Aphichart Karnchanatat

Purification and characterization of a xylanase from the endophytic fungus Alternaria alternata isolated from the Thai medicinal plant, Croton oblongifolius Roxb.

1 Biotechnology Program, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok 10330, Thailand. 2 Department of Microbiology, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok 10330, Thailand. 3 Department of Botany, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok 10330, Thailand. 4 Department of Chemistry, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok 10330, Thailand. 5 Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok 10330, Thailand.

Salt stress enhances choline uptake in the halotolerant cyanobacterium Aphanothece halophytica

The uptake of [14C]choline by a suspension of exponential-phase Aphanothece halophytica under various conditions has been studied. Salt stress was found to enhance the uptake of choline. The kinetics of choline transport followed the Michaelis-Menten relationship with apparent K(m) values of 272 and 286 microM, maximum rates of transport (V(max)) of 18 and 37 nmol/min/mg protein for unstressed and salt-stressed cells, respectively. Choline uptake under salt stress was significantly reduced in chloramphenicol-treated cells, suggesting that the activation by salt stress occurred via an inducible transport system. This was corroborated by the existence of the periplasmic choline binding protein, whose content was higher in cells grown under salt-stress condition. Exogenously provided choline significantly increased the growth rate of cells grown under salt stress, although less efficiently than glycine betaine. The presence of 1 mM choline in the growth medium conferred tolerance to high salinity on A. halophytica with the maintenance of high growth up to 1.5 M NaCl. The uptake of choline was Na(+)-dependent, sensitive to various metabolic inhibitors as well as thiol-reactive agents. The results of competition studies suggested that N-methyl on one end of molecule and on the other end either an aldehyde, an alcohol or a neutral group were important features for substrate recognition.

A proteomic analysis of Curcuma comosa Roxb. rhizomes

BackgroundThe similarly in plant physiology and the difficulty of plant classification, in some medicinal plant species, especially plants of the Zingiberaceae family, are a major problem for pharmacologists, leading to mistaken use. To overcome this problem, the proteomic base method was used to study protein profiles of the plant model, Curcuma comosa Roxb., which is a member of the Zingiberaceae and has been used in traditional Thai medicine as an anti-inflammatory agent for the treatment of postpartum uterine bleeding.ResultsDue to the complexity of protein extraction from this plant, microscale solution-phase isoelectric focusing (MicroSol-IEF) was used to enrich and improve the separation of Curcuma comosa rhizomes phenol-soluble proteins, prior to resolving and analyzing by two-dimensional polyacrylamide gel electrophoresis and identification by tandem mass spectrometry. The protein patterns showed a high abundance of protein spots in the acidic range, including three lectin proteins. The metabolic and defense enzymes, such as superoxide dismutase (SOD) and ascorbate peroxidase, that are associated with antioxidant activity, were mainly found in the basic region. Furthermore, cysteine protease was found in this plant, as had been previously reported in other Zingiberaceae plants.ConclusionThis report presents the protein profiles of the ginger plant, Curcuma comosa. Several interesting proteins were identified in this plant that may be used as a protein marker and aid in identifying plants of the Zingiberaceae family.

ZINGIPAIN, A CYSTEINE PROTEASE FROM Zingiber ottensii VALETON RHIZOMES WITH ANTIPROLIFERATIVE ACTIVITIES AGAINST FUNGI AND HUMAN MALIGNANT CELL LINES

The objective of this study was to investigate the activity of a protein identified as cysteine protease, purified from Zingiber ottensii Valeton rhizomes, in terms of antiproliferation against fungi, bacteria, and human malignant cell lines. By means of buffer extraction followed by (NH4)2SO4 precipitation and ion-exchange chromatography, the obtained dominant protein (designated F50) was submitted to non-denaturing and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), where a single band and three bands were revealed from eletrophoretic patterns, respectively. It could be concluded at this point that the F50 was potentially a heterotrimer or heterodimer composed of either two small (∼13.8 and ∼15.2 kD) subunits or these two together with a larger (∼32.5 kD) one. In-gel digestion was carried out for the most intense band from reducing SDS-PAGE, and to the resulting material was applied liquid chromatography (LC)–mass spectroscopy (MS)/MS. The main F50 subunit was found to contain fragments with 100% similarity to zingipain-1, a cysteine protease first discovered in Zingiber officinale. The activity corresponding to the identified data, cysteine protease, was then confirmed in the F50 by azocasein assay and a positive result was obtained. The F50 then was further investigated for antiproliferation against three plant pathogenic fungi species by disk diffusion test, four bacterial species by direct exposure in liquid culture and dish diffusion tests, and five human malignant cell lines by tissue culture assay. It was found that a dose of 23.6 µg F50/0.3 cm2 of paper disk exhibited the best inhibitory effect against Collectotrichum cassiicola, while lesser effects were found in Exserohilum turicicum and Fusarium oxysporum, respectively. No inhibitory effect against bacterial proliferation was detected in all studied bacterial strains. However, relatively strong antiproliferative effects were found against five human cell lines, with IC50 values ranging from 1.13 µg/mL (hepatoma cancer; HEP-G2) to 5.37 µg/mL (colon cancer; SW620). By periodic acid–Schiffs staining and phenol–sulfuric acid assay, the F50 was determined as a glycoprotein containing 26.30 ± 1.01% (by weight) of carbohydrate. Thus, a new glycoprotein with protease activity was successfully identified in Zingiber ottensii rhizome. The glycoprotein also contained antiproliferative activity against some plant pathogenic fungi and human cancer cell lines.

A thermostable lectin from the rhizomes of Kaempferia parviflora.

BACKGROUND Kaempferia parviflora, or black galingale (Kra-Chai-Dam), belongs to the Zingiberaceae family and is used as both a food ingredient and a medicinal plant. There are diverse reports on the biological activities of compounds extracted from the plant, such as antimalarial, antifungal and an effective sexual-enhancing role, but not on the lectins. RESULTS A lectin was isolated from the rhizomes of Kaempferia parviflora using affinity chromatography on Concanavalin A followed by gel filtration chromatography on Sephacryl S-100. The molecular weight of the purified lectin was about 41.7 kDa. This lectin showed haemagglutinating activity against erythrocytes from several sources, with the highest level being against those from rabbits. Moreover, the lectin was thermostable, with significant haemagglutinating activity detectable up to 75 degrees C. The results of trypsin digestion and liquid chromatography/tandem mass spectrometry analysis suggested that this protein could be a member of the lectin/endochitnase1 family. CONCLUSION A lectin that showed thermotolerant haemagglutinating activity against erythrocytes from several sources was successfully purified from K. paviflora rhizomes. Peptide sequence analysis indicated that this lectin is similar to lectin/endochitinase 1 (Urtica dioica) or Hevein-like protein (Hevea brasiliensis).

Superoxide dismutase isozyme detection using two-dimensional gel electrophoresis zymograms

Superoxide dismutases (SODs) are ubiquitous antioxidant enzymes involved in cell protection from reactive oxygen species. Their antioxidant activities make them of interest to applied biotechnology industries and are usually sourced from plants. SODs are also involved in stress signaling responses in plants, and can be used as indicators of these responses. In this article, a suitable method for the separation of different SOD isoforms using two-dimensional-gel electrophoresis (2D-GE) zymograms is reported. The method was developed with a SOD standard from bovine erythrocytes and later applied to extracts from Stemona tuberosa. The first (non-denaturing isoelectric focusing) and second (denaturing sodium dodecylsulphate-polyacrylamide gel electrophoresis) dimensions of duplicate 2D-GE gels were stained with either Coomassie brilliant blue G-250 for total protein visualization, or SOD activity (zymogram) using riboflavin/nitroblue tetrazolium. For confirmation, putative SOD activity positive spots were subject to trypsin digestion and nano-liquid chromatography tandem mass spectrometry, followed by searching the MASCOT database for potential identification. The method could separate different SOD isoforms from a plant extract and at least partially maintain or allow renaturation to the native forms of the enzyme. Peptide sequencing of the 2D-GE suggested that the SODs were resolved correctly, identifying the control CuZn-SOD from bovine erythrocytes. The two SODs from S. tuberosa tubers were found to be likely homologous of a CuZn-SOD. SOD detection and isoform separation by 2D-GE zymograms was efficient and reliable. The method is likely applicable to SOD detection from plants or other organisms. Moreover, a similar approach could be developed for detection of other important enzymes in the future.

An extracellular lipase from the endophytic fungi Fusarium oxysporum isolated from the Thai medicinal plant, Croton oblongifolius Roxb.

From 65 endophytic fungal isolates, ten were found to produce extracellular lipase activity, with Fusarium oxysporum isolate PTM7, isolated from the leaves of Croton oblongifolius Roxb. (Plao yai), yielding the highest level. The lipase activity in the basal culture medium of PTM7 was highest with 1% (v/v) olive oil, 1% (w/v) peptone and 0.5% (w/v) sodium nitrate as the carbon, organic and inorganic nitrogen sources, respectively. A 37.4 kDa lipase was enriched with 41.4-fold to apparent homogeneity from PTM7 culture media using 80% saturation ammonium sulfate precipitation, DEAE-cellulose anion exchange and Superdex-75 gel filtration chromatography, but at a final yield of only 2.21%. The enriched lipase showed optimal activity at pH 8 and 30 o

Cell proliferative effect of polyxyloses extracted from the rhizomes of wild turmeric, Curcuma aromatica.

Hot water-soluble crude polysaccharides were extracted from the rhizomes of wild turmeric, Curcuma aromatica Salisb. (Zingiberaceae), using dry grinding, boiling water extraction, and then ethanol precipitation. The crude polysaccharide extract was then fractionated by DEAE-cellulose ion exchange column chromatography, and subsequently further purified by Superdex G-200 gel filtration column chromatography, giving two relatively abundant polysaccharide fractions, called P11 and P21, and a much less common fraction P22 obtained in insufficient amounts for further analysis. The two main polysaccharide fractions were evaluated for monosaccharide composition by acid hydrolysis and high performance liquid chromatography (HPLC), whilst the molecular weight and functional groups were determined by gel permeable chromatography (GPC) and FT-IR, respectively. Fractions P11 and P21 were found to be polyxyloses with molecular weight-averages of 469,171 and 157,665 Da, respectively. P11 (100 μg/mL) could significantly induce human gingival fibroblast cells proliferation by 30%, while P21 (100 μg/mL) could significantly inhibit gingival fibroblast cells proliferation by 92%. The in vitro human primary gingival fibroblast cell proliferation in cell culture at a concentration of 100 μg/mL.

INHIBITION OF NITRIC OXIDE PRODUCTION IN THE MACROPHAGE-LIKE RAW 264.7 CELL LINE BY PROTEIN FROM THE RHIZOMES OF ZINGIBERACEAE PLANTS

Nitric oxide (NO) plays a key role in the pathogenesis of inflammation and has been implicated in endotoxin-induced tissue injury. Zingiberaceae is a family of indigenous plants of tropical regions, many of which have traditionally been used as anti-inflammatory agents. Here, the ability of crude protein extracts from the rhizomes of 15 Zingiberaceae species to inhibit NO production in the RAW 264.7 cell line after co-stimulation with lipopolysaccharide (LPS) and interferon-gamma (IFN-γ) was evaluated. The crude protein extract of Zingiber ottensii Valeton exhibited the highest inhibitory activity, with an IC50 value of 38.6 ± 0.34 µg protein/mL, and also suppressed the LPS- and rm-interferon (IFN)-γ-mediated increase in the inducible nitric oxide synthase (iNOS), interleukin (IL)-6, and tumor necrosis factor (TNF)-α mRNA transcript expression levels, suggesting the interference was mediated at the transcriptional level. This strong anti-inflammatory activity may have the potential to be developed as a therapeutic compound. Analytical sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry revealed four main protein bands, including a likely lectin, superoxide dismutase, and cysteine protease, in the fractions related to the antioxidant activity.

A SUPEROXIDE DISMUTASE PURIFIED FROM THE ROOTS FROM Stemona tuberosa

Proteins from the fresh roots of Stemona tuberosa (Stemonaceae) were extracted into 20 mM phosphate buffer, pH 7.2/0.1 M NaCl, precipitated with 90% saturation ammonium sulfate, and enriched by diethylaminoethanol (DEAE) cellulose. The protein eluted as a single main peak from the unbound fractions (ST-1), and appeared as a single band with superoxide dismutase (SOD) activity after native polyacrylamide gel electrophoresis (PAGE) resolution and zymogram development. ST-1 was classified as SOD due to its strong inhibition by HCN and H2O2. The amino acid sequence of three tryptic peptides of ST-1 matched with the SOD isozymes from Ananas comosus and Solanum lycopersicum. The SOD consisted of at least two heterologous protein subunits with molecular mass of 17.6 and 31.5 kD, respectively, and had an optimal SOD activity at pH 5 and over a temperature range of 0–50°C. MgCl2, MnCl2, and HgCl2 were strongly inhibitory at all concentrations tested. The SOD activity was completely negated in the presence of 0.5 mM SDS or 5 mM HgCl2. The relationship between riboflavin and nitroblue tetrazolium (NBT) on SOD activity was linear, giving K m and V max values of the purified SOD of 62.414 ± 0.015 M and 101.010 ± 0.022 µmol/min/mg protein for NBT and 27.389 ± 0.032 M and 38.167 ± 0.021 µmol/min/mg protein for riboflavin, respectively.