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Immunochemical characterization of edible bird's nest allergens

Goh DL, Chua KY, Chew FT, Liang RC, Seow TK, Ou KL, et al.

J Allergy Clin Immunol, 2001 Jun;107(6):1082-7.
PMID: 11398089

BACKGROUND: We have previously described anaphylaxis induced by edible bird's nest (BN) and demonstrated that this condition is IgE mediated.
OBJECTIVES: This study aimed at describing the immunochemical properties of the BN allergens. Comparative studies between 3 commercially available sources (according to the country of origin) of BN were also made.
METHODS: Crude extracts of commercially available processed BN from Sarawak (Malaysia), Thailand, and Indonesia and fresh unprocessed BN from the caves of Sarawak were obtained by means of aqueous extraction. Specific IgE toward these sources were determined by using fluorescence allergosorbent tests (FASTs). Cross-reactivity studies between the 3 sources of commercially available processed BN were carried out by means of FAST inhibition. Immunochemical characterization by means of IgE immunoblot, periodate treatment, and heat stability studies were carried out on fresh unprocessed BN from Sarawak.
RESULTS: Serum from allergic patients showed differences in IgE binding to the 3 sources of commercially available BN, with the highest levels of specific IgE recorded with the Sarawak source (P

Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Neuraminidase treatment abrogates the binding abnormality of IgA1 from IgA nephropathy patients and the differential charge distribution of its alpha-heavy chains

Hashim OH, Shuib AS, Chua CT

Nephron, 2001 Dec;89(4):422-5.
PMID: 11721160

We have studied the interaction of the Gal-GalNAc-reactive champedak lectin-C with neuraminidase-treated and untreated IgA1 from IgA nephropathy patients. The binding ability of the lectin to untreated IgA1 from IgA nephropathy patients was significantly lower as compared to the untreated IgA1 from normal controls. This differential lectin-binding capacity was abrogated when the experiment was performed on neuraminidase-treated sera. Treatment of the serum IgA1 with neuraminidase also abrogated the differential charge distribution between the alpha-heavy chains of IgA nephropathy patients and normal controls.

Matched MeSH terms: Electrophoresis, Gel, Two-Dimensional
Epidemiology and molecular characterization of nosocomially transmitted multidrug-resistant Klebsiella pneumoniae

Parasakthi N, Vadivelu J, Ariffin H, Iyer L, Palasubramaniam S, Arasu A

Int J Infect Dis, 2000;4(3):123-8.
PMID: 11179914

OBJECTIVES: To describe the epidemiology, antimicrobial susceptibility, genomic profiles, and control of a nosocomial outbreak of multidrug-resistant Klebsiella pneumoniae (MRKP) that occurred in the pediatric oncology unit of the University of Malaya Medical Centre in Kuala Lumpur.
MATERIALS AND METHODS: A prospective epidemiologic and microbiologic study was conducted of MRKP isolated from the blood and wound of a boy with necrotizing fasciitis after a 7-day course of ceftazidime and amikacin. In the following 2 weeks, phenotypically similar MRKP were isolated from the blood cultures of four other patients and rectal swabs of another three patients and two liquid soap samples located in the same ward.
RESULTS: Antimicrobial profiles demonstrated that all the isolates were resistant to ceftazidime, sensitive to imipenem and ciprofloxacin, and confirmed to be extended-spectrum beta-lactamase producers. Plasmids of varying molecular weights were present in all isolates. In eight of these isolates, which included four from blood, there were common large molecular weight plasmids ranging from 80 kb to 100 kb. Pulsed-field gel electrophoresis analysis using XbaI demonstrated six different DNA profiles, A to F. Profile A was shared by two blood culture isolates and were related by 91%. Profile B was found in one rectal swab isolate and one isolate from liquid soap and were related by 94%. Profile C was shared by one blood isolate and one liquid soap isolate and showed 100% relatedness. Profiles D, E, and F each were demonstrated by one blood isolate and two rectal swab isolates, respectively. These showed only 65% relatedness.
CONCLUSIONS: The MRKP strains in this outbreak were not clonal in origin. The decline of the outbreak after 4 weeks was attributed to the reemphasis of standard infection control procedures and the implementation of a program that addressed sites of environmental contamination.

Matched MeSH terms: Electrophoresis, Gel, Pulsed-Field
Mild beta-thalassemia intermedia caused by compound heterozygosity for (G)gamma((A)gammadeltabeta)(o)/beta-thalassemia and molecular characterization of the defect in four Chinese families

Tan JAMA, Yap SF, Tan KL, Wong YC, Wee YC, Kok JL

Acta Haematol., 2003;109(4):169-75.
PMID: 12853688 DOI: 10.1159/000070965

Molecular characterization of the compound heterozygous condition - (G)gamma((A)gammadeltabeta)(o)/beta-thalassemia - in four families showing mild beta-thalassemia intermedia was carried out using DNA amplification techniques. Using the Amplification Refractory Mutation System (ARMS) to confirm the beta-mutations and DNA amplification to detect the 100-kb Chinese-specific (G)gamma((A)gammadeltabeta)(o)-deletion, ()two families were confirmed to possess (G)gamma((A)gammadeltabeta)(o)/beta-thalassemia with the IVSII No. 654 beta(+)-allele. In the third family, the (G)gamma((A)gammadeltabeta)(o)-deletion was confirmed in the father and the mother was a beta-thalassemia carrier with the cd 41-42 beta(o)-allele. Their affected child with (G)gamma((A)gammadeltabeta)(o)/beta-thalassemia was found to be transfusion dependent. The same (G)gamma((A)gammadeltabeta)(o)-deletion and beta-thalassemia (cd 41-42) was also confirmed in a fourth family. In addition, the mother was also diagnosed with Hb H disease (genotype -alpha(3.7)/-(SEA)). Both the children were found to possess (G)gamma((A)gammadeltabeta)(o)/beta-thalassemia but they were not transfusion dependent and this could be due to co-inheritance of alpha-thalassemia-2 (genotype-alpha(3.7)/alphaalpha) in the children together with their compound heterozygous condition.

Matched MeSH terms: Blood Protein Electrophoresis
Multidrug-resistant strains of Salmonella enterica serotype typhi are genetically homogenous and coexist with antibiotic-sensitive strains as distinct, independent clones

Thong KL, Bhutta ZA, Pang T

Int J Infect Dis, 2000;4(4):194-7.
PMID: 11231181

OBJECTIVE: The goal of this study was to report the molecular analysis of antibiotic-sensitive and multidrug-resistant (MDR) strains of Salmonella typhi, using pulsed-field gel electrophoresis (PFGE), with a particular emphasis on the coexistence of these strains in a typhoid-endemic region of Karachi, Pakistan.
METHODS: One hundred isolates of S. typhi in humans (50 MDR and 50 antibiotic-sensitive isolates) from sporadic cases of typhoid fever were analyzed by Vi-phage typing, antibiograms and PFGE.
RESULTS: The MDR S. typhi strains were resistant to ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole. Analysis by PFGE showed that 50 MDR isolates of S. typhi had a single, homogenous PFGE profile, which was distinctly different from that of 50 antibiotic-sensitive isolates obtained in the same time frame from the same area. This latter group of isolates showed much greater diversity of PFGE profiles, as has been observed in other endemic regions.
CONCLUSIONS: Multidrug-resistant and antibiotic-susceptible strains of S. typhi can coexist in endemic areas as epidemiologically independent pathogens and are not in competition for continued persistence and transmission.

Matched MeSH terms: Electrophoresis, Gel, Pulsed-Field
Isolation, identification and characterization of chicken anaemia virus in Malaysia

Chowdhury SM, Omar AR, Aini I, Hair-Bejo M, Jamaluddin AA, Kono Y, et al.

J. Biochem. Mol. Biol. Biophys., 2002 Aug;6(4):249-55.
PMID: 12186740

A study was conducted to isolate and identify chicken anaemia virus (CAV) from field samples of clinically infected broiler chickens in Malaysia. A total of 125 samples were collected from chickens aged 2-6 weeks with clinically depressed and retarded growth, of which five samples were found positive to CAV directly by polymerase chain reaction (PCR). Later, five isolates of CAV from the respective five PCR positive samples were isolated in MDCC-MSB1 cells at passage 4 based on cytopathic effects, PCR and indirect immunofluorescent antibody test. The isolates were identified as BL-1, BL-2, BL-3, BL-4 and BL-5. These CAV isolates were found to resist treatment with chloroform and heat at 37 degrees C for 2 h, 56 degrees C for 30 min and 70 degrees C for 5 min. One of the isolates, BL-5 produced significant reduction (p < 0.001) of hematocrit values (9-19%), pale bone marrow, thymus atrophy and haemorrhages in skin/muscle when inoculated into 1-day old SPF chickens. Restriction enzyme digestion of 926 bp genomic fragments of all the isolates including Cux-1 isolate with HindIII exhibited a similar pattern of bands in 2% agarose gel. The present findings confirmed the presence of CAV in Malaysia.

Matched MeSH terms: Electrophoresis, Agar Gel
Intraspecific variation of Taenia taeniaeformis as determined by various criteria

Azuma H, Okamoto M, Oku Y, Kamiya M

Parasitol Res, 1995;81(2):103-8.
PMID: 7731915

The intraspecific variation of four laboratory-reared isolates of Taenia taeniaformis the SRN and KRN isolates from Norway rats, Rattus norvegicus, captured in Japan and Malaysia, respectively; the BMM isolated from a house mouse, Mus musculus, captured in Belgium; and the ACR isolate from a gray red-backed vole, Clethrionomys rufocanus bedfordiae, captured in Japan was examined by various criteria. Eggs of each of the four isolates were orally inoculated into several species of intermediate host. They were most infective to the rodent species from which the original metacestode of each isolate had been isolated in the field, and only the ACR isolate was infective to the gray red-backed vole. Although little difference was found between the SRN, KRN, and BMM isolates by the other criteria, including the morphology of rostellar hooks, the protein composition of the metacestode, and restriction endonuclease analysis of DNA, the ACR isolate was clearly different from the others. It was considered that the ACR isolate was independent as a strain distinct from the other three isolates.

Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Detection of antibodies against Salmonella typhi outer membrane protein (OMP) preparation in typhoid fever patients

Verdugo-Rodriguez A, Gam LH, Devi S, Koh CL, Puthucheary SD, Calva E, et al.

Asian Pac J Allergy Immunol, 1993 Jun;11(1):45-52.
PMID: 8216558

An indirect ELISA was used to detect antibodies against outer membrane protein preparations (OMPs) from Salmonella typhi. Sera from patients with a definitive diagnosis of typhoid fever (TF) gave a mean absorbance reading, at 414 nm, of 1.52 +/- 0.23 as compared to 0.30 +/- 0.11 for sera from healthy individuals. This gave a positive to negative ratio of absorbance readings of approximately 5.1. Suspected TF patients (no isolation of S. typhi), with positive and negative Widal titers had mean absorbance readings of 1.282 +/00.46 and 0.25 +/- 0.19, respectively. Sera from patients with leptospirosis, rickettsial typhus, dengue fever, and other infections gave mean absorbances of 0.20 +/- 0.08, 0.24 +/- 0.08, 0.27 +/- 0.08, and 0.31 +/- 0.16, respectively. The sensitivity, specificity, positive and negative predictive values were 100%, 94%, 80% and 100%, respectively. The antibody response detected in the definitive TF cases was predominantly IgG in nature and no cross-reactivity was seen with OMP preparations extracted from E. coli. Variable reactivity was noted with OMP preparations obtained from other Salmonella spp. Three major OMPs are presented in the antigen preparation and strong binding of positive sera was detected to all three bands.

Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Latex allergy diagnosis: in vivo and in vitro standardization of a natural rubber latex extract

Turjanmaa K, Palosuo T, Alenius H, Leynadier F, Autegarden JE, André C, et al.

Allergy, 1997 Jan;52(1):41-50.
PMID: 9062628

For the diagnosis of IgE-mediated (immediate) hypersensitivity to natural rubber latex (NRL), skin prick testing with extracts of latex gloves has been widely used, but such extracts are difficult to standardize. The present study aimed to produce on an industrial scale an NRL extract from freshly collected NRL and to evaluate, calibrate, and standardize the extract by both in vivo and in vitro testing. The source material, latex of the rubber tree, Hevea brasiliensis (clone RRIM 600), was frozen immediately after collection in Malaysia and shipped in dry ice to Stallergènes SA, France. Protein and allergen profiles were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, isoelectric focusing (IEF), crossed immunoelectrophoresis (CIE), and crossed radioimmunoelectrophoresis (CRIE). Allergen quantification was effected by RAST inhibition. The capacity of the preparation to elicit immediate hypersensitivity reactions in vivo was measured by skin prick testing in 46 latex-allergic patients and 76 nonallergic control subjects. SDS-PAGE and immunoblot profiles of the extract and an NRL standard (E8) provided by the US Food and Drug Administration were almost identical, disclosing several distinct IgE-binding proteins with apparent molecular weights of 14, 20, 27, 30, and 45 kDa, conforming to reported molecular weights of several significant NRL allergens. An arbitrary index of reactivity (IR) of 100 was assigned to the extract at 1:200 dilution (w/v), having a protein content of 22 micrograms/ml. Skin prick testing of latex-allergic patients and controls using the extract at 100 IR revealed 93% sensitivity, 100% specificity, 100% negative predictive value, and 96% positive predictive value. In conclusion, a skin prick test reagent for diagnosis of type I NRL allergy was successfully standardized. The reagent was demonstrated to contain most, if not all, of the currently known clinically significant NRL allergens, and it showed high sensitivity and specificity.

Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Immunogenicity of purified lipopolysaccharide or protein-oligosaccharide conjugates of Pasteurella multocida type 6:B in mice

Muniandy N, Love DN, Mukkur TK

Comp Immunol Microbiol Infect Dis, 1998 Oct;21(4):257-79.
PMID: 9775357

Purified lipopolysaccharide (LPS) of Pasteurella multocida type 6:B, while toxic at higher doses, was protective at lower dose levels against experimentally-induced pasteurellosis in mice. However, the observed protection was abrogated if such LPS was digested with proteinase K prior to use in immunisation. The O-antigen polysaccharide side-chain (OS) of LPS did not appear to contribute to the observed protection as judged by the fact that immunisation of mice with purified OS or OS-protein conjugates, all of which were nontoxic, failed to confer protection against challenge with homologous virulent organisms. This was despite generation of significant levels of OS-specific antibodies, predominantly either of the IgM or IgG isotypes, in immunised mice.

Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Isolation and characterization of the major phospholipase A2 from the venom of Trimeresurus purpureomaculatus (shore pit viper)

Nget Hong Tan, Chon Seng Tan, Hun Teck Khor

Int. J. Biochem., 1989;21(12):1421-6.
PMID: 2612728

1. The major phospholipase A2 (PLA-DE4) of the venom of Trimeresurus purpureomaculatus (shore pit viper) has been purified to electrophoretic homogeneity. 2. The isoelectric point of the purified enzyme was determined to be 4.20, and the mol. wt was 31,700 as estimated by Sephadex G-75 gel filtration chromatography; and 14,000 as estimated by SDS-polyacrylamide gel electrophoresis. The purified enzyme hydrolyzed phosphatidylcholine (PC) faster than phosphatidylethanolamine (PE), whereas phosphatidylserine (PS) was not hydrolyzed at all (PC greater than PE greater than PS =0). However, in reaction system consisted of mixtures of PC and PS, phosphatidylserine was effectively hydrolyzed by the enzyme. 4. The phospholipase A2 exhibited edema-forming activity but not hemolytic, hemorrhagic or anticoagulant activities. It was not lethal to mice at a dosage of 10 micrograms/g by i.v. route.

Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Aberrant expression of acute-phase reactant proteins in sera and breast lesions of patients with malignant and benign breast tumors

Doustjalali SR, Yusof R, Yip CH, Looi LM, Pillay B, Hashim OH

Electrophoresis, 2004 Jul;25(14):2392-401.
PMID: 15274022

We have analyzed unfractionated sera of newly diagnosed patients (n=10) with breast carcinoma (BC), prior to treatment, and patients (n=5) with fibrocystic disease of the breast (FDB) by two-dimensional gel electrophoresis (2-DE) and silver staining. The patients' 2-DE serum protein profiles obtained were then subjected to image analysis and compared to similar data generated from sera of normal healthy female controls (n=10) of the same range of age. The relative expression of alpha1-antichymotrypsin (ACT), clusterin, and complement factor B was significantly higher in all BC patients as compared to normal controls. However, the expression of alpha1-antitrypsin (AAT) in BC patients was apparently lower than that of the controls. Similar differential expression of ACT was detected in the FDB patients. The aberrant expression of the serum acute-phase proteins of patients with BC and FDB was confirmed by competitive enzyme-linked immunosorbent assay (ELISA). Similar altered proteins expression was also observed from immunohistochemical studies of malignant (n=5) and benign (n=5) breast lesions of the respective patients performed using antisera to the aberrantly expressed proteins. However, the malignant breast lesions were instead positively stained for AAT. The differential expression of the serum proteins was apparently abrogated when a six-month follow-up study was performed on nine of the BC patients subsequent to treatment.

Matched MeSH terms: Electrophoresis, Gel, Two-Dimensional
Macrolide resistance and genotypic characterization of Streptococcus pneumoniae in Asian countries: a study of the Asian Network for Surveillance of Resistant Pathogens (ANSORP)

Song JH, Chang HH, Suh JY, Ko KS, Jung SI, Oh WS, et al.

J Antimicrob Chemother, 2004 Mar;53(3):457-63.
PMID: 14963068

To characterize mechanisms of macrolide resistance among Streptococcus pneumoniae from 10 Asian countries during 1998-2001.

Matched MeSH terms: Electrophoresis, Gel, Pulsed-Field
Fulltext Unstable haemoglobin Köln disease in members of a Malay family

Eng LL, Lopez CG, Eapen JS, Eravelly J, Wiltshire BG, Lehmann H

J Med Genet, 1972 Sep;9(3):340-3.
PMID: 5079107 DOI: 10.1136/jmg.9.3.340
Matched MeSH terms: Electrophoresis, Starch Gel
Purification and characterisation of an F16L mutant of a thermostable lipase

Ali MS, Yun CC, Chor AL, Rahman RN, Basri M, Salleh AB

Protein J, 2012 Mar;31(3):229-37.
PMID: 22350313 DOI: 10.1007/s10930-012-9395-8

A mutant of the lipase from Geobacillus sp. strain T1 with a phenylalanine to leucine substitution at position 16 was overexpressed in Escherichia coli strain BL21(De3)pLysS. The crude enzyme was purified by two-step affinity chromatography with a final recovery and specific activity of 47.4 and 6,315.8 U/mg, respectively. The molecular weight of the purified F16L lipase was approximately 43 kDa by 12% SDS-PAGE analysis. The F16L lipase was demonstrated to be a thermophilic enzyme due its optimum temperature at 70 °C and showed stability over a temperature range of 40-60 °C. The enzyme exhibited an optimum pH 7 in phosphate buffer and was relatively stable at an alkaline pH 8-9. Metal ions such as Ca(2+), Mn(2+), Na(+), and K(+) enhanced the lipase activity, but Mg(2+), Zn(2+), and Fe(2+) inhibited the lipase. All surfactants tested, including Tween 20, 40, 60, 80, Triton X-100, and SDS, significantly inhibited the lipolytic action of the lipase. A high hydrolytic rate was observed on long-chain natural oils and triglycerides, with a notable preference for olive oil (C18:1; natural oil) and triolein (C18:1; triglyceride). The F16L lipase was deduced to be a metalloenzyme because it was strongly inhibited by 5 mM EDTA. Moderate inhibition was observed in the presence of PMSF at a similar concentration, indicating that serine residues are involved in its catalytic action. Further, the activity was not impaired by water-miscible solvents, including methanol, ethanol, and acetone.

Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Cold-adapted RTX lipase from antarctic Pseudomonas sp. strain AMS8: isolation, molecular modeling and heterologous expression

Ali MS, Ganasen M, Rahman RN, Chor AL, Salleh AB, Basri M

Protein J, 2013 Apr;32(4):317-25.
PMID: 23645400 DOI: 10.1007/s10930-013-9488-z

A new strain of psychrophilic bacteria (designated strain AMS8) from Antarctic soil was screened for extracellular lipolytic activity and further analyzed using molecular approach. Analysis of 16S rDNA showed that strain AMS8 was similar to Pseudomonas sp. A lipase gene named lipAMS8 was successfully isolated from strain AMS8, cloned, sequenced and overexpressed in Escherichia coli. Sequence analysis revealed that lipAMS8 consist of 1,431 bp nucleotides that encoded a polypeptide consisting of 476 amino acids. It lacked an N-terminal signal peptide and contained a glycine- and aspartate-rich nonapeptide sequence at the C-terminus, which are known to be the characteristics of repeats-in-toxin bacterial lipases. Furthermore, the substrate binding site of lipAMS8 was identified as S(207), D(255) and H(313), based on homology modeling and multiple sequence alignment. Crude lipase exhibited maximum activity at 20 °C and retained almost 50 % of its activity at 10 °C. The molecular weight of lipAMS8 was estimated to be 50 kDa via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal expression level was attained using the recombinant plasmid pET32b/BL21(DE3) expressed at 15 °C for 8 h, induced by 0.1 mM isopropyl β-D thiogalactoside (IPTG) at E. coli growth optimal density of 0.5.

Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Detection of immunoglobulin antibodies in the sera of patients using purified latex allergens

Kurup VP, Yeang HY, Sussman GL, Bansal NK, Beezhold DH, Kelly KJ, et al.

Clin Exp Allergy, 2000 Mar;30(3):359-69.
PMID: 10691894

BACKGROUND: Latex allergy is largely an occupational allergy due to sensitization to natural rubber latex allergens present in a number of health care and household products. Although several purified allergens are currently available for study, information on the usefulness of these purified, native or recombinant allergens in the demonstration of specific immunoglobulin (Ig) E in the sera of patients is lacking.
OBJECTIVE: To evaluate the purified latex allergens and to demonstrate specific IgE antibody in the sera of health care workers and spina bifida patients with clinical latex allergy.
METHODS: Two radioallergosorbent and an enzyme-linked immunosorbent assay (ELISA) using latex proteins Hev b 1, 2, 3, 4, 6 and 7 along with two glove extracts and Malaysian nonammoniated latex (MNA) were evaluated to demonstrate IgE in the sera of health care workers and spina bifida with latex allergy and controls with no history of latex allergy.
RESULTS: ELISA using the purified latex allergens demonstrated specific IgE in 32-65% health care workers and 54-100% of spina bifida patients with latex allergy. The corresponding figures for RAST were 13-48 and 23-85 for RAST-1 and 19-61 and 36-57 for RAST-2. These results were comparable with the results obtained with glove extracts and crude rubber latex proteins.
CONCLUSIONS: When used simultaneously, latex proteins Hev b 2 and Hev b 7 reacted significantly with specific serum IgE in 80% of health care workers and 92% of spina bifida patients with latex allergy by ELISA technique, while this combination gave lower positivity when the RASTs were used. By the addition of Hev b 3, specific IgE was detected in all spina bifida patients with latex allergy. Both RASTs failed to show specific IgE in the control subjects, while the ELISA showed significant latex-specific IgE in 22% of controls.

Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Neuroprotective effects of glucomoringin-isothiocyanate against H2O2-Induced cytotoxicity in neuroblastoma (SH-SY5Y) cells

Jaafaru MS, Nordin N, Rosli R, Shaari K, Bako HY, Saad N, et al.

Neurotoxicology, 2019 12;75:89-104.
PMID: 31521693 DOI: 10.1016/j.neuro.2019.09.008

Neurodegenerative diseases (NDDs) are pathological conditions characterised by progressive damage of neuronal cells leading to eventual loss of structure and function of the cells. Due to implication of multi-systemic complexities of signalling pathways in NDDs, the causes and preventive mechanisms are not clearly delineated. The study was designed to investigate the potential signalling pathways involved in neuroprotective activities of purely isolated glucomoringin isothiocyanate (GMG-ITC) against H2O2-induced cytotoxicity in neuroblastoma (SH-SY5Y) cells. GMG-ITC was isolated from Moringa oleifera seeds, and confirmed with NMR and LC-MS based methods. Gene expression analysis of phase II detoxifying markers revealed significant increase in the expression of all the genes involved, due to GMG-ITC pre-treatment. GMG-ITC also caused significant decreased in the expression of NF-kB, BACE1, APP and increased the expressions of IkB and MAPT tau genes in the differentiated cells as confirmed by multiplex genetic system analysis. The effect was reflected on the expressed proteins in the differentiated cells, where GMG-ITC caused increased in expression level of Nrf2, SOD-1, NQO1, p52 and c-Rel of nuclear factor erythroid factor 2 (Nrf2) and nuclear factor kappa-B (NF-kB) pathways respectively. The findings revealed the potential of GMG-ITC to abrogate oxidative stress-induced neurodegeneration through Nrf2 and NF-kB signalling pathways.

Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Genetic characterization of Escherichia coli O157: H7/- strains carrying the stx2 gene but not producing Shiga toxin 2

Koitabashi T, Vuddhakul V, Radu S, Morigaki T, Asai N, Nakaguchi Y, et al.

Microbiol. Immunol., 2006;50(2):135-48.
PMID: 16490932

Nine Escherichia coli O157: H7/- strains isolated primarily from non-clinical sources in Thailand and Japan carried the stx(2) gene but did not produce Stx2 toxin in a reversed passive latex agglutination (RPLA) assay. A strain (EDL933) bearing a stx(2) phage (933W) was compared to a strain (Thai-12) that was Stx2-negative but contained the stx(2) gene. To study the lack of Stx2 production, the Thai-12 stx(2) gene and its upstream nucleotide sequence were analyzed. The Thai-12 stx(2) coding region was intact and Stx2 was expressed from a cloned stx(2) gene using a plasmid vector and detected using RPLA. A lacZ fusion analysis found the Thai-12 stx(2) promoter non-functional. Because the stx(2) gene is downstream of the late promoter in the stx(2) phage genome, the antitermination activity of Q protein is essential for strong stx(2) transcription. Thai-12 had the q gene highly homologous to that of Phi21 phage but not to the 933W phage. High-level expression of exogenous q genes demonstrated Q antitermination activity was weak in Thai-12. Replication of stx(2) phage was not observed in Stx2-negative strains. The q-stx(2) gene sequence of Thai-12 was well conserved in all Stx2-negative strains. A PCR assay to detect the Thai-12 q-stx(2) sequence demonstrated that 30% of O157 strains from marketed Malaysian beef carried this sequence and they produced little or no Stx2. These results suggest that stx(2)-positive O157 strains that produce little or no Stx2 may be widely distributed in the Asian environment.

Matched MeSH terms: Electrophoresis, Gel, Pulsed-Field
A transient assay to evaluate the expression of polyhydroxybutyrate genes regulated by oil palm mesocarp-specific promoter

Omidvar V, Siti Nor Akmar A, Marziah M, Maheran AA

Plant Cell Rep, 2008 Sep;27(9):1451-9.
PMID: 18563415 DOI: 10.1007/s00299-008-0565-2

The promoter of the oil palm metallothionein-like gene (MT3-A) demonstrated mesocarp-specific activity in functional analysis using transient expression assay of reporter gene in bombarded oil palm tissue slices. In order to investigate the tissue-specific expression of polyhydroxybutyrate (PHB) biosynthetic pathway genes, a multi-gene construct carrying PHB genes fused to the oil palm MT3-A promoter was co-transferred with a construct carrying GFP reporter gene using microprojectile bombardment targeting the mesocarp and leaf tissues of the oil palm. Transcriptional analysis using RT-PCR revealed successful transcription of all the three phbA, phbB, and phbC genes in transiently transformed mesocarp but not in transiently transformed leaf tissues. Furthermore, all the three expected sizes of PHB-encoded protein products were only detected in transiently transformed mesocarp tissues on a silver stained polyacrylamide gel. Western blot analysis using polyclonal antibody specific for phbB product confirmed successful translation of phbB mRNA transcript into protein product. This study provided valuable information, supporting the future engineering of PHB-producing transgenic palms.

Matched MeSH terms: Electrophoresis, Polyacrylamide Gel

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