Biomedical subjects

Tao Sun

Publications and source records attributed to Tao Sun.

At least 55 records · Page 3Linked to original sources

PubMed2005

[Calculation of environmental flows in river reaches based on ecological objectives].

Based on the identifying the ecological objectives, environmental flows in river reaches is calculated after the relation between parameters of objectives, and river discharges is determined. The ecological objectives are determined in a two step process: theobjective will be determined for the critical period of the year, then the temporal variation will be defined. Considering the compatibility between the different kinds of environmental flow requirements, the strictest objective is settled to be the ecological objectives for the critical period of the year. The temporal variation of the natural river discharge monthly is settled to be the temporal variation of ecological objectives. In the studies of the environmental flows in the river reaches downstream for Guanting reservoir in the Yongding River of Haihe River Basin, the requirements of velocities for spawning of the fish in April are regarded as the ecological objectives in the most critical period because this also is the period of highest water demand for irrigation. The relation between objectives and river discharge is identified using historical data at the river station. The results indicate that the minimum, medium and ideal lever of annual environmental flow requirements are 1.56 x 10(8) m3, 5.97 x 10(8) m3 and 11.02 x 10(8) m3, about 7.19%, 27.51% and 50.78 % of the natural river discharge respectively. The ratio of water requirements monthly should be 20 % in the flood period (Aug.) and be 20% in biological propagation period in spring (Apr. - Jun.).

Conservation of Natural Resources↗

PubMed2004

A yeast two-hybrid technology-based system for the discovery of PPARgamma agonist and antagonist.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is an important therapeutic drug target against several diseases such as diabetes, inflammation, dyslipidemia, hypertension, and cancer. Ligand binding to PPARgamma is responsible for controlling the biological functions, and developing new technology to measure ligand-PPARgamma binding is significant for both the function study of the receptor and ligand discovery. In this study, we exploited an efficient approach for the discovery of PPARgamma agonist and antagonist via a yeast two-hybrid system based on the fact that PPARgamma interacts with the coactivator CBP (CREP-binding protein) ligand-dependently. We employed the MEL1 reporter gene instead of the traditionally used LacZ gene to evaluate the protein-protein interactions by conducting a convenient alpha-galactosidase assay in the yeast strain AH109 with genes of PPARgamma-LBD (ligand-binding domain) and CBP N terminus introduced. With this built screening platform, the EC(50) values of the PPARgamma agonists rosiglitazone, troglitazone, pioglitazone, indomethacin, 15-deoxy-Delta12,14-prostaglandin J(2) (15d-PGJ(2)), and GI262570 were investigated, and the quantitatively antagonistic effect by IC(50) of the PPARgamma typical antagonist GW9662 on the rosiglitazone agonistic activity was fully examined. The reliability of this presented system evaluated by the comparable agreement of EC(50) and IC(50) values for the test compounds with the reported ones indicated that this yeast two-hybrid-based approach is powerful for PPARgamma agonist and antagonist screening. In addition, because this screening system is designed for use in a microtiter plate format where numerous chemicals could be readily screened, it is hoped that this yeast two-hybrid screening approach may be adaptable for high-throughput settings.

Anilides↗

PubMed2004

In vitro biochemical and thermodynamic characterization of nucleocapsid protein of SARS.

The major biochemical and thermodynamic features of nucelocapsid protein of SARS coronavirus (SARS_NP) were characterized by use of non-denatured gel electrophoresis, size-exclusion chromatographic and surface plasmon resonance (SPR) techniques. The results showed that SARS_NP existed in vitro as oligomer, more probably dimer, as the basic functional unit. This protein shows its maximum conformational stability near pH 9.0, and it seems that its oligomer dissociation and protein unfolding occur simultaneously. Thermal-induced unfolding for SARS_NP was totally irreversible. Both the thermal and chemical denaturant-induced denaturation analyses showed that oligomeric SARS_NP unfolds and refolds through a two-state model, and the electrostatic interactions among the charge groups of SARS_NP made a significant contribution to its conformational stability.

Chromatography, Gel↗

PubMed2004

Inhibitor-resistant class A beta-lactamases: consequences of the Ser130-to-Gly mutation seen in Apo and tazobactam structures of the SHV-1 variant.

A bacterial response to the clinical use of class A beta-lactamase inhibitors such as tazobactam and clavulanic acid is the expression of variant beta-lactamases with weaker binding affinities for these mechanism-based inhibitors. Some of these inhibitor-resistant variants contain a glycine mutation at Ser130, a conserved active site residue known to be adventitiously involved in the inhibition mechanism. The crystallographic structure of a complex of tazobactam with the Ser130Gly variant of the class A SHV-1 beta-lactamase has been determined to 1.8 A resolution. Two reaction intermediates are observed. The primary intermediate is an acyclic species bound to the reactive Ser70. It is poorly primed for catalytic hydrolysis because its ester carbonyl group is completely displaced from the enzyme's oxyanion hole. A smaller fraction of the enzyme contains a Ser70-bound aldehyde resulting from hydrolytic loss of the triazoyl-sulfinyl amino acid moiety from the primary species. This first structure of a class A beta-lactamase lacking Ser130, the side chain of which functions in beta-lactam binding and possibly in catalysis, gives crystallographic evidence that the acylation step of beta-lactam turnover can occur without Ser130. Unexpectedly, the crystal structure of the uncomplexed Ser130Gly enzyme, also determined to 1.8 A resolution, shows that a critical Glu166-activated water molecule is missing from the catalytic site. Comparison of this uncomplexed variant with the wild-type structure reveals that Ser130 is required for orienting the side chain of Ser70 and ensuring the hydrogen bonding of Ser70 to both Lys73 and the catalytic water molecule.

Alanine↗

PubMed2004

Severe acute respiratory syndrome coronavirus 3C-like proteinase N terminus is indispensable for proteolytic activity but not for enzyme dimerization. Biochemical and thermodynamic investigation in conjunction with molecular dynamics simulations.

Severe acute respiratory syndrome (SARS) coronavirus is a novel human coronavirus and is responsible for SARS infection. SARS coronavirus 3C-like proteinase (SARS 3CL(pro)) plays key roles in viral replication and transcription and is an attractive target for anti-SARS drug discovery. In this report, we quantitatively characterized the dimerization features of the full-length and N-terminal residues 1-7 deleted SARS 3CL(pro)s by using glutaraldehyde cross-linking SDS-PAGE, size-exclusion chromatography, and isothermal titration calorimeter techniques. Glutaraldehyde cross-linking SDS-PAGE and size-exclusion chromatography results show that, similar to the full-length SARS 3CL(pro), the N-terminal deleted SARS 3CL(pro) still remains a dimer/monomer mixture within a wide range of protein concentrations. Isothermal titration calorimeter determinations indicate that the equilibrium dissociation constant (K(d)) of the N-terminal deleted proteinase dimer (262 microm) is very similar to that of the full-length proteinase dimer (227 microm). Enzymatic activity assay using the fluorescence resonance energy transfer method reveals that N-terminal deletion results in almost complete loss of enzymatic activity for SARS 3CL(pro). Molecular dynamics and docking simulations demonstrate the N-terminal deleted proteinase dimer adopts a state different from that of the full-length proteinase dimer, which increases the angle between the two protomers and reduces the binding pocket that is not beneficial to the substrate binding. This conclusion is verified by the surface plasmon resonance biosensor determination, indicating that the model substrate cannot bind to the N-terminal deleted proteinase. These results suggest the N terminus is not indispensable for the proteinase dimerization but may fix the dimer at the active state and is therefore vital to enzymatic activity.

Binding Sites↗

PubMed2004

Nucleocapsid protein of SARS coronavirus tightly binds to human cyclophilin A.

Severe acute respiratory syndrome coronavirus (SARS-CoV) is responsible for SARS infection. Nucleocapsid protein (NP) of SARS-CoV (SARS_NP) functions in enveloping the entire genomic RNA and interacts with viron structural proteins, thus playing important roles in the process of virus particle assembly and release. Protein-protein interaction analysis using bioinformatics tools indicated that SARS_NP may bind to human cyclophilin A (hCypA), and surface plasmon resonance (SPR) technology revealed this binding with the equilibrium dissociation constant ranging from 6 to 160nM. The probable binding sites of these two proteins were detected by modeling the three-dimensional structure of the SARS_NP-hCypA complex, from which the important interaction residue pairs between the proteins were deduced. Mutagenesis experiments were carried out for validating the binding model, whose correctness was assessed by the observed effects on the binding affinities between the proteins. The reliability of the binding sites derived by the molecular modeling was confirmed by the fact that the computationally predicted values of the relative free energies of the binding for SARS_NP (or hCypA) mutants to the wild-type hCypA (or SARS_NP) are in good agreement with the data determined by SPR. Such presently observed SARS_NP-hCypA interaction model might provide a new hint for facilitating the understanding of another possible SARS-CoV infection pathway against human cell.

Amino Acid Sequence↗

PubMed2004

Different hydroxyl radical scavenging activity of water-soluble beta-alanine C60 adducts.

Three C(60) derivatives [C(60) (NHCH(2)CH(2)COONa)(n)(H)(n)], n=1, 5, 9] (A, B, C) with different additional number of beta-alanine were synthesized by the control of relative amount of C(60) and beta-alanine added. Hydroxyl radical scavenging activity of the adducts was evaluated in a copper-catalyzed Haber-Weiss reaction by chemiluminescence technology. The 50% inhibition concentrations (IC(50)'s) of A, B, and C were 147.2 micromol/L, 76.3 micromol/L, and 96.2 micromol/L, respectively. The difference should be closely related to the numbers of residual C=C bonds in C(60), steric effect and electron-withstanding effect of amino group especially.

Dose-Response Relationship, Drug↗

PubMed2004

Cloning and functional characterization of the murine mastermind-like 1 (Maml1) gene.

The Notch signaling pathway controls cell fate decisions and plays a critical role in normal development and diseases. The human mastermind-like (MAML) family members (MAML1, 2 and 3) encode critical transcriptional co-activators for Notch receptors. In this study, we cloned a murine cDNA that is highly homologous to the human MAML1 gene, Maml1. Mouse Maml1 encodes a nuclear protein, binds to the ankyrin repeat domain of Notch receptors, forms a ternary complex with the intracellular domain of Notch (ICN) and the DNA binding protein CSL, and enhances Notch-induced transcription of the target gene, HES-1. Therefore, Maml1 is the murine homologue for human MAML1 and functions as a transcriptional co-activator for Notch signaling. We also characterized the organization of the mouse Maml1 gene: It spans at least 35 kilobases (kb) on chromosome 11 and contains five exons and four introns. Analysis of the 5' flanking region revealed that the promoter is TATA-less, and contains consensus binding sites for transcription factors such as Sp1, glucocorticoid receptor (GR), activating transcription factor (ATF) and cAMP response element-binding protein (CREB). Moreover, we examined Maml1 expression during early mouse development and found that Maml1 gene is expressed widely but selectively in several tissues. There seems to be close correlation of the spatial and temporal expression among Maml1, Notch1 and Hes1 in the central nervous system (CNS) during early development, implicating a role for the Maml1 gene in neurogenesis.

Amino Acid Sequence↗

PubMed2004

Photo-crosslinkable microcapsules formed by polyelectrolyte copolymer and modified collagen for rat hepatocyte encapsulation.

New anionic polyelectrolyte tetra-copolymers with photo-crosslinkable 4-(4-methoxycinnamoyl)phenyl methacrylate monomer in addition to a HEMA-MMA-MAA ter-copolymer system were synthesized. The tetra-copolymers were used to form photo-crosslinkable microcapsules with modified collagen by complex coacervation for rat hepatocytes encapsulation. The hepatocytes were encapsulated within a two-layered membrane comprising of modified collagen as the inner core and an outer photo-crosslinkable copolymer shell. Upon photo-crosslinking of the microcapsules with UV-Vis light irradiation, the mechanical strength and chemical stability of the microcapsules, and the cellular functions of the encapsulated hepatocytes were enhanced. Particularly, the mechanical stability of the microcapsules was dramatically strengthened. The new photo-crosslinkable tetra-copolymer formulation described in this article has opened a way to the development of hepatocyte microencapsulation technology for bioartifical liver assist device.

Animals↗

PubMed2004

Localization of infection-related epitopes on the non-structural protein 3ABC of foot-and-mouth disease virus and the application of tandem epitopes.

By means of overlapping peptides expressed in Escherichia coli in combination with Western-blotting, infection specific linear epitopes were identified on the non-structural protein 3ABC of FMDV. The epitopes reacted with sera from pigs or guinea pigs infected with different serotypes of FMDV, but not with sera from normal or vaccinated animals. A protein was constructed by tandem repeat of the epitope covering amino acid residues 141-190 on 3ABC. An ELISA based on the protein with tandem epitopes could be used as a diagnostic antigen for differentiating infected pigs from vaccinated ones.

Animals↗

PubMed2003

Cross-repressive interaction of the Olig2 and Nkx2.2 transcription factors in developing neural tube associated with formation of a specific physical complex.

In developing neural tube, the basic helix-loop-helix (bHLH) transcription factor Olig2 interacts with the homeodomain transcription factor Nkx2.2 at two distinct stages. During neuronogenesis, a cross-repressive interaction appears to establish the precise boundary between the p3 and pMN domains. At later times, a cooperative interaction is noted because Nkx2.2 promotes maturation of oligodendrocyte progenitor cells specified by expression of Olig2. We show here that the Olig2 protein can form a physical complex with Nkx2.2 protein in mammalian cells and yeast two-hybrid trap assay. This interaction is specific because Olig2 does not bind to a biologically irrelevant homeodomain transcription factor (Nkx6.1), and Nkx2.2 does not interact with a biologically irrelevant bHLH protein (NeuroD). Deletion mapping analysis suggests that formation of an Olig2-Nkx2.2 physical complex is insufficient for the induction of oligodendrocyte progenitors in developing spine; however, the protein-protein interaction observed might be important for the cross-repressive interaction between Olig2 and Nkx2.2 that helps to establish the pMN-p3 boundary in the developing spinal cord.

Animals↗

PubMed2003

Molecular cloning, expression, purification, and mass spectrometric characterization of 3C-like protease of SARS coronavirus.

Severe acute respiratory syndrome (SARS) is an acute respiratory illness, which has broken out in China. It has been known that SARS coronavirus (SARS_CoV) is a novel human coronavirus and is responsible for SARS infection. Belonging to one of the major proteins associated with SARS_CoV, SARS 3C-like protease (SARS_3CL(pro)) functions as a cysteine protease engaging in the proteolytic cleavage of the viral precursor polyprotein to a series of functional proteins required for coronavirus replication and is considered as an appealing target for designing anti-SARS agents. To facilitate the studies regarding the functions and structures of SARS_3CL(pro), in this report the synthetic genes encoding 3CL(pro) of SARS_CoV were assembled, and the plasmid was constructed using pQE30 as vector and expressed in Escherichia coli M15 cells. The highly yielded ( approximately 15mg/L) expressed protease was purified by use of NTA-Ni(2+) affinity chromatography and FPLC system, and its sequence was determined by LC/MS with the residue coverage of 46.4%.

Amino Acid Sequence↗

PubMed2003

Use of ultrathin shell microcapsules of hepatocytes in bioartificial liver-assist device.

We previously encapsulated hepatocytes in ultrathin shell microcapsules and showed them to have enhanced differentiated functions over cells cultured in monolayer. Here we have used these microencapsulated hepatocytes in a bioartificial liver-assisted device (BLAD) with a rat hepatectomy model. Primary rat hepatocytes were encapsulated in 150- to 200-microm microcapsules, using an electrostatic droplet generator. The microencapsulated hepatocytes exhibited good in vitro urea synthesis activity in plasma from rats with fulminant hepatic failure (FHF). The ex vivo hemoperfusion was conducted in FHF rats by perfusing plasma at a rate of 1-2 mL/min through 1.5-2 x 10(8) encapsulated hepatocytes packed into a packed-bed bioreactor. Hemoperfusion with the bioreactor was initiated 5 h after operative induction of liver failure and continued for 7 h. The BLAD-treated rats showed improvements over the control groups in survival time and metabolic indicators, including ammonia and total bilirubin levels. Furthermore, expanded bed adsorption (EBA) detoxification technology using Streamline-SP resin was explored to complement the bioreactor with microencapsulated hepatocytes. In vitro experiments indicated that serum ammonia could be specifically removed in dose-dependent manner, whereas the total serum proteins were unaffected by the resin. In ex vivo experiments, hemoperfusion with the resin was initiated 3 h after operative induction of liver failure and continued for 7 h. The resin-treated rats showed obvious serum ammonia removal with no observable total blood protein and blood cell adsorption. Therefore, Streamline-SP resin can potentially be integrated into a BLAD for improved efficacy.

Ammonia↗

PubMed2003

Comparison of beta-lactamases of classes A and D: 1.5-A crystallographic structure of the class D OXA-1 oxacillinase.

The crystallographic structure of the Escherichia coli OXA-1 beta-lactamase has been established at 1.5-A resolution and refined to R = 0.18. The 28.2-kD oxacillinase is a class D serine beta-lactamase that is especially active against the penicillin-type beta-lactams oxacillin and cloxacillin. In contrast to the structures of OXA-2, OXA-10, and OXA-13 belonging to other subclasses, the OXA-1 molecule is monomeric rather than dimeric and represents the subclass characterized by an enlarged Omega loop near the beta-lactam binding site. The 6-residue hydrophilic insertion in this loop cannot interact directly with substrates and, instead, projects into solvent. In this structure at pH 7.5, carboxylation of the conserved Lys 70 in the catalytic site is observed. One oxygen atom of the carboxylate group is hydrogen bonded to Ser 120 and Trp 160. The other oxygen atom is more exposed and hydrogen bonded to the Ogamma of the reactive Ser 67. In the overlay of the class D and class A binding sites, the carboxylate group is displaced ca. 2.6 A from the carboxylate group of Glu 166 of class A enzymes. However, each group is equidistant from the site of the water molecule expected to function in hydrolysis, and which could be activated by the carboxylate group of Lys 70. In this ligand-free OXA-1 structure, no water molecule is seen in this site, so the water molecule must enter after formation of the acyl-Ser 67 intermediate.

Amino Acid Sequence↗

PubMed2003

A 3D model of SARS_CoV 3CL proteinase and its inhibitors design by virtual screening.

AIM: To constructed a three-dimensional (3D) model for the 3C like (3CL) proteinase of SARS coronavirus (SARS-CoV), and to design inhibitors of the 3CL proteinase based on the 3D model. METHODS: Bioinformatics analyses were performed to search the homologous proteins of the SARS-CoV 3CL proteinase from the GenBank and PDB database. A 3D model of the proteinase was constructed by using homology modeling technique. Targeting to the 3D model and its X-ray crystal structure of the main proteinase (Mpro) of transmissible gastroenteritis virus (TGEV), virtual screening was performed employing molecular docking method to identify possible 3CL proteinase inhibitors from small molecular databases. RESULTS: Sequence alignment indicated that the SARS-CoV 3CL proteinase was extremely homologous to TGEV Mpro, especially the substrate-binding pocket (active site). Accordingly, a 3D model for the SARS-CoV 3CL proteinase was constructed based on the crystal structure of TGEV Mpro. The 3D model adopts a similar fold of the TGEV Mpro, its structure and binding pocket feature are almost as same as that of TGEV Mpro. The tested virtual screening indicated that 73 available proteinase inhibitors in the MDDR database might dock into both the binding pockets of the TGEV Mpro and the SARS-CoV 3CL proteinase. CONCLUSIONS: Either the 3D model of the SARS-CoV 3CL proteinase or the X-ray crystal structure of the TGEV Mpro may be used as a starting point for design anti-SARS drugs. Screening the known proteinase inhibitors may be an appreciated shortcut to discover anti-SARS drugs.

Computational Biology↗

PubMed2003

[Plasma polypeptide hormone levels in rats with gastric ulcer after exposure to intense noise].

OBJECTIVE: To observe changes of plasma polypeptide hormone levels in rats with gastric ulcer after exposure to intense noise, and to discuss their mechanism. METHODS: 80 Wistar rats were used in the study. Plasma levels of rat gastrin (GAS), motilin (MTL), osteocalcin (BGP), substance P (SP), neurotensin (NT) and somatostatin (SS) in rats were measured by radioimmunoassay. RESULTS: (1) In non-noise-exposure but with gastric ulcer group, the plasma MTL [(160.70 +/- 40.34) pg/ml] and BGP [(27.63 +/- 13.13) pg/ml] levels on 10 d after gastric ulcer model operation were remarkably higher than those in control group [(89.21 +/- 49.94) pg/ml, (9.10 +/- 1.38) pg/ml respectively] (P < 0.05 and P < 0.01), while the GAS level was remarkably descended [(107.00 +/- 21.75) vs (158.48 +/- 20.92) pg/ml] (P < 0.01). (2) In noise-exposure but without gastric ulcer group, the plasma MTL [(312.80 +/- 207.42) pg/ml] and BGP [(17.76 +/- 12.33) pg/ml] levels on 10 d were also significantly increased as compared with the control group (P < 0.01 and P < 0.05 respectively), while the GAS levels didn't change. (3) In noise-exposure + gastric ulcer group, the areas of gastric ulcer on 10 d and 40 d after noise and operation [(15.33 +/- 7.26) and (15.11 +/- 12.45) mm(2) respectively] were significantly larger than those of the control [(8.22 +/- 6.66), (3.67 +/- 9.90) mm(2)] (P < 0.05). The plasma MTL levels on 10 d and 40 d [(244.44 +/- 68.11) and (191.20 +/- 60.50) pg/ml respectively] were higher than those in control group [(160.70 +/- 40.34) and (93.10 +/- 52.90) pg/ml respectively] (P < 0.01). CONCLUSION: Intense noise exposure may make the rat gastric ulcer worsened and induce negative effect on healing of it. The gastrointestinal endocrine would be disturbed by combined effect of intense noise exposure with gastric ulcer in rats.

Animals↗

PubMed2002

Common developmental requirement for Olig function indicates a motor neuron/oligodendrocyte connection.

The oligodendrocyte lineage genes Olig1 and Olig2 encode related bHLH proteins that are coexpressed in neural progenitors. Targeted disruption of these two genes sheds light on the ontogeny of oligodendroglia and genetic requirements for their development from multipotent CNS progenitors. Olig2 is required for oligodendrocyte and motor neuron specification in the spinal cord. Olig1 has roles in development and maturation of oligodendrocytes, evident especially within the brain. Both Olig genes contribute to neural pattern formation. Neither Olig gene is required for astrocytes. These findings, together with fate mapping analysis of Olig-expressing cells, indicate that oligodendrocytes are derived from Olig-specified progenitors that give rise also to neurons.

Animals↗

PubMed2002

Identification of a family of mastermind-like transcriptional coactivators for mammalian notch receptors.

The molecular mechanisms by which Notch receptors induce diverse biological responses are not fully understood. We recently cloned a mammalian homologue of the Mastermind gene of Drosophila melanogaster, MAML1 (Mastermind-like-1 molecule) and determined that it functions as a transcriptional coactivator for Notch receptors. In this report, we characterize two additional genes in this Mastermind-like gene family: MAML2 and MAML3. The three MAML genes are widely expressed in adult tissues but exhibit distinct expression patterns in mouse early spinal cord development. All MAML proteins localize to nuclear bodies, share a conserved basic domain in their N termini that binds to the ankyrin repeat domain of Notch, and contain a transcriptional activation domain in their C termini. Moreover, as determined by using coimmunoprecipitation assays, each MAML protein was found to be capable of forming a multiprotein complex with the intracellular domain of each Notch receptor (ICN1 to -4) and CSL in vivo. However, MAML3 bound less efficiently to the ankyrin repeat domain of Notch1. Also, in U20S cells, whereas MAML1 and MAML2 functioned efficiently as coactivators with each of the Notch receptors to transactivate a Notch target HES1 promoter construct, MAML3 functioned more efficiently with ICN4 than with other forms of ICN. Similarly, MAML1 and MAML2 amplified Notch ligand (both Jagged2 and Delta1)-induced transcription of the HES-1 gene, whereas MAML3 displayed little effect. Thus, MAML proteins may modify Notch signaling in different cell types based on their own expression levels and differential activities and thereby contribute to the diversity of the biological effects resulting from Notch activation.

Amino Acid Sequence↗