Lysosomal enzymes are decreased in the kidney of diabetic rats

The objective of the present study was to investigate the expression and activities of lysosomal enzymes that act upon proteins and sulfated polysaccharides in diabetic rat kidney. Cathepsins, glycosidases and sulfatases were studied on the 10th (DM-10) and on the 30th (DM-30) day of streptozotocin-induced diabetes mellitus (DM). the activity of cathepsin B, the main kidney cysteine protease, was decreased both in DM-10 and DM-30. Gel filtration chromatography of urinary proteins has shown the prevalence of low molecular weight peptides in normal and DM-10 urine, in contrast to the prevalence of high molecular weight peptides and intact proteins in DM-30. These results show that the decrease in lysosomal proteases could explain, at least in part, the increased albuminuria detected by radial immunodiffusion (RID), due to the excretion of less degraded or intact albumin. Concerning sulfated polysaccharides, the activities of beta-glucuronidase, N-acetyl-beta-D-glucosaminidase, and N-acetyl-beta-D-galactosaminidase were also decreased in DM-30, while aryl sulfatases did not vary. Increased toluidine blue metachromatic staining of the tissue suggests that the lower activities of glycosidases could lead to intracellular deposition of partially digested molecules, and this could explain the decreased urinary excretion and increased tissue buildup of these molecules. the main morphological changes observed in kidney were proximal convoluted tubules with thinner walls and thinner brush border. Immunohistochemistry revealed that most of cathepsin B was located in the brush border of proximal tubular cells, highlighting the involvement of proximal convoluted tubules in diabetic nephropathy. (C) 2012 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo, Disciplina Biol Mol, Dept Bioquim, Escola Paulista Med, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, Escola Paulista Med, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Morfol, Escola Paulista Med, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Disciplina Biol Mol, Dept Bioquim, Escola Paulista Med, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, Escola Paulista Med, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Morfol, Escola Paulista Med, BR-04044020 São Paulo, BrazilFAPESP: 2010/16022-5FAPESP: 2009/11817-2CNPq: 308642/2010-4Web of Scienc

Homeopathic preparations and separation anxiety in dogs: a pilot study

Separation Anxiety (SA) is a behavioral syndrome that may affect dogs of different ages and that is characterized by intense clinical signs. Traditional veterinary clinic efforts rely on harmful side effect drugs. Overall, homeopathy handles individual idiosyncrasies and susceptibilities and deal with them using a single medicine through the law of similarity. This study aimed to determine whether individualized homeopathic medicines have a greater effect than placebo for dogs suffering from SA or not, assessing its relation to behavioral settings, cortisol levels, and blood cells count before and after therapy. It also focused on setting a demographic profile of these dogs. Owners filled out a score questionnaire. Twenty-one dogs were recruited and repertorized in accordance to classical homeopathy. A pharmacist was responsible to randomize and dispense verum medicine or placebo. On the 30th day, reappraisal of owners were allowed altering the dispensed medicine. The final assessment occurred on the 60th day. In verum group, destructive behavior analysis had a significant statistical difference intra-group over the trial compared to the placebo group. The mean of cortisol levels in the placebo group was significantly higher on the 60th day of the trial when compared to the verum group, whose levels were sustained over the same period. Although evidenced behavioral improvements could be related to homeopathic preparations, it was not feasible to set any connection between homeopathic interventions, behavioral issues, and plasma component

Highly Diluted Glyphosate Mitigates Its Effects on Artemia salina: Physicochemical Implications.

Glyphosate is an herbicide widely used in agriculture but can present chronic toxicity in low concentrations. Artemia salina is a common bio-indicator of ecotoxicity; it was used herein as a model to evaluate the effect of highly diluted-succussed glyphosate (potentized glyphosate) in glyphosate-based herbicide (GBH) exposed living systems. Artemia salina cysts were kept in artificial seawater with 0.02% glyphosate (corresponding to 10% lethal concentration or LC10) under constant oxygenation, luminosity, and controlled temperature, to promote hatching in 48 h. Cysts were treated with 1% (v/v) potentized glyphosate in different dilution levels (Gly 6 cH, 30 cH, 200 cH) prepared the day before according to homeopathic techniques, using GBH from the same batch. Controls were unchallenged cysts, and cysts treated with succussed water or potentized vehicle. After 48 h, the number of born nauplii per 100 µL, nauplii vitality, and morphology were evaluated. The remaining seawater was used for physicochemical analyses using solvatochromic dyes. In a second set of experiments, Gly 6 cH treated cysts were observed under different degrees of salinity (50 to 100% seawater) and GBH concentrations (zero to LC 50); hatching and nauplii activity were recorded and analyzed using the ImageJ 1.52, plug-in Trackmate. The treatments were performed blind, and the codes were revealed after statistical analysis. Gly 6 cH increased nauplii vitality (p = 0.01) and improved the healthy/defective nauplii ratio (p = 0.005) but delayed hatching (p = 0.02). Overall, these results suggest Gly 6cH treatment promotes the emergence of the more GBH-resistant phenotype in the nauplii population. Also, Gly 6cH delays hatching, another useful survival mechanism in the presence of stress. Hatching arrest was most marked in 80% seawater when exposed to glyphosate at LC10. Water samples treated with Gly 6 cH showed specific interactions with solvatochromic dyes, mainly Coumarin 7, such that it appears to be a potential physicochemical marker for Gly 6 cH. In short, Gly 6 cH treatment appears to protect the Artemia salina population exposed to GBH at low concentrations

Cathepsin K induces platelet dysfunction and affects cell signaling in breast cancer - molecularly distinct behavior of cathepsin K in breast cancer

Background: Breast cancer comprises clinically and molecularly distinct tumor subgroups that differ in cell histology and biology and show divergent clinical phenotypes that impede phase III trials, such as those utilizing cathepsin K inhibitors. Here we correlate the epithelial-mesenchymal-like transition breast cancer cells and cathepsin K secretion with activation and aggregation of platelets. Cathepsin K is up-regulated in cancer cells that proteolyze extracellular matrix and contributes to invasiveness. Although proteolytically activated receptors (PARs) are activated by proteases, the direct interaction of cysteine cathepsins with PARs is poorly understood. In human platelets, PAR-1 and -4 are highly expressed, but PAR-3 shows low expression and unclear functions. Methods: Platelet aggregation was monitored by measuring changes in turbidity. Platelets were immunoblotted with anti-phospho and total p38, Src-Tyr-416, FAK-Tyr-397, and TGF beta monoclonal antibody. Activation was measured in a flow cytometer and calcium mobilization in a confocal microscope. Mammary epithelial cells were prepared from the primary breast cancer samples of 15 women with Luminal-B subtype to produce primary cells. Results: We demonstrate that platelets are aggregated by cathepsin K in a dose-dependent manner, but not by other cysteine cathepsins. PARs-3 and -4 were confirmed as the cathepsin K target by immunodetection and specific antagonists using a fibroblast cell line derived from PARs deficient mice. Moreover, through co-culture experiments, we show that platelets activated by cathepsin K mediated the up-regulation of SHH, PTHrP, OPN, and TGF beta in epithelial-mesenchymal-like cells from patients with Luminal B breast cancer. Conclusions: Cathepsin K induces platelet dysfunction and affects signaling in breast cancer cells.Associacao Beneficente de Coleta de Sangue (Colsan)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Univ Fed Sao Paulo, Dept Gynecol, BR-04024002 Sao Paulo, SP, BrazilCOLSAN, Charitable Assoc Blood Collect, BR-04080006 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Biophys, BR-04024002 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Biochem, BR-04024002 Sao Paulo, SP, BrazilAntonio Prudente Fdn, AC Camargo Canc Ctr, AC Camargo Hosp Biobank, Dept Pathol, BR-01509010 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Cellular Gynecol Lab, Dept Gynecol, Rua Napoleao Barros 608, BR-04024002 Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Gynecol, BR-04024002 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Biophys, BR-04024002 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Biochem, BR-04024002 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Cellular Gynecol Lab, Dept Gynecol, Rua Napoleao Barros 608, BR-04024002 Sao Paulo, BrazilFAPESP: 2012/19780-3FAPESP: 2012/19851-8FAPESP: 2009/53766-5Web of Scienc

Diabetes mellitus increases the susceptibility to encephalitozoonosis in mice.

Microsporidiosis are diseases caused by opportunistic intracellular fungi in immunosuppressed individuals, as well as in transplanted patients, the elderly and children, among others. Diabetes mellitus (DM) is a metabolic disease characterized by hyperglycemia and decreased T cell response, neutrophil function, humoral immunity failure, increasing the susceptibility to infections. Here, we investigated the susceptibility of streptozotocin (STZ)-induced type I diabetic and/or immunosuppressed mice to encephalitozoonosis by Encephalitozoon cuniculi. Microscopically, granulomatous hepatitis, interstitial pneumonia and pielonephritis were observed in all infected groups. STZ treatment induced an immunossupressor effect in the populations of B (B-1 and B2) and CD4+ T lymphocytes. Moreover, infection decreased CD4+ and CD8+ T lymphocytes and macrophages of DM mice. Furthermore, infection induced a significant increase of IL-6 and TNF-α cytokine serum levels in DM mice. IFN-γ, the most important cytokine for the resolution of encephalitozoonosis, increased only in infected mice. In addition to the decreased immune response, DM mice were more susceptible to encephalitozoonosis, associated with increased fungal burden, and symptoms. Additionally, cyclophosphamide immunosuppression in DM mice further increased the susceptibility to encephalitozoonosis. Thus, microsporidiosis should be considered in the differential diagnosis of comorbidities in diabetics

Spleen immune cell analysis.

<p>Evaluation of B-2 (CD23⁺CD19⁺) cells, CD4⁺ (CD19^-CD8^-CD4+), and CD8⁺ (CD19^-CD4^-CD8⁺) T lymphocytes and macrophages (CD19^-F4/80⁺CD11b⁺) in spleen Cy immunosuppressed and STZ-induced DM mice infected with <i>E</i>. <i>cuniculi</i> compared with its controls. Two ways variance analysis (ANOVA) revealed * p<0,05.</p

Peritoneal immune cell analysis.

<p>Evaluation of B-1 (CD23^-CD19⁺), B-2 (CD23⁺CD19⁺), Pre-B-1CDP (CD19⁺CD11b⁺F4/80⁺) cells, CD4⁺ (CD19^-CD8^-CD4+), CD8⁺ (CD19^-CD4^-CD8⁺) T lymphocytes and macrophages (CD19^-F4/80⁺CD11b⁺) from PerC of STZ-induced DM mice infected with <i>E</i>. <i>cuniculi</i> compared with its controls. Two ways variance analysis (ANOVA) revealed * p<0,05.</p