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Biomedical subjects

D Yang

Publications and source records attributed to D Yang.

At least 19 recordsLinked to original sources
PubMed2001

Expression of the REB transcriptional activator in rice grains improves the yield of recombinant proteins whose genes are controlled by a Reb-responsive promoter.

The gene encoding the rice transcription factor, REB (rice endosperm bZIP) was cloned from a bacterial artificial chromosome library of rice. The cloned 6,227-bp-long Reb gene is composed of six exons and five introns and is flanked by a 1.2-kb 5' promoter and a 1.2-kb 3' terminator region. The function of the Reb gene was explored by a transient assay by using a rice immature endosperm system. The effector constructs containing the native gene or fusion genes linking Reb to the rice actin (Act) or globulin (Glb) gene promoters and the reporter gene construct Glb-beta-glucuronidase (GUS) were used in this study. When these effector constructs were cotransferred with the reporter uidA gene encoding GUS under the control of the Glb promoter into immature rice endosperm cells, the Glb promoter was activated. The transient GUS expression was 2.0 to 2.5-fold higher with the effector construct than without. When the upstream activation sequence containing the GCCACGT(A/C)AG motifs of the Glb promoter was deleted, the activation by REB was abolished. On the other hand, a gain-of-function experiment showed that inserting the upstream activation sequence into the glutelin-1 (Gt1) promoter made it responsive to activation by REB. When cotransformed with Reb gene, mature transgenic rice grains containing the human lysozyme gene driven by the Glb promoter produced 3.7-fold more lysozyme. Accumulation of recombinant lysozyme in mature seed ranged from 30.57 to 279.61 microg.mg(-1) total soluble protein in individual transformants from 30 independent transformation events. Thus, our results show that REB is not only a transcriptional activator, it can also be used to increase the expression of recombinant protein in transgenic rice grains.

Actins↗
PubMed2001

Binding of a SART3 tumor-rejection antigen to a pre-mRNA splicing factor RNPS1: a possible regulation of splicing by a complex formation.

We recently reported the identification of a human SART3 gene that encodes a tumor-rejection antigen recognized by cytotoxic T lymphocytes (CTLs). The squamous-cell carcinoma antigen recognized by T cells-3 (SART3) is an RNA-binding protein expressed in the nucleus of the majority of proliferating cells, including normal cells and malignant cells, but not in normal tissues except for the testes and fetal liver. To determine its biologic function, we employed a 2-hybrid screening in yeast for proteins interacting with SART3, and this method yielded a pre-mRNA splicing factor (RNA-binding protein prevalent during the S phase or RNA-binding protein with a serine-rich domain [RNPS1]) that activated both constitutive and alternative splicing of pre-mRNA in vitro. Interaction of SART3 with RNPS1 through the physical association of N-terminal domains of RNPS1 was confirmed by both in vitro pull-down assay and immunoprecipitation assay. Cotransfection of the 2 genes changed the distribution pattern of SART3 from diffuse nucleoplasmic spreading to nuclear speckled regions in which the RNPS1 was colocalized, suggesting a complex formation of the 2 proteins. In cooperation with RNPS1, SART3 stimulated the proximal alternative 3' splicing of a calcitonin-dihydrofolate reductase chimeric minigene pre-mRNA. These results suggest that SART3 is involved in the regulation of mRNA splicing probably via its complex formation with RNPS1.

Antigens, Neoplasm↗
PubMed2001

EGFR tyrosine kinase inhibitor AG1478 inhibits cell proliferation and arrests cell cycle in nasopharyngeal carcinoma cells.

Nasopharyngeal carcinoma (NPC), which occurs with a high incidence in southern China and southeast Asia, is of epithelial origin with overexpression of EGF receptor. To study the effect of inhibition of EGFR signaling on nasopharyngeal carcinoma cell proliferation and cell cycle distribution, EGFR tyrosine kinase inhibitor AG1478 was employed to treat Nasopharyngeal Carcinoma CNE2 cells. The results showed that AG1478 inhibited proliferation of CNE2 cells. Immunoblot showed that AG1478 inhibited EGFR phosphorylation in CNE2 cells without reduced expression of EGFR protein. The activation of Akt and MAPK which are downstream molecules of EGFR signaling pathway, were also inhibited by AG1478. AG1478 induced cell cycle arrest in G1 phase, and the levels of protein p27 were significantly up-regulated. We concluded that inhibition of the EGFR signaling induced cell cycle arrest in G1 phase in CNE2 cells and p27 up-regulation was involved in this process. The EGFR kinase specific inhibitor is of potential to be developed into drugs for NPC treatment.

Cell Cycle↗
PubMed2001

RyR3 amplifies RyR1-mediated Ca(2+)-induced Ca(2+) release in neonatal mammalian skeletal muscle.

The neonatal mammalian skeletal muscle contains both type 1 and type 3 ryanodine receptors (RyR1 and RyR3) located in the sarcoplasmic reticulum membrane. An allosteric interaction between RyR1 and dihydropyridine receptors located in the plasma membrane mediates voltage-induced Ca(2+) release (VICR) from the sarcoplasmic reticulum. RyR3, which disappears in adult muscle, is not involved in VICR, and the role of the transiently expressed RyR3 remains elusive. Here we demonstrate that RyR1 participates in both VICR and Ca(2+)-induced Ca(2+) release (CICR) and that RyR3 amplifies RyR1-mediated CICR in neonatal skeletal muscle. Confocal measurements of intracellular Ca(2+) in primary cultured mouse skeletal myotubes reveal active sites of Ca(2+) release caused by peripheral coupling between dihydropyridine receptors and RyR1. In myotubes lacking RyR3, the peripheral VICR component is unaffected, and RyR1s alone are able to support inward CICR propagation in most cells at an average speed of approximately 190 microm/s. With the co-presence of RyR1 and RyR3 in wild-type cells, unmitigated radial CICR propagates at 2,440 microm/s. Because neonatal skeletal muscle lacks a well developed transverse tubule system, the RyR3 reinforcement of CICR seems to ensure a robust, uniform, and synchronous activation of Ca(2+) release throughout the cell body. Such functional interplay between RyR1 and RyR3 can serve important roles in Ca(2+) signaling of cell differentiation and muscle contraction.

Animals↗
PubMed2001

Asymmetric epoxidation of olefins catalyzed by chiral iminium salts generated in situ from amines and aldehydes.

[reaction: see text] A new approach for catalytic asymmetric epoxidation of olefins was developed that utilized chiral iminium salts, generated in situ from chiral amines and aldehydes, as catalysts. Epoxidation reactions can be conducted with 20 mol % of amines and aldehydes. The enantioselectivity of epoxides can be up to 65%. This modular approach obviates the difficulties inherent in the preparation and isolation of unstable exocyclic iminium salts.

Journal Article↗
PubMed2001

Murine six-transmembrane epithelial antigen of the prostate, prostate stem cell antigen, and prostate-specific membrane antigen: prostate-specific cell-surface antigens highly expressed in prostate cancer of transgenic adenocarcinoma mouse prostate mice.

To identify genes that are differentially up-regulated in prostate cancer of transgenic adenocarcinoma mouse prostate (TRAMP) mice, we subtracted cDNA isolated from mouse kidney and spleen from cDNA isolated from TRAMP-C1 cells, a prostate tumor cell line derived from a TRAMP mouse. Using this strategy, cDNA clones that were homologous to human six-transmembrane epithelial antigen of the prostate (STEAP) and prostate stem cell antigen (PSCA) were isolated. Mouse STEAP (mSteap) is 80% homologous to human STEAP at both the nucleotide and amino acid levels and contains six potential membrane-spanning regions similar to human STEAP. Mouse PSCA (mPsca) shares 65% homology with human PSCA at the nucleotide and amino acid levels. mRNA expression of mSteap and mPsca is largely prostate-specific and highly detected in primary prostate tumors and metastases of TRAMP mice. Both mSteap and mPsca map to chromosome 5. Another known gene coding for mouse prostate-specific membrane antigen (mPsma) is also highly expressed in both primary and metastatic lesions of TRAMP mice. These results indicate that the TRAMP mouse model can be used to effectively identify genes homologous to human prostate-specific genes, thereby allowing for the investigation of their functional roles in prostate cancer. mSteap, mPsca, and mPsma constitute new tools for preventative and/or therapeutic vaccine construction and immune monitoring in the TRAMP mouse model that may provide insights into the treatment of human prostate cancer.

Adenocarcinoma↗
PubMed2001

Macrocyclization in the design of a conformationally constrained Grb2 SH2 domain inhibitor.

Grubbs' olefin metathesis reaction was utilized to prepare a macrocyclic variant of a linear Grb2 SH2 domain antagonist in an attempt to induce a beta-bend conformation known to be required for high affinity binding. In extracellular Grb2 SH2 domain binding assays, the macrocyclic analogue exhibited an approximate 100-fold enhancement in binding potency relative to its linear counterpart. The macrocycle was not as effective in whole cell binding assays as would be expected based on its extracellular binding potency.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Ruthenium-catalyzed oxidative cleavage of olefins to aldehydes.

Three oxidation protocols have been developed to cleave olefins to carbonyl compounds with ruthenium trichloride as catalyst (3.5 mol %). These methods convert olefins that are not fully substituted to aldehydes rather than carboxylic acids. While aryl olefins were cleaved to aromatic aldehydes in excellent yields by using the system of RuCl3-Oxone-NaHCO3 in CH3CN-H2O (1.5:1), aliphatic olefins were converted into alkyl aldehydes with RuCl3-NaIO4 in 1,2-dichloroethane-H2O (1:1) in good to excellent yields. It is noteworthy that terminal aliphatic olefins were cleaved to the corresponding aldehydes in excellent yields by using RuCl3-NaIO4 in CH3CN-H2O (6:1).

Journal Article↗
PubMed2001

Kinetic resolution of acyclic secondary allylic silyl ethers catalyzed by chiral ketones.

Kinetic resolution of acyclic secondary allylic silyl ethers by chiral dioxiranes generated in situ from chiral ketones (R)-1 and (R)-2 and Oxone was investigated. An efficient and catalytic method has been developed for kinetic resolution of those substrates with a CCl(3), tert-butyl, or CF(3) group at the alpha-position. In particular, high selectivities (S up to 100) were observed for kinetic resolutions of racemic alpha-trichloromethyl allylic silyl ethers 7 and 9-15 catalyzed by ketones (R)-2. Both the recovered substrates and the resulting epoxides were obtained in high enantiomeric excess. On the basis of steric and electrostatic interactions between the chiral dioxiranes and the racemic substrates, a model was proposed to rationalize the enantioselectivities and diastereoselectivities in the chiral ketone-catalyzed kinetic resolution process.

Journal Article↗
PubMed2001

First enantioselective syntheses of (+)- and (-)-wilforonide by using chiral auxiliaries derived from the same chiral source.

[see structure]. The first enantioselective syntheses of both (+)-wilforonide (>98% ee) and (-)-wilforonide (>98% ee) have been accomplished by employing chiral auxiliaries derived from the same chiral source, (R)-pulegone. The bicyclic skeleton of wilforonide was constructed by using Mn(III)-based oxidative radical cyclization reactions of chiral beta-keto esters. The absolute configuration of natural wilforonide has been established to be (5aR,9aR).

Chromatography, High Pressure Liquid↗
PubMed2001

Measurement of (13)C(alpha)-(13)C(beta) dipolar couplings in (15)N,(13)C,(2)H-labeled proteins: application to domain orientation in maltose binding protein.

TROSY-based HN(CO)CA 2D and 3D pulse schemes are presented for measurement of (13)C(alpha)-(13)C(beta) dipolar couplings in high molecular weight (15)N,(13)C,(2)H-labeled proteins. In one approach, (13)C(alpha)-(13)C(beta) dipolar couplings are obtained directly from the time modulation of cross-peak intensities in a set of 2D (15)N-(1)HN correlated spectra recorded in both the presence and absence of aligning media. In a second approach 3D data sets are recorded with (13)C(alpha)-(13)C(beta) couplings encoded in a frequency dimension. The utility of the experiments is demonstrated with an application to an (15)N,(13)C,(2)H-labeled sample of the ligand free form of maltose binding protein. A comparison of experimental dipolar couplings with those predicted from the X-ray structure of the apo form of this two-domain protein establishes that the relative orientation of the domains in solution and in the crystal state are very similar. This is in contrast to the situation for maltose binding protein in complex with beta-cyclodextrin where the solution structure can be generated from the crystal state via a 11 degrees domain closure.

Carcinogens↗
PubMed2001

Repetitive nerve stimulation and stimulation single fiber electromyography studies in rats intoxicated with single or mixed insecticides.

The function of the neuromuscular transmission in rats dosed with phoxim (P), methomyl (M), fenvalerate (F), and mixtures of P+M and P+F was studied by using both the stimulation single fiber electromyography (SSFEMG) and repetitive nerve stimulations (RNS) to determine the single muscle fiber action potential and compound muscle action potential (CMAP) respectively. The results showed that the mean consecutive difference (MCD) in SSFEMG was significantly prolonged in P, P+M and P+F intoxicated rats during the presence of myasthenia, but not in rats dosed with F and M when stimuli were given at 10 Hz or 20 Hz, thus indicating a transmission blocking at the neuromuscular junction (NMJ) induced by P. The frequency of neuromuscular transmission abnormalities detected by SSFEMG was significantly higher than those detected by RNS. This study demonstrated that the neuromuscular junction (NMJ) blocking is more frequently seen in P, P+M and P+F poisoning than in M and F poisoning, and that SSFEMG is a more sensitive electrophysiological method than RNS for detecting neuromuscular transmission blockage in myasthenia rats with acute insecticides poisoning.

Acetylcholinesterase↗
PubMed2001

Differential regulation of formyl peptide receptor-like 1 expression during the differentiation of monocytes to dendritic cells and macrophages.

Monocytes are the common precursors for myeloid dendritic cells (DC) and macrophages. Identification of chemotactic receptors expressed by myeloid DC, macrophages, and their precursors in the course of differentiation and maturation is important not only for elucidation of their in vivo trafficking, but also for understanding of the functional distinction between DC and macrophages. We chose to study formyl peptide receptor like-1 (FPRL1), a chemotactic receptor known to interact with several endogenous agonists that are involved in inflammatory and host defense responses. Here we show that FPRL1 is down-regulated as monocytes differentiate into DC. This down-regulation occurs at both mRNA and functional levels. Therefore, the interaction of FPRL1 with its agonists is more likely to regulate the in vivo trafficking of DC precursors than DC. In contrast, FPRL1 expression is maintained at both mRNA and functional levels as monocytes differentiate into macrophages. Thus, our results demonstrate further distinctions between myeloid DC and macrophages, albeit they share a common precursor. The fact that macrophages rather than myeloid DC express functional FPRL1 suggests that this chemotactic receptor may be more involved in inflammatory reactions and innate host defense than in adaptive immune responses.

Animals↗
PubMed2001

Chiral auxiliaries for asymmetric radical cyclization reactions: application to the enantioselective synthesis of (+)-triptocallol.

[figure: see text] A series of epimeric 8-aryl menthyl derivatives 5a-d and 6a-l, prepared from the same chiral source (R)-pulegone, were employed as chiral auxiliaries in the asymmetric radical cyclization reactions of beta-keto esters mediated by Mn(OAc)3. Chiral precursors 8c and 8d provided the cyclization products 10c and 10d, respectively, as single isomers (dr > 99:1), whereas the cyclization of precursor 9k gave 13k with good stereoselectivity (dr = 24:1). Diastereomer 13e was employed as the key intermediate in the enantioselective synthesis of (+)-triptocallol in 90% ee.

Biological Factors↗
PubMed2001

The role of mammalian antimicrobial peptides and proteins in awakening of innate host defenses and adaptive immunity.

Since we live in a dirty environment, we have developed many host defenses to contend with microorganisms. The epithelial lining of our skin, gastrointestinal tract and bronchial tree produces a number of antibacterial peptides, and our phagocytic neutrophils rapidly ingest and enzymatically degrade invading organisms, as well as produce peptides and enzymes with antimicrobial activities. Some of these antimicrobial moieties also appear to alert host cells involved in both innate host defense and adaptive immune responses. The epithelial cells are a source of constitutively produced beta defensin (HBD1) and proinflammatory cytokine-inducible beta defensins (HBD2 and -3) and cathelicidin (LL37). The neutrophils-derived antimicrobial peptides are released on demand from their cytoplasmic granules. They include the enzymes cathepsin G and chymase, azurocidin, a defensins and cathelicidin. In contrast, C5a and C3b are produced by activation of the serum complement cascade. The antimicrobial moieties direct the migration and activate target cells by interacting with selected G-protein-coupled seven-transmembrane receptors (GPCRs) on cell surfaces. The beta defensins interact with the CCR6 chemokine GPCRs, whereas cathelicidins interact with the low-affinity FPRL-1 receptors. The neutrophil-derived cathepsin G acts on the high-affinity FMLP receptor (GPCR) known as FPR, while the receptors for chymase and azurocidin have not been identified as yet. The serum-derived C5a uses a GPCR known as C5aR to mediate its chemotactic and cell-activating effects. Consequently, all these ligand-receptor interactions in addition to mediating chemotaxis also activate receptor-expressing cells to produce other mediators of inflammation.

Animals↗
PubMed2001

Rice ESTs with disease-resistance gene- or defense-response gene-like sequences mapped to regions containing major resistance genes or QTLs.

The chromosomal locations of 109 rice expressed sequence tags (ESTs) in the rice genome were determined using a doubled haploid mapping population. These ESTs show high similarity to disease resistance genes or to defense response genes. Nine of the ESTs were mapped to three regions that contain genetically defined resistance genes on chromosomes 6 and 11. Clustering of the ESTs in the rice genome was observed at several chromosomal regions. Some of the clusters were located in regions where quantitative trait loci (QTL) associated with partial resistance to rice blast, bacterial blight and sheath blight are known to lie. Three ESTs that were mapped to the regions containing blast resistance genes Pi2 and Pia were chosen for Northern analysis after inoculation of plants with the blast fungus. Two of them, which code for a receptor-like kinase and a putative membrane channel protein, respectively, and were mapped to the Pi2 locus, were induced by rice blast infection as early as 4 h after inoculation. Transcription of another EST, which codes for a homolog of a putative human tumor suppressor and was mapped to the region containing Pia, was repressed after blast infection. These findings demonstrate that the candidate-gene approach is an efficient way of mapping resistance genes or resistance QTLs in rice.

Chromosome Mapping↗