Biomedical subjects

Ying Chen

Publications and source records attributed to Ying Chen.

At least 235 records · Page 13Linked to original sources

PubMed2003

The absorption behavior of cyclosporin A lecithin vesicles in rat intestinal tissue.

The purpose of the study was to investigate the absorption behavior of lecithin vesicles of cyclosporin A (CsA-VES), prepared by the rotary evaporation method and treated further with sonication. The everted gut sac technique and in situ circulation method were used to examine: (1) relationship between the CsA-VES absorption velocity and the CsA-VES content; (2) the influence of the intestinal mucus, blank vesicles, concentration of Na(+), energy inhibitor and P-gp inhibitor on the absorption of CsA-VES; and (3) the respective accumulated content of CsA in the incubating medium and the sacs after incubation with Sandimmum Neoral((R))(CsA-NEO) and CsA-VES. Our results showed there was a saturated absorption of CsA. Most CsA-VES accumulated in the mucus before it reached the intestinal tissue. There was no significant difference in the accumulated absorption content in the incubating medium and the sacs of CsA-NEO and CsA-VES. The addition of blank vesicles and concentration of Na(+) had no significant influence on the accumulated absorption of CsA (P>0.05). The energy inhibitor and P-gp inhibitor influenced the accumulated absorption of CsA significantly (P<0.05). CsA-VES may be transported by phagocytosis. Mucus was a barrier blocking the diffusion of CsA-VES. CsA-NEO and CsA-VES showed equal absorption levels in the intestine.

Animals↗

PubMed2003

General and efficient synthesis of arylamino- and alkylamino-substituted diphenylporphyrins and tetraphenylporphyrins via palladium-catalyzed multiple amination reactions.

A series of arylamino- and alkylamino-substituted diphenylporphyrins and tetraphenylporphyrins were efficiently synthesized by reactions of brominated porphyrin precursors with amines via palladium-catalyzed amination. The multiple amination reactions are general and suitable for a variety of amines, affording the desired aminoporphyrins in good to excellent yields. Examples include aromatic and aliphatic amines, primary and secondary amines, electron-rich, -neutral, and -poor amines as well as heteroaromatic amines and imines.

Journal Article↗

PubMed2003

Loss of inhibitor of apoptosis proteins as a determinant of polyamine analog-induced apoptosis in human melanoma cells.

We have previously shown that the clinically relevant polyamine analog N1,N11-diethylnorspermine (DENSPM) causes rapid apoptosis in human melanoma SK-MEL-28 cells via a series of events that include mitochondrial release of cytochrome c and activation of the caspase cascade. Upstream to these events, DENSPM downregulates polyamine biosynthesis and potently upregulates polyamine catabolism at the level of spermidine/spermine N1-acetyltransferase (SSAT). In searching for downstream effectors that either contribute to or abrogate the apoptotic response, we observed that DENSPM treatment of SK-MEL-28 cells for 30 h led to cytosolic release of Smac/Diablo, a mitochondrial protein known to bind and inhibit the function of inhibitor of apoptosis proteins (IAPs). Subsequently, we found that DENSPM markedly lowered survivin and ML-IAP protein (but not XIAP) levels by 18 h via an apparently Smac/Diablo-independent pathway. Proteasome inhibitors fully prevented survivin and ML-IAP protein loss as well as apoptosis, suggesting that the proteasome-mediated degradation of survivin and ML-IAP is causally linked to the cellular outcome. We also observed that structural analogs of DENSPM which differentially induced SSAT and apoptosis lowered survivin and ML-IAP levels in a manner that correlated with enzyme activity. The linkage between IAPs and SSAT was more directly established by the finding that selective prevention of SSAT induction by small interfering RNA prevented survivin and ML-IAP loss as well as apoptosis during DENSPM treatment. Among the melanoma cell lines (SK-MEL-28, MALME-3M, A375 and LOX), survivin degradation correlated temporally with the onset of DENSPM induced apoptosis or growth inhibition. By contrast, ML-IAP degradation occurred only during rapid apoptosis seen in SK-MEL-28 cells. These data suggest a sequence of events whereby DENSPM induction of SSAT leads to loss of IAP proteins and a more fulminate apoptotic response. The findings implicate survivin and ML-IAP as important determinants of polyamine analog drug action in melanoma cells.

Antineoplastic Agents↗

PubMed2003

Genomic identification and biochemical characterization of a second spermidine/spermine N1-acetyltransferase.

In the polyamine back-conversion pathway, spermine and spermidine are first acetylated by spermidine/spermine N(1) -acetyl-transferase (SSAT-1) and then oxidized by polyamine oxidase to produce spermidine and putrescine respectively. Herein we apply homology-search methods to identify novel sequences belonging to a second SSAT, SSAT-2, with a chromosomal location at 17p13.1, which is distinct from SSAT-1 at Xp22. Human SSAT-2 cDNA derived from small-cell lung carcinoma was deduced to encode a 170-amino-acid protein having 46% sequence identity and 64% sequence similarity with SSAT-1. When transiently transfected into HEK-293 cells, SSAT-1 decreased spermidine and spermine pools by approximately 30%, while, at the same time, significantly increasing putrescine, N (1)-acetylspermidine, N (1)-acetylspermine and N (1), N (12)-diacetylspermine pools. By contrast, transfected SSAT-2 had no effect on intracellular polyamine or acetylated polyamine pools. When enzyme activity was assayed on enzyme extracts from transfected cells, both SSAT-1 and SSAT-2 demonstrated much higher acetylating activity than vector-transfected cells. The data suggest that, in intact cells, SSAT-2 may be compartmentalized or it may be inefficient at low intracellular polyamine concentrations. By substituting candidate substrates in the enzyme assay, we determined that SSAT-1 shows the substrate preference norspermidine=spermidine>>spermine> N (1)-acetylspermine>putrescine, whereas SSAT-2 shows the preference norspermidine>spermidine=spermine>> N (1)-acetylspermine=putrescine. Analysis of mRNA levels in cell lines and ESTs (expressed sequence tags) from various tissues by digiNorthern (a web-based tool for virtually displaying expression profiles of query genes based on EST sequences) indicated that SSAT-1 tends to be more widely and highly expressed than SSAT-2. While SSAT-1 mRNA was inducible by polyamine analogues in a variety of cell lines, SSAT-2 was not. The existence of an active, but possibly sequestered, SSAT-2 enzyme suggests that, under certain conditions, it may be recruited into basal or perturbed polyamine metabolism.

Acetyltransferases↗

PubMed2003

The coactivator p/CIP/SRC-3 facilitates retinoic acid receptor signaling via recruitment of GCN5.

p/CIP/SRC-3 is a member of a family of steroid receptor coactivators/nuclear receptor coactivators (SRC/NCoA) proteins that mediate the transcriptional effects of nuclear hormone receptors (NRs). Using deletion analysis we have mapped the location of two distinct activation domains in p/CIP (AD1 and AD2) capable of activating transcription in mammalian cells when fused to the Gal4-DNA binding domain. In addition to AD1 being coincident with the interaction domain for CBP, we demonstrate a novel in vivo interaction between the AD1 and GCN5. Overexpression of a Gal4-AD1 fusion protein in yeast leads to growth arrest that is relieved by mutation of genes encoding components of the SAGA complex including GCN5, ADA3, and SPT7. In addition, the AD1 of p/CIP and the ADA3 gene are shown to be essential for retinoic acid receptor alpha-dependent transcription in yeast. Transient transfection assays in mammalian cells indicate that GCN5 cooperates with p/CIP as a coactivator of RAR alpha-dependent transcription. Down-regulation of GCN5 using small interfering RNA in mammalian cells indicates that the AD1 domain and the RAR beta promoter activity are dependent, in part, on GCN5. Mutational analysis of AD1 has identified two helical motifs that are required for interactions with GCN5 and CBP. Taken together, these results support a model by which p/CIP functions as a ligand-dependent adapter, through specific protein-protein interactions with AD1, to recruit members from at least two distinct families of acetyltransferase proteins to NRs.

Acetyltransferases↗

PubMed2003

Efficient and stereoselective synthesis of beta-trifluoromethyl alpha,beta-unsaturated esters via iron(III) porphyrin-catalyzed olefination of ketones.

beta-Trifluoromethyl alpha,beta-unsaturated esters were efficiently prepared by reactions of fluorine-containing ketones with diazo compounds via metalloporphyrin-catalyzed olefination in the presence of triphenylphosphine. The commercially available Fe(III)(TPP)Cl (TPP: tetraphenylporphyrin) is effective for catalyzing the olefination of a variety of trifluoromethyl ketones with different diazoacetate esters under mild conditions. The reactions proceeded with high yields (up to 95% isolated yield) and high stereoselectivity (up to 99% (E)-selectivity).

Journal Article↗

PubMed2003

Ginsenoside Rg1 attenuates dopamine-induced apoptosis in PC12 cells by suppressing oxidative stress.

In Parkinson's disease, neuroprotective therapy to rescue dopamine neurons has been proposed. Ginsenoside Rg1, one of the biologically active ingredients of ginseng, may be a candidate neuroprotective drug. In the present study, the mechanism underlying the neuroprotection provided by ginsenosde Rg1 was studied against apoptosis induced by exogenous dopamine in PC12 cells. Pretreatment with ginsenoside Rg1 markedly reduced the generation of dopamine-induced reactive oxygen species and the release of mitochondrial cytochrome c into the cytosol, and subsequently inhibited the activation of caspase-3. In addition, Rg1 pretreatment also reduced inducible nitric oxide (NO) synthase protein level and NO production. These results suggested that ginsenoside Rg1 may attenuate dopamine-induced apoptotic cell death through suppression of intracellular oxidative stress, and that it may rescue or protect dopamine neurons in Parkinson's disease.

Animals↗

PubMed2003

Acid-promoted olefination of ketones by an iron(III) porphyrin complex.

[reaction: see text] The acid-promoted olefination of unactivated ketones with diazo reagents in the presence of triphenylphosphine can be catalyzed by the commercially available iron(III) porphyrin complex Fe(TPP)Cl. The reactions were effectively carried out under mild conditions in a one-pot fashion with the use of a stoichiometric diazo reagents and substoichiometric benzoic acid. Examples include aromatic, aliphatic, cyclic, and unsaturated ketones with ethyl diazoacetate or tert-butyl diazoacetate.

Journal Article↗

PubMed2003

Familial hypertrophic cardiomyopathy mutations in troponin I (K183D, G203S, K206Q) enhance filament sliding.

A major cause of familial hypertrophic cardiomyopathy (FHC) is dominant mutations in cardiac sarcomeric genes. Linkage studies identified FHC-related mutations in the COOH terminus of cardiac troponin I (cTnI), a region with unknown function in Ca(2+) regulation of the heart. Using in vitro assays with recombinant rat troponin subunits, we tested the hypothesis that mutations K183Delta, G203S, and K206Q in cTnI affect Ca(2+) regulation. All three mutants enhanced Ca(2+) sensitivity and maximum speed (s(max)) of filament sliding of in vitro motility assays. Enhanced s(max) (pCa 5) was observed with rabbit skeletal and rat cardiac (alpha-MHC or beta-MHC) heavy meromyosin (HMM). We developed a passive exchange method for replacing endogenous cTn in permeabilized rat cardiac trabeculae. Ca(2+) sensitivity and maximum isometric force did not differ between preparations exchanged with cTn(cTnI,K206Q) or wild-type cTn. In both trabeculae and motility assays, there was no loss of inhibition at pCa 9. These results are consistent with COOH terminus of TnI modulating actomyosin kinetics during unloaded sliding, but not during isometric force generation, and implicate enhanced cross-bridge cycling in the cTnI-related pathway(s) to hypertrophy.

Actin Cytoskeleton↗

PubMed2003

The role of mitogen-activated protein kinase activation in determining cellular outcomes in polyamine analogue-treated human melanoma cells.

The clinically relevant polyamine analogue N(1),N(11)-diethylnorspermine (DENSPM) inhibits cell growth by down-regulating polyamine biosynthesis, up-regulating polyamine catabolism at the level of spermidine/spermine N(1)-acetyltransferase (SSAT), and depleting intracellular polyamine pools. Among human melanoma cell lines, the analogue causes rapid apoptosis in SK-MEL-28 cells and a sharp G(1) arrest in MALME-3M cells. This study reveals that DENSPM potently activates the mitogen-activated protein kinase (MAPK) pathways in melanoma cells and investigates the role of this response in determining cellular outcomes. Onset of apoptosis was preceded by an intense phosphorylation of the MAPKs, including extracellular signal-regulated kinase 1/2, c-Jun NH(2)-terminal kinase, and p38 in both SK-MEL-28 and MALME-3M cells. A panel of DENSPM analogues differing only in their ability to induce SSAT was used to show that MAPK activation was causally linked to induction of SSAT activity and related oxidative events. The latter was confirmed with the polyamine oxidase inhibitor MDL-75275 and the antioxidant N-acetyl-L-cysteine, which when used in combination with DENSPM, decreased MAPK activation and as previously shown, reduced apoptosis. The MAP/extracellular signal-regulated kinase-1 inhibitor PD 98059 reduced activation of all three kinases but failed to alter apoptosis in DENSPM-treated SK-MEL-28 cells. By contrast, the inhibitor prevented p21(waf1/cip1) induction and enhanced apoptosis in MALME-3M cells as indicated by accelerated caspase-3 activation and positive annexin V staining. The generality of this effect was demonstrated in DENSPM-treated A375 and LOX human melanoma cells. Taken together, the importance of the MAPK pathways in determining the biological response to DENSPM treatment is dependent on the genetic environment of the cell.

Antineoplastic Agents↗

PubMed2003

High-resolution structure of the soluble, respiratory-type Rieske protein from Thermus thermophilus: analysis and comparison.

The structure of the soluble Rieske protein from Thermus thermophilus has been determined at a resolution of 1.3 A at pH 8.5 using multiwavelength anomalous dispersion (MAD) techniques. This is the first report of a Rieske protein from a menaquinone-utilizing organism. The structure shows an overall fold similar to previously reported Rieske proteins. A novel feature of this crystal form appears to be a shared hydrogen between the His-134 imidazole ring ligated to Fe2 of the [2Fe-2S] cluster and its symmetry partner, His-134', one being formally an imidazolate anion, Fe2-(His-134)N(epsilon)(-)...H-N(epsilon')(His-134')-Fe2', in which crystallographic C(2) axes pass equidistant between N(epsilon)...N(epsilon') and normal to the line defined by N(epsilon)...N(epsilon'). This provides evidence for a stable, oxidized cluster with a His(-) ligand and lends support to a previously proposed mechanism of coupled proton and electron transfer. A detailed comparison of the Thermus Rieske protein with six other Rieske and Rieske-type proteins indicates: (a) The cluster binding domain is tightly conserved. (b) The 3-D structure of the 10 beta-strand fold is conserved, even among the most divergent proteins. (c) There is an approximately linear relation between acid-pH redox potential and number of H-bonds to the cluster. (d) These proteins have two faces, one points into the larger complex (bc(1), b(6)f, or other), is involved in the proton coupled electron transfer function, and is highly conserved. The second is oriented toward the solvent and shows wide variation in charge, sequence, length, hydrophobicity, and secondary elements in the loops that connect the beta-sheets.

Amino Acid Sequence↗

PubMed2003

Facile and efficient synthesis of meso-arylamino- and alkylamino-substituted porphyrins via palladium-catalyzed amination.

meso-Arylamino- and alkylamino-substituted porphyrins were efficiently synthesized by reactions of meso-halogenated porphyrins with amines via palladium-catalyzed amination. The combination of palladium acetate and the commercially available phosphine ligand bis(2-diphenylphosphinophenyl) ether (DPEphos) is effective for catalyzing the couplings of both [5-bromo-10,20-diphenyl porphyrino]zinc(II) and [5,15-dibromo-10,20-diphenylporphyrino]zinc(II) with amines to give the corresponding monoamino- and diamino-substituted porphyrins in high yields under mild conditions. The corresponding halogenated free-base porphyrins also underwent the cross-coupling reactions efficiently under similar catalytic conditions.

Journal Article↗

PubMed2003

Iron(III) and ruthenium(II) porphyrin complex-catalyzed selective olefination of aldehydes with ethyl diazoacetate.

The commercially available Fe(III) and Ru(II) porphyrin complexes Fe(TPP)Cl and Ru(TPP)(CO) are efficient catalysts for selective olefination of a variety of aldehydes with ethyl diazoacetate in the presence of triphenylphosphine. The reactions were carried out under mild conditions in a one-pot fashion with the use of a stoichiometrical amount of EDA, which proceeded with excellent yields and high (E)-selectivity. Air atmosphere, low catalyst loadings, and functional group tolerance were also demonstrated.

Journal Article↗

PubMed2003

Synthesis and bioactivity of 4,10-dimethyl-pyridino[2,3-h]quinolin-2(1H)-one-9-carboxylic acid and its esters.

4,10-Dimethyl-pyridino[2,3-h]quinolin-2(1H)-one-9-carboxylic acid (1) was synthesized by a new approach via the key intermediate 7-[1-aza-2-(dimethylamino)vinyl]-4-methylquinolin-2(1H)-one (4). Compound 1 and its esters were evaluated in cytotoxicity and anti-HIV assays. The 9-carboxyl (1s)-endo-(-)-borneol ester (9) showed marginal cytotoxic activity in CAK1-1, HOS, KB, and HCT-8 cells.

Anti-HIV Agents↗

PubMed2003

Protein engineering of the tissue inhibitor of metalloproteinase 1 (TIMP-1) inhibitory domain. In search of selective matrix metalloproteinase inhibitors.

Studies of the structural basis of the interactions of tissue inhibitors of metalloproteinases (TIMPs) and matrix metalloproteinases (MMPs) may provide clues for designing MMP-specific inhibitors. In this paper we report combinations of mutations in the major MMP-binding region that enhance the specificity of N-TIMP-1. Mutants with substitutions for residues 4 and 68 were characterized and combined with previously studied Thr(2) mutations to generate mutants with improved selectivity or binding affinity to specific MMPs. Some combinations of mutations had non-additive effects on DeltaG of binding to MMPs, suggesting interactions between subsites in the reactive site. The T2L/V4S mutation generates an inhibitor that binds to MMP-2 20-fold more tightly than to MMP-3(DeltaC) and over 400-fold more tightly than to MMP-1. The T2S/V4A/S68Y mutant is the strongest inhibitor for stromelysin-1 among all mutants characterized to date, with an apparent K(i) for MMP-3(DeltaC) in the picomolar range. A third mutant, T2R/V4I, has no detectable inhibitory activity for MMP-1 but is an effective inhibitor of MMP-2 and -3. These selective TIMP variants may provide useful tools for investigation of biological roles of specific MMPs and for possible therapy of MMP-related diseases.

Animals↗

PubMed2003

Tie2 receptor tyrosine kinase, a major mediator of tumor necrosis factor alpha-induced angiogenesis in rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is an inflammatory disease and an angiogenic disease. However, the molecular mechanisms promoting angiogenesis in RA are not clearly identified. Our objective was to study the role of an endothelium-specific receptor tyrosine kinase, Tie2, in angiogenesis of inflammatory arthritis. METHODS: Expression of Tie2 and its ligand, angiopoietin 1 (Ang1), in human synovium was examined by immunohistochemistry and Western blot. A novel synovium vascular window model was established to study the role of Tie2 in angiogenesis in vivo. Primary cultured endothelial cells and synoviocytes were used to study tumor necrosis factor alpha (TNF alpha)-induced Tie2 and Ang1 expression. RESULTS: Tie2 was implicated in pathologic angiogenesis. We observed that Tie2 and Ang1 were elevated in human RA synovium. Using a novel collagen-induced arthritis synovial window model, we demonstrated that Tie2 signaling regulated arthritis angiogenesis in vivo. We also showed that Tie2 mediated TNF alpha-induced angiogenesis in a mouse cornea assay. In addition, we observed that TNF alpha can regulate Tie2 activation in multiple ways that may involve interactions between endothelial cells and synoviocytes. TNF alpha up-regulates Tie2 in endothelial cells through nuclear factor kappa B, and it up-regulates Ang1 in synoviocytes. These findings suggest paracrine regulation of angiogenesis between endothelial cells and synoviocytes. CONCLUSION: This study demonstrates that Tie2 regulates angiogenesis in inflammatory synovium. Tie2 signaling is an important angiogenic mediator that links the proinflammatory cytokine TNF alpha to pathologic angiogenesis.

Angiogenesis Inducing Agents↗

PubMed2003

T-box binding site mediates the dorsal activation of myf-5 in Xenopus gastrula embryos.

Myf-5, a member of the muscle regulatory factor family of transcription factors, plays an important role in the determination, development, and differentiation of the skeletal muscle. Factors that regulate the expression of myf-5 itself are not well understood. We show here that a T-box binding site in the Xenopus myf-5 promoter mediated the activation of myf-5 expression through specific interaction with nuclear proteins of gastrula embryos. The T-box binding site could be bound by and respond to T-box proteins. T-box genes could induce Xmyf-5. The results suggest that T-box proteins are involved in the specification of myogenic mesoderm and muscle development.

Animals↗

PubMed2003

Ca2+ regulation of rabbit skeletal muscle thin filament sliding: role of cross-bridge number.

We investigated how strong cross-bridge number affects sliding speed of regulated Ca(2+)-activated, thin filaments. First, using in vitro motility assays, sliding speed decreased nonlinearly with reduced density of heavy meromyosin (HMM) for regulated (and unregulated) F-actin at maximal Ca(2+). Second, we varied the number of Ca(2+)-activatable troponin complexes at maximal Ca(2+) using mixtures of recombinant rabbit skeletal troponin (WT sTn) and sTn containing sTnC(D27A,D63A), a mutant deficient in Ca(2+) binding at both N-terminal, low affinity Ca(2+)-binding sites (xxsTnC-sTn). Sliding speed decreased nonlinearly as the proportion of WT sTn decreased. Speed of regulated thin filaments varied with pCa when filaments contained WT sTn but filaments containing only xxsTnC-sTn did not move. pCa(50) decreased by 0.12-0.18 when either heavy meromyosin density was reduced to approximately 60% or the fraction of Ca(2+)-activatable regulatory units was reduced to approximately 33%. Third, we exchanged mixtures of sTnC and xxsTnC into single, permeabilized fibers from rabbit psoas. As the proportion of xxsTnC increased, unloaded shortening velocity decreased nonlinearly at maximal Ca(2+). These data are consistent with unloaded filament sliding speed being limited by the number of cycling cross-bridges so that maximal speed is attained with a critical, low level of actomyosin interactions.

Actin Cytoskeleton↗