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Y Chen

Publications and source records attributed to Y Chen.

At least 1,063 records · Page 59Linked to original sources
PubMed1999

Group-specific assays that distinguish between the four major types of mammalian phospholipase A2.

Phospholipase A2 (PLA2) constitutes a diverse superfamily of enzymes which catalyze the deacylation of phospholipids. At least four types of PLA2 are potentially involved in arachidonic acid release in cells and tissues. Since all of them catalyze the same enzymatic reaction, it is difficult to distinguish between them in mixtures of enzymes normally present in biological samples. Utilizing specific properties of each PLA2, we have designed distinct assay procedures which selectively and sensitively detect each type: Group VI Ca2+-independent PLA2, Group IV cytosolic Ca2+-dependent PLA2, Groups V and IIA secreted PLA2s. Each specific assay procedure is selective for a particular PLA2 type by at least fourfold and as high as four orders of magnitude relative to the other three enzymes. All assays can detect PLA2 activity with as low as subnanogram quantities of enzyme. Importantly, these assays are able to differentiate and quantitate the biochemically and structurally related enzymes, Group IIA and V sPLA2s in crude biological samples. Employing this system, we have found that iPLA2 is the dominant PLA2 in rat brain, and cPLA2 is the most abundant PLA2 in P388D1 macrophages and human amnionic WISH cells.

Animals↗
PubMed1999

The application of an artificial neural network and pharmacokinetic simulations in the design of controlled-release dosage forms.

The objective of this work is to use an artificial neural network (ANN) and pharmacokinetic simulations in the design of controlled-release formulations with predictable in vitro and in vivo behavior. Seven formulation variables and three other tablet variables (moisture, particle size and hardness) for 22 tablet formulations of a model sympathomimetic drug were used as the ANN model input, and in vitro dissolution-time profiles at ten different sampling times were used as output. An ANN model was constructed by selecting the optimal number of iterations and model structure (the number of hidden layers and number of hidden layer nodes). The optimized ANN model was used for prediction of formulations based on two desired target in vitro dissolution-time profiles and two desired bioavailability profiles. For three of the four predicted formulations there was very good agreement between the ANN predicted and the observed in vitro and simulated in vivo properties. This work illustrates the potential for an artificial neural network, along with pharmacokinetic simulations, to assist in the development of complex dosage forms.

Biological Availability↗
PubMed1999

Use of a mixed-mode packing and voltage tuning for peptide mixture separation in pressurized capillary electrochromatography with an ion trap storage/reflectron time-of-flight mass spectrometer detector.

A mixed-mode (reversed-phase/anion-exchange) stationary phase has been used as the capillary column packing for investigation of the separation of peptide mixtures in pressurized capillary electrochromatography (pCEC). This stationary phase contains both octadecylsilanes and dialkylamines. The amine groups of the stationary phase determine the charge density on the surface of the packing and can produce a strong and constant electroosmotic flow (EOF) at low pH. A comparison was made in terms of the capability of separating tryptic digests between the mixed-mode phase and C18 reversed phase. In addition, the constant EOF enabled the tuning of the retention and the selectivity of the separation by adjusting the mobile phase pH from 2 to 5. Furthermore, the magnitude and the polarity of the electric voltage were demonstrated to greatly influence the elution profiles of the peptides in pCEC. An ion trap storage/reflectron time-of-flight mass spectrometer was used as an on-line detector in these experiments due to its ability to provide rapid and accurate mass detection of the sample components eluting from the separation column.

Chromatography, Liquid↗
PubMed1999

Formation of reactive cyclopentenone compounds in vivo as products of the isoprostane pathway.

Cyclopentenone prostaglandins A2 and J2 are reactive compounds that possess unique biological activities. However, the extent to which they are formed in vivo remains unclear. In this study, we explored whether D2/E2-isoprostanes undergo dehydration in vivo to form A2/J2-isoprostanes. Oxidation of arachidonic acid in vitro generated a series of compounds that were confirmed to be A2/J2-isoprostanes by mass spectrometric analyses. A2/J2-isoprostanes were detected in vivo esterified to lipids in livers from normal rats at a level of 5. 1 +/- 2.3 ng/g, and levels increased dramatically by a mean of 24-fold following administration of CCl4. An A2-isoprostane, 15-A2t-isoprostane, was obtained and found to readily undergo Michael addition with glutathione and to adduct covalently to protein. A2/J2-isoprostanes could not be detected in the circulation, even following CCl4 administration, which we hypothesized might be explained by rapid formation of adducts. This was supported by finding that essentially all the radioactivity excreted into the urine following infusion of radiolabeled 15-A2t-isoprostane into a human volunteer was in the form of a polar conjugate(s). These data identify a new class of reactive compounds that are produced in vivo as products of the isoprostane pathway that can exert biological effects relevant to the pathobiology of oxidant injury.

Animals↗
PubMed1999

DNA binding by the VH domain of anti-Z-DNA antibody and its modulation by association of the VL domain.

mAb Z22 is a highly selective IgG anti-Z-DNA Ab from an immunized C57BL/6 mouse. Previous studies showed that heavy chain CDR3 amino acids are critical for Z-DNA binding by the single chain variable fragment (scFv) comprising both V region heavy chain (VH) and V region light chain (VL) of mAb Z22 and that the VH domain alone binds Z-DNA with an affinity similar to that of whole variable fragment (Fv). To determine whether Z-DNA binding by VH alone and by Fv involves identical complementarity determining region residues, we tested effects of single or multiple amino acid substitutions in recombinant VH, scFv, and associated VH-VL heterodimers. Each recombinant product was a fusion protein with a B domain of Staphylococcal protein A (SPA). Z22VH-SPA alone was not highly selective; it bound strongly to other polynucleotides, particularly polypyrimidines, and ssDNA as well as to Z-DNA. In contrast, scFv-SPA or associated VH-VL dimers bound only to Z-DNA. VL-SPA domains bound weakly to Z-DNA; SPA alone did not bind. Introduction of multiple substitutions revealed that the third complementarity determining region of the heavy chain (CDR3H) was critical for both VH and scFv binding to Z-DNA. However, single substitutions that eliminated or markedly reduced Z-DNA binding by scFv instead caused a modest increase or no reduction in binding by VH alone. Association of VH-SPA with Z22VL-SPA restored both the effects of single substitutions and Z-DNA selectivity seen with Fv and intact Ab. Polypyrimidine and ssDNA binding by the isolated VH domain of immunization-induced anti-Z-DNA Ab resembles the activity of natural autoantibodies and suggests that VH-dependent binding to a ligand mimicked by polypyrimidines may play a role in B cell selection before immunization with Z-DNA.

Amino Acid Substitution↗
PubMed1999

[Absorbable mesh wrapping of injured kidney: a renal salvage technique].

Blunt trauma to the kidney is a rare cause of hospitalization, found in only 3% of patients hospitalized for trauma. Surgery is seldom required, but if necessary there are a variety of surgical techniques to preserve renal function. Organ-wrapping with absorbable mesh is a procedure previously described, but renal-wrapping has not gained the same acceptance as splenic or hepatic wrapping. We describe a 30-year-old man with grade III blunt renal trauma, in whom this technique was applied. He was discharged after 10 days without complications.

Adult↗
PubMed1999

Tamoxifen inhibits acidification in cells independent of the estrogen receptor.

Tamoxifen has been reported to have numerous physiological effects that are independent of the estrogen receptor, including sensitization of resistant tumor cells to many chemotherapeutic agents. Drug-resistant cells sequester weak base chemotherapeutics in acidic organelles away from their sites of action in the cytosol and nucleus. This work reports that tamoxifen causes redistribution of weak base chemotherapeutics from acidic organelles to the nucleus in drug-resistant cells. Agents that disrupt organelle acidification (e.g., monensin, bafilomycin A1) cause a similar redistribution. Measurement of cellular pH in several cell lines reveals that tamoxifen inhibits acidification of endosomes and lysosomes without affecting cytoplasmic pH. Similar to monensin, tamoxifen decreased the rate of vesicular transport though the recycling and secretory pathways. Organellar acidification is required for many cellular functions, and its disruption could account for many of the side effects of tamoxifen.

Anti-Bacterial Agents↗
PubMed1999

Thyroid hormone, T3-dependent phosphorylation and translocation of Trip230 from the Golgi complex to the nucleus.

Trip230 is a novel coactivator of the thyroid hormone receptor that is negatively regulated by the retinoblastoma tumor-suppressor protein. In an examination of its subcellular distribution, Trip230 localized predominantly to the vicinity of the Golgi instead of the nucleus, as other nuclear hormone receptor coactivators. Using a series of deletion mutants, a critical region identified for Golgi area targeting coincided with a previously defined thyroid hormone receptor-binding domain of Trip230. During cell cycle progression, the expression level of Trip230 is constant and a significant portion is imported into the nucleus at S phase. Within an hour of treating cells with T3, Trip230 immunofluorescence transiently colocalized with TR in prominent subnuclear structures. T3-dependent nuclear import of Trip230 does not require new protein synthesis. Coincident with T3 treatment and nuclear import, newly phosphorylated residue(s) appeared in Trip230, suggesting that phosphorylation may be involved in its nuclear import. These findings provided a novel mechanism for the regulation of nuclear hormone transcription factors by hormone-responsive phosphorylation and nuclear import of cytoplasmically located coactivators.

Animals↗
PubMed1999

Psychophysical isolation of a motion-processing deficit in schizophrenics and their relatives and its association with impaired smooth pursuit.

Schizophrenia patients and many of their relatives show impaired smooth pursuit eye tracking. The brain mechanisms underlying this impairment are not yet known, but because reduced open-loop acceleration and closed-loop gain accompany it, compromised perceptual processing of motion signals is implicated. A previous study showed that motion discrimination is impaired in schizophrenia patients. Motion discrimination can make use of position and contrast as well as velocity cues. Here, we report that the motion discrimination deficit, which occurs in both schizophrenic patients and in their first-degree relatives, involves a failure of velocity detection, which appears when judging intermediate target velocities. At slower and faster velocities, judgments of velocity discrimination seemed normal until we experimentally disentangled velocity cues from nonmotion cues. We further report that compromised velocity discrimination is associated with sluggish initiation of smooth pursuit. These findings point to specific central nervous system correlates of schizophrenic pathophysiology.

Adult↗
PubMed1999

[A comparative study of HLA-A locus in northern and southern Chinese by means of PCR/SSOP typing]

OBJECTIVE: To compare the HLA-A locus in a population selected from Beijing (northern group) with that in a population selected from Guangzhou (southern group). METHODS: HLA typing was performed by using PCR/SSOP method. A pair of primer and 54 probes were used. RESULTS: In 18 alleles identified, the differences in gene frequency between northern and southern Chinese were found. In northern group, A*0205, 0210 and 2901 were absent, and the frequencies of A*2601, 3001 and 3101 were higher than those of southern group; while in southern group, A*3103, 3201 and 6801 were absent, the frequencies of A*0203 and 1101 were higher than those of northern group; and in addition, six subtypes of A2 serological specificity, namely A*0201, 0203, 0205, 0206, 0207 and 0210, were found, in which A*0201 predominated. CONCLUSION: There are some differences in the genetic background of northern and southern Chinese. A2 subtypes have important implications for unrelated-donor transplantation.

Journal Article↗
PubMed1999

Positive and negative regulation of IkappaB kinase activity through IKKbeta subunit phosphorylation.

IkappaB [inhibitor of nuclear factor kappaB (NF-kappaB)] kinase (IKK) phosphorylates IkappaB inhibitory proteins, causing their degradation and activation of transcription factor NF-kappaB, a master activator of inflammatory responses. IKK is composed of three subunits-IKKalpha and IKKbeta, which are highly similar protein kinases, and IKKgamma, a regulatory subunit. In mammalian cells, phosphorylation of two sites at the activation loop of IKKbeta was essential for activation of IKK by tumor necrosis factor and interleukin-1. Elimination of equivalent sites in IKKalpha, however, did not interfere with IKK activation. Thus, IKKbeta, not IKKalpha, is the target for proinflammatory stimuli. Once activated, IKKbeta autophosphorylated at a carboxyl-terminal serine cluster. Such phosphorylation decreased IKK activity and may prevent prolonged activation of the inflammatory response.

Amino Acid Sequence↗
PubMed1999

Antioxidant mechanisms in apolipoprotein E deficient mice prior to and following closed head injury.

Apolipoprotein E deficient mice have distinct memory deficits and neurochemical derangements and their recovery from closed head injury is impaired. In the present study, we examined the possibility that the neuronal derangements of apolipoprotein E deficient mice are associated with oxidative stress, which in turn affects their ability to recover from close head injury. It was found that brain phospholipid levels in apolipoprotein E deficient mice are lower than those of the controls (55+/-15% of control, P<0. 01), that the cholesterol levels of the two mice groups are similar and that the levels of conjugated dienes of the apolipoprotein E deficient mice are higher than those of control mice (132+/-15% of P<0.01). Brains of apolipoprotein E deficient mice had higher Mn-superoxide dismutase (134+/-7%), catalase (122+/-8%) and glutathione reductase (167+/-7%) activities than control (P<0.01), whereas glutathione peroxidase activity and the levels of reduced glutathione and ascorbic acid were similar in the two mouse groups. Closed head injury increased catalase and glutathione peroxidase activities in both mouse groups, whereas glutathione reductase increased only in control mice. The superoxide dismutase activity was unaffected in both groups. These findings suggest that the antioxidative metabolism of apolipoprotein E deficient mice is altered both prior to and following head injury and that antioxidative mechanisms may play a role in mediating the neuronal maintenance and repair derangements of the apolipoprotein E deficient mice.

Animals↗
PubMed1999

DNA microarray technology: the anticipated impact on the study of human disease.

One can imagine that, one day, there will be a general requirement that relevant array data be deposited, at the time of publication of manuscripts in which they are described, into a single site made available for the storage and analysis of array data (modeled after the GenBank submission requirements for DNA sequence information). With this system in place, one can anticipate a time when data from thousands of gene expression experiments will be available for meta-analysis, which has the potential to balance out artifacts from many individual studies, thus leading to more robust results and subtle conclusions. This will require that data adhere to some type of uniform structure and format that would ideally be independent of the particular expression technology used to generate it. The pros and cons of various publication modalities for these large electronic data sets have been discussed elsewhere [12], but, practical difficulties aside, general depositing must occur for this technology to reach the broadest range of investigators. Finally, as mentioned at the beginning of this review, it is unfortunate that this important research tool remains largely restricted to a few laboratories that have developed expertise in this area and to a growing number of commercial interests. Ultimately the real value of microarray technology will only be realized when this approach is generally available. It is hoped that issues including platforms, instrumentation, clone availability, and patents [20] will be resolved shortly, making this technology accessible to the broadest range of scientists at the earliest possible moment.

Cell Line↗
PubMed1999

3-Bromotyrosine and 3,5-dibromotyrosine are major products of protein oxidation by eosinophil peroxidase: potential markers for eosinophil-dependent tissue injury in vivo.

Detection of specific reaction products is a powerful approach for dissecting out pathways that mediate oxidative damage in vivo. Eosinophil peroxidase (EPO), an abundant protein secreted from activated eosinophils, has been implicated in promoting oxidative tissue injury in conditions such as asthma, allergic inflammatory disorders, cancer, and helminthic infections. This unique heme protein amplifies the oxidizing potential of H2O2 by utilizing plasma levels of Br- as a cosubstrate to form potent brominating agents. Brominated products might thus serve as powerful tools for identifying sites of eosinophil-mediated tissue injury in vivo; however, structural identification and characterization of specific brominated products formed during EPO-catalyzed oxidation have not yet been reported. Here we explore the role of EPO and myeloperoxidase (MPO), a related leukocyte protein, in promoting protein oxidative damage through bromination and demonstrate that protein tyrosine residues serve as endogenous traps of reactive brominating species forming stable ring-brominated adducts. Exposure of the amino acid L-tyrosine to EPO, H2O2, and physiological concentrations of halides (100 mM Cl-, </=100 microM Br-) produced two new major products with distinct retention times on reverse phase HPLC. The products were identified as 3-bromotyrosine and 3, 5-dibromotyrosine by electrospray ionization mass spectrometry and multinuclear (1H and 15N) NMR spectroscopy. Formation of the ring-brominated forms of the amino acid occurred readily at neutral pH with the enzymatic system and a variety of reactive brominating species, including HOBr/OBr-, N-bromoamines, and N,N-dibromoamines. Addition of primary amines (e.g., Nalpha-acetyllysine and taurine) to L-tyrosine exposed to either HOBr/OBr- or the EPO-H2O2-Br- system enhanced phenolic ring bromination, suggesting N-bromoamines are preferred brominating intermediates in these reactions. Reduction of N-bromoamines (e.g., Nalpha-acetyl,Nepsilon-bromolysine) by L-tyrosine was shown to result in the loss of reactive halogen with a near stoichiometric increase in the level of tyrosine ring bromination (i.e., carbon-bromine bonds). Although both EPO and MPO could use Br- to halogenate protein tyrosine residues in vitro, only EPO effectively brominated the aromatic amino acid at physiological levels of halides and H2O2. Collectively, these results suggest that 3-bromotyrosine and 3,5-dibromotyrosine are attractive candidates for serving as molecular markers for oxidative damage of proteins by reactive brominating species in vivo. They also suggest that in biological mixtures where amine groups are abundant, the trapping of EPO-generated HOBr/OBr- as N-bromoamines will serve to effectively "funnel" reactive brominating equivalents to stable ring-brominated forms of tyrosine.

Animals↗
PubMed1999

Iron overload upregulates haem oxygenase 1 in the lung more rapidly than in other tissues.

Haem oxygenase-1 is upregulated by numerous insults, including oxidative stress, and under such circumstances it is considered to be a protective stratagem. We have measured the haem oxygenase-1 expression in heart, lung and liver tissues of control and iron-overloaded rats. Lung tissue from iron-overloaded rats displayed a significant increase in the haem oxygenase-1 protein but no changes in haem oxygenase-1 mRNA. Conversely, heart tissue showed a significant increase in haem oxygenase-1 mRNA but no changes in haem oxygenase-1 protein. We conclude that during oxidative stress caused by iron overload, lung tissue responds with a rapid upregulation of haem oxygenase-1 levels.

Animals↗
PubMed1999

L-764406 is a partial agonist of human peroxisome proliferator-activated receptor gamma. The role of Cys313 in ligand binding.

Insulin-sensitizing thiazolidinedione (TZD) compounds are high affinity ligands for a member of the nuclear receptor family, peroxisome proliferator-activated receptor (PPAR) gamma. A scintillation proximity assay for measurement of 3H-radiolabeled TZD binding to human PPARgamma under homogeneous conditions was developed. Using this approach, a novel non-TZD compound (L-764406) was shown to be a potent (apparent binding IC50 of 70 nM) PPARgamma ligand. Preincubation of PPARgamma with L-764406 prevented binding of the [3H]TZD, suggesting a covalent interaction with the receptor; in addition, structurally related analogues of L-764406, which would be predicted not to interact with PPARgamma in a covalent fashion, did not displace [3H]TZD binding to PPARgamma. Covalent binding of L-764406 was proven by an observed molecular weight shift of a tryptic PPARgamma ligand binding domain (LBD) peptide by mass spectrometric analysis. A specific cysteine residue (Cys313 in helix 3 of hPPARgamma2) was identified as the attachment site for this compound. In protease protection experiments, the liganded receptor adopted a typical agonist conformation. L-764406 exhibited partial agonist activity in cells expressing a chimeric receptor containing the PPARgamma LBD and a cognate reporter gene and also induced the expression of the adipocyte-specific gene aP2 in 3T3-L1 cells. In contrast, L-764406 did not exhibit activity in cells transfected with chimeric receptors containing PPARalpha or PPARdelta LBDs. The partial agonist properties of L-764406 were also evident in a co-activator association assay, indicating that the increased transcription in cells was co-activator mediated. Thus, L-764406 is a novel non-TZD ligand for PPARgamma and is also the first known partial agonist for this receptor. The results suggest a critical functional role for Cys313, and helix 3, in contributing to ligand binding and subsequent agonist-induced conformational changes.

Amino Acid Sequence↗
PubMed1999

Resolution of a signal transfer region from a general binding domain in gbeta for stimulation of phospholipase C-beta2.

Signaling by guanine nucleotide-binding proteins (G proteins) involves sequential protein-protein interactions. G protein-betagamma subunit (Gbetagamma) interactions with phospholipase C-beta2 (PLC-beta2) were studied to determine if all Gbeta contacts are required for signaling. A peptide encoding Gbeta amino acid residues 86 to 105 stimulated PLC-beta2. Six residues (96 to 101) within this sequence could transfer signals and thus constitute a core signal transfer region. Another peptide, encoding Gbeta amino acid residues 115 to 135, did not substantially stimulate PLC-beta2 by itself but inhibited Gbetagamma stimulation, indicating that residues 115 to 135 constitute a general binding domain. Resolution of signal transfer regions from general binding domains indicates that all protein-protein contacts are not required for signal transfer and that it may be feasible to synthesize agonists and antagonists that regulate intracellular signal flow.

Amino Acid Substitution↗