The first trimeric Galanthus nivalis agglutinin-related lectin of Orchidaceae was found in Dendrobium pendulum: purification, characterization, and effects of stress factors.

KEY MESSAGE: Trimeric Galanthus nivalis agglutinin-related lectin of Orchidaceae with two conformational forms was first studied in Dendrobium pendulum . It was highly expressed by stress factors. Using mannan-agarose column chromatography, a mannose-binding protein was purified from Dendrobium pendulum Roxb. pseudobulb. After heating in the presence of sodium dodecyl sulfate (SDS) with or without 2-mercaptoethanol, the protein showed one band with molecular mass of 14.0 kDa on SDS-polyacrylamide gel electrophoresis (PAGE). Without heating, three bands were found at positions of 14.0, 39.4, and 41.5 kDa, but a higher amount of 39.4 and 41.5 kDa protein bands were seen in the presence of 2-mercaptoethanol. Liquid chromatography-tandem mass spectrometry and database search indicated that the 14.0 kDa protein band contained three peptide fragments identical to parts of a lectin precursor from Dendrobiu m findleyanum Parish & Rchb.f. Native-PAGE and Ferguson plot showed that the purified protein had two native forms with molecular masses of 44.2 and 45.3 kDa, indicating three 14.0 kDa polypeptide subunits. The purified protein exhibited the agglutination activity with trypsinized chicken erythrocytes. It was then recognized as a Galanthus nivalis agglutinin-related lectin and named D. pendulum agglutinin (DPA). Using reverse transcription-polymerase chain reaction and DNA sequencing, the deduced amino acid sequence of DPA precursor showed the highest homology (96.4%) with a lectin precursor of D. findleyanum and contained three mannose-binding sites. Greater amounts of DPA were found when the pseudobulbs were treated with stress factors including ultraviolet light, abscisic acid, hydrogen peroxide, and acetylene gas.

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.

Isolation of heat-tolerant myoglobin from Asian swamp eel Monopterus albus.

Myoglobin from Asian swamp eel Monopterus albus was purified from fish muscle using salt fractionation followed by column chromatography and molecular filtration. The purified Mb of 0.68 mg/g wet weight of muscle was determined for its molecular mass by MALDI-TOF-MS to be 15,525.18 Da. Using isoelectric focusing technique, the purified Mb showed two derivatives with pI of 6.40 and 7.12. Six peptide fragments of this protein identified by LC-MS/MS were homologous to Mbs of sea raven Hemitripterus americanus, yellowfin tuna Thunnus albacores, blue marlin Makaira nigicans, common carp Cyprinus carpio, and goldfish Carassius auratus. According to the Mb denaturation, the swamp eel Mb had thermal stability higher than walking catfish Clarias batrachus Mb and striped catfish Pangasius hypophthalmus Mb, between 30 and 60 (°)C. For the thermal stability of Mb, the swamp eel Mb showed a biphasic behavior due to the O(2) dissociation and the heme orientation disorder, with the lowest increase in both Kd(f) and Kd(s). The thermal sensitivity of swamp eel Mb was lower than those of the other Mbs for both of fast and slow reaction stages. These results suggest that the swamp eel Mb globin structure is thermally stable, which is consistent with heat-tolerant behavior of the swamp eel particularly in drought habitat.

Dendrobium findleyanum agglutinin: production, localization, anti-fungal activity and gene characterization.

The recently reported Dendrobium findleyanum agglutinin (DFA) was identified and determined in different parts of D. findleyanum pseudobulbs by using Western blot analysis, LC-MS/MS, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and histochemical procedure. Western blot analysis of crude protein extract with horseradish peroxidase (HRP), a mannose-rich glycoprotein, showed only one band at 14.5 kDa, which had the same molecular mass as DFA. This band was a major band when the membrane was stained with Coomassie Brilliant Blue. The protein profiles from SDS-PAGE showed higher band intensity of the 14.5 kDa mannose-binding protein in nearly mature and mature stages, compared to very young and young stages of the orchid. In addition, the band intensity was to a great extent different between the swollen and the non-swollen internode of the pseudobulb. Using LC-MS/MS, the sequence tags of the 14.5-kDa protein bands from the node, swollen internode and non-swollen internode revealed that the protein was DFA. Histochemical procedure in the transverse section of the pseudobulbs demonstrated major HRP binding sites, which reflected the location of DFA, in periphery of parenchymal cells. The purified DFA showed anti-fungal activity against Alternaria alternata and Collectotrichum sp. Using reverse transcription polymerase chain reaction and DNA sequencing, the deduced amino acid sequence of the DFA precursor revealed 94% homology with a lectin precursor from D. officinale. N-terminal sequencing demonstrated the processing site between residues 24 and 25 of the DFA precursor.

Thermostable mannose-binding lectin from Dendrobium findleyanum with activities dependent on sulfhydryl content.

A mannose-binding lectin was purified from Dendrobium (D.) findleyanum pseudobulb using mannan-agarose column chromatography. After heating in the presence of SDS with or without 2-mercaptoethanol on SDS-PAGE with a continuous gradient of 8%-20% acrylamide, the purified lectin showed only one protein band with a molecular mass of 14.5 kDa. Without heating, two bands were seen on the gel at the positions of 14.5 kDa and 53.7 kDa, but a higher amount of the 53.7 kDa protein was observed in the presence of 2-mercaptoethanol. Protein identification of both protein bands by liquid chromatography-tandem mass spectrometry showed three peptide fragments identical to parts of a lectin precursor from D. officinale; the lectin was named D. findleyanum agglutinin (DFA). Using various concentrations of native-PAGE and Ferguson plot, only one protein band revealed a molecular mass of 56.2 kDa, indicating four 14.5 kDa polypeptide subunits in the DFA. Isoelectric focusing revealed that the DFA had three conformational forms with an isoelectric point of 5.18, 4.87 and 4.72, whereas 2-mercaptoethanol-treated DFA showed only one band with an isoelectric point of 5.18. DFA exhibited specificity towards mannose using the solid-phase method. The binding activity, anti-fungal activity and hemagglutination activity of DFA were not affected by heat, but were increased by free sulfhydryl groups.

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).

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.

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.

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 animal's 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.

Detection of Babesia bovis in cattle by PCR-ELISA.

We established a new highly sensitive method, PCR-ELISA, for the dectection of Babesia bovis in cattle for farms in Thailand. The detection of around 2.4 x 10(-8)% parasitemia (equivalent to 1 infected erythrocyte per 2 ml) was achieved by PCR amplification followed by the ELISA detection of a biotin tagged gene. When comparing the sensitivity of PCR-ELISA with the microscopic method, our PCR-ELISA method is more sensitive than thin blood smears by at least 1,000 times. The established PCR-ELISA also showed high specificity to B. bovis with no cross reaction to other endemic parasites except for A. marginale. Regarding the detection threshold for B. bovis, the PCR-ELISA method could detect parasites inoculated into splenectomized calves at least 1 week earlier than the thin blood microscopic method. The PCR-ELISA method is a valuable screening technique for B. bovis and applicable for the routine detection of carrier states and automated analysis.

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. METHODS: The active compound was obtained from C. zedoaroides by extraction with acetone followed by purification using column chromatography; its X-ray structure was determined. Its inhibition of venom lethality was studied in vitro in rat phrenic nerve-hemidiaphragms and in vivo in mice. KEY FINDINGS: The acetone extract of the Curcuma rhizomes contained a C20 dialdehyde, [2-(5,5,8a-trimethyl-2-methylene-decahydro-naphthalen-1-yl)-ethylidene]-succinaldehyde, as the major component. The isolated curcuma dialdehyde was found active in vitro and in vivo for antivenin activity against the King cobra venom. Using isolated rat phrenic nerve-hemidiaphragm preparations, a significant antagonistic effect on the inhibition of neuromuscular transmission was observed in vitro. Inhibition on muscle contraction, produced by the 4 microg/ml venom, was reversed by 2-16 microg/ml of Curcuma dialdehyde in organ bath preparations over a period of 2 h. Mice intraperitoneally injected with 0.75 mg/kg venom and dialdehyde at 100 mg/kg had a significantly increased survival time. Injection of Curcuma dialdehyde (100 mg/kg) 30 min before the subcutaneous injection of the venom resulted in a 100% survival time after 2 h compared with 0% for the control group. CONCLUSIONS: The in vitro and in vivo evaluation confirmed the medicinal use of traditional snake plants against snakebites. The bioactivity is linked to an isolated molecule and not a result of synergistic effects of a mixture. The active compound was isolated and the structure fully elucidated, including its stereochemistry. This dialdehyde is a versatile chemical building block and can be easily obtained from this plant source.

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, gamma-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 Mn(2+) 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. Mn(2+) showed different effects on the two activities.

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.

Genetic relationships of langur species using AFLP markers.

Cytogenetic studies of five langur species using conventional banding pattern were investigated. All species studied have an identical number of 44 diploid chromosomes, they are assumed to have common evolutionary relationships. For in depth study, molecular markers were assessed using the Amplified Fragment Length Polymorphism (AFLP) method. With seven successful primer combinations, a total of 1043 scorable bands were generated. The percentage of polymorphic bands for each primer ranged from 48.60 to 94.12%. The resulting bands were used for dendrogram construction. From the dendrogram, the individuals of Trachypithecus species are grouped into two major clusters, T. phayrei is clustered with T. obscurus, while T. cristatus is clustered with T. francoisi. The bootstrap value between two groups is 94%. The other cluster, Presbytis femoralis is separated from the Trachypithecus species with a bootstrap value of 94%. Averages of inter-specific genetic similarity values among all langur species studied are 70.16% (between T. obscurus and P. femoralis) to 88.12% (between T. phayrei and T. obscurus). In summary T. phayrei might be a subspecies of T. obscurus. The development of specific molecular markers of a species is beneficial for genetic differentiation of this group of primates.

Analysis of the genetic relationships among Thai gibbon species using AFLP markers.

Cytogenetic studies of three gibbon species using conventional banding patterns were investigated showing an identical number of 44 diploid chromosomes. They are assumed to have common evolutionary relationships. For in depth study, molecular markers were assessed using the Amplified Fragment Length Polymorphism (AFLP) method. With seven successful primer combinations, a total of 1669 scorable bands were generated. The resulting bands were used for dendrogram construction. From the dendrogram, the individuals of Hylobates lar are closely related to H. agilis more than H. pileatus with a bootstrap value of 78%. Averages of inter-specific genetic similarity values among all gibbon species studied are 67.15% (between H. lar and H. pileatus) to 71.03% (between H. lar and H. agilis). In summary three gibbon species show genetic stability within a species. The development of specific molecular markers of a species is beneficial for genetic differentiation of this group of primates.

Genetic relationships among wild Felidae in Thailand using AFLP markers.

The cytogenetics of eight Felidae species in Thailand were investigated by the colchicines-hypotonic fixation-air drying technique followed by a conventional technique. All species studied have an identical number of 38 diploid chromosomes, indicating a close genetic relationship among species. At a deep study level, the genetic relationships of eight Felidae species were accessed by the AFLP method. Blood samples were collected from sources locating in their original regions for DNA extraction. With ten successful primer combinations, a total of 4208 scorable bands were generated. Of these bands, 18.91% are polymorphic. Percentages of Polymorphic Bands (PPB) for each primer combination range from 15.00 to 23.59%. The generating bands were used for dendrogram construction. The average genetic similarity values among all Felidae species are 68.20% (between Panthera tigris and Neofelis nebulosa) to 85.53% (between Prionailurus bengalensis and Prionailurus viverrinus). The dendrogram shows that the eight Felidae species were clustered together and the subfamily Pantherinae and Felinae with Neofelis nebulosa are distinguished. The Felinae, Prionailurus bengalensis, Prionailurus viverrinus, Catopuma temminckii, Felis chaus, Pardofelis marmorata and Neofelis nebulosa were clustered together with 91% bootstrap support and the Pantherinae, Panthera pardus is clustered with Panthera tigris with 92% bootstrap support. In summary, the ten successful primer combinations can be used to determine genetic differences among eight Thailand Felidae species.

Synthesis and antibacterial activities of 5-hydroxy-4-amino-2(5H)-furanones.

Starting from the mucohalogen acids 1a and b 5-hydroxy-2(5H)-furanones 2a-h have been prepared and tested. These novel 4-amino-5-hydroxy 2(5H)-furananones have shown a broad antibiotic activity against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 in the micromolar range. A one step synthesis from mucohalogen acids towards the antibacterials 2a-h was developed, in which the target was obtained from 1a and b under reflux in toluene in presence of a catalytic amount of sulfuric acid. The derivatives 2b and c displayed a MIC and MBC of 4/8mug/ml, against Staphylococcus aureus with a selectivity towards the resistant strains.

Screening of plants containing Naja naja siamensis cobra venom inhibitory activity using modified ELISA technique.

Enzyme-linked immunosorbent assay (ELISA) has been modified for screening plants with antagonistic activity to Naja naja siamensis cobra venom. Aqueous extracts from plants were investigated for their inhibitory effects on the binding of anti-cobra venom antibody to antigen, cobra venom, fixed onto 96-well microtiter plates. Ingredients in extracts were allowed to react with immobilized venom before the subsequent addition of antivenom antibody. Venom components affected by exposure to the extracts, unable to interact with their specific antibody, were predicted to be unable to bind to their native destinations or natural receptors. Curcuma cf. zedoaria, an old Thai medicinal plant, showed clear inhibitory activity in the ELISA test. Neurotoxin and protein degradative enzymes, major components in venom, were identified as targets of this extract in Western immunoblotting analysis. Ingredients in the extract showed high affinity to the toxin in competition assay by immunoprecipitation. The extract attenuated toxin activity by extending contraction time of diaphragm muscle after envenomation and had a potency to protect cellular proteins from venom degradative enzymes. Curcuma parviflora, with less activity in ELISA, exhibited acceptable results in two experiments but negative results in two experiments, whereas Curcuma longa, having low activity in the ELISA test, never showed any favorable results. Screening of 36 samples could classify plants into an inhibition range of 0 to 86%. This modified ELISA is recommended as a preliminary screening method for inhibitors with a large number of samples.