Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 26
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
J Thromb Haemost ; 17(11): 1798-1807, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31271700

RESUMO

BACKGROUND: Congenital disorders of glycosylation are rare inherited diseases affecting many different proteins. The lack of glycosylation notably affects the hemostatic system and leads to deficiencies of both procoagulant and anticoagulant factors. OBJECTIVE: To assess the hemostatic balance in patients with multiple coagulation disorders by using a thrombin generation assay. METHOD: We performed conventional coagulation assays and a thrombin generation assay on samples from patients with congenital disorder of glycosylation. The thrombin generation assay was performed before and after activation of the protein C system by the addition of soluble thrombomodulin. RESULTS: A total of 35 patients were included: 71% and 57% had low antithrombin and factor XI levels, respectively. Protein C and protein S levels were abnormally low in 29% and 26% of the patients, respectively, whereas only 11% displayed low factor IX levels. Under baseline conditions, the thrombin generation assay revealed a significantly higher endogenous thrombin potential and thrombin peak in patients, relative to controls. After spiking with thrombomodulin, we observed impaired involvement of the protein C system. Hence, 54% of patients displayed a hypercoagulant phenotype in vitro. All the patients with a history of stroke-like episodes or thrombosis displayed this hypercoagulant phenotype. CONCLUSION: A thrombin generation assay revealed a hypercoagulant in vitro phenotype under baseline condition; this was accentuated by impaired involvement of the protein C system. This procoagulant phenotype may thus reflect the risk of severe vascular complications. Further research will have to determine whether the thrombin generation assay is predictive of vascular events.

2.
Genet Med ; 2018 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-30293989

RESUMO

PURPOSE: PMM2-CDG is the most common congenital disorder of glycosylation (CDG), which presents with either a neurologic or multisystem phenotype. Little is known about the longitudinal evolution. METHODS: We performed data analysis on PMM2-CDG patients' clinical features according to the Nijmegen CDG severity score and laboratory data. Seventy-five patients (28 males) were followed up from 11.0 ± 6.91 years for an average of 7.4 ± 4.5 years. RESULTS: On a group level, there was no significant evolution in overall clinical severity. There was some improvement in mobility and communication, liver and endocrine function, and strabismus and eye movements. Educational achievement and thyroid function worsened in some patients. Overall, the current clinical function, the system-specific involvement, and the current clinical assessment remained unchanged. On follow-up there was improvement of biochemical variables with (near) normalization of activated partial thromboplastin time (aPTT), factor XI, protein C, antithrombin, thyroid stimulating hormone, and liver transaminases. CONCLUSION: PMM2-CDG patients show a spontaneous biochemical improvement and stable clinical course based on the Nijmegen CDG severity score. This information is crucial for the definition of endpoints in clinical trials.

4.
Nat Commun ; 9(1): 3087, 2018 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-30082715

RESUMO

Skeletal dysplasia with multiple dislocations are severe disorders characterized by dislocations of large joints and short stature. The majority of them have been linked to pathogenic variants in genes encoding glycosyltransferases, sulfotransferases or epimerases required for glycosaminoglycan synthesis. Using exome sequencing, we identify homozygous mutations in SLC10A7 in six individuals with skeletal dysplasia with multiple dislocations and amelogenesis imperfecta. SLC10A7 encodes a 10-transmembrane-domain transporter located at the plasma membrane. Functional studies in vitro demonstrate that SLC10A7 mutations reduce SLC10A7 protein expression. We generate a Slc10a7-/- mouse model, which displays shortened long bones, growth plate disorganization and tooth enamel anomalies, recapitulating the human phenotype. Furthermore, we identify decreased heparan sulfate levels in Slc10a7-/- mouse cartilage and patient fibroblasts. Finally, we find an abnormal N-glycoprotein electrophoretic profile in patient blood samples. Together, our findings support the involvement of SLC10A7 in glycosaminoglycan synthesis and specifically in skeletal development.

6.
Electrophoresis ; 2018 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-29869806

RESUMO

Congenital disorders of glycosylation (CDG) are rare autosomal genetic diseases affecting the glycosylation of proteins and lipids. Since CDG-related clinical symptoms are classically extremely variable and nonspecific, a combination of electrophoretic, mass spectrometric, and gene sequencing techniques is often mandatory for obtaining a definitive CDG diagnosis, as well as identifying causative gene mutations and deciphering the underlying biochemical mechanisms. Here, we illustrate the potential of integrating data from capillary electrophoresis of transferrin, two-dimensional electrophoresis of N- and O-glycoproteins, mass spectrometry analyses of total serum N-linked glycans and mucin core1 O-glycosylated apolipoprotein C-III for the determination of various culprit CDG gene mutations. "Step-by-step" diagnosis pathways of four particular and new CDG cases, including MGAT2-CDG, ATP6V0A2-CDG, SLC35A2-CDG, and SLC35A3-CDG, are described as illustrative examples.

7.
Mol Genet Metab ; 124(3): 228-235, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29759592

RESUMO

Congenital disorders of glycosylation (CDG) linked to defects in Golgi apparatus homeostasis constitute an increasing part of these rare inherited diseases. Among them, COG-CDG, ATP6V0A2-CDG, TMEM199-CDG and CCDC115-CDG have been shown to disturb Golgi vesicular trafficking and/or lumen pH acidification. Here, we report 3 new unrelated cases of CCDC115-CDG with emphasis on diagnosis difficulties related to strong phenotypic similarities with mitochondriopathies, Niemann-Pick disease C and Wilson Disease. Indeed, while two individuals clinically presented with early and severe liver fibrosis and cirrhosis associated with neurological symptoms, the other one "only" showed isolated and late severe liver involvement. Biological results were similar to previously described patients, including hypercholesterolemia, elevated alkaline phosphatases and defects in copper metabolism. CDG screening and glycosylation study finally led to the molecular diagnosis of CCDC115-CDG. Besides pointing to the importance of CDG screening in patients with unexplained and severe liver disease, these reports expand the clinical and molecular phenotypes of CCDC115-CDG. The hepatic involvement is particularly addressed. Furthermore, hypothesis concerning the pathogenesis of the liver disease and of major biological abnormalities are proposed.

8.
J Chromatogr A ; 1532: 238-245, 2018 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-29221866

RESUMO

ApolipoproteinC-III (ApoC-III) is a human plasma glycoprotein whose O-glycosylation can be altered as a result of congenital disorders of glycosylation (CDG). ApoC-III exhibits three major glycoforms whose relative quantification is of utmost importance for the diagnosis of CDG patients. Considering the very close structures of these glycoforms and their tendency to adsorb on the capillary, a thorough optimization of capillary electrophoresis (CE) parameters including preconditioning and in-between rinsing procedures was required to efficiently separate all the ApoC-III glycoforms. Permanent coatings did not contribute to high resolution separations. A fast and reliable method based on a bare-silica capillary combining the effect of urea and diamine additives allowed to separate up to six different ApoC-III forms. We demonstrated by a combination of MALDI-TOF mass spectrometry (MS) analyses and CE of intact and neuraminidase-treated samples that this method well resolved glycoforms differing not only by their sialylation degree but also by carbamylation state, an undesired chemical modification of primary amines. This method allowed to demonstrate the carbamylation of ApoC-III glycoforms for the first time. Our CZE method proved robust and accurate with excellent intermediate precision regarding migration times (RSDs < 0.7%) while RSDs for peak areas were less than 5%. Finally, the quality of three distinct batches of commercial ApoC-III obtained from different suppliers was assessed and compared. Quite similar but highly structurally heterogeneous ApoC-III profiles were observed for these samples.


Assuntos
Apolipoproteína C-III/análise , Artefatos , Eletroforese Capilar/métodos , Glicoproteínas/análise , Aminoácidos/química , Tampões (Química) , Glicosilação , Humanos , Neuraminidase/metabolismo , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Raios Ultravioleta
9.
J Med Internet Res ; 19(11): e360, 2017 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-29117929

RESUMO

BACKGROUND: The prevalence of abdominal obesity and type 2 diabetes mellitus (T2DM) is a public health challenge. New solutions need to be developed to help patients implement lifestyle changes. OBJECTIVE: The objective of the study was to evaluate a fully automated Web-based intervention designed to help users improve their dietary habits and increase their physical activity. METHODS: The Accompagnement Nutritionnel de l'Obésité et du Diabète par E-coaching (ANODE) study was a 16-week, 1:1 parallel-arm, open-label randomized clinical trial. Patients with T2DM and abdominal obesity (n=120, aged 18-75 years) were recruited. Patients in the intervention arm (n=60) had access to a fully automated program (ANODE) to improve their lifestyle. Patients were asked to log on at least once per week. Human contact was limited to hotline support in cases of technical issues. The dietetic tool provided personalized menus and a shopping list for the day or the week. Stepwise physical activity was prescribed. The control arm (n=60) received general nutritional advice. The primary outcome was the change of the dietary score (International Diet Quality Index; DQI-I) between baseline and the end of the study. Secondary endpoints included changes in body weight, waist circumference, hemoglobin A1c (HbA1c) and measured maximum oxygen consumption (VO2 max). RESULTS: The mean age of the participants was 57 years (standard deviation [SD] 9), mean body mass index was 33 kg/m² (SD 4), mean HbA1c was 7.2% (SD 1.1), and 66.7% (80/120) of participants were women. Using an intention-to-treat analysis, the DQI-I score (54.0, SD 5.7 in the ANODE arm; 52.8, SD 6.2 in the control arm; P=.28) increased significantly in the ANODE arm compared to the control arm (+4.55, SD 5.91 vs -1.68, SD 5.18; between arms P<.001). Body weight, waist circumference, and HbA1c changes improved significantly in the intervention. CONCLUSIONS: Among patients with T2DM and abdominal obesity, the use of a fully automated Web-based program resulted in a significant improvement in dietary habits and favorable clinical and laboratory changes. The sustainability of these effects remains to be determined. TRIAL REGISTRATION: ClinicalTrials.gov NCT02343107; http://clinicaltrials.gov/ct2/show/NCT02343107 (Archived by WebCite at http://www.webcitation.org/6uVMKPRzs).


Assuntos
Diabetes Mellitus Tipo 2/terapia , Educação a Distância/métodos , Hemoglobina A Glicada/metabolismo , Internet/estatística & dados numéricos , Estilo de Vida , Obesidade Abdominal/terapia , Telemedicina/métodos , Adolescente , Adulto , Idoso , Feminino , Hemoglobina A Glicada/análise , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
10.
J Med Genet ; 54(12): 843-851, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28954837

RESUMO

BACKGROUND: Phosphomannomutase 2-congenital disorder of glycosylation (PMM2-CDG) is a multisystem inborn error of metabolism. OBJECTIVES: To better characterise the natural history of PMM2-CDG. METHODS: Medical charts of 96 patients with PMM2-CDG (86 families, 41 males, 55 females) were retrospectively reviewed. Data on clinical, laboratory and molecular parameters at diagnosis were analysed. Follow-up data at last examination were reported for 25 patients. RESULTS: The patients were born between 1963 and 2011. Diagnosis of PMM2-CDG was made at a mean (SD) age of 6.8 (8.5) years. The presenting signs were mostly neurological (hypotonia, intellectual disability, cerebellar syndrome) and observed in almost all the patients. A total of 38 patients (14 males, 24 females) exhibited, in addition to neurological signs, visceral features including at least one of these: feeding difficulty requiring a nutritional support (n=23), cardiac features (n=20; pericarditis: 14, cardiac malformation: 9, cardiomyopathy: 2), hepato-gastrointestinal features (n=12; chronic diarrhoea: 7, protein-losing enteropathy: 1, ascites: 3, liver failure: 1, portal hypertension: 1), kidney features (n=4; nephrotic syndrome: 2, tubulopathy: 2) and hydrops fetalis (n=1). Twelve patients died at a mean age of 3.8 years (especially from pericarditis and other cardiac issues). Laboratory abnormalities mostly included elevated transaminases and abnormal coagulation parameters. High thyreostimulin levels, hypocholesterolemia, hypoalbuminemia and elevated transaminases were associated with the visceral phenotype. Besides the common Arg141His PMM2 variant harboured by half of the patients, 45 different variants were observed. CONCLUSIONS: PMM2-CDG clinical phenotype is heterogeneous in terms of clinical course, with no clear division between neurological and visceral presentations.


Assuntos
Defeitos Congênitos da Glicosilação/diagnóstico , Defeitos Congênitos da Glicosilação/genética , Estudos de Associação Genética , Fosfotransferases (Fosfomutases)/genética , Adolescente , Alelos , Substituição de Aminoácidos , Criança , Pré-Escolar , Defeitos Congênitos da Glicosilação/mortalidade , Feminino , Seguimentos , Humanos , Lactente , Masculino , Mutação , Fenótipo , Fosfotransferases (Fosfomutases)/metabolismo
11.
Clin Chim Acta ; 470: 70-74, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28457853

RESUMO

Congenital disorders of glycosylation (CDGs) are rare inherited disorders affecting glycosylation of proteins and lipids and sharing very heterogeneous multivisceral symptoms. The biochemical screening of these diseases is currently limited to electrophoresis or HPLC separation/quantification of serum transferrin glycoforms and is relatively frequently hampered by genetic polymorphism. Further, it has been shown that transferrin glycosylation can be very poorly affected in confirmed CDGs. We developed a fast and simple two-dimensional (2-DE) Western-blot analysis applied to the simultaneous detection of various serum glycoproteins, i.e. haptoglobin, α1-anti-trypsin, transferrin and α1-acid glycoprotein, and applied it to a large cohort of CDGs and secondary glycosylation disorders. When separated using 2-DE, haptoglobin ß glycoforms showed clear abnormalities in all interpretable CDG type I and CDG type II patterns. Although secondary glycosylation defects such as alcoholism, untreated fructosemia and bacterial neuraminidase remain to be excluded, we showed that 2-DE pattern of haptoglobin ß glycoforms thus constitute a very reliable additional biomarker of all types of CDGs. Coupled with common screening techniques and glycans mass spectrometry, it can orientate and facilitate the way towards CDG molecular diagnostic.


Assuntos
Defeitos Congênitos da Glicosilação/metabolismo , Eletroforese em Gel Bidimensional , Haptoglobinas/metabolismo , Biomarcadores/metabolismo , Glicosilação , Haptoglobinas/isolamento & purificação , Humanos , Transferrina/metabolismo
12.
Ann Biol Clin (Paris) ; 74(5): 607-612, 2016 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-27707675

RESUMO

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease considered as an autoimmune disease. To identify new biomarkers of PBC, serum profiling analysis using Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) was employed. Twelve patients with either asymptomatic PBC (group 1, n=6) or PBC with a poor response to UDCA (group 2, n=6), were compared to healthy controls (group 3, n=6). Analysing the 18 sera by using four SELDI-TOF arrays under various conditions, we found four biomarkers of PBC at 5.9, 8.6, 8.9 and 9.0 kDa. The combination of the two arrays IMAC-40/Zn2+ and CM-10/pH 7 improved the positive diagnosis of this disease. We also found a biomarker of severity of PBC at 95.2 kDa on LSAX-30 array which characterized patients with a bad prognosis. In conclusion, our study identified several serum proteomics signatures as potential biomarkers of PBC for its diagnosis or prognosis.


Assuntos
Biomarcadores/sangue , Proteínas Sanguíneas/metabolismo , Cirrose Hepática Biliar/diagnóstico , Proteômica/métodos , Adulto , Idoso , Proteínas Sanguíneas/análise , Estudos de Casos e Controles , Feminino , Humanos , Cirrose Hepática Biliar/sangue , Masculino , Pessoa de Meia-Idade , Prognóstico , Análise Serial de Proteínas/métodos
13.
Proteomics Clin Appl ; 9(7-8): 787-93, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25641685

RESUMO

PURPOSE: The O-glycan abnormalities accompanying some congenital disorders of glycosylation, namely conserved oligomeric Golgi-congenital disorders of glycosylation (COG-CDGs) and ATP6V0A2-CDGs, are mainly detected using electrophoresis methods applied to circulating apolipoprotein C-III. The objective of this study was to evaluate the reliability of MALDI-TOF MS of apoC-III for the detection and characterization of CDG-associated O-glycan defects. EXPERIMENTAL DESIGN: plasmas from CDG-negative, COG-CDG, and ATP6V0A2-CDG patients were analyzed and results were compared to those obtained using 2DE followed by Western blot. RESULTS: MALDI-TOF of apoC-III allowed to detect various significant O-glycan abnormalities in CDG-patients with emphasis to COG-CDG. Furthermore, in CDG samples, comparison study between 2DE and MALDI-TOF showed a particular behavior of monosialylated apoC-III in the mass spectrometer that could be related to an abnormal O-glycan structure. CONCLUSIONS AND CLINICAL RELEVANCE: MALDI-TOF MS appears as a powerful technique for the analysis of apoC-III glycoforms for potential routine screening of COG- and ATP6V0A2-CDGs.


Assuntos
Apolipoproteína C-III/metabolismo , Defeitos Congênitos da Glicosilação/metabolismo , Eletroforese em Gel Bidimensional/métodos , Glicoproteínas/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Western Blotting , Humanos , Mucinas/química , Mucinas/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo
14.
Transfusion ; 55(6 Pt 2): 1563-71, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25556575

RESUMO

BACKGROUND: Pneumococcal hemolytic uremic syndrome (P-HUS) is a rare but severe complication of invasive pneumococcal disease (IPD) in young children. Consensual biologic diagnosis criteria are currently lacking. STUDY DESIGN AND METHODS: A prospective study was conducted on 10 children with culture-confirmed IPD. Five presented with full-blown P-HUS, three had an incomplete form with hemolytic anemia and mild or no uremia (P-HA), and two had neither HUS nor HA. Thomsen-Friedenreich (T), Th, and Tk cryptantigens and sialic acid expression were determined on red blood cells (RBCs) with peanut (PNA), Glycine soja (SBA), Bandeiraea simplicifolia II, and Maackia amurensis lectins. Plasma concentrations of the major endogenous T-antigen-binding protein, galectin-3 (Gal-3), were analyzed. RESULTS: We found that RBCs strongly reacted with PNA and SBA lectins in all P-HUS and P-HA patients. Three P-HUS and three P-HA patients showed also concomitant Tk activation. Direct antiglobulin test (DAT) was positive in three P-HUS (one with anti-C3d and two with anti-IgG) and two P-HA patients (one with anti-C3d and one with anti-IgG). RBCs derived from the two uncomplicated IPD patients reacted with PNA but not with SBA lectin. Gal-3 plasma concentrations were increased in all P-HUS patients. CONCLUSIONS: The results indicate high levels of neuraminidase activity and desialylation in both P-HUS and P-HA patients. T-antigen activation is more sensitive than DAT for P-HUS diagnosis. Combining PNA and SBA lectins is needed to improve the specificity of T-antigen activation. High concentrations of Gal-3 in P-HUS patients suggest that Gal-3 may contribute to the pathogenesis of P-HUS.


Assuntos
Anemia Hemolítica/microbiologia , Antígenos Glicosídicos Associados a Tumores/metabolismo , Eritrócitos/metabolismo , Galectina 3/sangue , Síndrome Hemolítico-Urêmica/microbiologia , Infecções Pneumocócicas/complicações , Streptococcus pneumoniae/fisiologia , Anemia Hemolítica/sangue , Anemia Hemolítica/imunologia , Antígenos Glicosídicos Associados a Tumores/imunologia , Teste de Coombs , Eritrócitos/imunologia , Feminino , Síndrome Hemolítico-Urêmica/sangue , Síndrome Hemolítico-Urêmica/imunologia , Humanos , Lactente , Masculino , Neuraminidase/metabolismo , Infecções Pneumocócicas/sangue , Infecções Pneumocócicas/imunologia , Estudos Retrospectivos
15.
J Biol Chem ; 289(17): 11816-28, 2014 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-24619423

RESUMO

Beyond its presence in stable microtubules, tubulin acetylation can be boosted after UV exposure or after nutrient deprivation, but the mechanisms of microtubule hyperacetylation are still unknown. In this study, we show that this hyperacetylation is a common response to several cellular stresses that involves the stimulation of the major tubulin acetyltransferase MEC-17. We also demonstrate that the acetyltransferase p300 negatively regulates MEC-17 expression and is sequestered on microtubules upon stress. We further show that reactive oxygen species of mitochondrial origin are required for microtubule hyperacetylation by activating the AMP kinase, which in turn mediates MEC-17 phosphorylation upon stress. Finally, we show that preventing microtubule hyperacetylation by knocking down MEC-17 affects cell survival under stress conditions and starvation-induced autophagy, thereby pointing out the importance of this rapid modification as a broad cell response to stress.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Acetiltransferases/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Acetiltransferases/genética , Animais , Sequência de Bases , Linhagem Celular , Humanos , Camundongos , Microtúbulos/metabolismo , RNA Interferente Pequeno
16.
Malar J ; 12: 250, 2013 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-23866736

RESUMO

BACKGROUND: Safety surveillance of widely used artemisinin-based combination therapy (ACT) is essential, but tolerability data in the over five years age group are largely anecdotal. METHODS: Two open-label, randomized trials were conducted in Nimba County, Liberia: i) the main tolerability trial with 1,000 Plasmodium falciparum malaria patients aged over five years (Study-T), and, ii) an efficacy trial with a secondary objective of collecting tolerability data among 300 children age six to 59 months (Study-E). In both studies patients were randomized to fixed-dose artesunate-amodiaquine (ASAQ Winthrop®) or artemether-lumefantrine (AL, Coartem®), respectively. Clinical- and laboratory-adverse events (AEs) were recorded until day 28. RESULTS: Study-T: most patients experienced at least one AE. Severe AEs were few, primarily asymptomatic blood system disorders or increased liver enzyme values. No treatment or study discontinuation occurred. Mild or moderate fatigue (39.8% vs 16.3%, p < 0.001), vomiting (7.1% vs 1.6%, p < 0.001), nausea (3.2% vs 1.0%, p = 0.01), and anaemia (14.9% vs 9.8%, p = 0.01) were more frequently recorded in the ASAQ versus AL arm. Study-E: mild or moderate AEs were common, including anaemia, fatigue, vomiting or diarrhoea. The few severe events were asymptomatic blood system disorders and four clinical events (pneumonia, malaria, vomiting and stomatitis). CONCLUSION: Both ASAQ and AL were well tolerated in patients of all age groups. No unexpected AEs occurred. Certain mild or moderate AEs were more frequent in the ASAQ arm. Standardised safety surveillance should continue for all forms of ACT. TRIAL REGISTRATION: The protocols were registered with Current Controlled Trials, under the identifier numbers ISRCTN40020296, ISRCTN51688713, (http://www.controlled-trials.com).


Assuntos
Amodiaquina/efeitos adversos , Antimaláricos/efeitos adversos , Artemisininas/efeitos adversos , Etanolaminas/efeitos adversos , Fluorenos/efeitos adversos , Malária Falciparum/tratamento farmacológico , Adolescente , Adulto , Amodiaquina/uso terapêutico , Antimaláricos/uso terapêutico , Combinação Arteméter e Lumefantrina , Artemisininas/uso terapêutico , Criança , Pré-Escolar , Combinação de Medicamentos , Etanolaminas/uso terapêutico , Feminino , Fluorenos/uso terapêutico , Humanos , Lactente , Malária Falciparum/epidemiologia , Masculino , Pessoa de Meia-Idade , Adulto Jovem
17.
J Clin Lab Anal ; 26(5): 384-97, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23001985

RESUMO

BACKGROUND: Dimension Vista® analyzer combines four technologies (photometry, nephelometry, V-LYTE® integrated multisensor potentiometry, and LOCI® chemiluminescence) into one high-throughput system. METHODS: We assessed analytical performance of assays routinely performed in our emergency laboratory according to the VALTEC protocol, and practicability. RESULTS: Precision was good for most parameters. Analytical domain was large and suitable for undiluted analysis in most clinical settings encountered in our hospital. Data were comparable and correlated to our routine analyzers (Roche Modular DP®, Abbott AXSYM®, Siemens Dimension® RxL, and BN ProSpec®). Performance of nephelometric and LOCI modules was excellent. Functional sensitivity of high-sensitivity C-reactive protein and cardiac troponin I were 0.165 mg/l and 0.03 ng/ml, respectively (coefficient of variation; CV < 10%). The influence of interfering substances (i.e., hemoglobin, bilirubin, or lipids) was moderate, and Dimension Vista® specifically alerted for interference according to HIL (hemolysis, icterus, lipemia) indices. Good instrument performance and full functionality (no reagent or sample carryover in the conditions evaluated, effective sample-volume detection, and clot detection) were confirmed. Simulated routine testing demonstrated excellent practicability, throughput, ease of use of software and security. CONCLUSION: Performance and practicability of Dimension Vista® are highly suitable for both routine and emergency use. Since no volume detection and thus no warning is available on limited sample racks, pediatric samples require special caution to the Siemens protocol to be analyzed in secured conditions. Our experience in routine practice is also discussed, i.e., the impact of daily workload, "manual" steps resulting from dilutions and pediatric samples, maintenances, flex hydration on instrument's performance on throughput and turnaround time.


Assuntos
Automação/instrumentação , Técnicas de Laboratório Clínico/instrumentação , Técnicas de Laboratório Clínico/métodos , Fotometria/instrumentação , Integração de Sistemas , Automação/normas , Coagulação Sanguínea , Proteína C-Reativa/análise , Técnicas de Laboratório Clínico/normas , Fibrina/análise , Fibrina/metabolismo , Humanos , Limite de Detecção , Modelos Teóricos , Fotometria/normas , Reprodutibilidade dos Testes , Troponina I/sangue
18.
Naunyn Schmiedebergs Arch Pharmacol ; 384(4-5): 407-19, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21541759

RESUMO

NME/NDPK family proteins are involved in the control of intracellular nucleotide homeostasis as well as in both physiological and pathological cellular processes, such as proliferation, differentiation, development, apoptosis, and metastasis dissemination, through mechanisms still largely unknown. One family member, NME1/NDPK-A, is a metastasis suppressor, yet the primary physiological functions of this protein are still missing. The purpose of this study was to identify new NME1/NDPK-A-dependent biological functions and pathways regulated by this gene in the liver. We analyzed the proteomes of wild-type and transgenic NME1-null mouse livers by combining two-dimensional gel electrophoresis and mass spectrometry (matrix-assisted laser desorption/ionization time of flight and liquid chromatography-tandem mass spectrometry). We found that the levels of three proteins, namely, phenylalanine hydroxylase, annexin IV, and elongation factor 1 Bα (EF-1Bα), were strongly reduced in the cytosolic fraction of NME1(-/-) mouse livers when compared to the wild type. This was confirmed by immunoblotting analysis. No concomitant reduction in the corresponding messenger RNAs or of total protein level was observed, however, suggesting that NME1 controls annexin IV and EF-1Bα amounts by post-translational mechanisms. NME1 deletion induced a change in the subcellular location of annexin IV in hepatocytes resulting in enrichment of this protein at the plasma membrane. We also observed a redistribution of EF-1Bα in NME1(-/-) hepatocytes to an intracytoplasmic compartment that colocalized with a marker of the reticulum endoplasmic. Finally, we found reduced expression of annexin IV coincident with decreased NME1 expression in a panel of different carcinoma cell lines. Taken together, our data suggest for the first time that NME1 might regulate the subcellular trafficking of annexin IV and EF-1Bα. The potential role of these proteins in metastatic dissemination is discussed.


Assuntos
Anexina A4/metabolismo , Fígado/enzimologia , Nucleosídeo NM23 Difosfato Quinases/fisiologia , Fator 1 de Elongação de Peptídeos/genética , Fator 1 de Elongação de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Anexina A4/genética , Western Blotting , Linhagem Celular Tumoral , Citosol/enzimologia , Citosol/metabolismo , Eletroforese em Gel Bidimensional , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/metabolismo , Humanos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Nucleosídeo NM23 Difosfato Quinases/genética , Transporte Proteico , Proteômica , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem
19.
Nat Protoc ; 2(3): 481-5, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17406610

RESUMO

We describe a protocol for easy isolation and culture of human umbilical vein endothelial cells (HUVECs) to supply every researcher with a method that can be applied in cell biology laboratories with minimum equipment. Endothelial cells (ECs) are isolated from umbilical vein vascular wall by a collagenase treatment, then seeded on fibronectin-coated plates and cultured in a medium with Earles' salts and fetal calf serum (FCS), but without growth factor supplementation, for 7 days in a 37 degrees C-5% CO2 incubator. Cell confluency can be monitored by phase-contrast microscopy; ECs can be characterized using cell surface or intracellular markers and checked for contamination. Various protocols can be applied to HUVECs, from simple harvesting to a particular solubilization of proteins for proteomic analysis.


Assuntos
Técnicas de Cultura de Células/métodos , Células Endoteliais/citologia , Veias Umbilicais/citologia , Colagenases , Humanos , Microscopia de Contraste de Fase
20.
Genomics Proteomics Bioinformatics ; 4(2): 134-6, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16970552

RESUMO

PHProteomicDB is a PHP-written module to help researchers in proteomics to share two-dimensional electrophoresis gel data using personal web sites. No technical or PHP knowledge is necessary except a few basics about web site management. PHProteomicDB has a user-friendly administration interface to enter and update data. It creates web pages on the fly displaying gel characteristics, gel pictures, and numbered gel spots with their related identifications pointing to their reference pages in protein databanks. The module is freely available at http://www.huvec.com/index.php3?rub=Download.


Assuntos
Bases de Dados de Proteínas , Internet , Proteoma/análise , Proteômica , Animais , Eletroforese em Gel Bidimensional/métodos , Humanos , Processamento de Imagem Assistida por Computador/métodos , Proteômica/métodos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA