Insulin temporal sensitivity and its signaling pathway in the rat pineal gland

Aims: In our previous work, we reported that the insulin potentiating effect on melatonin synthesis is regulated by a post-transcriptional mechanism. However, the major proteins of the insulin signaling pathway (ISP) and the possible pathway component recruited on the potentiating effect of insulin had not been characterized. A second question raised was whether windows of sensitivity to insulin exist in the pineal gland due to insulin rhythmic secretion pattern. Main methods: Melatonin content from norepinephrine(NE)-synchronized pineal gland cultures was quantified by high performance liquid chromatography with electrochemical detection and arylalkylamine-N-acetyltransferase (AANAT) activity was assayed by radiometry. Immunoblotting and immunoprecipitation techniques were performed to establish the ISP proteins expression and the formation of 14-3-3: AANAT complex, respectively. Key findings: The temporal insulin susceptibility protocol revealed two periods of insulin potentiating effect, one at the beginning and another one at the end of the in vitro induced ""night"". In some Timed-insulin Stimulation (TSs), insulin also promoted a reduction on melatonin synthesis, showing its dual action in cultured pineal glands. The major ISP components, such as IR beta, IGF-1R, IRS-1, IRS-2 and PI3K(p85), as well tyrosine phosphorylation of pp85 were characterized within pineal glands. Insulin is not involved in the 14-3-3:AANAT complex formation. The blockage of PI3K by LY 294002 reduced melatonin synthesis and AANAT activity. Significance: The present study demonstrated windows of differential insulin sensitivity, a functional ISP and the PI3K-dependent insulin potentiating effect on NE-mediated melatonin synthesis, supporting the hypothesis of a crosstalk between noradrenergic and insulin pathways in the rat pineal gland. (C) 2010 Elsevier Inc. All rights reserved.

Visual signaling and reproductive biology in a nocturnal treefrog, genus Hyla (Anura: Hylidae)

We investigated reproductive activity, courtship behavior, call structure, body size, clutch size, oviposition site, and reproductive mode of Hyla sp. (aff. ehrhardti). Males called in all months, but showed a peak of activity during the rainy season. Three pair formations were observed and courtship involved stereotyped behavioral sequences, including visual signaling. Males emitted three different vocalization types: advertisement calls, courtship calls, and a vocalization of unknown function. Females attained larger body sizes than males and deposited an average of 74.5 unpigmented eggs per clutch. Early larval stages are aquatic but restricted to water in constructed subterranean nests; subsequent to flooding of nests, exotrophic tadpoles live in ponds or streams. Courtship behavior in Hyla sp. (aff. ehrhardti) is complex and the diverse repertoire of limb movements, exhibited by males, probably provide visual stimuli to females in this nocturnal treefrog. Hyla sp. (aff. ehrhardti) belongs to the H. albomarginata group. Considering the reproductive modes in this group, the complexes of H. albosignata and of H. albofrenata can be considered more close related to each other than to the H. albomarginata complex. © Koninklijke Brill NV, Leiden, 2004.

Signaling Pathways Activation by Primary Endodontic Infectious Contents and Production of Inflammatory Mediators

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

Structure of importin-a from a filamentous fungus in complex with a classical nuclear localization signal

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

Characterization of the procera Tomato Mutant Shows Novel Functions of the SlDELLA Protein in the Control of Flower Morphology, Cell Division and Expansion, and the Auxin-Signaling Pathway during Fruit-Set and Development

procera (pro) is a tall tomato (Solanum lycopersicum) mutant carrying a point mutation in the GRAS region of the gene encoding SlDELLA, a repressor in the gibberellin (GA) signaling pathway. Consistent with the SlDELLA loss of function, pro plants display a GA-constitutive response phenotype, mimicking wild-type plants treated with GA(3). The ovaries from both nonemasculated and emasculated pro flowers had very strong parthenocarpic capacity, associated with enhanced growth of preanthesis ovaries due to more and larger cells. pro parthenocarpy is facultative because seeded fruits were obtained by manual pollination. Most pro pistils had exserted stigmas, thus preventing self-pollination, similar to wild-type pistils treated with GA(3) or auxins. However, Style2.1, a gene responsible for long styles in noncultivated tomato, may not control the enhanced style elongation of pro pistils, because its expression was not higher in pro styles and did not increase upon GA(3) application. Interestingly, a high percentage of pro flowers had meristic alterations, with one additional petal, sepal, stamen, and carpel at each of the four whorls, respectively, thus unveiling a role of SlDELLA in flower organ development. Microarray analysis showed significant changes in the transcriptome of preanthesis pro ovaries compared with the wild type, indicating that the molecular mechanism underlying the parthenocarpic capacity of pro is complex and that it is mainly associated with changes in the expression of genes involved in GA and auxin pathways. Interestingly, it was found that GA activity modulates the expression of cell division and expansion genes and an auxin signaling gene (tomato AUXIN RESPONSE FACTOR7) during fruit-set.

The IgA1 immune complex-mediated activation of the MAPK/ERK kinase pathway in mesangial cells is associated with glomerular damage in IgA nephropathy

IgA nephropathy (IgAN), the most common primary glomerulonephritis worldwide, has significant morbidity and mortality as 20-40% of patients progress to end-stage renal disease within 20 years of onset. In order to gain insight into the molecular mechanisms involved in the progression of IgAN, we systematically evaluated renal biopsies from such patients. This showed that the MAPK/ERK signaling pathway was activated in the mesangium of patients presenting with over 1 g/day proteinuria and elevated blood pressure, but absent in biopsy specimens of patients with IgAN and modest proteinuria (<1 g/day). ERK activation was not associated with elevated galactose-deficient IgA1 or IgG specific for galactose-deficient IgA1 in the serum. In human mesangial cells in vitro, ERK activation through mesangial IgA1 receptor (CD71) controlled pro-inflammatory cytokine secretion and was induced by large-molecular-mass IgA1-containing circulating immune complexes purified from patient sera. Moreover, IgA1-dependent ERK activation required renin-angiotensin system as its blockade was efficient in reducing proteinuria in those patients exhibiting substantial mesangial activation of ERK. Thus, ERK activation alters mesangial cell-podocyte crosstalk, leading to renal dysfunction in IgAN. Assessment of MAPK/ERK activation in diagnostic renal biopsies may predict the therapeutic efficacy of renin-angiotensin system blockers in IgAN. Kidney International (2012) 82, 1284-1296; doi:10.1038/ki.2012.192; published online 5 September 2012

Signaling Events During Cyclic Guanosine Monophosphate-Regulated Pigment Aggregation in Freshwater Shrimp Chromatophores

Crustacean color change results partly from granule aggregation induced by red pigment concentrating hormone (RPCH). In shrimp chromatophores, both the cyclic GMP (3', 5'-guanosine monophosphate) and Ca2+ cascades mediate pigment aggregation. However, the signaling elements upstream and downstream from cGMP synthesis by GC-S (cytosolic guanylyl cyclase) remain obscure. We investigate post-RPCH binding events in perfused red ovarian chromatophores to disclose the steps modulating cGMP concentration, which regulates granule translocation. The inhibition of calcium/calmodulin complex (Ca2+/CaM) by N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W7) induces spontaneous aggregation but inhibits RPCH-triggered aggregation, suggesting a role in pigment aggregation and dispersion. Nitric oxide synthase inhibition by N omega-nitro-L-arginine methyl ester hydrochloride (L-NAME) strongly diminishes RPCH-induced aggregation; protein kinase G inhibition (by rp-cGMPs-triethylamine) reduces RPCH-triggered aggregation and provokes spontaneous dispersion, disclosing NO/PKG participation in aggregation signaling. Myosin light chain phosphatase inhibition (by cantharidin) accelerates RPCH-triggered aggregation, whereas Rho-associated protein kinase inhibition (by Y-27632, H-11522) reduces RPCH-induced aggregation and accelerates dispersion. MLCP (myosin light chain kinase) and ROCK (Rho-associated protein kinase) may antagonistically regulate myosin light chain (MLC) dephosphorylation/phosphorylation during pigment dispersion/aggregation. We propose the following general hypothesis for the cGMP/Ca2+ cascades that regulate pigment aggregation in crustacean chromatophores: RPCH binding increases Ca2+ (int), activating the Ca2+/CaM complex, releasing NOS-produced nitric oxide, and causing GC-S to synthesize cGMP that activates PKG, which phosphorylates an MLC activation site. Myosin motor activity is initiated by phosphorylation of an MLC regulatory site by ROCK activity and terminated by MLCP-mediated dephosphorylation. Qualitative comparison reveals that this signaling pathway is conserved in vertebrate and invertebrate chromatophores alike.

Extensive cross-talk and global regulators identified from an analysis of the integrated transcriptional and signaling network in Escherichia coli

To understand the regulatory dynamics of transcription factors (TFs) and their interplay with other cellular components we have integrated transcriptional, protein-protein and the allosteric or equivalent interactions which mediate the physiological activity of TFs in Escherichia coli. To study this integrated network we computed a set of network measurements followed by principal component analysis (PCA), investigated the correlations between network structure and dynamics, and carried out a procedure for motif detection. In particular, we show that outliers identified in the integrated network based on their network properties correspond to previously characterized global transcriptional regulators. Furthermore, outliers are highly and widely expressed across conditions, thus supporting their global nature in controlling many genes in the cell. Motifs revealed that TFs not only interact physically with each other but also obtain feedback from signals delivered by signaling proteins supporting the extensive cross-talk between different types of networks. Our analysis can lead to the development of a general framework for detecting and understanding global regulatory factors in regulatory networks and reinforces the importance of integrating multiple types of interactions in underpinning the interrelationships between them.

Curcumin requires tumor necrosis Factor α signaling to alleviate cognitive impairment elicited by Lipopolysaccharide

A decline in cognitive ability is a typical feature of the normal aging process, and of neurodegenerative disorders such as Alzheimer’s, Parkinson’s and Huntington’s diseases. Although their etiologies differ, all of these disorders involve local activation of innate immune pathways and associated inflammatory cytokines. However, clinical trials of anti-inflammatory agents in neurodegenerative disorders have been disappointing, and it is therefore necessary to better understand the complex roles of the inflammatory process in neurological dysfunction. The dietary phytochemical curcumin can exert anti-inflammatory, antioxidant and neuroprotective actions. Here we provide evidence that curcumin ameliorates cognitive deficits associated with activation of the innate immune response by mechanisms requiring functional tumor necrosis factor α receptor 2 (TNFR2) signaling. In vivo, the ability of curcumin to counteract hippocampusdependent spatial memory deficits, to stimulate neuroprotective mechanisms such as upregulation of BDNF, to decrease glutaminase levels, and to modulate N-methyl- D –aspartate receptor levels was absent in mice lacking functional TNFRs. Curcumin treatment protected cultured neurons against glutamate-induced excitotoxicity by a mechanism requiring TNFR2 activation. Our results suggest the possibility that therapeutic approaches against cognitive decline designed to selectively enhance TNFR2 signaling are likely to be more beneficial than the use of anti-inflammatory drugs per se.

Modulation of epidermial growth factor receptor signaling in colon adenocarcinoma

The use of agents targeting EGFR represents a new frontier in colon cancer therapy. Among these, monoclonal antibodies (mAbs) and EGFR tyrosine kinase inhibitors (TKIs) seemed to be the most promising. However they have demonstrated low utility in therapy, the former being effective at toxic doses, the latter resulting inefficient in colon cancer. This thesis work presents studies on a new EGFR inhibitor, FR18, a molecule containing the same naphtoquinone core as shikonin, an agent with great anti-tumor potential. In HT-29, a human colon carcinoma cell line, flow cytometry, immunoprecipitation, and Western blot analysis, confocal spectral microscopy have demonstrated that FR18 is active at concentrations as low as 10 nM, inhibits EGF binding to EGFR while leaving unperturbed the receptor kinase activity. At concentration ranging from 30 nM to 5 μM, it activates apoptosis. FR18 seems therefore to have possible therapeutic applications in colon cancer. In addition, surface plasmon resonance (SPR) investigation of the direct EGF/EGFR complex interaction using different experimental approaches is presented. A commercially available purified EGFR was immobilised by amine coupling chemistry on SPR sensor chip and its interaction to EGF resulted to have a KD = 368 ± 0.65 nM. SPR technology allows the study of biomolecular interactions in real-time and label-free with a high degree of sensitivity and specificity and thus represents an important tool for drug discovery studies. On the other hand EGF/EGFR complex interaction represents a challenging but important system that can lead to significant general knowledge about receptor-ligand interactions, and the design of new drugs intended to interfere with EGFR binding activity.

Insulin-like growth factor-I treatment in primary growth hormone insensitivity: effect of recombinant human IGF-I (rhIGF-I) and rhIGF-I/rhIGF-binding protein-3 complex

Growth hormone insensitivity syndrome (GHIS) is a rare cause of growth retardation characterized by high serum GH levels, and low serum insulin-like growth factor I (IGF-I) levels associated with a genetic defect of the GH receptor (GHR) as well post-GHR signaling pathway. Based on clinical, as well as biochemical characteristics, GHIS can be genetically classified as classical/Laron's syndrome and nonclassical/atypical GHIS. Recombinant human IGF-I (rhIGF-I) treatment is effective in promoting growth in subjects who have GHIS. Further, pharmacological studies of a IGF-I compound containing a 1:1 molar complex of rhIGF-I and rhIGF-binding protein-3 (BP-3) demonstrated that the complex was effective in increasing levels of circulating total and free IGF-I and that the administration in patients with GHIS should be safe, well-tolerated and more effective than rhIGF-I on its own.

Regulation of GPCR signaling in hypertension

Hypertension represents a complex, multifactorial disease and contributes to the major causes of morbidity and mortality in industrialized countries: ischemic and hypertensive heart disease, stroke, peripheral atherosclerosis and renal failure. Current pharmacological therapy of essential hypertension focuses on the regulation of vascular resistance by inhibition of hormones such as catecholamines and angiotensin II, blocking them from receptor activation. Interaction of G-protein coupled receptor kinases (GRKs) and regulator of G-protein signaling (RGS) proteins with activated G-protein coupled receptors (GPCRs) effect the phosphorylation state of the receptor leading to desensitization and can profoundly impair signaling. Defects in GPCR regulation via these modulators have severe consequences affecting GPCR-stimulated biological responses in pathological situations such as hypertension, since they fine-tune and balance the major transmitters of vessel constriction versus dilatation, thus representing valuable new targets for anti-hypertensive therapeutic strategies. Elevated levels of GRKs are associated with human hypertensive disease and are relevant modulators of blood pressure in animal models of hypertension. This implies therapeutic perspective in a disease that has a prevalence of 65million in the United States while being directly correlated with occurrence of major adverse cardiac and vascular events. Therefore, therapeutic approaches using the inhibition of GRKs to regulate GPCRs are intriguing novel targets for treatment of hypertension and heart failure.

Cancer therapy modulates VEGF signaling and viability in adult rat cardiac microvascular endothelial cells and cardiomyocytes

This work was motivated by the incomplete characterization of the role of vascular endothelial growth factor-A (VEGF-A) in the stressed heart in consideration of upcoming cancer treatment options challenging the natural VEGF balance in the myocardium. We tested, if the cytotoxic cancer therapy doxorubicin (Doxo) or the anti-angiogenic therapy sunitinib alters viability and VEGF signaling in primary cardiac microvascular endothelial cells (CMEC) and adult rat ventricular myocytes (ARVM). ARVM were isolated and cultured in serum-free medium. CMEC were isolated from the left ventricle and used in the second passage. Viability was measured by LDH-release and by MTT-assay, cellular respiration by high-resolution oxymetry. VEGF-A release was measured using a rat specific VEGF-A ELISA-kit. CMEC were characterized by marker proteins including CD31, von Willebrand factor, smooth muscle actin and desmin. Both Doxo and sunitinib led to a dose-dependent reduction of cell viability. Sunitinib treatment caused a significant reduction of complex I and II-dependent respiration in cardiomyocytes and the loss of mitochondrial membrane potential in CMEC. Endothelial cells up-regulated VEGF-A release after peroxide or Doxo treatment. Doxo induced HIF-1α stabilization and upregulation at clinically relevant concentrations of the cancer therapy. VEGF-A release was abrogated by the inhibition of the Erk1/2 or the MAPKp38 pathway. ARVM did not answer to Doxo-induced stress conditions by the release of VEGF-A as observed in CMEC. VEGF receptor 2 amounts were reduced by Doxo and by sunitinib in a dose-dependent manner in both CMEC and ARVM. In conclusion, these data suggest that cancer therapy with anthracyclines modulates VEGF-A release and its cellular receptors in CMEC and ARVM, and therefore alters paracrine signaling in the myocardium.

The ubiquitin ligase XIAP recruits LUBAC for NOD2 signaling in inflammation and innate immunity

Nucleotide-binding and oligomerization domain (NOD)-like receptors constitute a first line of defense against invading bacteria. X-linked Inhibitor of Apoptosis (XIAP) is implicated in the control of bacterial infections, and mutations in XIAP are causally linked to immunodeficiency in X-linked lymphoproliferative syndrome type-2 (XLP-2). Here, we demonstrate that the RING domain of XIAP is essential for NOD2 signaling and that XIAP contributes to exacerbation of inflammation-induced hepatitis in experimental mice. We find that XIAP ubiquitylates RIPK2 and recruits the linear ubiquitin chain assembly complex (LUBAC) to NOD2. We further show that LUBAC activity is required for efficient NF-κB activation and secretion of proinflammatory cytokines after NOD2 stimulation. Remarkably, XLP-2-derived XIAP variants have impaired ubiquitin ligase activity, fail to ubiquitylate RIPK2, and cannot facilitate NOD2 signaling. We conclude that XIAP and LUBAC constitute essential ubiquitin ligases in NOD2-mediated inflammatory signaling and propose that deregulation of NOD2 signaling contributes to XLP-2 pathogenesis.

Metformin inhibits human androgen production by regulating steroidogenic enzymes HSD3B2 and CYP17A1 and complex I activity of the respiratory chain

Metformin is treatment of choice for the metabolic consequences seen in polycystic ovary syndrome for its insulin-sensitizing and androgen-lowering properties. Yet, the mechanism of action remains unclear. Two potential targets for metformin regulating steroid and glucose metabolism are AMP-activated protein kinase (AMPK) signaling and the complex I of the mitochondrial respiratory chain. Androgen biosynthesis requires steroid enzymes 17α-Hydroxylase/17,20 lyase (CYP17A1) and 3β-hydroxysteroid dehydrogenase type 2 (HSD3B2), which are overexpressed in ovarian cells of polycystic ovary syndrome women. Therefore, we aimed to understand how metformin modulates androgen production using NCI-H295R cells as an established model of steroidogenesis. Similar to in vivo situation, metformin inhibited androgen production in NCI cells by decreasing HSD3B2 expression and CYP17A1 and HSD3B2 activities. The effect of metformin on androgen production was dose dependent and subject to the presence of organic cation transporters, establishing an important role of organic cation transporters for metformin's action. Metformin did not affect AMPK, ERK1/2, or atypical protein kinase C signaling. By contrast, metformin inhibited complex I of the respiratory chain in mitochondria. Similar to metformin, direct inhibition of complex I by rotenone also inhibited HSD3B2 activity. In conclusion, metformin inhibits androgen production by mechanisms targeting HSD3B2 and CYP17-lyase. This regulation involves inhibition of mitochondrial complex I but appears to be independent of AMPK signaling.