HIV and syphilis knowledge, perceptions, and practices among Myanmar migrant workers in Samut Sakhon Province, Thailand.

BACKGROUND: Thailand has been one of the largest migration hubs in Southeast Asia for the past four decades and keeps attracting migrants from neighboring countries. Due to difficulties associated with their status, migration can place individuals at a heightened risk for sexually transmitted diseases. This study aimed to examine factors influencing HIV and syphilis preventive behaviors among Myanmar migrants in Samut Sakhon, Thailand. METHODS: A cross-sectional mixed-method study was conducted among Myanmar migrants aged 18 years and above in Samut Sakhon Province, Thailand. To fulfill this study's aims, four hundred seventy-three respondents completed a survey to provide quantitative data, and eight participants completed in-depth qualitative interviews. The factors associated with protective sexual behaviors were identified with multiple logistic regression analysis of the quantitative study data and thematic analysis of the qualitative data. RESULTS: The respondents showed good knowledge of HIV and syphilis (50.1%), but over half (55.6%) exhibited the negative perception of sexually transmitted disease prevention; about 81.4% of male respondents never used a condom when they had sexual intercourse in the past year. Based on multivariate analysis, income expenses-balance (adjusted odds ratio (AOR) = 2.379, 95% confidence interval (CI): 1.002-5.731, p = 0.049), number of sex partners (AOR = 3.044, 95% CI: 1.339-6.924, p = 0.008), and having sex with a prostitute (AOR = 6.085, 95% CI: 1.28-28.918, p = 0.023) were all statistically associated with unprotected sex. In the qualitative analysis, knowledge, understanding, beliefs; the influence of culture, community, and environment; and condom perceptions were also important factors. CONCLUSION: Low use of condoms in sexual practice was identified, and the appropriate intervention or approach to improve the utilization of condoms in the community was provided.

Molecular detection of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in school children at the Thai-Myanmar border.

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.

Expression and characterization of a recombinant stevioside hydrolyzing ß-glycosidase from Enterococcus casseliflavus.

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.

Associations among serum folate, waist-to-hip ratio, lipid profile, and eating habits with homocysteine in an elderly Thai population.

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.

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.

Cocultures of Enterococcus faecium and Aeromonas veronii Induce the Secretion of Bacteriocin-like Substances against Aeromonas.

Lactic acid bacteria (LAB) were screened from Lutjanus russellii (red sea bass), and their antimicrobial activities were evaluated against two Aeromonas species isolated from the Nile tilapia, namely, Aeromonas veronii (AV) and Aeromonas jandaei (AJ). Three LAB isolates, Enterococcus faecium MU8 (EF_8), Enterococcus faecalis MU2 (EFL_2), and E. faecalis MU9 (EFL_9), were found to inhibit both AV and AJ; however, their cell-free supernatant (CFS) did not do so. Interestingly, bacteriocin-like substances (BLS) induced by cocultures of EF_8 with AV exhibited the highest antimicrobial activity against both Aeromonas sp. The size of BLS was less than 1.0 kDa; the purified BLS were susceptible to proteinase K digestion, indicating that they are peptides. BLS contained 13 identified peptides derived from E. faecium, as determined by liquid chromatography-tandem mass spectrometry. Cocultures of Gram-positive-producing and -inducing LAB strains have been used to increase bacteriocin yields. To our knowledge, this is the first report describing inducible BLS produced by cocultures of Gram-positive-producing and Gram-negative-inducing strains.

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.

Prevalence and Subtype Distribution of Blastocystis Isolated from School-Aged Children in the Thai-Myanmar Border, Ratchaburi Province, Thailand.

Blastocystis is one of the most common enteric protozoa that inhabits the intestinal tract of humans and different animals. Moreover, it has a worldwide geographic distribution. Its main mode of transmission is via the fecal-oral route. At present, 26 subtypes are widely distributed across both humans and animals. The current study aimed to determine the prevalence and subtype distribution of Blastocystis among school-aged children living on the Thai-Myanmar border, Ratchaburi province, Thailand. In total, 508 samples were collected from children at six schools. The prevalence of Blastocystis infection was amplified and sequenced in the 600 bp barcode region of the small-subunit ribosomal RNA (SSU rRNA). The overall prevalence of Blastocystis infection was 3.35% (17/508). ST3 (11/17) was the most predominant subtype, followed by ST1 (5/17) and ST2 (1/17). A phylogenetic tree was constructed based on the Tamura92+G+I model using the maximum-likelihood algorithm. Surprisingly, all sequences of the ST3-positive samples were closely correlated with the cattle-derived sequence. Meanwhile, all sequences of the Blastocystis ST1-positive samples were closely correlated with the human-derived sequence. Nevertheless, further studies should be conducted to validate the zoonotic transmission of Blastocystis. Based on our findings, personal hygiene and sanitation should be improved to promote better health in children in this area.

The Effects of Crocodile Blood Supplementation on Delayed-Onset Muscle Soreness.

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.

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 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.

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.

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 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.

Yeast ß-Glucan Modulates Inflammation and Waist Circumference in Overweight and Obese Subjects.

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.

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

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.

Furostanol glycoside 26-O-beta-glucosidase from the leaves of Solanum torvum.

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.

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

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.

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.

Inhibitory effects of Thai plants beta-glycosides on Trichomonas vaginalis.

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.

beta-Glucosidase catalyzing specific hydrolysis of an iridoid beta-glucoside from Plumeria obtusa.

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.