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Biomedical subjects

W Tan

Publications and source records attributed to W Tan.

At least 37 records · Page 2Linked to original sources
PubMed2001

Biochemically functionalized silica nanoparticles.

In this report, we demonstrate the biochemical modification of silica based nanoparticles. Both pure and dye-doped silica nanoparticles were prepared, and their surfaces were modified with enzymes and biocompatible chemical reagents that allow them to function as biosensors and biomarkers. The nanoparticles produced in this work are uniform in size with a 1.6% relative standard deviation. They have a pure silica surface and can thus be modified easily with many biomolecules for added biochemical functionality. Specifically, we have modified the nanoparticle surfaces with enzyme molecules (glutamate dehydrogenase (GDH) and lactate dehydrogenase (LDH)) and a biocompatible reagent for cell membrane staining. Experimental results show that the silica nanoparticles are a good biocompatible solid support for enzyme immobilization. The immobilized enzyme molecules on the nanoparticle surface have shown excellent enzymatic activity in their respective enzymatic reactions. The nanoparticle surface biochemical functionalization demonstrates the feasibility of using nanoparticles for biosensing and biomarking applications.

Biosensing Techniques↗
PubMed2001

Relationship between dopamine-stimulated phospholipid methylation and the single-carbon folate pathway.

In a previous study we demonstrated the ability of dopamine (DA) to stimulate phospholipid methylation (PLM) via a novel mechanism involving the D4 dopamine receptor (D4R) in which single-carbon folates appeared to be the primary source of methyl groups. To further understand the relationship between D4R-mediated PLM and folate metabolism, we examined the effect of several folate pathway interventions on the level of basal and DA-stimulated incorporation of [14C]-labeled formate into phospholipids in cultured SH-SY5Y neuroblastoma cells. These interventions included: (i) Overexpression of methenyltetrahydrofolate synthetase (MTHFS). (ii) Treatment with 5-formylTHF. (iii) Treatment with the MTHFS inhibitor 5-formyltetrahydrohomofolic acid (5-formylTHHF). (iv) Growth in nucleoside-free media. 31P-NMR was also used to follow DA-induced changes in cell phospholipid composition. MTHFS overexpression and 5-formylTHHF treatment, both of which lower 5-methylTHF levels, each reduced basal PLM and its stimulation by DA. In contrast, 5-formylTHF, which increases 5-methylTHF, caused a dose-dependent increase in both basal and DA-stimulated PLM. Growth in nucleoside-free media caused time-dependent changes in PLM, which were due to the absence of purine nucleosides. While basal PLM was maintained at a reduced level, DA-stimulated PLM was initially increased followed by a later decrease. Together, these findings indicate a close functional relationship between single-carbon folate metabolism and DA-stimulated PLM, consistent with a role for 5-methylTHF as the methyl donor for the D4R-mediated process.

Carbon Radioisotopes↗
PubMed2001

Evaluation of nanostructured composite collagen--chitosan matrices for tissue engineering.

The development of suitable three-dimensional matrices for the maintenance of cellular viability and differentiation is critical for applications in tissue engineering and cell biology. The structure and composition of the extracellular matrix (ECM) has been shown to modulate cell behavior with respect to shape, movement, proliferation, and differentiation. Although collagen and chitosan have separately been proposed as in vitro ECM materials, the influence of chitosan--collagen composite matrices on cell morphology, differentiation, and function is not well studied. To this end, gel matrices of different proportions of collagen and chitosan were examined ultrastructurally and characterized for their ability to regulate cellular activity. A three-chamber system with circulating hydraulic fluids was used to evaluate the gel stability under fluid force. Results indicated that overall matrix integrity increased with the proportion of chitosan. Scanning electron microscopy indicated that the addition of chitosan greatly influences ultrastructure and changes collagen fiber cross-linking, reinforcing the structure and increasing pore size. K562 cells cultured in three-dimensional gels were examined for cell proliferation and differentiation. Although cell proliferation was inhibited with an increasing proportion of chitosan, cell function based on cytokine-release was greatly augmented. Results suggest that a hybrid chitosan--collagen matrix may have potential biological and mechanical benefits for use as a cellular scaffold.

Animals↗
PubMed2001

CYP 1A1 polymorphism and risk of lung cancer in relation to tobacco smoking: a case-control study in China.

The impact of genetic polymorphisms in CYP1A1 on susceptibility to lung cancer has received particular interest in recent years since this enzyme plays a central role in activation of major classes of tobacco carcinogens. Several polymorphisms in the CYP1A1 locus have been identified and their genotypes appear to exhibit population frequencies that depend on ethnicity. We have assessed the role of CYP1A1 genotype in lung cancer risk in the Chinese population via a case-control study. Three polymorphisms, m1 (MSP:I), m2 (exon 7 Ile-->Val) and m4 (exon 7 Thr-->Asn), were determined by PCR-RFLP in 404 controls and 217 lung cancer cases. While no polymorphic alleles were detectable in the m4 site among our study subjects, the allele frequencies for CYP1A1 m1 and CYP1A1 m2 were found to be 35.6 and 25.6% among controls, compared with 42.6 and 34.2% among cases. Multivariate analysis showed an elevated risk for lung cancer in subjects having at least one m1 allele [odds ratio (OR) = 2.0, 95% confidence interval (CI) = 1.4-2.8] or having at least one m2 allele (OR = 1.9, 95% CI = 1.3-2.7). However, this increased risk was limited to squamous cell carcinoma (SCC), but not adenocarcinoma or other histological types of lung cancer. Stratified analysis indicated a multiplicative interaction between tobacco smoking and variant CYP1A1 m1 genotypes on the risk of SCC. The ORs of SCC for the variant CYP1A1 m1 genotype, tobacco smoking and both factors combined were 2.8, 9.1 and 29.9, respectively. When the data was stratified by the pack-year values, this joint effect was consistent and stronger among the heaviest smokers. The interaction between tobacco smoking and the variant CYP1A1 m2 genotypes followed the same pattern. Our findings support the conclusion that CYP1A1 m1 and CYP1A1 m2 polymorphisms are associated with smoking-related lung cancer risk in Chinese.

Adenocarcinoma↗
PubMed2001

Development of novel dye-doped silica nanoparticles for biomarker application.

We report the development of novel luminescent nanoparticles composed of inorganic luminescent dye, Tris(2,2'-bipyridyl) dichlororuthenium (II) hexahydrate, doped inside a silica network. These dye doped silica (DDS) nanoparticles have been synthesized using a water-in-oil microemulsion technique in which controlled hydrolysis of the tetraethyl orthosilicate leads to the formation of monodispersed nanoparticles. They are prepared with a variety of sizes: small (5+/-1 nm), medium (63+/-4 nm), and large (400+/-10 nm), which shows the efficiency of the microemulsion technique for the synthesis of uniform nanoparticles. All these nanoparticles are suitable for biomarker application since they are much smaller than cellular dimension. These nanoparticles are highly photostable in comparison to most commonly used organic dyes. These nanoparticles have been characterized by various microscopic and spectroscopic techniques. The amount of dye content in these nanoparticles has been optimized to eliminate self-quenching. It has been observed that maximum luminescence intensity is achieved when the dye content is around 20 wt%. Silica surface of DDS nanoparticles is available for surface modification and bioconjunction. For demonstration as a biomarker, the DDS nanoparticle's surface has been biochemically modified to attach membrane-anchoring groups and applied successfully to stain human leukemia cells.

2,2'-Dipyridyl↗
PubMed2001

Ultrasensitive optical DNA biosensor based on surface immobilization of molecular beacon by a bridge structure.

A novel biotinylated molecular beacon (MB) probe was developed to prepare a DNA biosensor using a bridge structure. MB was biotinylated at the quencher side of the stem and linked on a biotinylated glass cover slip through streptavidin, which acted as a bridge between MB and glass matrix. An efficient fluorescence microscope system was constructed to detect the fluorescence change caused by the conformation change of MB in the presence of complementary DNA target. The proposed biosensor was used to directly detect, in real-time, the target DNA molecules. The bridge immobilization method caused the proposed DNA biosensor to have a faster and more stable response. Under the optimal conditions, the newly developed DNA biosensor showed a linear response toward ssDNA in the range of 5-100 nM with a detection limit of 2 nM. It was interesting to note that the described biosensor was reproducible after being regenerated by urea.

Biosensing Techniques↗
PubMed2001

[Detection of malondialdehyde-DNA adduct level by 32P-postlabeling assay in normal human esophageal epithelium and esophageal squamous cell carcinoma].

OBJECTIVE: To study whether the main malondialdehyde-DNA adduct (M1-dG) produced by lipid peroxidation is involved in the carcinogenesis of esophagus. METHODS: DNA samples were isolated from normal esophageal epithelium (n = 32) obtained by biopsy and esophageal squamous cell carcinoma specimens (n = 30) obtained by surgery. All tissue samples came from individuals living in Linxian, Henan, a high-risk area of esophageal cancer. Contents of M1-dG adducts were detected by 32P-postlabeling method. RESULTS: M1-dG adducts were detectable both in the normal and cancerous tissue samples. However, normal esophageal epithelial tissues exhibited significantly lower levels of M1-dG adducts (median 3.4, range 1.7/10(8)-55.4/10(8) nucleotides) than those found in esophageal cancer tissues (median 14.1, range 1.4/10(8)-59.0/10(8) nucleotides, P < 0.0001). The adduct levels were neither associated with gender, age, tobacco smoking status or genetic polymorphism in the CYP2E1, an enzyme participating in the oxidation of ethanol to form reactive free radicals. CONCLUSIONS: Our findings provide evidence that DNA damage, resulted from lipid peroxidation, can accumulate in the normal human esophageal tissue and reach relatively high level in cancer tissue which suggests that M1-dG adducts may be involved in the initiation and progression of cancer with its mutagenic and carcinogenic effects.

Carcinoma, Squamous Cell↗
PubMed2000

Natural animal coloration can Be determined by a nonfluorescent green fluorescent protein homolog.

It is generally accepted that the colors displayed by living organisms are determined by low molecular weight pigments or chromoproteins that require a prosthetic group. The exception to this rule is green fluorescent protein (GFP) from Aequorea victoria that forms a fluorophore by self-catalyzed protein backbone modification. Here we found a naturally nonfluorescent homolog of GFP to determine strong purple coloration of tentacles in the sea anemone Anemonia sulcata. Under certain conditions, this novel chromoprotein produces a trace amount of red fluorescence (emission lambda(max) = 595 nm). The fluorescence demonstrates unique behavior: its intensity increases in the presence of green light but is inhibited by blue light. The quantum yield of fluorescence can be enhanced dramatically by single amino acid replacement, which probably restores the ancestral fluorescent state of the protein. Other fluorescent variants of the novel protein have emission peaks that are red-shifted up to 610 nm. They demonstrate that long wavelength fluorescence is attainable in GFP-like fluorescent proteins.

Amino Acid Sequence↗
PubMed2000

Molecular beacons for DNA biosensors with micrometer to submicrometer dimensions.

Ultrasensitive molecular beacon (MB) DNA biosensors, with micrometer to submicrometer sizes, have been developed for DNA/RNA analysis. The fluorescence-based biosensors have been applied in DNA/ RNA detection without the need for a dye-labeled target molecule or an intercalation reagent in the testing solution. Molecular beacons are hairpin-shaped oligonucleotides that report the presence of specific nucleic acids. We have designed a surface-immobilizable biotinylated ssDNA molecular beacon for DNA hybridization at a liquid-solid interface. The MBs have been immobilized onto ultrasmall optical fiber probes through avidin-biotin binding. The MB DNA biosensor has been used directly to detect, in real time, its target DNA molecules without the need for a competitive assay. The biosensor is stable and reproducible. The MB DNA biosensor has selectivity with single base-pair mismatch identification capability. The concentration detection limits and mass detection limits are 0.3 nM and 15 amol for a 105-microm biosensor, and 10 nM and 0.27 amol for a submicrometer biosensor, respectively. We have also prepared molecular beacon DNA biosensor arrays for simultaneous analysis of multiple DNA sequences in the same solution. The newly developed DNA biosensors have been used for the precise quantification of a specific rat gamma-actin mRNA sequence amplified by the polymerase chain reaction.

Actins↗
PubMed2000

Using molecular beacons to probe molecular interactions between lactate dehydrogenase and single-stranded DNA.

The interactions between two key macromolecular species, nucleic acids and proteins, control many important biological processes. There have been limited effective methodologies to study these interactions in real time. In this work, we have applied a newly developed molecular beacon (MB) DNA probe for the analysis of an enzyme, lactate dehydrogenase (LDH), and for the investigation of its properties of binding with single-stranded DNA. Molecular beacons are single-stranded oligonucleotide probes designed to report the presence of specific complementary nucleic acids by fluorescence detection. The interaction between LDH and MB has resulted in a significant fluorescence signal enhancement, which is used for the elucidation of MB/LDH binding properties. The processes of binding between MB and different isoenzymes of LDH have been studied. The results show that the stoichiometry of LDH-5/MB binding is 1:1, and the binding constant is 1.9 x 10(-7) M(-1). We have also studied salt effects, binding sites, temperature effects, pH effects, and the binding specificities for different isoenzymes. Our results demonstrate that MB can be effectively used for sensitive protein quantitation and for efficient protein-DNA interaction studies. MB has a signal transduction mechanism built within the molecule and can thus be used for the development of rapid protein assays and for real-time measurements.

DNA, Single-Stranded↗
PubMed2000

Using molecular beacons as a sensitive fluorescence assay for enzymatic cleavage of single-stranded DNA.

Traditional methods to assay enzymatic cleavage of DNA are discontinuous and time consuming. In contrast, recently developed fluorescence methods are continuous and convenient. However, no fluorescence method has been developed for single-stranded DNA digestion. Here we introduce a novel method, based on molecular beacons, to assay single-stranded DNA cleavage by single strand-specific nucleases. A molecular beacon, a hairpin-shaped DNA probe labeled with a fluorophore and a quencher, is used as the substrate and enzymatic cleavage leads to fluorescence enhancement in the molecular beacon. This method permits real time detection of DNA cleavage and makes it easy to characterize the activity of DNA nucleases and to study the steady-state cleavage reaction kinetics. The excellent sensitivity, reproducibility and convenience will enable molecular beacons to be widely useful for the study of single-stranded DNA cleaving reactions.

DNA Probes↗
PubMed2000

Molecular beacons: a novel DNA probe for nucleic acid and protein studies.

A new concept has been introduced for molecular beacon DNA molecules. Molecular beacons are a new class of oligonucleotides that can report the presence of specific nucleic acids in both homogeneous solutions and at the liquid-solid interface. They emit an intense fluorescent signal only when hybridized to their target DNA or RNA molecules. Biotinylated molecular beacons have been designed and used for the development of ultrasensitive DNA sensors and for DNA molecular interaction studies at a solid-liquid interface. Molecular beacons have also been used to study protein-DNA interactions. They have provided a variety of exciting opportunities in DNA/RNA/protein studies.

Animals↗
PubMed2000

Direct observation of single-molecule generation at a solid-liquid interface

Direct observation of single-molecule generation from a chemical reaction was achieved at a solid-liquid interface. The reaction between fluorescamine and immobilized N'-(3-trimethoxysilylpropyl)diethylenetriamine (DETA) was studied at the single-molecule level. Time-lapse fluorescence images of single-molecule products, excited by the evanescent field generated at a quartz-liquid interface, were recorded to follow the chemical reaction to its completion. The reactions were restricted to the approximately 1 nm thick layer nearest to the interface. Analysis of the photoelectron intensity of the fluorescent product of the reaction and its distribution shows that the reaction kinetics goes through a transition from zeroth-order to first-order as the reaction proceeds. This approach offered a novel means to study single-molecule reactions at the solid-liquid interface. It also enabled the investigation of reaction kinetics and chemical mapping of surface heterogeneity at the single-molecule level.

Journal Article↗
PubMed2000

Herpes simplex viral and amplicon vector-mediated gene transfer into glia and neurons in organotypic spinal cord and dorsal root ganglion cultures.

The progression of neurodegenerative diseases and secondary consequences of spinal cord injury may be diminished by introducing transgenes to glia, spinal neurons, and/or sensory neurons. Organotypic cultures of spinal cord slices and dorsal root ganglia proved to be an excellent system in which to compare the relative neurotropism of a replication-defective recombinant herpes simplex virus and herpes virus-derived amplicon vectors. Hundreds of beta-galactosidase-expressing cells, transduced by the viral vectors, were observed in spinal cord slices 3 and 8 days postinfection. Immunostaining to identify the infected cell type indicated that oligodendrocytes were permissive for viral vector transduction of beta-galactosidase in the spinal cord slice, whereas neurons were not. Heparan sulfate proteoglycan, the initial receptor for herpes contact with cells, was highly expressed in the white matter of the spinal cord slice, but was negligible in the gray matter. In contrast to the spinal cord, many fewer cells were infected in the dorsal root ganglia (DRG) by these vectors, but a majority of infected cells were identified as sensory neurons. Heparan sulfate proteoglycan expression was abundant in the sensory fibers emanating from the DRG and also surrounded each neuron within the ganglion. Our results demonstrate HSV-induced transgene expression that is amenable to ex vivo assessment of its physiological impact.

Animals↗
PubMed2000

Attachment kinetics of Vero cells onto CT-3 microcarriers.

The effects of cell seeding density, microcarrier concentration, agitation speed and age of seeding cells on the rate of cell attachment to the surface of CT-3 microcarriers were investigated. It was shown that the attachment followed first-order kinetics. When either the cell seeding density or the microcarrier concentration was increased, the kinetic constant increased due to an increase in the probability of collision between cells and microcarriers. However, at higher microcarrier concentrations, the increase in the kinetic constant with increasing microcarrier concentration was not significant. Cell attachment was decelerated upon increasing the agitation speed because of the shorter cell-microcarrier contact time. In addition, it was also demonstrated that cell attachment occurred more efficiently when seeding cells from the middle or early exponential growth phase were used. The process of cell attachment onto CT-3 microcarriers was investigated, and our results showed that the attachment stage was the rate-limiting step. These results will facilitate the optimization of the Vero cell culture process.

Journal Article↗
PubMed2000

The mRNA export in Caenorhabditis elegans is mediated by Ce-NXF-1, an ortholog of human TAP/NXF and Saccharomyces cerevisiae Mex67p.

Human TAP and Saccharomyces cerevisiae Mex67p belong to a family of proteins that mediate mRNA export. Computer searches identified previously two Caenorhabditis elegans genes, C15H11.3 and C115H11.6, that encode putative homologs of hTAP and Mex67p (Segref et al., EMBO J, 1997, 16:3256-3271). Using RNA interference experiments in C. elegans, we found that functional knockout of C15H11.3 resulted in nuclear accumulation of poly(A)-containing RNAs and was lethal for both embryos and adult nematodes. No embryonic or progeny abnormality was observed in functional knockout of C15H11.6. Taken together, these data established that the C15H11.3 gene product is an ortholog of hTAP and Mex67p; thus, it was named Ce-NXF-1. Ce-NXF-1 binds RNA directly and is a nucleocytoplasmic shuttle protein accumulating in the nucleoplasm and at the nuclear rim. The rim association is mediated via unique signals present in the C-terminal portion of all TAP/NXF and Mex67p proteins. This region was shown to interact with the FG-repeat domains of nucleoporins Nup98, Nup153, and Nup214, indicating that the rim association occurs through components of the nuclear pore complex. In summary, Ce-NXF-1 belongs together with hTAP and Mex67p to a family of proteins that participate in mRNA export and can provide a direct molecular link between mRNAs and components of the nuclear pore complex. Therefore, despite differences in mRNA metabolism between these species, they utilize a conserved mRNA transport mechanism.

ATP Binding Cassette Transporter, Subfamily B, Mem↗