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Biomedical subjects

Z Wu

Publications and source records attributed to Z Wu.

At least 649 records · Page 36Linked to original sources
PubMed1994

Characterization of a region in phage Mu transposase that is involved in interaction with the Mu B protein.

Mu A protein, the 75-kDa phage transposase, consists of three domains: a 30-kDa NH2 terminus, a 35-kDa central domain, and a 10-kDa COOH terminus (Nakayama, C., Teplow, D. B., and Harshey, R. M. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1809-1813). Genetic and biochemical experiments have demonstrated that the COOH-terminal domain must be present for functional interaction with Mu B protein. To further investigate the COOH-terminal domain of Mu A, we fused this 89-amino acid region to the glutathione S-transferase gene to facilitate subsequent expression and purification. We show that either the glutathione S-transferase-peptide fusion protein or the COOH-terminal peptide severed from glutathione S-transferase is active in Mu B interaction. Addition of the COOH-terminal domain to the in vitro strand transfer reaction inhibits intermolecular strand transfer by a mechanism previously characterized for intact Mu A protein (Baker, T. A., Mizuuchi, M., and Mizuuchi, K. (1991) Cell 65, 1003-1013), although the COOH-terminal domain is 70 times less effective than intact Mu A. The transient interaction between the COOH-terminal domain and Mu B does not inhibit Mu B stimulation of the strand cleavage and intramolecular strand transfer activity of Mu A. Deletion analysis has shown that the last 36 amino acids are sufficient for interaction with Mu B, but that removal of as few as 4 amino acids from the COOH terminus renders the peptide inactive. The recovery of an active COOH-terminal domain of Mu A will facilitate future structure/function studies of the Mu transposase.

Bacteriophage mu↗
PubMed1994

Synergistic activation of the type I adenylyl cyclase by Ca2+ and Gs-coupled receptors in vivo.

The type I adenylyl cyclase is directly stimulated by Ca2+ and calmodulin in vivo (Choi, E. J., Wong, S. T., Hinds, T. R. and Storm, D. R. (1992) J. Biol. Chem. 267, 12440-12442; Wu, Z., Wong, S. T., and Storm, D. R. (1993) J. Biol. Chem. 268, 23766-23768). In this study, we examined the sensitivity of the type I adenylyl cyclase expressed in HEK-293 cells to beta-adrenergic agonists or glucagon when intracellular Ca2+ was elevated by Ca2+ ionophore or carbachol. Although previous studies have shown that this enzyme can be directly stimulated by activated Gs in vitro, we demonstrate that it is not stimulated by Gs-coupled receptors in vivo. However, the enzyme was stimulated by Gs-coupled receptors in vivo when it was activated by intracellular Ca2+. For example, the Ca2+ ionophore A23187 stimulated the enzyme 3 +/- 0.5-fold (n = 9) and isoproterenol alone did not stimulate the enzyme, but the combination of the two stimulated type I adenylyl cyclase 13 +/- 2-fold (n = 9) in vivo. Similarly, 500 nM glucagon alone did not stimulate the enzyme but the combination of A23187 and glucagon activated the enzyme 90 +/- 8-fold (n = 4). Synergistic stimulation of type I adenylyl cyclase activity was also obtained with combinations of carbachol and isoproterenol or glucagon. This phenomenon was not observed with a mutant enzyme that is insensitive to Ca2+ and calmodulin, suggesting that conformational changes caused by binding of calmodulin to the type I adenylyl cyclase enhance binding or coupling to activated Gs. These data illustrate that this adenylyl cyclase can couple Ca2+ and neurotransmitter signals to generate optimal cAMP levels, a property of the enzyme that may be important for its role in learning and memory in mammals.

Adenylyl Cyclases↗
PubMed1994

Interleukin 2 receptor gamma chain expression on resting and activated lymphoid cells.

The interleukin 2 receptor (IL-2R) is known to be comprised of at least three genetically distinct subunits termed alpha, beta, and gamma. These chains can be expressed individually or in various combinations resulting in distinct receptors with different affinities for IL-2. In contrast to alpha and beta, the cell surface expression of the gamma chain protein previously has not been well-characterized. To examine cell surface expression of IL-2R gamma on hematopoietic cells, we developed two new monoclonal antibodies (mAbs) specific for this protein. Both 1A11 (immunoglobulin [IgG1]) and 3G11 (IgM) specifically reacted with murine cells transfected with IL-2R gamma cDNA, and immunoprecipitation studies indicated that both antibodies precipitated a protein of approximately 62-65 kD. Scatchard analysis of IL-2 binding to murine cells transfected with cDNA-encoding combinations of IL-2R components demonstrated that neither beta nor gamma chain bind IL-2 with measurable affinity, but coexpression of both beta and gamma is sufficient to form an intermediate affinity receptor. In the absence of gamma chain, beta chain interacts with alpha chain to form a "pseudo-high" affinity receptor. In contrast, gamma chain does not appear capable of interacting with alpha in the absence of beta chain. Thus, gamma chain appears to interact only with beta, but beta chain is capable of interacting with both alpha and gamma. Using the newly developed mAbs to examine cell surface expression by immunofluorescence, resting T cells were found to express low levels of gamma chain without detectable alpha or beta. Early after mitogen stimulation, T cells expressed higher levels of alpha, beta, and gamma. However, at later time points, T cells expressed alpha and gamma in marked excess over beta. Thus, formation of high affinity IL-2R on activated T cells was primarily limited by beta chain expression. In contrast, resting natural killer (NK) cells constitutively expressed IL-2R beta without detectable alpha or gamma. After activation with either IL-2 or IL-12, expression of both alpha and gamma transiently increased and then returned to very low levels. Expression of functional IL-2R on resting and activated NK cells, therefore, appeared to be primarily limited by the expression of gamma chain. IL-2 binding studies with resting NK cells confirmed the results of immunofluorescence studies indicating the presence of very low numbers of intermediate affinity (beta gamma) receptors for IL-2 on these cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Expression and ligand binding characterization of the beta-subunit (p75) ectodomain of the interleukin-2 receptor.

The baculovirus-mediated eukaryotic insect cell expression system was used to prepare large quantities of the beta-subunit ectodomain of the high-affinity interleukin-2 receptor (IL-2R beta x). We describe the expression, purification, and biophysical characterization of this ligand binding domain. The human cDNA encoding IL-2R beta x was inserted into baculovirus transfer vectors. High titer recombinant baculovirus was produced in Spodoptera frugiperda (Sf9) insect cells, and the viral supernatants were subsequently used to infect monolayers of Trichoplusia ni (High Five) insect cells in serum-free culture. Maximal expression of the recombinant protein excreted into the cell culture supernatants was determined by SDS/PAGE analysis, where a band migrating with an apparent molecular mass of 31 kDa was identified by immunostaining. One-step purification was achieved by affinity chromatography on either a monoclonal antibody (TIC-1) column or an IL-2 column, with a final yield of approximately 5 mg/L of culture supernatant. Interestingly, partial purification was also demonstrated using metal chelate affinity chromatography. Amino-terminal sequence analysis of the protein matched the published sequence. Both equilibrium sedimentation analysis and gel filtration chromatography indicated that IL-2R beta x remains monomeric. Deconvolution of far-UV circular dichroism (CD) spectra indicated the predominant secondary structural element to be beta-sheet, consistent with structural analysis and predictions for other members of the hematopoietic receptor family. A dissociation constant (Kd) for IL-2R beta x in solution of 5.3 x 10(-7) M was calculated from competitive receptor binding assays.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Pentalenene synthase. Purification, molecular cloning, sequencing, and high-level expression in Escherichia coli of a terpenoid cyclase from Streptomyces UC5319.

Pentalenene synthase, which catalyzes the cyclization of farnesyl diphosphate (1) to the tricyclic sesquiterpene hydrocarbon pentalenene (2), was purified from Streptomyces UC5319. A 450-bp hybridization probe, generated by PCR amplification of genomic DNA using primers based on N-terminal and internal tryptic peptide sequence data for pentalenene synthase, was used to screen both plasmid and phage DNA libraries of Streptomyces genomic DNA, resulting in the isolation and sequencing of the complete pentalenene synthase gene. PCR was used to insert the pentalenene synthase gene into the T7 expression vector pLM1. Cloning of the resulting construct in the expression host Escherichia coli BL21 (DE3) gave transformants that expressed pentalenene synthase as greater than 10% of soluble protein. The recombinant enzyme has been purified, and initial physical and kinetic characterization has been performed. The recombinant enzyme appears to be identical in every respect with the native Streptomyces synthase and exhibits the following steady-state kinetic parameters: Km = 0.31 +/- 0.05 microM, kcat = 0.32 +/- s-1, KI(PPi) = 3.2 +/- 0.6 microM. Both enzymes have an absolute requirement of Mg2+ for catalysis and an optimum pH of 8.2-8.4. Both proteins have M(r) values of 41-42 kDa, as determined by SDS-PAGE.

Amino Acid Sequence↗
PubMed1994

Identification of the DNA-binding site for the phosphorylated VanR protein required for vancomycin resistance in Enterococcus faecium.

The vancomycin resistance operon of Enterococcus faecium encodes a two-component regulatory system comprising VanS and VanR. In vitro experiments showed that about 5% of a labile phosphorylated VanR (P-VanR) was accumulated from ATP and a maltose-binding protein-VanS fusion protein (MBP-VanS). Alternatively, about an 8% abundance of P-VanR was produced with acetyl phosphate. In such incubations, gel shift experiments revealed that P-VanR selectively bound to a 254-bp DNA fragment that contains the vanH promoter for the vanH, vanA, and vanX structural genes. When VanS was added with a mole ratio for VanS:VanR of higher than 1:1, VanS competed with DNA for P-VanR and abolished the gel shift. P-VanR bound 500-fold more tightly to the vanH promoter region, with an estimated EC50 of 40 nM, than the unphosphorylated VanR. A second DNA fragment of 197 bp containing the proposed vanR promoter for the vanR and vanS regulatory genes also exhibited gel shift, but with much lower affinities. A mutant VanR(D53A) was shown to be incompetent for phosphorylation by phosphorylated MBP-VanS or by acetyl phosphate; however, it still bound DNA specifically, albeit with low affinity. DNase I footprinting by P-VanR revealed that a ca. 80-bp region was protected on the vanH promoter and a ca. 40-bp region was protected on the vanR promoter. The unphosphorylated VanR footprinted the same 80 bp on the vanH promoter, but only 20 bp on the vanR promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylation↗
PubMed1994

A Bayesian approach to subvoxel tissue classification in NMR microscopic images of trabecular bone.

NMR microscopy is currently being used as an investigational tool for the evaluation of micromorphometric parameters of trabecular bone as a possible means to assess its strength. Since, typically, the image voxel size is not significantly smaller than individual trabecular elements, partial volume blurring can be a major complication for accurate tissue classification. In this paper, a Bayesian segmentation technique is reported that achieves improved subvoxel tissue classification. Each voxel is subdivided either into eight subvoxels twice the original resolution, or up to four subvoxels along the transaxial direction and the subvoxels optimally classified as volume blurring, the likelihood for the number of marrow subvoxels in each voxel can be computed on the basis of its measured signal. To resolve the ambiguity of the location of the marrow subvoxels, a Gibbs distribution is introduced to model the interaction between the subvoxels. Neighboring subvoxel pairs with the same tissue label are encouraged, and pairs with distinct labels are penalized. The segmentation is achieved by maximizing the a posteriori probability of the label image using the block ICM (iterative conditional mode) algorithm. The potential of the proposed technique is demonstrated in real and synthetic NMR microscopic images.

Algorithms↗
PubMed1994

Automated processing for proton spectroscopic imaging using water reference deconvolution.

Automated formation of MR spectroscopic images (MRSI) is necessary before routine application of these methods is possible for in vivo studies; however, this task is complicated by the presence of spatially dependent instrumental distortions and the complex nature of the MR spectrum. A data processing method is presented for completely automated formation of in vivo proton spectroscopic images, and applied for analysis of human brain metabolites. This procedure uses the water reference deconvolution method (G. A. Morris, J. Magn. Reson. 80, 547(1988)) to correct for line shape distortions caused by instrumental and sample characteristics, followed by parametric spectral analysis. Results for automated image formation were found to compare favorably with operator dependent spectral integration methods. While the water reference deconvolution processing was found to provide good correction of spatially dependent resonance frequency shifts, it was found to be susceptible to errors for correction of line shape distortions. These occur due to differences between the water reference and the metabolite distributions.

Artifacts↗
PubMed1994

Suppression by isoproterenol of endothelial cell morphology and barrier function changes induced by platelet-activating factor.

Using a model to study vascular permeability on hydrostatically perfused bovine pulmonary artery endothelial cell (EC) monolayers and software to analyze cell morphological parameters automatically in a computer image workstation, we studied the effects of isoproterenol (IPN) on platelet-activating factor (PAF)-induced changes in EC monolayer permeability and cell morphological parameters. Albumin has fortifying effects on endothelial barrier function. As albumin concentration in the perfusate increased (0, 1, 5, 10, 20 mg/ml), EC monolayer hydraulic conductivity (Lp) decreased gradually while Lp of the filter membranes did not change. After treatment of the EC monolayer with PAF 10(-8) mol/liter for 30 min, transmonolayer fluid flow, protein clearance rate, and Lp value increased noticeably. At the same time, cell area decreased and intercellular distance and percentage of intercellular space area in total cell monolayer increased. Pretreatment with 10(-4) mol/liter IPN blocked PAF-induced EC permeability and morphological changes, suggesting that EC contraction and intercellular gap formation are important mechanisms for PAF-induced high vascular permeability. IPN inhibits the effects of PAF via stabilization of EC morphology, protection of intercellular junction, and blockade of intercellular gap formation.

Albumins↗
PubMed1994

The impact of a media campaign in the reduction of risk-taking behavior on the part of drivers.

As part of an ongoing media educational campaign to enhance pedestrian safety in the Victoria (Canada) metropolitan area, multimedia campaigns have been targeted at drivers. This paper evaluates the campaign that focused on encouraging left-turning drivers to yield. Observers monitored five signalized intersections during five separate weeks. Logistic regression analyses indicate that this media campaign produced a long-term effect of increasing drivers' yielding behaviors.

Accidents, Traffic↗
PubMed1994

Applications of least-squares FIR filters to virtual acoustic space.

A virtual acoustic space (VAS) employs the localization cues specified by the direction-dependent 'free-field to eardrum transfer function' (FETF) to synthesize sound-pressure waveforms present near the tympanum. The combination of a VAS and the earphone delivery of synthesized waveforms is useful to study parametrically the neural mechanisms of directional hearing. The VAS-earphone procedure requires accurate FETF estimation from free-field measurements and appropriate compensation for the undesirable spectral characteristics of the closed-field earphone sound delivery and measurement systems. Here we describe how specially designed finite-impulse-response (FIR) filters improve these two operations. The coefficients of an FIR filter are determined using a least-squares error criterion. The least-squares FIR filter is implemented entirely in the time domain and avoids the usual problems with division inherent in a frequency domain approach. The estimation of an FETF by a least-squares FIR filter is veracious since its impulse response can recover signals that were recorded near the eardrum in the free field with a very high fidelity. The correlation coefficient between recorded and recovered time waveforms typically exceeds 0.999. Similarly, least-squares FIR filters prove excellent in compensating closed-field sound systems since comparisons of waveforms delivered by a compensated earphone to their corresponding predistorted signals yield correlation coefficients that exceed 0.99 on average.

Acoustic Stimulation↗
PubMed1994

Human p80-coilin is targeted to sphere organelles in the amphibian germinal vesicle.

Cultured vertebrate cells often display one or more coiled bodies in their nuclei. These are spherical structures approximately 0.5-1.0 micron in diameter that contain high concentrations of small nuclear ribonucleoproteins (snRNPs); they are distinct from nuclear speckles and nucleoli, the other major sites of snRNP concentration. Coiled bodies in human cells contain a unique protein, p80-coilin, that has an M(r) = 80 kDa. Cloned p80-coilin cDNA encodes 576 amino acids with a calculated molecular weight of 62.6 kDa. To determine which of several snRNP-containing structures in the amphibian germinal vesicle (GV) might be the homologue of coiled bodies, we injected myc-tagged transcripts of full-length human p80-coilin into the cytoplasm of Xenopus oocytes and followed the fate of the translated proteins with an antibody specific for the tag. Western blots of GV proteins showed rapid appearance of both full-length and truncated p80-coilin in the nucleus. Immunofluorescent staining of spread GV contents demonstrated specific uptake of p80-coilin by the sphere organelle within 1 h after injection. Similar experiments were performed with a series of deletion constructs that lacked progressively longer segments from the carboxy terminus. A construct that contained only the first 102 amino acids (18% of the molecule) was specifically targeted to the sphere organelle. Conversely, a construct lacking the first 92 amino acids failed to localize, although it was imported into the GV. Thus, a relatively short region at the amino terminus of human p80-coilin is both necessary and sufficient for localization in the sphere organelle. Sphere organelles in the GV and coiled bodies in somatic nuclei are clearly related in composition. We suggest that they are homologous organelles with similar functions in preassembly and sorting of RNA processing components. Differences in their composition suggest functional specialization in the two cell types.

Amino Acid Sequence↗