Human versus chimpanzee chromosome-wide sequence comparison and its evolutionary implication.
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Amino Acid Substitution↗
Biomedical subjects
G P Zhao
Publications and source records attributed to G P Zhao.
Dendritomas formed by fusion of mature dendritic cells with allogenic human hepatocellular carcinoma cells activate autologous cytotoxic T lymphocytes.
Mature dendritic cells (DCs) have highly expressed CD1a, MHC class I, MHC class II, B7-1, B7-2 and ICAM-I molecules, all of which are essential for activation of naïve T cells. In this study, dendritomas were formed by fusion of hepatocellular carcinoma (HCC) SMMC-7721 cells with autologous DCs in vitro. DCs were obtained from adherent monocytes cultured in the presence of GM-CSF and IL-4 and were matured in monocyte-conditioned media. Expression of MHC class II and HCC-specific antigen by these dendritomas were determined using a specific murine anti-HCC monoclonal antibody (mAb) specific for HCC cell line SMMC-7721, and a murine anti-human HLA-DR mAb, and was also confirmed using bi-dimensional flow cytometry and immuno-histostaining. Dendritomas were co-cultured with autologous T cells, resulting in activation of T cell proliferation and priming of naïve T cells to induce MHC class I restricted lysis of HCC SMMC-7721 cells. The results imply that these dendritomas may have potential for use in HCC immunotherapy.
Carcinoma, Hepatocellular↗
[Establishment of detective systems for GL-7ACA acylase expression].
Glutaryl-7-amino cephalosporanic acid (GL-7ACA) acylase catalyzes the conversion of GL-7ACA to 7-amino cephalosporanic acid (7-ACA). The product 7-ACA is a starting compound for semi-synthetic cephalosporin antibiotics in industry. In order to detect the expression and specific activity of protein-engineered GL-7ACA acylase accurately, two useful detective systems for its expression has been established, in which reporter genes xylE and lacZ were fused to the downstream the GL-7ACA acylase gene acy respectively and the activity of catechol dioxygenase or beta-galactosidase could indicate the amount of acy expression.
Catechol 2,3-Dioxygenase↗
[An exploration of animal behavior screen platform for novel gene function in central nervous system].
For the purpose of large-scale screening of novel gene functions in mammalian nervous system, we have developed an animal behavior-monitoring platform employing antisense-oligo technology. Twenty genes of different categories were chosen from a low abundant gene (c)DNA sub-library of rat brain. Antisense oligo-nucleotides of these genes were designed and synthesized according to the homologues of the genes in mouse for mouse behavior tests. These antisense oligos were injected into the lateral ventricles of mouse brain using a Hamilton micro-syringe, with saline and oligos of scramble sequences as controls. These mice were tested with the following behavior model paradigms: metabolism, open field behavior, tail flick latency, and step-down test. Out of the 20 genes tested, 14 genes showed significant behavioral differences from the control groups at the level of P value less than 0.05 or 0.001 in different behavior animal models.
Animals↗
Mutagenesis of N-terminal Amino Acid Residues in beta-subunit of Glutaryl-7-amino-cephalosporanic Acid Acylase C130.
In many GL-7ACA acylases, the first Ser residue at the N-terminal of beta-subunit is the catalytic center. In order to investigate relationship between the N-terminal structure and catalytic activities, peptide replacement and site-directed mutagenesis were performed at the N-terminal of beta-subunit of GL-7ACA acylase C130. When the N-terminal 8 amino acid residues of C130 were replaced by the corresponding sequence of penicillin acylases PAC and PGA, respectively, the first mutant B8PAC lost the activity of the acylase, and the second mutant B8PGA had lower activity with the K(m) value increasing from 0.44x10(-3)mol.L(-1) to 0.55x10(-3) mol.L(-1), and the k(cat) decreasing from 4.92 s(-1) to 1.64 s(-1). Although the substitution of Trp (beta4) by Tyr did not change the K(m) value, the k (cat) decreased to 2.29 s(-1). When the Trp was substitued by Leu, both the K ( m ) and k ( cat ) values decreased. Compared with the wild type, mutations of Ser (beta3) to Met, Ala and Cys caused decrease of K(m) values by 52.27%, 43.18% and 38.64%, respectively. Mutation of Asn (beta2) to Gln caused the K ( m ) value being increased by 5-fold, and k ( cat ) decreased by 10-fold. These results suggested that the N-terminal amino acid residues of beta-subunit in GL-7ACA acylase C130 are important for enzyme function.
Journal Article↗
Expression in Escherichia coli, purification and kinetic analysis of the aspartokinase and aspartate semialdehyde dehydrogenase from the rifamycin SV-producing Amycolatopsis mediterranei U32.
The operon encoding aspartokinase and aspartate semialdehyde dehydrogenase was cloned and sequenced from rifamycin-SV-producing Amycolatopsis mediterranei U32 previously. In the present work, these two genes were introduced into the auxotrophic Escherichia coli strain CGSC5074 (ask-) and E. coli X6118 (asd-), respectively. The A. mediterranei U32 asparto-kinase and aspartate semialdehyde dehydrogenase genes can be functionally expressed in E. coli and the gene products are able to substitute for the E. coli enzymes. Histidine-tagged aspartokinase and aspartate semialdehyde dehydrogenase were partially purified from E. coli cellular extracts and their kinetic characteristics were studied. Both aspartokinase and aspartate semialdehyde dehydrogenase showed typical Michaelis-Menten type substrate saturation patterns. Aspartokinase has Km values of 3.4 mM for aspartate and 2.3 mM for ATP, while aspartate semialdehyde dehydrogenase has Km values of 1.25 mM for DL-aspartate semialdehyde and 0.73 mM for NADP, respectively. Aspartokinase was inhibited by L-threonine, L-lysine, and L-methionine, but not by L-isoleucine and diaminopimelate. Aspartate semialdehyde dehydrogenase was not inhibited by any of the end-product amino acids at a concentration of less than 5 mM. Hill plot analysis suggested that asparto-kinase was subject to allosteric control by L-threonine. Repression of both aspartokinase and aspartate semi-aldehyde dehydrogenase gene transcription in A. mediterranei U32 by L-lysine, L-methionine, L-threonine, and L-isoleucine were found. The network of regulation of aspartokinase and aspartate semialdehyde dehydrogenase in rifamycin SV-producing A. mediterranei U32 is presented.
Actinomycetales↗
Synechocystis PCC 6803 contains a single gene for the beta subunit of tryptophan synthase with strong homology to the trpB genes of Arabidopsis and maize (Zea mays L.).
We report the sequence of the trpB gene of the cyanobacterium Synechocystis sp. PCC 6803. This gene was cloned from a plasmid library by functional complementation of a trpB mutant of Escherichia coli K-12. Among the known trpB sequences, the Synechocystis gene bears the greatest homology to the duplicated trpB genes of Arabidopsis thaliana and Zea mays. Southern and northern blotting analyses suggest that Synechocystis contains only a single trpB gene. In contrast to all other prokaryotes, Synechocystis has a trpB gene that is monocistronic. Attempts to construct a trpB null mutant of Synechocystis by standard techniques were unsuccessful, suggesting that this organism is unable to concentrate tryptophan from the external medium.
Amino Acid Sequence↗
An amino acid switch (Gly281-->Arg) within the "hinge" region of the tryptophan synthase beta subunit creates a novel cleavage site for the OmpT protease and selectively diminishes affinity toward a specific monoclonal antibody.
The in vitro susceptibility to endogenous proteases of the beta subunit of Escherichia coli tryptophan synthase was studied immunochemically. Whereas the wild-type beta subunit was apparently very stable, the missense mutant beta(B8), carrying an amino acid switch from Gly to Arg at residue 281, underwent specific proteolytic cleavage. Polyclonal chicken antibodies and monoclonal antibodies specific for the N terminus (monoclonal antibody (mAb) 15-1), the C terminus (mAb 93-6), and the "hinge" region (mAb 164-2) were used to study the hydrolysis of the beta(B8) polypeptide. Cleavage products of 30 kDa, from the N terminus, and 13 kDa, from the C terminus, were observed. These two polypeptides correspond to the well characterized F1 (N-terminal) and F2 (C-terminal) fragments that are generated during the limited tryptic proteolysis of the wild-type beta subunit. The outer membrane-associated protease OmpT was shown to be responsible for the cleavage of the beta(B8) mutant protein. Proteolytic cleavage, observed only under neutral non-denaturing conditions, was specific for the peptide bond between Arg281 and Met282. The Arg-Met peptide bond has not previously been reported to be susceptible to cleavage by the OmpT protease. The beta(B8) polypeptide had dramatically reduced affinity for mAb 164-2. This antibody interacted more strongly with the OmpT-generated F1-like fragment than with the intact beta(B8) protein. These results strongly suggest that the G281R mutation alters the conformation of the hinge region of the mutant beta subunit, particularly the beta-turn around Gly281. The implications with respect to the epitope recognized by mAb 164-2 are discussed.
Amino Acid Sequence↗
A single amino acid switch within the "hinge" region of the tryptophan synthase beta subunit of Escherichia coli that leads to diminished association with alpha subunit and arrested conversion of ESII to product.
The trpB8 mutation of Escherichia coli causes a major conformational change within the beta subunit of tryptophan synthase. The basis of this effect is a replacement of glycine 281 by arginine within a structurally important "hinge" region. The mutant subunit, beta(B8), is catalytically active only under certain conditions, both in vivo and in vitro. Physiologically, the availability of wild type alpha subunit is the most important determinant of catalytic proficiency (Zhao, G.-P., and Somerville, R. L. (1992) J. Biol. Chem. 267, 526-541; Zhao, G.-P., and Somerville, R. L. (1993) J. Biol. Chem. 268, 14912-14920). Through enzyme activity titration experiments it was shown that the alpha subunit of tryptophan synthase dramatically stimulates catalysis by the beta 2(B8) mutant enzyme. However, by size exclusion high performance liquid chromatography, the stability of the alpha.beta 2(B8) complex was markedly reduced in comparison with wild type. The alpha-mediated stimulation of catalysis by the beta 2(B8) mutant enzyme was enhanced by polyethylene glycol, a volume excluder. By absorption spectroscopy, it was shown that catalysis by the beta(B8) mutant protein is blocked in at least one step after the formation of a particular Schiff base intermediate (ESII). Either the alpha subunit or ammonium ion was able to overcome this block. The microenvironment of the ESII catalytic intermediate was examined by fluorescence spectroscopy. The data are consistent with a less hydrophobic environment for ESII in the beta 2(B8) mutant protein than in the wild type protein. These lines of evidence not only support a conformational switch model of open versus closed states within the beta subunit during the catalytic cycle but also suggest a functional role for the hinge region in the process of conformational switching.
Arginine↗
Genetic and biochemical characterization of the trpB8 mutation of Escherichia coli tryptophan synthase. An amino acid switch at the sharp turn of the trypsin-sensitive "hinge" region diminishes substrate binding and alters solubility.
The trpB8 mutation of Escherichia coli tryptophan synthase is unique in that the cells bearing this lesion are not only capable of utilizing indole for growth, but they also accumulate indole, under conditions of tryptophan limitation. The lesion was shown by DNA sequencing to be a G to C transversion at nucleotide 5528 of the trp operon, resulting in a Gly to Arg switch at codon 281. Gly-281, within the trypsin-sensitive "hinge" region, is invariant among all known beta polypeptides. The catalytic activity of the mutant beta 2(B8) protein is dramatically stimulated by alpha subunit, both in vivo and in vitro. In the absence of alpha subunit, ammonium ion effectively stimulated the activity in an apparently cooperative manner. The pH optimum for the mutant subunit was 9.8, which is 2 units higher than that of wild type. In contrast to the wild-type subunit, beta(B8) partially aggregated within cells upon overexpression. At the optimal concentration of ammonium ions (2.25 M), the beta 2(B8) mutant enzyme displayed lower affinity than wild-type enzyme toward indole and L-serine, but the Vmax was almost unchanged. The physicochemical behavior of beta 2(B8) is supported by computer graphic modeling studies. An open versus closed model of conformational change within the beta 2 protein is proposed. A plausible role for the hinge region is discussed.
Amino Acid Sequence↗
[The correlation between hue discrimination and color brightness sensitivity in glaucoma].
Based on studies of 17 eyes with ocular hypertension and 72 eyes with glaucoma, it was shown that (1) significant correlation existed between hue discrimination and blue brightness sensitivity in patients with ocular hypertension and glaucoma, and (2) very significant correlation existed between hue discrimination and green brightness sensitivity in patients with glaucoma. The significance of the relationship between the two color visual functions was discussed.
Adult↗