Biomedical subjects

Weiping Yang

Publications and source records attributed to Weiping Yang.

15 recordsLinked to original sources

PubMed2006

Cyclooxygenase-independent effects of aspirin on HT-29 human colon cancer cells, revealed by oligonucleotide microarrays.

Aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) inhibit proliferation of human colon cancer cells in vitro. Transmission electron microscope detected morphological features of apoptosis in the aspirin-treated (5 mM, 72 h) HT-29 cells in which cyclooxygenoase-2 is catalytically inactive. We investigated aspirin-induced genome-wide expression changes in HT-29 cells and further studied the time- and concentration-dependent expression changes in 374 apoptosis-related genes, which is the first to show stimulation of genome-wide expression of HT-29 cells by aspirin. The most marked effects of aspirin are on ribosome assembly and rRNA metabolism, which could explain why the quasi-apoptotic morphological changes are not accompanied by a classical DNA ladder. These findings demonstrate that aspirin induces apoptosis in HT-29 cells, bolstering the hypothesis that apoptosis may be a mechanism by which NSAIDs inhibit colon carcinogenesis.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed2006

Structural studies of constitutive nitric oxide synthases with diatomic ligands bound.

Crystal structures are reported for the endothelial nitric oxide synthase (eNOS)-arginine-CO ternary complex as well as the neuronal nitric oxide synthase (nNOS) heme domain complexed with L: -arginine and diatomic ligands, CO or NO, in the presence of the native cofactor, tetrahydrobiopterin, or its oxidized analogs, dihydrobiopterin and 4-aminobiopterin. The nature of the biopterin has no influence on the diatomic ligand binding. The binding geometries of diatomic ligands to nitric oxide synthase (NOS) follow the {MXY}(n) formalism developed from the inorganic diatomic-metal complexes. The structures reveal some subtle structural differences between eNOS and nNOS when CO is bound to the heme which correlate well with the differences in CO stretching frequencies observed by resonance Raman techniques. The detailed hydrogen-bonding geometries depicted in the active site of nNOS structures indicate that it is the ordered active-site water molecule rather than the substrate itself that would most likely serve as a direct proton donor to the diatomic ligands (CO, NO, as well as O(2)) bound to the heme. This has important implications for the oxygen activation mechanism critical to NOS catalysis.

Biopterins↗

PubMed2006

Genome-wide analysis of the expression profile of Saccharomyces cerevisiae in response to treatment with the plant isoflavone, wighteone, as a potential antifungal agent.

The limitations of currently available antifungal agents and the rapid emergence of drug-resistant strains necessitate more efficient approaches to screening and developing novel antifungal drugs. The antifungal activity of the natural products of a series of plants was evaluated and wighteone, 5, 7, 4'-trihydroxy-6-(gamma,gamma-dimethylallyl)isoflavone showed excellent anti-yeast activity (MIC against Saccharomyces cerevisiae was 4 microg/ml). Transcriptome profiling of wighteone-treated S. cerevisiae indicated that wighteone is different from commonly used antifungal compounds in its mode of action.

Antifungal Agents↗

PubMed2005

Exploring the binding conformations of bulkier dipeptide amide inhibitors in constitutive nitric oxide synthases.

A series of L-nitroarginine-based dipeptide inhibitors are highly selective for neuronal nitric oxide synthase (nNOS) over the endothelial isoform (eNOS). Crystal structures of these dipeptides bound to both isoforms revealed two different conformations, curled in nNOS and extended in eNOS, corresponding to higher and lower binding affinity to the two isoforms, respectively. In previous studies we found that the primary reason for selectivity is that Asp597 in nNOS, which is Asn368 in eNOS, provides greater electrostatic stabilization in the inhibitor complex. While this is the case for smaller dipeptide inhibitors, electrostatic stabilization may no longer be the sole determinant for isoform selectivity with bulkier dipeptide inhibitors. Another residue farther away from the active site, Met336 in nNOS (Val106 in eNOS), is in contact with bulkier dipeptide inhibitors. Double mutants were made to exchange the D597/M336 pair in nNOS with N368/V106 in eNOS. Here we report crystal structures and inhibition constants for bulkier dipeptide inhibitors bound to nNOS and eNOS that illustrate the important role played by residues near the entry to the active site in isoform selective inhibition.

Animals↗

PubMed2005

Pharmacokinetic study of trimebutine maleate in rabbit blood using in vivo microdialysis coupled to capillary electrophoresis.

In vivo microdialysis was used together with capillary electrophoresis (CE) to monitor the concentration of trimebutine maleate (TM) in rabbit blood. Dialysis probe was perfused at 3 microl/min resulting in relative recovery of 26.6+/-3.1% (n=3). After a one step sample preparation the samples were injected directly into the capillary. TM was detected on-column using UV detector at 214 nm. Separation of TM from other components in the dialysate was achieved within 15 min. Evaluation was based on the relative collected peak height (TM/IS). The response for TM in the blood dialysate was linear over the range of 0.5-100 microg/ml. The detection limit of TM in the blood dialysate was 0.1 microg/ml (S/N=3). This method has been successfully applied to the pharmacokinetic study of trimebutine maleate in rabbit blood following oral administration of 200 mg/kg. It provides a fast and simple technique for the pharmacokinetic study of TM in vivo.

Administration, Oral↗

PubMed2005

Parallel detection and quantification using nine immunoassays in a protein microarray for drug from serum samples.

A protein microarray system for detection and quantification of nine prohibited drugs in serum is described. Chemically modified slides were chosen as the microarray substrates because of their suitable for drug-BSA printing. The developed protein microarray was able to preserve the biological function of the haptens, when immobilized on the microarray surface and demonstrated binding with their corresponding antibodies. The microarray could also be used for quantitative analysis when mouse IgG was chosen as an internal control for data processing. There was no qualitative difference between the results obtained using the protein microarray and ELISA. The protein microarray technology should be applicable to performing, simultaneously, large scale screening tests for many different analytes in serum.

Blood Chemical Analysis↗

PubMed2005

Genomic analysis of anti-hepatitis B virus (HBV) activity by small interfering RNA and lamivudine in stable HBV-producing cells.

Hepatitis B virus (HBV) causes acute and chronic hepatitis and hepatocellular carcinoma. Small interfering RNA (siRNA) and lamivudine have been shown to have anti-HBV effects through different mechanisms. However, assessment of the genome-wide effects of siRNA and lamivudine on HBV-producing cell lines has not been reported, which may provide a clue to interrogate the HBV-cell interaction and to evaluate the siRNA's side effect as a potential drug. In the present study, we designed seven siRNAs based on the conserved HBV sequences and tested their effects on the expression of HBV genes following sorting of siRNA-positive cells. Among these seven siRNAs, siRNA-1 and siRNA-7 were found to effectively suppress HBV gene expression. We further addressed the global gene expression changes in stable HBV-producing cells induced by siRNA-1 and siRNA-7 by use of human genome-wide oligonucleotide microarrays. Data from the gene expression profiling indicated that siRNA-1 and siRNA-7 altered the expression of 54 and 499 genes, respectively, in HepG2.2.15 cells, which revealed that different siRNAs had various patterns of gene expression profiles and suggested a complicated influence of siRNAs on host cells. We further observed that 18 of these genes were suppressed by both siRNA-1 and siRNA-7. Interestingly, seven of these genes were originally activated by HBV, which suggested that these seven genes might be involved in the HBV-host cell interaction. Finally, we have compared the effects of siRNA and lamivudine on HBV and host cells, which revealed that siRNA is more effective at inhibiting HBV expression at the mRNA and protein level in vitro, and the gene expression profile of HepG2.2.15 cells treated by lamivudine is totally different from that seen with siRNA.

Antiviral Agents↗

PubMed2004

Development of miniaturized competitive immunoassays on a protein chip as a screening tool for drugs.

BACKGROUND: Doping in sports has become a serious problem. Gas chromatography-mass spectrometry (GC-MS) serves as an effective reference method, but it is limited by low throughput and is therefore not suitable for large-scale screening. Use of protein chips for high-throughput screening of all athletes for prohibited substances could become an important complementary tool to GC-MS. METHODS: We developed a protein chip based on an aldehyde-activated glass slide containing 10 physically isolated arrays. The chip was used to screen urine from 1347 athletes for prohibited substances and to screen a negative control group consisting of 200 females and 120 males. Urine samples from 66 individuals known to be abusers, provided by the China Doping Control Center (CDCC), and 129 standard prohibited substances were tested as positive controls. RESULTS: All 1347 urine samples screened by means of the protein chips were also subjected to reference analysis by GC-MS at the CDCC. There was no qualitative difference between the results obtained with the two methods. The correlation coefficient (r(2)) for the quantitative results obtained with the protein chip and GC-MS was 0.991. CONCLUSIONS: The protein chip could be used to screen for a series of 16 prohibited drugs in urine samples. This system has the potential to become an effective screening method to test substances prohibited by the International Olympic Committee.

Doping in Sports↗

PubMed2004

Preparation of steroid antibodies and parallel detection of multianabolic steroid abuse with conjugated hapten microarray.

A conjugated hapten microarray based on miniature immunoassay for fast and multiplex detection of anabolic steroids is reported for the first time. This preliminary study investigated the possibility of using a microarray technology as a multisteroid detection assay. The microarray system used eight monoclonal antibodies raised against three steroid conjugates, 4-androsten-4-chloro-17beta-ol-3-one, 1,5alpha-androsten-1beta-methyl-17beta-ol-3-one, and 5beta-androsten-1-en-17beta-ol-3-one, which were conjugated to BSA by the active ester method. In addition to 4 commercial conjugated haptens, 18 steroid-BSA conjugates were synthesized and from all these a conjugated hapten microarray was fabricated. The analyzed substances included 42 types of anabolic steroid reference materials and 28 positive urine samples. Of these, 24 anabolic steroids and 12 positive urines were successfully detected.

Anabolic Agents↗

PubMed2004

Structures of the neuronal and endothelial nitric oxide synthase heme domain with D-nitroarginine-containing dipeptide inhibitors bound.

In a continuing effort to unravel the structural basis for isoform-selective inhibition of nitric oxide synthase (NOS) by various inhibitors, we have determined the crystal structures of the nNOS and eNOS heme domain bound with two D-nitroarginine-containing dipeptide inhibitors, D-Lys-D-Arg(NO)2-NH(2) and D-Phe-D-Arg(NO)2-NH(2). These two dipeptide inhibitors exhibit similar binding modes in the two constitutive NOS isozymes, which is consistent with the similar binding affinities for the two isoforms as determined by K(i) measurements. The D-nitroarginine-containing dipeptide inhibitors are not distinguished by the amino acid difference between nNOS and eNOS (Asp 597 and Asn 368, respectively) which is key in controlling isoform selection for nNOS over eNOS observed for the L-nitroarginine-containing dipeptide inhibitors reported previously [Flinspach, M., et al. (2004) Nat. Struct. Mol. Biol. 11, 54-59]. The lack of a free alpha-amino group on the D-nitroarginine moiety makes the dipeptide inhibitor steer away from the amino acid binding pocket near the active site. This allows the inhibitor to extend into the solvent-accessible channel farther away from the active site, which enables the inhibitors to explore new isoform-specific enzyme-inhibitor interactions. This might be the structural basis for why these D-nitroarginine-containing inhibitors are selective for nNOS (or eNOS) over iNOS.

Amino Acid Substitution↗

PubMed2003

Structural basis for dipeptide amide isoform-selective inhibition of neuronal nitric oxide synthase.

Three nitric oxide synthase (NOS) isoforms, eNOS, nNOS and iNOS, generate nitric oxide (NO) crucial to the cardiovascular, nervous and host defense systems, respectively. Development of isoform-selective NOS inhibitors is of considerable therapeutic importance. Crystal structures of nNOS-selective dipeptide inhibitors in complex with both nNOS and eNOS were solved and the inhibitors were found to adopt a curled conformation in nNOS but an extended conformation in eNOS. We hypothesized that a single-residue difference in the active site, Asp597 (nNOS) versus Asn368 (eNOS), is responsible for the favored binding in nNOS. In the D597N nNOS mutant crystal structure, a bound inhibitor switches to the extended conformation and its inhibition of nNOS decreases >200-fold. Therefore, a single-residue difference is responsible for more than two orders of magnitude selectivity in inhibition of nNOS over eNOS by L-N(omega)-nitroarginine-containing dipeptide inhibitors.

Amides↗

PubMed2003

A novel protein-mineral interface.

Transferrins transport Fe3+ and other metal ions in mononuclear-binding sites. We present the first evidence that a member of the transferrin superfamily is able to recognize multi-nuclear oxo-metal clusters, small mineral fragments that are the most abundant forms of many metals in the environment. We show that the ferric ion-binding protein from Neisseria gonorrhoeae (nFbp) readily binds clusters of Fe3+, Ti4+, Zr4+ or Hf4+ in solution. The 1.7 A resolution crystal structure of Hf-nFbp reveals three distinct types of clusters in an open, positively charged cleft between two hinged protein domains. A di-tyrosyl cluster nucleation motif (Tyr195-Tyr196) is situated at the bottom of this cleft and binds either a trinuclear oxo-Hf cluster, which is capped by phosphate, or a pentanuclear cluster, which in turn can be capped with phosphate. This first high-resolution structure of a protein-mineral interface suggests a novel metal-uptake mechanism and provides a model for protein-mediated mineralization/dissimilation, which plays a critical role in geochemical processes.

Animals↗

PubMed2002

The novel binding mode of N-alkyl-N'-hydroxyguanidine to neuronal nitric oxide synthase provides mechanistic insights into NO biosynthesis.

A series of N-alkyl-N'-hydroxyguanidine compounds have recently been characterized as non-amino acid substrates for all three nitric oxide synthase (NOS) isoforms which mimic NO formation from N(omega)-hydroxy-L-arginine. Crystal structures of the nNOS heme domain complexed with either N-isopropyl-N'-hydroxyguanidine or N-butyl-N'-hydroxyguanidine reveal two different binding modes in the substrate binding pocket. The binding mode of the latter is consistent with that observed for the substrate N(omega)-hydroxy-L-arginine bound in the nNOS active site. However, the former binds to nNOS in an unexpected fashion, thus providing new insights into the mechanism on how the hydroxyguanidine moiety leads to NO formation. Structural features of substrate binding support the view that the OH-substituted guanidine nitrogen, instead of the hydroxyl oxygen, is the source of hydrogen supplied to the active ferric-superoxy species for the second step of the NOS catalytic reaction.

Alkylation↗

PubMed2002

Synergistic anion and metal binding to the ferric ion-binding protein from Neisseria gonorrhoeae.

The 34-kDa periplasmic iron-transport protein (FBP) from Neisseria gonorrhoeae (nFBP) contains Fe(III) and (hydrogen)phosphate (synergistic anion). It has a characteristic ligand-to-metal charge-transfer absorption band at 481 nm. Phosphate can be displaced by (bi)carbonate to give Fe.CO(3).nFBP (lambda(max) 459 nm). The local structures of native Fe-PO(4)-nFBP and Fe.CO(3).nFBP were determined by EXAFS at the FeK edge using full multiple scattering analysis. The EXAFS analysis reveals that both phosphate and carbonate ligands bind to FBP in monodentate mode in contrast to transferrins, which bind carbonate in bidentate mode. The EXAFS analysis also suggests an alternative to the crystallographically determined position of the Glu ligand, and this in turn suggests that an H-bonding network may help to stabilize monodentate binding of the synergistic anion. The anions oxalate, pyrophosphate, and nitrilotriacetate also appear to serve as synergistic anions but not sulfate or perchlorate. The oxidation of Fe(II) in the presence of nFBP led to a weak Fe(III).nFBP complex (lambda(max) 471 nm). Iron and phosphate can be removed from FBP at low pH (pH 4.5) in the presence of a large excess of citrate. Apo-FBP is less soluble and less stable than Fe.nFBP and binds relatively weakly to Ga(III) and Bi(III) but not to Co(III) ions, all of which bind strongly to apo-human serum transferrin.

Anions↗

PubMed2002

[Whether the impairment of grafted liver was induced by the inflammatory cells in cold and warm ischemia after transplantation].

OBJECTIVE: To investigate whether the impairment of grafted liver after transplantation was induced by the same inflammatory cells in cold and warm ischemia. METHODS: Male SD rats were divided into two groups randomly, 24 grafted livers in each group were stored for 120 or 240 min at 4 degrees Centigrade Ringer's solution. Also male SD rats were divided into three groups, in which 24 grafted livers in each group were experienced warm ischemia ranged from 90, 120 to 150 min from non-heart-beating donor. The recipients were killed after 1, 3, 6, and 24 hours of transplantation for sample collection. RESULTS: Along with the prolongation of cold and warm ischemia time, the serum ALT and AST levels were increased gradually after transplantation. Light microscopy showed some necroses in hepatocytes after 3 and 6 hours of transplantation in cold ischemia, and some neutrophilic infiltration in sinusoids. There were a large number of hepatocytes necroses after 3, 6 hours of transplantation in warm ischemia from non-heart-beating donor and a lot of lymphocytic infiltration in sinusoids. The findings in electron microscopy were as the same as those found in light microscopy, and the lymphocytes which infiltrated in sinusoids in warm ischemia were identified as T lymphocytes in electron microscopy. CONCLUSIONS: The impairment of grafted livers after transplantation seems to be induced by two different inflammatory cells in cold and warm ischemia, that is, neutrophils mediate the cold ischemia-reperfusion, and T lymphocytes mediate the warm ischemia-reperfusion from non-heart-beating donor.

Alanine Transaminase↗