Displaying publications 21 - 40 of 72 in total

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  1. The protective effect of Mucuna pruriens seeds against snake venom poisoning
    Tan NH, Fung SY, Sim SM, Marinello E, Guerranti R, Aguiyi JC
    J Ethnopharmacol, 2009 Jun 22;123(2):356-8.
    PMID: 19429384 DOI: 10.1016/j.jep.2009.03.025
    The seed, leaf and root of Mucuna pruriens have been used in traditional medicine for treatments of various diseases. In Nigeria, the seed is used as oral prophylactics for snakebite.
    Matched MeSH terms: Immunoblotting
  2. Antigenic types of Orientia tsutsugamushi in Malaysia
    Tay ST, Rohani MY, Ho TM, Devi S
    Southeast Asian J. Trop. Med. Public Health, 2002 Sep;33(3):557-64.
    PMID: 12693591
    The seroprevalence of various Orientia tsutsugamushi (OT) strains among Malaysian patients with suspected scrub typhus infections was determined using an indirect immunoperoxidase (IIP) assay. IgG against a single OT strain were detected in six sera (3 Karp, 1 Gilliam and 2 TC586), whereas IgM antibodies against a single OT strain (Gilliam) were noted in 3 sera (Gilliam). IgG reactive to all OT strains were present in 33 (47.1%) of the 70 sera and IgM reactive to all OT strains were present in 22 (78.6%) of the 28 sera. The fact that most sera were reactive to multiple OT strains suggests that group-specific antigens are involved in scrub typhus infections, whereas very few were due to strain-specific epitopes present on these strains. Peak IgG and IgM titers were noted more frequently against Gilliam, Karp, and TA763 strains: this suggests that these strains may be the commonest infecting strains among Malaysian patients. Two predominant OT polypeptides consistently reacted with patients' sera were the 70 kDa and 56 kDa proteins.
    Matched MeSH terms: Immunoblotting
  3. Detection of immunoglobulins M and G using culture filtrate antigen of Burkholderia pseudomallei
    Chenthamarakshan V, Vadivelu J, Puthucheary SD
    Diagn Microbiol Infect Dis, 2001 Jan;39(1):1-7.
    PMID: 11173184
    IgM and IgG based ELISA systems were developed using the culture filtrate antigen (CFA) of Burkholderia pseudomallei. The assays were evaluated using 95 sera from 66 septicemic cases and 47 sera from 20 cases with localized melioidosis. In addition 65 sera from culture negative cases that were also serologically negative for other endemic infections clinically suspected of melioidosis were included. These were compared with sera from 260 non-melioidosis cases, 169 sera from individuals with high risk of acquiring the infection and 48 sera from healthy controls. The IgG-ELISA was 96% sensitive and 94% specific. All sera from cases with septicemic and localized infections and 61 of 63 sera from clinically suspected melioidosis cases were positive for IgG antibody. The geometric mean titre index (GMTI) values of IgG antibody in melioidosis cases were significantly higher (p < 0.0005) compared to that of healthy subjects, high risk group and subjects with non-melioidosis infections. The sensitivity and specificity of IgM ELISA was 74 and 99% respectively. The GMTI value of IgM antibody in the sera of melioidosis cases was significantly higher as compared to that of non-melioidosis disease controls (p < or = 0.001). These results demonstrate that the detection of IgG is a better indicator of the disease in the diagnosis of melioidosis.
    Matched MeSH terms: Immunoblotting
  4. Fulltext A serological study of Japanese encephalitis virus infections in northern Peninsular Malaysia
    Cardosa MJ, Choo BH, Zuraini I
    Southeast Asian J. Trop. Med. Public Health, 1991 Sep;22(3):341-6.
    PMID: 1667957
    This study describes the status of viral encephalitis in Perak, Malaysia during the year 1990. In addition, 14 cases selected from Penang and Perak during the years 1989 and 1990 are presented, with data showing titers of neutralizing antibodies against Japanese encephalitis virus (JEV) and dengue 2 virus, titers of antibodies against JEV and dengue virus antigens as determined by DEIA, and a comparison of these with the presence of IgM to JEV and dengue virus. These data show that there probably is far more viral encephalitis due to JEV in Malaysia than the national figures reflect.
    Matched MeSH terms: Immunoblotting
  5. Detection and precipitation of hepatitis B core antigen using a fusion bacteriophage
    Hasmoni SS, Yusoff K, Tan WS
    J Gen Appl Microbiol, 2005 Apr;51(2):125-31.
    PMID: 15942873
    The nucleocapsids of hepatitis B virus (HBV) are made of 180 or 240 subunits of core proteins or known as core antigens (HBcAg). A fusion bacteriophage bearing the WSFFSNI sequence that interacts tightly to HBcAg was employed as a diagnostic reagent for the detection of the antigen using the phage-enzyme-linked immunosorbent (phage-ELISA), dot blot and immunoprecipitation assays. The results from phage-ELISA and dot blot assay showed that as low as 10 ng of HBcAg can be detected optimally by 1.0x10(12) pfu/ml fusion M13 bacteriophage. The sensitivity of the dot blot assay corresponds with that of the phage-ELISA. HBcAg in HBV positive serum samples can also be detected using the fusion phage via the phage-ELISA and phage-dot blot assay. The phage cross-linked to cyanogen bromide (CNBr) activated agarose can also be used to precipitate HBcAg in bacterial lysate. The optimum amount of phage needed for cross-linking to 1 g of agarose is about 7.0x10(6) pfu/ml which could also precipitate purified and unpurified HBcAg in bacterial lysate. This study demonstrates the potential of fusion bacteriophage bearing the sequence WSFFSNI as a diagnostic reagent and a ligand for the detection and purification of HBcAg respectively.
    Matched MeSH terms: Immunoblotting
  6. Fulltext Moringa oleifera Flower Extract Suppresses the Activation of Inflammatory Mediators in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages via NF-κB Pathway
    Tan WS, Arulselvan P, Karthivashan G, Fakurazi S
    Mediators Inflamm, 2015;2015:720171.
    PMID: 26609199 DOI: 10.1155/2015/720171
    Aim of Study. Moringa oleifera Lam. (M. oleifera) possess highest concentration of antioxidant bioactive compounds and is anticipated to be used as an alternative medicine for inflammation. In the present study, we investigated the anti-inflammatory activity of 80% hydroethanolic extract of M. oleifera flower on proinflammatory mediators and cytokines produced in lipopolysaccharide- (LPS-) induced RAW 264.7 macrophages. Materials and Methods. Cell cytotoxicity was conducted by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Nitric oxide (NO) production was quantified through Griess reaction while proinflammatory cytokines and other key inflammatory markers were assessed through enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Results. Hydroethanolic extract of M. oleifera flower significantly suppressed the secretion and expression of NO, prostaglandin E2 (PGE2), interleukin- (IL-) 6, IL-1β, tumor necrosis factor-alpha (TNF-α), nuclear factor-kappa B (NF-κB), inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2). However, it significantly increased the production of IL-10 and IκB-α (inhibitor of κB) in a concentration dependent manner (100 μg/mL and 200 μg/mL). Conclusion. These results suggest that 80% hydroethanolic extract of M. oleifera flower has anti-inflammatory action related to its inhibition of NO, PGE2, proinflammatory cytokines, and inflammatory mediator's production in LPS-stimulated macrophages through preventing degradation of IκB-α in NF-κB signaling pathway.
    Matched MeSH terms: Immunoblotting
  7. Fulltext Identification of parvalbumin and two new thermolabile major allergens of Thunnus tonggol using a proteomics approach
    Rosmilah M, Shahnaz M, Meinir J, Masita A, Noormalin A, Jamaluddin M
    Int Arch Allergy Immunol, 2013;162(4):299-309.
    PMID: 24193115 DOI: 10.1159/000354544
    The longtail tuna (Thunnus tonggol) is widely consumed in Asia. Parvalbumin, the main major allergen of fish, has been well identified in multiple fish species, yet little is known about the allergenic proteins in T. tonggol. Thus, the aim of this study was to characterize the major allergens of T. tonggol using a proteomics approach.
    Matched MeSH terms: Immunoblotting
  8. Fulltext IgE-binding residues analysis of the house dust mite allergen Der p 23
    Pang SL, Matta SA, Sio YY, Ng YT, Say YH, Ng CL, et al.
    Sci Rep, 2021 01 13;11(1):921.
    PMID: 33441720 DOI: 10.1038/s41598-020-79820-y
    House dust mites (HDMs) are one of the major causes of allergies in the world. The group 23 allergen, Der p 23, from Dermatophagoides pteronyssinus, is a major allergen amongst HDM-sensitized individuals. This study aims to determine the specific immunoglobulin E (sIgE) binding frequency and IgE-binding residues of recombinant Der p 23 (rDer p 23) allergen amongst a cohort of consecutive atopic individuals in a tropical region. We performed site-directed mutagenesis and carried out immuno-dot blot assays using 65 atopic sera. The immuno-dot blot assays results indicated that the two residues K44 and E46 which are located at the N-terminal region are the major IgE-binding residues. The rDerp-23 sIgE titers are strongly correlated to the number of IgE-binding residues for rDer p 23 (P 
    Matched MeSH terms: Immunoblotting
  9. Regional and accelerated molecular evolution in group I snake venom gland phospholipase A2 isozymes
    Chuman Y, Nobuhisa I, Ogawa T, Deshimaru M, Chijiwa T, Tan NH, et al.
    Toxicon, 2000 Mar;38(3):449-62.
    PMID: 10669032
    In accordance with detection of a few phospholipase A2 (PLA2) isozyme genes by Southern blot analysis, only two cDNAs, named NnkPLA-I , and NnkPLA-II, encoding group I PLA2s, NnkPLA-I and NnkPLA-II, respectively, were isolated from the venom gland cDNA library of Elapinae Naja naja kaouthia of Malaysia. NnkPLA-I and NnkPLA-II showed four amino acid substitutions, all of which were brought about by single nucleotide substitution. No existence of clones encoding CM-II and CM-III, PLA2 isozymes which had been isolated from the venom of N. naja kaouthia of Thailand, in Malaysian N. naja kaouthia venom gland cDNA library was verified by dot blot hybridization analysis with particular probes. NnkPLA-I and NnkPLA-II differed from CM-II and CM-III with four and two amino acid substitutions, respectively, suggesting that their molecular evolution is regional. The comparison of NnkPLA-I, NnkPLA-II and cDNAs encoding other group I snake venom gland PLA2s indicated that the 5'- and 3'-untranslated regions are more conserved than the mature protein-coding region and that the number of nucleotide substitutions per nonsynonymous site is almost equal to that per synonymous site in the protein-coding region, suggesting that accelerated evolution has occurred in group I venom gland PLA2s possibly to acquire new physiological functions.
    Matched MeSH terms: Immunoblotting
  10. Fulltext Sero-prevalence study of IgE responses to allergens from Malaysian house dust (HDM) and storage mites (SM)
    Chong KT, Wong SF, Mak JW, Loh LC, Ho TM
    Trop Biomed, 2015 Sep;32(3):524-39.
    PMID: 26695214 MyJurnal
    Allergens of Dermatophagoides and Blomia species are well-characterized but not for other species. This study was conducted to determine the prevalence of allergic sensitization to house dust (HDM) and storage mites (SM). One hundred adult subjects (aged ≥ 18) were recruited. The mite specific IgE of all allergic subjects were higher compared with healthy subjetcs despite being not statistically significant except for D. farinae and G. malaysiensis. The mean serum IgE levels against HDM and SM for allergic subjects were significantly higher compared with those in healthy subjects. They were mainly sensitized to Dermatophagoides farinae (35%) and Glycycometus malaysiensis (37%). Immunoblots revealed not all allergic subjects showed positive immuno-reactivity against the mites tested. Single or multiple bands were observed for different species. The subjects were commonly sensitized to Group 2 (9-12 kDa), 10 (38 kDa) and 18 (40-48 kDa) allergens. Twenty-one out of 60 allergic subjects were sensitized to either one or more species. The majority of them (71%) were sensitized to single species. The allergic subjects were mainly sensitized to D. pteronyssinus, followed by Tyrophagus putrecentiae and Aleuroglyphus ovatus. Seven were solely sensitized to HDM while 10 were solely sensitized to SM. Four subjects were sensitized to both. Pre-adsorption study revealed no cross-reactivity. There was difference between the prevalence and reactivity to allergens of HDM and SM in these subjects. Both ELISA and immunoblot did not correlate well but can complement each other in improving the detection of mite allergens to the species level.
    Matched MeSH terms: Immunoblotting
  11. Magnoflorine Enhances LPS-Activated Pro-Inflammatory Responses via MyD88-Dependent Pathways in U937 Macrophages
    Haque MA, Jantan I, Harikrishnan H, Abdul Wahab SM
    Planta Med, 2018 Nov;84(17):1255-1264.
    PMID: 29906814 DOI: 10.1055/a-0637-9936
    Magnoflorine, a major bioactive metabolite isolated from Tinospora crispa, has been reported for its diverse biochemical and pharmacological properties. However, there is little report on its underlying mechanisms of action on immune responses, particularly on macrophage activation. In this study, we aimed to investigate the effects of magnoflorine, isolated from T. crispa on the pro-inflammatory mediators generation induced by LPS and the concomitant NF-κB, MAPKs, and PI3K-Akt signaling pathways in U937 macrophages. Differentiated U937 macrophages were treated with magnoflorine and the release of pro-inflammatory mediators was evaluated through ELISA, while the relative mRNA expression of the respective mediators was quantified through qRT-PCR. Correspondingly, western blotting was executed to observe the modulatory effects of magnoflorine on the expression of various markers related to NF-κB, MAPK and PI3K-Akt signaling activation in LPS-primed U937 macrophages. Magnoflorine significantly enhanced the upregulation of TNF-α, IL-1β, and PGE2 production as well as COX-2 protein expression. Successively, magnoflorine prompted the mRNA transcription level of these pro-inflammatory mediators. Magnoflorine enhanced the NF-κB activation by prompting p65, IκBα, and IKKα/β phosphorylation as well as IκBα degradation. Besides, magnoflorine treatments concentration-dependently augmented the phosphorylation of JNK, ERK, and p38 MAPKs as well as Akt. The immunoaugmenting effects were further confirmed by investigating the effects of magnoflorine on specific inhibitors, where the treatment with specific inhibitors of NF-κB, MAPKs, and PI3K-Akt proficiently blocked the magnoflorine-triggered TNF-α release and COX-2 expression. Magnoflorine furthermore enhanced the MyD88 and TLR4 upregulation. The results suggest that magnoflorine has high potential on augmenting immune responses.
    Matched MeSH terms: Immunoblotting
  12. Detection of IgM and IgG antibodies to Cryptococcus neoformans proteins in blood donors and HIV patients with active cryptococcosis
    Chai HC, Tay ST
    Mycoses, 2009 Mar;52(2):166-70.
    PMID: 18643920 DOI: 10.1111/j.1439-0507.2008.01549.x
    The serological responses to Cryptococcus neoformans proteins of blood donors and HIV patients with active cryptococcosis from a tropical region were investigated in this study. Exposure to C. neoformans, an organism ubiquitous in the environment, contributes to the antibody responses observed in the blood donors. IgG responses to cryptococcal proteins were stronger than IgM responses in most sera tested in this study. A 53-kDa cryptococcal protein fragment was identified as the most immunoreactive protein on the IgM immunoblots of both blood donors and patients. Overall, there was no obvious difference in IgG responses of patients when compared with blood donors. Some immunogenic protein fragments (27.5, 76, 78 and 91.5 kDa) were detected at least two times more frequently on IgM immunoblots of patients compared with those of blood donors. It is yet to be investigated whether the proteins identified in this study may have any potential to be used as biomarker for cryptococcosis.
    Matched MeSH terms: Immunoblotting
  13. Fulltext Antibody responses of dengue fever patients to dengue 2 (New Guinea C strain) viral proteins
    AbuBakar S, Azmi A, Mohamed-Saad N, Shafee N, Chee HY
    Malays J Pathol, 1997 Jun;19(1):41-51.
    PMID: 10879241
    The present study was undertaken to investigate the antibody responses of dengue fever (DF) patients to specific dengue virus proteins. Partially purified dengue 2 New Guinea C (NGC) strain virus was used as antigen. Under the present experimental protocols, it was observed that almost all DF patients' sera had detectable presence of antibodies which recognize the dengue 2 envelope (E) protein. The convalescent-phase sera especially had significant detectable IgG, IgM and IgE against the protein. In addition, IgGs specific against the NS1 dimer and PrM were also detected. Antibody against the core (C) protein, however, was not detectable in any of the DF patients' sera. The substantial presence of IgG against the PrM in the convalescent-phase sera, and the presence of IgE specific for the E, reflect the potential importance of these antibody responses in the pathogenesis of dengue.
    Matched MeSH terms: Immunoblotting
  14. Fulltext Hepatitis C: an update
    Merican MI
    Med J Malaysia, 1992 Sep;47(3):158-69.
    PMID: 1283440
    The identification of the Hepatitis C virus using molecular cloning techniques, besides making the term Non-A Non-B Hepatitis obsolete, enables the development of specific assays for the detection of antibodies in HCV-infected individuals, thus making it possible to obtain sero-epidemiological data of the disease. The carriage of Hepatitis C antibody varies worldwide. The disease is most prevalent in intravenous drug abusers or haemophiliacs. Parenteral transmission is the most important route of transmission. Sexual, intra-familial and perinatal transmissions are uncommon. About 40% could be community-acquired (sporadic). Diagnostic tests include enzyme-linked immunosorbant (ELISA) anti-HCV assay, recombinant immunoblot assay, HCV-RNA by polymerase chain reaction and HCV-Ag. More than 50% of acute cases becomes chronic and runs a benign and indolent course. About 20% progress to cirrhosis and some of these develop hepatocellular carcinoma. Several published trials have consistently shown that treatment with interferon in some patients is useful. There is however a relapse rate of 50%. Further trials with interferon and other anti-viral agents like ribavirin are awaited for more effective treatment.
    Matched MeSH terms: Immunoblotting
  15. Demonstration of an antigenic protein specific for Salmonella typhi
    Ismail A, Hai OK, Kader ZA
    Biochem Biophys Res Commun, 1991 Nov 27;181(1):301-5.
    PMID: 1958200
    Current studies were undertaken to determine the presence of a specific antigenic protein on the outer membrane of Salmonella typhi. Immunoblot analysis using sera from patients with fevers revealed that the 50 kD band was specifically recognized only by typhoid sera. The 50 kD band located on the outer membrane is protein by nature and is not a Vi (capsular), dH (flagellar), or O9 (somatic) antigen of S. typhi. These results indicate the usefulness of the specific antigen in the development of a serodiagnostic test for typhoid fever since antibodies of both the IgM and IgG class responses were obtained.
    Matched MeSH terms: Immunoblotting
  16. Recombinant expression of Toxocara canis excretory-secretory antigen TES-120 in Escherichia coli
    Fong MY, Lau YL, Init I, Jamaiah I, Anuar AK, Rahmah N
    Southeast Asian J. Trop. Med. Public Health, 2003 Dec;34(4):723-6.
    PMID: 15115078
    The gene encoding the excretory-secretory antigen TES-120 of dog ascarid worm Toxocara canis was cloned into the bacterium Escherichia coli. The specificity of the recombinant TES-120 antigen produced by the bacterium was investigated. A total of 45 human serum samples from patients infected with differenthelminthes and protozoa, including 8 cases of toxocariasis, were tested against the recombinant antigens in immunoblot assays. The results from the assays revealed that the recombinant TES-120 antigen reacted with sera from toxocariasis patients only. This highly specific recombinant TES-120 antigen can potentially be used for the development of an inexpensive serodiagnostic assay for human toxocariasis.
    Matched MeSH terms: Immunoblotting
  17. Fulltext Hepatitis C infection and detection of antibodies, RNA and genotypes among female healthcare workers in Baghdad
    Waqar, A.K., Nik Shamsidah N.I., Nor Aini M.N., Waqar, Abd Alqahar Al –Kubaisy
    JUMMEC, 2018;21(1):14-20.
    MyJurnal
    Background: Hepatitis C Virus (HCV) is a major public health problem worldwide. About 130- 200 million people
    are infected with HCV worldwide leading to 500,000 deaths annually (WHO 2014). Healthcare workers (HCWs)
    have played an important role in the transmission of HCV infection, either as victims or as sources of infection.
    Objectives: To determine the prevalence of HCV, antibodies (Abs) RNA and genotypes among the female HCWs
    in Baghdad and to identify whether HCWs were infective or only infected.
    Subjects and Methods: A cross-sectional study involving 1001 women attending 17 health care centres in
    Baghdad, Iraq, was carried out. Information on type and duration of their occupation was obtained. HCV Abs
    (anti-HCV) were tested using a third generation enzyme immunoassay (EIA-3) and immunoblot assay (Lia
    Tek-111). Molecular analysis using RT-PCR and DNA enzyme immunoassay (DEIA) for HCV-RNA and genotype
    detections were carried out for 63 serum samples.
    Results: Only 160/1001 (15.98%) were HCWs. Anti-HCV and HCV- RNA seroprevalence were significantly higher
    (6.37%, p=0.0057, 88.83%, p= 0.011 respectively) among HCWs than non HCWs. HCWs were at a significantly
    higher risk of exposure to HCV infection (OR=2.75, 95% C.I. =1.31-5.79). There was no significant association
    between HCV genotypes and the HCWs. HCV-4 showed higher expression (62.5%) among HCWs.
    Conclusion: Female HCWs were infective and infected with HCV, thus there is a need for medical equipment
    to be sterilized and cleaned thoroughly.
    Matched MeSH terms: Immunoblotting
  18. Identification of Common and Novel Major Crab Allergens in Scylla tranquebarica and the Allergen Stability in Untreated and Vinegar-treated Crab
    Jasim HA, Misnan R, Yadzir ZHM, Abdullah N, Bakhtiar F, Arip M, et al.
    Iran J Allergy Asthma Immunol, 2021 Feb 11;20(1):76-87.
    PMID: 33639634 DOI: 10.18502/ijaai.v20i1.5414
    Crab allergy is reported as a serious form of food allergy in many countries. This study was aimed to identify the major allergens of the local mud crab, Scylla tranquebarica (S. tranquebarica), and subsequently, determine the effect of vinegar treatments on the crab allergens. Crab muscles were treated with synthetic and natural vinegar. Crab proteins were then extracted from the untreated and vinegar-treated crabs. All extracts were then fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and analyzed by immunoblotting; using sera from crab-allergic patients. The crab proteins were then further fractionated by two-dimensional electrophoresis (2-DE)and analyzed by mass spectrometry (MS). The untreated crab had 38 protein bands, while that was only a few bands between 18 to 73 kDa for the vinegar-treated crabs. Immunoblotting of untreated crab revealed 20 IgE-binding bands, whereas the vinegar-treated crabs could only retain a few IgE-binding bands. Five major allergens were identified with molecular weightsof38, 42, 49, 63, and 73 kDa in the untreated crab. In contrast, the vinegar-treated crabs had only a few major allergens with molecular weights of 38, 42, and 73 kDa. MS identified the 43 and 49 kDa as arginine kinase, while the 38, 63, and 73 kDa were identified as tropomyosin, actin, and hemocyanin, respectively. Inconclusion, we found three common major allergens for S. tranquebarica including tropomyosin, arginine kinase, and actin, and one novel allergen known as hemocyanin. All the major allergens could retain minimal allergenic capability in vinegar-treated crabs, suggesting that vinegar treatments might be useful to reduce crab allergenicity. These data would assist the clinicians in the management of crab-allergic patients worldwide.
    Matched MeSH terms: Immunoblotting
  19. In Vitro Functional Characterisation of Cytochrome P450 (CYP) 2C19 Allelic Variants CYP2C19*23 and CYP2C19*24
    Lau PS, Leong KV, Ong CE, Dong AN, Pan Y
    Biochem Genet, 2017 Feb;55(1):48-62.
    PMID: 27578295 DOI: 10.1007/s10528-016-9771-8
    Cytochrome P450 (CYP) 2C19 is essential for the metabolism of clinically used drugs including omeprazole, proguanil, and S-mephenytoin. This hepatic enzyme exhibits genetic polymorphism with inter-individual variability in catalytic activity. This study aimed to characterise the functional consequences of CYP2C19*23 (271 G>C, 991 A>G) and CYP2C19*24 (991 A>G, 1004 G>A) in vitro. Mutations in CYP2C19 cDNA were introduced by site-directed mutagenesis, and the CYP2C19 wild type (WT) as well as variants proteins were subsequently expressed using Escherichia coli cells. Catalytic activities of CYP2C19 WT and those of variants were determined by high performance liquid chromatography-based essay employing S-mephenytoin and omeprazole as probe substrates. Results showed that the level of S-mephenytoin 4'-hydroxylation activity of CYP2C19*23 (V max 111.5 ± 16.0 pmol/min/mg, K m 158.3 ± 88.0 μM) protein relative to CYP2C19 WT (V max 101.6 + 12.4 pmol/min/mg, K m 123.0 ± 19.2 μM) protein had no significant difference. In contrast, the K m of CYP2C19*24 (270.1 ± 57.2 μM) increased significantly as compared to CYP2C19 WT (123.0 ± 19.2 μM) and V max of CYP2C19*24 (23.6 ± 2.6 pmol/min/mg) protein was significantly lower than that of the WT protein (101.6 ± 12.4 pmol/min/mg). In vitro intrinsic clearance (CLint = V max/K m) for CYP2C19*23 protein was 85.4 % of that of CYP2C19 WT protein. The corresponding CLint value for CYP2C19*24 protein reduced to 11.0 % of that of WT protein. These findings suggested that catalytic activity of CYP2C19 was not affected by the corresponding amino acid substitutions in CYP2C19*23 protein; and the reverse was true for CYP2C19*24 protein. When omeprazole was employed as the substrate, K m of CYP2C19*23 (1911 ± 244.73 μM) was at least 100 times higher than that of CYP2C19 WT (18.37 ± 1.64 μM) and V max of CYP2C19*23 (3.87 ± 0.74 pmol/min/mg) dropped to 13.4 % of the CYP2C19 WT (28.84 ± 0.61 pmol/min/mg) level. Derived from V max/K m, the CLint value of CYP2C19 WT was 785 folds of CYP2C19*23. K m and V max values could not be determined for CYP2C19*24 due to its low catalytic activity towards omeprazole 5'-hydroxylation. Therefore, both CYP2C19*23 and CYP2C19*24 showed marked reduced activities of metabolising omeprazole to 5-hydroxyomeprazole. Hence, carriers of CYP2C19*23 and CYP2C19*24 allele are potentially poor metabolisers of CYP2C19-mediated substrates.
    Matched MeSH terms: Immunoblotting
  20. Fulltext Effects of Amyloid Precursor Protein Overexpression on NF-κB, Rho-GTPase and Pro-Apoptosis Bcl-2 Pathways in Neuronal Cells
    Chan HH, Leong YQ, Voon SM, Pan ML, Leong CO, Lim CL, et al.
    Rep Biochem Mol Biol, 2021 Jan;9(4):417-425.
    PMID: 33969135 DOI: 10.52547/rbmb.9.4.417
    Background: Alzheimer's disease (AD) is a neurodegenerative disorder that causes cognitive dysfunction. Previous studies have suggested that amyloid plaques, mainly comprising of amyloid-beta peptides, play a pivotal role in AD pathophysiology. This study focuses on the evaluation of the effects of amyloid precursor protein (APP) overexpression on NF-κB, Rho-GTPase and Bcl-2 mediated pro-apoptotic pathways in neuronal cells.

    Methods: A lentiviral transduction system was used to generate SH-SY5Y cells overexpressing APP. Immunoblotting was conducted to determine expression levels of NF-κB, Rho-GTPase, and Bcl-2 family proteins in the APP overexpressed cells.

    Results: In the NF-κB signaling pathway, APP-overexpressing SH-SY5Y cells showed that there was a reduction of p-NF-κB (p< 0.05) and IKKα. Subsequently, there was upregulation of protein expression of NF-Κb, IKKβ and IκBα. On the other hand, protein expression of RhoC (p< 0.05) and Rac1/2/3 was upregulated as compared to the control group. Meanwhile, a decrease in RhoA, Cdc42 (p< 0.05) and p-Rac1/cdc42 protein levels was observed in the APP-overexpressed group. Lastly, in the pro-apoptotic pathway, the expression of Bcl-2, Bid, Bok and Puma (p< 0.05) was up regulated in the APP-overexpressed group. Downregulation of Bad and Bim expression was observed in the APP-overexpressed as compared to the control group, and Bax expression remained unchanged in the APP-overexpressed group.

    Conclusion: APP overexpression regulated signaling in the NF-κB, Rho-GTPase and Bcl-2 family pathways in neuronal cells, suggesting that these are involved in promoting neuronal survival and modulating synaptic plasticity in AD. However, further studies are essential to elucidate the APP-mediated mechanism of action.

    Matched MeSH terms: Immunoblotting
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