Biomedical subjects

Guo-Ping Zhao

Publications and source records attributed to Guo-Ping Zhao.

At least 37 records · Page 2Linked to original sources

PubMed2004

His23beta and Glu455beta of the Pseudomonas sp. 130 glutaryl-7-amino cephalosporanic acid acylase are crucially important for efficient autoproteolysis and enzymatic catalysis.

Glutaryl-7-amino cephalosporanic acid acylase is a member of the N-terminal nucleophilic hydrolase family of enzymes. The crystal structure of the acylase reveals there is a Ser-His-Glu motif composed of Ser1beta, His23beta, and Glu455beta near the active site. This mimics the catalytic triad of Ser-His-Asp in serine proteases. Experiments prove that maturation of this enzyme involves autoproteolysis. It has been shown that Ser1beta is the catalytic residue for the autoproteolysis and catalytic reaction. Our works on site-directed mutagenesis followed by the characterization of mutant enzymes demonstrated that His23beta is essential for autoproteolysis whereas Glu455beta is responsible for the efficiency of the process. Neither His23beta nor Glu455beta is essential for the acylase activity, although they affect the catalytic efficiency.

Amino Acid Sequence↗

PubMed2004

Identification and analysis of genes present in Leptospira interrogans serovar lai but absent in L. biflexa serovar monvalerio.

Genes present in virulent bacterial strains but absent in avirulent close relatives can be of great biologic and clinical interest. This project aimed to identify strain specific DNA sequences of Leptospira interrogans serovar lai, which is absent in the saprophytic L. biflexa serovar monvalerio, via suppression subtractive hybridization with the former as the tester while the latter as the driver. The mixture of PCR amplified DNA fragments from two subtractive hybridization experiments were cloned into pMD18-T vector and the positive clones were identified by dot blotting against the chromosome DNA of the two strains individually. After DNA sequencing and analysis, the distribution of these genomic fragment sequences in a panel of pathogenic and nonpathogenic leptospires was investigated employing dot blot analysis. Among the 188 positive clones randomly chosen, 24 contained the tester strain specific genomic regions, of which, 5 were non-coding fragments while the others contained 23 distinct protein coding sequences. Besides 9 genes encoding functional proteins, 12 genes encode unknown proteins and the rest two genes encode proteins with recognizable domain structures, one for a putative leucine-rich repeats (LRR) family protein while the other as an outer-membrane protein. Our experiment results indicated that suppression subtractive hybridization is effective for screening specific DNA sequences between two leptospiral strains, and some of these sequences might be responsible for virulence determination. Further analysis of these DNA sequences will provide important information on the pathogenesis of Leptospira.

DNA, Bacterial↗

PubMed2004

Identification and characterization of hmr19 gene encoding a multidrug resistance efflux protein from Streptomyces hygroscopicus subsp. yingchengensis strain 10-22.

The hmr19 gene was cloned from Streptomyces hygroscopicus subsp. yingchengensis strain 10-22, a bacterium strain producing agricultural antibiotics. Sequence similarity comparison indicates that hmr19 gene may encode a predicted protein with 14 putative transmembrane alpha-helical spanners, belonging to the drug:H(+) antiporter-2 family of the major facilitator superfamily. The expression of hmr19 in the mycelium of strain 10-22 was detected by Western blotting analysis. Gene replacement technology was employed to construct an hmr19 disruption mutant. The growth inhibition test against different antibiotics indicated that the mutant strain was 5-20 fold more susceptible to tetracycline, vancomycin and mitomycin C than the parental wild type strain. The mutant took up tetracycline much faster and accumulated more antibiotics than the wild type strain 10-22. While with the addition of an energy uncoupler, carbonyl cyanide m-chlorophenylhydrazone, the characteristics of the accumulation of [(3)H]tetracycline in these two strains were almost the same. It was thus concluded that hmr19 encoded a multidrug resistance efflux protein.

Amino Acid Sequence↗

PubMed2004

[Study on the mechanism of traditional Chinese medicines in promoting gastrointestinal peristalsis].

Traditional Chinese medicine has accumulated rich experience in treating dysfunction of gastrointestinal peristalsis. In recent years, a large number of studies have been made on the mechanism and effects of traditional Chinese medicines on the gastrointestinal peristalsis, and the concept of "gastrointestinal promoting Chinese medicine" has been advocated. These traditional Chinese medicines can be divided into three types: promoting the gastrointestinal peristalsis, inhibiting the gastrointestinal peristalsis, and bi-directional modulating. The in vivo and/or in vitro experiments showed that some of the traditional Chinese medicines for activating blood or regulating qi could promote the stomach peristalsis, and the traditional Chinese medicines for moistening intestines to relieve constipation or invigorating spleen to promote digestion could accelerate the intestinal peristalsis. The mechanism lies in the neuroregulation and gut-peptide regulation. Further research on multi-regulation and of multi-target should be done, for the mechanism of the traditional Chinese medicines in regulating the gastrointestinal peristalsis is far more complicated.

Drugs, Chinese Herbal↗

PubMed2004

[Expression and significance of PCNA and p27 in benign prostate hypertrophy and prostate carcinoma].

OBJECTIVE: To investigate the expression of PCNA and p27 in human benign prostate hypertrophy (BPH) and prostate carcinoma (PCa) and their effect on the genesis and progression of the tumor. METHODS: The paraffin-embedded sections of 30 cases with BPH and 37 cases with PCa were collected. The expression of p27 and PCNA protein were examined by S-P immunohistochemical method. Comparative analysis for BPH and pathological grade and clinical stage of PCa was performed. RESULTS: The expression of PCNA in BPH (3.3%) was significantly lower than that in Pca (83.8%, P < 0.01). The expression of p27 in BPH (70.0%) was significantly higher than that in Pca (27.0%, P < 0.05). The expression of p27 was not correlated with histological grade and clinical stage in Pca (P > 0.05). An inverse correlation was found between p27 and PCNA expression in BPH (P < 0.01), while no correlation was found in Pca (P > 0.05). CONCLUSION: The loss or decreased expression of p27 protein may be related to the genesis of benign prostate hypertrophy, but not to the development of prostate carcinoma; the overexpression of PCNA may play an important role in the malignant behavior and progression of prostate carcinoma.

Adenocarcinoma↗

PubMed2003

Unique physiological and pathogenic features of Leptospira interrogans revealed by whole-genome sequencing.

Leptospirosis is a widely spread disease of global concern. Infection causes flu-like episodes with frequent severe renal and hepatic damage, such as haemorrhage and jaundice. In more severe cases, massive pulmonary haemorrhages, including fatal sudden haemoptysis, can occur. Here we report the complete genomic sequence of a representative virulent serovar type strain (Lai) of Leptospira interrogans serogroup Icterohaemorrhagiae consisting of a 4.33-megabase large chromosome and a 359-kilobase small chromosome, with a total of 4,768 predicted genes. In terms of the genetic determinants of physiological characteristics, the facultatively parasitic L. interrogans differs extensively from two other strictly parasitic pathogenic spirochaetes, Treponema pallidum and Borrelia burgdorferi, although similarities exist in the genes that govern their unique morphological features. A comprehensive analysis of the L. interrogans genes for chemotaxis/motility and lipopolysaccharide synthesis provides a basis for in-depth studies of virulence and pathogenesis. The discovery of a series of genes possibly related to adhesion, invasion and the haematological changes that characterize leptospirosis has provided clues about how an environmental organism might evolve into an important human pathogen.

Bacterial Adhesion↗

PubMed2003

Genome-based analysis of virulence genes in a non-biofilm-forming Staphylococcus epidermidis strain (ATCC 12228).

Staphylococcus epidermidis strains are diverse in their pathogenicity; some are invasive and cause serious nosocomial infections, whereas others are non-pathogenic commensal organisms. To analyse the implications of different virulence factors in Staphylococcus epidermidis infections, the complete genome of Staphylococcus epidermidis strain ATCC 12228, a non-biofilm forming, non-infection associated strain used for detection of residual antibiotics in food products, was sequenced. This strain showed low virulence by mouse and rat experimental infections. The genome consists of a single 2499 279 bp chromosome and six plasmids. The chromosomal G + C content is 32.1% and 2419 protein coding sequences (CDS) are predicted, among which 230 are putative novel genes. Compared to the virulence factors in Staphylococcus aureus, aside from delta-haemolysin and beta-haemolysin, other toxin genes were not found. In contrast, the majority of adhesin genes are intact in ATCC 12228. Most strikingly, the ica operon coding for the enzymes synthesizing interbacterial cellular polysaccharide is missing in ATCC 12228 and rearrangements of adjacent genes are shown. No mec genes, IS256, IS257, were found in ATCC 12228. It is suggested that the absence of the ica operon is a genetic marker in commensal Staphylococcus epidermidis strains which are less likely to become invasive.

Adhesins, Bacterial↗

PubMed2003

A high-resolution genetic and physical map of a mouse coat abnormality locus (Uncv).

More than a hundred of loci (genes) affect the development of mouse and human hair. A locus of Uncv (uncovered) has been confirmed to be involved in hairlessness for homozygote and sparse hair for heterozygote. Except hairlessness(or called uncovered coat), the homozygote was also accompanied by growth retard and puberty delay. Identification of the mutation in the gene will be important for understanding the related diseases in human. Although the uncovered locus (Uncv) has been mapped to the mouse distal chromosome 11(Chr11), the high-resolution genetic map and physical map of the locus has not been created. In this study, 2074 F2 mouse populations from backcross [BALB/c (Uncv/Uncv) x C3H (+/+)]x BALB/c (Uncv/Uncv) and [BALB/c (Uncv/Uncv) x C57BL/6 (+/+)] x BALB/c(Uncv/ Uncv) were genotyped using 16 polymorphic markers with an approximately 20 cM interval on mouse distal Chr11. By genetic linkage analysis, Uncv locus was mapped to an approximately 1.4 cM interval between markers D11Mit337 and D11Mit338 with the following order: proximal D11Mit338-D11Mit203 (Uncv)-D11Mit103 -D11Mit337 distal on mouse Chr11. And then, a contig of 35 BACs representing the Uncv-containing region was constructed. The contig covered 800-1000 kb region flanked by 189K10-SP6 and D11Mit103. Together, we have constructed the high-resolution genetic map and detailed physical map of the Uncv region. This will facilitate the identification of the Uncv loci.

Animals↗

PubMed2003

Putative hAPN receptor binding sites in SARS_CoV spike protein.

AIM: To obtain the information of ligand-receptor binding between the S protein of SARS-CoV and CD13, identify the possible interacting domains or motifs related to binding sites, and provide clues for studying the functions of SARS proteins and designing anti-SARS drugs and vaccines. METHODS: On the basis of comparative genomics, the homology search, phylogenetic analyses, and multi-sequence alignment were used to predict CD13 related interacting domains and binding sites in the S protein of SARS-CoV. Molecular modeling and docking simulation methods were employed to address the interaction feature between CD13 and S protein of SARS-CoV in validating the bioinformatics predictions. RESULTS: Possible binding sites in the SARS-CoV S protein to CD13 have been mapped out by using bioinformatics analysis tools. The binding for one protein-protein interaction pair (D757-R761 motif of the SARS-CoV S protein to P585-A653 domain of CD13) has been simulated by molecular modeling and docking simulation methods. CONCLUSION: CD13 may be a possible receptor of the SARS-CoV S protein, which may be associated with the SARS infection. This study also provides a possible strategy for mapping the possible binding receptors of the proteins in a genome.

Amino Acid Sequence↗

PubMed2003

Identification of probable genomic packaging signal sequence from SARS-CoV genome by bioinformatics analysis.

AIM: To predict the probable genomic packaging signal of SARS-CoV by bioinformatics analysis. The derived packaging signal may be used to design antisense RNA and RNA interfere (RNAi) drugs treating SARS. METHODS: Based on the studies about the genomic packaging signals of MHV and BCoV, especially the information about primary and secondary structures, the putative genomic packaging signal of SARS-CoV were analyzed by using bioinformatic tools. Multi-alignment for the genomic sequences was performed among SARS-CoV, MHV, BCoV, PEDV and HCoV 229E. Secondary structures of RNA sequences were also predicted for the identification of the possible genomic packaging signals. Meanwhile, the N and M proteins of all five viruses were analyzed to study the evolutionary relationship with genomic packaging signals. RESULTS: The putative genomic packaging signal of SARS-CoV locates at the 3' end of ORF1b near that of MHV and BCoV, where is the most variable region of this gene. The RNA secondary structure of SARS-CoV genomic packaging signal is very similar to that of MHV and BCoV. The same result was also obtained in studying the genomic packaging signals of PEDV and HCoV 229E. Further more, the genomic sequence multi-alignment indicated that the locations of packaging signals of SARS-CoV, PEDV, and HCoV overlaped each other. It seems that the mutation rate of packaging signal sequences is much higher than the N protein, while only subtle variations for the M protein. CONCLUSIONS: The probable genomic packaging signal of SARS-CoV is analogous to that of MHV and BCoV, with the corresponding secondary RNA structure locating at the similar region of ORF1b. The positions where genomic packaging signals exist have suffered rounds of mutations, which may influence the primary structures of the N and M proteins consequently.

Amino Acid Sequence↗

PubMed2003

[Expression and purification of recombinant SARS coronavirus spike protein].

A novel coronavirus (SARS-coronavirus, SARS-CoV) was discovered in association with cases of severe acute respiratory syndrome (SARS) recently. The first step in coronavirus infection is binding of the viral spike protein to certain receptor on host cells. The spike protein is the main surface antigen of the coronavirus and there should be antibodies against spike protein in patients serum. Thus, to develop and expression protein fragment from spike protein gene are the purposes of this experiment. Partial spike gene fragments (751-1925 bp, 2005-3410 bp, 1-1925 bp and 32-3659 bp) and its intact gene were cloned into pET32 or pGEX vectors, and transformed into competent Escherichia coli BL21(DE3) (pLysS), respectively. 63, 78, 98, 160 and 164 kD fusion proteins were successfully expressed with amounts of 35%, 34%, 24%, 17% and 5% of total cell protein. The soluble parts of the cell crude extract were then partially purified by GST affinity chromatography.

Blotting, Western↗

PubMed2003

[Sequence and functional analysis of the chromosome replication origin (oriC) of Streptoverticillum caespitosus ATCC27422].

Twenty-two DnaA boxes were identified in the chromosome replication origin (oriC) of Streptoverticillum caespitosus ATCC27422 based upon the characteristics of consensus sequences. The 21st and 22nd DnaA boxes overlapped 8 base pairs each other reversely. Compared with the oriC database of actinomycetes, similar overlapping DnaA boxes were recognized in several species of Streptomyces and Mycobacterium. These overlapping DnaA boxes were composed of the last two DnaA box (21st and 22nd) in the Streptomyces species, but the of 1st and 2nd ones in the Mycobacterium species. The consensus sequence of the overlapping DnaA box is CTGTGCACAA, one base longer than the normal DnaA box sequence presumably due to the overlapping structure. Although the DnaA boxes exist in the 189 792 bp region only, the 1 188 bp and 793 939 bp regions are also important to the DNA replication. Deletion of the 1 188 bp region may cause absolute loss of DNA replication initiation activity measured by the transformation efficiency of plasmids with truncated oriC. When the 793 939 bp region was truncated, the transformation efficiency reduced about 40%. If the oriC was cloned into a vector with partial flanking region sequences (partial dnaA and dnaN gene sequences), the transformation rate was about 4.3-fold lower than that of the construct containing the oriC region only. However, the transformants were much more similar to the host with respect to the morphology of colony and mycelium. The cis-regulatory functions of the flanking sequences, which may influence the initiation efficiency of the chromosome replication and/or the stability of replicon, are thus suggested.

Actinomycetales↗

PubMed2002

[Nucleotide sequence and protein sequence analysis of GL-7-ACA acylase from Pseudomonas sp. 130].

The nucleotide sequence and N-, C-terminal amino acid sequences of alpha,beta-subunit of glutaryl 7-ACA acylase C130 from Pseudomonas sp. 130 were determined. The alignment of the acylase C130 with the other acylases shows that it has high homology with the acylases from Pseudomonas sp. GK16 and C427, but low homology with the others. There is large difference in the N-terminal of alpha-subunit, while the N-terminal of beta-subunit has significant conservation.

Amino Acid Sequence↗

PubMed2002

[Construction of a proteomic map database].

The first proteomic map database of China will be published by Bioinformation Center and Research Center of Proteomics, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences.The database consists of physical layer, link layer and interfacial layer. As Java technique was introduced to construct the database system, the database does not depend on special working platform. With the search instruments users can easily look through the proteomic map and get concrete information of proteins they are interested in.

China↗

PubMed2002

[Establishment of gene replacement/disruption system through homologous recombination in Amycolatopsis mediterranei U32].

A gene replacement/disruption system of Amycolatopsis mediterranei U32 was developed based on the established electroporation conditions as well as appropriate selective markers. Through two-step selection, ahbas gene in U32 was replaced by a promoterless alpha-amylase gene constructed on the plasmid pDK110 of E. coli. The first single-crossover and the second double-crossover frequencies were approximately 0.5%-0.7% and 2%, respectively. Denaturation of the plasmid pDK110 increased the integration frequency about 7-10 folds, while electric shock treatment of the single-crossover recombinants increased the frequency of second crossover recombination about 5 folds. Employing denatured DNA fragments containing an apramycin-resistance gene flanked with regions of the respective genes, One-step disruption of rifO and amrA genes of U32 was also achieved with an efficiency of 30-50 transformants per microgram of DNA.

Actinomycetales↗

PubMed2002

[Cloning of the replication origin (oriC) of Streptoverticillum caespitosus and transformation of Streptomyces lividans ZX7].

Streptoverticillum caespitosus ATCC27422 is a producing strain of mitomycin A for cancer therapy. Taking the advantage of the conserved sequences of genes flanking the oriC of high G + C Gram-positive bacteria, a 1.3 kb DNA fragment containing oriC and its flanking region was cloned by PCR. Nuleotide sequence comparisons revealed that the cloned fragment is more than 80% identical to the same region of S. coelicolor. There are 22 DnaA-boxes in the oriC region, and the conserved sequence of DnaA-box is TTGTCCACA. The plasmid containing the oriC of S. caespitosus was constructed (pMJ9), and it was able to transform the protoplast of Streptomyces lividans ZX7 at the frequency of 3.2 x 10(2) transformants/micrograms plasmid DNA. The colony and mycelia's morphology of the transformants are normal. The constructed plasmid can exist stable in the host as a low copy extra-chromosome replicon. The high rate of the homology and the cross genus replication initiation activity suggests close relationship between Streptomyces and Streptoverticillum in the evolution. While the maximum likelihood phylogenetic tree based upon the oriC of S. caespitosus and several Streptomyces spp. revealed that S. caespitosus differed extensively from the Streptomyces spp. This result supports the separation of Streptoverticillum from Streptomyces.

Actinomycetales↗

PubMed2000

Cloning and Overexpression of the Mitomycin C Resistance Gene (mcr) from Streptoverticillium caespitosum.

Streptoverticillium caespitosum ATCC27422 is a major producer of an anti-cancer drug, mitomycin C. A 6.6 kb DNA fragment containing the mitomycin C resistance gene (mcr) was isolated from ATCC27422 by shotgun method in order to learn the molecular mechanism of mitomycin C resistance. By constructing a series of subclones from this 6.6 kb DNA fragment, the mitomycin C resistance gene was localized on a 3.1 kb DNA fragment. Sequence analysis revealed that the open reading frame of mcr gene was 1 347 bp in size, encoding 448 amino acids with ATG as initiation codon and TGA as termination codon. The mcr gene was specifically expressed under the control of T7 promoter in E.coli, and the resistance to mitomycin C in the transformant was over 100-fold higher than that in wild-type strain. The overexpression of mcr gene in E.coli is very helpful for the further research about the molecular mechanism of drug resistance.

Journal Article↗

PubMed1999

Site-directed Mutagenesis of the Active Center of Penicillin Acylase from E. coli ATCC 11105.

Site-directed mutagenesis and chemical modification were performed at Ser290 of the penicillin G acylase from E. coli ATCC11105. The Ser290 was substituted by Cys or Secys. Wild type and mutant proteins were purified, and the activities and kinetic constants of penicillin acylases for hydrolysis and synthesis were determined, respectively. Although their K(m) values were not changed, the k(cat) values of the thiol-PGA and seleno-PGA were decreased from 135s(-1) to 0.63s(-1) and 0.38s(-1) against NIPAB, and from 34.38s(-1) to 0.23s(-1) and 0.06s(-1) against penicillin G. Contrary to Choi's report(Choi K S (et al. J Bacteriology), 1992, 10 6270-6276), we found that hydrolysis activity was certainly kept in the mutant of penicillin acylase. In addition, the specific activities of synthesis were decreased by 5-fold and 20-fold, respectively.

Journal Article↗