Displaying publications 21 - 40 of 72 in total

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  1. 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
  2. Detection of virulent Newcastle disease virus using a phage-capturing dot blot assay
    Lee TC, Yusoff K, Nathan S, Tan WS
    J Virol Methods, 2006 Sep;136(1-2):224-9.
    PMID: 16797732
    Newcastle disease virus (NDV) strains can be classified as virulent or avirulent based upon the severity of the disease. Differentiation of the virus into virulent and avirulent is necessary for effective control of the disease. Biopanning experiments were performed using a disulfide constrained phage displayed heptapeptide library against three pathotypes of NDV strains: velogenic (highly virulent), mesogenic (moderately virulent) and lentogenic (avirulent). A phage clone bearing the peptide sequence SWGEYDM capable of distinguishing virulent from avirulent NDV strains was isolated. This phage clone was employed as a diagnostic reagent in a dot blot assay and it successfully detected only virulent NDV strains.
    Matched MeSH terms: Immunoblotting/methods*
  3. Development and evaluation of dot-immunobinding assay for detection of scrub typhus infection in Leptotrombidium fletcheri (Acari: Trombiculidae)
    Ho TM, Shara S, Koay AS, Cheong YM
    J Med Entomol, 1992 Jul;29(4):611-3.
    PMID: 1495069
    A dot-immunobinding assay (DIBA) was compared with a direct fluorescent antibody technique (DFAT) for the detection of Rickettsia tsutsugamushi infection in Leptotrombidium fletcheri (Womersley & Heaslip). Laboratory colonies of infected and noninfected chiggers were examined. The relative proportions of positive, negative, and indeterminate results were significantly different between DIBA and DFAT for infected but not for noninfected chiggers. DIBA was more sensitive and had a better negative predictive value and a lower false negative percentage than DFAT. It was concluded that DIBA is a suitable alternative to DFAT for detecting scrub typhus infection in chiggers.
    Matched MeSH terms: Immunoblotting*
  4. Fulltext Development and preliminary evaluation of an IgM dot-immunobinding assay (IgM-DIA) for rapid serodiagnosis of scrub typhus infection
    Koay AS, Tay ST, Cheong YM, Yasin RM
    Southeast Asian J. Trop. Med. Public Health, 1995 Jun;26(2):350-3.
    PMID: 8629074
    An IgM dot-immunobinding assay (IgM-DIA) was developed for the diagnosis of scrub typhus infection. The whole cell antigens of Karp, Kato and Gilliam strains of Rickettsia tsutsugamushi were immobilized onto nitrocellulose paper and reacted with patients sera. The presence of IgM R. tsutsugamushi specific antibody in the patient sera could be detected by the observation of a visible brown dot on the nitrocellulose paper. The IgM-DIA has a sensitivity of 90.4% and specificity of 81.4% as compared to the indirect immunoperoxidase test. The IgM-DIA is rapid, simple, cost-effective, does not require microscope or incubator. It is recommended as a rapid screening test for the diagnosis of scrub typhus infection in the field or rural area within the hyperendemic region.
    Matched MeSH terms: Immunoblotting/economics; Immunoblotting/methods*
  5. Fulltext Development of an Indirect ELISA and Dot-Blot Assay for Serological Detection of Rice Tungro Disease
    Henry Sum MS, Yee SF, Eng L, Poili E, Lamdin J
    Biomed Res Int, 2017;2017:3608042.
    PMID: 29201901 DOI: 10.1155/2017/3608042
    Rice tungro disease (RTD) is one of the most destructive diseases of rice in South and Southeast Asia. RTD is routinely detected based on visual observation of the plant. However, it is not always easy to identify the disease in the field as it is often confused with other diseases or physiological disorders. Here we report the development of two serological based assays for ease of detection of RTD. In this study we had developed and optimized an indirect ELISA and dot-blot assay for detection of RTD. The efficiency of both assays was evaluated by comparing the specificity and sensitivity of the assays to PCR assay using established primer sets. The indirect ELISA showed 97.5% and 96.6%, while the dot-blot assay showed 97.5% and 86.4% sensitivity and specificity, respectively, when compared to established PCR method. The high sensitivity and specificity of the two assays merit the use of both assays as alternative methods to diagnose RTD. Furthermore, the dot-blot assay is a simple, robust, and rapid diagnostic assay that is suitable for field test for it does not require any specialized equipment. This is a great advantage for diagnosing RTD in paddy fields, especially in the rural areas.
    Matched MeSH terms: Immunoblotting/methods*
  6. Dot immunogold filtration assay (DIGFA) for the rapid detection of specific antibodies against the rat lungworm Angiostrongylus cantonensis (Nematoda: Metastrongyloidea) using purified 31-kDa antigen
    Eamsobhana P, Gan XX, Ma A, Wang Y, Wanachiwanawin D, Yong HS
    J Helminthol, 2014 Dec;88(4):396-401.
    PMID: 23710755 DOI: 10.1017/S0022149X13000321
    A rapid dot immunogold filtration assay (DIGFA) was adopted for specific immunodiagnosis of human cerebral angiostrongyliasis, using purified 31-kDa glycoprotein specific to Angiostrongylus cantonensis as diagnostic antigen and protein A colloidal gold conjugate as antigen-antibody detector. A total of 59 serum samples were assayed - 11 samples from clinically diagnosed patients with detectable A. cantonensis-specific antibody in immunoblotting; 23 samples from patients with other related parasitic diseases, i.e. gnathostomiasis (n= 8), cysticercosis (n= 5), toxocariasis (n= 2), filariasis (n= 4), paragonimiasis (n= 2) and malaria (n= 2); and 25 samples from normal healthy subjects. The sensitivity and specificity of DIGFA to detect anti-A. cantonensis specific antibodies in serologically confirmed angiostrongyliasis cases, were both 100%. No positive DIGFA was observed in cases with other parasitic diseases, and the healthy control subjects. The 3-min DIGFA is as sensitive and specific as the 3-h immunoblot test in angiostrongyliasis confirmed cases that revealed a 31-kDa reactive band. The gold-based DIGFA is more rapid and easier to perform than the traditional enzyme-linked immunosorbent assay (ELISA). The test utilizing purified A. cantonensis antigen is reliable and reproducible for specific immunodiagnosis of human infection with A. cantonensis - thus can be applied as an additional routine test for clinical diagnostic support. Large-scale sero-epidemiological studies in endemic communities in north-east Thailand are under way to evaluate its usefulness under field conditions.
    Matched MeSH terms: Immunoblotting/methods*
  7. Effectiveness of a dot-blot immunoassay of anti-Rickettsia tsutsugamushi antibodies for serologic analysis of scrub typhus
    Weddle JR, Chan TC, Thompson K, Paxton H, Kelly DJ, Dasch G, et al.
    Am J Trop Med Hyg, 1995 Jul;53(1):43-6.
    PMID: 7625532
    We compared a commercially available dot-blot immunoassay system with the indirect immunofluorescence assay (IFA) in tests of known negative and known positive sera from scrub typhus cases. Using a panel of 100 sera from patients with various rickettsial and nonrickettsial infections, we observed that the IFA was 99% specific and the dipstick assay was 98% specific. In tests of 91 sera (30 negative and 61 positive for scrub typhus antibodies) from a study of febrile patients in Malaysia, using the standard of an IFA titer < 1:64 as negative, an IFA titer > 1:128 as positive, and an IFA titer = 1:64 as either positive or negative (supported by clinical records), dipsticks were 83% specific and 90% sensitive. The quantitative correlation of the dipsticks to IFA titers was confirmed by significant differences in geometric means of inverse IFA titers corresponding to the number of positive dipstick spots (no dots = 8.5, one dot = 43.3, two dots = 206.7, and three dots = 676.9). The assay would enable physicians and public health workers who deal with patients to quickly diagnose and appropriately treat most cases of the disease, especially in areas of high prevalence where the proportion of false-positive results to true-positive results would be low.
    Matched MeSH terms: Immunoblotting/methods*
  8. 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
  9. Eurycomanone suppresses expression of lung cancer cell tumor markers, prohibitin, annexin 1 and endoplasmic reticulum protein 28
    Wong PF, Cheong WF, Shu MH, Teh CH, Chan KL, AbuBakar S
    Phytomedicine, 2012 Jan 15;19(2):138-44.
    PMID: 21903368 DOI: 10.1016/j.phymed.2011.07.001
    Bioactive compounds from the medicinal plant, Eurycoma longifolia Jack have been shown to promote anti-proliferative effects on various cancer cell lines. Here we examined the effects of purified eurycomanone, a quassinoid found in Eurycoma longifolia Jack extract, on the expression of selected genes of the A549 lung cancer cells. Eurycomanone inhibited A549 lung cancer cell proliferation in a dose-dependent manner at concentrations ranging from 5 to 20 μg/ml. The concentration that inhibited 50% of cell growth (GI(50)) was 5.1 μg/ml. The anti-proliferative effects were not fully reversible following the removal of eurycomanone, in which 30% of cell inhibition still remained (p<0.0001, T-test). At 8 μg/ml (GI(70)), eurycomanone suppressed anchorage-independent growth of A549 cells by >25% (p<0.05, T-test, n=8) as determined using soft agar colony formation assay. Cisplatin, a chemotherapy drug used for the treatment of non small cell lung cancer on the other hand, inhibited A549 cells proliferation at concentrations ranging from 0.2 μg/ml to 15 μg/ml with a GI(50) of 0.58 μg/ml. The treatment with eurycomanone reduced the abundance expression of the lung cancer markers, heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1, p53 tumor suppressor protein and other cancer-associated genes including prohibitin (PHB), annexin 1 (ANX1) and endoplasmic reticulum protein 28 (ERp28) but not the house keeping genes. The mRNA expressions of all genes with the exception of PHB were significantly downregulated, 72 h after treatment (p<0.05, T-test, n=9). These findings suggest that eurycomanone at viable therapeutic concentrations of 5-20 μg/ml exhibited significant anti-proliferative and anti-clonogenic cell growth effects on A549 lung cancer cells. The treatment also resulted in suppression of the lung cancer cell tumor markers and several known cancer cell growth-associated genes.
    Matched MeSH terms: Immunoblotting
  10. Evaluation of bovine antibody responses to haemorrhagic septicaemia vaccine
    Johnson RB, Dawkins HJ, Spencer TL, Saharee AA, Bahaman AR, Ramdani, et al.
    Res Vet Sci, 1989 Sep;47(2):277-9.
    PMID: 2508206
    ELISA and immunoblotting techniques were used to examine the humoral immune response to Pasteurella multocida, in bovine sera from Indonesia and Malaysia. Elevated levels of antibody to a crude lipopolysaccharide preparation were found in vaccinated animals. In addition to the response to lipopolysaccharide, antibodies from the vaccinated cattle strongly labelled five to six of the 40 protein bands in this organism.
    Matched MeSH terms: Immunoblotting
  11. Expression of recombinant human epidermal growth factor in Escherichia coli and characterization of its biological activity
    Abdull Razis AF, Ismail EN, Hambali Z, Abdullah MN, Ali AM, Mohd Lila MA
    Appl Biochem Biotechnol, 2008 Mar;144(3):249-61.
    PMID: 18556814
    Recombinant human epidermal growth factor (EGF) was successfully expressed as a fusion protein in Escherichia coli system. This system was used OmpA signal sequence to produce soluble protein into the periplasm of E. coli. Human EGF (hEGF) synthesized in bacterial cell was found to be similar in size with the original protein and molecular weight approximately at 6.8 kDa. Cell proliferation assay was conducted to characterize the biological activity of hEGF on human dermal fibroblasts. The synthesized hEGF was found to be functional as compared with authentic hEGF in stimulating cell proliferation and promoting growth of cell. In comparison of biological activity between synthesized and commercial hEGF on cell proliferation, the results showed there was no significant different. This finding indicates the synthesized hEGF in E. coli system is fully bioactive in vitro.
    Matched MeSH terms: Immunoblotting
  12. Frequency and role of low molecular weight IgM in systemic lupus erythematosus. Study of patients from different ethnic origins
    Roberts-Thomson PJ, Shepherd K, Bradley J, Boey ML
    Rheumatol Int, 1990;10(3):95-8.
    PMID: 2392640
    Low molecular weight IgM (LMW IgM) is the monomeric subunit of the naturally occurring pentameric IgM. It is not seen in health but has been previously observed in systemic lupus erythematosus (SLE) particularly in those patients with active disease and may reflect an adverse prognostic finding. We have therefore studied the presence of LMW IgM in 33 Chinese or Malay SLE patients (Singapore) and 21 Caucasian patients (Adelaide). LMW IgM was measured using filtration chromatography or by a sensitive immunoblotting technique. LMW IgM was observed in all patients in the Adelaide group and in 32 patients in the Singapore group with slightly greater quantities being seen in the Adelaide group. LMW IgM constituted up to 15.3% of the total IgM and was frequently associated with the presence of other low molecular weight IgM oligomers. In both groups LMW IgM correlated significantly with the total IgM levels (P less than 0.01). In a more detailed study in the Singapore group LMW IgM also correlated significantly with the IgM anticardiolipin levels (P = 0.02) but not with IgG anticardiolipin or with IgG or IgM anti-DNA levels or with rheumatoid factor. Patients with more extensive organ involvement had higher levels of LMW IgM but not at a significant level. We conclude that circulating LMW IgM occurs almost universally in SLE, is closely related to the total IgM levels and appears independent of ethnic background. The significance of LMW IgM in this disorder is unclear.
    Matched MeSH terms: Immunoblotting
  13. Fruit-specific expression of papaya subtilase gene
    Othman R, Nuraziyan A
    J Plant Physiol, 2010 Jan 15;167(2):131-7.
    PMID: 19729222 DOI: 10.1016/j.jplph.2009.07.015
    Subtilisin-like serine proteases (EC 3.4.21) consist of a widespread family of enzymes that is involved in various processes including in plants. The full-length cDNA (CpSUB1) and the corresponding genomic DNA for papaya subtilase have been obtained using rapid amplification of cDNA ends (RACEs) and PCR primer walking techniques, respectively. The cDNA clone contains an open reading frame of 2316bp encoding 772 amino acids with a calculated molecular mass of 82.6kDa and an isoelectric point (pI) of 8.97. The CpSUB1 gene is composed of nine exons and eight introns. The amino acid sequence encoded by CpSUB1 shared high identity (>60%) with the amino acid sequence of other plant subtilisin-like proteases. Sequence analysis of CpSUB1 revealed the presence of a possible signal peptide (25 amino acid residues) and an NH(2)-terminal prosequence (88 amino acid residues). In addition, papaya subtilase possesses the characteristic subtilisin catalytic triad amino acids namely Asp, His and Ser, together with the substrate-binding site, Asn. DNA hybridization analysis showed that subtilase gene exists as a single copy in the papaya genome. RNA hybridization analyses showed that expression of the subtilase transcripts was only detected in mesocarp but not in non-fruit tissues. Gene expression in fruit tissues reached the highest level during the ripening stage at which the fruits undergo dramatic softening process. Subsequently, pro-subtilase ( approximately 80kDa) was expressed as recombinant pro-enzyme ( approximately 97kDa), which was used to generate antiserum against papaya subtilase, anti-sub. Protein gel blot analysis using anti-sub towards total protein extracted from all ripening stages revealed that a protein with a molecular mass of approximately 70kDa reacted with the antiserum. Hence both RNA hybridization and protein gel blot analyses confirmed the presence of subtilase during papaya fruit ripening, pointing to its possible involvement in this important process.
    Matched MeSH terms: Immunoblotting
  14. 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
  15. 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
  16. Heterologous expression of human cytochrome P450 (CYP) 2C19 in Escherichia coli and establishment of RP-HPLC method to serve as activity marker
    Pan Y, Mak JW, Ong CE
    Biomed Chromatogr, 2013 Jul;27(7):859-65.
    PMID: 23386533 DOI: 10.1002/bmc.2872
    In this study, a simple and reliable reverse-phase high-performance liquid chromatography (RP-HPLC) method was established and validated to analyze S-mephenytoin 4-hydroxylase activity of a recombinant CYP2C19 system. This system was obtained by co-expressing CYP2C19 and NADPH-CYP oxidoreductase (OxR) proteins in Escherichia coli (E. coli) cells. In addition to RP-HPLC, the expressed proteins were evaluated by immunoblotting and reduced CO difference spectral scanning. The RP-HPLC assay showed good linearity (r(2) = 1.00) with 4-hydroxymephenytoin concentration from 0.100 to 50.0 μm and the limit of detection was 5.00 × 10(-2) μm. Intraday and interday precisions determined were from 1.90 to 8.19% and from 2.20 to 14.9%, respectively. Recovery and accuracy of the assay were from 83.5 to 85.8% and from 95.0 to 105%. Enzyme kinetic parameters (Km , Vmax and Ki ) were comparable to reported values. The presence of CYP2C19 in bacterial membranes was confirmed by immunoblotting and the characteristic absorbance peak at 450 nm was determined in the reduced CO difference spectral assay. Moreover, the activity level of co-expressed OxR was found to be comparable to that of the literature. As a conclusion, the procedures described here have generated catalytically active CYP2C19 and the RP-HPLC assay developed is able to serve as CYP2C19 activity marker for pharmacokinetic drug interaction study in vitro.
    Matched MeSH terms: Immunoblotting
  17. Fulltext High Fat Diet Attenuates the Anticontractile Activity of Aortic PVAT via a Mechanism Involving AMPK and Reduced Adiponectin Secretion
    Almabrouk TAM, White AD, Ugusman AB, Skiba DS, Katwan OJ, Alganga H, et al.
    Front Physiol, 2018;9:51.
    PMID: 29479319 DOI: 10.3389/fphys.2018.00051
    Background and aim:
    Perivascular adipose tissue (PVAT) positively regulates vascular function through production of factors such as adiponectin but this effect is attenuated in obesity. The enzyme AMP-activated protein kinase (AMPK) is present in PVAT and is implicated in mediating the vascular effects of adiponectin. In this study, we investigated the effect of an obesogenic high fat diet (HFD) on aortic PVAT and whether any changes involved AMPK.Methods:Wild type Sv129 (WT) and AMPKα1 knockout (KO) mice aged 8 weeks were fed normal diet (ND) or HFD (42% kcal fat) for 12 weeks. Adiponectin production by PVAT was assessed by ELISA and AMPK expression studied using immunoblotting. Macrophages in PVAT were identified using immunohistochemistry and markers of M1 and M2 macrophage subtypes evaluated using real time-qPCR. Vascular responses were measured in endothelium-denuded aortic rings with or without attached PVAT. Carotid wire injury was performed and PVAT inflammation studied 7 days later.Key results:Aortic PVAT from KO and WT mice was morphologically indistinct but KO PVAT had more infiltrating macrophages. HFD caused an increased infiltration of macrophages in WT mice with increased expression of the M1 macrophage markersNos2andIl1band the M2 markerChil3. In WT mice, HFD reduced the anticontractile effect of PVAT as well as reducing adiponectin secretion and AMPK phosphorylation. PVAT from KO mice on ND had significantly reduced adiponectin secretion and no anticontractile effect and feeding HFD did not alter this. Wire injury induced macrophage infiltration of PVAT but did not cause further infiltration in KO mice.Conclusions:High-fat diet causes an inflammatory infiltrate, reduced AMPK phosphorylation and attenuates the anticontractile effect of murine aortic PVAT. Mice lacking AMPKα1 phenocopy many of the changes in wild-type aortic PVAT after HFD, suggesting that AMPK may protect the vessel against deleterious changes in response to HFD.
    Matched MeSH terms: Immunoblotting
  18. Fulltext Highly pathogenic avian influenza virus nucleoprotein interacts with TREX complex adaptor protein Aly/REF
    Balasubramaniam VR, Hong Wai T, Ario Tejo B, Omar AR, Syed Hassan S
    PLoS One, 2013;8(9):e72429.
    PMID: 24073193 DOI: 10.1371/journal.pone.0072429
    We constructed a novel chicken (Gallus gallus) lung cDNA library fused inside yeast acting domain vector (pGADT7). Using yeast two-hybrid screening with highly pathogenic avian influenza (HPAI) nucleoprotein (NP) from the strain (A/chicken/Malaysia/5858/2004(H5N1)) as bait, and the Gallus gallus lung cDNA library as prey, a novel interaction between the Gallus gallus cellular RNA export adaptor protein Aly/REF and the viral NP was identified. This interaction was confirmed and validated with mammalian two hybrid studies and co-immunoprecipitation assay. Cellular localization studies using confocal microscopy showed that NP and Aly/REF co-localize primarily in the nucleus. Further investigations by mammalian two hybrid studies into the binding of NP of other subtypes of influenza virus such as the swine A/New Jersey/1976/H1N1 and pandemic A/Malaysia/854/2009(H1N1) to human Aly/REF, also showed that the NP of these viruses interacts with human Aly/REF. Our findings are also supported by docking studies which showed tight and favorable binding between H5N1 NP and human Aly/REF, using crystal structures from Protein Data Bank. siRNA knockdown of Aly/REF had little effect on the export of HPAI NP and other viral RNA as it showed no significant reduction in virus titer. However, UAP56, another component of the TREX complex, which recruits Aly/REF to mRNA was found to interact even better with H5N1 NP through molecular docking studies. Both these proteins also co-localizes in the nucleus at early infection similar to Aly/REF. Intriguingly, knockdown of UAP56 in A549 infected cells shows significant reduction in viral titer (close to 10 fold reduction). Conclusively, our study have opened new avenues for research of other cellular RNA export adaptors crucial in aiding viral RNA export such as the SRSF3, 9G8 and ASF/SF2 that may play role in influenza virus RNA nucleocytoplasmic transport.
    Matched MeSH terms: Immunoblotting
  19. 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
  20. Fulltext Identification of major allergens of two species of local snappers: Lutjanus argentimaculatus (merah/ red snapper) and Lutjanus johnii (jenahak/ golden snapper)
    Rosmilah M, Shahnaz M, Masita A, Noormalin A, Jamaludin M
    Trop Biomed, 2005 Dec;22(2):171-7.
    PMID: 16883284 MyJurnal
    Fish has been recognized as a source of potent allergens both in food and occupational allergy. Lutjanus argentimaculatus (red snapper) and Lutjanus johnii (golden snapper) locally known as merah and jenahak, respectively, are among the most commonly consumed fish in Malaysia. The objective of this study is to identify the IgE-binding proteins and major allergens of these species of fishes. Extracts of both fish species were prepared and fractionated by sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE). IgE binding patterns were then demonstrated by immunoblotting using sera from patients allergic to the fishes. The raw extracts of both fish produced 26 protein bands. Both species of fishes had similar protein profiles. In cooked extracts, several protein bands in the range of about 40 to 90 kD which were present in the uncooked extracts appeared to be denatured and formed high molecular weight complexes. The immunoblotting of golden snapper and red snapper revealed 16 and 15 various IgE-binding bands, in the range of 151 to 12-11 kD, respectively. A 51 kD protein was identified as a major allergen for both fishes. A 46 kD protein was also demonstrated as a major allergen in golden snapper and a 42 kD protein was also seen as a major allergen in red snapper. A heat-resistant protein of ~12 kD which is equivalent in size with fish parvalbumin was demonstrated only as minor allergen for both fishes.
    Matched MeSH terms: Immunoblotting
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