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Biomedical subjects

H Wang

Publications and source records attributed to H Wang.

At least 307 records · Page 17Linked to original sources
PubMed2001

Identification and structural elucidation of lectin-binding oligosaccharides by bioaffinity matrix-assisted laser desorption/ionization Fourier transform mass spectrometry.

Cortical granule lectin (CGL) is released by the egg of the South African toad Xenopus laevis upon fertilization. The lectin binds to oligosaccharides in the extracellular matrix of the egg to form a physical block to prevent additional sperm penetration or polyspermy. To identify the oligosaccharides that bind to CGL, the lectin was immobilized on the surface of a matrix-assisted laser desorption/ionization probe. This bioaffinity probe was used to determine oligosaccharides that bind preferentially to CGL. Structural analyses based on collision-induced dissociation was used to determine that oligosaccharides with the sulfate esters at the nonreducing ends preferentially bind to the lectin.

Animals↗
PubMed2001

A VH12 transgenic mouse exhibits defects in pre-B cell development and is unable to make IgM+ B cells.

V(H)12 B cells undergo stringent selection at multiple checkpoints to favor development of B-1 cells that bind phosphatidylcholine. Selection begins with the V(H) third complementarity-determining region (CDR3) at the pre-B cell stage, in which most V(H)12 pre-B cells are selectively eliminated, enriching for those with V(H)CDR3s of 10 aa and a fourth position Gly (designated 10/G4). To understand this selection, we compared B cell differentiation in mice of two V(H)12 transgenic lines, one with the favored 10/G4 V(H)CDR3 and one with a non-10/G4 V(H)CDR3 of 8 aa and no Gly (8/G0). Both H chains drive B cell differentiation to the small pre-BII cell stage, and induce allelic exclusion and L chain gene rearrangement. However, unlike 10/G4 pre-B cells, 8/G0 pre-B cells are deficient in cell division and unable to differentiate to B cells. We suggest that this is due to poor 8/G0 pre-B cell receptor expression and to an inability to form an 8/G0 B cell receptor. Our findings also suggest that V(H)12 H chains have evolved such that association with surrogate and conventional L chains is most efficient with a 10/G4 CDR3. Thus, selection for phosphatidylcholine-binding B-1 cells is most likely the underlying evolutionary basis for the loss of non-10/G4 pre-B cells.

Alleles↗
PubMed2001

Detection of DNA adducts of benzo[a]pyrene using immunoelectrophoresis with laser-induced fluorescence. Analysis of A549 cells.

Detection of benzo[a]pyrene diol epoxide (BPDE)-damaged DNA in a human lung carcinoma cell line (A549) has been performed using free zone affinity capillary electrophoresis with laser-induced fluorescence (LIF). Using BPDE as a model carcinogenic compound, the speed, sensitivity and specificity of this technique was demonstrated. Under free zone conditions, an antibody bound adduct was baseline-resolved from an unbound adduct in less than 2 min. The efficiencies of separation were in excess of 6 x 10(5) and 1 x 10(6) plates per meter for the antibody-bound and unbound adducts, respectively. Separation using a low ionic strength buffer permitted the use of a high electric field (830 V/cm) without the loss of resolving power. Using LIF detection, a concentration detection limit of roughly 3 x 10(-10) M was achieved for a 90-mer oligonuleotide containing a single BDPE. The use of formamide in the incubation buffer to enhance denaturing of DNA did not affect the stability of the complex between the antibody and the adducts. Using a fluorescently labeled BPDE-modified DNA adduct probe, a competitive assay was established to determine the levels of BPDE-DNA adducts in A549 cells.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2001

Enhanced Kerr nonlinearity via atomic coherence in a three-level atomic system.

We measure the Kerr-nonlinear index of refraction of a three-level Lambda-type atomic system inside an optical ring cavity. The Kerr nonlinearity is modified and greatly enhanced near atomic resonant conditions for both probe and coupling beams. The Kerr nonlinear coefficient n(2) changes sign when the coupling beam frequency detuning switches sign, which can lead to interesting applications in optical devices such as all-optical switches.

Journal Article↗
PubMed2001

[Epitope mapping of anti-glomerular basement membrane antibodies].

OBJECTIVE: To map the epitopes of anti-glomerular basement membrane (GBM) antibodies and to study their clinical relevance. METHODS: 33 sera from patients with anti-GBM antibody mediated diseases were applied to inhibit monoclonal antibody against alpha 3 (IV) NC1 (Mab3), monoclonal antibody against alpha 5 (IV) NC1 (Mab5) and affinity purified, horseradish peroxidase conjugated, human anti-GBM antibodies (APab-HRP) in competitive inhibitory ELISA system using soluble human GBM proteins as solid phase ligands. The inhibition rates were evaluated in epitope mapping and used in comparison for patients with different clinical manifestations. RESULTS: All 33 sera inhibited Mab3, 30/33 sera inhibited APab-HRP, and 11/33 inhibited Mab5. However, the inhibition rates were not parallel. The mean inhibition rate for APab-HRP was 55% in 27 patients with anti-GBM antibody only and was 32% in 6 patients with both anti-GBM antibody and ANCA with a significant difference (P = 0.01). For patients with or without pulmonary hemorrhage, no significant differences could be found in the mean inhibition rates for Mab3, Mab5 or APab-HRP (P < 0.05). CONCLUSION: The anti-GBM antibodies from all patients in the present study recognize the common major epitopes on alpha 3 (IV) NC1, however, the epitopes recognized by different anti-GBM antibodies were not exactly the same. Only a small number of anti-GBM antibody positive sera recognizes alpha 5 (IV) NC1. The epitopes recognized by sera from patients with or without ANCA might be different.

Antibodies↗
PubMed2001

Chelerythrine and genistein inhibit the endothelin-1-induced increase in myofilament Ca(2+) sensitivity in rabbit ventricular myocytes.

We performed experiments to elucidate the cellular mechanism for the biphasic inotropic response to endothelin-1 of single rabbit ventricular myocytes loaded with a fluorescent dye, acetoxymethylester of indo-1. Endothelin-1 at 10 nM elicited a biphasic inotropic effect: a transient decrease in cell shortening and Ca(2+) transients followed by an increase in cell shortening without significant elevation of peak Ca(2+) transients. The selective endothelin ET(A) receptor antagonist FR139317 (2(R)-[2(R)-[2(S)-[(1-hexahydro-1H-azepinyl)]carbonyl]amino-4-methylpentanoyl]amino-3-[3-(1-methyl-1H-indolyl)propionyl]amino-3-(2-pyridyl)propionic acid) at 1 microM abolished the biphasic effect of endothelin-1 on cell shortening and Ca(2+) transients. The selective protein kinase C inhibitor chelerythrine at 1 microM and the tyrosine kinase inhibitor genistein at 5 microM inhibited the endothelin-1-induced increase in cell shortening without significantly affecting Ca(2+) transients and the transient decrease in cell shortening and Ca(2+) transients. The present results indicate that both protein kinase C and tyrosine kinase may contribute to the increase in myofilament Ca(2+) sensitivity induced by endothelin-1, whereas the decrease in Ca(2+) transients induced by endothelin-1 may be mediated by a signalling pathway different from that involved in the increase in cardiac contractility in rabbit ventricular myocytes.

Actin Cytoskeleton↗
PubMed2001

Galpha i2 enhances in vivo activation of and insulin signaling to GLUT4.

Heterotrimeric G-proteins, including Galpha(i2), have been implicated in modulating glucose disposal and insulin signaling. This cross-talk between G-protein-coupled and tyrosine kinase-coupled signaling pathways is a focal point for the study of integration of cell signaling. Herein we study the role of Galpha(i2) in modulating glucose transport, focusing upon linkages to insulin signaling. Utilizing mice harboring a transgene that directs the expression of a constitutively activated, GTPase-deficient mutant of Galpha(i2) (Q205L) in adipose tissue, skeletal muscle, and liver, we demonstrate that Galpha(i2) regulates the translocation of the insulin-sensitive GLUT4 glucose transporter in skeletal muscle and adipose tissue. The expression of Q205L Galpha(i2) increased glucose transport and translocation of GLUT4 to the plasma membrane in vivo in the absence of insulin stimulation. Adipocytes from the Q205L Galpha(i2) mice displayed enhanced insulin-stimulated glucose transport and GLUT4 translocation to the plasma membrane to levels nearly twice that of those from littermate controls. Phosphatidylinositol 3-kinase and Akt activities were constitutively activated in tissues expressing the Q205L Galpha(i2). Studies of adipocytes from wild-type mice displayed short term activation of phosphatidylinositol 3-kinase, Akt, and GLUT4 translocation in response to activation of Galpha(i2) by lysophosphatidic acid, a response sensitive to pertussis toxin. These data provide an explanation for the marked glucose tolerance of the Q205L Galpha(i2) mice and demonstrate a linkage between Galpha(i2) and GLUT4 translocation.

Adipose Tissue↗
PubMed2001

Inhibition of DNA replication by tirapazamine.

Tirapazamine (TPZ) is a hypoxia-selective cytotoxin that is currently being examined in Phase II and III clinical trials in combination with radiotherapy and cisplatin-based chemotherapy. Reductases convert TPZ to a cytotoxic radical that produces DNA damage under hypoxic conditions. Because one or more of the enzymes responsible for the bioactivation of TPZ is/are thought to be at or near the nuclear matrix, we hypothesized that TPZ may have a major affect on DNA replication, a process that is known to occur predominantly at the nuclear matrix. To assess the effect of TPZ on DNA replication, we measured the incorporation of radioactive thymidine into DNA of HCT116 human colon cancer cells and HeLa cells. We show that incorporation of radioactive thymidine is dramatically inhibited in cells that are pretreated with TPZ under hypoxic conditions. TPZ-induced inhibition of DNA synthesis was much greater than that produced by more toxic doses of ionizing radiation. We used the SV40-based in vitro DNA replication assay to study the mechanism of inhibition of DNA synthesis in cells treated with TPZ. Using this assay, we show that extracts prepared from cells treated with TPZ under hypoxic conditions had only 25-50% of the DNA replication activity measured in control cells. This reduction in DNA replication activity was associated with a reduction in levels of replication protein A (RPA) in cytoplasmic extracts used for the in vitro DNA replication assay and could be overcome by addition of recombinant human RPA. Furthermore, we show by indirect immunofluorescence that TPZ leads to a localization of the p34 subunit of RPA (RPA2) to small subnuclear foci. These results show that TPZ dramatically inhibits DNA replication and that the mechanism of inhibition, at least in part, involves changes in RPA that alter its cellular localization.

Antineoplastic Agents↗
PubMed2001

Electron-transfer reactivity and enzymatic activity of hemoglobin in a SP Sephadex membrane.

Hemoglobin can exhibit a direct electron-transfer reaction after being entrapped in a SP Sephadex membrane. A pair of stable and well-defined redox waves are obtained at a hemoglobin-SP sephadex modified pyrolytic graphite electrode. The anodic and cathodic peak potentials are located at -0.244 and -0.336 V (vs SCE), respectively. On the other hand, the peroxidase activity of the protein in the membrane is also greatly enhanced. The apparent Michaelis-Menten constant is calculated to be 1.9 mM, which shows a large catalytic activity of hemoglobin in the SP Sephadex membrane toward hydrogen peroxide (H2O2). According to the direct electron-transfer property and enhanced peroxidase activity of Hb in the membrane, a Hb/SP Sephadex membrane-based H2O2 biosensor is prepared, with a linear range approximately 5.0 x 10(-6) to 1.6 x 10(-4) mol/L.

Animals↗
PubMed2001

Structural features of nonpeptide prenyl pyrophosphates that determine their antigenicity for human gamma delta T cells.

Human Vgamma2Vdelta2(+) T cells proliferate in vivo during many microbial infections. We have found that Vgamma2Vdelta2(+) T cells recognize nonpeptide prenyl pyrophosphates and alkylamines. We now have defined structural features that determine the antigenicity of prenyl pyrophosphates by testing synthetic analogs for bioactivity. We find that the carbon chain closest to the pyrophosphate moiety plays the major role in determining bioactivity. Changes in this area, such as the loss of a double bond, abrogated bioactivity. The loss of a phosphate from the pyrophosphate moiety also decreased antigenicity 100- to 200-fold. However, nucleotide monophosphates could be added with minimal changes in bioactivity. Longer prenyl pyrophosphates also retained bioactivity. Despite differences in CDR3 sequence, Vgamma2Vdelta2(+) clones and a transfectant responded similarly. Ag docking into a Vgamma2Vdelta2 TCR model reveals a potential binding site in germline regions of the Vgamma2Jgamma1.2 CDR3 and Vdelta2 CDR2 loops. Thus, Vgamma2Vdelta2(+) T cells recognize a core carbon chain and pyrophosphate moiety. This recognition is relatively unaffected by additions at distal positions to the core Ag unit.

Adult↗
PubMed2001

N-(4-hydroxyphenyl)retinamide elevates ceramide in neuroblastoma cell lines by coordinate activation of serine palmitoyltransferase and ceramide synthase.

The retinoid N-(4-hydroxyphenyl)retinamide (4-HPR; fenretinide) is cytotoxic to a variety of cancer cell lines, and we previously showed an association between ceramide generation and 4-HPR cytotoxicity for neuroblastoma cell lines (B. J. Maurer et al., J. Natl. Cancer Inst. (Bethesda), 91: 1138-1146, 1999). Here we determine whether the increased ceramide mediated by 4-HPR in the CHLA-90 human neuroblastoma cell line results from de novo ceramide synthesis. Treatment of CHLA-90 with 4-HPR for 2 h, in the presence of [(3)H]palmitic acid, caused sequential formation of [(3)H]sphinganine (220% over control) and [(3)H]ceramide (160% over control), with sphinganine returning to baseline at 4 h, and ceramide continuing to increase (215% over control). 4-HPR treatment did not accelerate cellular decay of sphingomyelin. Preincubation of cells with either L-cycloserine, an inhibitor of serine palmitoyltransferase (SPT), or fumonisin B(1), an inhibitor of ceramide synthase, retarded ceramide formation in response to 4-HPR treatment, although sphinganine was still generated when 4-HPR and FB(1) were present. Data from in vitro enzyme assays using microsomes showed that preexposure of intact cells to 4-HPR resulted in a time (175% over control; 6 h)- and dose-dependent increase (173% over control; 10 microM) in SPT activity as well as a time (265% over control)- and dose-dependent increase (215% above control; 10 microM) in ceramide synthase activity. Our results show that 4-HPR-mediated ceramide generation is derived from the de novo synthetic pathway by coordinate activation of SPT and ceramide synthase. Knowledge of these biochemical events is of utility when downstream modulators of ceramide metabolism are used to heighten the cytotoxic response to chemotherapy.

Acyltransferases↗
PubMed2001

MinK-related peptide 1: A beta subunit for the HCN ion channel subunit family enhances expression and speeds activation.

The HCN family of ion channel subunits underlies the currents I(f) in heart and I(h) and I(q) in the nervous system. In the present study, we demonstrate that minK-related peptide 1 (MiRP1) is a beta subunit for the HCN family. As such, it enhances protein and current expression as well as accelerating the kinetics of activation. Because MiRP1 also functions as a beta subunit for the cardiac delayed rectifier I(Kr), these results suggest that this peptide may have the unique role of regulating both the inward and outward channels that underlie cardiac pacemaker activity. The full text of this article is available at http://www.circresaha.org.

Animals↗
PubMed2001

A quantitative description of the ground-state wave function of Cu(A) by X-ray absorption spectroscopy: comparison to plastocyanin and relevance to electron transfer.

To evaluate the importance of the electronic structure of Cu(A) to its electron-transfer (ET) function, a quantitative description of the ground-state wave function of the mixed-valence (MV) binuclear Cu(A) center engineered into Pseudomonas aeruginosa azurin has been developed, using a combination of S K-edge and Cu L-edge X-ray absorption spectroscopies (XAS). Parallel descriptions have been developed for a binuclear thiolate-bridged MV reference model complex ([(L(i)(PrdacoS)Cu)(2)](+)) and a homovalent (II,II) analogue ([L(i)(Pr2tacnS)Cu)(2)](2+), where L(i)(PrdacoS) and L(i)(Pr2tacnS) are macrocyclic ligands with attached thiolates that bridge the Cu ions. Previous studies have qualitatively defined the ground-state wave function of Cu(A) in terms of ligand field effects on the orbital orientation and the presence of a metal--metal bond. The studies presented here provide further evidence for a direct Cu--Cu interaction and, importantly, experimentally quantify the covalency of the ground-state wave function. The experimental results are further supported by DFT calculations. The nature of the ground-state wave function of Cu(A) is compared to that of the well-defined blue copper site in plastocyanin, and the importance of this wave function to the lower reorganization energy and ET function of Cu(A) is discussed. This wave function incorporates anisotropic covalency into the intra- and intermolecular ET pathways in cytochrome c oxidase. Thus, the high covalency of the Cys--Cu bond allows a path through this ligand to become competitive with a shorter His path in the intramolecular ET from Cu(A) to heme a and is particularly important for activating the intermolecular ET path from heme c to Cu(A).

Azurin↗
PubMed2001

Specific residues within the alpha 2 integrin subunit cytoplasmic domain regulate migration and cell cycle progression via distinct MAPK pathways.

The alpha(2) integrin subunit cytoplasmic domain is necessary for epidermal growth factor (EGF)-stimulated chemotactic migration and insulin-dependent entry into S-phase of mammary epithelial cells adherent to type I collagen. Truncation mutants revealed that the seven amino acids, KYEKMTK, in addition to the GFFKR motif were sufficient for these functions. Mutation of tyrosine 1134 to alanine inhibited the ability of the cells to phosphorylate p38 MAPK and to migrate in response to EGF but had only a modest effect on the ability of the cells to induce sustained phosphorylation of the ERK MAPK, to up-regulate cyclin E and cdk2 expression, and to enter S-phase when adherent to type I collagen. Conversely, mutation of the lysine 1136 inhibited the ability of the cells to increase cyclin E and cdk2 expression, to maintain long term phosphorylation of the ERK MAPK, and to enter S-phase but had no effect on the ability of the cells to phosphorylate the p38 MAPK or to migrate on type I collagen in response to EGF. Methionine 1137 was essential for both migration and entry into S-phase. Thus, distinctly different structural elements of the alpha(2) integrin cytoplasmic domain are required to engage the signaling pathways leading to cell migration or cell cycle progression.

Amino Acid Sequence↗
PubMed2001

The proapoptotic protein Bad binds the amphipathic groove of 14-3-3zeta.

Through interaction with a multitude of target proteins, 14-3-3 proteins participate in the regulation of diverse cellular processes including apoptosis. These 14-3-3-interacting proteins include a proapoptotic Bcl-2 homolog, Bad (Bcl-2/Bcl-XL-associated death promoter). To understand how 14-3-3 interacts with Bad and modulates its function, we have identified structural elements of 14-3-3 necessary for 14-3-3/Bad association. 14-3-3 contains a conserved amphipathic groove that is required for binding to several of its ligands. We used peptides of known binding specificity as competitors to demonstrate that Bad interacts with 14-3-3zeta via its amphipathic groove. More detailed analysis revealed that several conserved residues in the groove, including Lys-49, Val-176, and Leu-220, were critical for Bad interaction. These results were applied to investigations of the ability of 14-3-3 to prevent Bad-induced cell death. When co-expressed with Akt, wild-type 14-3-3 could reduce the ability of Bad to cause death, however 14-3-3zetaK49E, which cannot bind Bad, failed to inhibit Bad. It seems that the amphipathic groove of 14-3-3 represents a general binding site for multiple ligands, raising issues related to competition of ligands for 14-3-3.

14-3-3 Proteins↗
PubMed2001

Development of inhibition by ephrin-A5 on outgrowth of embryonic spinal motor neurites.

The spinal motor pool maps systematically onto the surface of muscles. This map is detectable in rat embryonic muscles, and is partially restored after reinnervation. Recent evidence shows that either overexpression or deletion of the ephrin-A5 gene significantly disrupts the map, suggesting that ephrin-A5 plays a critical role in the formation of this topography. Several studies have demonstrated that ephrin-A5 is a repulsive molecule in the nervous system, including the neuromuscular system. To examine the development of sensitivity of ventral spinal axons to this inhibitory ligand, slices of E11 to E15 embryonic rat spinal cords were cocultured with membranes derived from ephrin-A5-expressing cell lines. We detected a progressive expression of inhibition by ephrin-A5 between E11 and E15. By E15, rostral and caudal spinal neurites showed clear differences in responsiveness to the ephrin-A5 ligand. Further, we found that at this age caudal neurites are more sensitive to changes of ephrin-A5 concentration along a gradient. In addition, growth cones of caudal, more than rostral, neurites tended to assume a collapsed shape in the presence of the ligand. These results demonstrate a progressive development of sensitivity to ephrin-A5, and suggest a divergence in this sensitivity between rostral and caudal spinal cord neurites. These results provide further insight into how subtle rostrocaudal differences in the development of sensitivity to ephrin-A5 may explain, in part, neuromuscular topography.

Animals↗
PubMed2001

Pds1 phosphorylation in response to DNA damage is essential for its DNA damage checkpoint function.

In Saccharomyces cerevisiae, Pds1 is an anaphase inhibitor and plays an essential role in DNA damage and spindle checkpoint pathways. Pds1 is phosphorylated in response to DNA damage but not spindle disruption, indicating distinct mechanisms delaying anaphase entry. Phosphorylation of Pds1 is Mec1 and Chk1 dependent in vivo. Here, we show that Pds1 is phosphorylated at multiple sites in vivo in response to DNA damage by Chk1. Mutation of the Chk1 phosphorylation sites on Pds1 abolished most of its DNA damage-inducible phosphorylation and its checkpoint function, whereas its anaphase inhibitor functions and spindle checkpoint functions remain intact. Loss of Pds1 phosphorylation correlates with APC-dependent Pds1 destruction in response to DNA damage. We also show that APC(Cdc20) is active in preanaphase arrested cells after DNA damage. This suggests that Pds1 is stabilized by phosphorylation in response to DNA damage, but APC(Cdc20) activity is not altered. Our results indicate that phosphorylation of Pds1 by Chk1 is the key function of Chk1 required to prevent anaphase entry.

Amino Acid Sequence↗