Dumrongkiet Arthan

Noncatalytic Function of ERK1/2 Can Promote Raf/MEK/ERK-mediated Growth Arrest Signaling

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 p21CIP1 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, p90RSK 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.

Leukemia inhibitory factor can mediate Ras/Raf/MEK/ERK-induced growth inhibitory signaling in medullary thyroid cancer cells

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.

Functional expression of a Bombyx mori cocoonase: potential application for silk degumming.

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.

Yeast β–Glucan Modulates Inflammation and Waist Circumference in Overweight and Obese Subjects

ABSTRACT Increased inflammation occurs with excessive adiposity and yeast β-glucan modulates immune responses. This study investigated the potential effect of yeast β-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 β–glucan 477 mg/capsule (n = 22) or placebo (n = 22) orally for six weeks. At weeks one to two, participants received 1 β–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. β-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. β-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). β-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 β-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, β-glucan might have the potential to decrease comorbid conditions associated with overweight/ obesity.

A Solanum torvum GH3 β-glucosidase expressed in Pichia pastoris catalyzes the hydrolysis of furostanol glycoside

Plant β-glucosidases are usually members of the glucosyl hydrolase 1 (GH1) or 3 (GH3) families. Previously, a β-glucosidase (torvosidase) was purified from Solanum torvum leaves that specifically catalyzed hydrolysis of two furostanol 26-O-β-glucosides, torvosides A and H. Furostanol glycoside 26-O-β-glucosides have been reported as natural substrates of some plant GH1 enzymes. However, torvosidase was classified as a GH3 β-glucosidase, but could not hydrolyze β-oligoglucosides, the natural substrates of GH3 enzymes. Here, the full-length cDNA encoding S. torvum β-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 β-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 β-glucosidase that hydrolyzes furostanol 26-O-β-glucosides, has not been previously reported in contrast to substrates of GH1 enzymes.

Adiponectin/ACP30, a collagen-like plasma protein in relation to anthropometric measurement in Thai overweight and obese subjects

Adiponectin, anthropometric parameters including weight, height, body mass index (BMI), arm circumference, triceps skinfold, subscapular skinfold, waist, hip circumferences and waist/hip ratio were recorded in 48 male and 166 female overweight and obese Thai volunteers (BMI ≥ 25.0 kg/m2), and in 26 male and 81 female normal subjects (BMI = 18.5 − 24.9 kg/m2). Thai volunteers were investigated. Statistically significantly lower adiponectin concentrations in overweight and obese subjects were found when compared with control subjects of both sexes. Anthropometric parameters, including weight, height, BMI, arm circumference, triceps skinfold, subscapular skinfold, waist, hip circumferences and waist/hip ratio, except arm span, were statistically significantly higher in overweight and obese subjects than in control subjects. The overweight and obese subjects had higher glucose concentrations than the control subjects. The BMI and glucose concentrations were found to be significantly related, under these conditions, to adiponectin.

Development and Validation of an Enzymatic Method To Determine Stevioside Content from Stevia rebaudiana

An enzymatic method for specific determination of stevioside content was established. Recombinant β-glucosidase BT_3567 (rBT_3567) from Bacteroides thetaiotaomicron HB-13 exhibited selective hydrolysis of stevioside at β-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.

Functional expression and molecular characterization of Culex quinquefasciatus salivary α-glucosidase (MalI)

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.

Three novel mutations in α-galactosidase gene involving in galactomannan degradation in endosperm of curd coconut

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.

Obesity prevalence and contributing factors among adolescents in secondary schools in Pemagatshel district, Bhutan

Abstract 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.