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Biomedical subjects

W Tan

Publications and source records attributed to W Tan.

At least 55 records · Page 3Linked to original sources
PubMed2000

The prevalence of BRCA1 mutations in Chinese patients with early onset breast cancer and affected relatives.

The purpose of this study was to determine the prevalence of BRCA1 mutations in Chinese breast cancer patients in Singapore. BRCA1 analysis was conducted in consecutive patients with breast cancer before the age of 40 years (76 women), or whose relatives had breast or ovarian cancer (16 women). Ten patients had both early onset breast cancer and affected relatives. Genomic DNA from peripheral mononuclear blood cells was studied by using the protein transcription-translation assay (exon 11) and single-strand conformational polymorphism, with subsequent DNA sequencing. All six disease-causing mutations occurred in women under 40 years (8.6%) with three occurring in patients under 35 years (three out of 22 patients, 13.6%). Mis-sense mutations of unknown significance were found in three patients. Two of the ten women with affected relatives under 40 years had BRCA1 mutations. The prevalence of BRCA1 mutations in Chinese patients with early onset breast cancer is similar to that observed in Caucasian women. Most Chinese patients with affected relatives were not carriers of BRCA1 mutations.

Adult↗
PubMed2000

Human CYP1B1 Leu432Val gene polymorphism: ethnic distribution in African-Americans, Caucasians and Chinese; oestradiol hydroxylase activity; and distribution in prostate cancer cases and controls.

Cytochrome P4501B1 (CYP1B1) is involved in the activation of many carcinogens and in the metabolism of steroid hormones, including 17beta-oestradiol (E2) and testosterone. We report a significant difference in the allele frequencies of two point mutations in the coding region of the CYP1B1 gene among Caucasian (n = 189), African-American (n = 52) and Chinese (Linxian) (n = 109) populations. A (C to G) transversion at position 1666 in exon 3, which results in an amino acid substitution of Leu432 to Val, was present in African-Americans with an allele frequency for Va1432 of 0.75, in Caucasians of 0.43, and in Chinese of 0.17. A (C to T) transition at position 1719 in exon 3, with no amino acid change (Asp449), appeared to be closely linked with the Val432 variant. Results using human lung microsomal preparations from individuals with the CYP1B1Val/Val and CYP1B1Leu/Leu genotypes indicate that Val432 variant may be a high activity allele and thus may contribute to the interindividual differences in CYP1B1 activity. Because CYP1B1 is involved in hormone and carcinogen metabolism, and given the disparate rates of prostate cancer among ethnic groups, we also evaluated the association of the CYP1B1 Leu432Val polymorphism with prostate cancer risk in a pilot case-control study. Among Caucasians, 34% of men with cancer (n = 50) were homozygous for the Val432 polymorphism, while only 12% of matched control subjects (n = 50) had this genotype. These preliminary data indicate that genetic polymorphisms in CYP1B1 might play an important role in human prostate carcinogenesis.

Adult↗
PubMed2000

Localized exocytosis detected by spatially resolved amperometry in single pancreatic beta-cells.

Spatially resolved measurements of exocytosis in pancreatic beta-cells were made using amperometry with 1-microm radius electrodes. These measurements revealed that certain portions of a cell actively undergo exocytosis following stimulation with depolarizing agents, but other regions are inactive. The amperometric measurements were compared to measurements made with the membrane indicator dye, FM1-43, which showed uneven increases in fluorescence around the surface of the cell, with amperometric secretion being detected only at the brightest regions. In some instances, a large number of exocytotic events were detected from one electrode position. The number of events was larger than what would be expected based on the number of vesicles that could fit under an electrode of the dimensions used. These results suggest a mechanism of vesicle traffic that allows multiple fusions at a small membrane area.

Animals↗
PubMed2000

Impact of genetic polymorphisms in cytochrome P450 2E1 and glutathione S-transferases M1, T1, and P1 on susceptibility to esophageal cancer among high-risk individuals in China.

Esophageal cancer, which is prevalent in China, is believed to be induced by environmental carcinogens such as nitrosamines and other agents. The disproportionate geographical distribution of this cancer among individuals suggests a role for gene-environment interactions in developing the disease. We have shown in our preliminary study that a genetic polymorphism in cytochrome P450 2E1 (CYP2E1) that is known to activate nitrosamines may be a susceptibility factor involved in the early events leading to the development of esophageal cancer (Lin et al., Cancer Epidemiol. Biomark. Prev., 7: 1013-1018, 1998). This relatively larger study was conducted to compare the results with our previous findings. One hundred and fifty cases with esophageal cancer, 146 cases with esophageal dysplasia, and 150 normal controls were residents of Linxian, China, a high-risk area. Genomic DNA samples were assayed for restriction fragment length polymorphisms in the CYP2E1 and GSTP1 loci by PCR amplification followed by digestion with RsaI and Alw26I, respectively. Deletion of the GSTM1 and GSTT1 genes was detected by multiplex PCR. The distribution of CYP2E1 c1/c1 allele frequency was found to be significantly different between controls (44.0%) and cases with cancer (71.3%) or cases with dysplasia (70.6%; P < 0.0001). Individuals having the c1/c1 genotype were at a 3.1-fold [95% confidence interval (CI), 2.4-3.9] increased risk of developing dysplasia and a 3.2-fold (95% CI, 2.5-4.1) increased risk of developing squamous cell carcinoma of the esophagus. Although polymorphisms in the GSTT1 and GSTP1 were not significantly different between cases with cancer or cases with dysplasia and controls, the frequency of the GSTM1 non-null (+/+ and +/0) genotypes appeared to be overrepresented in cases with cancer compared with controls (odds ratio, 2.3; 95% CI, 1.8-3.0). Furthermore, a joint effect of the CYP2E1 c1/c1 genotype and GSTM1 non-null genotype on the cancer risk was observed, showing an odds ratio of 8.5 (95% CI, 3.7-19.9). These results demonstrate that CYP2E1 and perhaps GSTM1 are genetic determinants in the development of squamous cell carcinoma of the esophagus.

Carcinoma, Squamous Cell↗
PubMed2000

[Genetic polymorphism in hOGG1 and susceptibility to esophageal cancer in Chinese].

OBJECTIVE: To examine the association between Ser326Cys polymorphism in hOGG1 gene, which is involved in the repair of 8-hydroxyguanine in damaged DNA, and investigate the risk of squamous cell carcinoma of the esophagus in Chinese. METHODS: Ser326Cys polymorphism in hOGG1 gene was determined by PCR-SSCP approach among 201 normal controls and 196 patients with squamous cell carcinoma of the esophagus. The association between this genetic polymorphism and the risk of the cancer was examined by a multivariate analysis. RESULTS: The Cys/Cys genotype of hOGG1 was found in 21.4% of patients with the cancer and in 13.4% of controls (P<0.05). Homozygosity for the Cys/Cys genotype significantly increased the risk of developing esophageal cancer, with the odds ratio adjusted for age, sex and smoking being 1.9(95% CI 1.3-2.6). Smoking also significantly increased esophageal cancer risk in this case-control study (adjusted OR 2.6; 95% CI 1.7- 3.9). No interaction between smoking and Cys/Cys genotype was observed for the risk of esophageal cancer. CONCLUSION: Polymorphism of hOGG1 Ser326Cys may play a role in esophageal carcinogenesis.

Adult↗
PubMed2000

[Susceptibility to lung cancer in Chinese is associated with genetic polymorphism in cytochrome P4502E1].

OBJECTIVE: To investigate the genetic polymorphism of cytochrome P4502E1 (CYP2E1) as a susceptibility factor for lung cancer in Chinese population. METHODS: The genotype frequencies of RsaI restriction fragment length polymorphisms were analyzed in 92 lung cancer cases and 137 frequency-matched normal controls. RESULTS: The CYP2E1 c1/c1 genotype was found in 72.8% of lung cancer cases, which was significantly higher (P < 0.01) than that in controls (54.7%). By multivariate analysis, this genotype was found to be associated with a 2.5-fold increased risk of lung cancer (adjusted OR 2.5, 95% CI 1.8-3.8). Stratified analysis suggested an interaction between CYP2E1 c1/c1 genotype and cigarette smoking. The odds ratio (OR) for the CYP2E1 c1/c1 genotype, cigarette smoking and both factors combined was 3.9 (95% CI 1.1-15.2), 4.1 (95% CI 1.1-15.9) and 7.9 (95% CI 2.4-29.4), respectively. The interaction between CYP2E1 c1/c1 genotype and pack-years of cigarette smoking followed the same pattern, with a markedly increased risk in the heaviest smokers. CONCLUSION: The CYP2E1 c1/c1 genotype is a susceptibility factor for development of lung cancer in Chinese and there is an apparent gene-environment interaction between the susceptible genotype and cigarette smoking.

Aged↗
PubMed2000

[Effects of IFN-alpha combined with IL-6 on cell growth and related genes expression and apoptosis of bone marrow cells from CGL patients].

OBJECTIVE: To investigate the effects of interferon-alpha (IFN-alpha) and IFN-alpha combined with interleukin-6 (IL-6) on cell growth and bcr/abl, bcl-2 and c-myc genes expression in the bone marrow mononuclear cells (MNC) from chronic granulocytic leukemia (CGL) patients. METHODS: MNCs were cultured in liquid medium at the presence of IFN-alpha (200 U/ml) or IFN-alpha (200 U/ml) plus IL-6 (100 ng/ml). The viable cells were counted and the expression levels of beta-actin, bcr/abl, bcl-2 and c-myc genes were quantitatively detected by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: The cell growth was markedly inhibited by IFN-alpha, but the extent of the inhibition was slightly decreased when IFN-alpha combined with IL-6. The expression levels of bcr/abl and bcl-2 gene were reduced by IFN-alpha or IFN-alpha plus IL-6. The expression of c-myc gene was inhibited by IFN-alpha but promoted by IL-6. CONCLUSIONS: Both IFN-alpha and IFN-alpha plus IL-6 can inhibit the expression of anti-apoptosis genes, and modulate the expression of c-myc. It is the possible mechanism of IFN-alpha therapy for CGL in chronic phase.

Adolescent↗
PubMed1999

A fiber-optic evanescent wave DNA biosensor based on novel molecular beacons.

We have prepared a novel optical fiber evanescent wave DNA biosensor using a newly developed molecular beacon DNA probe. The molecular beacons (MB) are oligonucleotide probes that become fluorescent upon hybridization with target DNA/RNA molecules. Biotinylated MBs have been designed and immobilized on an optical fiber core surface via biotin-avidin or biotin-streptavidin interactions. The DNA sensor based on a MB does not need labeled analyte or intercalation reagents. It can be used to directly detect, in real-time, target DNA/RNA molecules without using competitive assays. The sensor is rapid, stable, highly selective, and reproducible. We have studied the hybridization kinetics of the immobilized MB by changing the ionic strength of the hybridization solution and target DNA concentration. Our result shows divalent cations play a more important role than monovalent cations in stabilizing the MB stem hybrids and in accelerating the hybridization reaction with target DNA/RNA molecules. The concentration detection limit of the MB evanescent wave biosensor is 1.1 nM. The MB DNA biosensor has been applied to the analysis of specific gamma-actin mRNA sequences amplified by polymerase chain reaction.

Actins↗
PubMed1999

Probing intracellular dynamics in living cells with near-field optics.

Near-field optics (NFO) overcomes the diffraction limit of light microscopes and permits visualization of single molecules. However, despite numerous applications of NFO in the physical sciences, there is still a paucity of applications in the neurosciences. In this work, the authors have developed NFO probes to image intracellular dynamic processes in living cells. This is the first time a NFO probe has been inserted inside a living cell to deliver light to a spatially controlled region for optical measurements and to record cellular responses to external stimuli. Two different optical detection systems (CCD camera and avalanche photon detection) were developed to monitor cellular responses to drug administration in two different cell types. NG108-15 neuroblastoma cells and vascular smooth muscle cells (VSMC) were penetrated with NFO probes. Intracellular Ca2+ increases post drug stimulation were detected by NFO probes. The cells were loaded with either fura-2/AM or fluo-3/AM calcium dyes. VSMC were stimulated with angiotensin II, resulting in a precise area of intracellular Ca2+ increase. Different response profiles of Ca2+ increases were observed after ionomycin and bradykinin administration in NG108-15 cells. Responsive heterogeneities due to ionomycin among different cells of the same type were recorded. The results show that NFO probes make possible real-time visualization of intracellular events. With refinement, intracellular NFO probes offer the potential of probing cell function with fast temporal and excellent spatial resolutions.

Angiotensin II↗
PubMed1999

Automated detection of Salmonella spp. in foods.

An automated method to detect salmonellae in foods was developed and tested in food samples intentionally contaminated with the test organisms. Liquid eggs, shell eggs, dry eggs, skim milk and chicken were spiked with Salmonella enteritidis, S. typhimurium or S. newport to yield 2 to 25 CFU per 25 g or ml of sample. Following pre-enrichment in universal pre-enrichment broth at 42 degrees C for 6 h (eggs and milk) or 16 h (chicken), Salmonella cells were captured by immunomagnetic beads coated with Salmonella antibody (Vicam, Watertown, MA). The beads were transferred to selective liquid media containing carbohydrate (dulcitol or xylose), amino acid (lysine or ornithine), and H2S indicator, and incubated at 42 degrees C in the BioSys instrument (MicroSys, Ann Arbor, MI). Salmonella positive samples were identified by black discoloration of the media during incubation, while negative samples remained colorless. These color changes were recorded by the instrument. All the artificially contaminated samples tested positive within 15-18 h, while control samples remained negative during 24 h incubation. The results agreed with standard identification procedures. A total of 24 h was required to detect 2 to 25 CFU of the pathogen in 25 g or ml of eggs and milk, and up to 36 h in chicken, compared to 72 h in the standard methods.

Animals↗
PubMed1999

D4 dopamine receptor-mediated phospholipid methylation and its implications for mental illnesses such as schizophrenia.

Previous studies have shown D2-like dopamine receptor involvement in the regulation of phospholipid methylation (PLM), while others have documented impaired methionine and folate metabolism in schizophrenia. Utilizing [14C]formate labeling in cultured neuroblastoma cell lines, we now show that D4 dopamine receptors (D4R) mediate the stimulatory effect of dopamine (DA) on PLM. The effect of DA was potently blocked by highly D4R-selective antagonists and stimulated by the D4R-selective agonist CP-226269. DA-stimulated PLM was dependent upon the activity of methionine cycle enzymes, but DA failed to increase PLM in [3H]methionine labeling studies, indicating that a methionine residue in the D4R might be involved in mediating PLM. A direct role for MET313, located on transmembrane helix No. 6 immediately adjacent to phospholipid headgroups, was further suggested from adenosylation, site-directed mutagenesis and GTP-binding results. A comparison of PLM in lymphocytes from schizophrenia patients vs control samples showed a four-fold lower activity in the schizophrenia group. These findings reveal a novel mechanism by which the D4R can regulate membrane composition. Abnormalities in D4R-mediated PLM may be important in psychiatric illnesses such as schizophrenia.

Amino Acid Sequence↗
PubMed1999

Single cell studies of enzymatic hydrolysis of a tetramethylrhodamine labeled triglucoside in yeast.

Several hundred molecules of enzyme reaction products were detected in a single spheroplast from yeast cells incubated with a tetramethylrhodamine (TMR) labeled triglucoside, alpha-d-Glc(1-->2)alpha-d-Glc(1-->3)alpha-d-Glc-O(CH2)8CONHCH2- CH2NH- COTMR. Product detection was accomplished using capillary electrophoresis and laser induced fluorescence following the introduction of a single spheroplast into the separation capillary. The in vivo enzymatic hydrolysis of the TMR-trisaccharide involves at least two enzymes, limited by processing alpha-glucosidase I, producing TMR-disaccharide, TMR-monosaccharide, and the free TMR-linking arm. Hydrolysis was reduced by preincubation of the cells with the processing enzyme inhibitor castanospermine. Confocal laser scanning microscopy studies confirmed the uptake and internalization of fluorescent substrate. This single cell analysis methodology can be applied for the in vivo assay of any enzyme with a fluorescent substrate.

Cytological Techniques↗
PubMed1999

Identification of novel import and export signals of human TAP, the protein that binds to the constitutive transport element of the type D retrovirus mRNAs.

The nuclear export of the unspliced type D retrovirus mRNA depends on the cis-acting constitutive transport RNA element (CTE) that has been shown to interact with the human TAP (hTAP) protein promoting the export of the CTE-containing mRNAs. We report here that hTAP is a 619-amino-acid protein extending the previously identified protein by another 60 residues at the N terminus and that hTAP shares high homology with the predicted rat and mouse TAP proteins. We found that hTAP is a nuclear protein that accumulates in the nuclear rim and the nucleoplasm. We further demonstrated that hTAP is able to shuttle between the nucleus and the cytoplasm. Identification of the signals responsible for nuclear import (NLS) and export (NES) revealed that they are distinct but partially overlapping. NLS and NES of hTAP are active transferable signals that do not share similarities with known elements. The C-terminal portion contributes further to hTAP's nuclear retention and contains a signal(s) for nuclear rim association. Taken together, our data show that hTAP is a dynamic protein capable of bidirectional trafficking across the nuclear envelope. These data further support hTAP's role as an export factor of the CTE-containing mRNAs.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Expression and purification of a secreted functional mouse/human chimaeric antibody against bacterial endotoxin in baculovirus-infected insect cells.

We have created a mouse/human chimaeric antibody by taking antigen-binding fragment (Fab) genes of a mouse antibody-producing hybridoma with specificity for bacterial endotoxin and joining them to human Ig crystallizing-fragment (Fc) genes using recombinant DNA techniques. This chimaeric antibody has been expressed in Sf21 and High Fivetrade mark (BTI-TN-5B1-4) insect cells using the baculovirus expression system, which may allow the mass production of secretory recombinant antibodies. This was achieved by using infection with a double-recombinant virus containing cDNAs of both the Ig heavy-chain (HC) and light-chain (LC) genes. Prior to recombination, each gene was cloned into the dual-expression baculovirus transfer vector pPLSP2, which permitted the insertion of the LC gene in-frame with the signal peptide of honey bee melittin downstream of the polyhedrin promoter, and of the HC gene in-frame with the signal peptide of Bombyx mori larval serum protein downstream of the p10 promoter. Our results showed that the polypeptide chains were secreted by insect cells and correctly assembled into H(2)L(2) heterodimers containing N-linked carbohydrate at the heavy chain. Furthermore, the recombinant chimaeric antibody exhibited a similar antigen specificity to that of the monoclonal antibody. More importantly, it provides a generic method for the high-level expression of antibodies.

Amino Acid Sequence↗
PubMed1999

Influence of FL on ex vivo expansion of hematopoietic cells from cord blood in long-term liquid cultures.

The use of umbilical cord blood for stem cell transplantation has numerous advantages, but a major deficiency is the rather low cell number available. Therefore, ex vivo expansion were proposed to overcome this limitation. In this paper the effects of flt-3 ligand (FL), stem cell factor (SCF), interleukin(IL)-3, IL-6, granulocyte colony-stimulating factor (G-CSF) and granulocyte- monocyte colony-stimulating factor (GM-CSF) on long-term ex vivo expansion and differentiation of cord blood hematopoietic cells were investigated. In the culture containing FL + SCF + G-CSF + GM-CSF, the total cell expansion ratio reached the maximum (385.30 +/- 163.51-fold) at 28 days, whereas in the culture with FL + SCF + IL-3 + IL-6, CFU-GMs expansion ratio reached a plateau (409.52 +/- 189.50-fold) at 28 days. FL synergized with SCF and other cytokines. In all the conditions investigated, cultures with FL obtained an increase of fold expansion in both total cells and CFU-GMs. The combination of FL + SCF does not benefit the proliferation of the total cells, however, the expansion of CFU-GM was kept at a high level. Apparently, both cytokines maintained the activities of stem/progenitor cells and hampered differentiation. In the presence of G-CSF and GM-CSF, total cells enhanced quickly, and had the most expansion of total cells in comparison with other combinations. However, CFU-GM output peaked at 18 days and subsequently dropped to 0 promptly, suggesting that G-CSF and GM-CSF inspired differentiation.

Cell Culture Techniques↗
PubMed1999

cDNA cloning and sequence analysis of hepatitis G virus genome isolated from a Chinese blood donor.

OBJECTIVE: To obtain full-length sequence of a Chinese hepatitis G virus (HGV) strain (HGVch) and investigate the genetic characteristic of HGVch and its identity to other isolates. METHODS: Reverse transcription (RT) and nested-PCR were used to screen HGV RNA positive serum and amplify cDNA fragments. A positive serum without known hepatitis virus markers was selected for isolating HGV RNA template. The HGV genome was divided into 12 overlapping fragments and directly cloned into pGEM-T vector. Sequences were determined by dideoxy terminus-end method of DNA sequencing and then analyzed by computer. RESULTS: The twelve fragments of HGVch cover 9213 nucleotides in length, containing a large open reading frame (ORF) encoding 2873 animo acids polyprotein that began with a methonine residue and ended at termination codon. HGVch is about 86.5%-89.5% identical to other known HGV isolates at the nucleotide level and about 93.9%-96.2% at the deduced animo acid level. CONCLUSION: HGV is a non-A-E hepatitis causal agent, proved to be related with posttransfusion hepatitis in all over the world. Chinese HGV isolate has very close relationship to other isolates from Africa, Europe, Japan, without significant difference across the entire genome. It is suggested that the sequences of HGV isolates are very conservative and the evolution is very slow.

Blood Donors↗
PubMed1999

[The mechanism and effect of erythromycin in development of pulmonary fibrosis].

OBJECTIVE: To observe the effect of erythromycin on the development of pulmonary fibrosis and explore the new therapy in pulmonary fibrosis. METHODS: By evaluating the pathologic changes, the synthesis of collagen protein, the level of activity and expression of tumor necrosis factor-alpha (TNF-alpha), the effect of erythromycin was investigated on the rat model of pulmonary fibrosis induced by bleomycin. RESULTS: The alveolitis alleviated in the first week in treatment group comparing with the control group (142.5 +/- 13.9 mg/g), the synthesis of collagen protein decreased in the fourth week treatment group (96.8 +/- 4.8 mg/g) (q Test P < 0.01). CONCLUSION: Erythromycin may alleviate the process of rat pulmonary fibrosis induced by bleomycin.

Animals↗