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Y Xu

Publications and source records attributed to Y Xu.

At least 1,063 records · Page 59Linked to original sources
PubMed1994

Substrate binding domains in pyruvate phosphate dikinase.

Proteolysis of Clostridium symbiosum pyruvate phosphate dikinase (PPDK) in its free or phosphorylated state with subtilisin Carlsberg followed two different cleavage pathways. The major pathway involved initial cleavage of the holoenzyme (93 kDa) into a stable 25-kDa N-terminal fragment and transiently stable 67-kDa C-terminal fragment. The 67-kDa fragment was cleaved to generate a stable 35-kDa fragment and an unstable 30-kDa fragment (containing the catalytic histidine). Proteolytic cleavage via the minor pathway divided the holoenzyme into an unstable 37-kDa N-terminal piece (which was further cleaved to the stable 25-kDa fragment produced in the major pathway) and a transiently stable 55-kDa C-terminal fragment. The 55-kDa fragment was then cleaved to produce the stable 35-kDa fragment produced by the major pathway. The cleavage pattern of PPDK complexed with the ATP analog adenyl imidodiphosphate was identical to that of the free enzyme, only the rate of cleavage as slower. In contrast, proteolysis of the phosphorylenzyme-oxalate complex generated the 55-kDa fragment indicating that oxalate binding induces a change in protein conformation. Treatment of PPDK with [1-14C]bromopyruvate followed by proteolysis revealed selective radiolabeling of the stable 35-kDa fragment while similar experiments with [14C]2',3'-dialdehyde adenosine 5'-monophosphate resulted in selective radiolabeling of the stable 25-kDa fragment. These results were interpreted to suggest that PPDK contains several structural domains and that the catalytic histidine, the pyruvate binding site, and the ATP binding site may be located on different domains.

Adenosine Triphosphate↗
PubMed1994

Residue at position 331 in the IgG1 and IgG4 CH2 domains contributes to their differential ability to bind and activate complement.

A conserved proline residue is found at position 331 in the CH2 domains of human IgG subclasses which fix complement. This residue is replaced by a serine in IgG4 which is inactive. To determine the role of residue 331 in the differential ability of human IgGs to activate the complement cascade, a pair of genetically engineered anti-dinitrophenol IgG1 and IgG4 antibodies with reciprocal mutations at position 331 were tested for their hemolytic activity as well as for their ability to bind C1q, activate C1 and cleave C4. The IgG1 Ser331 mutant was virtually unable to mediate the lysis of trinitrobenzene-sulfonic acid-derivatized sheep red blood cells as a result of a marked defect in C1q binding activity. In contrast, the substitution of Pro for Ser331 in IgG4 bestowed partial hemolytic activity (40%) to the IgG4 Pro331 variant. Under low ionic strength conditions, this mutant was found to be approximately 50 and 75% as active as wild-type IgG1 in the C1q binding and C4b deposition assays, respectively. These results indicate that residue Pro331, which folds into close proximity to a previously identified C1q binding motif (Duncan, A. R., and Winter, G. (1988) Nature 332, 738-740), contributes to the architecture of the IgG1 C1q binding site and that its replacement by a serine residue in IgG4 is largely responsible for the functional inactivity of this isotype.

Amino Acid Sequence↗
PubMed1994

Retention of unassembled components of integral membrane proteins by calnexin.

Quality control mechanisms prevent the cell surface expression of incompletely assembled multisubunit receptors such as the T cell receptor (TCR). The molecular chaperone function of calnexin (IP90, p88), a 90-kilodalton protein that resides in the endoplasmic reticulum (ER), in the retention of representative chains of the TCR-CD3 complex in the ER was tested. Truncation mutants of calnexin, when transiently expressed in COS cells, were exported from the ER and either accumulated in the Golgi or progressed to the cell surface. CD3 epsilon chains cotransfected with the forms of calnexin that were not retained in the ER exited the ER and colocalized with calnexin. Since engineered calnexin determined the intracellular localization of the proteins associated with it, it is concluded that calnexin interacts with incompletely assembled TCR components and retains them in the ER.

Amino Acid Sequence↗
PubMed1994

Regulation of adhesion molecule expression in Kaposi's sarcoma cells.

Kaposi's sarcoma (KS) is a neoplasm with multifocal vascular lesions that is often seen in homosexual HIV-infected individuals. Infiltrates of leukocytes are characteristic components of KS lesions, and the products of leukocytes have been shown to enhance the proliferation of KS cells in vitro and most likely are crucial for the development of KS lesions in vivo. It is therefore likely that the expression of cellular adhesion molecules (CAM) is a critical determinant in the pathogenesis of KS by dictating the numbers and types of leukocytes that accumulate in areas predisposed to KS. We report that in the absence of inducers, KS cells in culture expressed low levels of ICAM-1 and undetectable VCAM-1 and E-selectin. ICAM-1, VCAM-1, and E-selectin were all induced by dsRNA (poly (I:C)), IL-1 beta, TNF-alpha, and LPS in KS cells. All of these agents increased NF-kappa B binding activity in nuclear extracts from KS cells. Neither human dermal fibroblasts nor human aortic smooth muscle cells had detectable VCAM-1 protein expression in response to conditions that led to high levels of VCAM-1 expression in KS cells. Although E-selectin expression was induced in KS cells, the peak cell surface protein levels were less than 25% the levels achieved on HUVEC or human dermal microvascular endothelial cells (HMEC). These low levels resembled the levels that were induced in HMEC immortalized with SV 40 large T Ag. These data indicate that multiple proinflammatory agents can induce NF-kappa B binding activity and can enhance ICAM-1, VCAM-1, and E-selectin expression in KS cells. The increased CAM expression enhances leukocyte binding to KS cells. Thus, the induction of CAM expression could be an early event in the development of KS by recruiting leukocytes into KS lesions, thereby providing factors that could potentiate the development of KS.

Base Sequence↗
PubMed1994

Surface coil cardiac tagging and 31P spectroscopic localization with B1-insensitive adiabatic pulses.

A technique is presented for MRI tagging in the presence of inhomogeneous B1 fields. A rectangular tagging grid is produced with B1-insensitive adiabatic pulses in a magnetization preparation period that precedes image acquisition. Phantom results demonstrate that the method is well-suited to surface coil experiments. The technique is applied to a canine model of myocardial ischemia to track the spatially dependent wall motion of the left ventricle during the cardiac cycle. Transmural 31P spectra are acquired from the same double-tuned surface coil, with tagging and spectroscopy performed for the first time, during normal, ischemic, and recovery conditions for the same animal.

Adenosine Triphosphate↗
PubMed1994

Design of a cryogenic videocamera-recorder and image processing system and calculation of volumes of red cells.

Since Rowe reported that the storage technique for red cells at very low temperatures had been realized successfully, many experts who work in the fields of cryobiology and medicine have turned their attention to this storage technique for tissues and organs. Since the first quantitative cryomicroscope was made successfully about 20 years ago, it has been possible to observe changes in shape and phase. Particularly the image processing technique has laid the foundation for quantitative analysis of the relationship between changes in shape and damage from freezing of cells and cooling rate and storage temperature. In this work, we constructed a system consisting of a cryomicroscope, a videocamera-recorder, and an image processor. We carried out many experiments with red cells in cold storage and have established a model for calculating the volumes of red cells. In our experiments we also dynamically traced the shape changes of cells with various experimental parameters.

Blood Preservation↗
PubMed1994

Modulation of edema by dizocilpine, kynurenate, and NBQX in respiring brain slices after exposure to glutamate.

Brain edema caused by glutamate excitotoxicity was studied in well oxygenated neonatal cerebrocortical brain slices (350 mu thick). Slices exposed to 60 minutes of 2 mM glutamate, with or without glutamate antagonists (dizocilpine, kynurenate, or NBQX), were allowed to recover for 60 minutes. The protocol was identical to that in noninvasive multinuclear NMR spectroscopy studies (31P/1H/19F) of live slices. Percent water and swelling were determined invasively in isolated slices by wet and dry weight measurements before and after glutamate exposure. Edema was detectable within minutes in all experiments with glutamate exposures, but not in untreated control slices. Dizocilpine, kynurenate, and NBQX differently affected swelling, which correlated with PCr and ATP loss in separate NMR studies. Synaptic glutamate receptor activation appears to initiate events causing both edema and energy failure. Multiple glutamate receptor types seem to be involved. No glutamate antagonist provided greater protection against both edema and energy loss than dizocilpine. Dizocilpine might also block voltage-dependent Na+ channels, and provide protection via mechanisms other than NMDA-receptor dependent channel antagonism.

Animals↗
PubMed1994

Production of trichothecene mycotoxins by Fusarium graminearum and Fusarium culmorum on barley and wheat.

Wheat cultivars (Stoa, MN87150, SuMai-3, YMI-6, Wheaton) and barley cultivars (Robust, Excel, Chevron, M69) were inoculated in the field with isolates of Fusarium graminearum and F. culmorum. The disease (Fusarium head blight) kernels were analyzed for deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-ADON) and nivalenol (NIV). F. culmorum produced all three trichothecenes on all cultivars tested whereas F. graminearum only produced DON and 15-ADON. There was no well defined correlation between DON production in the host and resistance although the data tended to favor SuMai-3 as having definitive resistance to both F. graminearum and F. culmorum.

Animal Feed↗
PubMed1994

An ultra-sensitive electrochemical enzyme immunoassay for thyroid stimulating hormone in human serum.

A sensitive heterogeneous electrochemical enzyme immunoassay has been developed for thyroid stimulating hormone (TSH) by modifying a commercially available two-site immunoenzymometric assay. p-Aminophenyl phosphate (PAPP) was used as the substrate of alkaline phosphatase, and hydrolysed to p-aminophenol (PAP). The amount of PAP produced from the assay was proportional to the amount of TSH in the sample. Detection of PAP was done by oxidative amperometry in a flow injection system. The working electrode was a glassy carbon electrode whose potential was held at +325 mV (vs Ag/AgCl). The amperometric detection of PAP required only 1 microliter of sample (the range of linearity: 50.0 fmol-100 pmol PAP, the limit of detection: 10.9 fmol PAP). Intra-assay precision over the assay range of linearity (0.02-60 mIU l-1 or 0.02-60 pIU TSH) showed a maximum RSD of 8.0%, and a low detection limit of 0.01 mIU l-1 or 0.01 pIU TSH. The study also indicates that this two-site electrochemical enzyme immunoassay correlates well with the Bio-Rad's immunoradiometric assay currently used in our medical center (r = 0.992, slope = 1.53, n = 43) and a highly sensitive immunochemiluminometric assay in the Nichols Institute (r = 0.986, slope = 0.499, n = 23).

Electricity↗
PubMed1994

Regulation of plasminogen activator activity in transitional carcinoma cell lines by wound site growth factors.

The effect of two growth factors ellicited in response to surgical woundings (transforming growth factor alpha [TGF alpha] and beta 1) on the regulation of cellular plasminogen activator activity (PAA) in human transitional carcinoma cell lines, with differing baseline PAA (253J--high; 647V--low), was studied. mRNA transcript levels of PA regulatory proteins in both cell lines were responsive to TGF alpha and TGF beta 1. However, both the magnitude and nature of the response differed markedly between the two lines. TGF alpha increased uPA transcript levels in both cell lines in a dose-dependent fashion. In the case of uPA receptor, exogenous TGF alpha concentrations which increased receptor levels over fivefold in the 253J line had no effect on this transcript in the 647V line. This differential responsiveness was even more pronounced for TGF beta 1. TGF beta 1 appeared to increase uPA transcript in the 253J line in a dose dependent manner while decreasing transcript levels in the 647V line. uPAr mRNA in 253J cells increased over a 19-fold range in response to TGF beta 1 while this same transcript was decreased 14-fold in 647V cells. The most pronounced effect of TGF beta 1 was seen on PAI1 transcript levels in the 253J line. This transcript increased in a concentration dependent fashion from non-detectable levels. These findings demonstrate that growth factors ellicited by surgical wounding may alter the biology of neoplastic cells. Both the growth factor milieu, and intrinsic cellular regulatory mechanism, appear important in determing net PAA in transitional carcinoma cell lines.

Carcinoma, Transitional Cell↗
PubMed1994

Functionalized depsipeptides, substrates and inhibitors of beta-lactamases and DD-peptidases.

A series of derivatives of phenyl phenylacetylglycinates (aryl phenaceturates) with a carboxylate substituent meta to the oxygen of the phenoxide leaving group and a functionalized methylene group in the ortho- or para-position have been synthesized. These molecules possess a latent o- or p-quinone methide electrophile which could be unmasked during enzymic turnover and could react with an active site nucleophile. This chemistry does seem to occur in solution where a common hydrolysis product, independent of the benzylic leaving group, presumably o- or p-hydroxymethylphenol, was observed. These depsipeptides are substrates of class A and C beta-lactamases, particularly of the latter, comparable with the parent m-carboxyphenyl phenaceturate. They also have modest inhibitory activity against these enzymes and against the serine DD-peptidase of Streptomyces R61. The inhibition of a class C beta-lactamase was turnover dependent, as expected of mechanism-based inhibitor, but the small leaving group dependence of the inhibition suggested that the quinone methide, if it was in fact responsible for the inhibition, was generated in solution subsequent to release of the product phenol from the active site.

Anti-Bacterial Agents↗
PubMed1994

Modulation of glutamate-induced intracellular energy failure in neonatal cerebral cortical slices by kynurenic acid, dizocilpine, and NBQX.

The severity and rapidity of acute, glutamate-induced energy failure were compared in live cerebral cortical slices. In each experiment 80 live cerebral cortical slices (350 microns thick) were obtained from neonatal Sprague-Dawley rats, suspended and perfused in a nuclear magnetic resonance (NMR) tube, and studied at 4.7 T with interleaved 31P/1H NMR spectroscopy. NMR spectra, obtained continually, were determined as 5-min averages. Slices were perfused for 60 min with artificial cerebrospinal fluid (ACSF) containing either glutamate alone or glutamate mixed with one of three glutamate-receptor antagonists: kynurenate, dizocilpine (MK-801), and 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(F)quinoxaline (NBQX). Dose-dependent decreases in high-energy phosphates were studied during glutamate exposure (0.5 to 10 mM), with and without antagonist protection. Energy recovery after glutamate exposures was measured during a 60-min washout with glutamate-free, antagonist-free ACSF. Reversible and irreversible energy failures were characterized by changes in intracellular pH, and by changes in relative concentrations of ATP, phosphocreatine (PCr), and inorganic phosphate. No changes were observed in intracellular levels of N-acetylaspartate and lactate. Some special studies were also done using R-(-)-2-amino-5-phosphonovaleric acid (100 microM) and tetrodotoxin (1 mM) to examine glutamate receptor specificity in this tissue model. Dizocilpine (150 microM) best ameliorated the energy failure caused by 2.0 mM glutamate. With dizocilpine the maximum ATP decrease was only 6 +/- 5%, instead of 35 +/- 7%.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1994

Inactivation of free and cell-associated human immunodeficiency virus in platelet suspensions by aminomethyltrimethylpsoralen and ultraviolet light.

BACKGROUND: It has previously been reported that 40 micrograms per mL of aminomethyltrimethylpsoralen (AMT) plus 2.4 to 7.2 J per cm2 of ultraviolet A (UVA) light inactivated 4 to 6 log10 of several model viruses in platelet suspensions. This inactivation was achieved while satisfactory levels of platelet count, pH, morphology, aggregation, and hemostatic effectiveness were maintained. STUDY DESIGN AND METHODS: The efficacy of this procedure for inactivating free and intracellular human immunodeficiency virus (HIV), including integrated proviral sequences, was studied. RESULTS: The kinetics of inactivation for free HIV (4-5 log10 kill with 1.2-4.8 J/cm2) were similar to those obtained for the previously studied viruses. For studies on cell-associated virus, H9 cells productively infected with HIV were added to platelet suspensions and treated with the above regimen of AMT and UVA. The phototreated cells were then cocultivated with uninfected H9 cells for 4 weeks and supernatants were assayed by enzyme-linked immunosorbent assay for HIV p24. No evidence of HIV replication was detectable for cells receiving as little as 2.4 J per cm2 of UVA irradiation in the presence of AMT. Further, it has been demonstrated that stably integrated sequences from the HIV proviral env gene can no longer be amplified by polymerase chain reaction after 1.2 J per cm2 of UVA (with 40 micrograms/mL AMT) exposure. CONCLUSION: These data suggest that AMT and UVA is an effective antiviral treatment for free and cell-associated HIV in platelet suspensions.

Base Sequence↗
PubMed1994

Cellular origins of tenascin in the developing nervous system.

We have used in situ hybridization and reverse transcriptase polymerase chain reaction (PCR) to study the origins of the extracellular matrix glycoprotein tenascin during the development of the central and peripheral nervous systems. Previous studies have shown that neural crest cells migrate along pathways that are lined with tenascin. In situ hybridization, PCR, and western blotting reveal that these cells themselves are a major source of tenascin both in vitro and in the embryo. Thus, tenascin is probably not acting as a guidance molecule but is more likely to be promoting neural crest cell motility in a more general way. Similarly, subpopulations of proliferating and migrating glia make tenascin in the developing central nervous system, as do the radial glia that are used as a substratum for migrating neuronal cell bodies. In the adult, tenascin continues to be expressed in the cerebellum by Golgi epithelial cells. This expression, as well as the expression of tenascin in connective tissue, indicates that this molecule may also be playing a role in regulating differentiation. Finally, the distribution of tenascin transcripts in the developing brain and spinal cord is similar to the distribution of mRNAs encoding receptors for platelet-derived growth factor-AA and basic fibroblast growth factor. In vitro studies indicate that both of these factors are potential regulators of tenascin expression.

Animals↗
PubMed1994

Purification and properties of cow splenic biliverdin reductase.

Biliverdin reductase was purified from cow spleen. The specific activity of the final enzyme preparation was 24.01 u/mg, representing 686-fold purification as measured with NADPH. The yield was 3 grams of enzyme per 100 grams of cow spleen. The purified enzyme was a monomeric protein with an apparent molecular weight of about 34,000 and an isoelectric point of about 6.2. The biliverdin reductase was specific for biliverdin and reduced IX alpha faster than the biliverdin isomers IX beta, IXr, or IX delta. The purified enzyme could utilize both NADH and NADPH, but the kinetic properties of the NADH-dependent and the NADPH-dependent enzyme activities were different: the time course of the NADPH-dependent reaction displayed a sigmoidal curve, whereas that of the NADH-dependent reaction did not. Km for biliverdin IX alpha was 4 x 10(-4) mM in the NADPH system, while it was 1.5 x 10(-3) mM in the NADH system. Both enzyme activities were inhibited by excess biliverdin, but the inhibition of the NADPH-dependent enzyme activity was more pronounced. The pH optimum was 7.0 with NADH, and 6.8 with NADPH.

Animals↗
PubMed1994

Constructing gene models from accurately predicted exons: an application of dynamic programming.

This paper presents a computationally efficient algorithm, the Gene Assembly Program III (GAP III), for constructing gene models from a set of accurately-predicted 'exons'. The input to the algorithm is a set of clusters of exon candidates, generated by a new version of the GRAIL coding region recognition system. The exon candidates of a cluster differ in their presumed edges and occasionally in their reading frames. Each exon candidate has a numerical score representing its 'probability' of being an actual exon. GAP III uses a dynamic programming algorithm to construct a gene model, complete or partial, by optimizing a predefined objective function. The optimal gene models constructed by GAP III correspond very well with the structures of genes which have been determined experimentally and reported in the Genome Sequence Database (GSDB). On a test set of 137 human and mouse DNA sequences consisting of 954 true exons, GAP III constructed 137 gene models using 892 exons, among which 859 (859/954 = 90%) are true exons and 33 (33/892 = 3%) are false positive. Among the 859 true positives, 635 (74%) match the actual exons exactly, and 838 (98%) have at least one edge correct. GAP III is computationally efficient. If we use E and C to represent the total number of exon candidates in all clusters and the number of clusters, respectively, the running time of GAP III is proportional to (E x C).

Algorithms↗
PubMed1994

Identification of the Ped gene at the molecular level: the Q9 MHC class I transgene converts the Ped slow to the Ped fast phenotype.

The Ped (preimplantation embryo development) gene, which maps to the Q region of the mouse major histocompatibility complex (MHC), controls the rate of cleavage division of preimplantation mouse embryos and subsequent embryonic survival. Of the ten known MHC class I genes in the Q region of the mouse MHC, four--Q6, Q7, Q8, and Q9--are almost identical and encode similar proteins, all called the Qa-2 antigen. Previous studies have suggested that the Q9 gene encodes the Ped gene. To test this directly, one-cell embryos from the CBA/Ca strain (Ped slow) that is missing the Q9 gene and the Qa-2 antigen were injected with the Q9 gene from the C57BL/10 strain (Ped fast) that possesses the Q9 gene and expresses the Qa-2 antigen. The resulting Q9 transgenic mice were found to express the Qa-2 antigen. In addition, it was found that introduction of the Q9 gene converted the Ped gene phenotype of the recipient strain from slow to fast. Therefore, the Ped gene product is the Qa-2 antigen encoded by the Q9 gene.

Animals↗