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Yu-Min Mao

Publications and source records attributed to Yu-Min Mao.

18 recordsLinked to original sources
PubMed2004

Analysis of gene expression profiles in human HL-60 cell exposed to cantharidin using cDNA microarray.

Cantharidin is a natural toxin that has antitumor properties and causes leukocytosis as well as increasing sensitivity of tumor cells resistant to other chemotherapeutic agents. There is limited information, however, on the molecular pharmacological mechanisms of cantharidin on human cancer cells. We have used cDNA microarrays to identify gene expression changes in HL-60 promyeloid leukemia cells exposed to cantharidin. Cantharidin-treated cells not only decreased expression of genes coding for proteins involved in DNA replication (e.g., DNA polymerase delta), DNA repair (e.g., FANCG, ERCC), energy metabolism (e.g., isocitrate dehydrogenase alpha, ADP/ATP translocase), but also decreased expression of genes coding for proteins that have oncogenic activity (e.g., c-myc, GTPase) or show tumor-specific expression (e.g., phosphatidylinositol 3-kinase). In contrast, these treated cells overexpressed several genes that encode intracellular and secreted growth-inhibitory proteins (e.g., BTG2, MCP-3) as well as proapoptotic genes (e.g., ATL-derived PMA-responsive peptide). Our findings suggest that alterations in specific genes functionally related to cell proliferation or apoptosis may be responsible for cantharidin-mediated cytotoxicity. We also found that exposure of HL-60 cells to cantharidin resulted in the decreased expression of multidrug resistance-associated protein genes (e.g., ABCA3, MOAT-B), suggesting that cantharidin may be used as an oncotherapy sensitizer, and the increased expression of genes in modulating cytokine production and inflammatory response (e.g., NFIL-3, N-formylpeptide receptor), which may partly explain the stimulating effects on leukocytosis. Our data provide new insight into the molecular mechanisms of cantharidin.

Cantharidin↗
PubMed2004

Over-expression of Bim alpha3, a novel isoform of human Bim, result in cell apoptosis.

Bim proteins are essential factors of apoptosis. Nine isoforms of Bim have been submitted to GenBank database. In order to improve the understanding of the regulation of Bims' proapoptotic activity, we screened a multiple tissue cDNA panels for Bim isoforms and tested their proapoptotic activity by over-expression. Two novel cDNA isoforms of Bim family are generated by alternative splicing. One isoform encodes a 79 residue putative protein with a BH3 domain, named Bim alpha3. There is not any significant ORF found in another isoform, named Bim beta5. Subcellular localized analysis of EGFP-Bim fusion protein suggests Bim alpha3 distributed to both plasma and nucleus of HEK 293 cell. HEK 293 cells transfected with pcDNA-Bim alpha3 presented a similar proapoptotic activity (32.05 +/- 6.42%) with Bim alpha2 (30.14 +/- 2.66%). The proapoptotic activity of Bim alpha3 was obviously weaker than that of Bim S (46.52 +/- 5.07%) and Bim L (55.53 +/- 1.99%). Anti-sense over-expression of Bim in HEK 293 cells results in a weak down-regulated proapoptotic level. Expression pattern analysis reveals that both the novel cDNAs are expressed widely in normal tissue just like the other reported isoforms. The expression pattern of Bim isoforms shows tissue specific obviously. The results suggest that BH3 domain is sufficiency for proapoptotic activity of Bim proteins. The functional state of Bims might be regulated both in the transcript and post transcript process.

Apoptosis↗
PubMed2004

Cloning and expression of human GTDC1 gene (glycosyltransferase-like domain containing 1) from human fetal library.

The glycosyltransferases (GTs) catalyze the synthesis of the carbohydrate portions of glycoproteins, glycolipids, and proteoglycans. Here we report the cloning and characterization of a novel human GTDC1 (glycosyltransferase-like domain containing 1) gene, which locates on human chromosome 2q22. The GTDC1 cDNA is 2954 bp in length, encoding a putative protein of 458 amino acids. At protein level human GTDC1 has 75 and 37% identity with its homologous counterparts in the mouse and fruitfly, respectively. RT-PCR analysis revealed its relatively high expression level in the adult lung, spleen, testis, and peripheral blood leukocyte.

Amino Acid Sequence↗
PubMed2004

[Detecting genetic polymorphisms of CYP1 A1 and GSTM1 simultaneously with oligonucleotide microarray].

Cytochrome P4501A1 plays a major role in the bioactivation of a number of tobacco procarcinogens. Glutathione S-transferase( GSTM1), a member of the class of GST gene family, has been shown to be polymorphic because of gene deletion resulting in a failure to express the GSTM1 gene in 50% approximately 60% of individuals. Some CYP1 A1/GSTM1 null genotype combinations seem to predispose the lung, esophagus, and oral cavity of smokers to an even higher risk for cancer or DNA damage, requiring, however, confirmation. An easy and reliable oligonuleotide microarray approach validated through direct sequencing method is developed in order to accurately detect single nucleotide polymorphisms of CYP1 A1 gene and discriminate the presence and absence of GSTM1 gene. The m1 (Msp I) and m2 (Ile462Val) polymorphisms of CYP1 A1 gene and GSTM1 null genotype were also determined in a random population of 84 healthy, unrelated volunteers with developed microarray-based method. Of 84 cases, 47.6% were calssified as GSTM1 null, close to the published data. It's interesting that there lack three genotypes of m1 -m2 locus in the population: TT-AG, TT-GG and TC-GG. However, according to the data of the genotype frequencies independently happened at both m1 and m2 site, the combination frequencies of above three genotypes are 11.4%, 2.6%, and 3.1% respectively. Therefore we assume that the haplotypes of m1 -m2 are only T-A, C-A and C-G, but not T-G, as it were,there is no recombination happened between m1 site and m2 site. The frequencies of three haplotypes of T-A, C-A and C-G, calculated through corresponding genotypes, are 69.6%, 7.7% and 22.6% respectively.

Base Sequence↗
PubMed2003

Purification, crystallization and preliminary X-ray studies of GMP reductase 2 from human.

GMP reductase 2 from human has been expressed in Escherichia coli, purified and crystallized. The crystals belong to space group P3(2)21, with unit-cell parameters a = b = 110.6, c = 209.8 A, alpha = beta = 90, gamma = 120 degrees. Diffraction data were collected to 3.0 A with a completeness of 100% (100% for the last shell), an R(merge) value of 0.089 (0.189) and an I/sigma(I) value of 7.3 (3.2).

Crystallization↗
PubMed2003

A novel cDNA encodes a putative hRALY-like protein, hRALYL.

High throughput cDNA sequencing and 5'-rapid amplification of cDNA ends (5'RACE) isolated two cDNAs that shared the same open reading fragment (ORF). Northern blot analysis with the fetal brain mRNA blots detected two transcripts with the length of 3.2 kb and 2.2 kb respectively. The ORF encodes a 291 residues putative protein that shares great homology with hRALY and hnRNPC. So it was named hRALY like protein, hRALYL. Expression pattern was detected by multiple-issue cDNA (MTC) panel based RT-PCR. It revealed that the transcripts of hRALYL were expressed ubiquitously in human tissues with different intensities. The transcript is highest in brain. Blast analysis found the cDNA corresponding to a contig NT_008292, which revealed the gene containing at least 9 exons and located the gene on human chromosome 8q21.13-8q21.2. hRALYL might be a member of hnRNPC subfamily.

Amino Acid Sequence↗
PubMed2003

Cloning and identification of a novel cDNA which encodes a putative protein with a DnaJ domain and a thioredoxin active motif, human macrothioredoxin.

A 3345 bp cDNA was isolated from the fetal brain cDNA library by high throughput cDNA sequencing. The cDNA with an open reading fragment (ORF) of 2241 bp encodes a 747 amino acids putative protein with a DnaJ N-terminus domain and four thioredoxin active sets. So it is named human macrothioredoxin (hMTHr). We used Northern blot to detect a band with a length of about 5 kb, which was ubiquitously expressed in human adult tissues with different intensities. The expression pattern was verified by RT-PCR, revealing that the transcripts were ubiquitously expressed in fetal tissues and human tumor tissues also. The transcripts in fetal tissues were more numerous than in adult tissues. The transcripts were high in adult testis, adult pancreas, fetal thymus, and fetal kidney. We have found three splice isoforms with lengths of 3483, 3345, and 2982 bp in the sequencing analysis of the RT-PCR products. They encode three putative proteins with 793, 747, and 275 amino acids, respectively. The first two putative proteins contain a DnaJ domain and four thioredoxin domains. The third putative protein only contains an N-terminus DnaJ domain and one thioredoxin active set. Blast analysis against the NCBI database revealed that the gene spans a genome sequence of about 75 kb that contains at least 25 exons and is located in human chromosome 2q32.1. The organization of the functional motifs of hMTHr suggests that the protein might be a member of a molecular chaperone family.

Amino Acid Sequence↗
PubMed2003

Purification, crystallization and preliminary X-ray studies of a p-nitrophenylphosphatase from Bacillus stearothermophilus.

Thermostable p-nitrophenylphosphatase from Bacillus stearothermophilus has been expressed in Escherichia coli, purified and crystallized. The crystals belong to space group C(2), with unit-cell parameters a = 67.17 A, b = 57.84 A, c = 62.49 A and alpha = 90.0 degrees, beta = 95.4 degrees, gamma = 90.0 degrees. Diffraction data were collected to 1.40 A resolution with a completeness of 94.7% (96.6% for the last shell), an R(fac) value of 0.074 (0.341) and an I/sigma (I) value of 30.1 (2.67).

4-Nitrophenylphosphatase↗
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

[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

[RAPD study of populations of Glycine tabacina].

The ecological genetic research on Glycine tabacina populations was based on RAPD technique, which revealed 100% polymorphisms, with minimum value of 0.41 in population MM, and maximum value of 0.82 in population PT. Cluster analysis showed that the populations' genetic variation was correlation to the environment gradient when the geographic distance among populations was big. In small geographic range,however, no correlation exists between genetic structure and ecological factors because of random genetic drift.

English Abstract↗
PubMed2002

Cloning and expression of a novel human galectin cDNA, predominantly expressed in placenta(1).

A novel human galectin cDNA (PPL13) was isolated by screening a human 18-week fetal brain library. The mRNA was predominantly expressed in placenta, while the expression of it was not or barely detectable in heart, brain, lung, liver, skeletal muscle, kidney, and pancreas by Northern blot. COS-7 cells transfected with cDNA encoding human PPL13 sequestered the protein in nuclei although it lacked any known nuclear localization signal. STS of Unigene Hs. 24236 placed the cDNA to human chromosome 19q13.2.

Amino Acid Sequence↗
PubMed2002

Cloning and expression pattern of a spermatogenesis-related gene, BEX1, mapped to chromosome Xq22.

Through screening a human fetal brain cDNA library, a cDNA similar to the mouse Bex1 was isolated. This new gene was named brain expressed X-linked protein 1 (BEX1). Northern blot analysis revealed a 1.0 kb transcript highly expressed in brain, pancreas, testis, and ovary, with lower levels present in heart, placenta, liver, kidney, spleen, thymus, prostate, small intestine, colon (no mucus), thyroid, spinal cord, and adrenal gland. No hybridization signal was seen in lung, skeletal muscle, peripheral blood leukocyte, stomach, lymph node, trachea, and bone marrow. The BEX1 gene was localized to chromosome band Xq22 between markers between DXS990 and DXS1059 by screening Stanford radiation hybrid G3 panels. In situ hybridization of mouse testis using BEX1 as a probe detected the signal in the pachytene spermatocytes and spermatids but not in spermatogonia. Furthermore, it was not detected at 6, 9, and 12 days postpartum, was present in low amount on Days 15 and 18 and its expression increased sharply after the initiation of puberty (about 21 days) in mouse testis.

Amino Acid Sequence↗
PubMed2002

Cloning and identification of a cDNA that encodes a novel human protein with thrombospondin type I repeat domain, hPWTSR.

A cDNA was isolated from the fetal brain cDNA library by high throughput cDNA sequencing. The 2390 bp cDNA with an open reading fragment (ORF) of 816 bp encodes a 272 amino acids putative protein with a thrombospondin type I repeat (TSR) domain and a cysteine-rich region at the N-terminus, so it is named hPWTSR. We used Northern blot detected two bands with length of about 3 kb and 4 kb respectively, which expressed in human adult tissues with different intensities. The expression pattern was verified by RT-PCR, revealing that the transcripts were expressed ubiquitously in fetal tissues and human tumor tissues too. However, the transcript was detected neither in ovarian carcinoma GI-102 nor in lung carcinoma LX-1. Blast analysis against NCBI database revealed that the new gene contained at least 5 exons and located in human chromosome 6q22.33. Our results demonstrate that the gene is a novel member of TSR supergene family.

Adult↗
PubMed2001

Expression, Purification, Crystallization and Preliminary X-ray Diffraction Analysis of the Mutant Pro(229)Ser of Thermostable Catechol 2,3-dioxygenase.

The mutant Pro(229)Ser of thermostable catechol 2,3-dioxygenase (TC23O) was expressed and purified. Enzymatic analysis revealed that its thermostability was decreased, the temperature corresponding to 50% enzyme activity being about 10.2 degrees lower than that of the wild type TC23O. Its kinetic parameter k(cat)/K(m) value (4.89x 10(6) mol(-1).s(-1)) was lower than that of the wild type TC23O(6.97x10(6) mol(-1).s(-1)). By the hanging-drop vapor-diffusion method using polyethylene glycol 400 as a precipitant, the mutant Pro(229)Ser of TC23O crystallizedat 4 degrees. X-ray diffraction analysis revealed that the crystals belong to the orthorhombic space group I(222) with unit-cell parameters a 7.059 nm, b 7.415 nm, c 13.311 nm, and they diffracted to at least 0.24 nm resolution. Assuming the presence of 2 molecules of the mutant Pro(229)Ser in the asymmetric unit, the Matthews parameter (Vm) was calculated to be 2.49x10( 3) nm(3).D(-1), and the solventcontent was about 51%. The crystal structure determination is now in progress.

Journal Article↗
PubMed1998

Overexpression, Purification and Characterization of Thermostable Catechol 2,3-dioxygenase.

Catechol 2,3-dioxygenase(EC 1.13.11.2) from Bacillus stearothermophilus has been shown to be highly thermostable. In order to obtain sufficient enzyme for its characterization, and to analyze the molecular basis of its intrinsic stability, the gene coding for this enzyme was sub-cloned and overexpressed in E. coli. After heat denaturation, ammonium sulfate fractionation, DEAE-52 ion-exchange chromatography and phenyl-Sepharose CL-4B hydrophobic chromatography, the enzyme was purified to homogeneity and the yield was 16%. The enzyme is a homotetramer. The molecular weight of each subunit is 36.4 kD as determined by SDS-PAGE. Using the tyrosine difference spectrum method, the extinction coefficient of the enzyme at 279.2 nm is estimated as 89(mmol/L)(-1).cm(-1), the optimum temperature and pH of the enzyme is 60 degrees and 8.0, respectively. The K(m) for catechol of the enzyme is 1.24x10(-5) mol/L, K(cat) is 24 000 min(-1). Acetone competitively inhibited the enzyme activity to catechol, with an inhibition constant K(i) of about 165 mmol/L. The thermostability of the enzyme is reflected by its unaltered catalytic activity over 1 h at 65 degrees. Irreversible thermal denaturation becomes significant between 70-80 degrees. THE pH stability of the enzyme and its resistance toward chemical denaturation such as urea and SDS confirm the intrinsic stability of the enzyme. It was also studied that the effect of different pHs on the molecular structure of the enzyme, using circular dichroism spectra and intrinsic fluorescence analysis.

Journal Article↗
PubMedDate not supplied

Assignment of human sprouty 4 gene to chromosome segment 5q32 approximately 33 and analysis of its pattern of expression.

The human sprouty 4 (SPRY4) gene was localized to chromosome band 5q32 approximately 33 by screening the Stanford radiation hybrid G3 panel using a SPRY4-specific primer pair for PCR. Northern blot analysis revealed two different mRNAs (5 kb and 2 kb) in liver, skeletal muscle, heart, lung, kidney, spleen, placenta and small intestine. Reverse transcriptase-PCR analysis showed that SPRY4 was expressed in all tested tissues to different levels.

Blotting, Northern↗