Showing papers by "Sittiruk Roytrakul published in 2013"

The role of hydrogen peroxide in chitosan-induced resistance to osmotic stress in rice (Oryza sativa L.)

Abstract: Chitosan is a biopolymer with multiple agricultural applications. The objective of this research was to identify the mechanism required for the chitosan response. Chitosan clearly induced resistance to osmotic stress (a surrogate for drought stress) in the ‘Leung Pratew 123’ (‘LPT123’) rice (Oryza sativa L. ‘Leung Pratew123’) by enhancing plant growth and maintenance of the photosynthetic pigments during osmotic stress, but not in the derived mutated line, LPT123-TC171. Hydrogen peroxide (H2O2) was increased after osmotic stress in both lines, but higher levels were found in the LPT123 cultivar. Chitosan application did not affect the H2O2 or glutathione content under the osmotic stress condition in the LPT123 cultivar, but decreased H2O2 accumulation in the LPT123-TC171 line. The 20-fold lower glutathione level in the LPT123 cultivar suggested a low glutathione-ascorbate cycle activity that would lead to the higher H2O2 levels. Whereas, the chitosan-mediated reduction in glutathione levels in the LPT123-TC171 line during osmotic stress suggested a higher glutathione-ascorbate cycle activity leading to low H2O2 levels. Additionally, a higher peroxidase and catalase activity following chitosan treatment of the LPT123-TC171 line supports the lower observed H2O2 level. The lipid peroxidation after osmotic stress was decreased by chitosan treatment in LPT123, but not in LPT123-TC171. The exogenous H2O2 application with chitosan treatment in LPT123-TC171 could enhance plant growth during osmotic stress. It is concluded that the limited H2O2 level, the signal molecule for chitosan responses in the LPT123-TC171 line, resulted in no beneficial effects of chitosan application for osmotic stress. Therefore, H2O2 is proposed to be one of the key components for plant growth stimulation during osmotic (drought) stress by chitosan.

Solubilization and Identification of Hen Eggshell Membrane Proteins During Different Times of Chicken Embryo Development Using the Proteomic Approach

Abstract: A fertilized chicken egg is a unit of life During hatching, transport of nutrients, including calcium, have been reported from the egg components to the developing embryo Calcium is mobilized from the eggshell with the involvement of Ca2+-binding proteins In addition, other unknown proteins may also play some important roles during embryo developing process Therefore identification and prediction of biological functions of eggshell membrane (ESM) proteins during chick embryo development was conducted by proteome analysis Comparison of different lysis solutions indicated that the highest ability to extract ESM proteins could be obtained with 1 % sodium dodecyl sulfate in 5 mM Tris–HCl buffer pH 88 containing 01 % 2-mercaptoethanol In this study fertilized Cornish chicken eggs were incubated at 37 °C in humidified incubators for up to 21 days At selected times (days 1, 9, 15 and 21), samples were taken and the ESMs were carefully separated by hand, washed with distilled water, and air-dried at room temperature The ESM proteins were then solubilized and analyzed by proteome analysis Sodium dodecyl sulfate polyacrylamide gel electrophoresis combined with high performance liquid chromatography and mass spectrometry revealed 62 proteins in the ESM; only keratin is known ESM protein, 8 of which are egg white proteins and related while 53 others have not previously been reported Some differences in the types of proteins and their molecular functions were noted in ESM at different incubation times One protein which was present only at days 15 and 21 of egg incubation was identified as a calcium binding protein ie EGF like repeats and discoidin I like domain 3 (EDIL3 homologous protein)

Proteomic analysis to identify plasma orosomucoid 2 and kinesin 18A as potential biomarkers of cholangiocarcinoma

Abstract: Proteomic analysis was performed to search for the diagnostic biomarkers of the early stage of cholangiocarcinoma (CCA). For this purpose, CCA was experimentally induced in hamsters by the combination of N-nitrosodimethylamine (NDMA) treatment and Opisthorchis viverrini (OV) infection. Pooled plasma of normal control, NDMA-treated, OV-infected and OV+NDMA (ON) treated group was separated by 1-D PAGE, and the trypsin-digested bands were analyzed with LC-MS/MS. Among 82 overexpressed proteins, the study focused on 26 proteins overexpressed in ON group because CCA development was almost exclusively found in this group. A further selection was made based on the protein overexpression on day 21, the precancerous stage. Orosomucoid 2 (Orm2) was overexpressed in OV and ON groups and kinesin 18A (KIF18A) was overexpressed in the ON group. The overexpression levels were verified by real-time RT-PCR and western blotting in the liver and plasma. The transcription and translation levels of these two candidate molecules increased significantly at 21 days post-treatment before tumor development. Immunohistochemistry revealed KIF18A was expressed in the epithelial cells of newly formed small bile ducts, some inflammatory cells and hepatocytes. These results suggest that Orm2 and KIF18A could be the potential biomarkers for early diagnosis of CCA.

Brucin, an antibacterial peptide derived from fruit protein of Fructus Bruceae, Brucea javanica (L.) Merr

Abstract: UNLABELLED A novel antibacterial peptide specific to Streptococcus pyogenes was produced from dried fruit protein of Brucea javanica (L) Merr A mixture of active peptides from the fruit protein was produced in vitro by pepsin hydrolysis The hydrolysate was purified by reverse-phase HPLC, and antimicrobial peptides active against Gram-negative and Gram-positive bacteria were analysed using SDS-PAGE and nanoLC-MS/MS Here, four possible peptides were obtained and chemically synthesized for comparative study of the growth inhibition of Strep pyogenes One chemically synthesized peptide with a molecular mass of 116831 Da, His-Thr-Leu-Cys-Met-Asp-Gly-Gly-Ala-Thr-Tyr, showed the most potent antibacterial activity against Strep pyogenes This 11-amino acid peptide was named Brucin Its bacterial inhibitory activity was 16-fold and 125-fold higher than penicillin G and chloramphenicol, respectively, with a MIC value of 20 μmol l(-1) The results suggest that Brucin, a potent antibiotic peptide, may be developed as an alternative drug for the treatment of the disease caused by Strep pyogenes SIGNIFICANCE AND IMPACT OF THE STUDY An antibacterial peptide, named Brucin with specificity for Streptococcus pyogenes, was produced in vitro from dried fruit protein of Brucea javanica (L) Merr by pepsin-catalysed hydrolysis Its inhibitory activity towards the Gram-positive bacteria was higher than penicillin G and chloramphenicol The result suggested that Brucin may be applied for the treatment of the disease caused by Strep pyogenes(*)

Astakine 2—the Dark Knight Linking Melatonin to Circadian Regulation in Crustaceans

Abstract: Daily, circadian rhythms influence essentially all living organisms and affect many physiological processes from sleep and nutrition to immunity. This ability to respond to environmental daily rhythms has been conserved along evolution, and it is found among species from bacteria to mammals. The hematopoietic process of the crayfish Pacifastacus leniusculus is under circadian control and is tightly regulated by astakines, a new family of cytokines sharing a prokineticin (PROK) domain. The expression of AST1 and AST2 are light-dependent, and this suggests an evolutionarily conserved function for PROK domain proteins in mediating circadian rhythms. Vertebrate PROKs are transmitters of circadian rhythms of the suprachiasmatic nucleus (SCN) in the brain of mammals, but the mechanism by which they function is unknown. Here we demonstrate that high AST2 expression is induced by melatonin in the brain. We identify RACK1 as a binding protein of AST2 and further provide evidence that a complex between AST2 and RACK1 functions as a negative-feedback regulator of the circadian clock. By DNA mobility shift assay, we showed that the AST2-RACK1 complex will interfere with the binding between BMAL1 and CLK and inhibit the E-box binding activity of the complex BMAL1-CLK. Finally, we demonstrate by gene knockdown that AST2 is necessary for melatonin-induced inhibition of the complex formation between BMAL1 and CLK during the dark period. In summary, we provide evidence that melatonin regulates AST2 expression and thereby affects the core clock of the crustacean brain. This process may be very important in all animals that have AST2 molecules, i.e. spiders, ticks, crustaceans, scorpions, several insect groups such as Hymenoptera, Hemiptera, and Blattodea, but not Diptera and Coleoptera. Our findings further reveal an ancient evolutionary role for the prokineticin superfamily protein that links melatonin to direct regulation of the core clock gene feedback loops.

Salivary gland proteome of the human malaria vector, Anopheles campestris-like (Diptera: Culicidae)

Abstract: Anopheles campestris-like is proven to be a high-potential vector of Plasmodium vivax in Thailand. In this study, A. campestris-like salivary gland proteins were determined and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis, and nano-liquid chromatography-mass spectrometry. The total amount of salivary gland proteins in the mosquitoes aged 3-5 days was approximately 0.1 ± 0.05 μg/male and 1.38 ± 0.01 μg/female. SDS-PAGE analysis revealed at least 12 major proteins found in the female salivary glands and each morphological region of the female glands contained different major proteins. Two-dimensional gel electrophoresis showed approximately 20 major and several minor protein spots displaying relative molecular masses from 10 to 72 kDa with electric points ranging from 3.9 to 10. At least 15 glycoproteins were detected in the female glands. Similar electrophoretic protein profiles were detected comparing the male and proximal-lateral lobes of the female glands, suggesting that these lobes are responsible for sugar feeding. Blood-feeding proteins, i.e., putative 5'-nucleotidase/apyrase, anti-platelet protein, long-form D7 salivary protein, D7-related 1 protein, and gSG6, were detected in the distal-lateral lobes (DL) and/or medial lobes (ML) of the female glands. The major spots related to housekeeping proteins from other arthropod species including Culex quinquefasciatus serine/threonine-protein kinase rio3 expressed in both male and female glands, Ixodes scapularis putative sil1 expressed in DL and ML, and I. scapularis putative cyclophilin A expressed in DL. These results provide information for further study on the salivary gland proteins of A. campestris-like that are involved in hematophagy and disease transmission.

Bioactivities of Jc-SCRIP, a type 1 ribosome-inactivating protein from Jatropha curcas seed coat.

Abstract: In this study, a type 1 RIP, designated as Jc-SCRIP, was first isolated from the seed coat of Jatropha curcas Linn. It was purified by ammonium sulfate precipitation and chromatography on DEAE-Sephacel™ and CM-cellulose columns. Purification fold of Jc-SCRIP increased 113.8 times, and the yield was 1.13% of the total protein in the final step. It was shown to be a monomeric glycoprotein with a molecular mass of 38 938 Da, as determined by MALDI-TOF/MS. It exhibited hemagglutination activity and possessed strong N-glycosidase activity. The antimicrobial activity of Jc-SCRIP was tested against nine human pathogenic bacteria and one fungus; the most potent inhibitory activity was against Staphylococcus epidermidis ATCC 12228, with minimum inhibitory concentration value of 0.20 μm. Jc-SCRIP demonstrated in vitro cytotoxicity against human breast adenocarcinoma cell line (MCF-7), a colon adenocarcinoma (SW620), and a liver carcinoma cell line (HepG2), with IC50 values of 0.15, 0.25, and 0.40 mm, respectively. The results suggested that Jc-SCRIP may be a potential natural antimicrobial and anticancer agent in medical applications.

Proteomic evaluation of free fatty acid biosynthesis in Jatropha curcas L. (physic nut) kernel development

Abstract: Jatropha curcas L. is one of the economic crops that are cultivated for biodiesel production. Here, the fatty acid and protein profiles of J. curcas kernels were evaluated during their development. The fruits were divided into eight developmental stages (stages I to VIII) based on their age and morphology. The fatty acid content was analyzed at each stage using gas chromatography after conversion to methyl esters. Fatty acid levels were found to differ between all eight developmental stages, although the major fatty acid in each stage was oleic acid followed by linoleic, palmitic and stearic acids, respectively, except in stage I where linoleic acid was more common than oleic acid. All fatty acids showed a maximum content at stage III, a rapid decline at stage IV and another peak at stage VII before declining. Significant changes were found in the relative abundance of 22 proteins during seed development, of which the expression levels for transcripts encoding for four of these proteins, acetyl CoA carboxylase, phosphoenolpyruvate carboxylase, mercaptopyruvate sulfurtransferase and 4-coumarate: coenzyme A ligase, as evaluated by quantitative RT-PCR, were altered between the developmental stages of the kernels in a broadly similar pattern as the level of most fatty acids. Keywords : Jatropha curcas L., FAME, ACCase, PEPC, MST, 4CL, quantitative real time PCR African Journal of Biotechnology Vol. 12(21), pp. 3132-3142

Partial Purification of Cytotoxic Peptides against Gastric Cancer Cells from Protein Hydrolysate of Euphorbia hirta Linn.

Abstract: Protein hydrolysates prepared from a number of medicinal plants are promising sources of various bioactive peptides. In this work, proteins from dried whole plant of Euphorbia hirta Linn. were extracted and digested with pepsin for 12h. The hydrolysates of lesser than 3 KDa were fractionated by a cut-off membrane. The peptide hydrolysate was then purified by an anionexchange chromatography on DEAE-SephacelTM column and reverse-phase chromatography on Sep-pak C18 column, respectively. The cytotoxic effect of each peptide fraction against a gastric carcinoma cell line (KATO-III, ATCC No. HTB103) was investigated using colorimetric MTT viability assay. A human liver cell line (Chang Liver, CLS No. 300139) was used as a control normal cell line. Two purified peptide peaks, peak l and peak ll at 100μg peptides mL affected cell viability of the gastric cancer cell lines to 63.85±4.94 and 66.92±6.46%, respectively. Our result showed for the first time that the peptide fractions derived from protein hydrolysate of Euphorbia hirta Linn. have anti-gastric cancer activity, which offers a potential novel and natural anti-gastric cancer remedy. Keywords—Cytotoxic, peptides, Euphorbia hirta Linn., gastric carcinoma.

Proteomic profiling of Stemona alkaloids production response to chitosan elicitor.

Abstract: The study purposed to investigate the protein expression of Stemona alkaloids biosynthesis response to chitosan elicitor by 2D gel electrophoresis. The total proteins extraction of Stemona roots were performed for comparison with the control and chitosan treatments. It was found that 15 out of 150 protein spots exhibited different expression between control and chitosan culture treatment. The identified 15 protein spots were subjected to amino acid sequencing and two proteins appeared interesting for examining Stemona alkaloids biosynthesis. After treated with chitosan, glutathione S-transferase became down-regulated while heat shock protein up-regulated in relation to the control treatment. These proteins may play roles in alkaloids biosynthesis via plant defense metabolism from the presumptions that chitosan might weaken the detoxifying function of glutathione S-transferase, then, heat shock protein is probably produced to signal for tissue protection mechanism. Thus, Stemona alkaloids may be responsive to this stress.

Proteomics of Papaya ringspot virus-Infected Papaya Leaves

Abstract: The Papaya ringspot virus (PRSV) causes severe economic losses in both papaya and cucurbits throughout the tropical and subtropical regions. An understanding of the interaction between the papaya plant and PRSV can help to improve papaya production. The protein profi les of virus-infected and healthy papaya leaves were compared by two-dimensional polyacrylamide gel electrophoresis. Among the observed 490 protein spots, 227 were identifi ed using matrix-assisted laser desorption/ionizationtime of fl ight mass spectrometry and liquid chromatography-mass spectrometry. Forty-three proteins were found to be similar to those identifi ed in the Papaya EST and NCBI nr databases. They play roles in the areas of: photosynthesis (14%), photorespiration (5%), metabolism (20%), gene and biosynthesis (10%), defence related (7%), stress response (5%), signal transduction (10%), and unknown processes (29%). Spot intensity and transcription levels determined by real-time polymerase chain reaction showed ribulose-1, 5-bisphosphate carboxylase, Rieske protein ubiquinol cytochome C and chlorophyll A/B binding were down-regulated in infected plants. On the other hand, ubiquitin-like modifi ers, vascular processing enzyme and germin-like protein were up-regulated in infected plants at transcription and translation levels. The results showed the novel virus-responding mechanism of the papaya plant that might be essential for developing viral-tolerant papaya in the agricultural industries.

Proteomics Study of Palmitic Acid-induced Insulin Resistance in Adipocyte Secretome

Abstract: Insulin resistance is a major cause of type 2 diabetes. Early detection of insulin resistance will be useful to help screening and prevention of type 2 diabetes. The purpose of this study was to explore the differentially expressed secretome of insulin resistance induced 3T3-L1 adipocytes, by using 1 mM palmitic acid/1% BSA for 2 h, compared to control in order to find biomarker(s) for diagnosis of insulin resistance. The secreted proteins were analyzed by GeLC-MS/MS and the raw MS/MS data were further quantified by DecyderMS and identified by Mascot. Quantitative protein expression was obtained from 245 proteins (p < 0.05) 120 of which were found only in the induced group. These proteins are known to be involved in cellular processes, cellular component organization, regulation, development processes, cell proliferation, interaction with cells and organisms, metabolic processes and immune system proceses but most are associated with metabolic processes, cellular processes and regulation. These proteins are interesting for their potential as biomarker(s) of insulin resistance.

Lipidomics จากพลาสมาของผู้ป่วยมะเร็งท่อน้ าดีชนิดภายนอกตับโดย MALDI-TOF Lipidomics from Plasma of Extrahepatic Cholangiocarcinoma Patients by MALDI-TOF

Abstract: Cholangiocarcinoma can present as obstructive jaundice that should be discriminated from other benign conditions. The using of serum marker for diagnosis of cholangiocarcinoma is not suitable in clinical practice due to the non-specific of present markers. Lipidomics might be an approach for determination of new plasma biomarkers for cholangiocarcinoma. Lipidomics from plasma by MALDI-TOF showed peak patterns different between cholangiocarcinomas and controls. The identification of specific peak difference indicated that m/z 808.05 peak might be used for identify the specific lipids as marker for diagnosis of cholangiocarcinoma.