Oral cancer secretome: identification of cancer associated proteins
Hong-Yun Chang 1,2, Seen-Yii Hor 3, Kue-Peng Lim3, Rosnah Binti Zain 4, Sok-Ching Cheong 3,6, Mariati Abdul Rahman 5 and Saiful Anuar Karsani 1,2
1
Institute of Biological Sciences, Faculty of Science, 2 University of Malaya Centre for Proteomics Research (UMCPR), Department of Medical Biotechnology, Faculty of Medicine, University of Malaya, 3 Oral Cancer Research Team, Cancer Research Initiatives Foundation (CARIF), 2nd
Floor Outpatient Centre, Sime Darby Medical Centre, 4 Oral Cancer Research and Co-ordinating Centre & Faculty of Dentistry, University of
Malaya, 5 Department of Clinical Oral Biology, Faculty of Dentistry, Universiti Kebangsaan Malaysia, 6 Department of Oral and Maxillofacial
Surgery, Kuala Lumpur Hospital, Malaysia
ABSTRACT
Introduction:Early detection of oral cancer will lead to better patient management and prognosis. This can be achieved with reliable cancer associated biomarkers. Serum is an ideal sample from which to identify biomarkers. However, directly studying the serum is like looking for a needle in a haystack due to the large dynamic range of serum proteins. An alternative approach is to study the secretome of established cell lines as it may reflect proteins secreted by tumors. Thus this study aims to identify cancer associated proteins in the secretome of oral cancer cell lines.
Objective: To identify cancer associated proteins in the secretome of oral cancer cell lines.
Methods: Most research on the oral cancer secretome lacks a normal cell line control. We have successfully established four normal cell lines with a limited lifespan without hTERT immortalization. The secretome of these normal cell lines were compared with that of oral cancer cell lines using two-dimensional gel electrophoresis.
Results & Discussion: Thirty five protein spots were found to be differentially expressed. Unambiguous identification of these proteins was achieved by MALDI TOF/TOF. In silico analysis predicted that 24 of these proteins were secreted via classical or non-classical mechanisms. The mRNA expression of six genes was found to correlate with the corresponding protein expression. IPA core analysis revealed that the identified proteins were relevant in, and related to, cancer development with likely involvements in tumor growth, metastasis, hyperproliferation, tumorigenesis, neoplasia, hyperplasia, and cell transformation.
Proteomics and glycomics approaches in breast cancer biomarker discovery
Abdullah Saleh Saleh Abdullah, Mohd Nazri Ismail and Aishah A. Latiff
Doping Control Centre, Universiti Sains Malaysia, Penang, Malaysia
ABSTRACT
Introduction:Breast cancer is one of the most common types of invasive cancer in females and the main reason for mortality worldwide. Developing biomarkers is valuable to early diagnosis, molecular staging, prognosis and understanding the mechanisms in breast cancer. Recent proteomic technologies have offered a promising opportunity for the identification of new breast cancer biomarkers. Here, we compared the phosphoproteomic and glycomic profiles of breast tumor versus non-tumor (tissues and serum) in order to identify modulated proteins, which could represent potential markers associated to clinical features.
Objective: To analyse the samples from breast cancer tissues and serum for Glycomics and Phosphoproteomics profiles using mass spectrometry in order to find potential biomarkers for breast cancer that can be correlated with the different stages of the disease.
Methods: Protein extracts were obtained from clinical breast cancer samples. Samples were fractionated and separated. Proteins were subjected to reduction, carboxymethylation and tryptic digestion. Samples were then purified and divided for phosphoproteomics and glycomics analyses. Phosphopeptides were enriched on-line or off-line prior mass spectrometry. On the other hand, O- and N-glycans were released from glycoproteins and derivatised. Samples were analysed by using advanced mass spectrometric instruments including Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography-mass spectrometry (LC-MS).
Results & Discussion: Our methodologies proved to be reliable in extracting proteins and detecting the relevant post-translational modifications (PTMs) from serum and the mucinous breast tissues. Preliminary mass spectrometric data demonstrates discrepancies in the expression levels of the glycome and phosphoproteome between tumour and normal samples. Detail tandem mass spectrometric analysis in on-going to sequence the peptides and their PTMs in order to identify the specific proteins that are affected in the progression of cancer.
Plasma proteomic study for prediction of response to chemotherapy in acute
myeloid leukemia patients
Fatemeh B., Seow H.F., Chong P.P., Zainina S., Mariana N.S., Maha A., Chang K.M., Ong T.C., Lee C.D. and Yiau S.K.X.
Immunology unit, Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia
ABSTRACT
Introduction: A majority of acute myeloid leukemia (AML) patients initially respond to chemotherapy and achieve complete remission. However, resistance to chemotherapy is considered as a major problem in treatment of these patients ; in addition, available prognostic markers are not sufficient for treatment failure prediction.
Objective: To explore the presence of potential biomarkers of drug sensitivity in plasma of AML patients using two-dimensional gel electrophoresis.
Methods: In order to achieve this objective, before starting chemotherapy, peripheral blood samples from responsive (5 samples) and resistant (5 samples) AML patients referred to Ampang Hospital were collected. Plasma were isolated by Ficoll- Paque Plus using density gradient centrifugation. The 2d-electrophoresis method was used to analyze and select differentially expressed proteins in these samples.
Results & Discussion: The 221 spots in resistant and 283 spots in responsive gels were detected. Among them, nine proteins were significantly different in plasma of resistant and responsive patients. Four of these proteins were of low molecular weight (LMW) while the rest of higher molecular weight (HMW) were over-expressed in responsive and resistant AML pateints, respectively.
Modification response of cyclic AMP agonists upon Nedd4/2 and epithelial
sodium channel (ENaC) in glucocorticoid treated human epithelial lung cells
Noor A S Ismail1,2 and S M Wilson2
1
Biochemistry Department, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia,
2Centre of Cardiovascular and Lung Biology, University of Dundee, Dundee, United Kingdom
ABSTRACT
Introduction: Glucocorticoid (GC) hormones are important to the development of the lung’s Na+ -absorbing phenotype, however Na+ transport also subject to acute regulation via cAMP-coupled agonists. Previous works suggested that Na+ conductance is increased in favour of cAMP activation through PKA pathway (Ramminger et al. 2002, Clunes et al. 2004). cAMP dependant agonists are thought to stimulate epithelial sodium channel (ENaC) on the surface membrane. This proposed to be activated through a potential phosphorylation of ubiquitin ligase, Nedd4/2 at its three sites: Ser221, Ser327, Thr246.
Methods: Acute effect of cAMP agonists (10µM forskolin, 100 µM isobutylmethylxathine, 1mM N6 ,2’-O-dibutyryl adenosine 3’5’-cyclic monophosphate) was treated upon H441 cells prior to pre-treatment of
dexamethasone,a synthetic member of the glucocorticoid (0.2 µM) for 24h. ENaC subunits (α-, β-, γ-) were extracted through biotinylation label of the cell surface whilst Nedd4/2 were isolated by immunoprecipitation and subjected to western blotting.
Results & Discussion: Combination of dexamethasone (0.2 µM) and cAMP agonists increased the abundance of the Ser221- and Ser327-phosphorylated Nedd4-2 and also increased the overall Nedd4-2 expression. These responses resembled those seen in dexamethasone-treated cells; however the cAMP agonists did not increase the abundance of Thr246-phosphorylated Nedd4-2. The cAMP agonists did not further alter the surface-labelled proteins of α-ENaC in the membrane, but did augment β- and γ-ENaC. Since these responses also differed from those seen in GC-deprived cells, this indicates that chronic (24 h) exposure to dexamethasone modifies the effects of cAMP agonists on the phosphorylation / expression of Nedd4-2 and the surface expression of ENaC subunits.
The effects of glucoprivation on adrenal medulla function in SHR and WKY rats
Hanafi A Damanhuri1,2,Natasha N Kumar2, Peter R Dunkley3 and Ann K Goodchild2
1
Biochemistry Department, Faculty of Medicine, Universiti Kebangsaan Malaysia (UKM), Kuala Lumpur, Malaysi, 2 The Australian School of Advanced Medicine, Macquarie University, North Ryde, New South Wales, Australia, 3 School of Biomedical Sciences, The University of
Newcastle, Callaghan, New South Wales, Australia
ABSTRACT
Introduction: Our previous work has established that acute intraperitoneal injection of 2-deoxy-d-glucose (2DG) into Sprague-Dawley rats led to activation of the adrenal medulla chromaffin cells. Hypertension has been postulated to impact the basal sympathoadrenal function and there is evidence suggesting that the response to acute challenge of the sympathoadrenal system may be altered in hypertension. Thus we sought to determine the function of adrenal medulla in the spontaneously hypertensive rats (SHR) as the model of human essential hypertension as compared to wistar Kyoto (WKY) rats.
Objective: To determine whether adrenomedullary function in the SHR or its response to glucoprivation induced by 2DG are altered in hypertension.
Methods: The level of TH phosphorylation and activated protein kinases were determined using western blot. TH activity was measure using radioimmunoassay while the level of plasma catecholamine was determined using HPLC.
Results & Discussion: Prior to the treatment, no differences were evident between SHR and WKY despite a significant difference in the level of systolic blood pressure. Saline injection evoked no significant changes in any parameter measured in SHR, but evoked significant increases in pSer19TH, plasma adrenaline and blood glucose in WKY. Single episode of glucoprivation evoked increases in PKA and CDK/MAPK, pSer40TH, pSer31TH, TH activity and plasma adrenaline and plasma glucose in SHR, and additionally evoked increases in PKC, CaMPKII and pSer19TH in WKY.
Profiling of spontaneously hypertensive rats (SHR) serum proteins expression
using
surface enhanced laser desorption/ionisation – time of flight (SELDI-ToF)
mass spectrometry
Aminudin N.1, Abdullah N.H.1, Abdul-Khalil K.H.2 and Karsani S.A.1
1Institute of Biological Sciences, Faculty of Science and University of Malaya Centre for Proteomics Research (UMCPR),
Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia, 2Faculty of Applied Sciences, Universiti Teknologi MARA (UiTM), Shah
Alam, Selangor, Malaysia
ABSTRACT
Introduction: Hypertension is a major disease suffered by people all over the world contributing to a high rate of mortality. It has been known to cause the altered levels of serum or plasma proteins. A considerable number of proteins have been previously reported to be altered in levels in the sera of animals and human subjects. While some of the proteins were thought to be involved as anti-inflammatory and protective response, others were related to endothelial vascular repair, arterial smooth muscle cell growth and some may instead be the contributing factors of hypertension. In the present study, we have investigated the simultaneous expression of the high abundant serum proteins in the normotensive rats (NR) and compared it with those expressed in the sera of SHR.
Objective: To characterize the differences in serum protein expression levels in SHR as compared to NR using SELDI-ToF.
Methods: Serum samples were subjected to SELDI-ToF analysis using CM-10 ProteinChip Array (pH 4) and ProteinChip Reader System. Univariate analysis was done using Expression Dynamic Mapping (EDM). Results obtained were used for multivariate analysis using Biomarker Pattern Software™ 5.0.2
Results & Discussion: SELDI-TOF univariate analysis revealed that 16 low molecular weight (LMW) proteins expression were down-regulated with the fold changes between 1.9 and 32. SELDI-TOF multivariate analysis suggested that the LMW serum proteins with m/z values 4180.6 could be a potential biological indicator candidate for hypertension.
Identification and quantitation of type 1 diabetes associated MHC peptide epitopes
Chor Teck Tan, Nadine L. Dudek, Nicholas A. Williamson and Anthony W. Purcell
Bio21 Molecular Science and Biotechnology Institute, Department of Biochemistry and Molecular Biology, The University of Melbourne, Australia
ABSTRACT
Introduction: In Type 1 diabetes (T1D), the immune system mistakenly recognises peptides derived from normal cellular (self) proteins, triggering an attack against the insulin secreting pancreatic β cells, leading to β cell loss and insulin deficiency. Recognition of MHC-bound peptides is critical for both the initiation and progression of the disease. Identifying the epitopes targeted during diabetes remains a critical step in defining the molecular basis of the disease. Human-patient testing in combination with the use of the Non-Obese Diabetic (NOD) mice has lead to the characterisation of multiple antigens that are recognised by autoreactive lymphocytes. However, the inherent complexities of antigen processing and presentation, the polygenic nature of the disease, extensive polymorphism of the MHC and the technical hurdles in working with T cells, have made epitope discovery and quantitation in T1D a challenge.
Objective: To probe the presentation of MHC class I peptide epitopes in β cells of T1D.
Methods: Immunoaffinity purified peptide epitopes were sequenced and identified using an ABSCIEX Triple-TOF system connected to an EksigentNanoUltracHiPLC system. Epitope quantitation was achieved through coupling immunoaffinity purification with multiple reaction monitoring (MRM) in combination with an AQUA peptide strategy. In vitro experiments were first carried out on NIT cells, which are derived from the β cells of NOD mice.
Results & Discussion: Over 2000 MHC-bound peptides, including sequences from a number of known autoantigens, were identified from β cells. Quantitation of an immunodominant H-2Kd
-restricted epitope,
IGRP206-214, demonstrated an increase in epitope abundance with increasing period of interferon-γ
incubation.
Conclusion: This study makes use of sensitive mass spectrometry to directly quantitate peptide epitopes displayed on MHC molecules in relation to T1D disease progression. We found that peptide-epitope presentation level has a direct correlation with disease progression and autoimmunity in T1D, and that inflammation drives the presentation of key disease-associated epitopes.
Integration of metabolomics and proteomics for fertility molecular marker
discovery: unraveling differences in energy metabolism between fertile and
sub-fertile bull sperm
Ali Ashrafzadeh1, Sheila Nathan1, Iekhsan Othman2, Tee Ting Yee2 and Saiful Anuar Karsani3
1School of Biosciences and Biotechnology, Faculty of Science and Technology,Universiti Kebangsaan Malaysia, Selangor, Malaysia, 2School of Medicine and Health Sciences, Monash University, Sunway Campus, Kuala Lumpur, Malaysia,
3Institute of Biological Sciences, Faculty of Science,University of Malaya, Kuala Lumpur, Malaysia
ABSTRACT
Introduction: Malaysia is still reliant on importation of up to 80% of its dairy and beef requirements. In an attempt to improve local beef and dairy production, cross breeding with high producing breeds is generally favored. However, fertility of the crossbred progenies is low as a result of the high local humidity and temperature.
Objective: To identify cattle sperm proteins and metabolites with potentials fertility and heat tolerance marker(s).
Methods: We attempted to integrate metabolomics and proteomics approaches to compare sperm from Kedah Kelantan (Bosindicus) as a high fertility indigenous breed in Malaysia with sperm from Mafriwal
(Bostaurus×Bosindicus), a low fertility crossbreed of cattle. Frozen semen of three high performance
bulls from each breed was processed to obtain live and pure sperm. SDS-PAGE was conducted and gel bands were processed by in-gel tryptic digestion. For each of the replicate sperm samples, MS/MS data was acquired. The identified proteins in the sperm from the two breeds were compared based on GO (Gene Ontology) annotations (99% confidence level). For metabolomics analysis, metabolite extraction was performed and MS data was acquired from seven replicate samples. Significantly over-expressed metabolites were identified (99% confidence level) and pathway analysis was then performed. The motility of sperm from both breeds was also evaluated by CASA (Computer Assisted Semen Analysis).
Results & Discussion: Sperm motility evaluation confirmed significantly higher motility in Kedah Kelantan sperm compared to Mafriwal. Data from metabolomics and proteomics experiments suggest that carbohydrate metabolism is up-regulated in Kedah Kelantan; in contrast, lipid metabolism is significantly higher in Mafriwal sperm.
Mapping the protein interaction between the sperm and oocyte in humans
S. Sabetian F.J, H. Bostan and M.S. Shamsir
Faculty of Bioscience & Bioengineering, Universiti Teknologi Malaysia, Johor Bahru, Malaysia
ABSTRACT
Introduction: Sperm-oocyte fusion is one of the most impressive processes in sexual reproduction, and its molecular mechanism is of great interest to fertility researchers. However, the limitation of materials and difficulties in in-vitro analysis of the protein-protein interactions in human caused the molecular interactions that mediate sperm-egg membrane fusion to be poorly understood.
Objective: To map the sperm-oocyte protein interaction network and to identify the biological roles of involved proteins in sperm-oocyte fusion process.
Methods: We used Cytoscape 2.8.2 to create the novel protein-protein interaction (PPI) network for all proteins involved in interaction between sperm and oocyte and then classified this network using the Allegro- M code algorithm.
Results & Discussion: The network consists of 725 input proteins with 642 proteins forming 22 functional clusters. There are 2173 interactions in the sperm-egg network with the binding node being the most significant node as it involves 587 of the 642 proteins (91%). The gene ontology analysis identified a number of ontologies and gene that are likely to be involved in the complex mechanism of sperm-oocyte fusion. These include phosphoprotein binding, protein N-terminus binding and insulin receptor substrate binding among others.
N
-terminal derivatization improves peptides
de novo
sequencing by MALDI-TOF
MS/MS
Azwan Awang1, Rafiah Karim1 and Mitsui Toshiaki2
1
Malaysian Cocoa Board, Center for Cocoa Biotechnology Research, Sabah, Malaysia,
2Faculty of Agriculture, Niigata University, Niigata, Japan
ABSTRACT
Introduction: One way to identify proteins from an organism with unsequenced genome is to do de novo
sequence analysis of peptides by mass spectrometry (MS). Application of MALDI for de novo
sequencing, however, is initially limited due to the low signal to noise level, spectral complexity, and incomplete peptide ion fragmentation resulting in gaps of the sequence. Accordingly, deducing the amino acid sequence directly from MALDI MS/MS spectra is often difficult. We present here a technique to obtain simple MALDI-TOF MS/MS spectra for de novo sequencing of peptide and its application to identify proteins from cocoa (Theobroma cacao).
Objective: To evaluate the use of SPITC as a derivative to improve de novo sequencing by MALDI-TOF MS/MS
Methods: Protein spots were excised from 2-dimensional electrophoresis (2-DE) and subjected to trypsin digestion. Digestion solutions were chemical derivatized with 4-sulphophenyl-isothiocyanate (SPITC) by incubation at 56°C for 1 h. Derivatized samples were desalted using C18 ZipTip and subjected to MALDI-TOF MS/MS. Proteins were identified by cross species homology searches.
Results & Discussion: Derivatization of N-terminal of tryptic peptide with SPITC resulted in abundant of sequence information bearing y-ions series in the spectra after MALDI-TOF MS/MS. Accordingly, amino acid sequences can be deduced from MALDI-TOF MS/MS spectra by simply measuring the differences between adjacent fragment ion peaks of y-ion series. We could obtain from 5 up to 22 amino acid residues without a gap. Using de novo sequence analysis with cross species homology search, we have identified 137 proteins from cocoa pod husk.
Glycomics study of Tamm-Horsfall glycoprotein and uromodulin
1
Hong C.-Y., 2Grassi P, 1Wong N.-K. and 2Dell A.
1
School of Science and Technology, Universiti Malaysia Sabah, Kota Kinabalu, Sabah, Malaysia, 2 Division of Molecular Biosciences, Faculty of Natural Sciences, Imperial College London, London, United Kingdom
ABSTRACT
Introduction: Tamm-Horsfall glycoprotein (THP) and uromodulin are the most abundant glycoproteins discovered in non-pregnant women/ male and pregnant women urine, respectively. Both of the proteins have been shown to inhibit in vitro T cell proliferation induced by specific antigens, and uromodulin was demonstrated to be higher in immunosuppressive activity than THP. Therefore, characterization of pregnancy-associated changes in the glycosylation is done in this study, which is anticipated to be responsible for enhanced immunosuppressive activity of uromodulin as both THP and uromodulin have the same amino acid sequences.
Objective: To characterize the glycan structures of THP and uromodulin using ultra high sensitivity MALDI-TOF mass spectrometry.
Methods: THP and uromodulin were isolated using diatomaceous earth filtration from healthy non-pregnant woman and non-pregnant women urine, respectively. Oligosaccharides of the glycoproteins were released and permethylated before subjected for MALDI-TOF MS-MS analysis.
Results & Discussion: Structural analysis showed that uromodulin expressed different glycan profiles compared to THP. For complex-type N-glycans, uromodulin tend to express multiple degree of sialylation, in which up to four sialic acid residues were expressed on its tetra-antennary N-glycans. Sequencing of the glycans indicated that THP and uromodulin expressed both core 1 and core 2 O-glycans, with core 2 as the majority type. It is suggested that core 2 might suppress the immune activity, whist the extensive capping of sialic acid on uromodulin N-glycans induced immunomodulatory effects by binding to PHA-L and interrupt the interaction between PHA-L and T cells.
Proteomic analysis of oil palm root infected with
Ganoderma boninense
Syahanim,S., Abrizah, O., Mohamad Arif, A. M., Idris, A.S and Mohd Din, A.
Malaysian Palm Oil Board (MPOB), Kajang, Selangor, Malaysia
ABSTRACT
Introduction: Ganoderma boninense is major pathogen of oil palm in Malaysia that causes the basal stem rot disease. Plants produce various biochemical and physiological responses during pathogenicity. Understanding the defence mechanisms in oil palm against fungal infection is important. Pathogenesis-related (PR) proteins are triggered to combat the fungus infestation. Despite the extensive information on the involvement of PR-proteins in other plants, the accumulation of these proteins in oil palm during
Ganoderma infection is still lacking.
Objective: To characterize oil palm root response to Ganoderma infection at day-7 by proteomic analysis.
Methods: 12-months oil palms were infected with Ganoderma using root inoculation technique. Total protein was extracted from root based on Sheffield et al. (2006). The protein was quantified and 500 ug protein was cleaned using the 2-D Clean Up Kit (GE Healthcare). Two-dimensional electrophoresis was performed on 18-cm IPG strip pH 3-10. Gels were stained and images were normalized with PDQuest™ 2-D Analysis Software (Bio-Rad). Differentially expressed protein spots were cored out and de-stained, followed by tryptic digestion. Spots were identified via MALDI TOF/TOF MS/MS (Applied Biosystem/MDS SCIEX) and sequences from MS/MS mass spectra were submitted using the SWISS-PROT database.
Results & Discussion:Sixteen protein spots were found to be differentially expressed in oil palm root at day-7 post infection. MALDI TOF/TOF MSMS had successfully identified pathogenesis-related protein (beta-1,3-glucanase) and other proteins involved in oxidative stress (glutathione S-transferase and thioredoxin H2).
Demonstration of antigenic outer membrane proteins in clinical isolate
of S
higella sonnei
Hemavathy Harikrishnan1, Asma Ismail 2, Izana Ibrahim1 and Kirnpal-Kaur Banga Singh1
1
Department of Medical Microbiology & Parasitology, School of Medical Science, 2Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Kubang Kerian, Kelantan, Malaysia
ABSTRACT
Introduction: Shigellosis is an acute invasive enteric infection often characterized by abdominal pain, fever and bloody diarrhea. It is caused by infection with Shigella spp. In Malaysia, Shigella spp. is the third most common bacterial agent responsible for diarrhea. Current method of diagnosis still depends on the traditional culture method, which is laborious and takes 3 to 5 days to produce results. The low sensitivity of this routine test has called for an alternative diagnostic method for the early identification of the bacterium. As such, there is a need to identify reliable signatures of biomarkers that are useful in development of a rapid, sensitive and reliable diagnostic assay.
Objective: To determine the antigenic outer membrane proteins (OMPs) in clinical isolate of Shigella
sonnei.
Methods: Outer membrane proteins of clinical strain of S. sonnei SH080 was extracted from an overnight culture grown at 37°C. In comparison, OMPs were also extracted from a reference strain of S. sonnei
ATCC 25931. SDS-PAGE separated protein profiles were observed by Coomassie Blue staining. The antigenic OMPs proteins were determined by Western blot analysis to detect the presence of IgA and IgG against the proteins in selected cultured confirmed host serum. The OMPs were compared in terms of molecular weight and degree of expression.
Results & Discussion: Similar major OMPs were detected in both strains. A total of 23 and 20 OMPs were identified in clinical and reference strains, respectively. Majority of these proteins were over-expressed in the clinical strain. Few antigenic OMPs were identified, and were uniquely recognized by IgA and IgG in selected culture-confirmed patients’ sera.
Proteomic analysis of
Burkholderia pseudomallei
colony morphology variants pre-
and post- exposure to A549 at mid-logarithmic phase of growth
Al-Maleki A. R., Mariappan V., Vellasamy K. M., Kalaiselvam K., Loke, M. F., Tay, S. T. and Vadivelu J.
Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia
ABSTRACT
Introduction:Burkholderia pseudomallei, a Gram-nbacillus, is the causative agent of melioidosis. Colony morphology variation of B. pseudomallei is a negative intracellular otable feature of clinical cultures from patients with melioidosis. These colony morphology variants are associated with changes in the expression of a range of putative virulence factors.
Objective: To investigate protein expression profiles among B. pseudomallei; wild type (WT), and small colony variant (SCV) pre- and post- exposure to A549 cells in order to ascertain differences if any differences exist between colony variants.
Methods: Total proteins were extracted from the whole cells (mid-log phase); WT and SCV pre- and post- exposure to the A549. Proteomic analysis using two-dimensional gel electrophoresis and MALDI TOF/TOF mass spectrometry analyses were performed for protein identification.
Results & Discussion: A total of 263 and 260 protein spots were detected in the WT pre- and post-exposure to A549 cells, respectively, as compared to the SCV pre-post-exposure (258 spots) and post-post-exposure (259 spots) to A549 cells. Comparison between the WT and SCV pre-exposed to A549 cells detected 38 protein spots which were differentially expressed (12 up-regulated; 26 down-regulated), whereas 12 were differentially expressed post-exposure (4 up-regulated; 8 down-regulated). Similarly, comparison between the pre- and post-exposure of the strains to A549 cells detected 35 protein spots which were differentially expressed in the WT (25 up-regulated, 10 down-regulated) and 25 protein spots differentially expressed in the SCV (21 up-regulated, 4 down-regulated). The proteins identified were found to be involved in various functions including energy production, fatty acid synthesis, cell transport, DNA replication, transcription and translation. Several of these identified proteins were also implicated in bacterial pathogenesis.
Study of protein expression profiles in
S.
Typhi biofilm culture
Y.L. Lee and K.K. Phua
Institute for Research in Molecular Medicine (INFORMM), Health Campus, Universiti Sains Malaysia, Kota Bharu, Kelantan, Malaysia
ABSTRACT
Introduction: Salmonella Typhi is a human specific pathogen which causes typhoid fever, a major health problem in developing countries including Malaysia. Although prevention and medical treatment are available but typhoid still persist. Biofilm formation is considered one of the major factors contributing to the persistence through evading the host immune response, hiding within the human gallbladder. In order to understand the formation of biofilm, protein expression profiles were studied to uncover the protein dynamics.
Objective: To study S. Typhi biofilm protein expression profiles by comparing with planktonik state cells using 2D-PAGE and MALDI-TOF MS/MS.
Methods:S. Typhi biofilm was cultured in-vitro by mimicking the human gallbladder environment using nutrient broth containing different concentrations of bile. Proteins from both cell types were harvested and analyzed using 2D-PAGE. Unique protein sports were excised and further analysed using MALDI-TOF MS/MS.
Results & Discussion: 188 protein spots were identified from the gels from which 15 were unique to biofilm and 19 were unique to the planktonik stage. On the other hand, 10 protein spots were found up-regulated by at least 2 folds and 11 proteins were found down-up-regulated at least 2 folds. MALDI-TOF analysis showed that one of the novel biofilm proteins was tolC, which was found to be empirically upregulated in response to bile in the culture media.
Differential proteomic analyses of periodontal pathogens grown in polymicrobial
biofilm and planktonic states
Zamirah Zainal-Abidin, Paul D. Veith, Stuart G. Dashper, Ying Zhu, Deanne V. Catmull, Yu-Yen Chen, Deasy C. Heryanto, Dina Chen, James S. Pyke, Kheng Tan, Helen L. Mitchell and Eric C. Reynolds
Oral Health CRC, Melbourne Dental School and the Bio21 Institute, The University of Melbourne, Australia
ABSTRACT
Introduction: Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia exist in a polymicrobial biofilm associated with chronic periodontitis.
Objective: To culture these three species as a polymicrobial biofilm and to determine proteins important for bacterial interactions.
Methods: In a novel preparative fermentor, all three species attached and grew as a biofilm. For comparison, planktonic cultures of P. gingivalis and T. denticola were grown separately in continuous culture. Whole cell lysates were subjected to SDS-PAGE, followed by in-gel proteolytic H2
16
O/ H2 18
O labelling. The relative protein abundance of P. gingivalis and T. denticola grown in different states was calculated using sophisticated in-house developed macros.
Results & Discussion: 135 and 174 P. gingivalis proteins and 134 and 194 T. denticola proteins were quantified from two replicates by LC-MALDI TOF/TOF MS. The results suggest a change of strategy in iron acquisition by P. gingivalis due to differences in the abundance iron/haem transport systems. Significant changes in the abundance of peptidases and enzymes involved in glycine catabolism between the two bacteria species suggest syntrophy.
Analysis of human serum protein profiles of patients with amoebic liver abscess by
two-dimensional electrophoresis (2-DE)
Nurulhasanah Othman1, Zeehaida Mohamed2, Maya Mazuwin Yahya3, Leow Voon Meng4, Lim Boon Huat5 and Rahmah Noordin1*
1
Institute for Research in Molecular Medicine, Universiti Sains Malaysia (USM), Penang, 2Dept of Medical Microbiology and Parasitology, School of Medical Sciences, USM, Kelantan, 3Dept of Surgery, School of Medical Sciences, USM, Kelantan, 4 Advanced Medical & Dental
Institute, USM, Penang, 5School of Health Sciences, USM, Kelantan, Malaysia * Corresponding author
ABSTRACT
Introduction: Amoebic liver abscess (ALA) is the main extraintestinal disease caused by Entamoeba
histolytica. Comparison between human serum proteins of disease and non-disease groups was performed
to study the differences in their expression levels and to identify possible surrogate disease markers.
Objective: To study protein expression profiles of host serum proteins from patients with ALA and healthy individuals using 2-DE, protein expression patterns and MS/MS analysis.
Methods: Fifteen and ten serum samples from patients with ALA and healthy individuals respectively were resolved in 2-DE. Image analysis of protein expression patterns was performed using Progenesis SameSpots software. The up/down-regulated protein spots were excised from the gels and analysed by MS/MS.
Results & Discussion: The 2-DE was able to resolve almost all cluster spots of high abundant serum protein that could be visualized in sera from patients with ALA and healthy individuals. The protein expression patterns analysis showed four spots from haptoglobin cluster (average 1.75 fold), three spots from α1-antitrypsin cluster (average 1.5 fold), one spot from albumin cluster (1.4 fold), one spot each from unknown spots no. 8 (2.0 fold) and 9 (1.9 fold) to be significantly up-regulated (p<0.05) in sera of ALA patients when compared to those of healthy individuals. Two protein spots from the transferrin cluster were significantly down-regulated with average 1.9 fold in patients’ sera. The MS/MS analysis verified the presence of the up/down-regulated protein spots in the cluster proteins. The unknown protein spot no.8 was identified as human transcript and haptoglobin; and spot no. 9 was identified as albumin.
Leptospira interrogans
72kDa protein reactive with sera from
patients with acute leptospirosis
M. Riazi1, F.Z. Zainul 2, A.R.Bahaman3, F. Amran4 and A. Khalilpour5
1School of Pharmaceutical Sciences, Universiti Sains Malaysia (USM), Penang & School of Health Sciences, USM, Kelantan, 2School of Health
Sciences, USM & Institute for Research in Molecular Medicine, USM, Penang, 3Faculty of Veterinary Medicine, Universiti Putra Malaysia,
Selangor, 4Bacteriology Division, Institute for Medical Research, Kuala Lumpur, 5Institute for Research in Molecular Medicine, USM, Penang,
Malaysia
ABSTRACT
Introduction: Leptospirosis is the most widespread zoonotic disease in the world and a public health problem particularly in tropical and subtropical countries. Non-specific symptoms of the disease frequently lead to misdiagnosis and possible risk for life-threatening multi-organ complications. Therefore, early laboratory investigation using appropriate diagnostic approach is crucial.
Objective: To identify and evaluate potential protein marker for serodiagnosis of acute leptospirosis.
Methods:Leptospirainterrogans serovar icterohemorrhagiae (l44), which represents a prevalent serovar in malaysia, was used in the preparation of sequential protein extract (seq) using a combination of tris, urea and thiourea. Two-dimensional electrophoresis and igm immunoblotting were performed with a sera panel comprising confirmed cases of leptospirosis in malaysia (n=42) and controls (n=42). The potential reactive protein was characterized using mass spectrometer and the corresponding recombinant antigen was evaluated for its potential diagnostic value.
Results & Discussion: Among the antigenic components, a 72kDa protein band demonstrated sensitivity and specificity of 83.3% and 95.2% respectively for the detection of specific anti-leptospiral igm antibodies. Further characterization by two-dimensional electrophoresis and igm immunoblotting showed a pi of 5.25-6.00. The protein was identified by mass-spectrometry analysis as heat shock protein dnak of
l. Interrogans. The recombinant form of this protein (r72seq) showed 85% sensitivity and 80% specificity
for the detection of specific anti-leptospiral lgM antibodies.
Proteomic analysis of
Helicobacter pylori
antigens of diagnostic potential
Khalilpour A., Amutha S., Lee C.W., Sabariah O., Ahmad M.and Rahmah N.*
Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang, Malaysia *Corresponding author
ABSTRACT
Introduction: Helicobacter pylori is a spiral shaped microaerophilic gram negative bacterium that colonizes the apical side of human gastric epithelial cells and mucous layer. Infection by H. pylori affects approximately 50% of the population in many developing countries. The infections are associated with various gastric diseases such as active gastritis, peptic ulceration, duodenal ulcer and gastric carcinoma. Since there is wide genetic variation among the H. pylori strains, thus the sensitivity and specificity of the
H. pylori detection in patients from a particular region may be improved by using local strains.
Objective: To identify potential infection markers from local H. pylori strain for development of specific and sensitive diagnostic test for patients in Malaysia and regional countries.
Methods: Serum samples were obtained from culture-positive H. pylori infected patients with duodenal ulcer, gastric ulcer and gastritis (n=30). As controls, three kinds of sera (n=60) without detectable H.
pylori IgG antibodies were used. H. pylori was isolated from biopsy sample of a duodenal ulcer patient in
Malaysia, and antigen prepared. Isoelectric focusing was performed by OFFGEL fractionator (Agilent), followed by SDS-PAGE of the liquid fractions. Western blot analysis was then performed using the panel of serum samples and the peroxidase conjugated anti-human IgG. The selected antigen bands were excised from the gel, processed and then sent for mass spectrometry analysis using MALDI TOF-TOF.
Results & Discussion: The results showed that the most promising diagnostic candidates were five proteins with >70% sensitivity and >95% specificity namely 50S ribosomal protein (13 kDa), CagI (25 kDa), urease G accessory protein (25 kDa), UreB (63 kDa) and proline/pyrroline-5-carboxylate dehydrogenase (118 KDa).
Conclusion: These identified proteins, probably in combinations, are potentially useful for diagnosis of
Proteomic evaluation and validation of anti-inflammatory and anti-proliferative
properties of Zerumbone in atherosclerotic-induced New Zealand white rabbit
Hemn, H.O. 1*, Noordin, M.M.1 Hazilawati, H. 1, Heshu, S.R.1 and Zuki, A.B.Z2
1 Department of Pathology and Microbiology, 2Department of Preclinical Sciences, Faculty of Veterinary Medicine, Universiti Putra Malaysia,
Serdang, Malaysia *Corresponding author
ABSTRACT
Introduction: Atherosclerosis is a chronic degenerative, accumulative, and proliferative inflammatory response. The pathogenesis of atherosclerosis is a subject of much debate, but it is now generally thought to be a chronic inflammatory disease. Many natural herbs been studied and approved to reduce early development of atherosclerosis, however, the role of Zerumbone (ZER) a natural cyclic sesquiterpene isolated from Zingiber zerumbet smith in modulating inflammatory progression of atherosclerosis is less investigated.
Objective: The objectives of this exploration is to evaluate the antiproliferative and anti-inflammatory properties of zerumbone (ZER), in preventing and reducing macrophages assembly and smooth muscle cells proliferation in early-developed atherosclerotic lesions.
Methods: Thirty male rabbits were used in the present study randomly assigned into five experimental groups (EG)s as follows: EG-I considered as control group was fed a standard rabbit pellet diet, EG-II given a cholesterol-rich diet (1%), EG-III given ZER (0.4%), EG-IV given ZER (0.8%), two weeks before day 0 as preventive measure and thereafter with the course of cholesterol-rich diet and EG-V given Simvastatin (SIM) (20 mg/Kg) . Tissue samples were collected from the thoracic aorta and aortic arch at 12 weeks post-feeding for immunohistochemistry and immunoflourescent analysis. The following antibodies were used against cellular protein components of macrophages (RAM-11) and smooth muscle cells B-actin (HHF-35).
Results & Discussion: Most of the ZER treated groups showed marked dropping in plague development in contrast to cholesterol-rich diet group EG-II, probably due to the inflammatory and anti-proliferative upshot of ZER via suppression of inflammatory cytokines and growth factors.
Proteomic profiling of milk fat globule membrane proteins of ruminants and
humans
Thanes J.1, Hashim O.H.1, 2, Fung S.Y.1, 2 and Abdul-Rahman P.S.1, 2
1
Department of Molecular Medicine, Faculty of Medicine, 2University of Malaya Centre for Proteomic Research (UMCPR), University of Malaya, Kuala Lumpur, Malaysia
ABSTRACT
Introduction: Milk is a source of complete nutrient provided by mammals to nourish their young offspring. Milk proteins could be divided into three main fractions i.e. casein, whey and milk fat globule membrane (MFGM). Milk from different species of mammalian differs in terms of protein quality and concentration. Although the major fractions of milk such as whey and MFGM have been studied, functions of some of the MFGM proteins have yet to be deciphered. Identification of the proteins found on MFGM is highly informative and may reveal unknown pathways that are involved in the mammary glands.
Objective: This study was aimed to compare the MFGM proteome profiles in the milk of ruminants and humans.
Methods: MFGM protein extracts from bovine, caprine and human milk were subjected to two-dimensional gel electrophoresis (2DGE) and subsequently developed by silver staining. The respective 2DGE profiles were analysed using Image Master 2D Platinum software 7 and the resolved proteins were identified by mass spectrometry and database search.
Results & Discussion: Our results demonstrated that the 2DGE profiles of bovine and caprine MFGM were comparable but markedly different from those of the humans. Five proteins that were present in the human MFGM were apparently not detected in 2DGE profiles of bovine and caprine MFGM. Beta lactoglobulin, a potential allergic causing milk component which was exclusively detected in the both ruminant milk proteome profile was not expressed in the human milk proteome profile.
Identification of
Plasmodium falciparum
circulating proteins in sera of malaria
patients using proteomics approach
Nurul Shazalina Zainudin1, Amir Rabu2,3, Nurulhasanah Othman1, Asmahani Azira Abdu Sani3, Jamail Muhi4, Chew Ai Lan1 and Rahmah Noordin1*
1Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Penang, Malaysia, 2Faculty of Science & Technology,
Universiti Kebangsaan Malaysia, Selangor, Malaysia, 3Malaysian Genome Institute, Ministry of Science & Innovation, 4Sarawak Health Office, Sarawak, Malaysia * Corresponding author
ABSTRACT
Introduction: Malaria is one of the most important tropical diseases, and is caused by several species of
Plasmodium. P. falciparum is responsible for over a million deaths per year especially among children.
Proteomic studies in this area have been performed mainly to study the pathogenesis of the parasite. Identification of the parasite proteins in patients’ sera using proteomics approach may be useful to further understand the disease pathogenesis and improve diagnosis.
Objective: To identify circulating proteins of P. falciparum in sera of patients with malaria.
Methods: Pooled sera from 10 malaria patients were used. Depletion was performed using Proteoseek Albumin/IgG Removal kit followed by in-solution digest of the depleted sera. This was then analysed by LC-MS/MS (LC-MS/MS (nanoACQUITY UPLC and Synapt HDMS, Waters) and database search analysis was performed using ProteinLynx Global SERVER (PLGS).
Results & Discussion: The MS/MS analysis and database search using PLGS on the depleted sera identified several P. falciparum proteins which were above the PLGS cut-off score of 200. There were a number of mature erythrocyte surface antigens (MESA) and P. falciparum chloroquine resistance transporter (PfCRT). The MESA proteins of P. falciparum are antigenically variable, and found on the internal face of the erythrocyte membrane. MESAs are involved in phosphorylation and are also known as phosphoproteins. PfCRT is an integral membrane protein and is located on the membrane of the intra-erythrocytic digestive vacuole of the parasite.
An
in-vivo
induced antigen of
Toxoplasma gondii
with high sensitivity and
specificity for detection of anti-
Toxoplasma
IgM antibodies
Atefeh Amerizadeh1, Khoo Boon Yin1, Majid Golkar2, Muhammad Hafiznur Yunus1 and Rahmah Noordin1*
1
Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang, Malaysia, 2Molecular Parasitology Laboratory, Parasitology Department, Pasteur Institute of Iran, Tehran, Iran *Corresponding author
ABSTRACT
Introduction: Primary infection with T. gondii during pregnancy may result in congenital infection of the fetus. The majorities of congenitally infected newborns do not show clinical signs at birth but may later develop symptoms. This can be prevented by early diagnosis using a highly sensitive test for detection of
T. gondii-specific IgM antibodies, since this antibody cannot cross the placenta.
Objective: To identify T. gondii in-vivo induced antigen for sensitive and specific detection of IgM antibodies.
Methods: In-vivo induced antigen technology (IVIAT) using T. gondii cDNA phage library and IgM probe was performed by repeated immunoscreening using well pre-absorbed sera from patients with acute infection. The strongly reactive clones were sequenced, and those with high homology to T. gondii were subjected to expression analysis by realtime PCR. Selected clones were tested for sensitivity and specificity for detection of anti-T. gondii IgM detection. The best clone was subjected to medium scale liquid culture protein expression, and the recombinant protein was analysed by IgM western blot using a panel of Toxoplasma positive and negative serum samples.
Results & Discussion: Thirty clones with high homology sequences to T. gondii genes were identified. The realtime PCR expression analysis revealed that the protein of clone AM 15 was exclusively expressed in-vivo. This clone also showed high sensitivity and specificity for detection of T. gondii -specific IgM antibodies. IgM western blot of the recombinant protein reaffirmed its good diagnostic value.
Identification of paramyosin as a potential marker of human hydatid
disease
via
proteomic approach
Zohreh Kazemi Moghadam Kakhki1, Fatemeh Ghaffarifar 2, Farhanah Abdul Aziz1 and Rahmah Noordin1*
1
Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 2Department of Parasitology, School of Medical Sciences, Tarbiat Modarres University, Tehran, Iran * Corresponding author
ABSTRACT
Introduction: Protoscolex or larval stage of tapeworm Echinococcus granulosus causes hydatid disease in humans. The clinical manifestations are variable and non-specific, making diagnosis challenging. Imaging methods in combination with serology are important for diagnosis. Accurate immunodiagnosis of the infection requires highly specific and sensitive antigen.
Objective: To identify protein marker(s) for immunodiagnosis of hydatid disease.
Methods: Serum samples used in this study were from 49 patients with surgically-confirmed hydatid disease, 37 individuals infected with other parasitic infections and 30 healthy people (controls). Protoscoleces were isolated from hydatid cysts from livers of naturally infected sheep, and the antigen prepared. The first dimension of the 2-DE gel was run with IPG strips pH 3-10 on an OFF-GEL apparatus, followed by 12% SDS-PAGE. Protein bands were electrophoretically transferred onto nitrocellulose membrane, blocked, incubated with positive and negative human sera at 1:100, then incubated with monoclonal anti–human IgG4, 1:2000. The selected antigenic protein band was excised from the gel for mass-spectrometry analysis. The corresponding recombinant protein was expressed in E.
coli and column-purified using Ni-NTA resin. It was then evaluated by western blot analysis with the
above sera panel.
Results & Discussion: An antigenic band between 50-75 kDa with potential diagnostic value was found to demonstrate sensitivity of 82% with sera of hydatid cyst patients and 100% specificity with control sera. It was identified by MS-MS analysis as protoscolex tegument paramyosin. The corresponding recombinant protein also showed a high sensitivity of 86% with sera of hydatid cyst patients and 100% specificity with control sera.
Amplification, expression and purification of synthetic UvrD helicase gene
Sanggetha Periya Sepuloh Maniam1, Shaharum Shamsuddin2, Asma Ismail1 and Venugopal Balakrishnan1
1
Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Pulau Pinang, Malaysia, 2School of Health Sciences, Universiti Sains, Malaysia, Kubang Kerian, Kelantan, Malaysia
ABSTRACT
Introduction: Helicase-dependant amplification (HDA) method uses a DNA helicase to unwind double-stranded DNA into two single strands. This enables primers hybridization and subsequent extension of the DNA strand. This method is an isothermal amplification method. Hence, various temperatures provided through the conventional PCR can be omitted. In this study, we applied assembly PCR method to produce synthetic UvrD helicase gene.
Objective: To construct and express the synthetic UvrD helicase gene.
Methods: Sixty pairs of oligonucleotides (oligos), representing both positive and negative strand were designed based on Escherichia coli UvrD helicase nucleotide sequence retrieved from NCBI website. The oligos were assembled and assembled product was used as template to synthesize the full length helicase gene. The synthetic gene was ligated into pET28a vector and expressed in BL21 (DE3) expression host. Expressed protein was purified by nickel charged affinity column.
Results & Discussion: Full length synthetic UvrD helicase gene with ~2.1 kb nucleotides was successfully amplified, and cloned into pET28a vector. The recombinant protein was expressed with the induction of 1.0 mM IPTG for 3 hours at 37°C. The nickel Immuno metal-affinity chromatography was performed to purify the Histidine-tag UvrD protein. The protein was detected through western blotting and showed a single specific band at size ~75 kDa.
Analysis of proteins as biomarkers in
Acanthamoeba polyphaga
for ammonium
chloride contamination in the environment
1Nakisah, M. A, 1Siti Faezah, .S and 2Mohd Tahir, N.
1Department of Biological Sciences, 2Department of Chemical Sciences, Faculty of Science and Technology, Universiti Malaysia Terengganu,
Kuala Terengganu, Terengganu, Malaysia
ABSTRACT
Introduction: Acanthamoeba spp are abundant in our environment and they occupy in most habitats especially in the aquatic ecosystem. Being single-celled organisms, these free-living amoebae are suitable to be used as bio-indicator since the environmental pollutants can affect them directly, and their response are immediate both at their cellular and molecular levels. Therefore, proteins that are expressed in ammonium chloride-exposed amoebae can be analysed and identified as a biomarker for ammonium chloride contamination.
Objective: To analyse proteins in Acanthamoeba polyphaga as biomarker for ammonium chloride contamination.
Methods: 2-D electrophoresis was employed to analyse protein profile in Acanthamoeba polyphaga
(CCAP 1501/3A) after the amoeba was exposed for 72 h at various concentrations (ranging from 0.5mg to 3.0 mg) of the ammonium chloride. Pellets of amoebae were collected by centrifugation and were then sonicated and centrifuged to obtain protein samples from the amoeba. A supernatant part (protein sample) from treated and control amoebae was subjected to 2D electrophoresis. A control gel was used as a reference/master gel when protein profiles in treated amoebae were analysed using ImageMaster 2D Platinum.
Results & Discussion: Different protein profiles were observed in the 2D gels of Acanthamoeba after being treated with different concentrations of ammonium chloride. Total number of protein dots in control gel was 833. In treated amoeba, some of the protein dots were either missing (not expressed), over expressed or newly synthesized protein (were not observed in control amoeba). These proteins are suitable to be candidates as biomarkers in amoebae for ammonium chloride contamination in the aquatic ecosystem.
Comparative proteomic analysis of large and small colony morphology variants of
Burkholderia pseudomallei
Kaveena Kalaiselvam, Vanitha Mariappan, Kumutha Malar Vellasamy, Nur Siti Khadijah Ramli, Anis R Al-Maleki and Jamuna Vadivelu
Department of Medical Microbiology, Faculty of Medicine, University Malaya, Kuala Lumpur, Malaysia
ABSTRACT
Introduction: The gold standard for identification of a microbiological agent is culture. The causative agent of melioidosis, Burkholderia pseudomallei, on primary culture has demonstrated variation in colony morphology and this variation has been associated with modification/alteration in the expression of virulence factors that can enhance survival under adverse conditions.
Objective: The aim of this study was to compare and contrast the protein expression profiles of B.
pseudomallei large colony variant (LCV) and the small colony variant (SCV) in order to identify proteins
that are associated with modification/alteration in the expression of virulence factors.
Methods: Whole bacterial proteins from B. pseudomallei were collected to stationary phase in Luria-Bertani broth following overnight culture at 37ºC with shaking. The bacterial pellet was collected through centrifugation and sonication. Following centrifugation the total whole bacterial proteins were subjected to proteomic analysis using 2-DE and hot Coomassie staining method. The protein spots were analysed using the PDQuest software and will be identified using MALDI-TOF analysis.
Results & Discussion: A total of 267 and 208 proteins spots were detected in B. pseudomallei LCV and SCV, respectively. Of these, 56 proteins spots were found to be present and differently expressed in both the isolates. Approximately, 152 and 211 proteins spots were found to be unique to LCV and SCV, correspondingly. Similarities and differences observed may contribute to the virulence phenotypic characteristics of the isogenic strains.
Heat shock protein(s) of
Shigella flexneri
expressed
at 37°c, 38.5°c and 40°c
Hemavathy Harikrishnan1, Asma Ismail 2 and Kirnpal-Kaur Banga Singh1
1
Department of Medical Microbiology & Parasitology, School of Medical Sciences, 2Institute for Research in Molecular Medicine (INFORMM), Health Campus, Universiti Sains Malaysia, Kubang Kerian, Kelantan, Malaysia
ABSTRACT
Introduction: Bacteria are able to survive in diverse natural environments. To survive in adverse environment such as nutrient depletion and high temperature, bacteria must develop variety of survival mechanisms. Heat shock proteins are family of proteins which expression are enhanced in response to environmental stress. This study was conducted to investigate how outer membrane proteins (OMPs) of
Shigella flexneri respond to stress, especially during fever when the host’s body temperature is high.
Bacteria were cultured at 37oC (healthy body temperature), 38.5oC and 40oC. Protein profiles expressed at 38.5oC and 40oC were compared to those expressed at 37oC.
Objective: To demonstrate heat shock proteins in the OMPs of S. flexneri expressed at 37°C, 38.5°C and 40°C.
Methods: OMPs of S. flexneri ATCC 12022 and clinical isolate SH057 were extracted from an overnight culture grown at 37°C, 38.5°C and 40°C. Comparison of the expressed proteins under the different growth conditions were based on equal numbers of bacterial cells loaded in the SDS-PAGE gels. Separated proteins were stained with Coomassie brilliant blue.
Results & Discussion: Different degrees of expressions were demonstrated with different proteins expressed at 37°C compared to 38.5°C and 40°C. A total of 18 and 21 OMPs were observed in S. flexneri
ATCC 12022 and SH057, respectively.Upregulation of proteins expression were observed in majority of OMPs expressed in clinical isolate at higher temperature.
Expression, purification and evaluation of recombinant pyruvate phosphate
dikinase-(PPDK)
protein
of Entamoeba histolytica
Syazwan Saidin1, Nurulhasanah Othman1, Muhammad Hafiznur Yusof1, Lim Boon Huat2, Izzati Zahidah Abdul Karim1 and Rahmah Noordin1*
1
Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Penang, Malaysia
2
School of Health Sciences, Universiti Sains Malaysia, Kelantan, Malaysia *Corresponding author
ABSTRACT
Introduction: Amoebic liver abscess (ALA), caused by Entamoeba histolytica, is the most common extraintestinal manifestation of the infection. The use of recombinant protein is a promising approach to improve the serological diagnosis of ALA. We have previously identified E. histolytica pyruvate phosphate dikinase (PPDK) to be potentially useful as ALA diagnostic marker.
Objective: To express, purify the recombinant PPDK and evaluate its immunoreactivity with anti-His-HRP, and sera of ALA patients and healthy individuals.
Methods: The recombinant plasmids, pET28a (+)/PPDK, were transformed into competent E. coli strain BL21 (DE3) cells. The bacteria were grown at 37°C to OD600 of 0.4-0.6. Recombinant PPDK expression
was induced by adding 1mM IPTG and incubated for 4 hours at 30oC. The recombinant protein was column-purified by Ni-NTA resin and the concentration determined. PPDK recombinant protein was analysed by western blot using anti-His-HRP and sera from patients with ALA (n=10) and healthy individuals (n=10).
Results & Discussion: SDS-PAGE showed that the molecular weight of the purified recombinant PPDK (106 kDa) was similar with that calculated from the size of the gene encoding the PPDK protein. Western blot using anti-His-HRP verified the expression of recombinant PPDK. W. blot using sera of ALA patients and healthy individuals (control) showed that recombinant PPDK were immunoreactive with all the patients’ samples and not reactive with sera of healthy individuals.
Proteomic analysis of human lymphocytes from old and young individuals’
revealed age related changes associated with cell proliferation and cellular
structure stability
Mariati Abdul Rahman1, Hasnizawati Mohamed Dahlan2, Saiful Anuar Karsani3,Noor Aini Abdul Hamid4,A Gapor Md. Top5 and Wan Zurinah Wan Ngah2
1Department of Clinical Oral Biology, Faculty of Dentistry and 2Department of Biochemistry, Faculty of Medicine, Universiti Kebangsaan
Malaysia, Kuala Lumpur, Malaysia, 3Institute of Biological Sciences, Faculty of Sciences, University of Malaya & University of Malaya Centre
for Proteomics Research (UMPCR), Kuala Lumpur, Malaysia, 4Faculty of Medicine, Cyber Jaya University College of Medical Sciences, Cyberjaya, Malaysia, 5Malaysian Palm Oil Board, Kuala Lumpur, Malaysia
ABSTRACT
Introduction: There is a decline of the immune system and abnormality of lymphocyte function with advancing age. The molecular mechanism(s) underlying these age-related defects are not fully understood.
Objective: This study aims to determine the effect of aging on protein expression in human lymphocytes from two different age groups.
Methods: Protein expression profiles of human lymphocytes obtained from young (30-49 years old) and old (>50 years old) volunteers were assessed using two-dimensional gel electrophoresis. Differentially expressed proteins were then identified by MALDI-TOF/TOF tandem mass spectrometry.
Results & Discussion: Purine nucleoside phosphorylase and 10 kDa heat shock protein were up-regulated whereas Proliferating cell nuclear antigen, 14-3-3 epsilon, Ezrin and S-phase kinase associated protein 1A were down-regulated in the old group.
Proteomic determination of antibacterial constituent, L-amino acid oxidase from
Calloselasma rhodostoma
and
Ophiophagus hannah
using bioassay guided
purification
Sugita K.1, Wayne H.2, Sharifah S.H.1 and Iekhsan O.1
1Jeffrey Cheah School of Medicine and Health Sciences, Monash University Sunway Campus, Selangor Darul Ehsan, Malaysia, 2Faculty of
Medicine, Nursing and Health Sciences, Monash University Clayton Campus, Victoria, Australia
ABSTRACT
Introduction: Emerging diseases and growing resistance to currently available antibiotics are serious threats to global public health. The evolution of pathogenic strains such as vancomycin- and methicillin-resistant Staphylococcus aureus has exposed the dire need for alternative antibiotics. Recent researches have shown evidence of snake venoms, namely Calloselasma rhodostoma (CR) and Ophiophagus hannah
(OH) exhibiting strong antibacterial activity against Staphylococcus aureus.
Objective: To isolate and identify the active antibacterial constituent and to evaluate its antibacterial activity.
Methods: Antibacterial activity was assessed against several bacterial strains including methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus aureus using a hole-plate diffusion method. Combining successive purifications with the antibacterial assay (bioassay guided purification); the active constituent was obtained through gel filtration, ion-exchange and affinity-binding chromatography. The homogeneity and identity of the enzyme was determined by gel electrophoresis and LC-MS/MS.
Results & Discussion: The active antibacterial constituent for both venoms determined by LC-MS/MS was found to be a homodimeric enzyme, L-Amino Acid Oxidase (LAAO). SDS-PAGE analysis revealed a single band with a molecular weight of approximately 65kDa and 66kDa from OH and CR, respectively. Both LAAOs showed positive inhibition of several Gram positive bacteria including MRSA with a minimum inhibitory concentration (MIC) of less than 10µg/mL, and were less effective in inhibiting most Gram negative bacterium, which required more than 20µg/mL. Past research and the MIC results proposed that both LAAOs probably attach more strongly to Gram positive bacterial surface than to Gram negative bacterial surface and are therefore more effective in inhibiting Gram positive bacterial growth inhibition.
Optimization of protein from methicillin-resistant
Staphylococcus aureus
(MRSA)
after treatment with
Quercus infectoria
for 2-D gel electrophoresis
Dayang Fredalina B.1, Radhiah K. 1, Lee Seng A.1, Mariati A.R.2 and Wan Zurinah W.N. 3
1
School of Diagnostic & Applied Health Sciences, Faculty of Health Sciences, 2Department of Clinical Oral Biology, Faculty of Dentistry,
3Department of Biochemistry, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia
ABSTRACT
Introduction: The widespread emergence of methicillin-resistant Staphylococcus aureus (MRSA) has been an important clinical and epidemiological problem in hospital environments. The appearance of vancomycin-intermediate S. aureus and more recently, vancomycin-resistant S. aureus is of further concern.
Objective:The objective of this study is to determine the optimum concentration of sample proteins from MRSA after treatment with acetone extract Quercus infectoria. Q. infectoria is an oak tree of the family Fagaceae in which its twigs develop galls (‘biji manjakani’) resulting from the deposition of eggs by
Cynips gallae tinctoriae wasp.
Methods: Comparison of the protein expression profile (PEP) between the treated MRSA and untreated strain as control was obtained using 2-dimensional gel electrophoresis. The minimum inhibitory concentration (MIC) value of acetone extract of galls from Q. infectoria against two strains of MRSA; ATCC 33591 and PPUKM clinical isolate was determined by broth micro dilution method.
Results & Discussion: MIC value of acetone extracts against both strains of MRSA was 0.3125 mg/ml compared to 0.00195 mg/ml to that of vancomycin. The optimum concentration of MRSA protein that produced the best resolution was 100 µg. Manifold technique was observed to produce a better resolution gel and greater number of spot compared with the strip holder technique. This study showed that there were seven protein spots that represented the increase in the protein expression of more than two-fold in the MRSA treated with acetone extract of galls Q. infectoria compare to the untreated group.
