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Fook Tim Chew

Publications and source records attributed to Fook Tim Chew.

15 recordsLinked to original sources
PubMed2006

Absolute quantification of gene expression in biomaterials research using real-time PCR.

One major measurement of tissue-engineered constructs efficacy and performance is determining expression levels of genes of interest at the molecular level. This measurement is commonly carried out with reverse transcription-polymerase chain reaction (RT-PCR). In this study, we described a novel method in achieving absolute quantification of gene expression using real-time PCR (aqPCR). This novel method did not require molecular cloning steps to prepare the standards for quantification comparison. Standards were linear double-stranded DNA molecules instead of the typical gene-in-plasmid format. aqPCR could also be used to give relative quantification comparisons between samples simply by dividing the copy numbers readings of the gene of interest with that of the normalization gene. RNA was extracted from monolayer and from polycaprolactone scaffold cultures and assayed for beta-actin and osteocalcin genes. We compared our aqPCR method with end-point PCR since end-point PCR is still a common means of measuring gene expression in the biomaterials field. This study showed that aqPCR was a better method to quantify gene expression than end-point PCR. With our described linear DNA standards method, we were able to obtain not only relative quantification of osteocalcin and beta-actin expression level but also actual copy numbers of osteocalcin and beta-actin for the monolayer culture and to be 1.34 x 10(4) and 1.45 x 10(7) copies, respectively and for the scaffold cultures to be 772 and 2.83 x 10(5) copies, respectively per starting total RNA mass of 10 ng. The standards curves made from these linear DNA standards showed good linearity (R(2)=0.9964 and 0.9902 for osteocalcin and beta-actin standards graphs), ranged from 10 to 10(9) copies and of comparable accuracy to current absolute quantification real-time PCR methods (which used plasmid standards obtained through molecular cloning methods). Our method might be a viable and more user-friendly alternative to current absolute quantification real-time PCR protocols.

Biocompatible Materials↗
PubMed2006

Production and proteomic characterization of pharmaceutical-grade Dermatophagoides pteronyssinus and Dermatophagoides farinae extracts for allergy vaccines.

BACKGROUND: House dust mites (HDM) such as Dermatophagoides pteronyssinus and Dermatophagoides farinae represent a major cause of type 1 allergies worldwide. Hence large quantities of well-characterized HDM extracts are needed to prepare pharmaceutical-grade allergy vaccines. To this aim, the present study was undertaken to define optimal conditions for large-scale cultures. METHODS: D. pteronyssinus and D. farinae were grown on different media combining various proportions of wheat germ, yeast and synthetic amino acids (the latter resembling the composition of the human stratum corneum). Extracts thus obtained were analyzed for their total allergenic activity, as well as major allergen and protein contents, using immunosorbent assays, HPLC, immunoblotting, two-dimensional electrophoresis and peptide mass fingerprinting. RESULTS: An optimal culture medium (Stalmite APF) based on wheat germ, yeast and amino acids in defined proportion (42, 42 and 15% w/w, respectively) was selected to grow various HDM species with high yields. A detailed proteomic analysis revealed that D. pteronyssinus extracts generated under such conditions did not contain allergens originating from culture medium components and that major prevalent HDM allergens (i.e. groups 1, 2, 7, 10, 13 and 20) are found among the most abundant proteins in the D. pteronyssinus extract. Semiquantitative dot-blot assays confirmed the presence of Der p 3-10 as well as Der p 13 and 14 allergens within the extracts. CONCLUSIONS: We developed a well-defined medium allowing to grow various HDM species at an industrial scale in a highly reproducible manner. Extracts from mites produced under such pharmaceutical conditions contain all the relevant allergens for desensitization purposes and in vivo diagnosis.

Animals↗
PubMed2006

Bla g 6: a troponin C allergen from Blattella germanica with IgE binding calcium dependence.

BACKGROUND: The known cockroach allergens do not appear to account for the full repertoire of IgE responses. OBJECTIVE: To identify and investigate the importance of other Blattella germanica allergens contributing to cockroach allergy. METHODS: A B germanica cDNA library was screened with pooled sera from patients with cockroach allergy. Three isoallergens of troponin C (Bla g 6) were cloned and expressed in Pichia pastoris. Homology modeling was performed by using Swiss-Model. IgE responses to purified allergens were simultaneously measured in 104 sera by using a fluorescent multiplex array system. The effect of calcium on IgE binding was investigated by ELISA. RESULTS: Three isoallergens, Bla g 6.0101, Bla g 6.0201, and Bla g 6.0301, were identified which share homology with insect troponin Cs and vertebrate calmodulins (61% to 78% and 42% to 44% amino acid identity, respectively) and have 2 EF-hand calcium binding domains. Molecular models of Bla g 6 showed 2 structurally homologous lobes connected by a linker that confers flexibility to the allergen. The prevalence of IgE binding to recombinant Bla g 6 was 14%. Calcium depletion by 10 mmol/L ethyleneglycol-bis-(beta-aminoethylether)-N,N,N',N'-tetraacetic acid did not significantly affect IgE binding in most cases. Interestingly, addition of 10 mmol/L CaCl2 after calcium depletion increased IgE binding by approximately 2-fold, a finding not previously reported for calcium binding allergens. CONCLUSION: Bla g 6 is a troponin allergen with a calcium dependent IgE reactivity that may be involved in muscle contraction. CLINICAL IMPLICATIONS: Bla g 6 homologous allergens may occur among other insects and cause cosensitization or allergenic cross-reactivity.

Allergens↗
PubMed2006

Nuclear magnetic resonance structure-based epitope mapping and modulation of dust mite group 13 allergen as a hypoallergen.

IgE-mediated allergic response involves cross-linking of IgE bound on mast cells by specific surface epitopes of allergens. Structural studies on IgE epitopes of allergens are essential in understanding the characteristics of an allergen and for development of specific allergen immunotherapy. We have determined the structure of a group 13 dust mite allergen from Dermatophagoides farinae, Der f 13, using nuclear magnetic resonance. Sequence comparison of Der f 13 with homologous human fatty acid-binding proteins revealed unique surface charged residues on Der f 13 that may be involved in IgE binding and allergenicity. Site-directed mutagenesis and IgE binding assays have confirmed four surface charged residues on opposite sides of the protein that are involved in IgE binding. A triple mutant of Der f 13 (E41A_K63A_K91A) has been generated and found to have significantly reduced IgE binding and histamine release in skin prick tests on patients allergenic to group 13 dust mite allergens. The triple mutant is also able to induce PBMC proliferation in allergic patients with indices similar to those of wild-type Der f 13 and shift the secretion of cytokines from a Th2 to a Th1 pattern. Mouse IgG serum raised using the triple mutant is capable to block the binding of IgE from allergic patients to wild-type Der f 13, indicating potential for the triple mutant as a hypoallergen for specific immunotherapy. Findings in this study imply the importance of surface charged residues on IgE binding and allergenicity of an allergen, as was also demonstrated in other major allergens studied.

Allergens↗
PubMed2006

A luminance- and contrast-invariant edge-similarity measure.

A novel similarity measure for edge-detection that is robust to varying luminance and contrast is presented. It incorporates a regularization term and employs directional FIR edge filters with hyperbolic tangent profiles to ensure improved noise performance and edge localization compared to classical methods.

Algorithms↗
PubMed2006

Investigating the effects of preinduction on human adipose-derived precursor cells in an athymic rat model.

The osteogenic potential of human adipose-derived precursor cells seeded on medical-grade polycaprolactone-tricalcium phosphate scaffolds was investigated in this in vivo study. Three study groups were investigated: (1) induced--stimulated with osteogenic factors only after seeding into scaffold; (2) preinduced--induced for 2 weeks before seeding into scaffolds; and (3) uninduced--cells without any introduced induction. For all groups, scaffolds were implanted subcutaneously into the dorsum of athymic rats. The scaffold/cell constructs were harvested at the end of 6 or 12 weeks and analyzed for osteogenesis. Gross morphological examination using scanning electron microscopy indicated good integration of host tissue with scaffold/cell constructs and extensive tissue infiltration into the scaffold interior. Alizarin Red histology and immunostaining showed a heightened level of mineralization and an increase in osteonectin, osteopontin, and collagen type I protein expression in both the induced and preinduced groups compared with the uninduced groups. However, no significant differences were observed in these indicators when compared between the induced and preinduced groups.

Adipose Tissue↗
PubMed2006

Characterization of osteogenically induced adipose tissue-derived precursor cells in 2-dimensional and 3-dimensional environments.

Earlier reports on a putative precursor cell population in adipose tissue showed differentiation along several mesodermal lineages, leading some to think that adipose tissue can be a source of cells applicable in regenerative medicine. However, characterizations of these adipose-derived precursor cells (ADPC) in the 3-dimensional (3-D) environment, especially within the area of bone-specific composite scaffolds, have been lacking. In this study, ADPC plated on culture flasks or seeded on medical grade polycaprolactone-tricalcium phosphate scaffolds (mPCL-CaP) were able to differentiate along the osteogenic lineages in both 2-D and 3-D environments as assessed with immunohistochemistry of osteo-related proteins, reverse transcriptase-polymerase chain reactions and alkaline phosphatase assay. The mPCL-CaP scaffolds provided adipose-derived cells (ADC) with a suitable environment as determined by DNA and metabolic assays, light, confocal and scanning electron microscopy. Flow cytometry revealed ADC to be CD29+, CD44+, CD73+, CD90+ and CD14-, CD31-, CD34-, CD45-, CD71-, and therefore showed the absence of hematopoietic stem cells but possibly the presence of pericytes and mescenchymal stem cells with osteogenic potential. The results of this study demonstrated the potential of using ADPC in combination with mPCL-CaP scaffolds for bone regenerative medicine.

Adipose Tissue↗
PubMed2004

Proteome analysis of gentisate-induced response in Pseudomonas alcaligenes NCIB 9867.

Pseudomonas alcaligenes NCIB 9867 (P25X wild-type) is capable of degrading aromatic hydrocarbons via the gentisate pathway. Biochemical characterization of P25X mutants indicated that it has isofunctional enzymes for the mono- and dioxygenase-catalyzed reactions. One set of the enzymes is constitutive whereas the other is strictly inducible. To date, only the gene encoding the constitutively-expressed gentisate dioxygenase had been cloned and characterized. A mutant strain of P25X, designated G56, which had the constitutive copy of the gentisate 1,2-dioxygenase gene interrupted by a streptomycin/spectinomycin resistance gene cassette, was found to express gentisate dioxygenase, but only when the cells were induced by gentisate. The proteome profiles of P. alcaligenes P25X and mutant G56 cells grown in the presence and absence of gentisate were compared after two-dimensional polyacrylamide gel electrophoresis. Eight distinctive protein spots (designated M1-M8) which were observed only in induced cells of strain G56 but absent in noninduced cells were further analyzed by matrix-assisted laser desorption/ionization-time of flight, quadrupole-TOF and N-terminal sequencing. Of the 15 proteins (including seven up-regulated) examined, 13 showed sequence similarities to proteins with assigned functions in other microorganisms. The identification of protein M5 which showed high homology to a gentisate dioxygenase from Ralstonia sp. U2 indicated the putative function of this protein being consistent with the inducible gentisate 1,2-dioxygenase in P. alcaligenes. In addition, the induction of stress proteins and other adaptation phenomena were also observed.

Bacterial Proteins↗
PubMed2004

Sequence tag catalogs of dust mite-expressed genomes: utility in allergen and acarologic studies.

Dust mites are a major source of indoor allergens. They contain a large number of components that react with immunoglobulin (Ig) E in individuals with allergies and are capable of inducing sensitization, and allergic respiratory and cutaneous diseases. With a significant proportion of the population affected in some way by mite allergies, it is essential that we improve our understanding of these organisms so that control strategies could be defined and its allergens better understood. Thus, we have initiated a project using the expressed sequence tagging (EST) strategy to study the major species of dust mites associated with allergic diseases, in particular, the American house dust mite, Dermatophagoides farinae, as well as Blomia tropicalis, the most prevalent mite in domestic tropical dwellings. The work has recently been expanded to include 'storage' mites such as Tyrophagus putrescentiae, Acarus siro, Lepidoglyphus destructor, Glycyphagus domesticus, Suidasia medanensis, and Aleuroglyphus ovatus. More than 50% of the initial 3000 ESTs from the D. farinae and B. tropicalis dust mites showed significant matches to known genes and were categorized into eight functional groups (such as proteins involved in metabolism, gene expression, protein synthesis, cell signaling, etc.). Of specific interest, however, were the homologs to known mite allergens, in addition to a number of sequences bearing significant homology to allergens from non-mite sources previously not known to exist in mites. The availability of these allergen sequences has facilitated their expression and subsequent characterization in our laboratory in terms of their IgE-binding reactivity. The wealth of sequence information, generated via the EST project, has also facilitated the identification of polymorphic forms of allergens, the investigation of differential gene expression under various environmental conditions via DNA microarrays, as well as the analysis of protein level expression profiling via the proteomics approach. Additionally, ESTs have also ameliorated the understanding of the phylogenetic relationships between mites, and enabled the isolation of gene products crucial for life processes so that mite control strategies can be more effectively devised. Taken together, the utilization of the EST strategy has opened up numerous new avenues by which the allergist can engage more effectively in the study of dust mites with the ultimate aim of developing appropriate treatment regimens for mite-induced allergy.

Animals↗
PubMed2002

Detection of two orchid viruses using quartz crystal microbalance (QCM) immunosensors.

Quartz crystal microbalance (QCM) immunosensors are based on the principle that adsorption of substances on the surface of a quartz crystal changes its resonance oscillation frequency. A QCM immunosensor was developed for the detection of both cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV) by pre-coating the QCMs with virus-specific antibodies. Upon binding of virions in either purified form or crude sap of infected orchids with the immobilised virus antibodies, the increase in mass at the QCM surface resulted in a reduction in the frequency of resonance oscillation in a manner dependent upon the amount of virus bound. The QCM was able to detect as low as 1 ng each of the two orchid viruses. This detection sensitivity is comparable to enzyme linked immunosorbent assay (ELISA) but the assay is faster. This immunoassay was shown to be specific, sensitive, rapid and economical, thus providing a viable alternative to virus detection methods. This is the first report using QCM immunosensors to detect plant viruses.

Antibodies, Viral↗
PubMed2002

Purification and characterization of 31-kDa palm pollen glycoprotein (Ela g Bd 31 K), which is recognized by IgE from palm pollinosis patients.

A basic glycoprotein, which was recognized by IgE from oil palm pollinosis patients, has been purified from oil palm pollen (Elaeis guineensis Jacq.), which is a strong allergen and causes severe pollinosis in Malaysia and Singapore. Soluble proteins were extracted from defatted palm pollen with both Tris-HCl buffer (pH 7.8) and Na-acetate buffer (pH 4.0). The allergenic glycoprotein was purified from the total extract to homogeneity with 0.4% yield by a combination of DEAE- and CM-cellulose, SP-HPLC, and gel filtration. The purified oil palm pollen glycoprotein with molecular mass of 31 kDa was recognized by the beta1-2 xylose specific antibody, suggesting this basic glycoprotein bears plant complex type N-glycan(s). The palm pollen basic glycoprotein, designated Ela g Bd 31 K, was recognized by IgE of palm pollinosis patients, suggesting Ela g Bd 31 K should be one of the palm pollen allergens. The preliminary structural analysis of N-glycans linked to glycoproteins of palm pollens showed that the antigenic N-glycans having alpha1-3 fucose and alpha1-2 xylose residues (GlcNAc(2 to approximately 0)Man3Xyl1Fuc(1 to approximately 0)GlcNAc2) actually occur on the palm pollen glycoproteins, in addition to the high-mannose type structures (Man(9 to approximately 5)GlcNAc2).

Allergens↗
PubMedDate not supplied

Proteomics technology and therapeutics.

1. Proteins are the workhorses of biological systems and, thus, it is not surprising that the field of proteomics has flourished in recent years. The present review briefly surveys the general proteomic approaches taken to identify drug targets and to evaluate drug efficacy and toxicity in the preclinical and clinical settings. These generally fall into three basic categories: a profiling approach, a functional approach and a structural approach. 2. The current 'tools' used and applied to the drug-discovery process will be highlighted. These include the two-dimensional gel electrophoresis, liquid chromatography, mass spectrometry, isotope-coded affinity tag and protein biochips. 3. Clearly, proteomics has begun to set a foothold in every stage of the drug-discovery process. Future developments in this area will likely make a significant impact in our quest for better, safer and cost-effective drugs.

Animals↗