Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment.

Synaptic-vesicle exocytosis is mediated by the vesicular Ca(2+) sensor synaptotagmin-1. Synaptotagmin-1 interacts with the SNARE protein syntaxin-1A and acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP2). However, it is unclear how these interactions contribute to triggering membrane fusion. Using PC12 cells from Rattus norvegicus and artificial supported bilayers, we show that synaptotagmin-1 interacts with the polybasic linker region of syntaxin-1A independent of Ca(2+) through PIP2. This interaction allows both Ca(2+)-binding sites of synaptotagmin-1 to bind to phosphatidylserine in the vesicle membrane upon Ca(2+) triggering. We determined the crystal structure of the C2B domain of synaptotagmin-1 bound to phosphoserine, allowing development of a high-resolution model of synaptotagmin bridging two different membranes. Our results suggest that PIP2 clusters organized by syntaxin-1 act as molecular beacons for vesicle docking, with the subsequent Ca(2+) influx bringing the vesicle membrane close enough for membrane fusion.

Pathological Reorganization of NMDA Receptors Subunits and Postsynaptic Protein PSD-95 Distribution in Alzheimer's Disease.

In Alzheimer's disease (AD), synaptic alterations play a major role and are often correlated with cognitive changes. In order to better understand synaptic modifications, we compared alterations in NMDA receptors and postsynaptic protein PSD-95 expression in the entorhinal cortex (EC) and frontal cortex (FC; area 9) of AD and control brains. We combined immunohistochemical and image analysis methods to quantify on consecutive sections the distribution of PSD-95 and NMDA receptors GluN1, GluN2A and GluN2B in EC and FC from 25 AD and control cases. The density of stained receptors was analyzed using multivariate statistical methods to assess the effect of neurodegeneration. In both regions, the number of neuronal profiles immunostained for GluN1 receptors subunit and PSD-95 protein was significantly increased in AD compared to controls (3-6 fold), while the number of neuronal profiles stained for GluN2A and GluN2B receptors subunits was on the contrary decreased (3-4 fold). The increase in marked neuronal profiles was more prominent in a cortical band corresponding to layers 3 to 5 with large pyramidal cells. Neurons positive for GluN1 or PSD-95 staining were often found in the same localization on consecutive sections and they were also reactive for the anti-tau antibody AD2, indicating a neurodegenerative process. Differences in the density of immunoreactive puncta representing neuropile were not statistically significant. Altogether these data indicate that GluN1 and PSD-95 accumulate in the neuronal perikarya, but this is not the case for GluN2A and GluN2B, while the neuropile compartment is less subject to modifications. Thus, important variations in the pattern of distribution of the NMDA receptors subunits and PSD-95 represent a marker in AD and by impairing the neuronal network, contribute to functional deterioration.

Ampakine CX546 bolsters energetic response of astrocytes: a novel target for cognitive-enhancing drugs acting as alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor modulators.

Glutamate was previously shown to enhance aerobic glycolysis i.e. increase glucose utilization and lactate production with no change in oxygen levels, in mouse cortical astrocytes by a mechanism involving glutamate uptake. It is reported here that a similar response is produced in both hippocampal and cerebellar astrocytes. Application of the cognitive-enhancing drug CX546 promoted further enhancement of glucose utilization by astrocytes from each brain area following glutamate exposure. alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors represent the purported molecular target of cognitive-enhancing drugs such as CX546, and the presence of AMPA receptor subunits GluR1-4 was evidenced in astrocytes from all three regions by immunocytochemistry. AMPA itself did not stimulate aerobic glycolysis, but in the presence of CX546, a strong enhancement of glucose utilization and lactate production was obtained in cortical, hippocampal and cerebellar astrocytes. The effect of CX546 was concentration-dependent, with an EC(50) of 93.2 microm in cortical astrocytes. AMPA-induced glucose utilization in the presence of CX546 was prevented by the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the negative modulator GYKI 52466. In addition, the metabolic effect of CX546 in the presence of AMPA was mimicked by the AMPA receptor modulator cyclothiazide. Our data suggest that astrocyte energetics represents a novel target for cognitive-enhancing drugs acting as AMPA receptor modulators.

Positive and negative roles of the trans-acting T cell factor-1 for the acquisition of distinct Ly-49 MHC class I receptors by NK cells.

Members of the Ly-49 gene family code for class I MHC-specific receptors that regulate NK cell function. Due to a combinatorial distribution of Ly-49 receptors, NK cells display considerable clonal heterogeneity. The acquisition of one Ly-49 receptor, Ly-49A is strictly dependent on the transcriptional trans-acting factor T cell-specific factor-1 (TCF-1). Indeed, TCF-1 binds to two sites in the Ly-49a promoter and regulates its activity, suggesting that the Ly-49a gene is a direct TCF-1 target. TCF-1 deficiency resulted in the altered usage of additional Ly-49 receptors. We show in this study, using TCF-1 beta(2)-microglobulin double-deficient mice, that these repertoire alterations are not due to Ly-49/MHC class I interactions. Our findings rather suggest a TCF-1-dependent, cell autonomous effect on the acquisition of multiple Ly-49 receptors. Besides reduced receptor usage (Ly-49A and D), we also observed no effect (Ly-49C) and significantly expanded (Ly-49G and I) receptor usage in the absence of TCF-1. These effects did not in all cases correlate with the presence of TCF binding sites in the respective proximal promoter. Therefore, besides TCF-1 binding to the proximal promoter, Ly-49 acquisition may also be regulated by TCF-1 binding to more distant cis-acting elements and/or by regulating the expression of additional trans-acting factors. Consistent with the observed differential, positive or negative role of TCF-1 for Ly-49 receptor acquisition, reporter gene assays revealed the presence of an inducing as well as a repressing TCF site in certain proximal Ly-49 promoters. These findings reveal an important role of TCF-1 for the formation of the NK cell receptor repertoire.

Mechanisms of B cell survival induced by BAFF, APRIL and their receptors

RESUME : BAFF est un membre de 1a famille du TNF qui contrôle l'homéostasie des lymphocytes B. BAFF lie les récepteurs TACI, BCMA et BAFF-R sur les cellules B, tandis qu'APRIL, son proche homologue, lie seulement TACI et BCMA. BAFF et APRIL sont des protéines transmembranaires pouvant -être relâchées sous forme de cytokines trimériques solubles suite à un clivage protéolytique. Le BAFF soluble peut s'assembler en 60-mère. Les rôles physiologiques des BAFF membranaires et solubles sont inconnnus. Nous avons étudié la capacité de diverses formes de BAFF et APRIL à activer différents récepteurs. BAFF-R répond à toutes les formes dé BAFF, tandis que TACI nécessite du BAFF ou de l'APRIL membranaire ou oligomérisé pour être activé et pour transmettre des signaux de survie dans les lymphocytes B primaires. TACI ne répond pas aux ligands trimériques bien qu'il puisse les lier. TACI est essentiel pour la réponse humorale aux antigènes présentant des épitoges répétitifs, une réponse qui est indépendante des lymphocytes T (réponse TI-2). Des souris exprimant moins de BAFF ont un pourcentage modérément réduit de lymphocytes B et leur réponse TI-2 est atténuée. Par contre, des souris qui n'expriment que du BAFF membranaire ont encore moins de cellules B mais répondent efficacement aux antigènes TI-2. Ces résultats suggèrent que le BAFF soluble est impliqué dans le maintien de la population des lymphocytes B, alors que le BAFF membranaire peut activer TACI lors d'are réponse TI-2. Le BAFF 60-mère est un autre activateur potentiel de TACI in vivo. Le BAFF 60-mère existe dans des surnageants de cellules productrices de BAFF mais n'est pas détecté dans le plasma de souris saines, même lorsqu'elles présentent des niveaux élevés de BAFF. BAFF 60-mère est néanmoins présent dans le plasma de souris transgéniques pour BAFF et de souris déficientes en TACI. Comme ces deux lignées présentent des signes d'autoimmunité, ces résultats suggèrent que la présence de BAFF 60-mère pourrait être liée à des conditions pathologiques. Summary : The TNF family ligand BAFF is essential for B cell homeostasis. BAFF binds to the receptors TACI, BCMA and BAFF-R on B cells, whereas its close homolog APRIL binds to TACI and BCMA only. BAFF and APRIL are transmembrane proteins, which can be proteolytically processed to release trimeric soluble cytokines. Soluble BAFF 3-mer can further assemble in a 60-mer. The physiological roles of membrane-bound and soluble BAFF are unknown. We studied the ability of various forms of BAFF and APRIL to signal through different receptors. BAFF-R responded to all forms of BAFF, but TACI required membrane-bound, cross-licked or oligomeric BAFF or APRIL in order to transmit productive signals in primary B cells. TACI was unresponsive to trimeric ligands, although it could bind them. TACI is essential for T-cell independent antibody responses to antigens with repetitive epitopes (TI-2 responses). Mice expressing lower than normal levels of BAFF displayed a moderate B cell reduction and impaired TI-2 responses, whereas mice expressing membrane-bound BAFF displayed severe B cell reduction, but unimpaired TI-2 responses. These results suggest that processed BAFF is involved in the maintenance of the B cell pool and that membrane-bound BAFF can activate TACI during T-cell independent humoral responses. BAFF 60-mer is another potential activator of TACI in vivo. BAFF 60-mer was detected in the supernatant of BAFF-producing cells, but not in the plasma of healthy mice with either norma1 or elevated BAFF levels. It was however present in sera of BAFF transgenic mice and TACI-/- mice, both of which suffer from autoimmunity, suggesting that GAFF 60-mer may be linked to pathogenic conditions.

An exogenous mouse mammary tumor virus with properties of Mls-1a (Mtv-7).

The classical minor lymphocyte stimulating (Mls) antigens, which induce a strong primary T cell response in vitro, are closely linked to endogenous copies of mouse mammary tumor viruses (MMTV). Expression of Mls genes leads to clonal deletion of T cell subsets expressing specific T cell receptor (TCR) V beta chains. We describe the isolation and characterization of a new exogenous (infectious) MMTV with biological properties similar to the Mls antigen Mls-1a. In vivo administration of either Mls-1a-expressing B cells or the infectious MMTV (SW) led to an increase of T cells expressing V beta 6 followed by their deletion. Surprisingly, different kinetics of deletion were observed with the exogenous virus depending upon the route of infection. Infection through the mucosa led to a slow deletion of V beta 6+ T cells, whereas deletion was rapid after subcutaneous infection. Sequence analysis of the open reading frames in the 3' long terminal repeat of both this exogenous MMTV (SW) and of Mtv-7 (which is closely linked to Mls-1a) revealed striking similarities, particularly in the COOH terminus, which has been implicated in TCR V beta recognition. The identification of an infectious MMTV with the properties of a strong Mls antigen provides a new, powerful tool to study immunity and tolerance in vivo.

Epidermal growth factor receptors, testosterone levels and parotid gland changes in rats infected with Trypanosoma cruzi

It has been demonstrated that parotid glands of rats infected with Trypanosoma cruzi present severe histological alterations; changes include reduction in density and volume of the acini and duct systems and an increase in connective tissue. We evaluated the association between morphological changes in parotid glands, circulating testosterone levels and epidermal growth factor receptor (EGF-R) expression in experimental Chagas disease in rats. Animals at 18 days of infection (acute phase) showed a significant decrease in body weight, serum testosterone levels and EGF-R expression in the parotid gland compared with a control group. Since decreases in body weight could lead to a reduction in circulating testosterone concentration, we believe that the reduction in EGF-R expression in parotid glands of infected rats is due to alterations in testosterone levels and atrophy of parotid glands is caused by changes in EGF-R expression. Additionally, at 50 days (chronic phase) of infection parotid glands showed a normal histological aspect likely due to the normalization of the body weight. These findings suggest that the testosterone-EGF-R axis is involved in the histological changes.

Helical tomotherapy (HT) for the treatment of anal canal cancer: preliminary clinical results, and dosimetric comparison between HT and intensity-modulated or 3D conformal radiotherapy

Background: To report a single-center experience in 19 patients (pts) with anal canal cancer treated with helical tomotherapy (HT) and concurrent chemotherapy, and compare the dosimetric results with fixed-field intensitymodulated radiotherapy (IMRT) and 3D conformal radiotherapy (3D RT). Materials and Methods: Between 2007 and 2008, 19 consecutive pts were treated with HT and concurrent CT for anal canal cancer. Median age was 59 years (range, 38−83), and female/male ratio was 14/5. The majority of the pts had T2 or T3 tumours (68.4%), and 52.6% had positive lymph nodes. In all 19 pts, pelvic and inguinal nodes, and tumour irradiation was given using HT upto a median dose of 36 Gy (1.8 Gy/fr) followed by a 1-week gap. A boost dose of 23.4 Gy (1.8 Gy/fr) was delivered to the tumour and involved nodes using 3DRT (n = 12), HT (n = 6), or IMRT (n = 1). Simultaneous integrated boost was used in none of the pts. All but one patient with a T1N0 tumour received concomitant mitomycin/5- fluorouracil (n = 12) or mitomycin/capecitabin (n = 7) CT. Toxicity was scored according to the Common Terminology Criteria for Adverse Events (NCICTCAE v3.0). HT plans and treatments were generated using Tomotherapy, Inc., software and hardware; and 3D or IMRT boost plans with the CMS treatment planning system (TPS), using 6−18 MV photons from a Siemens Primus accelerator. For dosimetric comparison, computed tomography data sets of 10 pts were imported into the TPS, and 3D and 5-field step-andshoot IMRT plans were generated for each case. Plans were optimized with the aim of assessing organs at risk (OAR) and healthy-tissue sparing while enforcing highly conformal target coverage, and evaluated by dose-volume histograms (DVH) of planning target volumes (PTV) and OAR. Results: With a median follow-up of 13 months (range, 3−18), all pts are alive and well; except one patient developing local recurrence at 12 months. No patient developed grade 3 or more acute toxicity. No unplanned treatment interruption was necessary because of toxicity. With 360-degree-of-freedom beam projection, HT showed an advantage over 3D or IMRT plans in terms of dose conformity around the PTV, and dose gradients were steeper outside the PTV, resulting in reduced doses to OARs. Using HT, acute toxicity was acceptable, and seemed to be better than historical standards. Conclusion: We conclude that HT combined with concurrent chemotherapy for anal canal cancer is effective and tolerable. Compared to 3DRT or 5-field IMRT, there is better conformity around the PTV, and OAR sparing.

The alpha 1a and alpha 1b-adrenergic receptor subtypes: molecular mechanisms of receptor activation and of drug action.

In this chapter we summarize some aspects of the structure-functional relationship of the alpha 1a and alpha 1b-adrenergic receptor subtypes related to the receptor activation process as well as the effect of different alpha-blockers on the constitutive activity of the receptor. Molecular modeling of the alpha 1a and alpha 1b-adrenergic receptor subtypes and computational simulation of receptor dynamics were useful to interpret the experimental findings derived from site directed mutagenesis studies.

Analysis of the expression of toll-like receptors 2 and 4 and cytokine production during experimental Leishmania chagasi infection

Toll-like receptors (TLRs) recognise pathogen-derived molecules and influence immunity to control parasite infections. This study aimed to evaluate the mRNA expression of TLRs 2 and 4, the expression and production of the cytokines interleukin (IL)-12, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, IL-17, IL-10 and transforming growth factor (TGF)-β and the production of nitric oxide (NO) in the spleen of mice infected with Leishmania chagasi. It also aimed to evaluate any correlations between mRNA expression TLR2 and 4 and cytokines and NO production. Infection resulted in increased TLR2-4, IL-17, TNF-α and TGF-β mRNA expression during early infection, with decreased expression during late infection correlating with parasite load. IFN-γ and IL-12 mRNA expression decreased at the peak of parasitism. IL-10 mRNA expression increased throughout the entire time period analysed. Although TGF-β, TNF-α and IL-17 were highly produced during the initial phase of infection, IFN-γ and IL-12 exhibited high production during the final phase of infection. IL-10 and NO showed increased production throughout the evaluated time period. In the acute phase of infection, there was a positive correlation between TLR2-4, TNF-α, IL-17, NO, IL-10 and TGF-β expression and parasite load. During the chronic phase of infection, there was a positive correlation between TLR2-4, TNF-α, IL-17 and TGF-β expression and parasite load. Our data suggest that infection by L. chagasi resulted in modulation of TLRs 2 and 4 and cytokines.

Ulcerative colitis impairs the acylethanolamide-based anti-inflammatory system reversal by 5-aminosalicylic acid and glucocorticoids

Studies in animal models and humans suggest anti-inflammatory roles on the N acylethanolamide (NAE)-peroxisome proliferators activated receptor alpha (PPARα) system in inflammatory bowel diseases. However, the presence and function of NAE-PPARα signaling system in the ulcerative colitis (UC) of humans remain unknown as well as its response to active anti-inflammatory therapies such as 5-aminosalicylic acid (5-ASA) and glucocorticoids. Expression of PPARα receptor and PPARα ligands-biosynthetic (NAPE-PLD) and -degrading (FAAH and NAAA) enzymes were analyzed in untreated active and 5-ASA/glucocorticoids/immunomodulators-treated quiescent UC patients compared to healthy human colonic tissue by RT-PCR and immunohistochemical analyses. PPARα, NAAA, NAPE-PLD and FAAH showed differential distributions in the colonic epithelium, lamina propria, smooth muscle and enteric plexus. Gene expression analysis indicated a decrease of PPARα, PPARγ and NAAA, and an increase of FAAH and iNOS in the active colitis mucosa. Immunohistochemical expression in active colitis epithelium confirmed a PPARα decrease, but showed a sharp NAAA increase and a NAPE-PLD decrease, which were partially restored to control levels after treatment. We also characterized the immune cells of the UC mucosa infiltrate. We detected a decreased number of NAAA-positive and an increased number of FAAH-positive immune cells in active UC, which were partially restored to control levels after treatment. NAE-PPARα signaling system is impaired during active UC and 5-ASA/glucocorticoids treatment restored its normal expression. Since 5-ASA actions may work through PPARα and glucocorticoids through NAE-producing/degrading enzymes, the use of PPARα agonists or FAAH/NAAA blockers that increases endogenous PPARα ligands may yield similar therapeutics advantages.

Exendin-(9-39) is an inverse agonist of the murine glucagon-like peptide-1 receptor: implications for basal intracellular cyclic adenosine 3',5'-monophosphate levels and beta-cell glucose competence.

The effect of exendin-(9-39), a described antagonist of the glucagon-like peptide-1 (GLP-1) receptor, was evaluated on the formation of cAMP- and glucose-stimulated insulin secretion (GSIS) by the conditionally immortalized murine betaTC-Tet cells. These cells have a basal intracellular cAMP level that can be increased by GLP-1 with an EC50 of approximately 1 nM and can be decreased dose dependently by exendin-(9-39). This latter effect was receptor dependent, as a beta-cell line not expressing the GLP-1 receptor was not affected by exendin-(9-39). It was also not due to the endogenous production of GLP-1, because this effect was observed in the absence of detectable preproglucagon messenger RNA levels and radioimmunoassayable GLP-1. Importantly, GSIS was shown to be sensitive to this basal level of cAMP, as perifusion of betaTC-Tet cells in the presence of exendin-(9-39) strongly reduced insulin secretion. This reduction of GSIS, however, was observed only with growth-arrested, not proliferating, betaTC-Tet cells; it was also seen with nontransformed mouse beta-cells perifused in similar conditions. These data therefore demonstrated that 1) exendin-(9-39) is an inverse agonist of the murine GLP-1 receptor; 2) the decreased basal cAMP levels induced by this peptide inhibit the secretory response of betaTC-Tet cells and mouse pancreatic islets to glucose; 3) as this effect was observed only with growth-arrested cells, this indicates that the mechanism by which cAMP leads to potentiation of insulin secretion is different in proliferating and growth-arrested cells; and 4) the presence of the GLP-1 receptor, even in the absence of bound peptide, is important for maintaining elevated intracellular cAMP levels and, therefore, the glucose competence of the beta-cells.

5

Retinoid X Receptors (RXR) were initially identified as nuclear receptors binding with stereo-selectivity the vitamin A derivative 9-cis retinoic acid, although the relevance of this molecule as endogenous activator of RXRs is still elusive. Importantly, within the nuclear receptor superfamily, RXRs occupy a peculiar place, as they are obligatory partners for a number of other nuclear receptors, thus integrating the corresponding signaling pathways. In this chapter, we describe the structural features allowing RXR to form homo- and heterodimers, and the functional consequences of this unique ability. Furthermore, we discuss the importance of studying RXR activity at a genome-wide level in order to comprehensively address the biological implications of their action that is fundamental to understand to what extent RXRs could be exploited as new therapeutic targets.

Pharmacological blockade of either cannabinoid CB1 or CB2 receptors prevents both cocaine-induced conditioned locomotion and cocaine-induced reduction of cell proliferation in the hippocampus of adult male rat.

Addiction to major drugs of abuse, such as cocaine, has recently been linked to alterations in adult neurogenesis in the hippocampus. The endogenous cannabinoid system modulates this proliferative response as demonstrated by the finding that pharmacological activation/blockade of cannabinoid CB1 and CB2 receptors not only modulates neurogenesis but also modulates cell death in the brain. In the present study, we evaluated whether the endogenous cannabinoid system affects cocaine-induced alterations in cell proliferation. To this end, we examined whether pharmacological blockade of either CB1 (Rimonabant, 3 mg/kg) or CB2 receptors (AM630, 3 mg/kg) would affect cell proliferation [the cells were labeled with 5-bromo-2'-deoxyuridine (BrdU)] in the subventricular zone (SVZ) of the lateral ventricle and the dentate subgranular zone (SGZ). Additionally, we measured cell apoptosis (as monitored by the expression of cleaved caspase-3) and glial activation [by analyzing the expression of glial fibrillary acidic protein (GFAP) and Iba-1] in the striatum and hippocampus during acute and repeated (4 days) cocaine administration (20 mg/kg). The results showed that acute cocaine exposure decreased the number of BrdU-immunoreactive (ir) cells in the SVZ and SGZ. In contrast, repeated cocaine exposure reduced the number of BrdU-ir cells only in the SVZ. Both acute and repeated cocaine exposure increased the number of cleaved caspase-3-, GFAP- and Iba1-ir cells in the hippocampus, and this effect was counteracted by AM630 or Rimonabant, which increased the number of BrdU-, GFAP-, and Iba1-ir cells in the hippocampus. These results indicate that the changes in neurogenic, apoptotic and gliotic processes that were produced by repeated cocaine administration were normalized by pharmacological blockade of CB1 and CB2. The restorative effects of cannabinoid receptor blockade on hippocampal cell proliferation were associated with the prevention of the induction of conditioned locomotion but not with the prevention of cocaine-induced sensitization.

High level expression of human neuropeptide Y receptors in mammalian cells infected with a recombinant vaccinia virus.

Neuropeptide Y (NPY) is a 36 amino acid peptide present in the central and peripheral nervous system. Numerous studies point to a role of NPY in cardiovascular regulation. NPY effects are mediated through stimulation of specific cell surface G protein-coupled receptors. To allow biochemical studies of the receptor and of its interaction with the ligand, we have developed a potent expression system for NPY receptors using a recombinant vaccinia virus. A human NPY receptor cDNA was fused to a strong vaccinia virus promoter and inserted into the viral genome by homologous recombination. Recombinant viruses were isolated and tested for their ability to induce NPY binding site expression following infection of mammalian cell lines. Using saturation and competition binding experiments we measured a Bmax of 5-10 x 10(6) NPY binding sites per cell. The Kd for the binding of NPY is about 20 nM. Labelling of infected cells with a fluorochrome-labelled NPY indicated that the recombinant protein integrates into the cell membrane.