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1.
Nutrients ; 13(7)2021 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-34371824

RESUMO

Delayed-onset muscle soreness (DOMS) is associated with increases in acute inflammatory and biochemical markers, muscle swelling, pain, and reduced functional performance. This study aimed to investigate the preventative effects of crocodile blood supplementation on DOMS induced by eccentric exercise. Sixteen healthy males were randomly allocated to either a crocodile blood (CB, n = 8) or a placebo (PL, n = 8) treatment. Participants receiving the CB treatment consumed four capsules of freeze-dried CB powder (1 g day-1) over 18 days. Participants receiving the other treatment were administered a placebo over the same period. An eccentric exercise protocol was performed, and functional performance, visual analogue scale (VAS)-measured pain, knee range of movement (ROM), thigh circumference (swelling), and cytokines, enzymes, and biochemical parameters were assessed immediately after exercise as well as after 24 h, 48 h, and 72 h. CB supplementation could significantly maintain maximum voluntary isometric contraction (MVIC) at 24 h (p = 0.001) and 48 h after exercise (p = 0.001) when comparing values at different times for the CB group. In the CB group, thigh circumference decreased only immediately after eccentric exercise (p = 0.031) in comparison with pre-eccentric exercise values. An 18-day supplementation (1 g day-1) of crocodile blood does aid in the maintenance of functional performance and muscle swelling after eccentric exercise. Our data indicate that 1 g day-1 of crocodile blood supplementation should be safe for human consumption.


Assuntos
Jacarés e Crocodilos/sangue , Suplementos Nutricionais , Exercício Físico/fisiologia , Doenças Musculares/prevenção & controle , Mialgia/prevenção & controle , Animais , Biomarcadores/análise , Método Duplo-Cego , Edema/etiologia , Edema/fisiopatologia , Edema/prevenção & controle , Voluntários Saudáveis , Humanos , Contração Isométrica/efeitos dos fármacos , Masculino , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiopatologia , Doenças Musculares/etiologia , Doenças Musculares/fisiopatologia , Mialgia/etiologia , Mialgia/fisiopatologia , Medição da Dor , Desempenho Físico Funcional , Amplitude de Movimento Articular/efeitos dos fármacos , Adulto Jovem
2.
Parasitol Res ; 120(8): 2887-2895, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34331137

RESUMO

Few data are available on the genetic identity of enteric protists Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in humans in Thailand. In this study, 254 stool samples were collected from primary school children from Ratchaburi Province at the Thai-Myanmar border and examined for Cryptosporidium spp., G. duodenalis, E. bieneusi and Cyclospora cayetanensis using PCR techniques. The genotype identity of the pathogens was determined by DNA sequence analysis of the PCR products. Cryptosporidium felis was found in 1 stool sample, G. duodenalis in 19 stool samples, and E. bieneusi in 4 stool samples. For G. duodenalis, sub-assemblage AII was the dominant genotype, but one infection with assemblage F was found. The E. bieneusi genotypes found included known genotypes D and J, and one novel genotype (HPTM1). Cyclospora cayetanensis was not detected in any samples. Results of the preliminary study indicate that children at the Thai-Myanmar border from Ratchaburi Province, Thailand are infected with diverse zoonotic genotypes of Cryptosporidium spp., G. duodenalis, and E. bieneusi.


Assuntos
Criptosporidiose , Cryptosporidium , Enterocytozoon , Giardia lamblia , Giardíase , Microsporidiose , Criança , Criptosporidiose/epidemiologia , Cryptosporidium/genética , Cryptosporidium/isolamento & purificação , Enterocytozoon/genética , Enterocytozoon/isolamento & purificação , Fezes , Genótipo , Giardia lamblia/genética , Giardia lamblia/isolamento & purificação , Giardíase/epidemiologia , Humanos , Microsporidiose/epidemiologia , Mianmar , Instituições Acadêmicas , Tailândia
3.
Protein Expr Purif ; 163: 105449, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31295559

RESUMO

The demand for steviol glycosides, non-caloric sweet components of Stevia rebaudiana Bertoni (stevia) leaves, has increased considerably as a benefit to enhance human health. However, the supply has remained challenging due to limited production, with the lack of a specific steviol glycoside hydrolyzing enzyme. In this study, a novel ß-glucosidase (EcBgl) from Enterococcus casseliflavus was cloned and expressed in Escherichia coli. An EcBgl consists of 721 amino acids corresponding to a molecular mass of 79.37 kDa. The EcBgl was purified to homogeneity, followed by enzyme characterization. The enzyme showed optimum pH and temperature at 6.0 and 37 °C, and exhibited the kinetic constants kcat/Km for pNPG and kcat/Km for stevioside of 8583 mM-1s-1 and 95.41 mM-1s-1, respectively. When compared to the stevioside hydrolyzing ß-glycosidases previously reported, EcBgl was found to be the most efficient enzyme. EcBgl also rendered hydrolysis of the stevioside to produce rubusoside, a rare steviol glycoside with a pharmaceutical solubilizing property, by cleaving at the glucose moiety. In addition, the enzyme demonstrated substantial resistance against amygdalin, so it served as a potential enzyme in agricultural and pharmaceutical applications.


Assuntos
Diterpenos do Tipo Caurano/metabolismo , Enterococcus/enzimologia , Glucosídeos/metabolismo , Glicosídeo Hidrolases/isolamento & purificação , Stevia/microbiologia , Clonagem Molecular , Enterococcus/genética , Estabilidade Enzimática , Escherichia coli/genética , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Metais/metabolismo , Especificidade por Substrato , Temperatura
4.
Int J Vitam Nutr Res ; 89(5-6): 246-254, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30982444

RESUMO

A lower serum folate level is common in older populations and is associated with increased serum homocysteine concentration. In turn, an elevated homocysteine level is associated with increased risk of cardiovascular disease and age-related diseases. Contemporary studies of folate and dietary risk factors for cardiovascular disease among the elderly population in Thailand are lacking. This cross-sectional study aimed to investigate the relationships among serum folate, homocysteine level, and nutritional status in the elderly Thai. Three hundred individuals, aged 60 years and over, underwent anthropometric and physiological measurements, and biochemical parameters, and eating habits were also determined. Folate insufficiency was found in approximately 35% of subjects. Folate and homocysteine showed a significant inverse correlation. Serum homocysteine levels rose with increasing age. Folate deficiency and high waist-to-hip ratio were associated with 7-fold and 2.5-fold increased risk for hyperhomocysteinemia, respectively. There were positive correlations between homocysteine and waist-to-hip ratio and systolic blood pressure, but a negative correlation between homocysteine and high-density lipoprotein (r = -0.239, p < 0.01), which are markers for cardiovascular disease risk. Folate negatively correlated with body mass index, waist-to-hip ratio, and diastolic blood pressure, but positively with high-density lipoprotein (r = 0.162, p < 0.01). Investigation of eating habits showed that low consumption of green leafy vegetables and high consumption of sugary foods were associated with high homocysteine levels. Given associations between nutritional status and cardiovascular disease confirmed in this study, nutrition education, holistic health promotion, and appropriate behavioral modification of eating habits represent important measures for preventing premature cardiovascular disease in the elderly Thai population.


Assuntos
Comportamento Alimentar , Idoso , Estudos Transversais , Ácido Fólico , Homocisteína , Humanos , Hiper-Homocisteinemia , Lipídeos , Pessoa de Meia-Idade , Tailândia , Vitamina B 12 , Relação Cintura-Quadril
5.
Phytochemistry ; 156: 33-42, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30172077

RESUMO

The deficiency of α-galactosidase activity in coconut endosperm has been reported to cause a disability to hydrolyze oligogalactomannan in endosperm resulting in curd coconut phenotype. However, neither the α-galactosidase encoding gene in coconut nor the mutation type has been identified and characterized in normal and curd coconuts. In this study, cDNA and genomic DNA encoding α-galactosidase gene alleles from a normal and two curd coconuts were successfully cloned and characterized. The deduced amino acid of wild type α-galactosidase contains 398 amino acid residues with a 17 N-terminal amino acids signal peptide sequence. Three mutant alleles, the first 19-amino acids from 67 to 85 (ADALVSTGLARLGYQYVNL) deletion with S137R and the second R216T, were identified from curd coconut plant no.1 while the third P250R was identified from curd coconut plant no. 10. All mutations of α-galactosidase gene were confirmed by the analysis of parental genomic DNA from normal and curd coconuts. Heterologous expression in Komagataella phaffii (Pichia pastoris) indicated that recombinant P250R, R216T and 19-amino acids deletion-S137R mutant proteins showed no α-galactosidase activity. Only the recombinant wild-type protein was able to detect for α-galactosidase activity. These results are in accordance with the no detection of α-galactosidase activity in developing curd coconut endosperms by tissue staining. While, the accumulation of enzyme activity was present in the solid endosperm of normal coconut. The full-length cDNA and parental genomic DNA sequences encoding α-galactosidase in normal coconut as well as identified curd coconut mutant alleles are reported in Genbank accession no. KJ957156 and KM001681-3. Transcription level of the α-galactosidase gene in mature curd coconut endosperm was at least 20 times higher than normal. In conclusion, absence of α-galactosidase activity caused by gene mutations associates with an accumulation of oligogalactomannan in endosperms, resulting in curd coconut phenotype.


Assuntos
Cocos/metabolismo , Endosperma/metabolismo , Mananas/metabolismo , Mutação , alfa-Galactosidase/genética , alfa-Galactosidase/metabolismo , Sequência de Aminoácidos , Cocos/enzimologia , Cocos/genética , Endosperma/enzimologia , Endosperma/genética , Alinhamento de Sequência
6.
Int J Adolesc Med Health ; 31(1)2017 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-28525348

RESUMO

Background The prevalence of obesity has increased globally, with childhood and adolescent obesity being more common in developed countries. There has been no study on teenage obesity in Bhutan. In this study, we aimed to assess the prevalence of obesity in Bhutan for the first time in order to provide a baseline for future researchers. Methods The investigation, which included 392 adolescents, aimed to identify the prevalence of overweight and obesity and its contributing factors. Anthropometric measurements, food recall and knowledge, attitude, behaviour and environment questionnaires were administered. The body mass index (BMI) cut-off points for adolescents matched with those of the US Centers for Disease Control and Prevention. Results The prevalence of overweight and obesity among the participants were 7.1% and 1.5%, respectively. The prevalence of obesity was 1.0% in females and 0.5% in males (p < 0.001). There were significant (p < 0.001) correlations between BMI and other variables; however, Pearson's χ2 test uncovered no significant associations. BMI also had no significant associations with attitude, behaviour, environment and distance travelled to school. Food recall results revealed the following findings for average food consumption: total energy intake, 3522.6 kcal; fat, 47.6 g; carbohydrate, 690.2 g; protein, 90.5 g; fibre, 20.3 g; and sodium, 12.5 g. Conclusion The results of this study clarified the prevalence of obesity among adolescents in Bhutan, who require appropriate strategies for combating overweight and obesity.

7.
J Agric Food Chem ; 65(15): 3223-3229, 2017 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-28343388

RESUMO

An enzymatic method for specific determination of stevioside content was established. Recombinant ß-glucosidase BT_3567 (rBT_3567) from Bacteroides thetaiotaomicron HB-13 exhibited selective hydrolysis of stevioside at ß-1,2-glycosidic bond to yield rubusoside and glucose. Coupling of this enzyme with glucose oxidase and peroxidase allowed for quantitation of stevioside content in Stevia samples by using a colorimetric-based approach. The series of reactions for stevioside determination can be completed within 1 h at 37 °C. Stevioside determination using the enzymatic assay strongly correlated with results obtained from HPLC quantitation (r2 = 0.9629, n = 16). The percentages of coefficient variation (CV) of within day (n = 12) and between days (n = 12) assays were lower than 5%, and accuracy ranges were 95-105%. This analysis demonstrates that the enzymatic method developed in this study is specific, easy to perform, accurate, and yields reproducible results.


Assuntos
Proteínas de Bactérias/química , Bacteroides thetaiotaomicron/enzimologia , Ensaios Enzimáticos/métodos , Extratos Vegetais/análise , Stevia/química , Edulcorantes/análise , beta-Glucosidase/química , Biocatálise , Colorimetria/métodos , Diterpenos do Tipo Caurano/análise , Glucose Oxidase/química , Glucosídeos/análise
8.
J Diet Suppl ; 14(2): 173-185, 2017 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-27715351

RESUMO

Increased inflammation occurs with excessive adiposity and yeast ß-glucan modulates immune responses. This study investigated the potential effect of yeast ß-glucan on inflammatory cytokines in overweight/obese people. A randomized, double blinded, placebo-controlled, clinical trial design enrolled 44 overweight/obese participants with body mass index ≥23 kg/m2, randomized to two groups receiving ß-glucan 477 mg/capsule (n = 22) or placebo (n = 22) orally for six weeks. At weeks one to two, participants received 1 ß-glucan or placebo capsule/day and at four weeks two tablets/day. Anthropometric changes, lipid profiles, liver and renal functions, and inflammatory cytokines were measured. ß-glucan reduced waist circumference (p = 0.037) and blood pressure (p = 0.006) compared with controls after six weeks of intervention. No statistical significance between groups was observed for triglyceride, cholesterol, lipid profile, liver and renal function, or energy and nutrient intake compared with controls at week six. ß-glucan increased interlukin-10 (IL-10), an anti-inflammatory cytokine, by 23.97% from baseline at week two (p < 0.001) and 31.12% at week six (p < 0.001) and was significantly increased compared with controls at week two (p < 0.001) until week six (p < 0.001). ß-glucan reduced pro-inflammatory cytokines IL-6 at week six (p = 0.005) and tumor necrosis factor-α at week two (p = 0.037) compared with controls. Supplementation of yeast ß-glucan for six weeks modulated pro-cytokines that accelerate overweight/obese comorbidities and reduced blood pressure as well as waist circumference, the strong risk factors for cardiovascular disease, in overweight/obese subjects. Thus, ß-glucan might have the potential to decrease comorbid conditions associated with overweight/ obesity.


Assuntos
Obesidade/tratamento farmacológico , Sobrepeso/tratamento farmacológico , Circunferência da Cintura/efeitos dos fármacos , Leveduras/química , beta-Glucanas/administração & dosagem , Adulto , Idoso , Pressão Sanguínea/efeitos dos fármacos , Índice de Massa Corporal , Suplementos Nutricionais , Método Duplo-Cego , Feminino , Humanos , Inflamação , Interleucina-10/sangue , Interleucina-6/sangue , Masculino , Pessoa de Meia-Idade , Obesidade/fisiopatologia , Sobrepeso/fisiopatologia , Fatores de Tempo , Fator de Necrose Tumoral alfa/sangue , Adulto Jovem
9.
Phytochemistry ; 127: 4-11, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27055587

RESUMO

Plant ß-glucosidases are usually members of the glucosyl hydrolase 1 (GH1) or 3 (GH3) families. Previously, a ß-glucosidase (torvosidase) was purified from Solanum torvum leaves that specifically catalyzed hydrolysis of two furostanol 26-O-ß-glucosides, torvosides A and H. Furostanol glycoside 26-O-ß-glucosides have been reported as natural substrates of some plant GH1 enzymes. However, torvosidase was classified as a GH3 ß-glucosidase, but could not hydrolyze ß-oligoglucosides, the natural substrates of GH3 enzymes. Here, the full-length cDNA encoding S. torvum ß-glucosidase (SBgl3) was isolated by the rapid amplification of cDNA ends method. The 1887bp ORF encoded 629 amino acids and showed high homology to other plant GH3 ß-glucosidases. Internal peptide sequences of purified native Sbgl3 determined by LC-MS/MS matched the deduced amino acid sequence of the Sbgl3 cDNA, suggesting that it encoded the natural enzyme. Recombinant SBgl3 with a polyhistidine tag (SBgl3His) was successfully expressed in Pichia pastoris. The purified SBgl3His showed the same substrate specificity as natural SBgl3, hydrolyzing torvoside A with much higher catalytic efficiency than other substrates. It also had similar biochemical properties and kinetic parameters to the natural enzyme, with slight differences, possibly attributable to post-translational glycosylation. Quantitative real-time PCR (qRT-PCR) showed that SBgl3 was highly expressed in leaves and germinated seeds, suggesting a role in leaf and seedling development. To our knowledge, a recombinant GH3 ß-glucosidase that hydrolyzes furostanol 26-O-ß-glucosides, has not been previously reported in contrast to substrates of GH1 enzymes.


Assuntos
Glicosídeos/metabolismo , Pichia/genética , Solanum/metabolismo , Esteróis/metabolismo , beta-Glucosidase/metabolismo , Hidrólise
10.
Protein Expr Purif ; 110: 145-50, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25746591

RESUMO

Salivary α-glucosidases (MalI) have been much less characterized when compared with midgut α-glucosidases, which have been studied in depth. Few studies have been reported on the partial characterization of MalI, but no clear function has been ascribed. The aim of this study is to purify and characterize the recombinant Culex quinquefasciatus (CQ) α-glucosidase expressed in Pichia pastoris. The cDNA encoding mature Cx. quinquefasciatus α-glucosidase gene with polyhistidine tag (rCQMalIHis) was successfully cloned into the expression vector, pPICZαB, designated as pPICZαB/CQMalIHis. The activity of recombinant rCQMalIHis expressed in P. pastoris could be detected at 3.75U/ml, under optimal culture conditions. The purified rCQMalIHis showed a single band of molecular weight of approximately 92kDa on SDS-PAGE. After Endoglycosidase H digestion, a single band at 69kDa was found on SDS-PAGE analysis, suggesting that rCQMalIHis is a glycoprotein. Additionally, tryptic digestion and LC-MALDI MS/MS analysis suggested that the 69kDa band corresponds to the Cx. quinquefasciatus α-glucosidase. Thus, rCQMalIHis is a glycoprotein. The rCQMalIHis exhibited optimum pH and temperature at 5.5 and 35°C, respectively. The catalytic efficiency (kcat/Km) of the purified rCQMalIHis for maltotriose is higher than those for sucrose, maltotetraose, maltose and p-nitrophenyl-α-glucoside, indicating that the enzyme prefers maltotriose. Additionally, the rCQMalIHis is significantly inhibited by d-gluconic acid δ-lactone, but not by Mg(2+), Ca(2+) and EDTA. The rCQMalIHis is strongly inhibited by acarbose with IC50 67.8±5.6nM, but weakly inhibited by glucose with IC50 115.9±7.3mM.


Assuntos
Culex/química , Glicoproteínas/genética , Proteínas de Insetos/genética , Proteínas Recombinantes de Fusão/genética , Glândulas Salivares/química , alfa-Glucosidases/genética , Acarbose/química , Animais , Clonagem Molecular , Culex/enzimologia , DNA Complementar/genética , DNA Complementar/metabolismo , Expressão Gênica , Glicoproteínas/antagonistas & inibidores , Glicoproteínas/química , Glicoproteínas/isolamento & purificação , Histidina/química , Histidina/genética , Concentração de Íons de Hidrogênio , Proteínas de Insetos/antagonistas & inibidores , Proteínas de Insetos/química , Proteínas de Insetos/isolamento & purificação , Cinética , Peso Molecular , Oligopeptídeos/química , Oligopeptídeos/genética , Pichia/genética , Pichia/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Glândulas Salivares/enzimologia , Especificidade por Substrato , Temperatura , Trissacarídeos/química , alfa-Glucosidases/química , alfa-Glucosidases/isolamento & purificação
11.
Acta Biochim Biophys Sin (Shanghai) ; 44(12): 974-83, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23169343

RESUMO

Cocoon, a shelter for larva development to silk moth, contains the fibrous protein fibroin, which is coated by the globular protein sericin. Emergence of the silk moth requires the action of cocoonase, a protease secreted by the pupa. The full-length prococoonase cDNA, with 780 bp open reading frame encoding 260 amino acids, was cloned by reverse transcription from total RNA of the head of 6-day-old Thai-silk Bombyx mori pupa. Only the gene fragment lacking the propeptide encoding sequence was successfully expressed in Pichia pastoris, yielding an extracellularly active cocoonase. The recombinant cocoonase was purified to homogeneity by 80% ammonium-sulfate fractionation and CM-Sepharose chromatography, and its internal peptide sequences were analyzed by nano liquid chromatography-mass spectrometry/mass spectrometry. This monomeric protein has native molecular weight of 26 kDa by gel exclusion analysis and 25 kDa subunit size by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The enzyme hydrolyses sericin but does not hydrolyse fibroin, as shown by radial diffusion on thin-layer enzyme assay (RD-TEA). Scanning electron microscopy showed that purified recombinant cocoonase could remove sericin from natural silk completely in 24 h, without damaging fibroin, using only 1 immobilized sericin unit (ISU) of enzyme as determined by RD-TEA. Natural cocoonase isolated from B. mori pupa could also digest sericin effectively, but required more enzymes (2 ISU) and longer time (48 h). In comparison, a commercial enzyme, alcalase, with the same activity not only showed less complete digestion of sericin but also caused damage of fibroin. These results suggest that recombinant B. mori cocoonase is potentially useful for silk degumming.


Assuntos
Bombyx/enzimologia , Proteínas de Insetos/metabolismo , Peptídeo Hidrolases/metabolismo , Sequência de Aminoácidos , Animais , Bombyx/genética , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Fibroínas/metabolismo , Expressão Gênica , Hidrólise , Proteínas de Insetos/genética , Proteínas de Insetos/ultraestrutura , Espectrometria de Massas , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Peso Molecular , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Pichia/genética , Pupa/enzimologia , Pupa/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Sericinas/metabolismo , Serina Proteases/química , Serina Proteases/genética , Serina Proteases/metabolismo , Seda/metabolismo , Seda/ultraestrutura
12.
Cancer Lett ; 297(1): 31-41, 2010 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-20570039

RESUMO

Medullary thyroid carcinoma (MTC) is a multiple endocrine neoplasia type 2 syndrome caused by mutations in extracellular receptor or intracellular kinase domains of the RET proto-oncogene. Activation of the Ras/Raf/MEK/ERK pathway can lead to growth arrest by secreting leukemia inhibitory factor (LIF) in MTC cells harboring a RET receptor domain mutation. Here, we report that Ras/Raf/MEK/ERK can also mediate, via LIF, growth inhibition in MTC cells harboring a RET kinase domain mutation. Ras/Raf/MEK/ERK activation was sufficient to induce growth inhibition and LIF expression in the human MTC line MZ-CRC-1. Presence of LIF-mediated signaling was determined by blocking the activity of culture medium conditioned by Raf-activated cells using anti-LIF neutralizing antibody. In addition, recombinant LIF effectively suppressed cell proliferation via cell cycle arrest in G0/G1 phase. Expression of dominant negative STAT3 abrogated LIF effects, indicating that LIF mediates its signaling through the JAK/STAT3 pathway. These results suggest that growth inhibition and activation of the autocrine/paracrine signaling through LIF/JAK/STAT may be a common response to Ras/Raf activation in different MTC types, and justify further evaluation of LIF as a potential anticancer agent for MTC.


Assuntos
Carcinoma Medular/enzimologia , Proliferação de Células , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator Inibidor de Leucemia/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Transdução de Sinais , Neoplasias da Glândula Tireoide/enzimologia , Proteínas ras/metabolismo , Comunicação Autócrina , Carcinoma Medular/genética , Carcinoma Medular/patologia , Morte Celular , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/metabolismo , Humanos , Janus Quinases/metabolismo , Mutação , Comunicação Parácrina , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/metabolismo , Proteínas Recombinantes/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Fatores de Tempo , Transfecção
13.
J Biol Chem ; 284(48): 33006-18, 2009 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-19805545

RESUMO

Kinase activity is known as the key biochemical property of MAPKs. Here, we report that ERK1/2 also utilizes its noncatalytic function to mediate certain signal transductions. Sustained activation of the Raf/MEK/ERK pathway induces growth arrest, accompanied by changes in cell cycle regulators (decreased retinoblastoma phosphorylation, E2F1 down-regulation, and/or p21(CIP1) up-regulation) and cell type-specific changes in morphology and expression of c-Myc or RET in the human tumor lines LNCaP, U251, and TT. Ablation of ERK1/2 by RNA interference abrogated all these effects. However, active site-disabled ERK mutants (ERK1-K71R, ERK2-K52R, and ERK2-D147A), which competitively inhibit activation of endogenous ERK1/2, could not block Raf/MEK-induced growth arrest as well as changes in the cell cycle regulators, although they effectively blocked phosphorylation of the ERK1/2 catalytic activity readouts, p90(RSK) and ELK1, as well as the cell type-specific changes. Because this indicated a potential noncatalytic ERK1/2 function, we generated stable lines of the tumor cells in which both ERK1 and ERK2 were significantly knocked down, and we further investigated the possibility using rat-derived kinase-deficient ERK mutants (ERK2-K52R and ERK2-T183A/Y185F) that were not targeted by human small hairpin RNA. Indeed, ERK2-K52R selectively restored Raf-induced growth inhibitory signaling in ERK1/2-depleted cells, as manifested by regained cellular ability to undergo growth arrest and to control the cell cycle regulators without affecting c-Myc and morphology. However, ERK2-T183A/Y185F was less effective, indicating the requirement of TEY site phosphorylation. Our study suggests that functions of ERK1/2 other than its "canonical" kinase activity are also involved in the pathway-mediated growth arrest signaling.


Assuntos
Proliferação de Células , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Transdução de Sinais/fisiologia , Catálise , Ciclo Celular , Linhagem Celular , Linhagem Celular Tumoral , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Immunoblotting , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mutação , Fosforilação , Interferência de RNA , Transfecção , Quinases raf/genética , Quinases raf/metabolismo
14.
Parasitol Res ; 103(2): 443-8, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18463895

RESUMO

Trichomoniasis is now an important health problem in developing countries. Although metronidazole has so far been widely used to treat this disease, the prevalence of metronidazole-resistant protozoa and unpleasant adverse effects have been found. In this study, natural products purified from Thai plants were, therefore, investigated for their effectiveness against Trichomonas vaginalis. The minimal inhibitory concentrations for all beta-glycosides against Trichomonas vaginalis at 24 h were in a range of 6.25-12.5 microM. In addition, torvoside A and H were found to be more potent than their corresponding aglycones, deglucosylated torvoside A and H, while other beta-glycosides were generally as active as their corresponding aglycones. The cytotoxicity of these compounds was also determined. Except for dalcochinin, none of the tested compounds showed cytotoxicity against Vero and cancer cell lines (KB and MCF-7), having IC(50) values greater than 50 microg/ml. In conclusion, beta-glycosides and several aglycones showed selective inhibition against Trichomonas vaginalis without harmful effect to mammalian cells.


Assuntos
Antitricômonas/farmacologia , Glicosídeos/farmacologia , Plantas Medicinais/química , Trichomonas vaginalis/efeitos dos fármacos , Animais , Antitricômonas/química , Antitricômonas/toxicidade , Linhagem Celular Tumoral/efeitos dos fármacos , Chlorocebus aethiops , Glicosídeos/química , Glicosídeos/toxicidade , Humanos , Concentração Inibidora 50 , Testes de Sensibilidade Parasitária , Tailândia , Células Vero/efeitos dos fármacos
15.
Acta Biochim Biophys Sin (Shanghai) ; 38(8): 563-70, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16894479

RESUMO

An iridoid beta-glucoside, namely plumieride coumarate glucoside, was isolated from the Plumeria obtusa (white frangipani) flower. A beta-glucosidase, purified to homogeneity from P. obtusa, could hydrolyze plumieride coumarate glucoside to its corresponding 13-O-coumarylplumieride. Plumeria beta-glucosidase is a monomeric glycoprotein with a molecular weight of 60.6 kDa and an isoelectric point of 4.90. The purified beta-glucosidase had an optimum pH of 5.5 for p-nitrophenol (pNP)-beta-D-glucoside and for its natural substrate. The Km values for pNP-beta-D-glucoside and Plumeria beta-glucoside were 5.04+/-0.36 mM and 1.02+/-0.06 mM, respectively. The enzyme had higher hydrolytic activity towards pNP-beta-D-fucoside than pNP-beta-D-glucoside. No activity was found for other pNP-glycosides. Interestingly, the enzyme showed a high specificity for the glucosyl group attached to the C-7' position of the coumaryl moiety of plumieride coumarate glucoside. The enzyme showed poor hydrolysis of 4-methylumbelliferyl-beta-glucoside and esculin, and did not hydrolyze alkyl-beta-glucosides, glucobioses, cyanogenic-beta-glucosides, steroid beta-glucosides, nor other iridoid beta-glucosides. In conclusion, the Plumeria beta-glucosidase shows high specificity for its natural substrate, plumieride coumarate glucoside.


Assuntos
Apocynaceae/enzimologia , Celulases/química , Ácidos Cumáricos/química , Iridoides/química , Catálise , Celulases/metabolismo , Ácidos Cumáricos/metabolismo , Hidrólise , Glucosídeos Iridoides , Iridoides/metabolismo , Cinética , Especificidade por Substrato
16.
Phytochemistry ; 67(1): 27-33, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16289258

RESUMO

A beta-glucosidase (torvosidase) was purified to homogeneity from the young leaves of Solanum torvum. The enzyme was highly specific for cleavage of the glucose unit attached to the C-26 hydroxyl of furostanol glycosides from the same plant, namely torvosides A and H. Purified torvosidase is a monomeric glycoprotein, with a native molecular weight of 87 kDa by gel filtration and a pI of 8.8 by native agarose IEF. Optimum pH of the enzyme for p-nitrophenyl-beta-glucoside and torvoside H was 5.0. Kinetic studies showed that Km values for torvoside A (0.06 3mM) and torvoside H (0.068 mM) were much lower than those for synthetic substrates, pNP-beta-glucoside (1.03 mM) and 4-methylumbelliferyl-beta-glucoside (0.78 mM). The enzyme showed strict specificity for the beta-d-glucosyl bond when tested for glycone specificity. Torvosidase hydrolyses only torvosides and dalcochinin-8'-beta-glucoside, which is the natural substrate of Thai rosewood beta-glucosidase, but does not hydrolyse other natural substrates of the GH1 beta-glucosidases or of the GH3 beta-glucosidase families. Torvosidase also hydrolyses C5-C10 alkyl-beta-glucosides, with a rate of hydrolysis increasing with longer alkyl chain length. The internal peptide sequence of Solanum beta-glucosidase shows high similarity to the sequences of family GH3 glycosyl hydrolases.


Assuntos
Folhas de Planta/enzimologia , Solanum/enzimologia , beta-Glucosidase/química , Sequência de Aminoácidos , Eletroforese em Gel de Poliacrilamida , Glicosídeos/síntese química , Glicosídeos/química , Hidrólise , Cinética , Conformação Molecular , Dados de Sequência Molecular , Folhas de Planta/química , Saponinas/síntese química , Saponinas/química , Alinhamento de Sequência , beta-Glucosidase/genética , beta-Glucosidase/isolamento & purificação
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