Nison Sattayasai

Genetic variation, population structure and identification of yellow catfish, Mystus nemurus (C&V) in Thailand using RAPD, ISSR and SCAR marker

Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to investigate the genetic structure of four subpopulations of Mystus nemurus in Thailand. The 7 RAPD and 7 ISSR primers were selected. Of 83 total RAPD fragments, 80 (96.39%) were polymorphic loci, and of 81 total ISSR fragments, 75 (92.59%) were polymorphic loci. Genetic variation and genetic differentiation obtained from RAPD fragments or ISSR fragments showed similar results. Percentage of polymorphic loci (%P), observed number of alleles, effective number of alleles, Nei’s gene diversity (H) and Shannon’s information index revealed moderate to high level of genetic variations within each M. nemurus subpopulation and overall population. High levels of genetic differentiations were received from pairwise unbiased genetic distance (D) and coefficient of differentiation. Mantel test between D or gene flow and geographical distance showed a low to moderate correlation. Analysis of molecular variance indicated that variations among subpopulations were higher than those within subpopulations. The UPGMA dendrograms, based on RAPD and ISSR, showing the genetic relationship among subpopulations are grouped into three clusters; Songkhla (SK) subpopulation was separated from the other subpopulations. The candidate species-specific and subpopulation-specific RAPD fragments were sequenced and used to design sequence-characterized amplified region primers which distinguished M. nemurus from other species and divided SK subpopulation from the other subpopulations. The markers used in this study should be useful for breeding programs and future aquacultural development of this species in Thailand.

Novel anti-bacterials against MRSA: synthesis of focussed combinatorial libraries of tri-substituted 2(5H)-furanones.

Mucobromic and mucochloric acid were used as building blocks for the construction of a chemical combinatorial library of 3,4,5-trisubstituted 2(5H)-furanones. With these 2 butenolide building blocks, and eight alcohols a sublibrary of 16 dihalogenated 5-alkoxy-2(5H)-furanones was prepared. This sublibrary of 5-alkoxylated furanones was reacted with 16 amines generating a full size focussed combinatorial library of 256 individual compounds. This three dimensional combinatorial library of 3-halogen-4-amino-5-alkoxy-2(5H)-furanones was prepared around the benzimida-zolyl furanone lead structure by applying a solution phase combinatorial chemistry concept. Typical representatives of the library were purified and fully characterized and one x-ray structures was recorded, additionally. The 3-bromo-4-benzimizazolyl-5-methoxy-2(5H)furanone, Br-A-l, showed an MIC of 8 microg/ml against the multiresistant Staphylococcus aureus (MRSA).

Dose-dependent effects of glutamate in pyridoxine-induced neuropathy

In order to explore the effects of glutamate in a pyridoxine megadose-induced neuropathy, rats were received glutamate either 0.5 or 1 g/kg/day orally with or without pyridoxine 0.8 g/kg/day intraperitoneally for 14 days. The animals motor coordination, the muscle power and the thermal threshold were observed daily. The nerve conduction velocity was measured at day 0 and day 15 of the treatment. Glutamate either 0.5 or 1 g/kg/day appeared to have no effect on motor coordination, the nerve conduction velocity and the muscle power score compared with control. However, the thermal response latency was significantly decreased (from day 9) in animals treated with 1 g/kg/day glutamate. In pyridoxine-induced neuropathy rats, glutamate 0.5 g/kg/day significantly decreased the effects of pyridoxine on the sciatic nerve conduction velocity, the muscle power score and the motor coordination. Interestingly, glutamate at a dose of 1 g/kg/day worsened the neurotoxic effects cause by pyridoxine.

In‐vitro and in‐vivo antivenin activity of 2‐[2‐(5,5,8a‐trimethyl‐2‐methylene‐decahydro‐naphthalen‐1‐yl)‐ethylidene]‐succinaldehyde against Ophiophagus hannah venom

Objectives Curcuma zedoaroides A. Chaveerach & T. Tanee, locally known as Wan‐Paya‐Ngoo‐Tua‐Mia, is commonly used in the North‐Eastern part of Thailand as a ‘snakebite antidote’. The aim of this study was to isolate the active compound from the rhizome of C. zedoaroides, to determine its structure and to assess its antagonistic activity in vitro and in vivo against King cobra venom.

Alanine Aminopeptidase and Dipeptidyl Peptidase IV in Saliva of Chronic Periodontitis Patients

BACKGROUND Alanine aminopeptidase (ALAP) and dipeptidyl peptidase IV (DPPIV) are ectopeptidases that play a role in collagen degradation and are thought to be involved in the destruction of periodontal tissue. This study compared the activities of salivary ALAP and DPPIV in patients with periodontitis and periodontally healthy subjects. The correlations of enzyme activities with clinical variables and the presence of Porphyromonas gingivalis were also evaluated. METHODS Whole saliva was collected from 30 periodontally healthy subjects, 30 localized chronic periodontitis (LCP) patients, and 30 generalized chronic periodontitis (GCP) patients to determine the activities of ALAP and DPPIV. The presence of P. gingivalis in subgingival plaque was detected by polymerase chain reaction. Periodontal clinical assessments included probing depth, clinical attachment level, and bleeding on probing. RESULTS The activities of DPPIV in the LCP and GCP groups were not significantly different from one another, but both groups had significantly higher enzyme activities than the periodontally healthy group (P = 0.001). DPPIV activity was positively correlated with all clinical parameters and the prevalence of P. gingivalis. The ALAP activities were not significantly different among the three study groups. There was no significant correlation of ALAP activity with any of the clinical and bacterial parameters. CONCLUSION DPPIV, but not ALAP, activity is associated with periodontitis and the presence of P. gingivalis.

A Biotin-Coupled Bifunctional Enzyme Exhibiting Both Glutamine Synthetase Activity and Glutamate Decarboxylase Activity

Purpose: To purify and study native form and enzymatic activity of the 42 kDa biotin-coupled protein (p42), which is related to glutamate action in chick retina. Methods: p42 was purified using molecular filtration in the presence of 0.7 M sodium chloride. Purity and identification of p42 were studied by SDS-PAGE, 2D-PAGE, LC-MS/MS, and MALDI-TOF MS. The native form of p42 was investigated using native-PAGE and Ferguson plot. Biotin-coupled property was examined by Western blot analysis. Enzymatic actions of p42 were studied using glutamate as substrate in the presence or absence of glutamine. Results: p42 was successfully purified from chick retinal protein solution using the molecular filtration. Western blot analysis with avidin showed that p42 was a biotin-coupled protein. Using SDS-PAGE, 2D-PAGE, LC-MS/MS, and MALDI-TOF MS, purified p42 was identified as a glutamine synthetase with four isoforms. Native-PAGE, followed by Ferguson plot analysis, showed two molecular forms of p42 corresponding to homotetramers and homooctamers. Enzymatic reaction followed by paper chromatography showed that p42 catalyzed the synthesis of glutamine from glutamate in the presence of ammonium ion, ATP, and magnesium ion. At prolonged reaction time, γ-aminobutyric acid (GABA) was also formed. With glutamate and glutamine present at equal concentrations in the reaction mixture, GABA could be rapidly detected, but GABA could not be detected when glutamate concentration was more than four-fold that of glutamine. The results indicated that p42 also had glutamate decarboxylase activity. Both enzymatic activities were inhibited by avidin. High concentrations of Mn2+ inhibited synthetase activity of p42 but not decarboxylase activity. Conclusion: p42 was purified from chick retinal protein solution using molecular filtration in the presence of sodium chloride. The protein was a biotin-coupled bifunctional enzyme that contained glutamine synthetase activity and glutamate decarboxylase activity. Biotin was possibly involved in these activities. Mn2+ showed different effects on the two activities.

Anticancer effects of Curcuma C20-dialdehyde against colon and cervical cancer cell lines

BACKGROUND Recent attention on chemotherapeutic intervention against cancer has been focused on discovering and developing phytochemicals as anticancer agents with improved efficacy, low drug resistance and toxicity, low cost and limited adverse side effects. In this study, we investigated the effects of Curcuma C20-dialdehyde on growth, apoptosis and cell cycle arrest in colon and cervical cancer cell lines. MATERIALS AND METHODS Antiproliferative, apoptosis induction, and cell cycle arrest activities of Curcuma C20-dialdehyde were determined by WST cell proliferation assay, flow cytometric Alexa fluor 488-annexin V/propidium iodide (PI) staining and PI staining, respectively. RESULTS Curcuma C20 dialdehyde suppressed the proliferation of HCT116, HT29 and HeLa cells, with IC50 values of 65.4±1.74 μg/ml, 58.4±5.20 μg/ml and 72.0±0.03 μg/ml, respectively, with 72 h exposure. Flow cytometric analysis revealed that percentages of early apoptotic cells increased in a dose-dependent manner upon exposure to Curcuma C20-dialdehyde. Furthermore, exposure to lower concentrations of this compound significantly induced cell cycle arrest at G1 phase for both HCT116 and HT29 cells, while higher concentrations increased sub-G1 populations. However, the concentrations used in this study could not induce cell cycle arrest but rather induced apoptotic cell death in HeLa cells. CONCLUSIONS Our findings suggest that the phytochemical Curcuma C20-dialdehyde may be a potential antineoplastic agent for colon and cervical cancer chemotherapy and/or chemoprevention. Further studies are needed to characterize the drug target or mode of action of the Curcuma C20-dialdehyde as an anticancer agent.

Antibacterial activity of 3-substituted pyrrole-2,5-diones against Pseudomonas aeruginosa

3-Substituted pyrrole-2,5-diones were synthesised from mucohalogen acids and the antibacterial activity was subsequently determined in biological assays. The minimum inhibitory concentration and the minimum bactericidal concentration of 2a were determined for a wide range of microorganisms in the low micromolar range. Protein identification using SDS-PAGE and LC/MS/MS demonstrated a partly degradation of OprF-related proteins giving an insight into the underlying mechanism of these novel antibacterial agents.

A non-mitochondrial carboxylase, related to glutamate action is synthesized in the retina of the chick embryo

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot of chick retinal proteins at 21 days after intravitreal injection with 5 or 10 microM/eye Glu showed decreases in 37, 42, 53 and 57 kDa proteins and increases in 35, 72 and >94 kDa proteins. These proteins were carboxylases except for the 35 and 37 kDa proteins. With > or =15 microM/eye Glu, non-specific loss of retinal proteins was observed. In embryonic retinas, the 42 kDa protein was seen a few days before hatching, with biotin incorporation on days 3-6 after hatching. Immunohistochemistry indicated that this protein was a component of both the inner nuclear layer and the photoreceptor. Immunocytochemistry located it to the cell surface.