Biomedical subjects

Yao Li

Publications and source records attributed to Yao Li.

70 records · Page 4Linked to original sources

PubMed2003

Molecular cloning and characterization of a novel dual-specificity phosphatase18 gene from human fetal brain.

Dual-specificity protein phosphatases (DSPs), a new family of protein tyrosine phosphatases (PTPs), are characterized by the ability to dephosphorylate both phospho-tyrosyl and phospho-seryl/threonyl residues. It has been known that most of the enzymes play important roles in the regulation of mitogenic signal transduction and control the cell cycle in response to extracellular stimuli. In this study, a novel human DSP gene named Dual-specificity Phosphatase18 (DUSP18) was isolated by large-scale sequencing analysis of a human fetal brain cDNA library. DUSP18 is localized at Chromosome 22 q12.1. Its cDNA is 2450 base pairs in length, encoding a 188-amino acid polypeptide in which a DSP motif but not a CH2 domain is included. RT-PCR revealed that the DUSP18 was widely expressed in different tissues. GST-DUSP18 fusion protein showed distinctive phosphatase activity toward p-nitrophenyl phosphate (pNPP), as well as oligopeptides containing pThr and pTyr, indicating that DUSP18 is a protein phosphatase with dual substrate specificity. The optimal condition for the reaction was pH 6.0 and 55 degrees C. Addition of Mn(2+) ions was able to enhance the enzyme activity while the activity was strongly inhibited by iodoaretic acid. Mutations in selected sites showed the importance of Asp-73, Cys-104, Arg-110 and Ser-111 in phosphatase activity of DUSP18.

Amino Acid Sequence↗

PubMed2003

Cloning and characterization of human synaptotagmin 10 gene.

Synaptotagmin (Syt) is a membrane protein family of secretory vesicles, abundant in neural and some endocrine cells. All the members of this family contain one transmembrane region and two conserved C2 domains. Here we reported a new human Syt 10 gene isolated when screening a human fetal brain cDNA library. This cDNA clone is 3287 bp and contains an open reading frame from 299 to 1870 encoding a putative protein of 523 amino acids. It shares 94.6 and 94.8% homology to rat Syt 10 and mouse Syt 10 at protein level, respectively. RT-PCR result showed that it is expressed only in pancreas, lung and kidney.

Amino Acid Sequence↗

PubMed2003

Mutations in the rpoB gene of multidrug-resistant Mycobacterium tuberculosis isolates from China.

Mutations in the 81-bp rifampin resistance determining region (RRDR) and mutation V176F locating at the beginning of the ropB gene were analyzed by DNA sequencing of 86 Mycobacterium tuberculosis clinical isolates (72 resistant and 14 sensitive) from different parts of China. Sixty-five mutations of 22 distinct kinds, 21 point mutations, and 1 insertion were found in 65 of 72 resistant isolates. The most common mutations were in codons 531 (41%), 526 (40%), and 516 (4%). Mutations were not found in seven (10%) of the resistant isolates. Six new alleles within the RRDR, along with five novel mutations outside the RRDR, are reported. None of isolates contained the V176 mutation.

Alleles↗

PubMed2003

[Optimization of T7-based RNA amplification system for cDNA microarray].

cDNA microarrays are powerful parallel tools for gene expression profiling analysis, which help us to understand the molecular mechanism of diseases and to identify potential targets for therapeutic intervention. However, their broader application are hampered by the large amount of RNA required: up to 200 microg of total RNA or 5 microg of mRNA for one chip, making analysis of small samples difficult. In this work, combined with a template switching effect, the T7 RNA linear amplification procedure was optimized, providing multiple copies of anti-sense RNA with reduced sample inputs: no more than 3 microg total RNA for one chip. Using the same RNA sample in all cases, the anti-sense RNA labeling method was compared with standard total RNA and mRNA methods by two sets of self-comparison experiments. Furthermore, the three methods in profiling analysis were compared with the same pair RNA samples. All the results indicated that the fidelity, reproducibility, and reliability showed no significant difference with conventional total RNA or mRNA microarrays.

DNA-Directed RNA Polymerases↗

PubMed2003

[The expression and activity of caspase-8 in the process of vitamin E succinate-induced apoptosis in human gastric carcinoma SGC-7901 cells].

OBJECTIVE: To investigate the role of caspase-8 in vitamin E succinate (VES)-induced apoptosis in human gastric carcinoma cells. METHODS: The expression and activity of caspase-8 were measured by the Western Blot method with fluorescence and DAPI staining. RESULTS: VES treatment induced the expression and activity of caspase-8 in a dose-dependent manner; the apoptosis ratio induced by VES was significantly decreased in SGC-7901 cells treated with caspase-8 inhibitor. CONCLUSION: Caspase-8 plays an important role in SGC-7901 cell apoptosis induced by VES.

Apoptosis↗

PubMed2003

[Development of oligoneucleotide arrays for HLA-DR53-associated group genotyping].

OBJECTIVE: To develop a method performed on an oligonucleotide array for HLA-DR53 group genotyping. METHODS: According to the specific allelic frequency and sequence of HLA-DRB loci in Chinese Han population, HLA-DR53 group typing probes which were immobilized on a glass supports were synthesized. A pair of group-special primers labeled by the Cy5-dCTP were designed, and the primers were used in the PCR, thus the PCR products were labeled with Cy5. The labeled PCR products were hybridized with array. The signals were scanned by scanner and analyzed by image software. The typing results were confirmed by standard DNA and PCR-SSO. One hundred and eleven samples were typed by this array. RESULTS: There were 72 HLA-DR53 group loci typed by oligonucleotide array. Among them, 34 loci were DR9, 25 were DR4, and 13 were DR7. No false positive or false negative typing results were observed. The specificity and reproducibility were 100% and the overall time of genotyping was 5 hours. CONCLUSION: The oligonucleotide array technique is a precise, rapid molecular method for HLA-DR53 genotyping, suited for clinical practice.

Genotype↗

PubMed2003

Using a cDNA microarray to study cellular gene expression altered by Mycobacterium tuberculosis.

OBJECTIVE: To examine the global effects of Mycobacterium tuberculosis (M. tuberculosis) infection on macrophages. METHODS: The gene expression profiling of macrophage U937, in response to infection with M. tuberculosis H(37)R(a), was monitored using a high-density cDNA microarray. RESULTS: M. tuberculosis infection caused 463 differentially expressed genes, of which 366 genes are known genes registered in the Gene Bank. These genes function in various cellular processes including intracellular signalling, cytoskeletal rearrangement, apoptosis, transcriptional regulation, cell surface receptors, cell-mediated immunity as well as a variety of cellular metabolic pathways, and may play key roles in M. tuberculosis infection and intracellular survival. CONCLUSIONS: M. tuberculosis infection alters the expression of host-cell genes, and these genes will provide a foundation for understanding the infection process of M. tuberculosis. The cDNA microarray is a powerful tool for studying pathogen-host cell interaction.

Cells, Cultured↗

PubMed2003

[Validation of cDNA microarray technology].

cDNA microarray is a technological approach that has the potential to globally measure changes in mRNA expression levels. Self-comparison experiments with the same kind of tissue and differential expression experiments with the different kinds of tissue have been done to verify the reproducibility and the accuracy of this technique. The parameter of the reliability and the reproducibility of the microarray data were analyzed by correlation coefficient (R), coefficient of variation (CV) and false positive rate (FPR) etc. Meanwhile, the error resource also has been inspected. These results showed that generally the correlation coefficient of data from this cDNA microarray system was more than 0.9, the coefficient of variation was about 15%, and the false positive rate was below 3%. The result proves the accuracy of the cDNA microarray data. Consistence rate (CR) was advanced here as a new parameter to evaluate the reproducibility of two replicate experiments. It has some advantages over correlation coefficient and coefficient of variation. The influence of some important factors in the experiments, such as different concentration of spotted DNA, mRNA and total RNA, different batches of slides and different processes of labeling, have been investigated by comparing the results. It was shown that most of the false position produced by the experiment system could be reduced by replicate experiments.

Humans↗

PubMed2003

[Differential expression of human macrophage genes encoding cytokines and their regulatory elements after Mycobacterium tuberculosis infection].

To investigate whether the genes encoding cytokines and their regulatory elements participated in the immune response to Mycobacterium tuberculosis infection. Expression microarray was employed to compare the avirulent strain and clinically isolated strains infection induced macrophage cytokine differential expression. Results were cytokines IFN, TNF, TGF, IL and their regulatory elements are involved in the immune reaction. IL-19 was first reported to be involved in the anti-Mtb immunity. Relative expression level of theses factors before and after infection were assayed too. The data provides clues for further scrutinize the role of cytokines and related elements in the interaction between Mycobacterium tuberculosis and host macrophage.

Cytokines↗

PubMed2002

Discovery and analysis of hepatocellular carcinoma genes using cDNA microarrays.

PURPOSE: Microarray analysis on a genomic scale was used to profile changes in gene expression accompanying hepatocellular carcinoma. METHODS: Gene expression profiles of liver tissues from twelve hepatocellular carcinoma samples relative to the gene expression profile of the normal liver tissue were analyzed using 4096 chips and 12800 chips. The results of microarray experiments were verified by the Northern blot technique. RESULTS: A group of 1,820 genes with altered expression were identified in more than 50% of the patients examined. This highly concordant expression profile included human genes encoding proteins involved in the function of peroxisomes, serum control, polycyclic aromatic hydrocarbon carcinogenesis, cell growth and differentiation, metastasis, the function of the immune system, apoptosis, and remodeling of the cytoskeleton. CONCLUSIONS: The newly identified genes afford a quantitative view of the changes that accompany liver cancer at the genomic level, enable deeper insights into the molecular basis of disease, and provide an extensive list of potential early-onset molecular markers for improved diagnosis.

Adult↗

PubMed2002

NADPH-dependent GMP reductase isoenzyme of human (GMPR2). Expression, purification, and kinetic properties.

GMP reductase (EC 1.6.6.8) is the only known metabolic step by which guanine nucleotides can be converted to the pivotal precursor of both adenine and guanine nucleotides. Human GMP reductase has been previously partially purified from erythrocytes and a chromosome 6-linked cDNA has been identified to correspond for encoding human GMP reductase. Here, we reported a distinct cDNA for human GMP reductase isoenzyme isolated from a human fetal brain library, and the GenBank accession number is AF419346. The deduced protein shows 90% identity with human GMP reductase reported (named GMPR1 compared with GMPR2 of this paper) and 69% with E. coli GMP reductase. Comparison of GMPR2 cDNA sequence with human genome indicates the corresponding gene spans about 6.6kb on chromosome 14, which encodes 348 amino acid residues. Northern hybridization analysis indicates a differential and disproportionate expression of mRNAs for GMPR1 and GMPR2, suggesting the existence of distinct molecular species of GMP reductase in human. The apparent Km of GMPR2 for NADPH and GMP are 26.6 and 17.4 microM, respectively. This is the first report suggesting the existence of two distinct types of human GMP reductase molecular species, which can be used to explain the bimodal saturation curve noted with the purified human erythrocyte GMP reductase.

Amino Acid Sequence↗

PubMed2002

Isolation of novel differentially expressed genes related to human glioma using cDNA microarray and characterizations of two novel full-length genes.

Identification of the genes that are differentially expressed between brain tumor and normal brain tissues is important for understanding the molecular basis of these nerve system tumors and for defining possible targets for therapeutic intervention. This investigation is intended to obtain differentially expressed genes related to human glioma using cDNA microarray. Total RNA was extracted from human glioma specimens and normal brain tissues, and mRNA was obtained by oligotex chromatography. The cDNA microarray contains 4366 novel cDNA clones. The results of hybridization were scanned using computer system. Two genes selected from the results of cDNA microarray hybridization were subsequently analyzed by bio-informatic approach, Northern blot, in situ hybridization and radiation hybridization. We demonstrated that at a differentially expressed ration of two to three times, 15 cDNA clones were considered differentially expressed. Two novel full-length genes were selected for further investigation, one named human PKIbeta gene (clone 436F11, GenBank with accession number: AF225513) was over-expressed in normal brain tissues and the other named human ribosomal protein L14.22 gene (clone 507E08, GenBank with accession number: AF329277) was over-expressed in gliomas. Furthermore, the 436F11 gene was located on 6q21-q23 between the D6S304 and D6S2156 markers, while the 507E08 gene was located between the D14S1066 and D14S265 markers. We realized that cDNA microarray technology can be successfully applied to identify differentially expressed genes in human glioma. This approach is superior to routine representational difference analysis, suppression subtractive hybridization and Northern blot for detection and isolation of differentially expressed genes in different tissues. At present, we have discovered two novel full-length genes related to human glioma and their characterizations have been partially clarified.

Amino Acid Sequence↗

PubMed2002

RRR-alpha-tocopheryl succinate inhibits human gastric cancer SGC-7901 cell growth by inducing apoptosis and DNA synthesis arrest.

AIM: To investigate the effects of growth inhibition of human gastric cancer SGC-7901 cell with RRR-alpha-tocopheryl succinate (VES), a derivative of natural Vitamin E, via inducing apoptosis and DNA synthesis arrest. METHODS: Human gastric cancer SGC-7901 cells were regularly incubated in the presence of VES at 5, 10 and 20mg x L(-1) (VES was dissolved in absolute ethanol and diluted in RPMI 1640 complete condition media correspondingly to a final concentration of VES and 1 mL x L(-1) ethanol), succinic acid and ethanol equivalents as vehicle (VEH) control and condition media only as untreated (UT) control. Trypan blue dye exclusion analysis and MTT assay were applied to detect the cell proliferation. Cells were pulsed with 37kBq of tritiated thymidine and (3H) TdR uptake was measured to observe DNA synthesis. Apoptotic morphology was observed by electron microscopy and DAPI staining. Flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay were performed to detect VES-triggered apoptosis. RESULTS: VES inhibited SGC-7901 cell growth in a dose-dependent manner. The growth curve showed suppression by 24.7%, 49.2% and 68.7% following 24h of VES treatment at 5, 10 and 20 mg x L(-1), respectively, similar to the findings from MTT assay. DNA synthesis was evidently reduced by 35%, 45% and 98% after 24h VES treatment at 20mg x L(-1) and 48 h at 10 and 20mg x L(-1), respectively. VES induced SGC-7901 cells to undergo apoptosis with typically apoptotic characteristics, including morphological changes of chromatin condensation, chromatin crescent formation/margination, nucleus fragmentation and apoptotic body formation, typical apoptotic sub-G1 peak by flow cytometry and increase of apoptotic cells by TUNEL assay in which 90% of cells underwent apoptosis after 48 h of VES treatment at 20 mg x L(-1). CONCLUSION: VES can inhibit human gastric cancer SGC-7901 cell growth by inducing apoptosis and DNA synthesis arrest. Inhibition of SGC-7901 cell growth by VES is dose- and time-dependent. Therefore VES can function as a potent chemotherapeutic agent against human gastric carcinogenesis.

Apoptosis↗

PubMed2002

Roles of Fas signaling pathway in vitamin E succinate-induced apoptosis in human gastric cancer SGC-7901 cells.

AIM: To investigate the roles of Fas signaling pathway in vitamin E succinate-induced apoptosis in human gastric cancer SGC-7901 cells. METHODS: Human gastric cancer SGC-7901 cells were treated with VES at 5, 10, 20 mg x L(-1), succinic acid and vitamin E as vehicle control and condition media only as untreated (UT) control. Apoptotic morphology was observed by DAPI staining. Western blot analysis was applied to measure the expression of Fas, FADD and caspase-8 proteins. After the cells were transiently transfected with Fas and FADD antisense oligonucleotides, respectively, caspase-8 activity was determined by flurometric method. RESULTS: The morphologically apoptotic changes were observed after VES treatment by DAPI staining. 23.7 % and 89.6 % apoptosis occurred after 24 h and 48 h of 20 mg x L(-1) VES treatment, respectively. The protein levels of Fas, FADD and caspase-8 were evidently increased in a dose-dependent manner after 24 h of VES treatment. The blockage of Fas by transfection with Fas antisense oligonucleotides obviously inhibited the expression of FADD protein. After SGC-7901 cells were transfected with Fas and FADD antisense oligonucleotides, caspase-8 activity was obviously decreased (P<0.01), whereas Fas blocked more than FADD. CONCLUSION: VES-induced apoptosis in human gastric cancer SGC-7901 cells involves Fas signaling pathway including the interaction of Fas, FADD and caspase-8.

Adaptor Proteins, Signal Transducing↗

PubMed2002

[Expression of cell adhesion molecules in acute leukemia cell].

OBJECTIVE: To investigate the role of cell adhesion molecule in the development and extramedullary infiltration (EI) of acute leukemia. METHODS: The expressions of neural cell adhesion molecule (NCAM) gene, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule (VCAM-1) genes in 25 acute leukemia patients bone marrow cells were detected by microarray and reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: The expressions of NCAM, ICAM-1 and VCAM-1 gene were significantly higher in acute leukemia cells and leukemia cells with EI than in normal tissues and leukemia cells without EI, respectively, both by cDNA microarray and by RT-PCR. CONCLUSION: The cDNA microarray is a powerful technique in analysis of acute leukemia cells associated genes. High expressions of cell adhesion molecule genes might be correlated with leukemia pathogenesis and infiltration of acute leukemia cell.

Acute Disease↗

PubMed2002

[cDNA microarray in the gene expression pattern in lymphatic metastasis of pancreatic carcinoma].

OBJECTIVE: Analysis of differential gene expression profiles by cDNA microarray in pancreatic carcinoma with or without lymphatic metastasis. METHODS: cDNA microarray was prepared by spotting polymerase chain reaction (PCR) products of 4 000 human genes onto specially treated glass slides. The cDNA probes were prepared by labeling normal tissue mRNA and cancer tissue mRNA with Cy3-dUTP and Cy5-dUTP, separately through reverse transcription. The mixed probes were, then, hybridized to the cDNA microarray. The chips were scanned by ScanArray 3000 laser scanner (General Scanning, Inc) on two wavelengths. The acquired image was analyzed by ImaGene 3.0 software (BioDiscovery, Inc). The intensity of each spot on the two wavelengths represented the quantity of Cy3-dUTP and Cy5-dUTP, with Cy5 to Cy3 ratio computed on each. RESULTS: Fifty-six genes (including 24 previously reported) exhibited differential expressions in 2 specimens of pancreatic carcinoma with lymphatic metastasis and 2 without. CONCLUSION: cDNA microarray provides an promising approach to specific gene expressions of the presence of lymphatic metastasis in human pancreatic carcinoma.

Aged↗