Biomedical subjects

Jia Chen

Publications and source records attributed to Jia Chen.

69 records · Page 4Linked to original sources

PubMed2004

Properties of pluripotent human embryonic stem cells BG01 and BG02.

Human ES (hES) cell lines have only recently been generated, and differences between human and mouse ES cells have been identified. In this manuscript we describe the properties of two human ES cell lines, BG01 and BG02. By immunocytochemistry and reverse transcription polymerase chain reaction, undifferentiated cells expressed markers that are characteristic of ES cells, including SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and OCT-3/4. Both cell lines were readily maintained in an undifferentiated state and could differentiate into cells of all three germ layers, as determined by expression of beta-tubulin III neuron-specific molecule (ectoderm), cardiac troponin I (cardiomyocytes, mesoderm), and alpha-fetoprotein (endoderm). A large-scale microarray (16,659 genes) analysis identified 373 genes that were expressed at three-fold or higher levels in undifferentiated BG01 and BG02 cells as compared with pooled human RNA. Ninety-two of these genes were also highly expressed in four other hES lines (TE05, GE01, GE09, and pooled samples derived from GE01, GE09, and GE07). Included in the list are genes involved in cell signaling and development, metabolism, transcription regulation, and many hypothetical proteins. Two focused arrays designed to examine transcripts associated with stem cells and with the transforming growth factor-beta superfamily were employed to examine differentially expressed genes. Several growth factors, receptors, and components of signaling pathways that regulate embryonic development, in particular the nodal signaling pathway, were detected in both BG01 and BG02. These data provide a detailed characterization and an initial gene expression profile for the BG01 and BG02 human ES cell lines.

Cell Differentiation↗

PubMed2004

Dopaminergic differentiation of human embryonic stem cells.

In this manuscript we report that human embryonic stem cells (hESCs) differentiated into dopaminergic neurons when cocultured with PA6 cells. After 3 weeks of differentiation, approximately 87% of hES colonies contained tyrosine hydroxylase (TH)-positive cells, and a high percentage of the cells in most of the colonies expressed TH. Differentiation was inhibited by exposure to BMP4 or serum. TH-positive cells derived from hESCs were postmitotic, as determined by bromodeoxyurindine colabeling. Differentiated cells expressed other markers of dopaminergic neurons, including the dopamine transporter, aromatic amino acid decarboxylase, and the transcription factors associated with neuronal and dopaminergic differentiation, Sox1, Nurr1, Ptx3, and Lmx1b. Neurons that had been differentiated on PA6 cells were negative for dopamine-beta-hydroxylase, a marker of noradrenergic neurons. PA6-induced neurons were able to release dopamine and 3,4-dihydroxphe-hylacetic acid (DOPAC) but not noradrenalin when depolarized by high K(+). When transplanted into 6-hydroxydopamine-treated animals, hES-derived dopaminergic cells integrated into the rat striatum. Five weeks after transplantation, surviving TH-positive cells were present but in very small numbers compared with the high frequency of TH-positive cells seen in PA6 coculture. Larger numbers of cells positive for smooth muscle actin, but no undifferentiated ES cells, were present after transplantation. Therefore, hESCs can be used to generate human dopaminergic cells that exhibit biochemical and functional properties consistent with the expected properties of mature dopaminergic neurons.

3,4-Dihydroxyphenylacetic Acid↗

PubMed2004

Polymorphism in the thymidylate synthase promoter enhancer region and risk of colorectal adenomas.

Thymidylate synthase (TS), a key one-carbon metabolizing gene, encodes an enzyme that converts dUMP to dTMP, the rate-limiting nucleotide in DNA synthesis. We recently reported that a promoter polymorphism in TS modified the risk of colorectal cancer as well as the survival rate after the disease. To explore whether TS may play an important role in colorectal carcinogenesis early in the multistaged pathogenic pathway, we investigated the relation between the TS promoter polymorphism and risk of colorectal adenoma in a nested case-control study within the prospective Health Professionals Follow-up Study. We ascertained the TS genotype from 373 incident colorectal adenoma cases and 720 control subjects. Although there was no overall association between the TS promoter polymorphism and adenoma risk, we observed a significant TS-alcohol interaction (P for interaction = 0.009); relative to low alcohol consumers with the 2R/2R genotype, those with high alcohol consumption (>30 g/d) were not at elevated risk if they had the 2R/2R genotype [relative risk (RR), 0.80; 95% confidence interval (95% CI), 0.34-1.90], but were at higher risk if they had the 2R/3R genotype (RR, 1.70; 95% CI, 0.87-3.31), and at the highest risk (RR, 3.16; 95% CI, 1.50-6.63) if they had the 3R/3R genotype. In addition, a significant interaction was observed between the TS promoter polymorphism and the 677C > T polymorphism of methylenetetrahydrofolate reductase (MTHFR; P for interaction = 0.007). These findings lend additional support that one-carbon metabolism is an important process in pathogenesis of colorectal cancer.

Adenoma↗

PubMed2004

BG01V: a variant human embryonic stem cell line which exhibits rapid growth after passaging and reliable dopaminergic differentiation.

PURPOSE: To explore a karyotypically abnormal variant human embryonic stem cell (hESC) line, BG01V, as a potential model for studies of dopaminergic neuronal differentiation. METHODS: The properties of BG01V cells were compared to those of normal BG01 cells using immunocytochemistry, RT-PCR, focused microarrays and in vitro differentiation, including dopaminergic differentiation, by culturing with the stromal cell line PA6. RESULTS: Despite the karyotypic abnormality (49, +12, +17 and XXY), undifferentiated BG01V cells expressed pluripotent ESC markers similar to BG01 cells, and retained the ability to differentiate into cell types characteristic of all three germ layers. When co-cultured with the stromal cell line PA6, BG01V cells differentiated into dopaminergic cells which exhibited properties similar to those of mature dopaminergic neurons. CONCLUSIONS: BG01V cells were easier to maintain in culture than karyotypically normal BG01 cells and can be used as an alternative pluripotent hESC type for in vitro developmental studies.

Blotting, Northern↗

PubMed2003

Ethanol inhibits fibroblast growth factor-induced proliferation of aortic smooth muscle cells.

OBJECTIVE: Epidemiological studies have demonstrated that moderate alcohol consumption reduces mortality associated with coronary artery disease. The protective effect is correlated with the amount of ethanol consumed but is unrelated to the form of alcoholic beverage. Adoption of a favorable lipoprotein profile accounts for about half of the protective action of alcohol, but the remaining causative factors remain conjectural. Fibroblast growth factors (FGFs) play important roles in mediating smooth muscle cell (SMC) proliferation and migration, which are key factors in the atherosclerotic process. In the present study, we examined the effect of ethanol on FGF-mediated SMC growth and signaling. METHODS AND RESULTS: Pharmacologically relevant concentrations of ethanol inhibited the proliferation of a rat aortic SMC line (SV40LT-SMCs) in response to FGF1 and FGF2. Human aortic SMC growth was similarly inhibited by ethanol. Transition into the G2/M phase was specifically affected. FGF-mediated phosphorylation of p42/p44 mitogen-activated protein kinase (MAPK) c-Raf, MAP kinase kinase kinase, MEK1/2 MAP kinase, kinase, stress-activated protein kinase/c-Jun-NH2-terminal kinase, and p38 MAPK were variably reduced by ethanol. The inhibition of intracellular signaling by ethanol was correlated with inhibition of FGF receptor autophosphorylation. By contrast, neither epidermal growth factor receptor autophosphorylation nor epidermal growth factor-mediated p42/p44 MAPK activation was affected by ethanol. CONCLUSIONS: The findings identify the FGF receptor as an inhibitory target for ethanol, which could account in part for the inhibitory actions of ethanol on SMC proliferation observed in vivo.

Animals↗

PubMed2003

Selective decreases of nicotinic acetylcholine receptors in PC12 cells exposed to fluoride.

In an attempt to elucidate the mechanism by which excessive fluoride damages the central nervous system, the effects of exposure of PC12 cells to different concentrations of fluoride for 48 h on nicotinic acetylcholine receptors (nAChRs) were characterized here. Significant reductions in the number of binding sites for both [3H]epibatidine and [125I]alpha-bungarotoxin, as well as a significant decrease in the B(max) value for the high-affinity of epibatidine binding site were observed in PC12 cells subjected to high levels of fluoride. On the protein level, the alpha 3 and alpha 7 subunits of nAChRs were also significantly decreased in the cells exposed to high concentrations of fluoride. In contrast, such exposure had no significant effect on the level of the beta 2 subunit. These findings suggest that selective decreases in the number of nAChRs may play an important role in the mechanism(s) by which fluoride causes dysfunction of the central nervous system.

Animals↗

PubMed2003

Increased influence of genetic variation on PON1 activity in neonates.

PON1 (paraoxonase-1) detoxifies organophosphates by cleavage of active oxons before they have a chance to inhibit cholinesterases. The corresponding gene PON1 has common polymorphisms in both the promoter (-909, -162, -108) and the coding region (L55M, Q192R). The five PON1 genotypes were determined for maternal blood (n= 402) and cord blood (n= 229) as part of a study of the effects of organophosphate pesticide exposure on infant growth and neurodevelopment. PON1 enzymatic activities were determined for a majority of subjects. The population contained Caucasians, Caribbean Hispanics, and African Americans. PON1 activity was strongly dependent upon the promoter alleles in both maternal and cord blood. For example, PON1 activities for position -108CC, CT, and TT mothers were 146, 128, and 109 arylesterase U/mL (analysis of variance, p< 0.0001), whereas the same PON1 activities for the respective cord bloods were 49.0, 32.4, and 23.2 U/mL (p < 0.0001). Compared with adults, neonates had lower PON1 activity, implying reduced capacity to detoxify organophosphates. In addition there was a larger difference in activity between genotype groups in neonates than in adults. Because the five polymorphisms in PON1 occur in a short stretch of DNA, they were tested for linkage disequilibrium (LD). Significant LD was found among all three promoter polymorphisms as well as between promoter polymorphisms and L55M, with the strongest LD for Caucasians and the weakest for African Americans. The Caribbean Hispanics fall between these two groups. Surprisingly, significant LD also was observed between the promoter polymorphisms and C311S in PON2. LD between the promoter polymorphisms and Q192R was not significant.

Adult↗

PubMed2003

Stable expression of hrGFP by mouse embryonic stem cells: promoter activity in the undifferentiated state and during dopaminergic neural differentiation.

Three promoters, cellular polypeptide chain elongation factor 1 alpha (EF1), cytomegalovirus (CMV), and Rous sarcoma virus (RSV) were examined for stable transgene expression in mouse embryonic stem (ES) cells and their progeny during dopaminergic neural differentiation. In undifferentiated ES cells the EF1 promoter was highly effective, while CMV had moderate activity. After 3 months in culture, expression of humanized renilla green fluorescent protein (hrGFP) was unchanged for the EF1 promoter and decreased for CMV. At the nestin-positive stage of differentiation, hrGFP and nestin were colocalized in about 20% of cells for EF1, in contrast to 80% of cells for the CMV promoter. In tyrosine hydroxylase (TH)-positive neurons neither the EF1 nor CMV promoter were effective. The RSV promoter was inactive in undifferentiated, nestin-positive, and TH-positive cells. Thus, EF1 and CMV are effective promoters for transgene expression in undifferentiated ES cells and nestin-positive neural precursors.

Animals↗

PubMed2003

Polymorphism in the thymidylate synthase promoter enhancer region modifies the risk and survival of colorectal cancer.

Thymidylate synthase (TS) converts dUMP to dTMP, the rate-limiting nucleotide in DNA synthesis. It is also the target for 5-flurorouracil, the most common chemotherapy agent for treatment of colorectal cancer (CRC). We designed a nested case-control study within the prospective Physicians' Health Study to investigate whether TS polymorphisms independently predict risk of CRC and simultaneously the overall survival after the disease in the same population. We also investigated influences of this polymorphism on plasma folate and homocysteine levels. The study consists of 270 incident CRC and 454 control subjects. Risk of CRC was estimated by use of conditional multiple logistic regression analysis. Survival was analyzed by Cox proportional hazards regression analysis. Compared with the TS 3R/3R genotype, the multivariate-adjusted risk ratio was 0.86 (0.59-1.25) for the 2R/3R genotype and 0.59 (0.36-0.98) for the 2R/2R genotype with P for trend of 0.03. The TS 2R/2R genotype was also associated with better survival, although the results were not significant. Compared with those with either the 3R/3R or 2R/3R genotypes, the age-adjusted hazard ratio for the 2R/2R genotype was 0.57 (0.30-1.07). Individuals with the 2R/2R genotype had significantly lower plasma folate levels than those with the 3R/3R genotype, whereas their plasma homocysteine levels were unaffected by the TS promoter polymorphism. The deletion polymorphism at the TS 3'-untranslated region did not influence the CRC risk and survival, nor did it modify the plasma folate and total homocysteine levels. Given that individuals with high plasma folate had a better survival outcome with a hazard ratio of 0.68 (0.45-1.03) compared with those with low plasma folate, we conclude that the TS promoter polymorphism may modify both the risk and the survival of CRC; however, these effects do not appear to be mediated through its modulation of biological folate levels.

Aged↗

PubMed2003

Methylenetetrahydrofolate reductase, alcohol dehydrogenase, diet, and risk of colorectal adenomas.

An increased occurrence of colorectal cancer and its adenoma precursor is observed among individuals with low intakes or circulating levels of folate, especially if alcohol intake is high, although results have not been statistically significant in all studies. We examined folate and alcohol intake and genetic polymorphisms in methylenetetrahydrofolate reductase [MTHFR 667-->T (ala-->val) and MTHFR 1298A-->C (gln-->ala)] (associated with reduced MTHFR activity) and in alcohol dehydrogenase 3 [ADH3 (2-2) associated with decreased alcohol catabolism] in relation to risk of colorectal adenoma in the Health Professionals Follow-Up Study. Among 379 cases and 726 controls, MTHFR genotypes were not appreciably related to risk of adenoma, but a suggestive interaction (P = 0.09) was observed between MTHFR 677C-->T and alcohol intake; men with TT homozygotes who consumed 30+ g/day of alcohol had an odds ratio (OR) of 3.52 [95% confidence interval (CI), 1.41-8.78] relative to drinkers of < or =5 g/day with the CC/CT genotypes. ADH3 genotype alone was not appreciably related to risk, but its influence was modified by alcohol intake. Compared with fast alcohol catabolizers [ADH3(1-1)] with low intakes of alcohol (< or =5 g/day), high consumers of alcohol (30+ g/day) had a marked increase in risk if they had the genotype associated with slow catabolism [ADH3(2-2); OR, 2.94; 95% CI, 1.24-6.92] or intermediate catabolism [ADH3(1-2)] of alcohol (OR, 1.83; 95% CI, 1.03-3.26) but not if they were fast catabolizers [ADH3(1-1); OR = 1.27; 95% CI = 0.63-2.53). In addition, an increased risk of colorectal adenoma (OR, 17.1; 95% CI, 2.1-137) was observed for those with the ADH3(2-2) genotype and high alcohol-low folate intake compared with those with low alcohol-high folate intake and the ADH3(1-1) genotype (P for interaction = 0.006). Our results indicate that high intake of alcohol is associated with an increased risk of colorectal adenoma, particularly among MTHFR 677TT and ADH3(2-2) homozygotes. The findings that alcohol interacts with a folate-related gene (MTHFR) and that the interaction between alcohol and ADH3 is stronger among those with low folate intake support the hypothesis that the carcinogenic influence of alcohol in the large bowel is mediated through folate status.

Adenoma↗

PubMed2002

Linkage disequilibrium between the 677C>T and 1298A>C polymorphisms in human methylenetetrahydrofolate reductase gene and their contributions to risk of colorectal cancer.

A common polymorphism in a folate-metabolizing gene, methylenetetrahydrofolate reductase (MTHFR) 677C>T has been associated with reduced risk of colorectal cancer. In this study, we investigated whether a second common polymorphism of the gene, MTHFR 1298A>C, is an independent risk factor for colorectal cancer and if it is associated with plasma folate and total homocysteine (tHcy) levels. We also examined whether the 677C>T and 1298A>C polymorphisms are in linkage disequilibrium and whether combined heterozygosity confers additional (or reduced) risk of colorectal cancer. We conducted a nested case-control study of 211 incident colorectal cancer cases and 343 controls in the prospective Physicians' Health Study. The MTHFR 677C>T and 1298A>C polymorphisms were in linkage disequilibrium in this population. Compared to MTHFR 1298AA genotype, multivariate-adjusted relative risk of colorectal cancer was 0.73 (95% CI 0.37-1.43) for the MTHFR 1298CC genotype. The slight reduction in risk was not a result of its linkage disequilibrium with the 677C>T polymorphism. This polymorphism was not significantly correlated with the plasma folate and tHcy levels. The combined heterozygosity did not modify the cancer risk; nor did it change the plasma folate and tHcy significantly. We conclude that the MTHFR 1298A>C polymorphism is a less substantial independent risk factor for colorectal cancer compared to the 677C>T polymorphism.

Case-Control Studies↗

PubMed2002

Kinetic polymerase chain reaction on pooled DNA: a high-throughput, high-efficiency alternative in genetic epidemiological studies.

The ideal technology for screening single-nucleotide polymorphisms requires high throughput with minimal cost per sample, minimal usage of valuable DNA resources, and maximal flexibility for assessment of new polymorphisms. We demonstrate here the feasibility of kinetic allele-specific PCR with DNA pooling (S. Germer et al., Genome Res., 10: 258-266, 2000) in a population study that satisfies all of the mentioned criteria and offers a powerful new tool for detecting meaningful polymorphic differences in candidate gene association studies and genome-wide linkage dysequilibrium scans. Three individuals prepared pooled DNA samples from 269 individuals separated into three racial/ethnic groups: Caucasians (n = 56), African-Americans (n = 86), and Hispanics (n = 127). We used kinetic allele-specific PCR to determine the allele frequencies of the common paraoxonase 1 polymorphism, PON1 Q191R, in these pools. Paraoxonase 1 is a critical enzyme for inactivating neurotoxic intermediates in the metabolism of organophosphates. In a blinded test of the technology, these nine pooled DNA samples were sent to Roche for genotyping by kinetic allele-specific PCR. The allele frequencies found were 0.266 +/- 0.011, 0.386 +/- 0.011, and 0.617 +/- 0.010, respectively, which were comparable to the frequencies of 0.269, 0.403, and 0.622 determined by PCR-restriction fragment length polymorphism analysis. These same samples were genotyped on two kinetic PCR platforms from different manufacturers, using three different DNA polymerases. The results were comparable between both platforms and among all three polymerases. The results demonstrate a powerful new technology for determining frequencies of single-nucleotide polymorphisms in an epidemiological study.

Alleles↗

PubMed2002

[The acute effects of dimethoate on the muscarinic-receptors of rat brains and the relationship between muscarinic-receptors and cholinesterase].

OBJECTIVE: To study the acute effects of dimethoate on the muscarinic-receptors(M1, M2) in the brain of rats. METHODS: 24 Sprague-Dawley rats were divided into 4 groups randomly. They were administered subcutaneously with 0, 25, 50, 100 mg/kg dimethoate, respectively. Brains were removed after 48 hours of administration. Radioligand binding assay was used to determine the density and affinity of M1 and M2 receptors. RESULTS: Rats in the treated group showed low density of M1 and M2 receptors compared with the control rats. The brain M1 receptor density of the rats in the highest dosage group was significantly lower than that in the control group while brain M2 receptors density had a decrease trend with increasing dosage, but the difference showed no significance. However, there were no differences of the affinity of both M1 and M2 among different treated groups. Correlation analysis showed there is positive relationship between cholinesterase activity and density of M1 receptors(r = 0.583, P < 0.01). CONCLUSION: M1 and M2 receptors density decreased with the increasing dosage of dimethoate. It is suggested that the alleviating of cholinergic symptoms may be due to the decrease of M1 and M2 receptors in rat brain.

Animals↗

PubMed1996

Identification of the Photoaffinity-labeled Abscisic Acid Binding Proteins from Maize Root Microsome.

ABA binding protein (ABA-BP) was solubilized with 0.2% (W/V) Triton X-100 from the microsomal membrane of maize root, with a specific binding activity of 0.56 pmol ABA/g FW. When ABA was cross-linked to its binding sites by irradiating the microsomal proteins with light of 330 nm after incubation with low levels of H(3)-(-/+)ABA, it was found that this kind of photoaffinity labeling was specific to ABA-BP. On PAGE and SDS-PAGE three major protein bands with molecular weights of 72 kD, 47 kD and 37 kD were found with high radioactivity.

Journal Article↗

PubMedDate not supplied

Chronic fluoride toxicity decreases the number of nicotinic acetylcholine receptors in rat brain.

In order to investigate the molecular mechanism(s) underlying brain dysfunction caused by chronic fluorosis, neuronal nicotinic acetylcholine receptors (nAChRs) in the brain of rats receiving either 30 or 100 ppm fluoride in their drinking water for 7 months were analyzed in the present study employing ligand binding and Western blotting. There was a significant reduction in the number of [3H]epibatidine binding sites in the brain of rats exposed 100 ppm of fluoride, but no alteration after exposed to 30 ppm. On the other hand, the number of [125I]alpha-BTX binding sites was significantly decreased in the brains of rats exposed to both levels of fluoride. Western blotting revealed that the level of the nAChR alpha4 subunit protein in the brains of rats was significantly lowered by exposure to 100 ppm, but not 30 ppm fluoride; whereas the expression of the alpha7 subunit protein was significantly decreased by both levels of exposure. In contrast, there was no significant change in the level of the beta2 subunit protein in the brains of rats administered fluoride. Since nAChRs play major roles in cognitive processes such as learning and memory, the decrease in the number of nAChRs caused by fluoride toxicity may be an important factor in the mechanism of brain dysfunction in the disorder.

Animals↗