Biomedical subjects

S X Yang

Publications and source records attributed to S X Yang.

At least 19 recordsLinked to original sources

PubMed2001

Activation of Galpha s mediates induction of tissue-type plasminogen activator gene transcription by epoxyeicosatrienoic acids.

The epoxyeicosatrienoic acids (EETs) are products of cytochrome P450 (CYP) epoxygenases that have vasodilatory and anti-inflammatory properties. Here we report that EETs have additional fibrinolytic properties. In vascular endothelial cells, physiological concentrations of EETs, particularly 11,12-EET, or overexpression of the endothelial epoxygenase, CYP2J2, increased tissue plasminogen activator (t-PA) expression by 2.5-fold without affecting plasminogen activator inhibitor-1 expression. This increase in t-PA expression correlated with a 4-fold induction in t-PA gene transcription and a 3-fold increase in t-PA fibrinolytic activity and was blocked by the CYP inhibitor, SKF525A, but not by the calcium-activated potassium channel blocker, charybdotoxin, indicating a mechanism that does not involve endothelial cell hyperpolarization. The t-PA promoter is cAMP-responsive, and induction of t-PA gene transcription by EETs correlated with increases in intracellular cAMP levels and, functionally, with cAMP-driven promoter activity. To determine whether increases in intracellular cAMP levels were due to modulation of guanine nucleotide-binding proteins, we assessed the effects of EETs on Galpha(s) and Galpha(i2). Treatment with EETs increased Galpha(s), but not Galpha(i2), GTP-binding activity by 3.5-fold. These findings indicate that EETs possess fibrinolytic properties through the induction of t-PA and suggest that endothelial CYP2J2 may play an important role in regulating vascular hemostasis.

8,11,14-Eicosatrienoic Acid↗

PubMed2001

HAL1 mediate salt adaptation in Arabidopsis thaliana.

The yeast HAL1 gene was introduced into Arabidopsis thaliana by Agrobacterium tumefaciens-mediated transformation with vacuum infiltration under the control of CaMV 35S promoter. Thirty-three individual kanamycin resistant plants were obtained from 75,000 seeds. Southern blotting analysis indicated that HAL1 gene had been integrated into all of the transgenic plants' genomes. The copy number of HAL1 gene in transgenic plants was mostly 1 to 3 by Southern analysis. Phenotypes of transgenic plants have no differences with wild type plants. Several samples of transformants were self-pollinated, and progenies from transformed and non-transformed plants (controls) were evaluated for salt tolerance and gene expression. Measurement of concentrations of intracellular K+ and Na+ showed that transgenic lines were able to retain less Na+ than that of the control under salt stress. Results from different tests indicated the expression of HAL1 gene promotes a higher level of salt tolerance in vivo in the transgenic Arabidopsis plants.

Adaptation, Physiological↗

PubMed2000

Quantitative, high-throughput cell-based assays for inhibitors of trkA receptor.

Two quantitative, high-throughput cell-based assays for evaluating inhibitors of NGF-stimulated trkA phosphorylation in trkA-transfected NIH3T3 cells have been established. Both assays involve capture of the trkA receptor from cell lysates in microtiter plates coated with an anti-trk antibody. The amount of trkA phosphorylation is then measured using either an anti-phosphotyrosine antibody with a colorimetric readout or a lanthanide (europium)-labeled anti-phosphotyrosine antibody with a fluorometric detection. The two assay formats exhibited at least a fivefold increase in phosphorylated trkA signal in trkA-transfected cells compared to vector control. Inhibition plots generated for trkA kinase inhibitors using the two detection systems yielded comparable IC(50) values. Overall, the two assays represent a marked improvement over the standard gel-based/western blot method in terms of throughput, quantitation, and amenability to automation.

3T3 Cells↗

PubMed2000

Image deconvolution for protein crystals.

The image deconvolution technique developed for non-biological samples, which is based on the weak-phase-object approximation and principle of maximum entropy, was applied to simulated images of the biotin-binding protein streptavidin. A slight modification of the technique was introduced to solve the problem caused by the special image formation condition for biological samples. It has been shown that the modified technique is effective.

Crystallography↗

PubMed2000

An efficient neural network approach to dynamic robot motion planning.

In this paper, a biologically inspired neural network approach to real-time collision-free motion planning of mobile robots or robot manipulators in a nonstationary environment is proposed. Each neuron in the topologically organized neural network has only local connections, whose neural dynamics is characterized by a shunting equation. Thus the computational complexity linearly depends on the neural network size. The real-time robot motion is planned through the dynamic activity landscape of the neural network without any prior knowledge of the dynamic environment, without explicitly searching over the free workspace or the collision paths, and without any learning procedures. Therefore it is computationally efficient. The global stability of the neural network is guaranteed by qualitative analysis and the Lyapunov stability theory. The effectiveness and efficiency of the proposed approach are demonstrated through simulation studies.

Artificial Intelligence↗

PubMed2000

Neuroprotection mediated by changes in the endothelial actin cytoskeleton.

Cerebral blood flow is regulated by endothelium-derived nitric oxide (NO), and endothelial NO synthase-deficient (eNOS-deficient; eNOS(-/-)) mice develop larger cerebral infarctions following middle cerebral artery (MCA) occlusion. We report that disruption of Rho-mediated endothelial actin cytoskeleton leads to the upregulation of eNOS expression and reduces the severity of cerebral ischemia following MCA occlusion. Mice treated with the Rho inhibitor Clostridium botulinum C3 transferase (10 microgram/d) or the actin cytoskeleton disrupter cytochalasin D (1 mg/kg) showed a two- to fourfold increase in vascular eNOS expression and activity. This increase in eNOS expression was not due to increases in eNOS gene transcription, but to prolongation of eNOS mRNA half-life from 10 +/- 3 hours to 24 +/- 4 hours. Indeed, endothelial cells overexpressing a dominant-negative Rho mutant (N19RhoA) exhibited decreased actin stress fiber formation and increased eNOS expression. Inhibition of vascular Rho guanosine-5'-triphosphate binding activity by the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor simvastatin increased cerebral blood flow to ischemic regions of the brain, and mice treated with simvastatin, C3 transferase, or cytochalasin D showed smaller cerebral infarctions following MCA occlusion. No neuroprotection was observed with these agents in eNOS(-/-) mice. These findings suggest that therapies which target the endothelial actin cytoskeleton may have beneficial effects in ischemic stroke.

Actins↗

PubMed1999

Activation of guanine nucleotide-binding proteins and induction of endothelial tissue-type plasminogen activator gene transcription by alcohol.

The mechanism by which moderate alcohol ingestion lowers the risk of cardiovascular disease is unknown but may be due, in part, to the ability of alcohol to increase the level of tissue-type plasminogen activator (t-PA). Human endothelial cells were treated with low concentrations of ethanol (0.25-25 mM, 0-24 h), which are associated with moderate alcohol consumption. Although treatment with ethanol alone did not affect t-PA gene transcription or mRNA expression, it augmented isoproterenol (ISO)-stimulated t-PA gene transcription and mRNA levels by 3.4- and 2.8-fold, respectively, and decreased plasminogen activator inhibitor-1 mRNA levels by 65%. These effects of ethanol correlated with 2.5- and 6.9-fold increases in ISO-stimulated cyclic AMP levels and 4x-cyclic AMP response element heterologous promoter activity, respectively. To determine whether alcohol-induced changes in agonist-stimulated cyclic AMP levels were because of modulation of guanine nucleotide-binding proteins (G proteins), we assessed the effects of ethanol on Galphas and Galphai2. Although ethanol did not affect the expression of Galphas or Galphai2, it increased ISO-stimulated Galphas GTPase and GTP binding activity by 2.2- and 2.9-fold and decreased UK14304-stimulated Galphai2 GTPase and GTP binding activity by 38 and 80%. These results indicate that treatment with relatively low concentrations of ethanol enhances agonist-stimulated cyclic AMP-dependent t-PA gene transcription in vascular endothelial cells through differential modulation of G protein.

Adenylyl Cyclases↗

PubMed1998

Kinetics of trkA tyrosine kinase activity and inhibition by K-252a.

The kinetic mechanism of the trk receptor-linked tyrosine kinase was determined using a baculovirus expressed trk kinase domain and a bacterially expressed phospholipase C-gamma/glutathione S-transferase (PLC-gamma/ GST) fusion protein as substrate. Product and dead-end inhibition studies indicate an ordered association of substrates to trkA kinase with the nucleotide ATP binding prior to the exogenous substrate PLC-gamma/GST, followed by release of the phosphorylated PLC-gamma/GST product prior to release of ADP (sequential ordered bi-bi mechanism). This is in contrast to the reported kinetic mechanisms of closely related EGF receptor and insulin receptor kinases which appear to proceed via a rapid equilibrium random mechanism. The indolocarbazole K-252a, which was previously shown to be a potent and relatively selective inhibitor of trk kinase activity, acts as a competitive inhibitor with respect to ATP. The data suggest that potent and selective kinase inhibitors can be rationally designed by exploring subtle variations surrounding the nucleotide binding sites of receptor tyrosine kinases.

Adenosine Diphosphate↗

PubMed1998

Comparative sequence analysis of open reading frames 2 to 7 of the modified live vaccine virus and other North American isolates of the porcine reproductive and respiratory syndrome virus.

To elucidate changes associated with the attenuated virulence in a modified live porcine reproductive and respiratory syndrome (PRRS) vaccine (Boehringer Ingelheim Animal Health, St. Joseph, MO), derived from an American prototype ATCC virus VR-2332, nucleotide sequence of 3' genome covering open reading frames (ORFs) 2 to 7 coding regions from the vaccine virus was determined by RT-PCR with two overlapping fragments. Comparisons showed 98 base changes (94 substitutions, 3 deletions, and 1 addition) out of 3318 nucleotides between the vaccine virus and its parental virus. There were 15, 26, 17, 29, 9, and 6 base substitutions in ORFs 2, 3, 4, 5, 6, and 7, respectively, resulting in 5, 13, 8, 13, 2, and 3 amino acid (a.a.) substitutions in their deduced proteins, respectively. Most of these a.a. substitutions were also present in 17 known virulent/wild type PRRS virus isolates from North America. However, there were 1, 4, 1, and 1 unique a.a. substitutions in the vaccine virus ORFs 2, 3, 4, and 5 deduced proteins, respectively. These unique amino substitutions may be responsible for the attenuated virulence in the vaccine virus.

Amino Acid Sequence↗

PubMed1996

Kinetics of nonenzymatic glycation of ribonuclease A leading to advanced glycation end products. Paradoxical inhibition by ribose leads to facile isolation of protein intermediate for rapid post-Amadori studies.

Nonenzymatic glycation (Maillard reaction) of long-lived proteins is a major contributor to the pathology of diabetes and possibly aging and Alzheimer's disease. We report here kinetic studies of the glycation of the model protein ribonuclease A by glucose and ribose leading to the formation of antigenic advanced glycation end products ("AGEs"), detectable by AGE-specific polyclonal antibodies, and pentosidine, an acid-stable fluorescent AGE. As anticipated, the kinetics of glycation by ribose were considerably faster than by glucose, and the rate of AGE formation initially increased with increasing sugar concentrations. However, ribose above 0.15 M appeared to paradoxically slow the kinetics of AGE formation, suggesting ribose inhibits the conversion of "early" Amadori rearrangement products to "late" AGEs and thus favors the accumulation of reactive Amadori intermediates. The facile isolation of such protein intermediates was achieved by an "interrupted glycation" protocol which free and reversibly bound (Schiff base) ribose was removed following a short (24h) initial incubation of 0.5 M ribose at 37 degrees C. The kinetics of buildup of the Amadori intermediates and the kinetics of their post-Amadori conversion to antigenic AGEs were independently studied. A rapid and reversible inhibition of the post-Amadori kinetics by free ribose was verified by direct re-addition of ribose to the isolated, sugar-free intermediate. The pH dependence of the kinetics of antigenic AGE formation from such intermediates was measured and exhibited an unusual bell-shaped profile over the pH range of 5.0-9.5 with a maximum near pH 8.0. Aminoguanidine, a pharmacological AGE inhibitor, was found to moderately or weakly inhibit antigenic AGE formation in such post- Amadori steps. The isolation of the glycated ribonuclease intermediate thus simplifies kinetic and mechanistic studies of AGE formation, permits AGE studies in the absence of complications arising from free or Schiff base bound sugar, and provides a novel methodology for evaluating the mechanism and efficacy of therapeutic agents that may inhibit AGE formation.

Antibodies↗

PubMed1996

Glycosylation in human thyroglobulin: location of the N-linked oligosaccharide units and comparison with bovine thyroglobulin.

The amino acid sequence established for human thyroglobulin (hTG) from its cDNA sequence contains 20 putative N-linked glycosylation sites. We have characterized the glycopeptides contained in a tryptic digest of hTG in order to determine which sites are actually linked to carbohydrate. In addition, the distribution of oligosaccharide type(s) at these confirmed sites of N-linked glycosylation has been examined. Glycopeptides were purified using gel permeation chromatography followed by several steps of HPLC. The purified tryptic glycopeptides were characterized by gas phase sequencing and carbohydrate analysis and located within the amino acid sequence of thyroglobulin. Each of the recovered glycopeptides contained a consensus sequence for N-linked glycosylation. Of the 20 putative N-linked glycosylation sites in the human thyroglobulin polypeptide chain, 16 were shown to be actually glycosylated in the mature protein. Eight of these confirmed glycosylation sites (at positions 57, 465, 510, 729, 797, 1696, 1754, and 2230) appear to be linked to complex-type oligosaccharide units containing fucose and galactose in addition to mannose and glucosamine. Five sites (at positions 1200, 1329, 1993, 2275, and 2562) contain high mannose type units and two sites (at positions 179 and 1345) are linked to oligosaccharide units containing galactose in addition to mannose and glucosamine but no fucose and may be either hybrid or complex structures. In addition, position 928 was found to be degenerate in oligosaccharide structure and very different oligosaccharide composition types were found associated with peptides containing the same amino acid sequence. A high probability of a beta turn which would include the glycosylated asparagine residue was predicted for the amino acid sequence found at 13 of the 16 sites. The glycosylation pattern in hTG was also compared with the data recently reported for bovine thyroglobulin (bTG) (27) and as has been recently reported for bTG, no oligosaccharides of the high mannose type were found in the N-terminal portion of hTG. Only four of the 20 putative sites the sequence of hTG, at asparagine residues 91, 477, 1849, and 2102 were not represented in the purified glycopeptide population and are presumed to escape significant glycosylation.

Amino Acid Sequence↗

PubMed1996

Analysis of the T cell receptor Vgamma region gene repertoire in bronchoalveolar lavage (BAL) and peripheral blood of atopic asthmatics and healthy subjects.

We analyzed the T cell receptor (TCR) Vgamma repertoire in BAL and peripheral blood (PBL) of three mild stable atopic asthmatics and two non-asthmatic controls. We used the polymerase chain reaction (PCR) to establish the expression of the four Vgamma families, and to detect oligo or monoclonal expansion of gammadelta+ T cells, we resolved the PCR projects on denaturing and non-denaturing gels to find the extent of junctional diversity arising from differences in the lengths of the V(D)J junctions. We also subcloned and sequenced the PCR products to characterize fully the sequence diversity. BAL T lymphocytes from two asthmatic patients (treated with inhaled steroids) expressed only VgammaII and, in one of them, VgammaIIJgamma usage was restricted to JgammaP and JgammaP1 gene segments, contrasting with the VgammaJgamma repertoire found in his respective PBL. Analyses in denaturing and non-denaturing gels showed that the BAL VgammaIIJgammaP and VgammaIIJgammaP1 PCR products resolved into few bands, suggesting deletions at the juctions due to oligoclonal expansion. BAL T lymphocytes from the third asthmatic (not receiving inhaled steroids) expressed VgammaI, II and III, and the sequences of the in-frame TCR transcripts from this asthmatic and one healthy volunteer who expressed a similar BAL VgammaTCR repertoire showed clonal expansion of T cells expressing all three Vgamma families. Our analyses showed that much of the GammaDeltaT cell population found in BAL fluid of humans derives from clonally expanded T cells.

Adult↗

PubMed1996

Analysis of T cell receptor gamma transcripts in right and left knee synovial fluids of patients with rheumatoid arthritis.

OBJECTIVE: To establish the extent of clonal expansion of T cell receptor (TCR) gamma delta + T cells in synovial fluid (SF), and the sharing of the clones between affected knee joints from patients with rheumatoid arthritis (RA). METHODS: We quantified, using the polymerase chain reaction (PCR), the level of expression from each of the 4 V gamma gene families. We resolved PCR products on denaturing polyacrylamide gels to measure the proportion of mRNA transcripts contributed by clonally expanded gamma delta T cells. We subcloned and sequenced 509 cDNA clones derived from 8 SF and one peripheral blood sample from 2 patients with RA and one patient with juvenile onset RA to fully characterize the populations of T cell receptor gamma mRNA sequences. RESULTS: We found in each patient disproportionate expression of a subpopulation of T cell receptor gamma mRNA transcripts. Some of these transcripts are expressed by T cells found in both joints. CONCLUSION: Synovial TCR gamma delta + T cells are oligoclonal and some of these T cell clones are common to SF of both joints. The finding of identical T cell sequences from SF of both affected joints from each patient points to a role for gamma delta + T cells in disease progression.

Amino Acid Sequence↗

PubMed1995

The insulin-like growth factor signaling system and ALS neurotrophic factor treatment strategies.

Because of its multi-faceted potential as a neurotrophic factor, insulin-like growth factor I (IGF-I) has been given to hundreds of ALS patients world-wide. Unlike some patients with post-polio syndrome and fragile elderly males, it is unclear whether any of these patients possess disturbances in IGF signaling. We found that about 25% of ALS patients in a controlled trial of human growth hormone (hGH) had lower or higher than normal IGF-I serum levels. Many ALS patients do have some of the characteristics of type II diabetes mellitus, where IGF-I therapy is also under way. In addition, in type I diabetes significant increase in a circulating molecule that binds IGF-I, IGF-I binding protein 1 (IGFBP-1), occurs along with reduced IGF-I, when neuropathic complications are prominent. We have studied the response of IGFBPs in ALS patients to subcutaneous rhIGF-I and found transient induction of IGFBP-1. Studies related to the IGFBPs have not been done in familial ALS (FALS) patients. However, the gene for another IGFBP, BP-2, co-localizes with the gene for juvenile ALS (ALSJ) on chromosome 2. IGF-I has been given to several models of motor neuron degeneration in the mouse, including motor neuron disease and wobbler, with beneficial effects. However, it is also not known whether any accepted genetic mouse model of motor neuron degeneration possesses any disturbance in the IGF signaling system.

Amyotrophic Lateral Sclerosis↗

PubMed1994

Insulin-like growth factor binding protein-1 at mouse neuromuscular synapses.

The insulin-like growth factor (IGF) signaling system includes the growth factors and their cell surface receptors, along with circulating IGF binding proteins (IGFBPs) that may alter and modulate the action of these neurotrophic hormones. These IGFBPs, along with IGFs and receptors, have been detected in various tissues including the brain. In this study, using polyclonal antibody to human IGFBP-1 or bovine IGFBP-2, we found that mouse muscle extracts contain similar-sized proteins that cross-react with these antibodies on Western immunoblots. After establishing that these antibodies reacted with the homologous murine IGFBPs, we performed immunocytochemistry to demonstrate the localization of IGFBP-1 at the neuromuscular junction, a model nicotinic, cholinergic synapse, as well as within intramuscular nerves. IGFBP-2, a distinct macromolecule, is present on the surface of muscle fibers and is not present within synapses or nerves.

Animals↗

PubMed1994

Insulin-like growth factor binding protein-1 is pre-synaptic at mouse neuromuscular synapses and is transported in nerve.

In a previous study, we localized insulin-like growth factor binding protein 1 (IGFBP-1) to mouse neuromuscular junctions, and intramuscular nerves. To determine if pre-synaptic accumulation of IGFBP-1 occurred, we used double ligation of sciatic nerve in adult mice at different time points. IGFBPs were detected by Western ligand blot (WLB) with 125I-IGF-I. WLB and Western immunoblot (WIB) analysis of extracts from double-ligated nerves showed a delayed (6 days) increase of IGFBP-1 in the soluble fraction between the ligatures and distal to the distal ligature. For comparison we evaluated transport of neurofilament components, using WIB and confirmed the primarily anterograde transport of these intraaxonal proteins. These data suggest that expression of IGFBP-1 is both by activated Schwann cells as well as retrograde axonal transport with likely entry into the axon at the synapse.

Animals↗

PubMed1994

Triptolide suppresses T-lymphocyte proliferation by inhibiting interleukin-2 receptor expression, but spares interleukin-2 production and mRNA expression.

The purpose of this study was to elucidate the mechanism of action of triptolide on the T-lymphocyte-mediated immune response. Lymphocytes were incubated with a suboptimal dose of Con A or PHA in the presence or absence of varying doses of triptolide to assess the effect of triptolide on lymphocyte proliferation, interleukin-2 (IL-2) production and IL-2 receptor expression. Then, Con A or PHA induced T-blast cells were cultured with a sufficient dose of recombinant human IL-2 in the presence or absence of triptolide to evaluate the effect of triptolide on the interaction of IL-2 and IL-2 receptors. The effect of triptolide on the immune response in vivo was also investigated. The results of these studies clearly demonstrated that triptolide selectively inhibited the T-lymphocyte proliferative response to Con A and PHA, but had less effect on LPS-induced B-lymphocyte proliferation. Triptolide also suppressed the expression of IL-2 receptors on PHA induced T-blast cells, but did not alter the production of IL-2 by mouse splenic cells and human tonsil lymphocytes. Furthermore, the results also showed that triptolide at higher concentration had a slight inhibitory effect on the interaction of IL-2 and IL-2 receptors, and addition of exogenous IL-2 did not reverse the inhibiting action of triptolide on T-cell proliferation. Taken together, these results suggest that triptolide inhibits T-lymphocyte proliferation mainly by affecting IL-2 receptor expression rather than IL-2 production.

Animals↗

PubMed1994

Enhancement of interleukin-2 production and its mRNA expression by dihydroartemisinin.

Immunoregulatory properties of a novel antimalarial drug dihydroartemisinin (DHA) were investigated in vitro. DHA 0.5-5 mumol.L-1 enhanced the lymphocyte proliferation induced by Con A. Interleukin 2 (IL-2) production and its mRNA expression by both Con A-stimulated mouse splenocytes and a T cell line LBRM-33-1A5 were also augmented by DHA. In contrast, DHA 0.5-5 mumol.L-1 did not show any effect on the lipopolysaccharides (LPS)-induced lymphocyte proliferation and the spontaneous and mitogen-induced proliferation of transformed T cells. These results indicated that DHA might regulate lymphocyte responses through the induction of IL-2 production and that the enhanced T cell proliferation and IL-2 production might be mediated through different pathways.

Animals↗