Displaying publications 61 - 80 of 226 in total

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  1. Expression and purification of the matrix protein of Nipah virus in baculovirus insect cell system
    Masoomi Dezfooli S, Tan WS, Tey BT, Ooi CW, Hussain SA
    Biotechnol Prog, 2016 Jan-Feb;32(1):171-7.
    PMID: 26519022 DOI: 10.1002/btpr.2192
    Nipah virus (NiV) causes fatal respiratory illness and encephalitis in humans and animals. The matrix (M) protein of NiV plays an important role in the viral assembly and budding process. Thus, an access to the NiV M protein is vital to the design of viral antigens as diagnostic reagents. In this study, recombinant DNA technology was successfully adopted in the cloning and expression of NiV M protein. A recombinant expression cassette (baculovirus expression vector) was used to encode an N-terminally His-tagged NiV M protein in insect cells. A time-course study demonstrated that the highest yield of recombinant M protein (400-500 μg) was expressed from 107 infected cells 3 days after infection. A single-step purification method based on metal ion affinity chromatography was established to purify the NiV M protein, which successfully yielded a purity level of 95.67% and a purification factor of 3.39. The Western blotting and enzyme-linked immunosorbent assay (ELISA) showed that the purified recombinant M protein (48 kDa) was antigenic and reacted strongly with the serum of a NiV infected pig.
    Matched MeSH terms: Blotting, Western
  2. Enhanced secretory production of hemolysin-mediated cyclodextrin glucanotransferase in Escherichia coli by random mutagenesis of the ABC transporter system
    Low KO, Mahadi NM, Abdul Rahim R, Rabu A, Abu Bakar FD, Abdul Murad AM, et al.
    J Biotechnol, 2010 Dec;150(4):453-9.
    PMID: 20959127 DOI: 10.1016/j.jbiotec.2010.10.001
    The hemolysin transport system was found to mediate the release of cyclodextrin glucanotransferase (CGTase) into the extracellular medium when it was fused to the C-terminal 61 amino acids of HlyA (HlyAs(61)). To produce an improved-secretion variant, the hly components (hlyAs, hlyB and hlyD) were engineered by directed evolution using error-prone PCR. Hly mutants were screened on solid LB-starch plate for halo zone larger than the parent strain. Through screening of about 1 × 10(4) Escherichia coli BL21(DE3) transformants, we succeeded in isolating five mutants that showed a 35-217% increase in the secretion level of CGTase-HlyAs(61) relative to the wild-type strain. The mutation sites of each mutant were located at HlyB, primarily along the transmembrane domain, implying that the corresponding region was important for the improved secretion of the target protein. In this study we describe the finding of novel site(s) of HlyB responsible for enhancing secretion of CGTase in E. coli.
    Matched MeSH terms: Blotting, Western
  3. Fulltext Isolation and partial characterization of Asian sea bass (Lates calcarifer) Vitellogenin
    Fazielawanie NM, Siraj SS, Harmin SA, Ina-Salwany MY
    Fish Physiol Biochem, 2013 Apr;39(2):191-200.
    PMID: 22878544 DOI: 10.1007/s10695-012-9690-5
    A study was conducted to isolate, partial characterize Asian sea bass (Lates calcarifer) vitellogenin (vtg). Two-year-old juvenile L. calcarifer (n = 10) were given three intraperitoneal injections of 17-β estradiol (E2) at a dose of 2 mg/kg body weight to induce vitellogenesis. Blood was collected 3 days after the last injection, and plasma was purified through gel filtration chromatography. A broad single symmetrical peak consisting of vtg molecule was produced. Protein concentration was 0.059 mg/ml as determined by Bradfrod assay using bovine serum albumin as a standard. The protein appeared as one circulating form in Native PAGE considering the dimeric form of putative vtg with molecular weight of 545 kDa. In SDS-PAGE under reducing conditions, two major bands appeared at 232.86 and 118.80 kDa and minor bands at 100.60, 85.80 and 39.92 kDa, respectively. The purified vtg was used to generate a polyclonal antibody, and the specificity of antibody was assessed by Western blot analysis. Two major bands were immunoreacted, but no cross-reactivity was observed with plasma from non-induced males. The protein was characterized as phosphoglycolipoprotein as it positively stained for the presence of lipid, phosphorus and carbohydrate using Sudan Black B, methyl green and periodic acid/Schiff reagent solution, respectively. The amino acid composition was analyzed by high sensitivity amino acid analysis that showed high percentage of non-polar amino acids (~48 %). The results suggest the potential utilization of vtg as a basis tool to further study about reproductive physiology of this important economical species.
    Matched MeSH terms: Blotting, Western/veterinary
  4. Protection of hydroquinone-induced apoptosis by downregulation of Fau is mediated by NQO1
    Siew EL, Chan KM, Williams GT, Ross D, Inayat-Hussain SH
    Free Radic. Biol. Med., 2012 Oct 15;53(8):1616-24.
    PMID: 22687461 DOI: 10.1016/j.freeradbiomed.2012.05.046
    The Fau gene (Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV)-associated ubiquitously expressed gene) was identified as a potential tumor suppressor gene using a forward genetics approach. Downregulation of Fau by overexpression of its reverse sequence has been shown to inhibit apoptosis induced by DNA-damaging agents. To address a potential role of Fau in benzene toxicity, we investigated the apoptotic effects of hydroquinone (HQ), a major benzene metabolite, in W7.2 mouse thymoma cells transfected with either a plasmid construct expressing the antisense sequence of Fau (rfau) or the empty vector (pcDNA3.1) as a control. HQ induced apoptosis via increased production of reactive oxygen species and DNA damage, measured using dihydroethidine (HE) staining and alkaline Comet assay, respectively, in W7.2 pcDNA3.1 cells. In contrast, when Fau was downregulated by the antisense sequence in W7.2 rfau cells, HQ treatment did not cause DNA damage and oxidative stress and these cells were markedly more resistant to HQ-induced apoptosis. Further investigation revealed that there was an upregulation of NAD(P)H: quinone oxidoreductase 1 (NQO1), a detoxification enzyme for benzene-derived quinones, in W7.2 rfau cells. Compromising cellular NQO1 by use of a specific mechanism-based inhibitor (MAC 220) and NQO1 siRNA resensitized W7.2 rfau cells to HQ-induced apoptosis. Silencing of Fau in W7.2 wild-type cells resulted in increased levels of NQO1, confirming that downregulation of Fau results in NQO1 upregulation which protects against HQ-induced apoptosis.
    Matched MeSH terms: Blotting, Western
  5. Fulltext Cloning and expression of Toxoplasma gondii dense granular protein 4 (GRA4) in Pichia pastoris
    Lau YL, Hasan MT, Thiruvengadam G, Idris MM, Init I
    Trop Biomed, 2010 Dec;27(3):525-33.
    PMID: 21399595
    GRA4 of Toxoplasma gondii has been shown to prompt IgG, IgM and IgA responses in previous studies and is thus considered one of the major immunogenic proteins from T. gondii that can be used for both diagnostics purposes and vaccine development. This study seeks to clone and express the GRA4 in Pichia pastoris, which has numerous advantages over other systems for expression of eukaryotic proteins. In order to achieve this, the gene was cloned into the pPICZα A expression vector, which was then incorporated into the P. pastoris genome via insertional integration for expression of the recombinant protein, under the AOX1 promoter. The antigen was expressed along with the prepro sequence of the α-factor of yeast so that it could be excreted out of the P. pastoris cells and obtained from the medium. Upon SDS-PAGE analysis it was found that the recombinant protein was expressed optimally as a 40 kDa protein after 96 hours of induction with 0.75% of methanol. The expressed GRA4 protein showed discrepancy in size with the calculated molecular mass. This may be attributed to the various posttranslational modifications including glycosylation and phosphorylation. Despite the difference in molecular weight, the recombinant protein was able to detect toxoplasmosis in Western blot format. The recombinant GRA4 was expressed with an intact polyhistidine-tag, which could be used for future purification of the antigen.
    Matched MeSH terms: Blotting, Western/methods
  6. Fulltext Neuroprotective effects of germinated brown rice against hydrogen peroxide induced cell death in human SH-SY5Y cells
    Ismail N, Ismail M, Fathy SF, Musa SN, Imam MU, Foo JB, et al.
    Int J Mol Sci, 2012;13(8):9692-708.
    PMID: 22949825 DOI: 10.3390/ijms13089692
    The neuroprotective and antioxidative effects of germinated brown rice (GBR), brown rice (BR) and commercially available γ-aminobutyric acid (GABA) against cell death induced by hydrogen peroxide (H(2)O(2)) in human neuroblastoma SH-SY5Y cells have been investigated. Results show that GBR suppressed H(2)O(2)-mediated cytotoxicity and induced G0/G1 phase cell cycle arrest in SH-SY5Y cells. Moreover, GBR reduced mitochondrial membrane potential (MMP) and prevented phosphatidylserine (PS) translocation in SH-SY5Y cells, key features of apoptosis, and subsequent cell death. GBR exhibited better neuroprotective and antioxidative activities as compared to BR and GABA. These results indicate that GBR possesses high antioxidative activities and suppressed cell death in SH-SY5Y cells by blocking the cell cycle re-entry and apoptotic mechanisms. Therefore, GBR could be developed as a value added functional food to prevent neurodegenerative diseases caused by oxidative stress and apoptosis.
    Matched MeSH terms: Blotting, Western
  7. Magnesium acetyltaurate protects against endothelin-1 induced RGC loss by reducing neuroinflammation in Sprague dawley rats
    Nor Arfuzir NN, Agarwal R, Iezhitsa I, Agarwal P, Ismail NM
    Exp Eye Res, 2020 05;194:107996.
    PMID: 32156652 DOI: 10.1016/j.exer.2020.107996
    Endothelin-1 (ET-1), a potent vasoconstrictor, plays a significant role in the pathophysiology of ocular conditions like glaucoma. Glaucoma is characterized by apoptotic loss of retinal ganglion cells (RGCs) and loss of visual fields and is a leading cause of irreversible blindness. In glaucomatous eyes, retinal ischemia causes release of pro-inflammatory mediators such as interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α and promotes activation of transcription factors such as nuclear factor kappa B (NFKB) and c-Jun. Magnesium acetyltaurate (MgAT) has previously been shown to protect against ET-1 induced retinal and optic nerve damage. Current study investigated the mechanisms underlying these effects of MgAT, which so far remain unknown. Sprague dawley rats were intravitreally injected with ET-1 with or without pretreatment with MgAT. Seven days post-injection, retinal expression of IL-1β, IL-6, TNF-α, NFKB and c-Jun protein and genes was determined using multiplex assay, Western blot and PCR. Animals were subjected to retrograde labeling of RGCs to determine the extent of RGC survival. RGC survival was also examined using Brn3A staining. Furthermore, visual functions of rats were determined using Morris water maze. It was observed that pre-treatment with MgAT protects against ET-1 induced increase in the retinal expression of IL-1β, IL-6 and TNF-α proteins and genes. It also protected against ET-1 induced activation of NFKB and c-Jun. These effects of MgAT were associated with greater RGC survival and preservation of visual functions in rats. In conclusion, MgAT prevents ET-1 induced RGC loss and loss of visual functions by suppressing neuroinflammatory reaction in rat retinas.
    Matched MeSH terms: Blotting, Western
  8. The effects of acute ethanol administration on ethanol withdrawal-induced anxiety-like syndrome in rats: A biochemical study
    Kumar J, Hapidin H, Get Bee YT, Ismail Z
    Alcohol, 2016 Feb;50:9-17.
    PMID: 26626323 DOI: 10.1016/j.alcohol.2015.10.001
    Withdrawal from long-term ethanol consumption results in overexcitation of glutamatergic neurotransmission in the amygdala, which induces an anxiety-like syndrome. Most alcoholics that suffer from such symptoms frequently depend on habitual drinking as self-medication to alleviate their symptoms. Metabotropic glutamate receptor subtype 5 (mGlu5) and protein kinase C (PKC) epsilon have been reported to mediate acute and chronic effects of ethanol. This study explores the changes in mGlu5 and PKC epsilon in the amygdala following acute administration of ethanol during ethanol withdrawal (EW) induced anxiety. Male Wistar rats were fed a modified liquid diet containing low-fat cow milk, sucrose, and maltodextrin, with a gradual introduction of 2.4%, 4.8% and 7.2% ethanol for 20 days. Six hours into EW, the rats were intraperitoneally injected with normal saline and ethanol (2.5 g/kg, 20% v/v), and exposed to open-field and elevated plus maze tests. Then, amygdala tissue was dissected from the rat brain for Western blot and gene expression studies. EW-induced anxiety was accompanied by a significant increase in mGlu5, total PKC epsilon, and phosphorylated PKC epsilon protein levels, and also of mRNA of mGlu5 (GRM5) in the amygdala. Acute administration of ethanol significantly attenuated EW-induced anxiety as well as an EW-induced increase in GRM5. The acute challenge of ethanol to EW rats had little effect on the phosphorylated and total protein levels of PKC epsilon in the amygdala. Our results demonstrate that amygdala PKC epsilon may not be directly involved in the development of anxiety following EW.
    Matched MeSH terms: Blotting, Western
  9. The effects of a synthetic curcuminoid analogue, 2,6-bis-(4-hydroxyl-3-methoxybenzylidine)cyclohexanone on proinflammatory signaling pathways and CLP-induced lethal sepsis in mice
    Tham CL, Lam KW, Rajajendram R, Cheah YK, Sulaiman MR, Lajis NH, et al.
    Eur J Pharmacol, 2011 Feb 10;652(1-3):136-44.
    PMID: 21114991 DOI: 10.1016/j.ejphar.2010.10.092
    We previously showed that 2,6-bis-(4-hydroxyl-3-methoxybenzylidine)cyclohexanone (BHMC), suppressed the synthesis of various proinflammatory mediators. In this study we explain the mechanism of action of BHMC in lipopolysaccharide (LPS)-induced U937 monocytes and further show that BHMC prevents lethality of CLP-induced sepsis. BHMC showed dose-dependent inhibitory effects on p38, JNK and ERK 1/2 activity as determined by inhibition of phosphorylation of downstream transcription factors ATF-2, c-Jun and Elk-1 respectively. Inhibition of these transcription factors subsequently caused total abolishment of AP-1-DNA binding. BHMC inhibited p65 NF-κB nuclear translocation and DNA binding of p65 NF-κB only at the highest concentration used (12.5μM) but failed to alter phosphorylation of JNK, ERK1/2 and STAT-1. Since the inhibition of p38 activity was more pronounced we evaluated the possibility that BHMC may bind to p38. Molecular docking experiments confirmed that BHMC fits well in the highly conserved hydrophobic pocket of p38 MAP kinase. We also show that BHMC was able to improve survival from lethal sepsis in a murine caecal-ligation and puncture (CLP) model.
    Matched MeSH terms: Blotting, Western
  10. Effects of 3-(2-Hydroxyphenyl)-1-(5-methyl-furan-2-y-l) propenone (HMP) upon signalling pathways of lipopolysaccharide-induced iNOS synthesis in RAW 264.7 cells
    Liew CY, Lam KW, Kim MK, Harith HH, Tham CL, Cheah YK, et al.
    Int Immunopharmacol, 2011 Jan;11(1):85-95.
    PMID: 21035434 DOI: 10.1016/j.intimp.2010.10.011
    We previously showed that 3-(2-hydroxyphenyl)-1-(5-methyl-furan-2-y-l)propenone (HMP), suppressed the synthesis of various proinflammatory mediators. In this study, HMP showed a dose-dependent inhibition of NO synthesis in the RAW 264.7 murine macrophage line. The inhibition of NO synthesis was related to inhibition of p38 phosphorylation and kinase activity that led to significant inhibition of phosphorylation of ATF-2. This effect in turn caused inhibition of AP-1-DNA binding which partially explains the inhibitory effect upon the synthesis of iNOS. HMP had no effect upon phosphorylation of JNK, ERK1/2 and STAT-1. Kinase activity of JNK and ERK1/2 was also not affected by HMP as determined by levels of phosphorylated c-jun and phosphorylated elk-1. Furthermore HMP failed to block phosphorylation of IκBα, and subsequent nuclear translocation and DNA-binding activity of p65 NF-κB in IFN-γ/LPS-induced RAW 264.7 cells. Molecular docking experiments confirmed that HMP fits well in the highly conserved hydrophobic pocket of p38 MAP kinase. We conclude that the synthetic HMP is a chalcone analogue that selectively inhibits the p38/ATF-2 and AP-1 signaling pathways in the NO synthesis by the macrophage RAW 264.7.
    Matched MeSH terms: Blotting, Western
  11. Fulltext The Protective Effects of a Synthetic Geranyl Acetophenone in a Cellular Model of TNF-α-Induced Pulmonary Epithelial Barrier Dysfunction
    Sim TY, Harith HH, Tham CL, Md Hashim NF, Shaari K, Sulaiman MR, et al.
    Molecules, 2018 Jun 05;23(6).
    PMID: 29874809 DOI: 10.3390/molecules23061355
    Alveolar epithelial barrier dysfunction contributes to lung edema and can lead to acute lung injury (ALI). The features include increased epithelial permeability, upregulation of inflammatory mediators and downregulation of junctional complex molecules; these changes are often induced by inflammation. tHGA is an acetophenone analogue with therapeutic potential in asthma. Its therapeutic potential in ALI is presently unknown. Herein, the effects of tHGA on epithelial barrier dysfunction were determined in TNF-α-induced human alveolar epithelial cells. The anti-inflammatory properties of tHGA were assessed by monocyte adhesion assay and analysis of MCP-1 and ICAM-1 expression. The epithelial barrier function was assessed by paracellular permeability and transepithelial electrical resistance (TEER) assays, and analysis of junctional complex molecules expression. To elucidate the mechanism of action, the effects of tHGA on the NF-κB and MAPK pathways were determined. Gene and protein expression were analyzed by RT-PCR and Western blotting or ELISA, respectively. tHGA suppressed leukocyte adhesion to TNF-α-induced epithelium and reduced MCP-1 and ICAM-1 gene expression and secretion. tHGA also increased TEER readings, reduced epithelial permeability and enhanced expression of junctional complex molecules (zona occludens-1, occludin and E-cadherin) in TNF-α-induced cells. Correspondingly, the NF-κB, ERK and p38 MAPK pathways were also inhibited by tHGA. These findings suggest that tHGA is able to preserve alveolar epithelial barrier function in response to acute inflammation, via its anti-inflammatory activity and stabilization of epithelial barrier integrity, mediated by NF-κB, ERK and p38 MAPK signaling.
    Matched MeSH terms: Blotting, Western
  12. Suppression of β-catenin and cyclooxygenase-2 expression and cell proliferation in azoxymethane-induced colonic cancer in rats by rice bran phytic acid (PA)
    Saad N, Esa NM, Ithnin H
    Asian Pac J Cancer Prev, 2013;14(5):3093-9.
    PMID: 23803085
    BACKGROUND: Phytic acid (PA) is a polyphosphorylated carbohydrate that can be found in high amounts in most cereals, legumes, nut oil, seeds and soy beans. It has been suggested to play a significant role in inhibition of colorectal cancer. This study was conducted to investigate expression changes of β-catenin and cyclooxygenase-2 (COX-2) and cell proliferation in the adenoma-carcinoma sequence after treatment with rice bran PA by immunocytochemistry.

    MATERIALS AND METHODS: Seventy-two male Sprague-Dawley rats were divided into 6 equal groups with 12 rats in each group. For cancer induction two intraperitoneal injections of azoxymethane (AOM) were given at 15 mg/kg bodyweight over a 2-weeks period. During the post initiation phase, two different concentrations of PA, 0.2% (w/v) and 0.5% (w/v) were administered in the diet.

    RESULTS: Results of β-catenin, COX-2 expressions and cell proliferation of Ki-67 showed a significant contribution in colonic cancer progression. For β-catenin and COX-2 expression, there was a significant difference between groups at p<0.05. With Ki-67, there was a statistically significant lowering the proliferating index as compared to AOM alone (p<0.05). A significant positive correlation (p=0.01) was noted between COX-2 expression and proliferation. Total β-catenin also demonstrated a significant positive linear relationship with total COX-2 (p=0.044).

    CONCLUSIONS: This study indicated potential value of PA extracted from rice bran in reducing colonic cancer risk in rats.

    Matched MeSH terms: Blotting, Western
  13. Differential effects of activated protein C on synovial fibroblasts in response to hypoxia and normoxia
    Park YG, Choi J, Song I, Park SY, Seol JW, Jackson CJ
    Sains Malaysiana, 2017;46:1895-1902.
    Rheumatoid arthritis (RA) is a chronic disease characterized by inflammation of the joints and their lining or synovium. Previous studies showed that the synovium in RA patients is more hypoxic than normal synovium. Activated protein C (APC) has anticoagulant and anti-inflammatory effects and is highly expressed in the joints of RA patients. We examined the effect of APC on RA and normal synovial fibroblasts under hypoxic conditions. Human synovial fibroblasts were isolated from the synovial tissues of RA patients and normal controls and cells were exposed to recombinant APC under normoxic (21% oxygen) or hypoxic (1% oxygen) conditions. Cell proliferation was measured using MTT assays. Cell lysates and conditioned media were collected and assayed for matrix metalloproteinase (MMP)-2, MMP-9 and p38 using zymography and western blots. Proliferation of both normal and RA synovial fibroblasts dose-dependently increased after APC treatment in normoxic conditions. Under hypoxia, APC enhanced RA cell proliferation but had no effect on normal fibroblasts. MMP-2 production and activation were significantly augmented by APC in both cell types under normoxia and hypoxia conditions. However, activated MMP-2 was more reduced in cells under hypoxia than normoxia. APC substantially reduced the phosphorylation of p38 in normal and RA synovial fibroblasts under hypoxia. No difference in p38 phosphorylation was observed under normoxia. The receptor for APC, endothelial protein C receptor (EPCR), was elevated in normal fibroblasts under hypoxic conditions whereas in RA cells, EPCR was highly expressed under both normoxic and hypoxic conditions. We found that hypoxia enhanced the effect of APC on RA synovial fibroblasts through activation of MMP2 and inhibition of p38 phosphorylation. Our results suggested that APC may suppress joint destruction and progression of inflammation in a hypoxic RA environment.
    Matched MeSH terms: Blotting, Western
  14. Fulltext Knockdown of Tousled‑like kinase 1 inhibits survival of glioblastoma multiforme cells
    Ibrahim K, Abdul Murad NA, Harun R, Jamal R
    Int J Mol Med, 2020 Aug;46(2):685-699.
    PMID: 32468002 DOI: 10.3892/ijmm.2020.4619
    Glioblastoma multiforme (GBM) is an aggressive type of brain tumour that commonly exhibits resistance to treatment. The tumour is highly heterogenous and complex kinomic alterations have been reported leading to dysregulation of signalling pathways. The present study aimed to investigate the novel kinome pathways and to identify potential therapeutic targets in GBM. Meta‑analysis using Oncomine identified 113 upregulated kinases in GBM. RNAi screening was performed on identified kinases using ON‑TARGETplus siRNA library on LN18 and U87MG. Tousled‑like kinase 1 (TLK1), which is a serine/threonine kinase was identified as a potential hit. In vitro functional validation was performed as the role of TLK1 in GBM is unknown. TLK1 knockdown in GBM cells significantly decreased cell viability, clonogenicity, proliferation and induced apoptosis. TLK1 knockdown also chemosensitised the GBM cells to the sublethal dose of temozolomide. The downstream pathways of TLK1 were examined using microarray analysis, which identified the involvement of DNA replication, cell cycle and focal adhesion signalling pathways. In vivo validation of the subcutaneous xenografts of stably transfected sh‑TLK1 U87MG cells demonstrated significantly decreased tumour growth in female BALB/c nude mice. Together, these results suggested that TLK1 may serve a role in GBM survival and may serve as a potential target for glioma.
    Matched MeSH terms: Blotting, Western
  15. Extracellular Vesicles Derived From Colorectal Cancer Affects CD8 T Cells: An Analysis Based on Body Mass Index
    Abu N, Othman N, Ab Razak NS, Bakarurraini NAAR, Nasir SN, Soh JEC, et al.
    Front Cell Dev Biol, 2020;8:564648.
    PMID: 33324632 DOI: 10.3389/fcell.2020.564648
    Colorectal cancer (CRC) is one of the most widely diagnosed cancers worldwide. It has been shown that the body-mass index (BMI) of the patients could influence the tumor microenvironment, treatment response, and overall survival rates. Nevertheless, the mechanism on how BMI affects the tumorigenesis process, particularly the tumor microenvironment is still elusive. Herein, we postulate that extracellular vesicles (EVs) from CRC patients and non-CRC volunteers with different BMI could affect immune cells differently, in CD8 T cells particularly. We isolated the EVs from the archived serum of CRC patients with high and low BMI, as well as healthy controls with similar BMI status. The EVs were further characterized via electron microscopy, western blot and dynamic light scattering. Then, functional analysis was performed on CD8 T cells including apoptosis, cell proliferation, gene expression profiling and cytokine release upon co-incubation with the different EVs. Our results suggest that CRC-derived EVs were able to regulate the CD8 T cells. In some assays, low BMI EVs were functionally different than high BMI EVs. This study highlights the possible difference in the regulatory mechanism of cancer patients-derived EVs, especially on CD8 T cells.
    Matched MeSH terms: Blotting, Western
  16. Effect of antisense oligodeoxynucleotides for ICAM-1 on renal ischaemia-reperfusion injury in the anaesthetised rat
    Kiew LV, Munavvar AS, Law CH, Azizan AN, Nazarina AR, Sidik K, et al.
    J Physiol, 2004 Jun 15;557(Pt 3):981-9.
    PMID: 15047774
    An antisense oligodeoxynucleotide (As-ODN) to the 3' untranslated region of the mRNA sequence expressing the intracellular adhesion molecule-1 (ICAM-1) was employed to determine ICAM-1's role in renal ischaemia-reperfusion injury in the rat. Wistar-Kyoto rats receiving i.v. either lipofectin-As-ODN (As-ODN group), lipofectin-reverse ODN (Rv-ODN group) or lipofectin (ischaemia control group) 8 h prior to study were anaesthetized and subjected to 30 min of renal artery occlusion. Renal haemodynamic and excretory parameters were monitored before and after renal ischaemia. On termination of the study renal tissue was subjected to histological and Western blot analysis. Renal blood flow decreased in the 3 h post-ischaemia period in the ischaemia control and Rv-ODN groups, but was maintained in the As-ODN group. Glomerular filtration rate was depressed initially but gradually increased to 10% above basal levels in the ischaemia control and Rv-ODN groups, but was below basal levels (20%) in the As-ODN group. There was a three- to fourfold increase in sodium and water excretion following ischaemia in the ischaemia control and reverse-ODN groups but not in the As-ODN treated group. The As-ODN ameliorated the histological evidence of ischaemic damage and reduced ICAM-1 protein levels to a greater extent in the medulla than cortex. These observations suggested that in the post-ischaemic period afferent and efferent arteriolar tone was increased with a loss of reabsorptive capacity which was in part due to ICAM-1. The possibility arises that the action of ICAM-1 at vascular and tubular sites in the deeper regions of the kidney contributes to the ischaemia-reperfusion injury.
    Matched MeSH terms: Blotting, Western
  17. Production of a monoclonal antibody against a mannose-binding protein of Acanthamoeba culbertsoni and its localization
    Kang AY, Park AY, Shin HJ, Khan NA, Maciver SK, Jung SY
    Exp Parasitol, 2018 Sep;192:19-24.
    PMID: 30031120 DOI: 10.1016/j.exppara.2018.07.009
    Amoebae from the genus Acanthamoeba are facultative pathogens of humans and other animals. In humans they most frequently infect the eye causing a sight threatening infection known as Acanthamoeba keratitis (AK), and also cause an often fatal encephalitis (GAE). A mannose-binding protein (MBP) has been identified as being important for Acanthamoeba infection especially in AK. This lectin has previously been characterized from Acanthamoeba castellanii as consisting of multiple 130 kDa subunits. MBP expression correlates with pathogenic potential and is expressed in a number of Acanthamoeba species. Here we report the purification of a similar lectin from Acanthamoeba culbertsoni and the production of a monoclonal antibody to it. The A. culbertsoni MBP was isolated by affinity chromatography using α-D-mannose agarose and has an apparent molecular weight of 83 kDa. The monoclonal antibody is an IgM that is useful in both western blots and immunofluorescence. We expect that this antibody will be useful in the study of the pathology of A. culbertsoni and in its identification in clinical samples.
    Matched MeSH terms: Blotting, Western
  18. 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: Blotting, Western
  19. Fulltext Recombinant human adiponectin as a potential protein for treating diabetic tendinopathy promotes tenocyte progenitor cells proliferation and tenogenic differentiation in vitro
    Rothan HA, Suhaeb AM, Kamarul T
    Int J Med Sci, 2013;10(13):1899-906.
    PMID: 24324367 DOI: 10.7150/ijms.6774
    Adiponectin is an adipocyte-secreting hormone that increases cell sensitivity to insulin. It has been previously demonstrated that this hormone protects against Type II Diabetes and, is found to concurrently promote cell proliferation and differentiation. It is postulated that diabetic patients who suffer from tendinopathy may benefit from using adiponectin, which not only improves the metabolism of diabetic ridden tenocytes but also promotes progenitor cell proliferation and differentiation in tendons. These changes may result in tendon regeneration, which, in diabetic tendinopathy, is difficult to treat. Considering that such findings have yet to be demonstrated, a study was thus conducted using diabetic ridden human tenocyte progenitor cells (TPC) exposed to recombinant adiponectin in vitro. TPC were isolated from tendons of diabetic patients and exposed to 10 μg/ml adiponectin. Cell proliferation rate was investigated at various time points whilst qPCR were used to determine the tenogenic differentiation potential. The results showed that adiponectin significantly reduced blood glucose in animal models. The proliferation rate of adiponectin-treated TPCs was significantly higher at 6, 8 and 10 days as compared to untreated cells (p<0.05). The levels of tenogenic genes expression (collagen I, III, tenomodulin and scleraxis) were also significantly upregulated; whilst the osteogenic (Runx2), chondrogenic (Sox9) and adipogenic (PPARУγ) gene expressions remained unaltered. The results of this study suggest that adiponectin is a potential promoter that not only improves diabetic conditions, but also increases tendon progenitor cell proliferation and differentiation. These features supports the notion that adiponectin may be potentially beneficial in treating diabetic tendinopathy.
    Matched MeSH terms: Blotting, Western
  20. Fulltext Oxidative Stress Down-Regulates MiR-20b-5p, MiR-106a-5p and E2F1 Expression to Suppress the G1/S Transition of the Cell Cycle in Multipotent Stromal Cells
    Tai L, Huang CJ, Choo KB, Cheong SK, Kamarul T
    Int J Med Sci, 2020;17(4):457-470.
    PMID: 32174776 DOI: 10.7150/ijms.38832
    Oxidative stress has been linked to senescence and tumorigenesis via modulation of the cell cycle. Using a hydrogen peroxide (H2O2)-induced oxidative stress-induced premature senescence (OSIPS) model previously reported by our group, this study aimed to investigate the effects of oxidative stress on microRNA (miRNA) expression in relation to the G1-to-S-phase (G1/S) transition of the cell cycle and cell proliferation. On global miRNA analysis of the OSIPS cells, twelve significantly up- or down-regulated miRNAs were identified, the target genes of which are frequently associated with cancers. Four down-regulated miR-17 family miRNAs are predicted to target key pro- and anti-proliferative proteins of the p21/cyclin D-dependent kinase (CDK)/E2F1 pathway to modulate G1/S transition. Two miR-17 miRNAs, miR-20-5p and miR-106-5p, were confirmed to be rapidly and stably down-regulated under oxidative stress. While H2O2 treatment hampered G1/S transition and suppressed DNA synthesis, miR-20b-5p/miR-106a-5p over-expression rescued cells from growth arrest in promoting G1/S transition and DNA synthesis. Direct miR-20b-5p/miR-106a-5p regulation of p21, CCND1 and E2F1 was demonstrated by an inverse expression relationship in miRNA mimic-transfected cells. However, under oxidative stress, E2F1 expression was down-regulated, consistent with hampered G1/S transition and suppressed DNA synthesis and cell proliferation. To explain the observed E2F1 down-regulation under oxidative stress, a scheme is proposed which includes miR-20b-5p/miR-106a-5p-dependent regulation, miRNA-E2F1 autoregulatory feedback and E2F1 response to repair oxidative stress-induced DNA damages. The oxidative stress-modulated expression of miR-17 miRNAs and E2F1 may be used to develop strategies to retard or reverse MSC senescence in culture, or senescence in general.
    Matched MeSH terms: Blotting, Western
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