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Biomedical subjects

W Tan

Publications and source records attributed to W Tan.

At least 19 recordsLinked to original sources
PubMed2002

Multiparameter flow cytometry as a tool for the detection of micrometastatic tumour cells in the sentinel lymph node procedure of patients with breast cancer.

AIM: To investigate whether multiparameter flow cytometry (MP-FCM) can be used for the detection of micrometastasis in sentinel lymph nodes (SLNs) in breast cancer. METHODS: Formalin fixed, paraffin wax embedded sentinel lymph nodes (n = 238) from 98 patients were analysed. For each lymph node, sections for haematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) for cytokeratin (MNF116) were cut at three levels with a distance of 500 microm. The intervening material was used for MP-FCM. Cells were immunostained with MNF116, followed by an incubation with fluorescein isothiocyanate (FITC) labelled goat antimouse immunoglobulin. DNA was stained using propidium iodide. From each lymph node 100,000 cells were analysed on the flow cytometer. RESULTS: Thirty eight of the 98 patients with breast carcinoma showed evidence of metastatic disease in the SLN by one ore more of the three methods. In 37 of 38 cases where metastatic cells were seen in the routine H&E and/or IHC, more than 1% cytokeratin positive cells were detected by MP-FCM. In 24 patients, metastatic foci were more than 2 mm (macrometastasis) and in 14 these foci were smaller than 2 mm (micrometastasis). In three of these 14 cases, MP-FCM revealed positive SLNs, although this was not seen at first glance in the H&E or IHC sections. After revision of the slides, one of these three remained negative. However, MP-FCM analysis of the cytokeratin positive cells showed an aneuploid DNA peak, which was almost identical to that of the primary breast tumour. Duplicate measurements, done in 41 cases, showed a 99% reproducibility. In five of 14 patients with micrometastasis, one or two metastatic foci were found in the non-SLN. However, in 15 of 24 macrometastases multiple non-SLNs were found to have metastatic tumour. All micrometastases except for the remaining negative one mentioned above showed only diploid tumour cells, despite the fact that their primary tumours contained both diploid and aneuploid tumour cells. In primary tumours with more than 60% aneuploid cells, predominantly aneuploid macrometastasis were found, whereas diploid primary tumours only showed diploid micrometastases or macrometastases in their SLN. Aneuploid SLN macrometastases were associated with non-SLN metastases in five of seven patients, whereas diploid cases showed additional non-SLN metastases in only seven of 16 patients. CONCLUSION: In all cases, MP-FCM was sufficient to detect micrometastatic tumour cells in a large volume of lymph node tissue from SLNs. In some cases it was superior to H&E and IHC staining. Approximately 30% of SLN micrometastases are accompanied by additional non-SLN metastases. The size of the aneuploid fraction (> 60%) in the primary tumour may influence the risk of having both SLN and non-SLN metastases.

Breast Neoplasms↗
PubMed2001

Molecular aptamer for real-time oncoprotein platelet-derived growth factor monitoring by fluorescence anisotropy.

Monitoring proteins in real time and in homogeneous solution has always been a difficult task. We have applied a fluorophore-labeled molecular probe based on a high-affinity platelet-derived growth factor (PDGF) aptamer for the ultrasensitive detection of PDGF in homogeneous solutions. The aptamer is labeled with fluorescein to specifically bind with the PDGF protein. Fluorescence anisotropy is used for the real-time monitoring of the binding between the aptamer and the protein. When the labeled aptamer is bound with its target protein, the rotational motion of the fluorophore attached to the complex becomes much slower because of an increased molecular weight after binding, resulting in a significant fluorescence anisotropy change. Using the anisotropy change, we are able to detect the binding events between the aptamer and the protein in real time and in homogeneous solutions (detection without separation). This assay is highly selective and ultrasensitive. It can detect PDGF in the subnanomolar range. The new method for protein detection is simple and inherits all of the advantages of molecular aptamers. Efficient oncoprotein detection using aptamer-based fluorescence anisotropy measurement will find wide applications in protein monitoring, in cancer diagnosis as well as other studies in which protein analysis is important.

Anisotropy↗
PubMed2001

Real-time monitoring of intracellular mRNA hybridization inside single living cells.

A molecular beacon, an oligonucleotide probe with inherent signal transduction mechanisms, is an optimal tool for visualizing real-time mRNA hybridization in single living cells. Each molecular beacon (MB) consists of a single-stranded DNA molecule in a stem-loop conformation with a fluorophore linked to the 5' end and a quencher at the 3' end. In this study, we demonstrate real-time monitoring of mRNA-DNA hybridization inside living cells using molecular beacons. A MB specific for beta-actin mRNA has been designed and synthesized. After microinjection into the cytoplasm of single living kangaroo rat kidney cells (PtK2 cells), the MB hybridizes with beta-actin mRNA as shown by fluorescence measurements over time. Hybridization dynamics have been followed. Strict control experiments have been carried out to confirm that the fluorescence signal increase is indeed due to the hybridization of mRNA inside single living cells. Variation in the MB/mRNA hybridization fluorescent signal has been observed for different PtK2 cells, which indicates the amount of mRNA in different cells is different. We have also monitored the beta-1 andrenergic receptor mRNA inside the PtK2 cells. These studies demonstrate the feasibility of using MBs and the ultrasensitivity achieved in our fluorescence imaging system for real-time detection of mRNA hybridization and for the visualization of oligonucleotide/mRNA interactions inside single living cells.

Actins↗
PubMed2001

Reversible glutathionylation regulates actin polymerization in A431 cells.

In response to growth factor stimulation, many mammalian cells transiently generate reactive oxygen species (ROS) that lead to the elevation of tyrosine-phosphorylated and glutathionylated proteins. While investigating EGF-induced glutathionylation in A431 cells, paradoxically we found deglutathionylation of a major 42-kDa protein identified as actin. Mass spectrometric analysis revealed that the glutathionylation site is Cys-374. Deglutathionylation of the G-actin leads to about a 6-fold increase in the rate of polymerization. In vivo studies revealed a 12% increase in F-actin content 15 min after EGF treatment, and F-actin was found in the cell periphery suggesting that in response to growth factor, actin polymerization in vivo is regulated by a reversible glutathionylation mechanism. Deglutathionylation is most likely catalyzed by glutaredoxin (thioltranferase), because Cd(II), an inhibitor of glutaredoxin, inhibits intracellular actin deglutathionylation at 2 microM comparable with its IC(50) in vitro. Moreover, mass spectral analysis showed efficient transfer of GSH from immobilized S-glutathionylated actin to glutaredoxin. Overall, this study revealed a novel physiological relevance of actin polymerization regulated by reversible glutathionylation of the penultimate cysteine mediated by growth factor stimulation.

Actins↗
PubMed2001

Conjugation of biomolecules with luminophore-doped silica nanoparticles for photostable biomarkers.

A new molecular conjugation method has been developed to label biomolecules with optically stable metalorganic luminophores, such as tris(2,2'-bipyridyl)dichlororuthenium(II) hexahydrate (Rubpy), which are otherwise not possible for direct linking with the biomolecules. Unique biochemical properties of the biomolecule can, thus, be associated with photostable luminophores. This opens a general way to conjugate desired biomolecules using a sensitive signal transduction method. It also promotes the application of excellent luminescent materials, especially those based on photostable metalorganic luminophores, in biochemical analysis and biomolecular interaction studies. The conjugation method is based on uniform luminophore-doped silica (LDS) nanoparticles (63 +/- 4 nm). These nanoparticles have been prepared using a water-in-oil (W/O) microemulsion method. The controlled hydrolysis of tetraethyl orthosilicate (TEOS) in W/O microemulsion leads to the formation of monodisperse LDS nanoparticles. The luminophores are doped inside the nanoparticles, and the particle's silica surfaces can be used to covalently bind with biomolecules. The luminophores are well-protected from the environmental oxygen when they are doped inside the silica network. As an example, we used an antibody for leukemia cell recognition. The antibody was first immobilized onto the luminophore-doped nanoparticle through silica chemistry and then was used for leukemia cell identification by an optical microscopy imaging technique. The leukemia cells were identified easily, clearly, and with high efficiency using these antibody-coated nanoparticles. The advantages of using small, uniform luminophore-doped nanoparticles are discussed.

Antibodies, Neoplasm↗
PubMed2001

Antiangiogenic potential of 10-hydroxycamptothecin.

To investigate the antiangiogenic potential of 10-hydroxycamptothecin (HCPT), the proliferation of human microvascular endothelial cells (HMEC) and seven human tumor cell lines were detected by SRB assay, and the endothelial cell migration and tube formation were assessed using two in vitro model systems. Also, inhibition of angiogenesis was determined with a modification of the chick embryo chorioallantoic membrane (CAM) assay in vivo. Morphological assessment of apoptosis was performed by fluorescence microscope. HCPT 0.313-5 micromol x L(-1) treatment resulted in a dose-dependent inhibition of proliferation, migration and tube formation in HMEC cells, and HCPT 6.25-25 nmol x egg(-1) inhibited angiogenesis in CAM assay. HCPT 1.25-5 micromol x L(-1) elicited typical morphological changes of apoptosis including condensed chromatin, nuclear fragmentation, and reduction in volume in HMEC cells. HCPT significantly inhibited angiogenesis both in vitro and in vivo at relatively low concentrations, and this effect was related with induction of apoptosis in HMEC cells. These results taken collectively suggest that HCPT may be a potent antiangiogenetic and cytotoxic drug and further investigation is warranted.

Angiogenesis Inhibitors↗
PubMed2001

Ser326Cys polymorphism in hOGG1 gene and risk of esophageal cancer in a Chinese population.

Ser326Cys polymorphism in the hOGG1 gene, which is involved in the repair of 8-hydroxyguanine in oxidatively damaged DNA, has been identified and the variant genotype appears to be related to susceptibility to certain cancers. We investigated the association between Ser326Cys polymorphism and squamous-cell carcinoma of the esophagus among a Chinese population. hOGG1 gene polymorphism was detected by PCR-based single-strand conformation polymorphism and DNA sequencing among 201 normal controls and 196 patients with esophageal cancer from Linxian, China, a high-risk area for the disease. The association between this genetic polymorphism and risk of the cancer was examined by a multivariate analysis. We found that the distribution of hOGG1 Ser326Cys genotypes among controls (Ser/Ser, 33.8%; Ser/Cys, 52.8%; and Cys/Cys, 13.4%) was significantly different from that among esophageal cancer cases (39.8%, 38.8% and 21.4%, respectively) (p < 0.05). Homozygosity for the Cys/Cys genotype significantly increased the risk of developing esophageal squamous-cell carcinoma, with the odds ratio (OR) adjusted for age, sex and smoking being 1.9 (95% confidence interval [CI] = 1.3-2.6). Although smoking alone also significantly increased esophageal cancer risk in this case-control study (adjusted OR = 2.6; 95% CI = 1.7-3.9), no significant interaction between smoking and the Cys/Cys genotype was observed in terms of risk. Our results suggest that the hOGG1 326Cys allele might play a role in the carcinogenesis of the esophagus.

Amino Acid Substitution↗
PubMed2001

Methylenetetrahydrofolate reductase polymorphisms increase risk of esophageal squamous cell carcinoma in a Chinese population.

Methylenetetrahydrofolate reductase (MTHFR) plays a central role in folate metabolism that affects DNA methylation and synthesis. Because germ-line mutations at nucleotides 677 (C-->T) and 1298 (A-->C) in the MTHFR gene cause diminished enzyme activity, and aberrant DNA methylation is oncogenic, we examined the relationship between these two MTHFR polymorphisms and susceptibility to esophageal squamous cell carcinoma (ESCC) in 240 ESCC cases and 360 age- and sex-matched controls in northern CHINA: We found that the allele frequency of MTHFR 677T was significantly higher among cases than among controls (63% versus 41%, P < 0.001). Subjects with the 677TT genotype had a more than 6-fold increased risk of developing ESCC [adjusted odds ratio (OR), 6.18; 95% confidence interval (CI), 3.32-11.51] compared with those who had the 677CC genotype. Furthermore, the elevated ESCC risk associated with the 677 polymorphism was in an allele-dose relationship (trend test, P = 0.0001) with ORs of 1.00, 3.14 (95% CI, 1.94-5.08), and 6.18 (95% CI, 3.32-11.51) for the CC, CT, and TT genotype, respectively, after adjustment for age, sex, smoking status, and the MTHFR 1298 polymorphism. The allele frequency for the MTHFR 1298C was 14% among cases and 17% among controls. The 1298CC genotype was extremely rare in both controls (1.4%) and cases (2.9%) and was also associated with an elevated risk of ESCC (adjusted OR, 4.43; 95% CI, 1.23-16.02) compared with the 1298AA genotype, whereas the 1298AC genotype had no effect on the risk of ESCC. Thus, our findings support the hypothesis that genetic polymorphisms in the MTHFR gene may contribute to susceptibility to carcinogenesis of the esophagus in the at-risk Chinese population.

Alleles↗
PubMed2001

Frequency of CYP2A6 gene deletion and its relation to risk of lung and esophageal cancer in the Chinese population.

Cytochrome P450 2A6 (CYP2A6) plays an important role in the oxidation of nicotine and in the activation of tobacco-related carcinogens, such as N-nitrosodimethylamine, N-nitrosodiethylamine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. It has been suggested that individuals with defective CYP2A6 alleles are at a lower risk of becoming smokers and of developing lung and other tobacco-related cancers. We examined the relationship between the CYP2A6 gene deletion and susceptibility to lung and esophageal cancer in a Chinese population via a hospital-based case-control study. The CYP2A6 gene deletion was determined by a PCR-based approach in 326 healthy controls, 149 patients with esophageal squamous-cell carcinoma and 151 patients with lung cancer. The allele frequency of the CYP2A6*4 deletion was 8.6% among controls compared with 8.4% among cases with esophageal squamous-cell carcinoma (p = 0.29) or 13.2% among cases with lung cancer (p < 0.01). Individuals who harbored at least one CYP2A6*4 deletion allele were at a 2-fold increased risk of developing lung cancer (95% confidence interval [CI] = 1.2-3.2) compared with those without a defective CYP2A6 allele. This effect was mainly limited to squamous-cell carcinoma and to non-smokers, although a joint effect of CYP2A6 deletion and tobacco smoking on lung cancer risk was observed among heavy smokers. The overall risk of esophageal cancer did not appear to be associated with this CYP2A6 genetic polymorphism (odds ratio [OR] = 1.2, 95% CI = 0.7-2.1). However, stratified analysis suggested an excess risk with borderline significance (OR = 2.1; 95% CI = 1.0-4.5) related to the CYP2A6*4 allele among non-smokers. The distribution of CYP2A6 genotype frequency was not significantly different (p = 0.40) between smokers (n = 174) and non-smokers (n = 152) in this study population. Our results demonstrate that the CYP2A6 gene deletion is associated with an increased risk of lung and esophageal cancer but not with a reduced tendency to smoke.

Aged↗
PubMed2001

Atomic force microscopy for the characterization of immobilized enzyme molecules on biosensor surfaces.

The development of biosensors has been one of the key areas in biotechnology and biomedical studies. Often it is difficult to investigate the immobilized biomolecules on the surfaces for biosensor optimization. Atomic force microscopy (AFM) should provide an ideal means for the visualization of biosensor surface and for the investigation of biomolecule activities. Therefore, AFM has been employed to study the surface topography of immobilized glutamate dehydrogenase (GDH) on two-dimensional glutamate biosensor surfaces. Correlation between the surface topography and the activity of the biosensor was investigated. Surface analysis has revealed that the enzymatic activity of the immobilized GDH molecules on the biosensor surface is linked to surface roughness, as measured by the peak-to-valley distance. Fractal dimension of the immobilization sensor surface was found to be a good parameter for judging the quality of the immobilized biosensors. As enzyme immobilization time increases, the biosensor has its maximum activity with around 18 h of immobilization in 10(-6) M GDH solution. Various biosensors prepared under different experimental conditions have been studied by AFM. This technique is shown to be an effective tool to characterize biosensor surfaces.

Animals↗
PubMed2001

A facile synthetic approach to prenylated flavanones: first total syntheses of (+/-)-bonannione A and (+/-)-sophoraflavanone A.

A facile and efficient approach for the syntheses of both C-8 and C-6 prenylated flavonoids has been developed that features a highly regioselective prenylation of 2,4,6-trihydroxyacetophenone and regioselective cyclization of prenylated polyhydroxy chalcones. Thus, the first efficient total syntheses of (+/-)-sophoraflavanone A (1) and (+/-)-bonannione A (2), two naturally occurring geranylated flavanones with antibacterial activities, have been achieved starting from the key intermediate 3 via regioselective cyclization of geranylated tetrahydroxychalcone 4.

Anti-Infective Agents↗
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↗