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Context: Insulin resistance, glucose alterations, arterial hypertension (HTN), and the renin-angiotensin-aldosterone system (RAAS) are related in adult obesity. This crosstalk is still unexplored in childhood. Objective: Characterize the relationships of fasting and postload glucose and insulin levels with new American Academy of Pediatrics classification of HTN and RAAS in pediatric obesity. Methods: This was a retrospective observational study; 799 pediatric outpatients (11.4 ± 3.1 years) at a tertiary center who were overweight or obese and not yet on diet were included. The main outcome measures were mean and correlations among parameters of a complete clinical and metabolic screening (body mass index, blood pressure, and glucose and insulin levels during an oral glucose tolerance test, and renin and aldosterone levels and their ratio). Results: 774 subjects had all the parameters, of whom 87.6% had HTN (5% elevated blood pressure, 29.2% stage I HTN, and 53.4% stage II HTN). Eighty subjects had 1 or more glucose alterations, and more frequently presented HTN. Blood pressure levels were higher in subjects with glucose alterations than in those with normal glucose levels. Fasting and stimulated glucose and insulin levels were directly related to the HTN stages, and insulin sensitivity was lower in HTN than in normal blood pressure. Aldosterone, renin, and aldosterone-renin ratio (ARR) were similar in sexes, whereas aldosterone was higher in prepubertal individuals. Subjects with impaired glucose tolerance (IGT) had higher renin and lower ARR. Renin was positively correlated with postload glucose, and ARR was negatively correlated with the Homeostatic Model Assessment for Insulin Resistance index. Conclusion: A close relationship exists among insulin resistance, glucose alterations, HTN, and renin in childhood obesity. Specific categories of risk could provide indicators for strict clinical surveillance.
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Trichophyton rubrum and Trichophyton mentagrophytes spp. are two of the most frequently isolated dermatophytes causing dermatophytosis worldwide. Since the incidence of resistance to antifungal agents is increasing, antifungal susceptibility tests are needed to successfully treat dermatophytoses. Most of the methods currently available are complicated, time-consuming and lack of reference procedures. The aim of this work was to establish a simple protocol to test the susceptibility of dermatophytes isolated from clinical samples against five antifungal drugs using E-test and disk diffusion methods. We used the E-test on non-supplemented Mueller-Hinton agar plates to determine the minimum inhibitory concentrations (MICs) of fluconazole, itraconazole, voriconazole and amphotericin B, and disk diffusion method to determine the interpretive MIC of terbinafine. Fifty dermatophytes-10 T. rubrum and 40 T. mentagrophytes spp.-were assessed after only 96 h of colony growth. Terbinafine was the most active antifungal agent with an inhibition diameter greater than 70 mm (sensitivity > 20 mm), followed by voriconazole, itraconazole and amphotericin B with MICs ranging from 0.032 to 0.38 µg/mL, from 0.006 to 0.125 µg/mL and from 0.5 to 1.5 µg/mL, respectively. All isolates were resistant to fluconazole. Collectively, the less laborious E-test and disk diffusion method were shown to be suitable and reliable to determine antifungal sensitivity of dermatophytes. This simple standard protocol could be employed in the routine of clinical laboratories.
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Antifúngicos/farmacologia , Testes de Sensibilidade Microbiana/métodos , Trichophyton/efeitos dos fármacos , Ágar , Meios de Cultura , Humanos , Tinha/microbiologia , Trichophyton/crescimento & desenvolvimentoRESUMO
INTRODUCTION: Chest pain and its clinical manifestations are the most common reasons for presentation to the emergency department (ED). Given that the prevalence of chest pain due to acute myocardial infarction (AMI) in the ED is modest, clinicians should use cardiac troponins to safely and rapidly rule out AMI, avoiding the delayed release of low risk patients. The study aims to develop and validate an algorithm to early rule-out of non-ST elevation myocardial infarction (NSTEMI) in subjects admitted to the ED with symptoms of myocardial infarction. METHODS: High sensitivity cardiac Troponin T (hs-cTnT) serial measurements (baseline, T0; after 1â¯h, T1; after 3â¯h, T3) were used to develop and validate the algorithm, respectively, in 6403 and 773 consecutive admissions suggestive of AMI. RESULTS: Patients were classified as having or not having NSTEMI according to clinical assessment, diagnostic imaging, and serial measurements ofhs-cTnT; ROC curve analysis allowed to find changes in consecutive hs-cTnT associated with diagnostic sensitivity close to 100%. Only patients with hs-cTnTat T0 lower than 14â¯ng/L resultedto be eligible for the safe rule-out of NSTEMI. CONCLUSIONS: Although some points remain to be improved, the results obtained indicate that algorithms for fast NSTEMI rule-out are feasible and safe.
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Algoritmos , Análise Química do Sangue/métodos , Serviço Hospitalar de Emergência , Limite de Detecção , Infarto do Miocárdio sem Supradesnível do Segmento ST/sangue , Infarto do Miocárdio sem Supradesnível do Segmento ST/diagnóstico , Troponina T/sangue , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Admissão do Paciente , Curva ROC , Fatores de TempoRESUMO
BACKGROUND: The use of phase contrast in urinalysis has been highly recommended. A new system, sediMAX conTRUST PRO, is now available providing simultaneous automated phase contrast and bright field microscopy. This study aimed to evaluate both analytical and diagnostic performance of this new analyzer. METHODS: Results from 504 samples evaluated with the sediMAX conTRUST PRO were compared to those obtained from the same samples by manual microscopy (MM). Analytical and diagnostic performance were assessed according to established guidelines. RESULTS: The concentration of red blood cells (RBCs)and white blood cells (WBCs) at which the LoQ satisfied a CV<â¯25% was 12 particles per µL (p/µL) and 8â¯p/µL, respectively. Within one grade of agreement concordance was quite high, 97.8% for RBCs and 98.0% for WBCs, and above 90% for all other particles. Overall, diagnostic sensitivity and specificity were good (>80%) for the particles considered, although lower sensitivities, 70.6% and 61.8%, were respectively found for hyaline and pathological casts. CONCLUSIONS: The sediMAX conTRUST PRO provides very good performance in terms of RBC and WBC recognition and enumeration, and quite good performance for all other particles. Hyaline cast and pathological cast identification is fine and comparable to other automated systems, but could use further improvement.
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Microscopia de Contraste de Fase , Urinálise/instrumentação , Automação , Humanos , Limite de Detecção , Modelos LinearesRESUMO
BACKGROUND: There are a limited number of studies investigating the type of serum proteins capable of differentiating intraductal papillary mucinous neoplasms from benign or malignant diseases of the pancreas. AIMS: To select proteins able to differentiate intraductal papillary mucinous neoplasms from benign and malignant pancreatic disease using semiquantitative proteomics. METHODS: Serum samples were obtained from 74 patients (19 with type II intraductal papillary mucinous neoplasms, 8 with type I/III intraductal papillary mucinous neoplasms, 24 with chronic pancreatitis, 23 with pancreatic ductal adenocarcinomas) and 21 healthy subjects. Small proteins and peptides were assayed by matrix-assisted laser desorption/ionization for the detection of differentially abundant species possibly related to tumor onset. Serum pancreatic amylase, lipase, carcinoembryonic antigen and carbohydrate antigen 19-9 (CA 19-9) were also assayed. RESULTS: Twenty-six of 84 peaks detected were dysregulated (7 more abundant and 19 less abundant in the type II intraductal papillary mucinous neoplasms, p < 0.05). Of the differentially abundant peaks, 17 were commonly dysregulated (3 peaks more abundant and 13 less abundant in type II intraductal papillary mucinous neoplasms, and one at m/z = 9961 at variance), indicating a protein fingerprint shared by types I/III and type II intraductal papillary mucinous neoplasms and pancreatic ductal adenocarcinomas. CONCLUSIONS: These results suggest that our approach can be used to differentiate type II intraductal papillary mucinous neoplasms from type I/III neoplasms, and type II intraductal papillary mucinous neoplasms from pancreatic ductal adenocarcinomas.
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Adenocarcinoma/diagnóstico , Carcinoma Ductal Pancreático/diagnóstico , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/diagnóstico , Pancreatite Crônica/sangue , Pancreatite Crônica/diagnóstico , Adenocarcinoma/sangue , Idoso , Biomarcadores/sangue , Carcinoma Ductal Pancreático/sangue , Estudos de Casos e Controles , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteômica , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Collagen VI is an extracellular matrix (ECM) protein playing a key role in skeletal muscles and whose deficiency leads to connective tissue diseases in humans and in animal models. However, most studies have been focused on skeletal muscle features. We performed an extensive proteomic profiling in two skeletal muscles (diaphragm and gastrocnemius) of wild-type and collagen VI null (Col6a1-/-) mice at different ages, from 6- (adult) to 12- (aged) month-old to 24 (old) month-old. While in wild-type animals the number of proteins and the level of modification occurring during aging were comparable in the two analyzed muscles, Col6a1-/- mice displayed a number of muscle-type specific variations. In particular, gastrocnemius displayed a limited number of dysregulated proteins in adult mice, while in aged muscles the modifications were more pronounced in terms of number and level. In diaphragm, the differences displayed by 6-month-old Col6a1-/- mice were more pronounced compared to wild-type mice and persisted at 12 months of age. In adult Col6a1-/- mice, the major variations were found in the enzymes belonging to the glycolytic pathway and the tricarboxylic acid (TCA) cycle, as well as in autophagy-related proteins. When compared to wild-type animals Col6a1-/- mice displayed a general metabolic rewiring which was particularly prominent the diaphragm at 6 months of age. Comparison of the proteomic features and the molecular analysis of metabolic and autophagic pathways in adult and aged Col6a1-/- diaphragm indicated that the effects of aging, culminating in lipotoxicity and autophagic impairment, were already present at 6 months of age. Conversely, the effects of aging in Col6a1-/- gastrocnemius were similar but delayed becoming apparent at 12 months of age. A similar metabolic rewiring and autophagic impairment was found in the diaphragm of 24-month-old wild-type mice, confirming that fatty acid synthase (FASN) increment and decreased microtubule-associated proteins 1A/1B light chain 3B (LC3B) lipidation are hallmarks of the aging process. Altogether these data indicate that the diaphragm of Col6a1-/- animal model can be considered as a model of early skeletal muscle aging.
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Obesity is a pathological condition caused by genetic and environmental factors, including vitamin D deficiency, which increases the risk of developing cardiovascular disorders and diabetes. This case-control study was designed to verify whether serum profiles could be identified differentiating obese and non-obese Saudis characterized by vitamin D deficiency and pathological levels of triglycerides, high-density lipoprotein cholesterol and high total cholesterol levels. The serum protein profiles of 64 vitamin D deficient (serum 25(OH)D < 50nmol/L) individuals with metabolic syndrome and with (n = 31; BMI ≥ 30) or without (n = 33; BMI < 30) obesity were analyzed by a quantitative label-free mass spectrometry approach (MALDI-profiling), combined with different serum immunodepletion strategies (Human7 and Human14 immuno-chromatographies), to analyze the intermediate- and low-abundant protein components. The analysis of intermediate-abundant proteins (Human7) in obese vs. non-obese subjects identified 14 changed peaks (p < 0.05) in the m/z range 1500-35000. Furthermore, the Human14 depletion provided new profiles related to obesity (121 changed peaks). Among changed peaks, 11 were identified in the m/z range 1500-4000 Da by high-resolution tandem mass spectrometry, belonging to apolipoprotein CIII, apolipoprotein B100, alpha-1-antichymotrypsin and complement C3. Data herein show that distinct protein profiles identify specific peptides belonging to lipid metabolism and inflammation processes that are associated with obesity and vitamin D deficiency.
Assuntos
Proteínas Sanguíneas/genética , Metabolismo dos Lipídeos/genética , Obesidade/sangue , Deficiência de Vitamina D/sangue , Adulto , Idoso , Glicemia , Índice de Massa Corporal , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Obesidade/complicações , Obesidade/genética , Obesidade/patologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Triglicerídeos/sangue , Vitamina D/sangue , Deficiência de Vitamina D/complicações , Deficiência de Vitamina D/genética , Deficiência de Vitamina D/patologiaRESUMO
In mammals, hypoxic stress management is under the control of the Hypoxia Inducible Factors, whose activity depends on the stabilization of their labile α subunit. In particular, the skeletal muscle appears to be able to react to changes in substrates and O2 delivery by tuning its metabolism. The present study provides a comprehensive overview of skeletal muscle metabolic adaptation to hypoxia in mice and in human subjects exposed for 7/9 and 19 days to high altitude levels. The investigation was carried out combining proteomics, qRT-PCR mRNA transcripts analysis, and enzyme activities assessment in rodents, and protein detection by antigen antibody reactions in humans and rodents. Results indicate that the skeletal muscle react to a decreased O2 delivery by rewiring the TCA cycle. The first TCA rewiring occurs in mice in 2-day hypoxia and is mediated by cytosolic malate whereas in 10-day hypoxia the rewiring is mediated by Idh1 and Fasn, supported by glutamine and HIF-2α increments. The combination of these specific anaplerotic steps can support energy demand despite HIFs degradation. These results were confirmed in human subjects, demonstrating that the TCA double rewiring represents an essential factor for the maintenance of muscle homeostasis during adaptation to hypoxia.
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Adaptação Fisiológica , Ciclo do Ácido Cítrico , Metabolismo Energético , Músculo Esquelético/metabolismo , Oxigênio/metabolismo , Animais , Autofagia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Expressão Gênica , Hexosaminas/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Redes e Vias Metabólicas , Modelos Biológicos , Proteoma , Proteômica/métodos , Roedores , Transdução de Sinais , Fatores de TempoRESUMO
In the diagnosis of Alzheimer's disease (AD) total tau (T-tau), tau phosphorylated at threonine 181 (P-tau181), and the 42 amino acid isoform of alpha ß-amyloid (Aß) are well established surrogate CSF markers. However, there is a constant need for new diagnostic markers to identify the disease at a very early stage. The identification of new molecules for AD diagnosis and monitoring in CSF is hampered by several "confounding" factors including intra- and inter-individual, pre-analytical and analytical variabilities. In an attempt to partially overcome patient's variability and to determine new molecules significantly dysregulated in CSF, we assessed the proteome profile of low molecular weight protein species in CSF and serum of the same patients. CSFs and sera from 36 ADs, 32 iNPHs (idiopathic normal pressure hydrocephalus) and 12 controls were compared by MALDI profiling (non-parametric statistics, CV<20%, AUC>0.750). After protein identification by mass spectrometry, the proteoform composition was assessed by 2-D DIGE/MS. Results indicated that CSF of iNPH can be used as control. Serum and CSF of AD patients shows a specific protein profile compared to iNPH samples. A variation (p<0.01) of Apo A-1 levels in AD, together with a specific dysregulation of Apo A-1 proteoforms was observed. The profiling of CSF and serum of the same patients, suggests that the decrement of total Apo A-1 occurs specifically in CSF. Serum and CSF of AD shows a characteristic Apo A-1 proteoform pattern suggesting it as potential marker which can support the clinical workflow adopted for AD diagnosis and progression.
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Doença de Alzheimer/diagnóstico , Apolipoproteína A-I/líquido cefalorraquidiano , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/sangue , Doença de Alzheimer/líquido cefalorraquidiano , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Apolipoproteína A-I/sangue , Apolipoproteína A-I/metabolismo , Área Sob a Curva , Estudos de Casos e Controles , Eletroforese em Gel Bidimensional , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Hidrocefalia/sangue , Hidrocefalia/líquido cefalorraquidiano , Hidrocefalia/diagnóstico , Masculino , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/metabolismo , Fosforilação , Análise de Componente Principal , Curva ROC , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Estatísticas não Paramétricas , Proteínas tau/líquido cefalorraquidianoRESUMO
Sphingolipids have hydrophilic and hydrophobic properties, different saturation and combination of the oligosaccharide chains and mass homology of species located in a narrow m/z region hampering their recognition. To target sphingolipids for diagnostic purposes, standardized methods for lipid extraction, quali- and quantitative assessments are required. In this study, HPTLC-MALDI MS was adopted to establish sphingolipid and glycosphingolipid profiles in muscle, brain and serum to create a database of molecules to be searched in the preclinical and clinical investigations. Specific protocols for lipid extraction were set up based on the characteristics of the tissue or/and fluids; this approach maximizes the HPTLC-MALDI MS analytical throughput both for lipids extracted in organic and aqueous phase. This study indicates that alkaline hydrolysis is necessary for the detection of low abundant species such as Gb3Cer and ceramides in serum and Gb4Cer, CerP and HexCer in muscle tissue. The high hydrophobicity of ceramides has been overcome by the development of HPTLC plate in chloroform:methanol/50:3.5, which increases the number and the intensity of low abundant Cer species. MS/MS analysis has been conducted directly on HPTLC plate allowing the molecular recognition; furthermore a dataset of spectra was acquired to create a database for future profiling of these molecules.
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Biomarcadores/metabolismo , Cromatografia em Camada Fina/métodos , Glicoesfingolipídeos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Biomarcadores/sangue , Encéfalo/metabolismo , Glicoesfingolipídeos/sangue , Hidrólise , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismoRESUMO
NEU3 sialidase has been shown to be a key player in many physio- and pathological processes, including cell differentiation, cellular response to hypoxic stress, and carcinogenesis. The enzyme, peculiarly localized on the outer leaflet of the plasma membrane, has been shown to be able to remove sialic acid residues from the gangliosides present on adjacent cells, thus creating cell to cell interactions. Nonetheless, herein we report that the enzyme localization is dynamically regulated between the plasma membrane and the endosomes, where a substantial amount of NEU3 is stored with low enzymatic activity. However, under opportune stimuli, NEU3 is shifted from the endosomes to the plasma membrane, where it greatly increases the sialidase activity. Finally, we found that NEU3 possesses also the ability to interact with specific proteins, many of which are different in each cell compartment. They were identified by mass spectrometry, and some selected ones were also confirmed by cross-immunoprecipitation with the enzyme, supporting NEU3 involvement in the cell stress response, protein folding, and intracellular trafficking.
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Neuraminidase/metabolismo , Membrana Celular/enzimologia , Chaperona BiP do Retículo Endoplasmático , Endossomos/enzimologia , Células HEK293 , Células HeLa , Proteínas de Choque Térmico/metabolismo , Humanos , Neuraminidase/química , Neuraminidase/genética , Dobramento de Proteína , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estresse Fisiológico , Regulação para CimaRESUMO
Aging is a universal phenomenon involving the whole body and is characterized by metabolic and physiological decline, leading to cardiovascular defects and heart failure. To characterize the molecular basis of physiological cardiac aging, the proteomic profiles of Sprague Dawley rat hearts of 6, 22 and 30 months were analysed by DIGE and immunoblotting. Results indicate changes in myosin binding protein C, aldehyde dehydrogenase, serpins and sirtuin-3 which protects from the opening of the mitochondrial permeability transition pore induced by cyclophilin D increment. Conversely, an increase of fusion, a decrease of mitochondrial fission and the activation of the non-canonical autophagy pathway were observed. These results support the hypothesis of successful aging in this rat model.
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In the skeletal muscle, the ageing process is characterized by a loss of muscle mass and strength, coupled with a decline of mitochondrial function and a decrease of satellite cells. This profile is more pronounced in hindlimb than in forelimb muscles, both in humans and in rodents. Utilizing light and electron microscopy, myosin heavy chain isoform distribution, proteomic analysis by 2D-DIGE, MALDI-TOF MS and quantitative immunoblotting, this study analyzes the protein levels and the nuclear localization of specific molecules, which can contribute to a preferential muscle loss. Our results identify the molecular changes in the hindlimb (gastrocnemius) and forelimb (triceps) muscles during ageing in rats (3- and 22-month-old). Specifically, the oxidative metabolism contributes to tissue homeostasis in triceps, whereas respiratory chain disruption and oxidative-stress-induced damage imbalance the homeostasis in gastrocnemius muscle. High levels of dihydrolipoyllysine-residue acetyltransferase (Dlat) and ATP synthase subunit alpha (Atp5a1) are detected in triceps and gastrocnemius, respectively. Interestingly, in triceps, both molecules are increased in the nucleus in aged rats and are associated to an increased protein acetylation and myoglobin availability. Furthermore, autophagy is retained in triceps whereas an enhanced fusion, decrement of mitophagy and of regenerative potential is observed in aged gastrocnemius muscle.
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Envelhecimento , Proteínas Musculares/análise , Músculo Esquelético/patologia , Doenças Musculares/patologia , Animais , Autofagia , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Musculares/metabolismo , Músculo Esquelético/fisiologia , Doenças Musculares/metabolismo , Cadeias Pesadas de Miosina/análise , Cadeias Pesadas de Miosina/metabolismo , Proteômica , Ratos Sprague-Dawley , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Eletroforese em Gel Diferencial BidimensionalRESUMO
Glioblastoma multiforme is the most aggressive astrocytoma characterized by the development of resistant cells to various cytotoxic stimuli. Nitric oxide (NO) is able to overcome tumor resistance in PTEN mutated rat C6 glioma cells due to its ability to inhibit cell growth by influencing the intracellular distribution of ceramide. The aim of this study is to monitor the effects of NO donor PAPANONOate on ceramide trafficking in human glioma cell lines, CCF-STTG1 (PTEN-mutated, p53-wt) and T98G (PTEN-harboring, p53-mutated), together with the assessment of their differential molecular signature by 2D-DIGE and MALDI mass spectrometry. In the CCF-STTG1 cell line, the results indicate that treatment with PAPANONOate decreased cell proliferation (<50%) and intracellular trafficking of ceramide, assessed by BODIPY-C5Cer, while these events were not observed in the T98G cell line. Proteomic results suggest that CCF-STTG1 cells are characterized by an increased expression of proteins involved in NO-associated ER stress (i.e. protein disulfide-isomerase A3, calreticulin, 78 kDa glucose-regulated protein), which could compromise ceramide delivery from ER to Golgi, leading to ceramide accumulation in ER and partial growth arrest. Conversely, T98G cell lines, resistant to NO exposure, are characterized by increased levels of cytosolic antioxidant proteins (i.e. glutathione-S-transferase P, peroxiredoxin 1), which might buffer intracellular NO. By providing differential ceramide distribution after NO exposure and differential protein expression of two high grade glioma cell lines, this study highlights specific proteins as possible markers for tumor aggressiveness. This study demonstrates that, in two different high grade glioma cell lines, NO exposure results in a different ceramide distribution and protein expression. Furthermore, this study highlights specific proteins as possible markers for tumor aggressiveness.
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Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Hidrazinas/farmacologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico/farmacologia , Proteoma/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Glioblastoma/patologia , Humanos , Hidrazinas/farmacocinética , Óxido Nítrico/metabolismo , Óxido Nítrico/farmacocinética , Doadores de Óxido Nítrico/farmacocinética , Proteoma/análise , ProteômicaRESUMO
BACKGROUND: Prostate cancer (PCa) is the second cause of mortality in men worldwide. The prostate-specific antigen (PSA) test is routinely adopted in diagnosis; nevertheless more reliable biomarkers are continuously under investigation by monitoring the release of molecules into the bloodstream. The serum protein profiles appear to provide cancer-specific fingerprints that help to discriminate patients (especially with low PSA level) from controls, improving the performance of existing clinical tests. METHODS: Samples from healthy controls and PCa patients with low (≤4 ng/mL) and high PSA (>4 ng/mL) levels were analyzed by MALDI profiling, and by a multi fractionation approach coupled to ESI-MS for peaks identification. RESULTS: MALDI profiling achieved to detect 10 and 14 changed peaks (p-value <0.05), respectively, in PCa patients with low and high PSA versus controls. In particular, a peak identified as C3f fragment, resulted overexpressed in low PSA PCa patients. CONCLUSIONS: PSA test, coupled to MALDI profiling, is able to detect changes, specifically related to PCa, in low molecular weight protein range. Furthermore, for the first time in prostate cancer research, the identification and quantification of the small peptide C3f appears to be relevant for the detection of false negatives, providing an additive diagnostic power to PSA (p<0.01) and suggesting its use in clinical tests.
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Adenocarcinoma/diagnóstico , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/diagnóstico , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Adenocarcinoma/sangue , Adenocarcinoma/imunologia , Idoso , Idoso de 80 Anos ou mais , Reações Falso-Negativas , Humanos , Masculino , Pessoa de Meia-Idade , Antígeno Prostático Específico/imunologia , Neoplasias da Próstata/sangue , Neoplasias da Próstata/imunologiaRESUMO
Owing to their exposure on the cell surface and the possibility of being directly recognized with specific antibodies, glycosphingolipids have aroused great interest in the field of stem cell biology. In the search for specific markers of the differentiation of human bone marrow mesenchymal stem cells (hBMSCs) toward osteoblasts, we studied their glycosphingolipid pattern, with particular attention to gangliosides. After lipid extraction and fractionation, gangliosides, metabolically (3)H-labeled in the sphingosine moiety, were separated by high-performance TLC and chemically characterized by MALDI MS. Upon induction of osteogenic differentiation, a 3-fold increase of ganglioside GD1a was observed. Therefore, the hypothesis of GD1a involvement in hBMSCs commitment toward the osteogenic phenotype was tested by comparison of the osteogenic propensity of GD1a-highly expressing versus GD1a-low expressing hBMSCs and direct addition of GD1a in the differentiation medium. It was found that either the high expression of GD1a in hBMSCs or the addition of GD1a in the differentiation medium favored osteogenesis, providing a remarkable increase of alkaline phosphatase. It was also observed that ganglioside GD2, although detectable in hBMSCs by immunohistochemistry with an anti-GD2 antibody, could not be recognized by chemical analysis, likely reflecting a case, not uncommon, of molecular mimicry.
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Biomarcadores/metabolismo , Diferenciação Celular , Gangliosídeos/metabolismo , Células-Tronco/metabolismo , Fosfatase Alcalina/genética , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Derme/citologia , Relação Dose-Resposta a Droga , Fibroblastos/citologia , Fibroblastos/metabolismo , Citometria de Fluxo , Gangliosídeos/farmacologia , Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Osteopontina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esfingolipídeos/metabolismo , Células-Tronco/citologiaRESUMO
Glycosphingolipids (GSLs) are a class of ubiquitous lipids characterized by a wide structural repertoire and a variety of functional implications. Importantly, altered levels have been correlated with different diseases, suggesting their crucial role in health. Conventional methods for the characterization and quantification are based on high-performance TLC (HPTLC) separation and comparison with the migration distance of standard samples or on MS. We set up and herein report the application of an ImagePrep method for glycosphingolipids qualitative and quantitative profiling through direct HPTLC-MALDI with particular application to wild-type and NEU3 sialidase-overexpressing C2C12 myoblasts. Lipids were analyzed by HPTLC, coupled with MALDI-TOF, and the resulting GSLs profiles were compared to the [³H]sphingolipids HPTLC patterns obtained after metabolic radiolabeling. GSLs detection by HPTLC-MALDI was optimized by testing different methods for matrix delivery and by performing quantitative analyses using serial dilutions of GSLs standards. Through this approach an accurate analysis of each variant of neutral and acidic GSLs, including the detection of different fatty-acid chain variants for each GSL, was provided and these results demonstrated that HPTLC-MALDI is an easy and high-throughput analytical method for GSLs profiling, suggesting its use for an early detection of markers in different diseases, including cancer and heart ischemia.
Assuntos
Glicoesfingolipídeos Acídicos/análise , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia em Camada Fina/métodos , Neuraminidase/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Glicoesfingolipídeos Acídicos/metabolismo , Animais , Área Sob a Curva , Linhagem Celular , Modelos Lineares , Camundongos , MioblastosRESUMO
The synthetic purine reversine has been shown to possess a dual activity as it promotes the de-differentiation of adult cells, including fibroblasts, into stem-cell-like progenitors, but it also induces cell growth arrest and ultimately cell death of cancer cells, suggesting its possible application as an anti-cancer agent. Aim of this study was to investigate the mechanism underneath reversine selectivity in inducing cell death of cancer cells by a comparative analysis of its effects on several tumor cells and normal dermal fibroblasts. We found that reversine is lethal for all cancer cells studied as it induces cell endoreplication, a process that malignant cells cannot effectively oppose due to aberrations in cell cycle checkpoints. On the other hand, normal cells, like dermal fibroblasts, can control reversine activity by blocking the cell cycle, entering a reversible quiescent state. However, they can be induced to become sensitive to the molecule when key cell cycle proteins, e.g., p53, are silenced.
Assuntos
Antineoplásicos/farmacologia , Morfolinas/farmacologia , Purinas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Benzotiazóis/farmacologia , Western Blotting , Caspases/metabolismo , Pontos de Checagem do Ciclo Celular , Morte Celular , Desdiferenciação Celular , Proliferação de Células , Forma Celular/efeitos dos fármacos , Sobrevivência Celular , Endorreduplicação , Ativação Enzimática , Fibroblastos/efeitos dos fármacos , Fibrossarcoma/genética , Fibrossarcoma/metabolismo , Fibrossarcoma/patologia , Citometria de Fluxo , Inativação Gênica , Células HeLa , Humanos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Tolueno/análogos & derivados , Tolueno/farmacologia , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/genéticaRESUMO
Exploring cellular mechanisms underlying beneficial and detrimental responses to hypoxia represents the object of the present study. Signaling molecules controlling adaptation to hypoxia (HIF-1α), energy balance (AMPK), mitochondrial biogenesis (PGC-1α), autophagic/apoptotic processes regulation and proteomic dysregulation were assessed. Responses to acute hypoxia (AH) and chronic hypoxia (CH) in mouse heart proteome were detected by 2-D DIGE, mass spectrometry and antigen-antibody reactions. Both in AH and CH, the results indicated a deregulation of proteins related to sarcomere stabilization and muscle contraction. Neither in AH nor in CH the HIF-1α stabilization was observed. In AH, the metabolic adaptation to lack of oxygen was controlled by AMPK activation and sustained by an up-regulation of adenosylhomocysteinase and acetyl-CoA synthetase. AH was characterized by the mitophagic protein Bnip 3 increment. PGC-1α, a master regulator of mitochondrial biogenesis, was down-regulated. CH was characterized by the up-regulation of enzymes involved in antioxidant defense, in aldehyde bio-product detoxification and in misfolded protein degradation. In addition, a general down-regulation of enzymes controlling anaerobic metabolism was observed. After 10 days of hypoxia, cardioprotective molecules were substantially decreased whereas pro-apoptotic molecules increased accompained by down-regulation of specific target proteins.