) are introduced. Mercury(II) was the identified target due to the environmental and health problems associated with its presence in water Thus following the synthesis and characterization of 1 and 2 in solution ((1)HNMR) and in the solid state (X-ray crystallography) the sequence of experimental events leading to cation complexation studies in acetonitrile and methanol ((1)H NMR, conductance, potentiometric, and calorimetric measurements) with the aim of assessing their behavior as mercury selective receptors are described. The cation selectivity pattern observed in acetonitrile follows the sequence Hg(II) > Cu(II) > Ag(I). In methanol 1 is also selective for Hg(II) relative to Ag(I) but no interaction takes place between this receptor and Cu(II) in this solvent. Based on previous results and experimental facts shown in this paper, it is concluded that the complexation observed with Cu(II) in acetonitrile occurs through the acetonitrile-receptor adduct rather than through the free ligand. Receptor 2 has an enhanced capacity for uptaking Hg(II) but forms metalate complexes with Cu(II). These studies in solution guided the inmobilization of receptor 1 into a silica support to produce a new and recyclable material for the removal of Hg(II) from water. An assessment on its capacity to extract this cation from water relative to Cu(II) and Ag (I) shows that the cation selectivity pattern of the inmobilized receptor is the same as that observed for the free receptor in methanol. These findings demonstrate that fundamental studies play a critical role in the selection of the receptor to be attached to silicates as well as in the reaction medium used for the synthesis of the new decontaminating agent.

On the Denaturation Mechanisms of the Ligand Binding Domain of Thyroid Hormone Receptors

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.

Fibroblast growth factor 2 restrains ras-driven proliferation of malignant cells by triggering RhoA-mediated senescence

Fibroblast growth factor 2 (FGF2) is considered to be a bona fide oncogenic factor, although results from our group and others call this into question. Here, we report that exogenous recombinant FGF2 irreversibly inhibits proliferation by inducing senescence in Ras-dependent malignant mouse cells, but not in immortalized nontumorigenic cell lines. We report the following findings in K-Ras-dependent malignant YI adrenocortical cells and H-Ras V12-transformed BALB-3T3 fibroblasts: (a) FGF2 inhibits clonal growth and tumor onset in nude and immunocompetent BALB/c mice, (b) FGF2 irreversibly blocks the cell cycle, and (c) FGF2 induces the senescence-associated -galactosidase with no accompanying signs of apoptosis or necrosis. The tyrosine kinase inhibitor PD173074 completely protected malignant cells from FGF2. In Yl adrenal cells, reducing the constitutively high levels of K-Ras-GTP using the dominant-negative RasN17 mutant made cells resistant to FGF2 cytotoxicity. In addition, transfection of the dominant-negative RhoA-N19 into either YI or 3T3-B61 malignant cell lines yielded stable clonal transfectants that were unable to activate RhoA and were resistant to the FGF2 stress response. We conclude that in Rasdependent malignant cells, FGF2 interacts with its cognate receptors to trigger a senescence-like process involving RboAGTP. Surprisingly, attempts to select FGF2-resistant cells from the Yl and 3T3-B61 cell lines yielded only rare clones that (a) had lost the overexpressed ras oncogene, (b) were dependent on FGF2 for proliferation, and (c) were poorly tumorigenic. Thus, FGF2 exerted a strong negative selection that Rasdependent malignant cells could rarely overcome.

Actions of octocoral and tobacco cembranoids on nicotinic receptors

Nicotinic acetylcholine receptors (AChRs) are pentameric proteins that form agonist-gated cation channels through the plasma membrane. AChR agonists and antagonists are potential candidates for the treatment of neurodegenerative diseases. Cembranoids are naturally occurring diterpenoids that contain a 14-carbon ring. These diterpenoids interact with AChRs in complex ways: as irreversible inhibitors at the agonist sites, as noncompetitive inhibitors, or as positive modulators, but no cembranoid was ever shown to have agonistic activity on AChRs. The cembranoid eupalmerin acetate displays positive modulation of agonist-induced currents in the muscle-type AChR and in the related gamma-aminobutyric acid (GABA) type A receptor. Moreover, cembranoids display important biological effects, many of them mediated by nicotinic receptors. Cembranoids from tobacco are neuroprotective through a nicotinic anti-apoptotic mechanism preventing excitotoxic neuronal death which in part could result from anti-inflammatory properties of cembranoids. Moreover, tobacco cembranoids also have anti-inflammatory properties which could enhance their neuroprotective properties. Cembranoids from tobacco affect nicotine-related behavior: they increase the transient initial ataxia caused by first nicotine injection into naive rats and inhibit the expression of locomotor sensitization to repeated injections of nicotine. In addition, cembranoids are known to act as anti-tumor compounds. In conclusion, cembranoids provide a promising source of lead drugs for many clinical areas, including neuroprotection, smoking-cessation, and anti-cancer therapies. (C) 2009 Elsevier Ltd. All rights reserved.

Metabotropic glutamate receptors transduce signals for neurite outgrowth after binding of the prion protein to laminin gamma 1 chain

The prion protein (PrP(C)) is highly expressed in the nervous system, and its abnormal conformer is associated with prion diseases. PrP(C) is anchored to cell membranes by glycosylphosphatidylinositol, and transmembrane proteins are likely required for PrP(C)-mediated intracellular signaling. Binding of laminin (Ln) to PrP(C) modulates neuronal plasticity and memory. We addressed signaling pathways triggered by PrP(C)-Ln interaction in order to identify transmembrane proteins involved in the transduction of PrP(C)-Ln signals. The Ln gamma 1-chain peptide, which contains the Ln binding site for PrP(C), induced neuritogenesis through activation of phospholipase C (PLC), Ca(2+) mobilization from intracellular stores, and protein kinase C and extracellular signal-regulated kinase (ERK1/2) activation in primary cultures of neurons from wild-type, but not PrP(C)-null mice. Phage display, coimmunoprecipitation, and colocalization experiments showed that group I metabotropic glutamate receptors (mGluR1/5) associate with PrP(C). Expression of either mGluR1 or mGluR5 in HEK293 cells reconstituted the signaling pathways mediated by PrP(C)-Ln gamma 1 peptide interaction. Specific inhibitors of these receptors impaired PrP(C)-Ln gamma 1 peptide-induced signaling and neuritogenesis. These data show that group I mGluRs are involved in the transduction of cellular signals triggered by PrP(C)-Ln, and they support the notion that PrP(C) participates in the assembly of multiprotein complexes with physiological functions on neurons.-Beraldo, F. H., Arantes, C. P., Santos, T. G., Machado, C. F., Roffe, M., Hajj, G. N., Lee, K. S., Magalhaes, A. C., Caetano, F. A., Mancini, G. L., Lopes, M. H., Americo, T. A., Magdesian, M. H., Ferguson, S. S. G., Linden, R., Prado, M. A. M., Martins, V. R. Metabotropic glutamate receptors trans-duce signals for neurite outgrowth after binding of the prion protein to laminin gamma 1 chain. FASEB J. 25, 265-279 (2011). www.fasebj.org

Associação do receptor toll-like 2 com o estado pró-inflamatório do diabetes tipo 1

Inflammation has been pointed out as an important factor in development of chronic diseases, as diabetes. Hyperglycemia condition would be responsible by toll-like receptors, TLR2 and TLR4, and, consequently by local and systemic inflammation induction. Thus, the objective of present study was to evaluate type 1 Diabetes mellitus (T1DM) pro-inflammatory state through mRNA expression of TLRs 2 and 4 and proinflammatory cytokines IL-1β, IL-6 and TNF-α correlating to diabetic nephropathy. In order to achieve this objective, 76 T1DM patients and 100 normoglycemic (NG) subjects aged between 6 and 20 years were evaluated. T1DM subjects were evaluated as a total group DM1, and considering glycemic control (good glycemic control DM1G, and poor glycemic control DM1P) and considering time of diagnosis (before achieving 5 years of diagnosis DM1< 5yrs, and after achieving 5 years of diagnosis DM1 <5yrs). Metabolic control was evaluated by glucose and glycated hemoglobin concentrations; to assess renal function serum urea, creatinine, albumin, total protein and urinary albumin-to-creatinine ratio were determined and to evaluate hepatic function, AST and ALT serum activities were measured. Pro-inflammatory status was assessed by mRNA expression of TLRs 2 and 4 and the inflammatory cytokines IL-1β, IL-6 and TNF-α. Except for DM1G group (18.4%), DM1NC patients (81.6%) showed a poor glycemic control, with glycated hemoglobin (11,2%) and serum glucose (225,5 md/dL) concentrations significantly increased in relation to NG group (glucose: 76,5mg/dL and glycated hemoglobin: 6,9%). Significantly enhanced values of urea (20%) and ACR (20,8%) and diminished concentrations of albumin (5,7%) and total protein (13,6%) were found in T1DM patients, mainly associated to a poor glycemic control (DM1P increased values of urea: 20% and ACR:49%, and diminished of albumin: 13,6% and total protein:13,6%) and longer disease duration (DM1 <5yrs - increased values of urea: 20% and ACR:20,8%, and diminished of albumin: 14,3% and total protein:13,6%). As regarding pro-inflammatory status evaluation, significantly increased mRNA expressions were presented for TLR2 (37,5%), IL-1β (43%), IL-6 (44,4%) and TNF-α (15,6%) in T1DM patients in comparison to NG, mainly associated to DM1P (poor glycemic control TLR2: 82%, IL-1β: 36,8% increase) and DM1 <5yrs (longer time of diagnosis TLR2: 85,4%, IL-1β: 46,5% increased) groups. Results support the existence of an inflammatory state mediated by an increased expression of TLR2 and pro-inflammatory cytokines IL-1β, IL-6 and TNF-α in T1DM

Involvement of serotoninergic receptors in the anteroventral preoptic region on hypoxia-induced hypothermia

Hypoxia causes a regulated decrease in body temperature (Tb). There is circumstantial evidence that the neurotransmitter serotonin (5-HT) in the anteroventral preoptic region (AVPO) mediates this response. However, which 5-HT receptor(s) is (are) involved in this response has not been assessed. Thus, we investigated the participation of the 5-HT receptors (5-HT(1), 5-HT(2), and 5-HT(7)) in the AVPO in hypoxic hypothermia. To this end, Tb of conscious Wistar rats was monitored by biotelemetry before and after intra-AVPO microinjection of methysergide (a 5-HT(1) and 5-HT(2) receptor antagonist, 0.2 and 2 mu g/100 nL), WAY-100635 (a 5-HT(1A) receptor antagonist, 0.3 and 3 mu g/100 nL), and SB-269970 (a 5-HT(7) receptor antagonist, 0.4 and 4 mu/100 nL), followed by 60 min of hypoxia exposure (7% O(2)). During the experiments, the mean chamber temperature was 24.6 +/- 0.7 degrees C (mean +/- SE) and the mean room temperature was 23.5 +/- 0.8 degrees C (mean +/- SE). Intra-AVPO microinjection of vehicle or 5-HT antagonists did not change Tb during normoxic conditions. Exposure of rats to 7% of inspired oxygen evoked typical hypoxia-induced hypothermia after vehicle microinjection, which was not affected by both doses of methysergide. However, WAY-100635 and SB-269970 treatment attenuated the drop in Tb in response to hypoxia. The effect was more pronounced with the 5-HT7 antagonist since both doses (0.4 and 4 mu g/0.1 mu L) were capable of attenuating the hypothermic response. As to the 5-HT(1A) antagonist, the attenuation of hypoxia-induced hypothermia was only observed at the higher dose. Therefore, the present results are consistent with the notion that 5-HT acts on both 5-HT(1A) and 5-HT7 receptors in the AVPO to induce hypothermia, during hypoxia. (c) 2005 Elsevier B.V All rights reserved.

Serotoninergic receptors in the anteroventral preoptic region modulate the hypoxic ventilatory response

Hypothalamus is a site of integration of the hypoxic and thermal stimuli on breathing and there is evidence that serotonin (5-HT) receptors in the anteroventral preoptic region (AVPO) mediate hypoxic hypothermia. Once 5-HT is involved in the hypoxic ventilatory response (HVR), we investigated the participation of the 5-HT receptors (5-HT1, 5-HT2 and 5-HT7) in the AVPO in the HVR. To this end, pulmonary ventilation (V-E) of rats was measured before and after intra-AVPO microinjection of methysergide (a 5-HT1 and 5-HT2 receptor antagonist), WAY-100635 (a 5-HT1A receptor antagonist) and SB-269970 (a 5-HT7 receptor antagonist), followed by 60 min of hypoxia exposure (7% O-2). Intra-AVPO microinjection of vehicles or 5-HT antagonists did not change VE during normoxic conditions. Exposure of rats to 7% O-2 evoked typical hypoxia-induced hyperpnea after vehicle microinjection, which was not affected by methysergide. WAY-100635 and SB-269970 treatment caused an increased HVR, due to a higher tidal volume. Therefore, the current data provide the evidence that 5-HT acting on 5-HT1A and 5-HT7 receptors in the AVPO exert an inhibitory modulation on the HVR. (c) 2005 Elsevier B.V. All rights reserved.

Ionotropic glutamatergic receptors in the rostral medullary raphe modulate hypoxia and hypercapnia-induced hyperpnea

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

Role of preoptic opioid receptors in the body temperature reduction during hypoxia

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

Involvement of median raphe nucleus 5-HT1A receptors in the regulation of generalized anxiety-related defensive behaviours in rats

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

Prostaglandin E-2 and the Suppression of Phagocyte Innate Immune Responses in Different Organs

The local and systemic production of prostaglandin E-2 (PGE(2)) and its actions in phagocytes lead to immunosuppressive conditions. PGE2 is produced at high levels during inflammation, and its suppressive effects are caused by the ligation of the E prostanoid receptors EP2 and EP4, which results in the production of cyclic AMP. However, PGE(2) also exhibits immunostimulatory properties due to binding to EP3, which results in decreased cAMP levels. The various guanine nucleotide-binding proteins (G proteins) that are coupled to the different EP receptors account for the pleiotropic roles of PGE(2) in different disease states. Here, we discuss the production of PGE(2) and the actions of this prostanoid in phagocytes from different tissues, the relative contribution of PGE(2) to the modulation of innate immune responses, and the novel therapeutic opportunities that can be used to control inflammatory responses.

Involvement of the midbrain periaqueductal gray 5-HT1A receptors in social conflict induced analgesia in mice

Recent results from our laboratory have shown that 30-bites social conflict in mice produces a high-intensity, short-term analgesia which is attenuated by systemically injected 5-HT1A receptor agonists, such as BAY R 1531 (6-methoxy-4-(di-n-propylamino)-1,3,4,5-tetrahydrobenz(c,d)indole hydrochloride) and gepirone. The present study investigated the effects of these drugs, as well as the 5-HT1A receptor antagonist WAY 100135 (N-tert-butyl-3-(4-(2-methoxyphenyl)piperazine-1-yl)-2-phenylpropanamide) injected into the midbrain periaqueductal gray matter of mice on 30-bites analgesia. Four to five days after guide-cannula implantation, each mouse received microinjection of gepirone (30 nmol/0.2 mu l), BAY R 1531 (10 nmol/0.2 mu l), WAY 100135 (10 nmol/0.2 mu l), saline (0.9% NaCl) or vehicle (saline + 4% Tween 80) 5 min before either an aggressive (30 bites) or a non-aggressive interaction. Nociception was assessed by the tail-flick test made before as well as 1, 5, 10 and 20 min after social interaction. The full 5-HT1A receptor agonist BAY R 1531 blocked, whereas, WAY 100135 and gepirone intensified 30-bites analgesia, Neither non-aggressive interaction, per se, nor the three compounds given after this type of social interaction significantly changed nociception. These results indicate that 5-HT1A receptors in the periaqueductal gray inhibit analgesia induced by social conflict in mice. (C) 1998 Elsevier B.V. B.V.

Activation of 5-HT2C receptors in the dorsal periaqueductal gray increases antinociception in mice exposed to the elevated plus-maze

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Blockade of 5-HT2 receptors in the periaqueductal grey matter (PAG) abolishes the anxiolytic-like effect of 5-HT1A receptor antagonism in the median raphe nucleus in mice

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