In our earlier study, an immunoblot analysis using sera from febrile patients revealed that a 50-kDa band from an outer membrane protein fraction of Salmonella enterica serovar Typhi was specifically recognized only by typhoid sera and not sera from other febrile illnesses. Here, we investigated the identities of the proteins contained in the immunogenic 50-kDa band to pinpoint antigens responsible for its immunogenicity. We first used LC-MS/MS for protein identification, then used the online tool ANTIGENpro for antigenicity prediction and produced recombinant proteins of the lead antigens for validation in an enzyme-linked immunosorbent assay (ELISA). We found that proteins TolC, GlpK and SucB were specific to typhoid sera but react to antibodies differently under native and denatured conditions. This difference suggests the presence of linear and conformational epitopes on these proteins.
Phytochemical investigation on rhizomes of Kaempferia angustifolia has afforded a new abietene diterpene, kaempfolienol (1) along with crotepoxide (2), boesenboxide (3), 2'-hydroxy-4,4',6'-trimethoxychalcone (4), zeylenol (5), 6-methylzeylenol (6), (24S)-24-methyl-5α-lanosta-9(11), 25-dien-3β-ol (7), sucrose, β-sitosterol, and its glycoside (8). The structures of the compounds were elucidated on the basis of spectroscopic methods (IR, MS, and NMR). Isolation of 6-methylzeylenol (6), (24S)-24-methyl-5α-lanosta-9(11), 25-dien-3β-ol (7), and β-sitosterol-3-O-β-D-glucopyranoside (8) from this plant species has never been reported previously. The spectroscopic data of (7) is firstly described in this paper. Cytotoxic screening indicated that most of the pure compounds tested showed significant activity with (4) showing the most potent activity against HL-60 (human promyelocytic leukemia) and MCF-7 (human breast cancer) cell lines. However, all extracts and most of the pure compounds tested were found to be inactive against HT-29 (human colon cancer) and HeLa (human cervical cancer) cell lines. Similarly, none of the extracts or compounds showed activity in the antimicrobial testing.
Bacteria realize the ability to communicate by production of quorum sensing (QS) molecules called autoinducers, which regulate the physiological activities in their ecological niches. The oral cavity could be a potential area for the presence of QS bacteria. In this study, we report the isolation of a QS bacterial isolate C10B from dentine caries. Preliminary screening using Chromobacterium violaceum CV026 biosensor showed that isolate C10B was able to produce N-acylhomoserine lactones (AHLs). This bacterium was further identified as a member of Burkholderia, an opportunistic pathogen. The isolated Burkholderia sp. was confirmed to produce N-hexanoyl-L-homoserine lactone (C6-HSL), N-octanoyl-L-homoserine lactone (C8-HSL), N-decanoyl-L-homoserine lactone (C10-HSL) and N-dodecanoyl-L-homoserine lactone (C12-HSL).
This study reports the concentrations and congener partners of polychlorinated biphenyls (PCBs) in commercially available paints. Polycyclic-type pigments containing dioxazine violet (pigment violet (PV) 23, PV37) and diketopyrrolopyrrole (PR254, PR255) were found to contain PCB-56, PCB-77, PCB-40, PCB-5, and PCB-12, and PCB-6, PCB-13, and PCB-15, respectively, as major congeners. Dioxazine violet is contaminated with by-products during synthesis from o-dichlorobenzene, which is used as a solvent during synthesis, and diketopyrrolopyrrole is contaminated with by-products during synthesis from p-chlorobenzonitrile. The concentration of PCBs in paint containing PV23 or PV37 was 0.050-29 mg/kg, and toxic equivalency (TEQ) values ranged 1.1-160 pg-TEQ/g. The concentration of PCBs in paint containing PR254 or PR255 was 0.0019-2.4 mg/kg. Naphthol AS is an azo-type pigment, and PCB-52 was detected in paint containing pigment red (PR) 9 with 2,5-dichloroaniline as its source. PCB-146, PCB-149, and PCB-153 were identified from paint containing PR112 produced from 2,4,5-trichloroaniline, as major congeners. These congeners have chlorine positions similar to aniline, indicating that these congeners are by-products obtained during the synthesis of pigments. The concentrations of PCBs in paints containing PR9 and PR112 were 0.0042-0.43 and 0.0044-3.8 mg/kg, respectively. The corresponding TEQ for PR112 was 0.0039-8.6 pg-TEQ/g.
Matched MeSH terms: Gas Chromatography-Mass Spectrometry
Colony morphology variation is a characteristic of Burkholderia pseudomallei primary clinical isolates, associated with variations in expression of virulence factors. Here, we performed comparative investigations on adhesion, invasion, plaque-forming abilities and protein profiles of B. pseudomallei wild-type (WT) and a small colony variant (SCV). The percentage of SCV adherence to A549 cells was significantly higher (2.73%) than WT (1.91%). In contrast, WT was significantly more efficient (0.63%) than SCV (0.31%) in invasiveness and in inducing cellular damage. Using 2-DE and MALDI TOF/TOF, 263 and 258 protein spots were detected in WT and SCV, respectively. Comparatively, 49 proteins were differentially expressed in SCV when compared with WT. Of these, 31 proteins were up-regulated, namely, nucleoside diphosphate kinase (Ndk), phosphoglycerate kinase (Pgk), thioredoxin (TrxA), putative ferritin DPS-family DNA-binding protein (DPS) and oxidoreductase (AhpC) that are known to be involved in adhesion, intracellular survival and persistence. However, among the 18 down-regulated proteins, enolase (Eno), elongation factor (EF-Tu) and universal stress-related proteins were associated with invasion and virulence. Differences observed in these protein profiles provide ample clues to their association with the morphotypic and phenotypic characteristics of colony variants, providing additional insights into the potential association of B. pseudomallei colony morphotypes with disease pathogenesis.
Leptospirosis is a widespread zoonotic disease and a public health problem, particularly in tropical and subtropical countries. Varied clinical manifestations of the disease frequently lead to misdiagnosis resulting in life-threatening multi-organ complications. Therefore, early laboratory investigation using an appropriate diagnostic approach is crucial. In the present study, a potential protein marker was identified and evaluated for its usefulness in the serodiagnosis of acute leptospirosis.
The periplasmic proteome of recombinant E. coli cells expressing human interferon-α2b (INF-α2b) was analysed by 2D-gel electrophoresis to find the most altered proteins. Of some unique up- and down-regulated proteins in the proteome, ten were identified by MS. The majority of the proteins belonged to the ABC transporter protein family. Other affected proteins were ones involved in the regulation of transcription such as DNA-binding response regulator, stress-related proteins and ecotin. Thus, the production of INF-α2b acts as a stress on the cells and results in the induction of various transporters and stress related proteins.
The present study was aimed at the identification of proteins that are differentially expressed in the urine of patients with prostate cancer (PCa), those with benign prostatic hyperplasia (BPH) and age-matched healthy male control subjects. Using a combination of 2DE and MS/MS, significantly lower expression of urinary saposin B and two different fragments of inter-alpha-trypsin inhibitor light chain (ITIL) was demonstrated in the PCa patients compared to the controls. However, only one of the ITIL fragments was significantly different between the PCa and BPH patients. When image analysis was performed on urinary proteins that were transferred onto NC membranes and detected using a lectin that binds to O-glycans, a truncated fragment of inter-alpha-trypsin inhibitor heavy chain 4 was the sole protein found to be significantly enhanced in the PCa patients compared to the controls. Together, these urinary peptide fragments might be useful complementary biomarkers to indicate PCa as well as to distinguish it from BPH, although further epidemiological evidence on the specificity and sensitivity of the protein candidates is required.
Protein profiling has revealed the presence of glacontryphan-M, a peptide toxin identified only in the sea snail genus Conus, in the wings of Hebomoia glaucippe (HG). The wings and body of HG were homogenized and the proteins were extracted and analyzed by 2D gel electrophoresis with subsequent in-gel digestion. Posttranslational protein modifications were detected and analyzed by nano-LC-MS/MS. An antibody was generated against glacontryphan-M, and protein extracts from the wings of HG samples from Malaysia, Indonesia, and the Philippines were tested by immunoblotting. Glacontryphan-M was unambiguously identified in the wings of HG containing the following posttranslational protein modifications: monoglutamylation at E55, methylation at E53, quinone modification at W61, cyanylation at C56, and amidation of the C terminus at G63. Immunoblotting revealed the presence of the toxin in the wings of HG from all origins, showing a single band for glacontryphan-M in HG samples from Malaysia and Philippines and a double band in HG samples from Indonesia. Intriguingly, sequence analysis indicated that the Conus glacontryphan is identical to that of HG. The toxin may function as a defense against diverse predators, including ants, mantes, spiders, lizards, green frogs, and birds.
The spatial distribution of 19 polycyclic aromatic hydrocarbons (tPAHs) was quantified in aquacultures located in intertidal mudflats of the west coast of Peninsular Malaysia in order to investigate bioaccumulation of PAH in blood cockles, Anadara granosa (A. granosa). Fifty-four samples from environmental matrices and A. granosa were collected. The sampling locations were representative of a remote area as well as PAH-polluted areas. The relationship of increased background levels of PAH to anthropogenic PAH sources in the environment and their effects on bioaccumulation levels of A. granosa are investigated in this study. The levels of PAH in the most polluted station were found to be up to ten-fold higher than in remote areas in blood cockle. These high concentrations of PAHs reflected background contamination, which originates from distant airborne and waterborne transportation of contaminated particles. The fraction and source identification of PAHs, based on fate and transport considerations, showed a mix of petrogenic and pyrogenic sources. The relative biota-sediment accumulation factors (RBSAF), relative bioaccumulation factors from filtered water (RBAFw), and from suspended particulate matter (SPM) (RBAFSP) showed higher bioaccumulations of the lower molecular weight of PAHs (LMWs) in all stations, except Kuala Juru, which showed higher bioaccumulation of the higher molecular weight of PAHs (HMWs). Calculations of bioaccumulation factors showed that blood cockle can accumulate PAHs from sediment as well as water samples, based on the physico-chemical characteristics of habitat and behaviour of blood cockles. Correlations among concentrations of PAHs in water, SPM, sediment and A. granosa at the same sites were also found. Identification of PAH levels in different matrices showed that A. granosa can be used as a good biomonitor for LMW of PAHs and tPAHs in mudflats. Considering the toxicity and carcinogenicity of PAHs, the bioaccumulation by blood cockles are a potential hazard for both blood cockles and their consumers.
Matched MeSH terms: Gas Chromatography-Mass Spectrometry
Crab is an important source of food allergen. Tropomyosin represents the main crab allergen and is responsible for IgE cross-reactivity between various species of crustaceans. Recently, other new crab allergens including arginine kinase have been identified. However, information on allergens of the local Portunidcrab is not available. Thus, the aim of this study was to identify the major allergens of Portunus pelagicus (blue swimming crab) using the allergenomics approach. Raw and cooked extracts of the crab were prepared from the crab meat. Protein profile and IgE binding pattern were demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using sera from 30 patients with crab allergy. The major allergens of the crab were then identified by two-dimensional electrophoresis (2-DE), followed by mass spectrometry analysis of the peptide digests. The SDS-PAGE of raw extract revealed approximately 20 protein fractions over a wide molecular weight range, while cooked extract demonstrated fewer protein bands. The raw extract also demonstrated a higher number of IgE reactive bands than the cooked extract. A heat-resistant protein of 36 kDa has been identified as the major allergen in both raw and cooked extracts. In addition, a heat-sensitive protein of 41 kDa was also recognized as a major allergen in raw crab. The 2-DE gel profile of the raw extract demonstrated about >100 distinct proteins spots and immunoblotting of the 2-DE profile demonstrated at least 12 different major IgE reactive spots with molecular masses between 13 to 250 kDa and isoelectric point (pI) values ranging from 4.0 to 7.0. The 36 and 41 kDa proteins were identified as the crab tropomyosin and arginine kinase, respectively by mass spectrometry. Therefore, this study confirmed that tropomyosin and arginine kinase are the major allergens of the local Portunid crab, P. pelagicus.
Protein phosphatase inhibition assay (PPIA), Neuroblastoma cell-based assay (Neuro-2a CBA) and LC-MS/MS analysis revealed for the first time the production of okadaic acid (OA) by a Prorocentrum rhathymum strain. Low amounts of OA were detected by LC-MS/MS analysis. Inhibition of PP2A activity and a weak toxicity to the Neuro-2a CBA were also observed.
An ELISA and a liquid chromatography-tandem mass spectrometry (LC-MS-MS) confirmation method were developed and validated for the identification and quantitation of ketamine and its major metabolite norketamine in urine samples. The Neogen ketamine microplate ELISA was optimized with respect to sample and enzyme conjugate volumes and the sample preincubation time before addition of the enzyme conjugate. The ELISA kit was validated to include an assessment of the dose-response curve, intra- and interday precision, limit of detection (LOD), and cross-reactivity. The sensitivity and specificity were calculated by comparison to the results from the validated LC-MS-MS confirmation method. An LC-MS-MS method was developed and validated with respect to LOD, lower limit of quantitation (LLOQ), linearity, recovery, intra- and interday precision, and matrix effects. The ELISA dose-response curve was a typical S-shaped binding curve, with a linear portion of the graph observed between 25 and 500 ng/mL for ketamine. The cross-reactivity of 200 ng/mL norketamine to ketamine was 2.1%, and no cross-reactivity was detected with 13 common drugs tested at 10,000 ng/mL. The ELISA LOD was calculated to be 5 ng/mL. Both intra- (n = 10) and interday (n = 50) precisions were below 5.0% at 25 ng/mL. The LOD for ketamine and norketamine was calculated statistically to be 0.6 ng/mL. The LLOQ values were also calculated statistically and were 1.9 ng/mL and 2.1 ng/mL for ketamine and norketamine, respectively. The test linearity was 0-1200 ng/mL with correlation coefficient (R(2)) > 0.99 for both analytes. Recoveries at 50, 500, and 1000 ng/mL range from 97.9% to 113.3%. Intra- (n = 5) and interday (n = 25) precisions between extracts for ketamine and norketamine were excellent (< 10%). Matrix effects analysis showed an average ion suppression of 5.7% for ketamine and an average ion enhancement of 13.0% for norketamine for urine samples collected from six individuals. A comparison of ELISA and LC-MS-MS results demonstrated a sensitivity, specificity, and efficiency of 100%. These results indicated that a cutoff value of 25 ng/mL ketamine in the ELISA screen is particularly suitable and reliable for urine testing in a forensic toxicology setting. Furthermore, both ketamine and norketamine were detected in all 34 urine samples collected from individuals socializing in pubs by the Royal Malaysian Police. Ketamine concentrations detected by LC-MS-MS ranged from 22 to 31,670 ng/mL, and norketamine concentrations ranged from 25 to 10,990 ng/mL. The concentrations of ketamine and norketamine detected in the samples are most ikely indicative of ketamine abuse.
In a preliminary screen, Aaptos aaptos showed significant cytotoxic activity towards a panel of cell lines and was thus subjected to bioassay-guided isolation of the bioactive constituents. In addition to the known aaptamine, two new derivatives of the alkaloid were isolated from the bioactive chloroform fraction of the crude methanolic extract. Detailed analysis by NMR and mass spectroscopy enabled their identification to be 3-(phenethylamino)demethyl(oxy)aaptamine and 3-(isopentylamino)demethyl(oxy) aaptamine. The cytotoxic activities of the three alkaloids were further evaluated against CEM-SS cells.
Trichoderma spp. are regularly found as a constituent of the mycoflora of many soils and are noted for their antagonistic activity against bacteria and other fungi. This latter property is the basis for the widespread interest in their use in the biological control of soil-borne fungal plant pathogens. This antagonism is partly based on their ability to produce an impressive inventory of secondary metabolites. An important group of bioactive metabolites produced by Trichoderma spp. are the non-ribosomal peptides (NRPs), especially the peptaibols. A virulent antagonistic strain, T. asperellum, which had been used in biological control strategies in Malaysia and previously examined for mycolytic enzyme production, has been studied for its potential for peptaibol production. The present research demonstrated the ability of T. asperellum to produce at least two metabolites which were identified as acid trichotoxin 1704E (Ac-Aib-Gly-Aib-Leu-Aib-Gln-Aib-Aib-Aib-Ala-Ala-Aib-Pro-Leu-Aib-Iva-Glu-Vol) and neutral trichotoxin 1717A (Ac-Aib-Gly-Aib-Leu-Aib-Gln-Aib-Aib-Aib-Ala-Aib-Aib-Pro-Leu-Aib-Iva-Gln-Vol). Addition of free Aib to the culture medium enhanced the production of trichotoxins. Biological activity of these substances was investigated against Bacillus stearothermophilus. The general characteristics of peptaibols, also found in the trichotoxins, include the presence of high proportions of the uncommon amino acid Aib, the protection of the N- and C-termini by an acetyl group and reduction of the C-terminus to 2-amino alcohols, respectively, amphipathy and microheterogeneity.
Detailed chemical studies on the roots of Piper sarmentosum and Piper nigrum have resulted in several alkaloids. The roots of P. sarmentosum gave a new aromatic compound, 1-nitrosoimino-2,4,5-trimethoxybenzene (1). Piper nigrum roots gave pellitorine (2), (E)-1-[3',4'-(methylenedioxy)cinnamoyl]piperidine (3), 2,4-tetradecadienoic acid isobutyl amide (4), piperine (5), sylvamide (6), cepharadione A (7), piperolactam D (8) and paprazine (9). Structural elucidation of these compounds was achieved through NMR and MS techniques. Cytotoxic activity screening of the plant extracts indicated some activity.
Toxoplasma gondii infection in pregnant women may result in abortion and foetal abnormalities, and may be life-threatening in immunocompromised hosts. To identify the potential infection markers of this disease, 2-DE and Western blot methods were employed to study the parasite circulating antigens and host-specific proteins in the sera of T. gondii-infected individuals. The comparisons were made between serum protein profiles of infected (n=31) and normal (n=10) subjects. Antigenic proteins were identified by immunoblotting using pooled sera and monoclonal anti-human IgM-HRP. Selected protein spots were characterised using mass spectrometry. Prominent differences were observed when serum samples of T. gondii-infected individuals and normal controls were compared. A significant up-regulation of host-specific proteins, α(2)-HS glycoprotein and α(1)-B glycoprotein, was also observed in the silver-stained gels of both active and chronic infections. However, only α(2)-HS glycoprotein and α(1)-B glycoprotein in the active infection showed immunoreactivity in Western blots. In addition, three spots of T. gondii proteins were detected, namely (i) hypothetical protein chrXII: 3984434-3 TGME 49, (ii) dual specificity protein phosphatase, catalytic domain TGME 49 and (iii) NADPH-cytochrome p450 reductase TGME 49. Thus, 2-DE approach followed by Western blotting has enabled the identification of five potential infection markers for the diagnosis of toxoplasmosis: three are parasite-specific proteins and two are host-specific proteins.
A field investigation was conducted at all water treatment plants throughout 11 states and Federal Territory in Peninsular Malaysia. The sampling points in this study include treatment plant operation, service reservoir outlet and auxiliary outlet point at the water pipelines. Analysis was performed by solid phase micro-extraction technique with a 100 microm polydimethylsiloxane fibre using gas chromatography with mass spectrometry detection to analyse 54 volatile organic compounds (VOCs) of different chemical families in drinking water. The concentration of VOCs ranged from undetectable to 230.2 microg/l. Among all of the VOCs species, chloroform has the highest concentration and was detected in all drinking water samples. Average concentrations of total trihalomethanes (THMs) were almost similar among all states which were in the range of 28.4--33.0 microg/l. Apart from THMs, other abundant compounds detected were cis and trans-1,2-dichloroethylene, trichloroethylene, 1,2-dibromoethane, benzene, toluene, ethylbenzene, chlorobenzene, 1,4-dichlorobenzene and 1,2-dichloro - benzene. Principal component analysis (PCA) with the aid of varimax rotation, and parallel factor analysis (PARAFAC) method were used to statistically verify the correlation between VOCs and the source of pollution. The multivariate analysis pointed out that the maintenance of auxiliary pipelines in the distribution systems is vital as it can become significant point source pollution to Malaysian drinking water.
Matched MeSH terms: Gas Chromatography-Mass Spectrometry
Studies on the stem of Garcinia mangostana have led to the isolation of one new xanthone mangosharin (1) (2,6-dihydroxy-8-methoxy-5-(3-methylbut-2-enyl)-xanthone) and six other prenylated xanthones, alpha-mangostin (2), beta-mangostin (3), garcinone D (4), 1,6-dihydroxy-3,7-dimethoxy-2-(3-methylbut-2-enyl)-xanthone (5), mangostanol (6) and 5,9-dihydroxy-8- methoxy-2,2-dimethyl-7-(3-methylbut-2-enyl)-2H,6H-pyrano-[3,2-b]-xanthene-6-one (7). The structures of these compounds were determined by spectroscopic methods such as 1H NMR, 13C NMR, mass spectrometry (MS) and by comparison with previous studies. All the crude extracts when screened for their larvicidal activities indicated very good toxicity against the larvae of Aedes aegypti. This article reports the isolation and identification of the above compounds as well as bioassay data for the crude extracts. These bioassay data have not been reported before.