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Biomedical subjects

Y Shi

Publications and source records attributed to Y Shi.

At least 109 records · Page 6Linked to original sources
PubMed2001

Determination of active component in silymarin by RP-LC and LC/MS.

Silybin, isosilybin, silydianin and silycristin in silymarin were separated and quantitatively determined by RP-HPLC. Two diastereoisomers of silybin and isosilybin were respectively separated by RP-HPLC and confirmed by LC/MS. Chromatographic condition consisted of column: Shim-pack VP-ODS (150 x 4.6mm i.d. 5 microm) and Pre-column (10 x 4.6 mm i.d. 5 microm); mobile phase: methanol and solvent mixture (water: dioxane=9:1) by gradient; flow rate: 1.5ml/min; column temp.: 40 degrees C; detector wavelength: 288 nm; The recovery of 99.66% for silycristin, 99.48% for silydianin, 100.0% for silybin and 98.72% for isosilybin was respectively obtained.

Antioxidants↗
PubMed2001

Selective regulation of gene expression by an orthogonal estrogen receptor-ligand pair created by polar-group exchange.

BACKGROUND: The nuclear and steroid hormone receptors function as ligand-dependent transcriptional regulators in eukaryotes. Hormone receptors have been engineered to selectively respond to synthetic ligands and used as remote regulators of gene expression for the study of gene function and as potential regulators of gene therapies. RESULTS: In this work, a new ligand-receptor engineering strategy called 'polar-group exchange' is used to create a mutant form of the estrogen receptor, ER(Glu353-->Ala), which lacks a carboxyl group critical for high-affinity binding of estradiol, but is able to transactivate in response to nanomolar concentrations of a carboxylate-functionalized estrogen analog, ES8. ES8 activates ER(Glu353-->Ala) at concentrations that do not appreciably activate the 'wild-type' receptor ER(wt). Two similar carboxylate-functionalized ligands, ES6 and ES7, do not induce transactivation function. Similar selectivities are observed in ligand-binding assays in vitro, which follow the trends predicted by molecular modeling. CONCLUSION: Polar-group exchange is an effective strategy for rationally engineering ligand-receptor pairs. The ER(E353A)/ES8 ligand-receptor pair should constitute a unique and functionally orthogonal ligand-dependent transcriptional regulator.

Cells, Cultured↗
PubMed2001

Structural analysis of a functional DIAP1 fragment bound to grim and hid peptides.

The inhibitor of apoptosis protein DIAP1 suppresses apoptosis in Drosophila, with the second BIR domain (BIR2) playing an important role. Three proteins, Hid, Grim, and Reaper, promote apoptosis, in part by binding to DIAP1 through their conserved N-terminal sequences. The crystal structures of DIAP1-BIR2 by itself and in complex with the N-terminal peptides from Hid and Grim reveal that these peptides bind a surface groove on DIAP1, with the first four amino acids mimicking the binding of the Smac tetrapeptide to XIAP. The next 3 residues also contribute to binding through hydrophobic interactions. Interestingly, peptide binding induces the formation of an additional alpha helix in DIAP1. Our study reveals the structural conservation and diversity necessary for the binding of IAPs by the Drosophila Hid/Grim/Reaper and the mammalian Smac proteins.

Amino Acid Sequence↗
PubMed2001

Crystal structure of a phosphorylated Smad2. Recognition of phosphoserine by the MH2 domain and insights on Smad function in TGF-beta signaling.

Ligand-induced phosphorylation of the receptor-regulated Smads (R-Smads) is essential in the receptor Ser/Thr kinase-mediated TGF-beta signaling. The crystal structure of a phosphorylated Smad2, at 1.8 A resolution, reveals the formation of a homotrimer mediated by the C-terminal phosphoserine (pSer) residues. The pSer binding surface on the MH2 domain, frequently targeted for inactivation in cancers, is highly conserved among the Co- and R-Smads. This finding, together with mutagenesis data, pinpoints a functional interface between Smad2 and Smad4. In addition, the pSer binding surface on the MH2 domain coincides with the surface on R-Smads that is required for docking interactions with the serine-phosphorylated receptor kinases. These observations define a bifunctional role for the MH2 domain as a pSer-X-pSer binding module in receptor Ser/Thr kinase signaling pathways.

Amino Acid Motifs↗
PubMed2001

A Schiff base is a major DNA adduct of crotonaldehyde.

Previous studies have demonstrated that the reaction of crotonaldehyde with DNA produces Michael addition products, and these have been detected in human tissues as well as tissues of untreated laboratory animals. A second class of crotonaldehyde-DNA adducts releases 2-(2-hydroxypropyl)-4-hydroxy-6-methyl-1,3-dioxane (paraldol, 12) upon hydrolysis, and these adducts are quantitatively more significant than the Michael addition adducts in vitro. In this study, we demonstrate that the major source of the paraldol-releasing DNA adducts of crotonaldehyde is a Schiff base. Reaction of crotonaldehyde with DNA, followed by treatment with NaBH(3)CN and enzyme hydrolysis, resulted in the formation of N(2)-(3-hydroxybutyl)dG (10), identified by its UV, MS, and proton NMR. Reactions of crotonaldehyde or paraldol with dG demonstrated that the Schiff base precursor to N(2)-(3-hydroxybutyl)dG is N(2)-(3-hydroxybutylidene)dG (7), identified by UV, LC-APCI-MS, and MS/MS. Four isomers of N(2)-(3-hydroxybutylidene)dG were observed. The (R)- and (S)-isomers were identified by reactions of chiral paraldol with dG; each existed as a pair of interconverting (E)- and (Z)-isomers. These data indicate that the structure of the major Schiff base DNA adduct in crotonaldehyde-treated DNA is N(2)-(3-hydroxybutylidene)dG (7). This adduct is unstable at the nucleoside level and accounts for more than 90% of the paraldol released from crotonaldehyde-treated DNA. However, the adduct is stable in DNA and therefore is a likely companion to the Michael addition adducts in human DNA.

Aldehydes↗
PubMed2001

Reactions of 2,6-dimethyl-1,3-dioxane-4-ol (aldoxane) with deoxyguanosine and DNA.

In a recent study, we identified several new DNA adducts of the carcinogen acetaldehyde, including N(2)-(2,6-dimethyl-1,3-dioxan-4-yl)deoxyguanosine (N(2)-aldoxane-dG, 2). Our goal in this study was to investigate further the formation of 2 by allowing 2,6-dimethyl-1,3-dioxane-4-ol (aldoxane, 5) to react with dG and DNA. Aldoxane is readily formed by trimerization of acetaldehyde. The reaction of aldoxane with dG and DNA produced diastereomers of N(2)-aldoxane-dG (2) as observed in the reactions of acetaldehyde with dG and DNA, supporting the intermediacy of aldoxane in their formation. Unexpectedly, however, an array of other adducts was formed in these reactions, including 3-(2-deoxyribos-1-yl)-5,6,7,8-tetrahydro-8-hydroxy-6-methylpyrimido[1,2-a]purine-10(3H)one (3), 2-amino-7,8-dihydro-8-hydroxy-6-methyl-3H-pyrrolo[2,1-f]purine-4(6H)one (13), N(2)-(3-hydroxybutylidene)dG (9), N(2)-[(2-hydroxypropyl)-6-methyl-1,3-dioxane-4-yl]dG (14), and N(2)-ethylidene-dG (1). Adduct 1 was the major product and was found to be quite stable in DNA. The adducts result from a cascade of aldehydes, e.g., 2-butenal (crotonaldehyde, 12), 3-hydroxybutanal (7) and its dimer (2-hydroxypropyl)-6-methyl-1,3-dioxane-4-ol (paraldol, 6), and acetaldehyde, produced from aldoxane under the reaction conditions. The reactions of aldoxane with dG and DNA were compared with those of paraldol. The paraldol reactions gave products resulting from reactions of dG and DNA with paraldol, 3-hydroxybutanal, and crotonaldehyde (adducts 3, 13, and 9) but the products of the aldoxane and acetaldehyde reactions (adducts 1 and 2) were not observed, indicating that paraldol is more stable under the reaction conditions than is aldoxane. The results of this study provide new insights about the formation of DNA adducts from aldehydes via condensation products of the latter.

Acetaldehyde↗
PubMed2001

Reactions of alpha-acetoxy-N-nitrosopyrrolidine with deoxyguanosine and DNA.

We investigated the reactions of alpha-acetoxy-N-nitrosopyrrolidine (alpha-acetoxyNPYR) with dGuo and DNA. Alpha-acetoxyNPYR is a stable precursor to the major proximate carcinogen of NPYR, alpha-hydroxyNPYR (3). Our goal was to develop appropriate conditions for the analysis of DNA adducts of NPYR formed in vivo. Products of the alpha-acetoxyNPYR-dGuo reactions were analyzed directly by HPLC or after treatment of the reaction mixtures with NaBH3CN. Products of the alpha-acetoxyNPYR-DNA reactions were released by enzymatic or neutral thermal hydrolysis of the DNA, then analyzed by HPLC. Alternatively, the DNA was treated with NaBH3CN prior to hydrolysis and HPLC analysis. The reactions of alpha-acetoxyNPYR with dGuo and DNA were complex. We have identified 13 products of the dGuo reaction-6 of these were characterized in this reaction for the first time. They were four diastereomers of N2-(3-hydroxybutylidene)dGuo (20, 21), 7-(N-nitrosopyrrolidin-2-yl)Gua (2), and 2-(2-hydroxypyrrolidin-1-yl)deoxyinosine (12). Adducts 20 and 21 were identified by comparison to standards produced in the reaction of 3-hydroxybutanal with dGuo. Adduct 2 was identified by its spectral properties while adduct 12 was characterized by comparison to an independently synthesized standard. With the exception of adduct 2, all products of the dGuo reactions were also observed in the DNA reactions. The major product in both the dGuo and DNA reactions was N2-(tetrahydrofuran-2-yl)dGuo (10), consistent with previous studies. Several other previously identified adducts were also observed in this study. HPLC analysis of reaction mixtures treated with NaBH3CN provided improved conditions for adduct identification, which should be useful for in vivo studies of DNA adduct formation by NPYR.

Carcinogens↗
PubMed2001

Incidence of second primary breast cancer among women with a first primary in Manitoba, Canada.

Second primary breast cancer is of importance because of the increasing incidence of first primary breast cancer and improved survival. There is lack of information on the disease in Canada. We studied 14,220 women with a first primary breast cancer (invasive only) diagnosed during the period from 1970 to 1997, in Manitoba, Canada. A breast cancer was considered to be a second primary if it was an invasive lesion diagnosed at least six months after the diagnosis of an initial primary breast cancer. The incidence rate of second primary breast cancer was approximately six per 1,000 person-years, and the cumulative incidence during the 28-year study period was 14.3%. Average age at diagnosis of first primary breast cancer had increased significantly since 1970. The incidence was higher in 1975-1979, and there was no significant difference among other study periods after adjustment for the age at diagnosis of first primary breast cancer. Standardized incidence ratios showed no significant difference among six age groups after adjusting for calendar year. However, women with a first primary breast cancer had an increased risk of developing a second primary breast cancer compared with the risk of developing a first primary breast cancer among the general female population. The SIR was markedly greater among women who had a first primary breast cancer before the age of 40 years, and decreased significantly with increasing age at diagnosis of first primary breast cancer. The data provide epidemiological information on second primary breast cancer risk for the general female population in Manitoba, Canada.

Adult↗
PubMed2001

A study of the influence of the hydrophobic core residues of yeast iso-2-cytochrome c on phosphate binding: a probe of the hydrophobic core-surface charge interactions.

To gain insight into the role of hydrophobic core-surface charge interactions in stabilizing cytochrome c, we investigated the influence of hydrophobic core residues on phosphate binding by mutating residues in yeast iso-2-cytochrome c to those corresponding to iso-l-cytochrome c in various combinations. Heat transition of ultraviolet CD was followed as a function of pH in the presence and absence of phosphate. Thermodynamic parameters were deduced. It was found that the I20V/V43A/M98L mutation in the hydrophobic core, whose locations are remote from the putative phosphate sites, modulates phosphate interactions. The modulation is pH dependent. The I20V/ M98L and V43A mutation effects are nonadditive. The results lead to a model analogous to that of Tsao, Evans, and Wennerstrom, where a domain associated with the ordered hydrophobic core is sensitive to the fields generated by the surface charges. Such an explanation would be in accord with the observed difference in thermal stability between iso-2 and horse cytochromes c.

Amino Acid Sequence↗
PubMed2001

Design of new selective inhibitors of cyclooxygenase-2 by dynamic assembly of molecular building blocks.

A method of dynamically assembling molecular building blocks - DycoBlock - has been proposed and tested by Liu et al. This method is based on multiple-copy stochastic dynamics simulation in the presence of a receptor molecule. In this method, a novel algorithm was used to dynamically assemble the molecular building blocks to form candidate compounds. Currently, some new improvements have been incorporated into DycoBlock to make it more efficient. In the new version of DycoBlock, the binding energy and solvent accessible surface area (SASA) can be used to screen the resulting compounds. A simple clustering algorithm based on molecular similarity was developed and used to classify the remaining compounds. The revised DycoBlock was tested by breaking SC-558 - a selective inhibitor of cyclooxygenase-2 (COX-2) - into building blocks and reassembling them in the active site of the enzyme. The accuracy of recovery grew to 58.8% while it was only 16.7% in the previous version. Then, thirty-three kinds of molecular building blocks were used in the design of novel inhibitors and the investigation of diversity. As a result, a total of 1441 compounds was generated with high diversity. After the first screening procedure, there remained 864 reasonable compounds. The results from clustering indicate that the structural motifs in the diarylheterocycle class of COX-2-selective inhibitors have been generated using the revised DycoBlock, and their binding modes were investigated.

Algorithms↗
PubMed2001

Structure-based ligand design for flexible proteins: application of new F-DycoBlock.

A method of structure-based ligand design - DycoBlock - has been proposed and tested by Liu et al. It was further improved by Zhu et al. and applied to design new selective inhibitors of cyclooxygenase 2. In the current work, we present a new methodology - F-DycoBlock that allows for the incorporation of receptor flexibility. During the designing procedure, both the receptor and molecular building blocks are subjected to the multiple-copy stochastic molecular dynamics (MCSMD) simulation, while the protein moves in the mean field of all copies. It is tested for two enzymes studied previously - cyclooxygenase 2 (COX-2) and human immunodeficiency type 1 (HIV-1) protease. To identify the applicability of F-DycoBlock, the binding protein structure was used as starting point to explore the conformational space around the bound state. This method can be easily extended to accommodate the flexibility in different degree. Four types of treatment of the receptor flexibility - all-atom restrained, backbone restrained, intramolecular hydrogen-bond restrained and active-site flexible - were tested with or without the grid approximation. Two inhibitors, SC-558 for COX-2 and L700417 for HIV-1 protease, are used in this testing study for comparison with previous results. The accuracy of recovery, binding energy, solvent accessible surface area (SASA) and positional root-mean-square (RMS) deviation are used as criteria. The results indicate that F-DycoBlock is a robust methodology for flexible drug design. It is particularly notable that the protein flexibility has been perfectly associated with each stage of drug design - search for the binding sites, dynamic assembly and optimization of candidate compounds. When all protein atoms were restrained, F-DycoBlock yielded higher accuracy of recovery than DycoBlock (100%). If backbone atoms were restrained, the same ratio of accuracy was achieved. Moreover, with the intramolecular hydrogen bonds restrained, reasonable conformational changes were observed for HIV- 1 protease during the long-time MCSMD simulation and L700417 was reassembled at the active site. It makes it possible to study the receptor motion in the binding process.

Computer Simulation↗
PubMed2001

In vitro binding of an orally active platinum antitumor drug, JM216 to metallothionein.

The in vitro binding of an orally active anticancer drug JM216 to metallothionein is firstly investigated in this paper. It is revealed that a redox reaction following a substitution reaction from JM216 with rabbit liver Zn7MT-II is presented. The reaction feature, the metal binding stoichiometry and the oxidation states of platinum and sulfur in the products are studied by UV-visible, chromatography and X-ray photoelectron spectroscopy methods. Parts of MT are oxidized to precipitate products with intra and intermolecular CyS-SCy disulfides linkages. Pt(IV) is reduced to its Pt(II) counterpart. And the reduced Pt(II) replace the metal ions in native MTs. Meanwhile it can also cause the dimerization of MT. Increasing the reaction ratio of JM216 to MT leads to a concomitant increase in the apparent yield of the precipitate and dimeric products and the elevation of the binding stoichiometry of Pt to the protein. Based on the experimental data, the reaction mechanism between JM216 and Zn7MT-II in vitro are discussed.

Administration, Oral↗
PubMed2001

Dentinogenesis imperfecta 1 with or without progressive hearing loss is associated with distinct mutations in DSPP.

Dentinogenesis imperfecta 1 (DGI1, MIM 125490) is an autosomal dominant dental disease characterized by abnormal dentin production and mineralization. The DGI1 locus was recently refined to a 2-Mb interval on 4q21 (ref. 1). Here we study three Chinese families carrying DGI1. We find that the affected individuals of two families also presented with progressive sensorineural high-frequency hearing loss (gene DFNA39). We identified three disease-specific mutations within the dentin sialophosphoprotein gene (DSPP) in these three families. We detected a G-->A transition at the donor-splicing site of intron 3 in one family without DFNA39, a mutation predicted to result in the skipping of exon 3. In two other families affected with both DGI1 and DFNA39, however, we identified two independent nucleotide transversions in exons 2 and 3 of DSPP, respectively, that cause missense mutations of two adjacent amino-acid residues in the predicted transmembrane region of the protein. Moreover, transcripts of DSPP previously reported to be expressed specifically in teeth are also detected in the inner ear of mice. We have thus demonstrated for the first time that distinct mutations in DSPP are responsible for the clinical manifestations of DGI1 with or without DFNA39.

Asian People↗
PubMed2001

A structural view of mitochondria-mediated apoptosis.

Mitochondria-mediated apoptosis plays a central role in animal development and tissue homeostasis, and its alteration results in a range of malignant disorders including cancer. Upon apoptotic stimuli, the mitochondrial proteins cytochrome c and Smac/DIABLO are released into the cytosol, where they synergistically activate caspases by activating Apaf-1 and relieving the apoptotic inhibition by IAPs. Recent biochemical and structural studies reveal a molecular basis for these important events and identify an evolutionarily conserved mechanism of apoptosis from fruit flies to mammals.

Amino Acid Sequence↗
PubMed2001

Safety of intracoronary administration of c-myc antisense oligomers after percutaneous transluminal coronary angioplasty (PTCA).

We wished to assess the clinical safety and pharmacokinetics of ascending doses of a synthetic oligodeoxynucleotide (LR-3280) administered after coronary angioplasty. Antisense oligodeoxynucleotides designed to hybridize with target messenger ribonucleic acid (mRNA) in a complementary fashion to inhibit the expression of corresponding protein also have the ability to bind to extracellular growth factors. LR-3280 has been shown to reduce c-myc expression, inhibit growth and collagen biosynthesis in human vascular cells, and reduce neointimal formation in animal models of vascular injury. After successful percutaneous transluminal coronary angioplasty (PTCA), 78 patients were randomized to receive either standard care (n = 26) or standard care and escalating doses of LR-3280 (n = 52) (doses from 1 to 24 mg), administered into target vessel through a guiding catheter. Overall safety was evaluated by clinical adverse events, laboratory tests, and electrocardiograms. Patency was evaluated by quantitative coronary angiography. There were no clinically significant differences between treated and control patients. No adverse effects of LR-3280 on the patency of dilated coronary arteries were observed. Pharmacokinetic data revealed that peak plasma concentrations of LR-3280 occurred at 1 minute over the studied dose range and rapidly decreased after approximately1 hour, with little LR-3280 detected in the urine between 0-6 hours and 12-24 hours. The intracoronary administration of LR-3280 is well tolerated at doses up to 24 mg and produces no adverse effects in dilated coronary arteries. These results provide the basis for the evaluation of local delivery of this phosphorothioate oligodeoxynucleotide for the prevention of human vasculoproliferative disease.

Aged↗
PubMed2001

Spatial and molecular organization of lymph node T cell cortex: a labyrinthine cavity bounded by an epithelium-like monolayer of fibroblastic reticular cells anchored to basement membrane-like extracellular matrix.

Naive T cells encounter antigen-presenting cells within the cortex of lymph nodes to initiate primary immune responses. Within this T cell cortex is the reticular network (RN)--a system of collagen fibers and extracellular matrix (ECM) wrapped by fibroblastic reticular cells (FRC). We have investigated the distribution of various molecules, including ECM proteins and proteoglycans, in the T cell cortex of both human and rodent lymph node. We confirm and extend reports of matrix elements in the RN. In addition, we find that staining for the laminin-alpha3 chain and for tenascin reveals a 'hollow' reticular pattern, consistent with localization to the basement membrane-like covering of reticular fibers. In contrast, keratan sulfate is observed in a fine linear pattern within the RN, suggesting it is localized to the core of the fibers. Staining with the marker ER-TR7 indicates that FRC cover all identifiable ECM surfaces of the T cell cortex. Based on these findings and previous reports, we conclude that cortical lymphocytes migrate within a 'labyrinthine cavity' free of fibrillar ECM, distinguishing the T cell cortex from other loose connective tissues, and that the FRC lining of the cavity constitutes an epithelium-like boundary. We propose that this spatial organization facilitates ameboid leukocyte crawling along preformed paths of least resistance and that the basement membrane-like ECM of the FRC may facilitate fluid transport within the RN by limiting leakage from the fiber.

Animals↗