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1.
PLoS One ; 15(12): e0242445, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33301490

RESUMO

Acyl-CoA dehydrogenase 10 (Acad10)-deficient mice develop impaired glucose tolerance, peripheral insulin resistance, and abnormal weight gain. In addition, they exhibit biochemical features of deficiencies of fatty acid oxidation, such as accumulation of metabolites consistent with abnormal mitochondrial energy metabolism and fasting induced rhabdomyolysis. ACAD10 has significant expression in mouse brain, unlike other acyl-CoA dehydrogenases (ACADs) involved in fatty acid oxidation. The presence of ACAD10 in human tissues was determined using immunohistochemical staining. To characterize the effect of ACAD10 deficiency on the brain, micro-MRI and neurobehavioral evaluations were performed. Acad10-deficient mouse behavior was examined using open field testing and DigiGait analysis for changes in general activity as well as indices of gait, respectively. ACAD10 protein was shown to colocalize to mitochondria and peroxisomes in lung, muscle, kidney, and pancreas human tissue. Acad10-deficient mice demonstrated subtle behavioral abnormalities, which included reduced activity and increased time in the arena perimeter in the open field test. Mutant animals exhibited brake and propulsion metrics similar to those of control animals, which indicates normal balance, stability of gait, and the absence of significant motor impairment. The lack of evidence for motor impairment combined with avoidance of the center of an open field arena and reduced vertical and horizontal exploration are consistent with a phenotype characterized by elevated anxiety. These results implicate ACAD10 function in normal mouse behavior, which suggests a novel role for ACAD10 in brain metabolism.


Assuntos
Acil-CoA Desidrogenase/genética , Ansiedade/genética , Encéfalo/enzimologia , Metabolismo Energético/genética , Mitocôndrias/enzimologia , Acil-CoA Desidrogenase/deficiência , Acil-CoA Desidrogenase/metabolismo , Animais , Ansiedade/enzimologia , Ansiedade/fisiopatologia , Comportamento Animal , Encéfalo/diagnóstico por imagem , Carnitina/análogos & derivados , Carnitina/metabolismo , Marcha/fisiologia , Humanos , Rim/enzimologia , Fígado/enzimologia , Pulmão/enzimologia , Imageamento por Ressonância Magnética , Aprendizagem em Labirinto , Camundongos , Camundongos Knockout , Músculo Esquelético/enzimologia , Pâncreas/enzimologia , Peroxissomos/enzimologia
2.
Am J Nurs ; 120(5): 58-62, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32332369

RESUMO

This is the seventh article in a new series about evidence-based practice (EBP) that builds on AJN's award-winning previous series-Evidence-Based Practice, Step by Step-published between 2009 and 2011 (to access the series, go to http://links.lww.com/AJN/A133). This follow-up series features exemplars illustrating the various strategies that can be used to implement EBP changes-one of the most challenging steps in the EBP process.


Assuntos
Fibrose Cística , Prática Clínica Baseada em Evidências , Adesão à Medicação , Pâncreas/enzimologia , Autogestão , Adolescente , Criança , Fibrose Cística/tratamento farmacológico , Fibrose Cística/enzimologia , Humanos , Inovação Organizacional , Participação dos Interessados , Inquéritos e Questionários , Aumento de Peso
3.
J Magn Reson Imaging ; 50(2): 417-423, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30637845

RESUMO

BACKGROUND: Pancreatic exocrine insufficiency is one of the causes of malabsorption syndrome. In many cases of malabsorption syndrome, pancreatic exocrine insufficiency can be treated with pancreatic enzyme replacement therapy. Therefore, it is important to detect pancreatic endocrine insufficiency as early and accurately as possible. Recent studies have shown that cine-dynamic MR cholangiopancreatography (MRCP) may be useful to evaluate pancreatic exocrine function PURPOSE: To identify abdominal symptoms that suggest decreased flow of pancreatic enzyme secretion for which cine-dynamic MRCP should be performed to diagnose pancreatic exocrine insufficiency. STUDY TYPE: Prospective. POPULATION: In all, 111 patients with various types of abdominal symptoms. FIELD STRENGTH/SEQUENCE: 5 T or 3 T, MRCP with spatially selective inversion recovery pulse (cine-dynamic MRCP). ASSESSMENT: Cine-dynamic MRCP was performed and an 18-question clinical questionnaire on abdominal symptoms was administered. The secretion grade derived from cine-dynamic MRCP was compared between those answering "yes" and "no" for all 18 items STATISTICAL TESTS: Univariate analysis and further analyzed using multiple regression analysis. The associations between the secretion grade and the items in the clinical questionnaire were analyzed by univariate analysis and further analyzed using multiple regression analysis. RESULTS: The following three items showed significantly negative correlations with secretion grade: Q9, Does your rectal gas smell foul? (ß = -0.44, P = 0.001); Q13, Is stool quantity large? (ß = -0.41, P = 0.001); and Q18, Are your stools soft? (ß = -0.53, P < 0.001). No significant correlations with exocrine pancreatic function measured by cine-dynamic MRCP were seen for the remaining 15 abdominal symptom items. DATA CONCLUSION: Abdominal symptoms that suggest decreased flow of pancreatic enzyme secretion were foul rectal gas, large stool, and soft stool. Pancreatic exocrine insufficiency due to decreased pancreatic enzyme flow may be suspected in patients with these abdominal symptoms. LEVEL OF EVIDENCE: 1 Technical Efficacy Stage: 3 J. Magn. Reson. Imaging 2019;50:417-423.


Assuntos
Colangiopancreatografia por Ressonância Magnética/métodos , Insuficiência Pancreática Exócrina/diagnóstico por imagem , Insuficiência Pancreática Exócrina/enzimologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pâncreas/diagnóstico por imagem , Pâncreas/enzimologia , Estudos Prospectivos , Adulto Jovem
4.
Asia Pac J Clin Nutr ; 27(3): 512-518, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29737796

RESUMO

BACKGROUND AND OBJECTIVES: Persistent diarrhea has been proven to cause pancreatic exocrine insufficiency, due to decreased stimulation to the pancreas caused by prolonged mucosal injury. Pancreatic enzyme replacement therapy (PERT) given in conjunction to regular treatment is thought to be beneficial in replacing this pancreatic enzyme deficiency, avoiding the need of elemental diet. This study aims to evaluate the benefit of PERT in chil-dren with persistent diarrhea. METHODS AND STUDY DESIGN: This is a randomized, two double-blind parallel group, placebo-controlled clinical trial to evaluate the effects of pancreatic enzyme supplementation in persistent diar-rhea. Children age 6-60 months were recruited from pediatric inpatient and outpatient units of five hospitals in Jakarta. Subjects was randomly assigned to either pancreatic enzyme 8371 USP unit of lipase or placebo, 3 times daily for 1 month, as an adjunctive therapy to standard treatment. Subjects were then reevaluated at 2 weeks and 4 weeks interval after administration of enzyme or placebo. Variables observed were length of diarrhea after the start of intervention, change in serum prealbumin, and change in FE-1 between week 0 and week 4. RESULTS: Pan-creatic enzyme supplementation shortens the length of diarrhea by 7 days in the intervention group compared to placebo (p=0.019). Serum prealbumin and FE-1 shows trend that favors the intervention group, although not sta-tistically significant (p>0.05). CONCLUSION: PERT is clinically effective in reducing the length of diarrhea, thus minimizing the need, accessibility and costs of an elemental diet.


Assuntos
Diarreia/tratamento farmacológico , Terapia de Reposição de Enzimas , Alimentos Formulados , Lipase/uso terapêutico , Pâncreas/enzimologia , Criança , Pré-Escolar , Método Duplo-Cego , Feminino , Humanos , Lactente , Lipase/administração & dosagem , Masculino , Terapia Nutricional
5.
Anal Bioanal Chem ; 410(17): 4213-4218, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29679114

RESUMO

Pancreatic α-amylase plays an important role in dietary starch hydrolysis in the small intestine and participates in enhanced glucose concentration after meals. It seems to be a problem for diabetic patients, who suffer from longer postprandial hyperglycemia after meal consumption than healthy people. There are commercially available drugs that inhibit α-amylase and thus reduce the postprandial hyperglycemia effect. However, these drugs may cause severe side effects. Conversely, some naturally occurring flavonoids were suggested to have an α-amylase-inhibiting effect without any side effects. There had been no rapid, undemanding method in terms of sample and reagent preparation that would enable screening of many potential inhibitors. Therefore, we developed an online capillary electrophoresis method to monitor α-amylase activity in the presence of an inhibitor. Each reaction constituent was introduced separately, directly into a capillary where the reagents were mixed by diffusion, which resulted in a 5-min analysis including conditioning of the capillary. We applied the method to test the inhibitory effect of flavonoid standards and their mixture and we investigated the inhibitory effect of ethanolic extract from Betula pendula bark. The developed method presents a faster and less expensive alternative to previously described offline methods. Graphical abstract Online CE screening of α-amylase inhibitors.


Assuntos
Eletroforese Capilar , Inibidores Enzimáticos/análise , alfa-Amilases , Eletroforese Capilar/economia , Eletroforese Capilar/métodos , Estabilidade Enzimática , Humanos , Sistemas On-Line , Pâncreas/enzimologia , Fatores de Tempo , alfa-Amilases/antagonistas & inibidores
7.
Biomed Res Int ; 2017: 5094592, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29082247

RESUMO

The present paper proposed an interactive segmentation method of pancreases in abdominal computed tomography (CT) images based on the anatomical knowledge of medical doctors and the statistical information of pancreas shapes. This segmentation method consisted of two phases: training and testing. In the training phase, pancreas regions were manually extracted from sample CT images for training, and then a probabilistic atlas (PA) was constructed from the extracted regions. In the testing phase, a medical doctor selected seed voxels for a pancreas and background in a CT image for testing by use of our graphical user interface system. The homography transformation was used to fit the PA to the seeds. The graph cut technique whose data term was weighted by the transformed PA was applied to the test image. The seed selection, the atlas transformation, and the graph cut were executed iteratively. This doctor-in-the-loop segmentation method was applied to actual abdominal CT images of fifteen cases. The experimental results demonstrated that the proposed method was more accurate and effective than the conventional graph cut.


Assuntos
Pâncreas/diagnóstico por imagem , Reconhecimento Automatizado de Padrão/métodos , Intensificação de Imagem Radiográfica/métodos , Tomografia Computadorizada por Raios X/métodos , Abdome/diagnóstico por imagem , Abdome/patologia , Algoritmos , Humanos , Fígado/diagnóstico por imagem , Fígado/enzimologia , Fígado/patologia , Modelos Teóricos , Pâncreas/enzimologia , Pâncreas/patologia , Pancrelipase/química , Pancrelipase/isolamento & purificação , Interpretação de Imagem Radiográfica Assistida por Computador
8.
Br J Nutr ; 117(2): 177-186, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28132656

RESUMO

Hyperlipidaemia is a major cause of atherosclerosis and related CVD and can be prevented with natural substances. Previously, we reported that a novel Bacillus-fermented green tea (FGT) exerts anti-obesity and hypolipidaemic effects. This study further investigated the hypotriglyceridaemic and anti-obesogenic effects of FGT and its underlying mechanisms. FGT effectively inhibited pancreatic lipase activity in vitro (IC50, 0·48 mg/ml) and ameliorated postprandial lipaemia in rats (26 % reduction with 500 mg/kg FGT). In hypertriglyceridaemic hamsters, FGT administration significantly reduced plasma TAG levels. In mice, FGT administration (500 mg/kg) for 2 weeks augmented energy expenditure by 22 % through the induction of plasma serotonin, a neurotransmitter that modulates energy expenditure and mRNA expressions of lipid metabolism genes in peripheral tissues. Analysis of the gut microbiota showed that FGT reduced the proportion of the phylum Firmicutes in hamsters, which could further contribute to its anti-obesity effects. Collectively, these data demonstrate that FGT decreases plasma TAG levels via multiple mechanisms including inhibition of pancreatic lipase, augmentation of energy expenditure, induction of serotonin secretion and alteration of gut microbiota. These results suggest that FGT may be a useful natural agent for preventing hypertriglyceridaemia and obesity.


Assuntos
Camellia sinensis , Metabolismo Energético/efeitos dos fármacos , Fermentação , Hiperlipidemias/sangue , Hipolipemiantes/farmacologia , Lipase/antagonistas & inibidores , Extratos Vegetais/farmacologia , Animais , Fármacos Antiobesidade/farmacologia , Fármacos Antiobesidade/uso terapêutico , Bacillus , Firmicutes , Microbioma Gastrointestinal/efeitos dos fármacos , Hiperlipidemias/tratamento farmacológico , Hipertrigliceridemia/sangue , Hipertrigliceridemia/tratamento farmacológico , Hipolipemiantes/uso terapêutico , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Mesocricetus , Camundongos , Camundongos Endogâmicos C57BL , Pâncreas/enzimologia , Fitoterapia , Extratos Vegetais/metabolismo , Extratos Vegetais/uso terapêutico , RNA Mensageiro/metabolismo , Serotonina/sangue , Chá , Triglicerídeos/sangue
9.
J Vet Intern Med ; 30(3): 764-70, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26968865

RESUMO

BACKGROUND: Feline pancreas-specific lipase (Spec fPL) is considered a useful test for the antemortem diagnosis of pancreatitis in cats. A recent study found good agreement between the results of the Spec fPL and catalytic 1,2-o-dilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin) ester (DGGR) lipase assay. Prospective studies evaluating their sensitivity and specificity are lacking. OBJECTIVES: To compare the results of the Spec fPL and the DGGR assays with a standardized histologic assessment of the pancreas. ANIMALS: Sixty client-owned cats presented for necropsy. PROSPECTIVE STUDY: Spec fPL concentrations and serum DGGR lipase activity were measured from the same blood sample. The pancreas was removed within 3 hours after euthanasia; serial transverse sections were made every 0.5 cm throughout the entire pancreas and reviewed using a histologic grading scheme. Sensitivity and specificity for the Spec fPL and DGGR assay results were determined. RESULTS: The sensitivity and specificity for the Spec fPL assay (cutoff value ≥5.4 µg/L) was 42.1 [95% confidence interval (95% CI), 29.4-55.9%] and 100% (95% CI, 31.0-100.0%). The sensitivity and specificity for the DGGR assay (cutoff value >26 U/L) was 36.8 (95% CI, 24.7-50.7%) and 100% (95% CI, 31.0-100.0%). When lymphocytic inflammation up to 10% of a section was considered normal, the sensitivity and specificity for Spec fPL assay (cutoff value ≥5.4 µg/L) was 61.1 (95% CI, 36.1-81.7%) and 69.0% (95% CI, 52.8-81.9%) and the sensitivity and specificity for the DGGR assay (cutoff value >26 U/L) was 66.7 (95% CI, 41.2-85.6%) and 78.6% (95% CI, 62.8-89.2%). CONCLUSIONS AND CLINICAL IMPORTANCE: Both lipase assays performed similarly well, but their agreement with histologic pancreatic inflammation was limited.


Assuntos
Azotemia/veterinária , Doenças do Gato/sangue , Glutaratos , Lipase/sangue , Oxazinas , Pâncreas/patologia , Pancreatite/veterinária , Animais , Azotemia/sangue , Bioensaio/veterinária , Doenças do Gato/diagnóstico , Doenças do Gato/enzimologia , Gatos , Feminino , Masculino , Pâncreas/enzimologia , Pancreatite/diagnóstico , Pancreatite/enzimologia , Sensibilidade e Especificidade
10.
Pancreas ; 43(6): 874-8, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24743380

RESUMO

OBJECTIVE: Benign pancreatic hyperenzymemia is characterized by a long-term increase of serum pancreatic enzymes in otherwise healthy subjects. This study was designed to determine (a) whether all pancreatic enzymes are elevated, (b) the extent of each enzyme increase, (c) the relative frequency of the familial form, and (d) the relative frequencies of pancreatic and salivary hyperamylasemia and macroamylasemia. METHODS: Two hundred seven asymptomatic subjects with benign pancreatic hyperenzymemia were studied during the 5-year period. Serum amylase, isoamylase, and lipase levels were assessed by immunoenzymatic assays. RESULTS: Most (n = 183; 88.4%) patients had benign pancreatic hyperenzymemia; 155 (74.9%) patients had an abnormal increase of all 3 enzymes, 15 (7.2%) patients of only lipase, and 13 (6.3%) patients of only amylase and pancreatic isoamylase. Lipase levels were the highest (1.1-21 times above upper limit). Of the 183 subjects, 72 were members of 35 different families, 15 (7.2%) had increased salivary amylase, and 9 (4.3%) had macroamylasemia. Wide day-to-day fluctuations of pancreatic enzymes, including falls within the reference ranges, were recorded. CONCLUSIONS: All enzymes were increased in benign pancreatic hyperenzymemia, with lipase showing the highest elevation. Doctors should reassure patients about the benign nature of this condition and limit repeating useless examinations.


Assuntos
Amilases/sangue , Isoamilase/sangue , Lipase/sangue , Pâncreas/enzimologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Enzimas/economia , Feminino , Seguimentos , Humanos , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Valores de Referência , Fatores de Tempo , Adulto Jovem
11.
J Colloid Interface Sci ; 417: 210-6, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-24407679

RESUMO

To investigate the surface effects of low cost mesoporous SiO2 on the properties of lipase, a series of modified mesoporous SiO2 was synthesized through various alkoxysilanes reacted with hydroxyl of SiO2 (C1-SiO2, C8-SiO2, C16-SiO2, SH-SiO2, Ph-SiO2, NH2-SiO2). Particularly, ionic liquids as novel alkoxysilane were synthesized and subjected to modify the low cost mesoporous SiO2 (CH3IL-SiO2 and COOHIL-SiO2). The porcine pancreas lipase (PPL) was immobilized on the prepared materials. The activity assay indicated that the activation of enzymatic activity site was benefit from the longer alkyl chain of alkoxysilane because of the growing hydrophobic nature. Nevertheless, the loading of lipase decreased from 69% for PPL-C1-SiO2 to 59% for PPL-C16-SiO2, reflecting the growing hydrophobic property limited the immobilization yield. The characteristic of alkoxysilane group (SH-SiO2, Ph-SiO2, NH2-SiO2) was another important factor to influence immobilization efficiency and enzymatic performance besides the alkyl chain length. The immobilization efficiency of PPL-SH-SiO2, PPL-Ph-SiO2 and PPL-NH2-SiO2 maintained at least 93%. Compared with conventional alkoxysilane, the activity of PPL-CH3IL-SiO2 improved to 2.60 folds of PPL-SiO2. The immobilization efficiency of PPL-COOHIL-SiO2 was up to 97% and the relative activity was above 62% after five recycles.


Assuntos
Enzimas Imobilizadas/química , Lipase/química , Dióxido de Silício/química , Adsorção , Animais , Reutilização de Equipamento , Interações Hidrofóbicas e Hidrofílicas , Líquidos Iônicos/química , Cinética , Modelos Moleculares , Pâncreas/química , Pâncreas/enzimologia , Porosidade , Silanos/química , Propriedades de Superfície , Suínos , Temperatura
13.
BMC Complement Altern Med ; 12: 145, 2012 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-22943464

RESUMO

BACKGROUND: Obesity is recognized as a social problem, associated with serious health risks and increased mortality. Numerous trials have been conducted to find and develop new anti-obesity drugs through herbal sources to minimize side effects associated with the present anti-obesity drugs. The present study was designed to evaluate the quality control parameters, quantitative phytochemical analysis (total phenolic, total flavonoids and total saponin content), and the anti-obesity effect of lyophilized juice (LJ) of Stellaria media (Linn.) Vill. by employing in vitro and in vivo models. METHODS: In vitro studies were performed to evaluate the inhibitory activity of LJ on pancreatic amylase and lipase. The in vivo pancreatic lipase activity was evaluated by measurement of plasma triacylglycerol levels after oral administration of lipid emulsion to swiss albino mice. Furthermore, the anti-obesity effect of LJ was assessed at two doses, 400 mg/kg and 900 mg/kg body weight in mice fed a high-fat-diet with or without LJ for 6 weeks. RESULTS: The LJ inhibited pancreatic amylase and lipase activity in vitro and elevated plasma triacylglycerol level in mice. LJ suppressed the increase in body weight, retroperitoneal adipose tissue, liver weights and serum parameters viz., total cholesterol, total triglyceride, LDL-cholesterol level at the dose of 900 mg/kg body weight of the mice fed with high fat diet. The total phenolic, flavonoid and saponin contents were found to be 0.26 mg/g, 1.4 mg/g and 1.19 µg/g respectively of LJ. CONCLUSION: The anti-obesity effects of LJ in high-fat-diet fed mice may be partly mediated through delaying the intestinal absorption of dietary fat and carbohydrate by inhibiting digestive enzymes.


Assuntos
Fármacos Antiobesidade/administração & dosagem , Obesidade/tratamento farmacológico , Extratos Vegetais/administração & dosagem , Stellaria/química , Amilases/metabolismo , Animais , Colesterol/sangue , Avaliação Pré-Clínica de Medicamentos , Lipase/metabolismo , Masculino , Camundongos , Obesidade/metabolismo , Tamanho do Órgão/efeitos dos fármacos , Pâncreas/efeitos dos fármacos , Pâncreas/enzimologia , Triglicerídeos/sangue
14.
J Sci Food Agric ; 92(3): 503-10, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21918995

RESUMO

BACKGROUND: A few common spices are known to stimulate secretion of bile with higher amount of bile acids which play a major role in digestion and absorption of dietary lipids. It would be appropriate to verify if these spices enable efficient digestion and absorption during high-fat intake. In this context, dietary ginger (0.05%), piperine (0.02%), capsaicin (0.015%), and curcumin (0.5%) were examined for their influence on bile secretion, digestive enzymes of pancreas and absorption of dietary fat in high-fat (30%) fed Wistar rats for 8 weeks. RESULTS: These spices enhanced the activity of pancreatic lipase, amylase, trypsin and chymotrypsin by 22-57%, 32-51%, 63-81% and 12-38%, respectively. Dietary intake of spices along with high-fat enhanced fat absorption. These dietary spices increased bile secretion with higher bile acid content. Stimulation of lipid mobilisation from adipose tissue was suggested by the decrease in perirenal adipose tissue weight by dietary capsaicin and piperine. This was also accompanied by prevention of the accumulation of triglyceride in liver and serum in high-fat fed rats. Activities of key lipogenic enzymes in liver were reduced which was accompanied by an increased activity of hormone-sensitive lipase. CONCLUSION: Thus, dietary ginger and other spice compounds enhance fat digestion and absorption in high-fat fed situation through enhanced secretion of bile salts and a stimulation of the activity pancreatic lipase. At the same time, the energy expenditure is facilitated by these spices to prevent the accumulation of absorbed fat.


Assuntos
Bile/metabolismo , Gorduras na Dieta/metabolismo , Digestão , Absorção Intestinal , Especiarias , Regulação para Cima , Zingiber officinale , Adiposidade , Alcaloides/administração & dosagem , Animais , Benzodioxóis/administração & dosagem , Bile/química , Ácidos e Sais Biliares/análise , Ácidos e Sais Biliares/metabolismo , Capsaicina/administração & dosagem , Curcumina/administração & dosagem , Dieta Hiperlipídica/efeitos adversos , Zingiber officinale/química , Hidrolases/química , Hidrolases/metabolismo , Índia , Metabolismo dos Lipídeos , Fígado/metabolismo , Masculino , Pâncreas/enzimologia , Pâncreas/metabolismo , Piperidinas/administração & dosagem , Alcamidas Poli-Insaturadas/administração & dosagem , Ratos Wistar , Rizoma/química , Especiarias/análise , Especiarias/economia
15.
Transplant Proc ; 43(9): 3171-5, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22099748

RESUMO

OBJECTIVES: Clostridium histolyticum expresses two classes of collagenases, C1 and C2. However, degradation of these enzymes by proteases during the fermentation or purification process may lead to numerous molecular forms that lead to inconsistent release of islets from human pancreata. This report defines the amino acid sequence of the truncated forms of C1 (C1b or C1c) that contain a single collagen-binding domain (CBD) and investigates the synergy between the different forms of C1 collagenase and C2 to degrade native collagen. METHODS: Highly purified collagenase isoforms were purified from C. histolyticum culture supernatants using established column chromatography techniques and analyzed using high-pressure liquid chromatograph (HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MS). The collagen-degrading activity (CDA) assay was used to investigate the synergy between different collagenase molecular forms. RESULTS: MS was used to confirm the sequence of full-length C2 and C1 from the reported gene sequence. These results were correlated with the molecular weights observed on the SDS- PAGE and elution after analytical anion-exchange HPLC. HPLC peaks designated as C1b and C1c were both confirmed to be C1 lacking the terminal CBD. The only difference being the cleavage site leading to a 12 amino acid difference between the two forms. A non-additive synergy in CDA relative to activity of individual collagenases was observed for C2 with each of the three C1 molecular forms. The C1 molecular forms did not display this synergy in the absence of C2. CONCLUSIONS: These observations support earlier reports that suggest the two collagenases bind to different portions of the collagen and have different specificities to cut native collagen. Although the implications of this are not yet understood, they are fundamental in advancing the understanding of how collagenases work together along with the neutral protease to breakdown the extracellular matrix for islet isolation.


Assuntos
Clostridium histolyticum/metabolismo , Colagenases/metabolismo , Bioquímica/métodos , Cromatografia Líquida de Alta Pressão/métodos , Colágeno/química , Colagenases/química , Eletroforese em Gel de Poliacrilamida/métodos , Humanos , Espectrometria de Massas/métodos , Colagenase Microbiana/química , Peso Molecular , Pâncreas/enzimologia , Peptídeo Hidrolases/química , Ligação Proteica
16.
Res Vet Sci ; 87(3): 367-72, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19386336

RESUMO

Isolation of pancreatic islets is necessary to study the molecular mechanisms underlying beta-cell demise in diabetic cats. Six collagenase-based methods of isolation were compared in 10 cat pancreata, including single and double course of collagenase, followed or not by Ficoll centrifugation or accutase, and collagenase plus accutase. Morphometric analysis was performed to measure the relative area of islet and exocrine tissue. Islet specific mRNA transcripts were quantified in isolates by real-time PCR. The single and double course of collagenase digestion was successful in each cat and provided similar islet-to-exocrine tissue ratio. Quantities of insulin mRNA did not differ between the two methods. However, on histological examination either method yielded only approximately 2% of pure islets. The other methods provided disrupted islets or insufficient samples in 1-7 cats. Although pancreas digestion with single and double course of collagenase was superior, further studies are needed to improve islet isolation in cats.


Assuntos
Gatos , Colagenases/metabolismo , Ilhotas Pancreáticas/citologia , Animais , Técnicas de Cultura de Células/veterinária , Pâncreas/enzimologia , Pâncreas/metabolismo
17.
Proteins ; 76(4): 861-81, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19291738

RESUMO

The multiple solvent crystal structures (MSCS) method uses organic solvents to map the surfaces of proteins. It identifies binding sites and allows for a more thorough examination of protein plasticity and hydration than could be achieved by a single structure. The crystal structures of bovine pancreatic ribonuclease A (RNAse A) soaked in the following organic solvents are presented: 50% dioxane, 50% dimethylformamide, 70% dimethylsulfoxide, 70% 1,6-hexanediol, 70% isopropanol, 50% R,S,R-bisfuran alcohol, 70% t-butanol, 50% trifluoroethanol, or 1.0M trimethylamine-N-oxide. This set of structures is compared with four sets of crystal structures of RNAse A from the protein data bank (PDB) and with the solution NMR structure to assess the validity of previously untested assumptions associated with MSCS analysis. Plasticity from MSCS is the same as from PDB structures obtained in the same crystal form and deviates only at crystal contacts when compared to structures from a diverse set of crystal environments. Furthermore, there is a good correlation between plasticity as observed by MSCS and the dynamic regions seen by NMR. Conserved water binding sites are identified by MSCS to be those that are conserved in the sets of structures taken from the PDB. Comparison of the MSCS structures with inhibitor-bound crystal structures of RNAse A reveals that the organic solvent molecules identify key interactions made by inhibitor molecules, highlighting ligand binding hot-spots in the active site. The present work firmly establishes the relevance of information obtained by MSCS.


Assuntos
Cristalografia por Raios X , Ribonuclease Pancreático/química , Ribonuclease Pancreático/metabolismo , Solventes/química , Animais , Domínio Catalítico , Bovinos , Simulação por Computador , Modelos Moleculares , Pâncreas/enzimologia , Ligação Proteica , Conformação Proteica , Solventes/metabolismo , Água/química , Água/metabolismo
18.
Clin Chim Acta ; 397(1-2): 87-91, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18706899

RESUMO

BACKGROUND: Disagreement continues regarding 2 fecal pancreatic elastase-1 (PE-1) ELISAs and their respective capabilities to assess pancreatic function. METHODS: The BioServ Diagnostics polyclonal PE-1 ELISA was validated and its performance characteristics compared to the previously validated ScheBo Biotech monoclonal PE-1 ELISA. Split sample study results were analyzed by Deming regression and Bland-Altman plot analysis. Data mining was utilized to explore PE-1 distribution and evaluate PE-1 and fecal fat correlation. RESULTS: Analysis demonstrates limited quantitative agreement; slope=0.9640, intercept=10.787, R(2)=0.633. Means were 228.8 and 226.2 microg PE-1/g stool for the polyclonal and monoclonal assays respectively. Bland-Altman analysis showed 91% of paired values within 2 SD of their means. There was good qualitative agreement when interpreted against established intervals with 91% of results equivalent in pancreatic function classification. The remaining 9% varied by one classification level with no bias evident. The distribution of PE-1 concentrations (n=400, 0-25 years) classified 78% of subjects with normal pancreatic function, 7% with moderate pancreatic insufficiency and 15% with severe insufficiency. There was little agreement between PE-1 and fecal fat results. CONCLUSIONS: The polyclonal PE-1 ELISA is an acceptable alternative to the monoclonal PE-1 ELISA. PE-1 is a potential substitute for fecal fat for evaluating pancreatic function.


Assuntos
Fezes/enzimologia , Pâncreas/fisiologia , Elastase Pancreática/análise , Testes de Função Pancreática , Adolescente , Adulto , Anticorpos/imunologia , Anticorpos Monoclonais/imunologia , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Humanos , Lactente , Recém-Nascido , Pâncreas/enzimologia , Elastase Pancreática/imunologia
20.
Med Wieku Rozwoj ; 7(2): 149-55, 2003.
Artigo em Polonês | MEDLINE | ID: mdl-12878785

RESUMO

OBJECTIVE: In pediatric patients, indirect tests are preferred because of their less invasive character. Among those, faecal elastase-1 test has so far been shown been shown to have the highest sensitivity and specificity. However, the role of the faecal lipase test in the diagnostic work up for pancreatic insufficiency in cystic fibrosis (CF) patients has not been defined. Therefore, the aim of the present study was to compare the sensitivity and the specificity of faecal lipase test to the faecal elastase-1 test in the assessment of exocrine pancreatic function. MATERIAL AND METHODS: 63 CF patients and 95 healthy subjects (HS) were evaluated. In all subjects, faecal elastase-1 concentration (ELISA) and lipase activity (ELISA) were measured. In 50 HS, sample-to-sample (n=3) variation from the same stool and day-to-day variation from three consecutive stools were determined twice. The presence of pancreatic insufficiency patients was documented in 55 pancreatic insufficient CF patients by the determination of faecal fat excretion and in 12 pancreatic sufficient patients by the direct test. The sensitivity and specificity of the faecal elastase-1 test and faecal lipase test were compared. RESULTS: The sample-to-sample variation (mean + SEM: 13.2+1.2% vs. 23.4+2.2%) and day-to-day variation (mean + SEM: 16.3+1.2% vs. 32.5+2.6%) were significantly lower (p<0.0001) for elastase-1 determinations than for lipase measurements. With the cut-off levels giving the same specificity for both tests (95.8%), the sensitivity of faecal elastase-1 test (87.3%) was significantly higher (p<0.04) than that of faecal lipase test (77.8%). IN CONCLUSION: Faecal lipase test is less useful in the assessment of exocrine pancreatic function sensitive than faecal elastase-1 test.


Assuntos
Fibrose Cística/enzimologia , Insuficiência Pancreática Exócrina/diagnóstico , Fezes/enzimologia , Pâncreas/enzimologia , Elastase Pancreática/análise , Testes de Função Pancreática/normas , Adolescente , Adulto , Estudos de Casos e Controles , Criança , Ensaio de Imunoadsorção Enzimática , Estudos de Avaliação como Assunto , Insuficiência Pancreática Exócrina/enzimologia , Fezes/química , Feminino , Humanos , Masculino , Análise de Regressão , Sensibilidade e Especificidade , Fatores de Tempo
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