Biomedical subjects

Y Wu

Publications and source records attributed to Y Wu.

At least 739 records · Page 41Linked to original sources

PubMed1997

Nuclear protein interactions with the human KDR/flk-1 promoter in vivo. Regulation of Sp1 binding is associated with cell type-specific expression.

The endothelial cell type-specific tyrosine kinase KDR/flk-1 is a receptor for vascular endothelial growth factor and a critical regulator of endothelial cell growth and development. To study mechanisms of endothelial cell differentiation and gene regulation, we have analyzed the topology of the proximal promoter of human KDR/flk-1. A protected sequence between base pairs -110 and -25 was defined by in vitro DNase I footprinting analysis in human umbilical vein endothelial cells (HUVECs). Purified Sp1 alone produced similar protection, and electrophoretic mobility shift assays demonstrated that Sp1 was indeed the major nuclear protein binding to this region. Despite the cell type specificity of KDR/flk-1 expression, no cell type differences were observed in DNA-protein interactions in vitro. In contrast, in vivo footprinting assays demonstrated marked differences in core promoter interactions between cell types. Protection of Sp1 binding sites was observed in HUVECs by in vivo DNase I footprinting, whereas in human fibroblasts and HeLa cells a pattern consistent with nucleosomal positioning was observed. In vivo dimethylsulfate footprinting confirmed that DNA-protein interactions occurred within Sp1 elements in HUVECs but not in nonendothelial cells. It is possible that distant elements coordinate Sp1 binding and chromatin structure to regulate cell type-specific expression of KDR/flk-1.

Base Sequence↗

PubMed1997

The carboxyl terminus of mouse delta-opioid receptor is not required for agonist-dependent activation.

The pharmacological effects caused by use of opiate are exerted through the opioid receptors (ORs). ORs couple to the inhibitory G protein (Gi) and result in decreased cAMP levels upon activation by specific agonists. To initiate study of the structure-function relationship during this process, we first ectopically expressed the wild-type delta OR and a C-terminally truncated mutant in CHO cells to investigate the necessity of its C-terminus. The binding potency of both the wild-type and truncated delta ORs to ligands including DPDPE, DSLET, DAGO, and U-50488 was compared. Their membrane localization and ability to mediate signal transduction were also studied. We conclude that the C-terminus of delta OR is not essential for plasma membrane targeting, ligand specificity, and agonist-dependent activation.

Amino Acid Sequence↗

PubMed1997

Roles of calnexin and Ig-alpha beta interactions with membrane Igs in the surface expression of the B cell antigen receptor of the IgM and IgD classes.

Membrane Igs (mIgs) of all five classes are associated with Ig-alpha beta dimers on the B cell surface. While mIgM requires the presence of these two associated molecules for its surface expression, mIgD does not. To study the structural basis for this differential Ig-alpha beta dependence, we created mutant mIgM and mIgD molecules (chimeras and those with reciprocal point mutations in their transmembrane sequences) and identified two amino acid residues in the transmembrane region of the mIg heavy chains responsible for this transport difference. Without Ig-alpha beta, mIgM and mutant mIgD molecules remained endoglycosidase H sensitive, consistent with endoplasmic reticulum (ER) localization. The molecular chaperone calnexin has previously been implicated in retaining unassembled mIgs in the ER. However, we found that inhibition of the association between calnexin and newly synthesized mIgs by castanospermine treatment did not lead to surface expression of normally retained mIgs. Therefore, calnexin cannot be the only retention molecule. Furthermore, for both wildtype and mutant mIgDs, association with calnexin was rather transient, thereby ruling out calnexin being a major ER retention molecule for mIgDs. Our study with castanospermine also showed that calnexin is required for wildtype mIgD surface expression only if Ig-alpha beta is absent, while the latter alone can function to promote mIg folding, assembly, and transport. Further study using our system will help to identify novel molecules and characterize their involvement in the control of mIg transport.

Amino Acid Sequence↗

PubMed1997

p53 and Bax activation in 6-hydroxydopamine-induced apoptosis in PC12 cells.

p53, Bax and Bcl-xL proteins have been implicated in apoptotic neuronal cell death. We have investigated whether those proteins are involved in 6-OHDA-induced PC12 cell death. After a 24-h exposure to the neurotoxin (100 microM), morphological evidence for apoptosis was observed in PC12 cells. Up-regulation of p53 and Bax proteins was demonstrated 4 and 6 h, respectively, after 6-OHDA treatment; in contrast, no change in Bcl-xL levels was found. These findings suggest that p53 and Bax could be relevant markers of neuronal apoptosis as previously described in kainic acid- or ischemia-induced neuronal cell death and may participate to neuronal degeneration in Parkinson's disease.

Animals↗

PubMed1997

Structural mapping of the catalytic mechanism for a mammalian phosphoinositide-specific phospholipase C.

The crystal structures of various ternary complexes of phosphoinositide-specific phospholipase C-delta 1 from rat with calcium and inositol phosphates have been determined at 2.30-2.95 A resolution. The inositol phosphates used in this study mimic the binding of substrates and the reaction intermediate and include D-myo-inositol-1,4,5-trisphosphate, D-myo-inositol-2,4, 5-trisphosphate. D-myo-inositol-4,5-bisphosphate, and D,1-myo-inositol-2-methylene-1,2-cyclićmonophosphonate. The complexes exhibit an almost invariant mode of binding in the active site, each fitting edge-on into the active site and interacting with both the enzyme and the catalytic calcium at the bottom of the active site. Most of the active site residues do not undergo conformational changes upon binding either calcium or inositol phosphates. The structures are consistent with bidentate liganding of the catalytic calcium to the inositol phosphate intermediate and transition state. The complexes suggest explanations for substrate preference, pH optima, and ratio of cyclic to acyclic reaction products. A reaction mechanism is derived that supports general acid/base catalysis in a sequential mechanism involving a cyclic phosphate intermediate and rules out a parallel mechanism where acyclic and cyclic products are simultaneously generated.

Animals↗

PubMed1997

Multiple classes of sulfhydryls modulate the skeletal muscle Ca2+ release channel.

Two sulfhydryl reagents, N-ethylmaleimide (NEM), an alkylating agent, and diamide, an oxidizing agent, were examined for effects on the skeletal muscle Ca2+ release channel. NEM incubated with the channel for increasing periods of time displays three distinct phases in its functional effects on the channel reconstituted into planar lipid bilayers; first it inhibits, then it activates, and finally it again inhibits channel activity. NEM also shows a three-phase effect on the binding of [3H]ryanodine by first decreasing binding (phase 1), followed by a recovery of the binding (phase 2), and then a final phase of inhibition (phase 3). In contrast, diamide 1) activates the channel, 2) enhances [3H]ryanodine binding, 3) cross-links subunits within the Ca2+ release channel tetramer, and 4) protects against phase 1 inhibition by NEM. All diamide effects can be reversed by the reducing agent, dithiothreitol. Diamide induces intersubunit dimer formation of both the full-length 565-kDa subunit of the channel and the 400-kDa generated by endogenous calpain digestion, suggesting that the cross-link does not involve sulfhydryls within the N-terminal 170-kDa fragment of the protein. NEM under phase 1 conditions blocks the formation of the intersubunit cross-links by diamide. In addition, single channels activated by diamide are further activated by the addition of NEM. Diamide either cross-links phase 1 sulfhydryls or causes a conformational change in the Ca2+ release channel which leads to inaccessibility of phase 1 sulfhydryls to NEM alkylation. The data presented here lay the groundwork for mapping the location of one of the sites of subunit-subunit contact in the Ca2+ release channel tetramer and for identifying the functionally important sulfhydryls of this protein.

Animals↗

PubMed1997

Activation of phosphatidylinositol-specific phospholipase C toward inositol 1,2-(cyclic)-phosphate.

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis catalyzes the hydrolysis of phosphatidylinositol (PI) in discrete steps: (i) an intramolecular phosphotransferase reaction to form inositol 1,2-(cyclic)-phosphate (cIP), followed by (ii) a cyclic phosphodiesterase activity that converts cIP to inositol 1-phosphate. Water-soluble cIP was used as the substrate to study the cyclic phosphodiesterase activity and interfacial behavior of PI-PLC. Different detergent micelles and phospholipid vesicles were used to examine if "interfacial activation" of the enzyme could occur toward a soluble substrate. Almost all detergents examined activated the enzyme at least 2-fold, with PC species yielding the largest increases in PI-PLC specific activity. Kinetic parameters were measured in the absence and presence of several representative detergents (e.g., Triton X-100 and diheptanoylphosphatidylcholine (diC7PC)). Gel filtration experiments showed that, under these conditions, the cIP did not partition to any measurable extent with these detergent micelles. The concentration at which half the maximum activation was observed occurred near the detergent CMC. Both Km and Vmax were altered by the presence of a surface: Km decreased to different degrees depending on the detergent, while Vmax increased substantially. The Km for cIP was 90 mM without detergent and decreased to 29 mM with diC7PC micelles added; Vmax increased almost 7-fold in the presence of diC7PC micelles. The enzyme efficiency (Vmax/Km) in the presence of diC7PC increased more than 21-fold, but it was still 20-fold lower than initial phosphotransferase activity for monomeric dihexanoylphosphatidylinositol. The poor efficiency of the cyclic phosphodiesterase activity is largely due to substrate binding affinity. The dependence of rate on substrate concentration exhibits cooperative behavior, especially without detergent. This cooperativity is discussed in terms of protein aggregation and ligand binding sites on the enzyme.

Bacillus subtilis↗

PubMed1997

Stereocontrolled syntheses of water-soluble inhibitors of phosphatidylinositol-specific phospholipase C: inhibition enhanced by an interface.

Three inositol 1,2-(cyclic)-phosphate analogs, inositol cyclic phosphonates with different stereochemistry at the C-2 position of the inositol ring, have been synthesized as water-soluble inhibitors of phosphatidylinositol-specific phospholipase C (PI-PLC). Their inhibition of both phosphotransferase and cyclic phosphodiesterase activities has been studied in the absence and presence of an interface. Key results include the following. (i) Only the analog with the same stereochemistry at the C-2 position of the inositol ring as the natural substrate, myo-inositol 1,2-(cyclic)-phosphate (cIP), exhibits effective inhibition of PI-PLC. (ii) The inhibition of the PI-PLC cyclic phosphodiesterase activity by this cIP analog is enhanced by the presence of an interface (Triton X-100 or diC7PC micelles). This is the first observation of detergent enhancing the effectiveness of a water-soluble inhibitor competing with a water-soluble substrate. (iii) For the cyclic phosphodiesterase activity measured in the presence of 8 mM of the best (e.g., most activating) interface, diC7PC, myo-inositol 1,2-(cyclic)-2-methylenephosphonate (cICH2P) was shown to be a competitive inhibitor with a Ki of 12.3 mM. (iv) The IC50 obtained for the same compound inhibiting the PI-PLC hydrolysis of PI dispersed in DiC7PC micelles was consistent with a Ki approximately 10 mM for the phosphotransferase activity. The similarity of Ki for both PI and cIP processing by PI-PLC suggests both reactions occur at the same site on the enzyme.

Bacillus subtilis↗

PubMed1997

From oligonucleotide shapes to genomic SELEX: novel biological regulatory loops.

The SELEX method and oligonucleotide combinatorial chemistry discovery process yields high-affinity/high-specificity ligands for virtually any molecular target. Typically, the enormous starting libraries used in the SELEX process contain 10(14)-10(15) sequences. We now ask if the smaller sequences, complexity of extant organisms, and evolutionary history provide useful interactions between oligonucleotides and at least some unexpected targets. That is, do organisms contain a robust "linkage map" between their oligonucleotides and proteins and/or small molecules that enriches life?

Base Sequence↗

PubMed1997

Microsample determination of lovastatin and its hydroxy acid metabolite in mouse and rat plasma by liquid chromatography/ionspray tandem mass spectrometry.

A sensitive and specific method was developed and validated to quantitate lovastatin and its hydroxy acid in mouse and rat plasma. This method employs a solid-phase extraction procedure to isolate lovastatin and its hydroxy acid metabolite from the biological matrices (0.1 ml of mouse or rat plasma). The reconstituted extracts were analyzed by liquid chromatography/ionspray tandem mass spectrometry (LC/MS/MS). Simvastatin and simvastatin hydroxy acid were used as internal standards for lovastatin and lovastatin hydroxy acid, respectively. The assay has a lower limit of quantitation (LLQ) of 0.50 ng ml-1 in mouse and rat plasma for both lovastatin and its hydroxy acid based on 0.1 ml aliquots of plasma. The intra- and inter-assay precision (RSD), calculated from quality control (QC) samples, was < 7% for lovastatin and < 6% for lovastatin hydroxy acid in both matrices. The inter-assay accuracy as determined from QC samples was less than 6% for lovastatin and less than 8% for lovastatin hydroxy acid in both matrices. The overall recovery of lovastatin was 54% in mouse plasma and 55% in rat plasma, and the overall recovery of lovastatin hydroxy acid was 100% in mouse plasma and 67% in rat plasma.

Animals↗

PubMed1997

Homotypic interaction of the heat-stable antigen is not responsible for its co-stimulatory activity for T cell clonal expansion.

The heat-stable antigen (HSA) is an important co-stimulatory molecule on antigen-presenting cells (APC). However, the receptor on T cells that receives the co-stimulatory signal from HSA has not been identified. Because the HSA is transiently expressed on T cells after the T cell receptor/CD3 complex is engaged, and because it can bind to itself in a homotypic fashion, it has been proposed that homotypic interaction of HSA is responsible for its co-stimulatory activity. Here we test this hypothesis using mice that have a targeted mutation of the HSA gene, as well as novel transgenic mice that constitutively express HSA on T cells. We show that HSA-deficient T cells remain responsive to co-stimulation by HSA. Furthermore, constitutive expression of HSA does not enhance T cell response to co-stimulatory by HSA. Taken together, our results demonstrate that homotypic interaction of HSA is not responsible for co-stimulation mediated by HSA expressed on APC.

Animals↗

PubMed1997

Application of color Doppler ultrasonic examination in deep vein valvular incompetence in lower limbs.

By use of color Doppler Flow Imaging (CDFI), the morphologic and hemodynamic parameters such as the diameters of femoral and popliteal veins, blood flow velocity and the reflux of valve area were examined in lower extremity deep valvular incompetence (DVI) and normal control groups. The purpose was to evaluate the value of CDFI in the diagnosis of DVI. The results demonstrated that the parameters between the two groups had a very significant difference (P < 0.001), indicating that the CDFI is non-invasive, cost-effective and safe and it might serve as a practical tool in the diagnosis of lower extremity deep valvular incompetence.

Adult↗

PubMed1997

Heat shock and the role of the HSPs during neural plate induction in early mammalian CNS and brain development.

We have investigated the early development expressional of the heat shock protein genes (hsps) and HSP synthesis and their role during neuroectoderm induction, differentiation and early CNS formation. The expression and kinetics of 90, 73/71, 47 and 27 HSPs on neuroectoderm differentiation was compared under normal and stressed conditions. The role of HSPs on neuroctoderm cell fate including thermotolerance and apoptosis using a whole in vitro embryo culture system was studied. Hsp expression appears closely linked in early mammalian development to critical differentiation and proliferation stages in early brain and heart formation. The hsps are developmentally activated around blastula stage and HSPs are constitutively expressed at high levels during neural tube closure and are heat shock responsive. Using both Northern analysis, confocal microscopy and whole mount in situ hybridisation we have identified the mRNA hsp transcripts and HSPs during organogenesis. HSPs were detected during neuroectoderm cell induction and differentiation with the hsp mRNA being tightly regulated during the cell cycle of neuroectoderm especially at early fore-, mid-, hindbrain and heart formation. The 'chaperone' functions of the HSPs are well known, recently during gastrulation the HSP47 and 27 have been shown to specifically bind and fold to nascent collagen and actin molecules respectively. This role is essential for the formation of the basement membrane, extra cellular matrix and neural crest migration during neural plate development. HSP function was observed by using anti-sense strategy, short '5 anti-sense cDNA' hsp oligonucleotides inhibited hsp expression during gastrulation in the whole embryo cultures. The developmental activation of the heat shock element (HSE) is essential to our understanding of the HSPs role in neuronal cell fate. Using specific polyclonal antibodies to HSF1 and 2 (Dr Nakai, Kyoto University) the expression of heat shock factors (HSFs) during neuroectoderm differentiation was examined. Using Western analysis, confocal microscopy and flow cytometry HSF1 and 2 were identified and studied under both normal and heat shocked conditions. During gastrulation higher levels of HSF1 and 2 were identified in the neuroectoderm layer especially in regions of the fore-, mid- and hindbrain. The heat shock response and activation of the HSPs 90, 70, 47 and 27 families have been correlated with HSF1 and 2. The HSF1 appears to be present in all early embryonic cells but appears not to bind to the HSE until early head fold stage at gastrulation when the presence of HSF2 is observed. During neuroectoderm differentiation the activation of HSF1 and 2 appears to correlate with high constitutive expression of many of the hsps specifically hsp90, 73, 71, 47 and 27 being tightly regulated by the cell cycle at neurulation.

Animals↗

PubMed1997

Absence of mutations in the RET gene in acute myeloid leukemia.

Expression of the tyrosine kinase receptor RET has previously been detected in normal hematopoietic cells, and especially in cells of the myeloid lineage. Furthermore, RET was shown to be differentially expressed in acute myeloid leukemia (AML), a disease characterized by excessive cell growth and aberrant maturation of cells, with the highest levels of expression in leukemias with monocytic differentiation. RET is known to be expressed in cells from the excretory system and from the developing central and peripheral nervous system. Both activating and inactivating aberrations in the RET gene have been detected in disorders derived from these tissues. To investigate whether the differential expression is a primary defect in AML, the presence of RET alterations was scanned by Southern blot analysis on DNA of blasts obtained from 17 AML patients. However, no RET gene aberrations were found. Subsequently, denaturing gradient gel electrophoresis (DGGE) analysis was performed on the DNA of blasts from ten selected cases. All five variants detected turned out to represent neutral DNA polymorphisms, including a novel polymorphism in exon 14. Since we were unable to detect mutations of RET in AML, it is unlikely that it plays an important role in leukemogenesis.

Blotting, Southern↗

PubMed1997

Neuronal cell death in the ischemic spinal cord: the effect of methylprednisolone.

BACKGROUND: Cell death occurs by either necrosis or apoptosis. The role of apoptosis in the neuronal degeneration after ischemia remains to be defined. We studied (1) the nature of neuronal death and (2) the neuroprotective action of methylprednisolone in a rat model of spinal cord ischemia. METHODS: Spinal cord ischemia was induced in adult Long-Evans rats by occluding the aortic arch for 14 minutes and simultaneously equilibrating the femoral artery pressure to the atmospheric pressure. Twenty rats were subjected to ischemia without treatment and another twenty to ischemia after treatment with methylprednisolone (30 mg/kg, 4 hours before ischemia). The animals were sacrificed and the lumbar spinal cords were examined on postoperative days 1 and 2. RESULTS: On day 1, neurons with morphology indicative of apoptosis were present in the gray matter. Their numbers increased from the ventral to the dorsal location. There were significantly fewer apoptotic neurons in the dorsal horn of the methylprednisolone-treated animals. DNA obtained from the spinal cord of untreated rats on days 1 and 2 showed laddering after electrophoresis, a feature of apoptosis. Pretreatment with methylprednisolone inhibited the development of DNA laddering. Methylprednisolone treatment was not associated with significantly improved neurologic function in the postoperative period. CONCLUSIONS: Apoptotic neuronal death occurs in the rat spinal cord after transient ischemia and is attenuated by pretreatment with methylprednisolone.

Animals↗

PubMed1997

China's health care sector in transition: resources, demand and reforms.

Economic development and reforms have had profound impacts on China's health care sector. As a result, the health care sector in China is in transition. This report reviews the major changes, and the possible policy response to these changes in China's health care sector. It discusses resource availability in the Chinese health sector, and analyses the trend of household demand for health care goods and services. This study also examines the trade and investment situations in China's health sector and investigates the major forces that are driving the transition in health care and comments on the potential policy responses.

China↗

PubMed1997

Quantitative acoustic characterization of a new surfactant-based ultrasound contrast agent.

The acoustic properties of a new ultrasound contrast agent, ST68, have been investigated. ST68 is a sonicated mixture of nonionic surfactants (Span-type and Tween-type) consisting of stabilized microbubbles with a mean diameter of 3.8 microns and a concentrations of 7.1 x 10(8) bubbles/mL. A pulsatile flow system was used to acquire data in vitro. The acoustic properties of ST68, as a function of time, frequency and dose, were calculated. Enhancement changed nonlinearly with contrast agent dose; maximum was 13.1 dB +/- 1.0 dB for a dose of 0.30 microL/mL of suspending medium. Attenuation reached approximately 11 dB/cm for dosages above 0.27 microL/mL and for frequencies between 2.5 and 6.0 MHz. In vivo, i.v. injections of ST68 were given to 4 rabbits (doses from 0.01 to 0.23 mL/kg). A clear increase in flow signal intensity was observed for 1 to 2 min. An in vivo dose-response curve was calculated from audio Doppler signals obtained with a 10-MHz cuff transducer placed around the distal aorta. Maximum enhancement was 18.3 dB +/- 3.13 dB for a 0.13 mL/kg dose. Moreover, ST68 appears to follow a simple relationship between in vivo enhancement and dose. In conclusion, ST68 is capable of producing marked vascular enhancement. Its acoustic properties have been characterized in vitro and in vivo.

Acoustics↗

PubMed1997

FP-21399 blocks HIV envelope protein-mediated membrane fusion and concentrates in lymph nodes.

The identification of fusin and other chemokine receptors as coreceptors for HIV-1 has renewed the interest in agents that may prevent viral entry. Polyanionic compounds such as dextran sulfate, curdian sulfate, and suramin act on the V3 loop of the viral envelope and may prevent its interaction with fusin. These agents show activity against a wide range of HIV-1 strains, but have undesirable circulating half-life, bioavailability, and toxicity. We have developed a small molecule inhibitor of HIV-1 that has several advantages over these other agents. FP-21399 is a novel compound of the bis(disulfonaphthalene) dimethoxybenzene class that blocks entry of HIV into CD4+ cells and blocks fusion of infected and noninfected CD4+ cells. This compound only weakly inhibits binding of CD4 and gp120, at concentrations much greater than are required to block viral entry. Furthermore, FP-21399 can block the interaction between gp120 and antibodies directed against the V3 loop, but does not block binding of antibodies directed against the V4 loop. Animal studies demonstrate that FP-21399 is concentrated in lymph nodes, making it a promising compound for anti-HIV therapy. In SCID mice reconstituted with human immune cells, maintenance of HIV-1 infection was blocked by a 5-day treatment with low doses of FP-21399, suggesting that lymph node accumulation may contribute to antiviral activity. Finally, attempts to generate drug-resistant virus in cell culture resulted in only weakly resistant variants with IC90 values that are much lower than concentrations of FP-21399 found in lymph nodes.

Animals↗