Biomedical subjects

Y M Mao

Publications and source records attributed to Y M Mao.

At least 19 recordsLinked to original sources

PubMed2001

Purification, crystallization and preliminary X-ray studies of thermostable alkaline phosphatase from Thermus sp. 3041.

Thermostable alkaline phosphatase from Thermus sp. 3041 has been expressed in Escherichia coli, purified and crystallized. The crystals belong to space group P2(1)22(1), with unit-cell parameters a = 57.7, b = 69.9, c = 111.5 A. Diffraction data were collected to 2.54 A with a completeness of 91.1% (87.8% for the last shell), an R(merge) value of 0.105 (0.312) and an I/sigma(I) value of 9.5 (3.6).

Alkaline Phosphatase↗

PubMed2001

[Effects of Pro229-->Ser and Glu243-->Gly on the characters of thermostable catechol 2, 3-dioxygenase].

In order to investigate the effects of amino acid replacement on the characters of thermostable catechol 2, 3-dioxygenase, two mutants (Pro229Ser and Glu243Gly) of this enzyme were obtained by using the method of PCR random mutagenesis. The wild type thermostable catechol 2, 3-dioxygenase and these two mutants (Pro229Ser, Glu243Gly) were over expressed in E. coli TG1 and purified. The enzymatic characters and thermostability of the wild type enzyme and the two mutants (Pro229Ser, Glu243Gly) were analyzed. The results revealed that the optimum enzymatic temperature of the two mutants were the same as that of the wild type enzyme (60 degrees C) and the Kcat/Km value of Pro229Ser and Glu243Gly (4.89 +/- 0.01 x 10(6) mol-1 s-1 and 5.88 +/- 0.01 x 10(6) mol-1 s-1, respectively) were reduced compared with the wild type enzyme (6.97 +/- 0.01 x 10(6) mol-1 s-1). However, the thermostability of Pro229Ser extremely decreased 10.2 degrees C and the thermostability of Glu243Gly slightly increased 1.5 degrees C. It was proposed that Pro229 played an important role on the thermostability of thermostable catechol 2, 3-dioxygenase.

Amino Acid Sequence↗

PubMed2001

[Investigation of genotyping HLA DRB1 gene using oligoneucleotide arrays].

We have developed a method performed on an oligoneucleotide array for genotyping HLA DRB1. The routine method Phenol-chloroform was used to extract genome DNA of standard samples. A pair of sense and antisense primers were designed according to the sequence of DRB1 exon2, then the primers and the Cy5-dCTP were used in the following PCR, thus the PCR products were labelled with Cy5. Many genotyping probes which were immobilized on the DNA Microarray made by APS-PDC method were designed. The labelled PCR products were hybridized with them, the signals were sanned by sanner and analyzed by Imagene software. We have genotyped 33 standard samples which have 12 DRB1 subtypes. The experimental results showed that the arrays we made and the method we used are accurate and sensitive. This proved that the DNA Microarray technique is good for DRB1 genotyping. Compared with PCR-SSP and PCR-SSO methods, the genotyping chip method is more intuitionistic and has the advantage of integration.

Genotype↗

PubMed2000

[A study of a mutant of thermostable alkaline phosphatase].

To reveal the mechanism of protein thermostability, we used in vivo random mutagenesis to generate variants of pTAP503F which contained thermostable alkaline phosphatase (FD-TAP). After screening about 5,000 clones, we obtained 4 temperature-sensitive mutants. The study of enzymatic properties of one mutant (TAPM3) showed that the thermostability of the mutant enzyme descended a lot, compared to the wild type, while the thermoactivity remained stable. DNA sequencing showed that the G-A transition in position 1,239 resulted in the substitution from glysine to serine in position 427. This mutation conspicuously affected thermostability, Michaelis constant and energy of activation. This suggests that only one substitution of amino acid will make great changes in thermostability and other properties, meanwhile, side-chain size, charge of residues and so on, which loosen the structure of protein, will result in the descent of thermostability.

Alkaline Phosphatase↗

PubMed2000

[Detection of differentially expressed genes in hepatocellular carcinoma using DNA microarray].

Large scale gene expression profiles between normal hepatocytes and hepatocellular carcinomas are detected with high throughput microarray. DNA microarrays were made by spotting PCR products of 4,096 human cDNAs onto a specially treated glass slides. The probes were prepared by labelling normal tissue mRNA and cancer tissue mRNA with Cy3-dUTP and Cy5-dUTP separately through reverse transcription. The arrays were then hybridized against the cDNA probe mixture and the fluorescent signals were scanned. The obtained data were analyzed from 4 repeated experiments. There are 903 genes of which the average ratios between Cy3 signal and Cy5 signal are greater than 3.0. These genes identified through this approach are potential candidates for hepatocellular carcinoma diagnosis and therapy.

Carcinoma, Hepatocellular↗

PubMed2000

[Studies of the active site, thermostability and thermophilicity of the thermostable alkaline phosphatase by site-directed mutagenesis].

Through PCR-mediated mutagenesis, three mutants E68S, S70A and E68SS70A around active site S(69) were obtained. Their enzymatic characteristics was determined. It was found that the specific activity of E68S ascended 8 times while its optional reactive temperature climbed 20 degrees C and its Tm descended 3 degrees C; the specific activity of S70A ascended 1 time while its optional reactive temperature climbed 5 degrees C and its Tm descended 2 degrees C; the specific activity of E68SS70A descended 50% while its optional reactive temperature climbed 5 degrees C and its Tm descended 19 degrees C. These result implied that the amino acids, beside the active site, were contributed not only to enzymatic activity but also to its thermostability and thermophilicity. The work provided the direction for mutation to improve enzymatic specific activity and studying the mechanism of thermostability and thermophilicity.

Alkaline Phosphatase↗

PubMed2000

[Ultrastructural observation of E. coli K12 treated with antibacterial peptide CM4].

The effect of antibacterial peptide CM4 of Bombyx mori against E. coli K12 was investigated using scanning electron microscopy(SEM) and transmission electron microscopy (TEM). The ultrastructural changes of E. coli K12 were observed by the challenge of the purified antibacterial peptide CM4. The results showed that the antibacterial peptide caused a series of pathological changes on E. coli. SEM and TEM revealed aggregates of bacteria and SEM revealed wrinkled bacterial surfaces in the early stage. Thereafter, plasmolysis was observed with irregular holes appearing in the two ends of bacteria and the cytoplasmic contents of the cells leaking out. Finally, bacteria became empty vesicles and disintegrated into small fragments subsequently. Comparatively, the bacterial membrane was normal and the bacterial structure remained intact in the control group.

Animals↗

PubMed1999

Aerosolization of P2Y(2)-receptor agonists enhances mucociliary clearance in sheep.

The purpose of this study was to determine whether aerosolized INS316 (UTP) stimulates lung mucociliary clearance (MCC) in sheep and, if so, to compare its effects with INS365, a novel P2Y(2)-receptor agonist. In the first series of studies, we used a previously described roentgenographic technique to measure tracheal mucus velocity (TMV), an index of MCC, before and for 4 h after aerosolization of INS316 (10(-1) M and 10(-2) M) and INS365 (10(-1) M and 10(-2) M), or normal saline in a randomized crossover fashion (n = 6). In a second series of studies, we compared the ability of these agents to enhance total lung clearance. For these tests, the clearance of inhaled technetium-labeled human serum albumin was measured serially over a 2-h period after aerosolization of 10(-1) M concentration of each agent (n = 7). Aerosolization of both P2Y(2)-receptor agonists induced significant dose-related increases in TMV (P < 0.05) compared with saline. The greatest increase in TMV was observed between 15 and 30 min after drug treatment. The highest dose (10(-1) M) of INS316 produced a greater overall stimulation of TMV than did INS365 (10(-1) M). Both compounds, compared with saline, induced a significant increase in MCC (P < 0.05) within 20 min of treatment. This enhancement in MCC began to plateau at 60 min. Although the response to INS316 started earlier, there was no significant difference between the clearance curves for the two compounds. We conclude that inhaled P2Y(2)-receptor agonists can increase lung MCC in sheep and that for P2Y(2)-receptor stimulation TMV accurately reflects changes in whole lung MCC.

Aerosols↗

PubMed1999

[A study of a derivative of Taq DNA polymerase].

Using the method of double primer oligonucleotide-mediated mutagenesis, the high expression plasmid of TaqND236, a derivative of Taq DNA polymerase, was constructed. To determine the frameshift mutation frequency of the in vitro DNA synthesis, we constructed a Gapped-DNA system using the pFDPM118 (a mutant of pUC118 with a -1 frameshift mutation on the lacZ gene) as template. By calculating the ratio of blue and white colonies on the X-gal plate after transforming E. coli TG1, the frameshift mutation frequency of Taq and TaqND236 was measured. It was found that the replication fidelity of the deleted Taq-TaqND236 increased more than 10 folds.

DNA Replication↗

PubMed1999

[Correlation analysis of (G + C)% of coding sequence and thermostability of xylose isomerase of thermophiles].

Statistical analysis about amino acids and coding sequence of xylose isomerase were performed in a number of thermophiles and mesophiles. It was found that there are correlations between the (G + C)% of the coding sequence and that of 1st, 2nd and 3rd position of the code of amino acids. There were also positive (for hydrophobic amino acids) and negative (for hydrophilic amino acids) correlation between the content of individual amino acids in the enzyme protein and the (G + C)% of their respective coding sequence. It speaks for the notion that high content of (G + C) in the coding sequence tends to increase the thermostability of the corresponding protein. The results in the statistical analysis of amino acid substitutions leading to change in thermostability of the protein may also be interpreted in the same way. An increase in the (G + C)% of DNA of a bacterium can therefore not only increase the thermostability of DNA itself but its proteins as well. Evolutionary consequence concerning thermophily and coding system are discussed.

Aldose-Ketose Isomerases↗

PubMed1998

Purification, crystallization and preliminary X-ray diffraction studies on the thermostable catechol 2,3-dioxygenase of Bacillus stearothermophilus expressed in Escherichia coli.

The thermostable catechol 2,3-dioxygenase of Bacillus stearothermophilus has been crystallized. The crystal is probably in the space group I222 with unit-cell dimensions of a = 70.87, b = 74.60 and c = 133.69 A. A native data set has been collected with a completeness of 96% at 2.22 A resolution and an Rmerge value of 0.091.

Catechol 2,3-Dioxygenase↗

PubMed1998

[Thermostable alkaline phosphatase from Thermus sp. FD3041: cloning of the gene and expression in Escherichia coli].

A genomic library of Thermus sp. FD3041 which produces thermostable alkaline phosphatase (FD-TAP) was constructed with the vector pUC118 and the host E. coli TG1. 3-10kb inserted fragments of foreign DNA were identified in 90 percent of the 12,000 clones thus obtained. Five positive clones were detected after screening the plated library by the method of colony coloration for TAP in situ. Preliminary analysis of the enzyme expressed from one recombinant plasmid pTAP362 showed that the properties of the recombinant enzyme, such as the thermal stability and optimal temperature of reaction, were identical to those of the native enzyme. The gene of FD-TAP was located on the 2.0kb BamHI-HindIII fragment of the pTAP362, determined by its physical map and the change of enzyme activity in different partially deleted plasmids. Results of thermostability experiment in PCR thermal cycle showed that the FD-TAP would be suitable for labelling of primers and detection of PCR amplified products.

Alkaline Phosphatase↗

PubMed1993

On two transposable elements from Bacillus stearothermophilus.

Two transposable elements, IS5376 and IS5377, were identified in the thermophile Bacillus stearothermophilus CU21 based upon the following criteria: (1) both were found to appear on different plasmids introduced into the same host CU21; (2) signals of homology were found between the genomic DNA of CU21 and each of them; (3) different numbers of Southern hybridization bands were found for the genomic DNA of different strains of B. stearothermophilus; and (4) characteristic inverted repeats at both ends and direct repeats of the target DNA adjacent to them were found for both IS5376 and IS5377. Two open reading frames (ORFs) were detected for IS5376 and one for IS5377. The putative coding products of the ORFs are homologous to those of known ISs from mesophiles and are considered to be transposases. The results of analyses of nucleotide sequence and the deduced amino acid sequence suggest that IS5376 is a member of the IS21 family and that IS5377 is a member of the IS4 family.

Amino Acid Sequence↗

PubMed1991

recA gene dependence of replication of the Escherichia coli chromosome initiated by plasmid pUC13 integrated at predetermined sites.

Plasmid pUC13 was used to clone DNA fragments of known sites from the chromosome of Escherichia coli. Each chimeric plasmid was introduced individually into the same dnaA46 mutant strain LC381 and suppressive integration (Sin) strains were selected. By means of cotransduction the null mutation recA56 was then introduced into each Sin strain and growth of each recA56 derivative at 42 degrees C was scored. Strains that failed to grow at 42 degrees C depended upon the recA gene for replication. Three factors were shown to limit the viability of LC381 harboring different chimeric plasmids and affect the degree of recA gene dependence of chromosome replication in the Sin strains at 42 degrees C. It is suggested that these three constraints are the consequence of the organization of the E. coli chromosome, particularly the unique ability of terC to retard the progression of replication forks. Two classes of hypotheses concerning the function of the recA gene are considered.

Chromosome Mapping↗

PubMed1991

[Through homologous recombination pathway recA gene takes part in chromosome replication of E. coli initiated by integrated plasmid].

We have reported in our previous paper that replication of E. coli chromosome initiated by the integrated F' plasmid depends on the recA gene. Here we report on work dealing with the role played by recA, recB, recC, lexA alleles in chromosome replication. Our results show that the recA gene takes part in chromosome replication through homologous recombination rather than SOS pathway and that Chi hot spot is not concerned with. They also show that the ATP-dependent dsDNA exonuclease activity of the RecBC enzyme has nothing to do with the recA gene in chromosome replication.

Bacterial Proteins↗

PubMed1991

[recA gene dependence of chromosome replication of Escherichia coli initiated by mini-F plasmids integrated at predetermined sites].

mini-F plasmids carrying particular chromosomal fragments were constructed. Through homologous recombination they were expected to integrate onto predetermined sites of the chromosome of dnaA46 strain LC381. Chromosome replication of suppressive integration (Sin) strains were recA dependent or independent according to the site of integration of the plasmid: chromosome replication of those with the plasmid integrated close to oriC were found to be recA dependent at 40 degrees C; those with the plasmid integrated between oriC and terC were recA dependent on rich medium but independent on minimal medium; those with the plasmid integrated close to terC were recA dependent even on minimal medium.

Chromosomes↗

PubMed1990

[Replication characteristics of plasmids and their dependence of recA gene for the initiation of Escherichia coli chromosome replication in the integrated state].

E. coli dnaA46 fails to grow and form colonies at 40 degrees C; integrated with plasmid enables it to grow at 40 degrees C or 42 degrees C. In a previous paper we reported that the replication of chromosome initiated by the integrated F' plasmid was recA gene dependent. In this paper we report further the behavior of 5 plasmids and 2 phages as to their dependence of recA gene in the integrated state. It was demonstrated that the dependence or independence of recA gene is not related to the direction of replication of the plasmids or phages in the free state. The hypothesis that the function of recA gene is to convert the unidirectionally replicating machinery in the free state to the bidirectionally replicating one in the integrated state is refuted accordingly.

Chromosomes, Bacterial↗

PubMed1990

[Dependence of recA gene for the replication of chromosome of Escherichia coli initiated by the integrated mini-F plasmid carrying IS1 sequence].

We have reported the dependence of recA gene for the replication of chromosome of E. coli initiated by the F' plasmid but not the F plasmid. Mini-F plasmids with ISI sequence and origin of replication from F and F' plasmid have been constructed. 20% of the integrative suppression strains of these mini-F plasmids were found to be recA dependent, irrespective of the origin of replication (F or F' plasmid) and the direction of replication (uni- or bidirectional). The reported experimental results tend to suggest that the site of integration is of primary importance in the dependence vs independence of recA gene for the replication of the chromosome initiated by the integrated plasmid.

Base Sequence↗