We determined the differential expression levels of proteins in peripheral blood mononuclear cells of patients with dengue fever (DF) and dengue hemorrhagic fever (DHF). Proteins were subjected to two-dimensional electrophoresis, mass spectrometry and Western blot analysis. We identified 8 proteins that were 2-fold or more up-regulated in patients compared to healthy control, three of which, aldolase, thioredoxin peroxidase and alpha tubulin, were related to dengue infection. Both thioredoxin peroxidase and alpha tubulin were over-expressed 4.9 and 3.3 times respectively in DHF compared to DF patients while aldolase was up-regulated 2.2 times in DF compared to DHF patients. Alpha tubulin and thioredoxin peroxidase have the potential to be utilized as biomarkers for DHF.
1'-(S)-1'-Acetoxychavicol acetate (ACA) isolated from the Malaysian ethno-medicinal plant Alpinia conchigera Griff. was investigated for its potential as an anticancer drug. In this communication, we describe the cytotoxic and apoptotic properties of ACA on five human tumour cell lines. Data from MTT cell viability assays indicated that ACA induced both time- and dose-dependent cytotoxicity on all tumour cell lines tested and had no adverse cytotoxic effects on normal cells. Total mortality of the entire tumour cell population was achieved within 30 hrs when treated with ACA at 40.0 µM concentration. Flow cytometric analysis for annexin-V and PI dual staining demonstrated that cell death occurred via apoptosis, followed by secondary necrosis. The apoptotic effects of ACA were confirmed via the DNA fragmentation assay, in which consistent laddering of genomic DNA was observed for all tumour cell lines after a 24 hrs post-treatment period at the IC(50) concentration of ACA. A cell cycle analysis using PI staining also demonstrated that ACA induced cell cycle arrest at the G(0)/G(1) phase, corresponding to oral tumour cell lines. In conclusion, ACA exhibits enormous potential for future development as a chemotherapeutic drug against various malignancies.
Waste from agricultural products represents a disposal liability, which needs to be addressed. Palm oil is the most widely traded edible oil globally, and its production generates 85 million tons of aqueous by-products annually. This aqueous stream is rich in phenolic antioxidants, which were investigated for their composition and potential in vitro biological activity. We have identified three isomers of caffeoylshikimic acid as major components of oil palm phenolics (OPP). The 2,2-diphenyl-1-picrylhydrazyl assay confirmed potent free radical scavenging activity. To test for possible cardioprotective effects of OPP, we carried out in vitro LDL oxidation studies as well as ex vivo aortic ring and mesenteric vascular bed relaxation measurements. We found that OPP inhibited the Cu-mediated oxidation of human LDL. OPP also promoted vascular relaxation in both isolated aortic rings and perfused mesenteric vascular beds pre-contracted with noradrenaline. To rule out developmental toxicity, we performed teratological studies on rats up to the third generation and did not find any congenital anomalies. Thus, these initial studies suggest that OPP is safe and may have a protective role against free radical damage, LDL oxidation and its attendant negative effects, as well as vascular constriction in mitigating atherosclerosis. Oil palm vegetation liquor thus represents a new source of phenolic bioactives.
Flavonoids are secondary metabolites synthesized by plants shown to exhibit health benefits such as anti-inflammatory, antioxidant, and anti-tumor effects. Thus, due to the importance of this compound, several enzymes involved in the flavonoid pathway have been cloned and characterized in Escherichia coli. However, the formation of inclusion bodies has become a major disadvantage of this approach. As an alternative, chalcone synthase from Physcomitrella patens was secreted into the medium using a bacteriocin release protein expression vector. Secretion of P. patens chalcone synthase into the culture media was achieved by co-expression with a psW1 plasmid encoding bacteriocin release protein in E. coli Tuner (DE3) plysS. The optimized conditions, which include the incubation of cells for 20 h with 40 ng/ml mitomycin C at OD(600) induction time of 0.5 was found to be the best condition for chalcone synthase secretion.
Microwave extraction of phytochemicals from medicinal plant materials has generated tremendous research interest and shown great potential. This research highlights the importance of microwave extraction in the analysis of flavonoids, isoflavonoid and phenolics and the antioxidant properties of extracts from three varieties of the Malaysian medicinal herb, Labisia pumila Benth. High and fast extraction performance ability, equal or higher extraction efficiencies than other methods, and the need for small samples and reagent volumes are some of the attractive features of this new promising microwave assisted extraction (MAE) technique. The aims of the present research were to determine the foliar phenolics and flavonoids contents of extracts of three varieties of L. pumila obtained by a microwave extraction method while flavonoid, isoflavonoid and phenolic compounds were analyzed using RP-HPLC. Furthermore, the antioxidant activities were measured by the DPPH and FRAP methods and finally, the chemical composition of the crude methanolic extracts of the leaves of all three varieties were analyzed by GS-MS.
Matched MeSH terms: Gas Chromatography-Mass Spectrometry
A new neolignan, 3,4-dimethoxy-3',4'-methylenedioxy-2,9-epoxy-6,7-cyclo-1,8-neolign-11-en-5(5H)-one, which has been named (+)-kunstlerone (1), together with six known alkaloids: (+)-norboldine (2), (+)-N-methylisococlaurine (3), (+)-cassythicine (4), (+)-laurotetanine (5), (+)-boldine (6) and (-)-pallidine (7), were isolated from the leaves of Beilschmiedia kunstleri. The structures were established through various spectroscopic methods notably 1D- and 2D-NMR, UV, IR and LCMS-IT-TOF. (+)- Kunstlerone (1) showed a strong antioxidant activity, with an SC(50) of 20.0 µg/mL.
Perfluorinated surfactants (PFSs) in Asian freshwater fish species were analyzed to investigate tissue distribution, temporal trends, extent of pollution, and level of PFS exposure through food intake. Freshwater fish species, namely carp, snakehead, and catfish, were collected in Japan, Vietnam, India, Malaysia, and Thailand, and 10 PFSs, including perfluorooctanesulfonate (PFOS) and perfluorooctanoate, were analyzed by liquid chromatography-tandem mass spectrometry. PFSs in carp in Tokyo were more concentrated in kidneys (Σ10 PFSs = 257 ± 95 ng/g wet weight [ww]) and livers (119 ± 36 ng/g ww) than in ovaries (43 ± 2 ng/g ww) and muscles (24 ± 17 ng/g ww). Concentrations of PFOS and its precursor, perfluorooctane sulfonamide, in livers of carp and in waters in Tokyo showed a dramatic decrease during the last decade, probably because of 3 M's phasing-out of the manufacture of perfluorooctanesulfonyl-fluoride-based products in 2000. In contrast, continuing contamination by long-chain perfluorocarboxylates (PFCAs) with ≥ 9 fluorinated carbons was seen in multiple media, suggesting that these compounds continue to be emitted. PFS concentrations in freshwater fish species in tropical Asian countries were generally lower than those in developed countries, such as Japan, e.g., for PFOS in muscle, Vietnam < 0.05-0.3 ng/g ww; India < 0.05-0.2 ng/g ww; Malaysia < 0.05-0.2 ng/g ww; Thailand < 0.05 ng/g ww; and Japan (Tokyo) = 5.1-22 ng/g ww. Daily intake of short-chain PFCAs with ≤ 8 fluorinated carbons from freshwater fish species in Japan was approximately one order of magnitude lower than that from drinking water, whereas daily intake of PFOS and long-chain PFCAs with ≥ 9 fluorinated carbons from freshwater fish species was comparable with or greater than that from drinking water. Because the risk posed by exposure to these compounds through intake of fish species is a matter of concern, we recommend the continued monitoring of PFS levels in Asian developing countries.
Although cervical cancer is preventable with early detection, it remains the second most common malignancy among women. An understanding of how proteins change in their expression during a particular diseased state such as cervical cancer will contribute to an understanding of how the disease develops and progresses. Potentially, it may also lead to the ability to predict the occurrence of the disease. With this in mind, we aimed to identify differentially expressed proteins in the plasma of cervical cancer patients. Plasma from control, cervical intraepithelial neoplasia (CIN) grade 3 and squamous cell carcinoma (SCC) stage IV subjects was resolved by two-dimensional gel electrophoresis and the resulting proteome profiles compared. Differentially expressed protein spots were then identified by mass spectrometry. Eighteen proteins were found to be differentially expressed in the plasma of CIN 3 and SCC stage IV samples when compared with that of controls. Competitive ELISA further validated the expression of cytokeratin 19 and tetranectin. Functional analyses of these differentially expressed proteins will provide further insight into their potential role(s) in cervical cancer-specific monitoring and therapeutics.
Malaria is caused by parasitic protozoans of the genus Plasmodium and is one of the most prevalent infectious diseases in tropical and subtropical regions. For this reason, effective and practical diagnostic methods are urgently needed to control the spread of malaria. The aim of the current study was to identify a panel of new malarial markers, which could be used to diagnose patients infected with various Plasmodium species, including P. knowlesi, P. vivax and P. falciparum. Sera from malaria-infected patients were pooled and compared to control sera obtained from healthy individuals using the isobaric tags for relative and absolute quantitation (iTRAQ) technique. Mass spectrometry was used to identify serum proteins and quantify their relative abundance. We found that the levels of several proteins were increased in pooled serum from infected patients, including cell adhesion molecule-4 and C-reactive protein. In contrast, the serum concentration of haptoglobin was reduced in malaria-infected individuals, which we verified by western blot assay. Therefore, these proteins might represent infectious markers of malaria, which could be used to develop novel diagnostic tools for detecting P. knowlesi, P. vivax and P. falciparum. However, these potential malarial markers will need to be validated in a larger population of infected individuals.
Palm kernel cake (PKC) is a useful source of protein and energy for livestock. Recently, it has been used as an ingredient in poultry feed. Mycotoxin contamination of PKC due to inappropriate handling during production and storage has increased public concern about economic losses and health risks for poultry and humans. This concern has accentuated the need for the evaluation of mycotoxins in PKC. Furthermore, a method for quantifying mycotoxins in PKC has so far not been established. The aims of this study were therefore (1) to develop a method for the simultaneous determination of mycotoxins in PKC and (2) to validate and verify the method. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using an electrospray ionisation interface (ESI) in both positive- and negative-ion modes was developed for the simultaneous determination of aflatoxins (AFB₁, AFB₂, AFG₁ and AFG₂), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), fumonisins (FB₁ and FB₂), T-2 and HT-2 toxin in PKC. An optimum method using a 0.2 ml min⁻¹ flow rate, 0.2% formic acid in aqueous phase, 10% organic phase at the beginning and 90% organic phase at the end of the gradient was achieved. The extraction of mycotoxins was performed using a solvent mixture of acetonitrile-water-formic acid (79:20:1, v/v) without further clean-up. The mean recoveries of mycotoxins in spiked PKC samples ranged from 81% to 112%. Limits of detection (LODs) and limits of quantification (LOQs) for mycotoxin standards and PKC samples ranged from 0.02 to 17.5 μg kg⁻¹ and from 0.06 to 58.0 μg kg⁻¹, respectively. Finally, the newly developed method was successfully applied to PKC samples. The results illustrated the fact that the method is efficient and accurate for the simultaneous multi-mycotoxin determination in PKC, which can be ideal for routine analysis.
N-Acyl homoserine lactone (AHL) serves as signaling molecule for quorum sensing (QS) in Gram-negative bacteria to regulate various physiological activities including pathogenicity. With the aim of isolating freshwater-borne bacteria that can cause outbreak of disease in plants and portrayed QS properties, environmental water sampling was conducted. Here we report the preliminary screening of AHL production using Chromobacterium violaceum CV026 and Escherichia coli [pSB401] as AHL biosensors. The 16S rDNA gene sequence of isolate M009 showed the highest sequence similarity to Pantoea stewartii S9-116, which is a plant pathogen. The isolated Pantoea sp. was confirmed to produce N-3-oxohexanoyl-L-HSL (3-oxo-C6-HSL) through analysis of high resolution mass tandem mass spectrometry.
Klebsiella pneumoniae is a healthcare-associated bacterial pathogen which causes severe diseases in immunocompromised individuals. Concanavalin A (conA), a lectin which recognizes proteins with mannose or glucose residues, has been reported to agglutinate K. pneumoniae and hence, is postulated to have therapeutical potential for K. pneumoniae-induced liver infection. This study investigated the conA binding properties of a large collection of clinical isolates of K. pneumoniae. ConA agglutination reaction was demonstrated by 94 (51.4%) of 183 K. pneumoniae isolates using a microtiter plate assay. The conA agglutination reactions were inhibited in the presence of 2.5 mg/ml D-mannose and 2.5 mg/ml glucose, and following pretreatment of the bacterial suspension with protease and heating at 80ºC. Majority of the positive isolates originated from respiratory specimens. Isolation of conA-binding proteins from K. pneumoniae ATCC 700603 strain was performed using conA affinity column and the conA binding property of the eluted proteins was confirmed by western blotting analysis using conA-HRP conjugates. Proteins with molecular weights ranging from 35 to 60 kDa were eluted from the conA affinity column, of which four were identified as outer membrane protein precursor A (37 kDa), outer membrane protein precursor C (40 kDa), enolase (45 kDa) and chaperonin (60 kDa) using mass spectrometry analysis. Several conA binding proteins (including 45 and 60 kDa) were found to be immunogenic when reacted with rabbit anti-Klebsiella antibody. The function and interplay of the conA binding proteins in bacterium-host cell relationship merits further investigation.
2,4',6-Trihydroxy-4-methoxybenzophenone was isolated from the ethyl acetate fraction of Phaleria macrocarpa (Scheff.) Boerl. fruits. It was found to inhibit cell proliferation in HT-29 human colon carcinoma cell line but caused little damage to WRL-68 normal human liver and MRC-5 normal human fibroblast lung cell lines. The compound was found to sharply affect the viability of HT-29 cells in a dose- and time-dependent manner. HT-29 cells treated with the compound showed morphological changes under microscopic examination such as cell shrinkage, membrane blebbing, DNA fragmentation, and the occurrence of apoptotic nuclei. The percentage of early apoptotic, late apoptotic, and dead or necrotic cells was determined by flow cytometry using annexin V-FTIC/PI staining. In addition, flow cytometry showed that, when the HT-29 cells were treated with 115 µM of the compound, it resulted in G0/G1 phase arrest in a time-dependent manner. Western blot revealed an upregulation of PUMA, Bak, Bcl-2, and Mcl-1 proteins suggesting that the compound induced apoptosis in HT-29 cells by regulating these proteins.
Matched MeSH terms: Gas Chromatography-Mass Spectrometry
The antioxidant and anti-diabetic properties of the sequential extracts of Vernonia amygdalina based on the chemical composition of the most effective anti-diabetic extract were studied. Using DPPH and ABTS radical scavenging as well as FRAP assays, the extracts showed a consistent dose-dependent trend of potent antioxidant activity in the following solvents: water extract>methanol extract>chloroform extract>and petroleum ether extracts. In the oral glucose tolerance test, the chloroform extract exerted the highest response (33.3%), similar to metformin (27.2%), after 2h compared to the control (50.8%, P<0.05). After a 14-day administration in diabetic rats, the chloroform extract recorded the highest blood (23.5%) and serum (21.4%) glucose-lowering effects (P<0.05). GC-MS analysis of the chloroform extract revealed high levels of linoleic acid (4.72%), α-linolenic acid (10.8%) and phytols (12.0%), as well as other compounds.
Matched MeSH terms: Gas Chromatography-Mass Spectrometry
Ardisia crispa (Family: Myrsinaceae) is an evergreen, fruiting shrub that has been traditionally used as folklore medicine. Despite a scarcity of research publications, we have succeeded in showing suppressive effects on murine skin papillomagenesis. In extension, the present research was aimed at determining the effect of a quinone-rich fraction (QRF) isolated from the same root hexane extract on both initiation and promotion stages of carcinogenesis, at the selected dose of 30 mg/kg. Mice (groups I-IV) were initiated with a single dose of 7,12-dimethylbenz(α)anthracene (DMBA, 100 μg/100 μl) followed by repeated promotion of croton oil (1%) twice weekly for 20 weeks. In addition, group I (anti-initiation) received QRF 7 days before and after DMBA; group II (anti-promotion) received QRF 30 minutes before each croton oil application; group III (anti-initiation/ promotion) was treated with QRF as a combination of group I and II. A further two groups served as vehicle control (group V) and treated control (group VI). As carcinogen control, group IV showed the highest tumor volume (8.79±5.44) and tumor burden (3.60±1.17). Comparatively, group III revealed only 20% of tumor incidence, tumor burden (3.00±1.00) and tumor volume (2.40±1.12), which were significantly different from group IV. Group II also showed significant reduction of tumor volume (3.11), tumor burden (3.00) and tumor incidence (11.11%), along with prominent increase of latency period of tumor formation (week 12). Group I, nonetheless, demonstrated marked increment of tumor incidence by 40% with prompted latency period of tumor formation (week 7). No tumor formation was observed in groups V and VI. This study provided clear evidence of inhibitory effects of QRF during promotion period which was in agreement with our previous findings. The mechanism(s) underlying such effects have yet to be elucidated.
Matched MeSH terms: Gas Chromatography-Mass Spectrometry
Essential oil from Cymbopogon nardus was evaluated for activity against Trypanosoma brucei brucei BS221 (IC50 = 0.31 ± 0.03 μg/mL) and cytotoxic effect on normal kidney (Vero) cells (IC50 = >100 μg/mL). The crude essential oil was subjected to various chromatography techniques afforded active sub fractions with antitrypanosomal activity; F4 (IC50 = 0.61 ± 0.06 μg/mL), F6 (IC50= 0.73 ± 0.33 μg/mL), F7 (IC50 = 1.15 ± 0 μg/mL) and F8 (IC50 = 1.11 ± 0.01 μg/mL). These active fractions did not exhibit any toxic effects against Vero cell lines and the chemical profiles investigation indicated presence of α-and γ-eudesmol, elemol, α-cadinol and eugenol by GC/MS analysis.
Matched MeSH terms: Gas Chromatography-Mass Spectrometry
Diabetes mellitus is normally characterized by chronic hyperglycemia associated with disturbances in the fat, carbohydrate, and protein metabolism. There is an increasing trend of using natural products instead of synthetic agents as alternative therapy for disorders due to their fewer side effects. In this study, antidiabetic and antioxidant activities of different Melicope lunu-ankenda (ML) ethanolic extracts were evaluated using inhibition of α-glucosidase and 2,2-diphenyl-l-picrylhydrazyl (DPPH) radicals scavenging activity, respectively; whereas, proton nuclear magnetic resonance ((1) H NMR) and ultra-high performance liquid chromatography-tandem mass spectrometric (UHPLC-MS/MS) techniques were used for metabolite profiling of ML leaf extracts at different concentrations of ethanol and water. Sixty percent of ethanolic ML extract showed highest inhibitory effect against α-glucosidase enzyme (IC50 of 37 μg/mL) and DPPH scavenging activity (IC50 of 48 μg/mL). Antidiabetic effect of ML extracts was also evaluated in vivo and it was found that the high doses (400 mg/Kg BW) of ML extract exhibited high suppression in fasting blood glucose level by 62.75%. The metabolites responsible for variation among ML samples with variable ethanolic levels have been evaluated successfully using (1) H-NMR-based metabolomics. The principal component analysis (PCA) and partial least squares(PLS) analysis scores depicted clear and distinct separations into 4 clusters representing the 4 ethanolic concentrations by PC1 and PC2, with an eigenvalue of 69.9%. Various (1) H-NMR chemical shifts related to the metabolites responsible for sample difference were also ascribed. The main bioactive compounds identified attributing toward the separation included: isorhamnetin, skimmianine, scopoletin, and melicarpinone. Hence, ML may be used as promising medicinal plant for the development of new functional foods, new generation antidiabetic drugs, as a single entity phytomedicine or in combinational therapy.
The purpose of this study was to evaluate the effect of different cooking methods on the allergenicity of cockle and to identify proteins most frequently bound by IgE antibodies using a proteomics approach. Raw, boiled, fried and roasted extracts of the cockle were prepared. The protein profiles of the extracts were obtained by separation using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and 2-dimensional gel electrophoresis (2-DE). IgE-immunoblotting was then performed with the use of individual sera from patients with cockle allergy and the major IgE-binding proteins were analyzed by mass-spectrometry. SDS-PAGE of raw extract showed 13 protein bands. Smaller numbers of protein bands were detected in the boiled, fried and roasted extracts. The 2-DE gel profile of the raw extract further separated the protein bands to ~50 protein spots with molecular masses between 13 to 180 kDa and isoelectric point (pI) values ranging from 3 to 10. Immunoblotting of raw extract exhibited 11 IgE-binding proteins with two proteins of 36 and 40 kDa as the major IgE-binding proteins, while the boiled extract revealed 3 IgE-binding proteins. Fried and roasted extracts only showed a single IgE-binding protein at 36 kDa. 2-DE immunoblotting of raw extract demonstrated 5 to 20 IgE reactive spots. Mass spectrometry analysis led to identification of 2 important allergens, tropomyosin (36 kDa) and arginine kinase (40 kDa). Heated extracts showed a reduction in the number of IgE-reactive bands compared with raw extract, which suggest that thermal treatment can be used as a tool in attempting to reduce cockle allergenicity. The degree of allergenicity of cockle was demonstrated in the order raw > boiled > fried ≈ roasted. Two important allergens reacting with more than 50% of patients' sera identified using mass spectrometric approaches were tropomyosin and arginine kinase. Thus, allergens found in this study would help in component based diagnosis, management of cockle allergic patients and to the standardisation of allergenic test products as tools in molecular allergology.
The radioprotective effect of Polyalthia longifolia was studied in mice. P. longifolia treatment showed improvement in mice survival compared to 100% mortality in the irradiated mice. Significant increases in hemoglobin concentration, and red blood cell, white blood cell and platelet counts were observed in the animals pretreated with leaf extract. Pre-irradiation administration of P. longifolia leaf extract also increased the CFU counts of the spleen colony and increased the relative spleen size. A dose-dependent decrease in lipid peroxidation levels was observed in the animals pretreated with P. longifolia. However, although the animals pretreated with P. longifolia exhibited a significant increase in superoxide dismutase and catalase activity, the values remained below normal in both liver and the intestine. Pre-irradiation administration of P. longifolia also resulted in the regeneration of the mucosal crypts and villi of the intestine. Moreover, pretreatment with P. longifolia leaf extract also showed restoration of the normal liver cell structure and a significant reduction in the elevated levels of ALT, AST and bilirubin. These results suggested the radioprotective ability of P. longifolia leaf extract, which is significant for future investigation for human applications in developing efficient, economically viable, non-toxic natural and clinically acceptable novel radioprotectors.
Brown spot disease, caused by Cochliobolus miyabeanus, is currently considered to be one of the most important yield reducers of rice (Oryza sativa L.). Despite its agricultural importance, little is known about the virulence mechanisms deployed by the fungus. Therefore, we set out to identify novel virulence factors with a role in disease development. This article reports, for the first time, the production of tentoxin by C. miyabeanus as a virulence factor during brown spot disease and the identification of the non-ribosomal protein synthetase (NRPS) CmNps3, responsible for tentoxin biosynthesis. We compared the chemical compounds produced by C. miyabeanus strains differing in virulence ability using ultra-high-performance liquid chromatography (UHPLC) coupled to high-resolution Orbitrap mass spectrometry (HRMS). The production of tentoxin by a highly virulent strain was revealed by principal component analysis of the detected ions and confirmed by UHPLC coupled to tandem-quadrupole mass spectrometry (MS/MS). The corresponding NRPS was identified by in silico genome analysis and confirmed by gene deletion. Infection tests with wild-type and Cmnps3 mutants showed that tentoxin acts as a virulence factor and is correlated with chlorosis development during the second phase of infection. Although rice has previously been classified as a tentoxin-insensitive plant species, our data demonstrate that tentoxin production by C. miyabeanus affects symptom development.