Diet-induced obesity impairs AKT signalling in the retina and causes retinal degeneration

Retinopathy, a common complication of diabetes, is characterized by an unbalanced production of nitric oxide (NO), a process regulated by nitric oxide synthase (NOS). We hypothesized that retinopathy might stem from changes in the insulin receptor substrate (IRS)/PI3K/AKT pathway and/or expression of NOS isoforms. Thus, we analysed the morphology and apoptosis index in retinas of obese rats in whom insulin resistance had been induced by a high-fat diet (HFD). Immunoblotting analysis revealed that the retinal tissue of HFD rats had lower levels of AKT1, eNOS and nNOS protein than those of samples taken from control animals. Furthermore, immunohistochemical analyses indicated higher levels of iNOS and 4-hydroxynonenal and a larger number of apoptotic nuclei in HFD rats. Finally, both the inner and outer retinal layers of HFD rats were thinner than those in their control counterparts. When considered alongside previous results, these patterns suggest two major ways in which HFD might impact animals: direct activity of ingested fatty acids and/or via insulin-resistance-induced changes in intracellular pathways. We discuss these possibilities in further detail and advocate the use of this animal model for further understanding relationships between retinopathy, metabolic syndrome and type 2 diabetes. © 2012 John Wiley & Sons, Ltd.

Interleucina-9 estimula a expressão de CCL17/TARC em células epiteliais de pulmão murino

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

Associação genômica ampla para características reprodutivas em bovinos da raça Nelore

Pós-graduação em Medicina Veterinária - FCAV

Melatonin attenuates Her-2, p38 MAPK, p-AKT, and mTOR levels in ovarian carcinoma of ethanol-preferring rats

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

Ovarian superstimulation using FSH combined with equine chorionic gonadotropin (eCG) upregulates mRNA encoding proteins involved with LH receptor intracellular signaling in granulosa cells from Nelore cows

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

The PI3K-Akt-eNOS pathway is involved in aortic hyporeactivity to Phenylephrine associated with late pregnancy in spontaneously hypertensive rats

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

Avaliação dos mecanismos moleculares envolvidos na instalação da resistência periférica à insulina em camundongos portadores de caquexia tumoral

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Exercise training decreases mitogen-activated protein kinase phosphatase-3 expression and suppresses hepatic gluconeogenesis in obese mice

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

Optimal Conditions for Biomass and Recombinant Glycerol Kinase Production Using the Yeast Pichia pastoris

The extracellular glycerol kinase gene from Saccharomyces cerevisiae (GUT]) was cloned into the expression vector pPICZ alpha. A and integrated into the genome of the methylotrophic yeast Pichia pastoris X-33. The presence of the GUT1 insert was confirmed by PCR analysis. Four clones were selected and the functionality of the recombinant enzyme was assayed. Among the tested clones, one exhibited glycerol kinase activity of 0.32 U/mL, with specific activity of 0.025 U/mg of protein. A medium optimized for maximum biomass production by recombinant Pichia pastoris in shaker cultures was initially explored, using 2.31 % (by volume) glycerol as the carbon source. Optimization was carried out by response surface methodology (RSM). In preliminary experiments, following a Plackett-Burman design, glycerol volume fraction (phi(Gly)) and growth time (t) were selected as the most important factors in biomass production. Therefore, subsequent experiments, carried out to optimize biomass production, followed a central composite rotatable design as a function of phi(Gly) and time. Glycerol volume fraction proved to have a significant positive linear effect on biomass production. Also, time was a significant factor (at linear positive and quadratic levels) in biomass production. Experimental data were well fitted by a convex surface representing a second order polynomial model, in which biomass is a function of both factors (R(2)=0.946). Yield and specific activity of glycerol kinase were mainly affected by the additions of glycerol and methanol to the medium. The optimized medium composition for enzyme production was: 1 % yeast extract, 1 % peptone, 100 mM potassium phosphate buffer, pH=6.0, 1.34 % yeast nitrogen base (YNB), 4.10(-5) % biotin, 1 %, methanol and 1 %, glycerol, reaching 0.89 U/mL of glycerol kinase activity and 14.55 g/L of total protein in the medium after 48 h of growth.

Changes in sodium, potassium-ATPase induced by repeated fencamfamine: the roles of cyclic AMP-dependent protein kinase and the nitric oxide-cyclic GMP pathway

Fencamfamine (FCF) is an indirect dopamine agent with effects similar to amphetamine and cocaine. In the present study, we investigate changes in Na,K-ATPase, cyclic AMP-dependent protein kinase (PKA) and nitric oxide synthase (NOS) activity and cyclic GMP levels in the nucleus accumbens (NAc) and striatum (ST) of animals acutely or repeatedly treated with FCF (3.5 mg/kg). Na,K-ATPase had a similar activity in control and repeatedly treated animals, but was reduced in the NAc of the acute group. This enzyme was reduced in the ST in acute and repeatedly treated animals, compared to the control group. Expression of the alpha(1,2,3)-Na,K-ATPase isoforms in the NAc and the ST was not altered in all groups studied. Acute FCF induced a significant increase in PKA activity in both the ST and the NAc. Repeatedly treated animals showed a higher increase in PKA activity in the NAc, but not in the ST, when compared to the acute group. There was also an increase in both NOS activity and cyclic GMP levels only in the NAc of FCF repeatedly treated animals compared to the acute and control groups. We suggest that chronic FCF treatment is linked to a modification in Na,K-ATPase activity through the PKA and NO-cyclic GMP pathway. (C) 2003 Elsevier Ltd. All rights reserved.

Actinobacillus actinomycetemcomitans lipopolysaccharide induces interleukin-6 expression through multiple mitogen-activated protein kinase pathways in periodontal ligament fibroblasts

Actinobacillus actinomycetemcomitans plays a major role in the pathogenesis of aggressive periodontitis. Lipopolysaccharide (LPS) derived from A. actinomycetemcomitans is a key factor in inflammatory cytokine generation within periodontal tissues. In this study, we identify major mitogen-activated protein kinase (MAPK) signaling pathways induced by A. actinomycetemcomitans LPS, Escherichia coli LPS and interleukin-1 beta (IL-1 beta) in a murine periodontal ligament (mPDL) fibroblast cell line. Immunoblot analysis was used to assess the phosphorylated forms of p38, extracellular-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) MAPK following stimulation with A. actinomycetemcomitans LPS, E. coli LPS and IL-1 beta. IL-6 mRNA induction was detected via reverse transcription-polymerase chain reaction, while protein levels were quantified via enzyme-linked immunosorbent assays (ELISA). We utilized biochemical inhibitors of p38, ERK and JNK MAPK to identify the MAPK signaling pathways needed for IL-6 expression. Additional use of stable mPDL cell lines containing dominant negative mutant constructs of MAPK kinase-3 and -6 (MKK-3/6) and p38 null mutant mouse embryonic fibroblast (MEF) cells were used to substantiate the biochemical inhibitor data. Blocking p38 MAPK with SB203580 reduced the induction of IL-6 mRNA by A. actinomycetemcomitans LPS, E. coli LPS and IL-1 beta by > 70%, > 95% and similar to 60%, respectively. IL-6 ELISA indicated that blocking p38 MAPK reduced the IL-6 protein levels induced by A. actinomycetemcomitans LPS, E. coli LPS and IL-1 beta by similar to 60%, similar to 50% and similar to 70%, respectively. All MAPK inhibitors significantly reduced the IL-6 protein levels induced by A. actinomycetemcomitans LPS, E. coli LPS and IL-1 beta whereas only p38 inhibitors consistently reduced the A. actinomycetemcomitans LPS, E. coli LPS and IL-1 beta induction of IL-6 mRNA steady-state levels. The contribution of p38 MAPK LPS-induced IL-6 expression was confirmed using MKK-3/6 dominant negative stable mPDL cell lines. Wild-type and p38 alpha(-/-) MEF cells provided additional evidence to support the role of p38 alpha MAPK in A. actinomycetemcomitans LPS-stimulated IL-6. Our results indicate that induction of IL-6 by E. coli LPS, IL-1 beta and A. actinomycetemcomitans LPS requires signaling through MKK-3-p38 alpha ERK, JNK and p38 MAPK in mPDL cells.

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.

MAP Kinase Phosphatase-1 Protects against Inflammatory Bone Loss

The mitogen-activated protein (MAP) kinase phosphatase (MKP) family plays an important function in regulating the pro-inflammatory cytokines by deactivating MAP kinases. MKP-1 is essential for the dephosphorylation of p38 MAP kinase that regulates expression of IL-6, TNF-alpha, and IL-1 beta. We hypothesized that MKP-1 regulates inflammatory bone loss in experimental periodontitis. Wild-type and Mkp-1(-/-) mice received A. actinomycetemcomitans LPS injection in the palatal region or PBS control 3 times/wk for 30 days. Mice were killed, and maxillae were assessed by microcomputed tomography, histological analysis, and TRAP staining for measurement of bone loss, extent of inflammation, and degree of osteoclastogenesis. Results indicated that, in LPS-injected Mkp-1(-/-) mice, significantly greater bone loss occurred with more inflammatory infiltrate and a significant increase in osteoclastogenesis compared with Mkp-1(-/-) control sites or either wild-type group. Analysis of these data indicates that MKP-1 plays a key role in the regulation of inflammatory bone loss.

Interaction of shikimic acid with shikimate kinase (Retracted Article. See vol 334, pg 967, 2005)

The crystal structure of shikimate kinase from Mycobacterium tuberculosis (MtSK) complexed with MgADP and shikimic acid (shikimate) has been determined at 2.3 Angstrom resolution, clearly revealing the amino acid residues involved in shikimate binding. In MtSK, the Glu61 strictly conserved in SK forms a hydrogen bond and salt-bridge with Arg58 and assists in positioning the guanidinium group of Arg58 for shikimate binding. The carboxyl group of shikimate interacts with Arg58, Gly81, and Arg136, and hydroxyl groups with Asp34 and Gly80. The crystal structure of MtSK-MgADP-shikimate will provide crucial information for elucidation of the mechanism of SK-catalyzed reaction and for the development of a new generation of drugs against tuberculosis. (C) 2004 Elsevier B.V. All rights reserved.

Structure of shikimate kinase from Mycobacterium tuberculosis reveals the binding of shikimic acid

Tuberculosis made a resurgence in the mid-1980s and now kills approximately 3 million people a year. The re-emergence of tuberculosis as a public health threat, the high susceptibility of HIV-infected persons and the proliferation of multi-drug-resistant strains have created a need to develop new drugs. Shikimate kinase and other enzymes in the shikimate pathway are attractive targets for development of non-toxic antimicrobial agents, herbicides and anti-parasitic drugs, because the pathway is essential in these species whereas it is absent from mammals. The crystal structure of shikimate kinase from Mycobacterium tuberculosis (MtSK) complexed with MgADP and shikimic acid ( shikimate) has been determined at 2.3 Angstrom resolution, clearly revealing the amino-acid residues involved in shikimate binding. This is the first three-dimensional structure of shikimate kinase complexed with shikimate. In MtSK, the Glu61 residue that is strictly conserved in shikimate kinases forms a hydrogen bond and salt bridge with Arg58 and assists in positioning the guanidinium group of Arg58 for shikimate binding. The carboxyl group of shikimate interacts with Arg58, Gly81 and Arg136 and the hydroxyl groups interact with Asp34 and Gly80. The crystal structure of MtSK-MgADP-shikimate will provide crucial information for the elucidation of the mechanism of the shikimate kinase-catalyzed reaction and for the development of a new generation of drugs against tuberculosis.