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Ricotti L.,Piaggio | Das Neves R.P.,Center for Neurosciences and Cell Biology | Ciofani G.,Italian Institute of Technology | Canale C.,Italian Institute of Technology | And 5 more authors.
Journal of Nanoparticle Research | Year: 2014

F/G-actin ratio modulation is known to have an important role in many cell functions and in the regulation of specific cell behaviors. Several attempts have been made in the latest decades to finely control actin production and polymerization, in order to promote certain cell responses. In this paper we demonstrate the possibility of modulating F/G-actin ratio and mechanical properties of normal human dermal fibroblasts by using boron nitride nanotubes dispersed in the culture medium and by stimulating them with ultrasound transducers. Increasing concentrations of nanotubes were tested with the cells, without any evidence of cytotoxicity up to 10 μg/ml concentration of nanoparticles. Cells treated with nanoparticles and ultrasound stimulation showed a significantly higher F/G-actin ratio in comparison with the controls, as well as a higher Young's modulus. Assessment of Cdc42 activity revealed that actin nucleation/polymerization pathways, involving Rho GTPases, are probably influenced by nanotube-mediated stimulation, but they do not play a primary role in the significant increase of F/G-actin ratio of treated cells, such effect being mainly due to actin overexpression. © 2014 Springer Science+Business Media Dordrecht.

Price N.L.,Harvard University | Gomes A.P.,Harvard University | Gomes A.P.,Center for Neurosciences and Cell Biology | Ling A.J.Y.,Harvard University | And 23 more authors.
Cell Metabolism | Year: 2012

Resveratrol induces mitochondrial biogenesis and protects against metabolic decline, but whether SIRT1 mediates these benefits is the subject of debate. To circumvent the developmental defects of germline SIRT1 knockouts, we have developed an inducible system that permits whole-body deletion of SIRT1 in adult mice. Mice treated with a moderate dose of resveratrol showed increased mitochondrial biogenesis and function, AMPK activation, and increased NAD + levels in skeletal muscle, whereas SIRT1 knockouts displayed none of these benefits. A mouse overexpressing SIRT1 mimicked these effects. A high dose of resveratrol activated AMPK in a SIRT1-independent manner, demonstrating that resveratrol dosage is a critical factor. Importantly, at both doses of resveratrol no improvements in mitochondrial function were observed in animals lacking SIRT1. Together these data indicate that SIRT1 plays an essential role in the ability of moderate doses of resveratrol to stimulate AMPK and improve mitochondrial function both in vitro and in vivo. © 2012 Elsevier Inc.

Moura R.A.,University of Lisbon | Cascao R.,University of Lisbon | Perpetuo I.,University of Lisbon | Canhao H.,University of Lisbon | And 16 more authors.
Rheumatology | Year: 2011

Objectives: B cells play an important role in the perpetuation of RA, particularly as autoantibody-producing cells. The ICs that further develop deposit in the joints and aggravate the inflammatory process. However, B-cell contribution in the very early stage of the disease remains unknown. The main goal of this work was to determine the concentration of cytokines potentially relevant for B-cell activation in serum from very early polyarthritis patients, with <6 weeks of disease duration, who latter on evolved into very early RA (VERA).Methods. A proliferation-inducing ligand (APRIL), B-cell activating factor (BAFF) and IL-21 levels were measured by ELISA in the serum of VERA, other very early arthritis (VEA), established RA patients and controls. SF samples of established RA were also analysed. Results: VERA patients have higher levels of APRIL and BAFF as compared with VEA, established RA and controls. Furthermore, APRIL and BAFF levels are also significantly elevated in RA-SF when compared with serum. Conclusions: The increased levels of APRIL and BAFF in VERA patients suggests that B-cell activation and the development of autoreactive B-cell responses might be crucial in early phases of RA. Therefore, APRIL and BAFF could be promising targets for therapy in the early phase of RA. © The Author 2010. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved.

Coelho M.,University of Coimbra | Valente-Silva P.,University of Coimbra | Tylki-Szymanska A.,Childrens Memorial Health Institute | Henriques T.,University of Coimbra | And 4 more authors.
Magnetic Resonance in Medicine | Year: 2016

Purpose Enrichment of glucose position 5 (H5) from deuterated water (2H2O) is widely used for quantifying gluconeogenesis. Exchanges of hexose and triose phosphates mediated by transaldolase have been postulated to enrich H5 independently of gluconeogenesis, but to date this mechanism has not been proven. We determined the enrichment of glucose-6-phosphate (G6P), the immediate precursor of endogenously produced glucose, from 2H2O in erythrocyte hemolysate preparations. Here, transaldolase exchange is active but gluconeogenesis is absent. Methods Hemolysates were prepared from human erythrocytes and incubated with a buffer containing 5% [U-13C]G6P, unlabeled fructose 1,6-bisphosphate, and 10% 2H2O. G6P 2H-enrichment and 13C-isotopomer distributions were analyzed by 2H and 13C NMR following derivatization to monoacetone glucose. Results 2H NMR analysis revealed high 2H-enrichment of G6P hydrogens 2, 4, and 5; low enrichment of hydrogen 3, and residual enrichments of hydrogens 1, 6R, and 6S. 13C NMR isotopomer analysis revealed that [U-13C]G6P was converted to [1,2,3-13C3]G6P, a predicted product of transaldolase-mediated exchange, as well as [1,2-13C2]G6P and [3-13C]G6P, predicted products of combined transaldolase and transketolase exchanges. Conclusion Hydrogen 5 of G6P was enriched from 2H2O through exchanges mediated by transaldolase. These studies prove that G6P can be enriched in hydrogen 5 by 2H2O independently of gluconeogenesis. © 2015 Wiley Periodicals, Inc.

Gomes A.P.,Harvard University | Gomes A.P.,Center for Neurosciences and Cell Biology | Gomes A.P.,University of Coimbra | Price N.L.,Harvard University | And 20 more authors.
Cell | Year: 2013

Ever since eukaryotes subsumed the bacterial ancestor of mitochondria, the nuclear and mitochondrial genomes have had to closely coordinate their activities, as each encode different subunits of the oxidative phosphorylation (OXPHOS) system. Mitochondrial dysfunction is a hallmark of aging, but its causes are debated. We show that, during aging, there is a specific loss of mitochondrial, but not nuclear, encoded OXPHOS subunits. We trace the cause to an alternate PGC-1α/β-independent pathway of nuclear-mitochondrial communication that is induced by a decline in nuclear NAD+ and the accumulation of HIF-1α under normoxic conditions, with parallels to Warburg reprogramming. Deleting SIRT1 accelerates this process, whereas raising NAD+ levels in old mice restores mitochondrial function to that of a young mouse in a SIRT1-dependent manner. Thus, a pseudohypoxic state that disrupts PGC-1α/β-independent nuclear-mitochondrial communication contributes to the decline in mitochondrial function with age, a process that is apparently reversible. © 2013 Elsevier Inc.

Jones J.,Center for Neurosciences and Cell Biology | Kahl S.,Institute of Clinical Diabetology | Kahl S.,Heinrich Heine University Düsseldorf | Kahl S.,German Center for Diabetes Research | And 5 more authors.
Analytical Biochemistry | Year: 2015

Measurement of acetaminophen glucuronide (AG) 2H enrichment from deuterated water (2H2O) by 2H nuclear magnetic resonance (NMR) analysis of its monoacetone glucose (MAG) derivative provides estimation of gluconeogenic and glycogenolytic contributions to endogenous glucose production (EGP). However, AG derivatization to MAG is laborious and unsuitable for high-throughput studies. An alternative derivative, 5-O-acetyl monoacetone glucuronolactone (MAGLA), was tested. Eleven healthy subjects ingested 2H2O to 0.5% body water enrichment and 500 mg of acetaminophen. Plasma glucose and urinary glucuronide positional 2H enrichments were measured by 2H NMR spectroscopy of MAG and MAGLA, respectively. A Bland-Altman analysis indicated agreement at the 95% confidence level between glucose and glucuronide estimates. © 2015 Elsevier Inc.

PubMed | Heinrich Heine University Düsseldorf and Center for Neurosciences and Cell Biology
Type: | Journal: Analytical biochemistry | Year: 2015

Measurement of acetaminophen glucuronide (AG) (2)H enrichment from deuterated water ((2)H2O) by (2)H nuclear magnetic resonance (NMR) analysis of its monoacetone glucose (MAG) derivative provides estimation of gluconeogenic and glycogenolytic contributions to endogenous glucose production (EGP). However, AG derivatization to MAG is laborious and unsuitable for high-throughput studies. An alternative derivative, 5-O-acetyl monoacetone glucuronolactone (MAGLA), was tested. Eleven healthy subjects ingested (2)H2O to 0.5% body water enrichment and 500 mg of acetaminophen. Plasma glucose and urinary glucuronide positional (2)H enrichments were measured by (2)H NMR spectroscopy of MAG and MAGLA, respectively. A Bland-Altman analysis indicated agreement at the 95% confidence level between glucose and glucuronide estimates.

Mega C.,University of Coimbra | Mega C.,Polytechnic Institute of Viseu | Vala H.,Polytechnic Institute of Viseu | Rodrigues-Santos P.,University of Coimbra | And 7 more authors.
Diabetology and Metabolic Syndrome | Year: 2014

Background: The purpose of this study was to investigate some of the possible mechanisms underlying the protective effects of a dipeptidyl peptidase IV (DPP-IV) inhibitor, sitagliptin, on pancreatic tissue in an animal model of type 2 diabetes mellitus (T2DM), the Zucker Diabetic Fatty (ZDF) rat, focusing on glycaemic, insulinic and lipidic profiles, as well as, on apoptosis, inflammation, angiogenesis and proliferation mediators. Methods. Male obese diabetic ZDF (fa/fa) rats, aged 20 weeks, were treated with sitagliptin (10 mg/kg bw/day) during 6 weeks and compared to untreated diabetic and lean control littermates. Metabolic data was evaluated at the beginning and at the end of the treatment, including glycaemia, HbA1c, insulinaemia, HOMA-beta and TGs. Endocrine and exocrine pancreas lesions were assessed semiquantitatively by histopathological methods. Pancreas gene (mRNA) and protein expression of mediators of apoptotic machinery, inflammation and angiogenesis/proliferation (Bax, Bcl2, IL-1β, VEGF, PCNA and TRIB3) were analyzed by RT-qPCR and/or by immunohistochemistry. Results: Sitagliptin treatment for 6 weeks (between 20 and 26 week-old) was able to significantly (p < 0.001) ameliorate all the metabolic parameters, by preventing the increase in blood glucose and in serum TGs contents (16.54% and 37.63%, respectively, vs untreated), as well as, by preventing the decrease in serum insulin levels and in the functional beta cells capacity accessed via HOMA-beta index (156.28% and 191.74%, respectively, vs untreated). Sitagliptin-treated diabetic rats presented a reduced pancreas Bax/Bcl2 ratio, suggestive of an antiapoptotic effect; in addition, sitagliptin was able to completely reduce (p < 0.001) the pancreas overexpression of IL-1β and TRIB3 found in the untreated diabetic animals; and promoted a significant (p < 0.001) overexpression of VEGF and PCNA. Conclusion: In this animal model of obese T2DM (the ZDF rat), sitagliptin prevented β-cell dysfunction and evolution of pancreatic damage. The protective effects afforded by this DPP-IV inhibitor may derive from improvement of the metabolic profile (viewed by the amelioration of glucose and TGs levels and of insulin resistance) and from cytoprotective properties, such as antiapoptotic, anti-inflammatory, pro-angiogenic and pro-proliferative. © 2014 Mega et al.; licensee BioMed Central Ltd.

Sereno J.,University of Coimbra | Rodrigues-Santos P.,University of Coimbra | Rodrigues-Santos P.,Center for Neurosciences and Cell Biology | Vala H.,Polytechnic Institute of Viseu | And 8 more authors.
International Journal of Molecular Sciences | Year: 2014

Cyclosporin A (CsA), a calcineurin inhibitor, remain the cornerstone of immunosuppressive regimens, regardless of nephrotoxicity, which depends on the duration of drug exposure. The mechanisms and biomarkers underlying the transition from CsA-induced renal dysfunction to nephrotoxicity deserve better elucidation, and would help clinical decisions. This study aimed to clarify these issues, using a rat model of shortand long-term CsA (5 mg/kg bw/day) treatments (3 and 9 weeks, respectively). Renal function was assessed on serum and urine; kidney tissue was used for histopathological characterization and gene and/or protein expression of markers of proliferation, fibrosis and inflammation. In the short-term, creatinine and blood urea nitrogen (BUN) levels increased and clearances decreased, accompanied by glomerular filtration rate (GFR) reduction, but without kidney lesions; at that stage, CsA exposure induced proliferating cell nuclear antigen (PCNA), transforming growth factor beta 1 (TGF-β1), factor nuclear kappa B (NF-κβ) and Tumor Protein P53 (TP53) kidney mRNA up-regulation. In the long-term treatment, renal dysfunction data was accompanied by glomerular and tubulointerstitial lesions, with remarkable kidney mRNA up-regulation of the mammalian target of rapamycin (mTOR) and the antigen identified by monoclonal antibody Ki-67 (Mki67), accompanied by mTOR protein overexpression. Transition from CsA-induced renal dysfunction to nephrotoxicity is accompanied by modification of molecular mechanisms and biomarkers, being mTOR one of the key players for kidney lesion evolution, thus suggesting, by mean of molecular evidences, that early CsA replacement by mTOR inhibitors is indeed the better therapeutic choice to prevent chronic allograft nephropathy. © 2014 by the authors; licensee MDPI, Basel, Switzerland.

Martins-Neves S.R.,University of Coimbra | Lopes T.O.,University of Coimbra | do Carmo A.,Center for Neurosciences and Cell Biology | do Carmo A.,University of Coimbra | And 4 more authors.
BMC Cancer | Year: 2012

Background: Osteosarcoma is a bone-forming tumor of mesenchymal origin that presents a clinical pattern that is consistent with the cancer stem cell model. Cells with stem-like properties (CSCs) have been identified in several tumors and hypothesized as the responsible for the relative resistance to therapy and tumor relapses. In this study, we aimed to identify and characterize CSCs populations in a human osteosarcoma cell line and to explore their role in the responsiveness to conventional therapies.Methods: CSCs were isolated from the human MNNG/HOS cell line using the sphere formation assay and characterized in terms of self-renewal, mesenchymal stem cell properties, expression of pluripotency markers and ABC transporters, metabolic activity and tumorigenicity. Cell's sensitivity to conventional chemotherapeutic agents and to irradiation was analyzed and related with cell cycle-induced alterations and apoptosis.Results: The isolated CSCs were found to possess self-renewal and multipotential differentiation capabilities, express markers of pluripotent embryonic stem cells Oct4 and Nanog and the ABC transporters P-glycoprotein and BCRP, exhibit low metabolic activity and induce tumors in athymic mice. Compared with parental MNNG/HOS cells, CSCs were relatively more resistant to both chemotherapy and irradiation. None of the treatments have induced significant cell-cycle alterations and apoptosis in CSCs.Conclusions: MNNG/HOS osteosarcoma cells contain a stem-like cell population relatively resistant to conventional chemotherapeutic agents and irradiation. This resistant phenotype appears to be related with some stem features, namely the high expression of the drug efflux transporters P-glycoprotein and BCRP and their quiescent nature, which may provide a biological basis for resistance to therapy and recurrence commonly observed in osteosarcoma. © 2012 Martins-Neves et al; licensee BioMed Central Ltd.

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