Biomedical subjects
Y Xu
Publications and source records attributed to Y Xu.
Contribution of the complement control protein modules of C2 in C4b binding assessed by analysis of C2/factor B chimeras.
To identify the complement control protein (CCP) module(s) of C2 that are required for C4b recognition, we constructed a panel of C2/factor B chimeras by substituting intact or partial factor B CCP modules for the corresponding ones of C2. Epitope mapping indicated that the anti-C2b mAb 3A3.3, which inhibits binding of C2 to C4b, reacts with the second CCP of C2 and similarly the anti-Ba mAb HA4-1A, which inhibits binding of factor B to C3b, reacts with the second CCP of factor B. The hemolytic activity of the chimeras CP1, CP2, and CP3a containing CCP1, CCP2, and a fragment of CCP3 of factor B, respectively, was substantially decreased compared with that of wild-type C2. The CP3 and CP1-3 chimeras, in which CCP3 and all three CCP modules of factor B, respectively, were substituted, had no hemolytic activity. Loss of activity could be attributed to the resistance of these two chimeras to C1s cleavage, which was probably due to conformational changes of the cleavage site. The combined results indicate that all three CCP modules of C2 contribute structural elements to the C4b-binding site of C2b. This site has been shown previously to be necessary for the initial binding of C2 to C4b which leads to the formation of the classical pathway C3 convertase.
A dendritic-cell-derived C-C chemokine that preferentially attracts naive T cells.
Dendritic cells form a system of highly efficient antigen-presenting cells. After capturing antigen in the periphery, they migrate to lymphoid organs where they present the antigen to T cells. Their seemingly unique ability to interact with and sensitize naive T cells gives dendritic cells a central role in the initiation of immune responses and allows them to be used in therapeutic strategies against cancer, viral infection and other diseases. How they interact preferentially with naive rather than activated T lymphocytes is still poorly understood. Chemokines direct the transport of white blood cells in immune surveillance. Here we report the identification and characterization of a C-C chemokine (DC-CK1) that is specifically expressed by human dendritic cells at high levels. Tissue distribution analysis demonstrates that dendritic cells present in germinal centres and T-cell areas of secondary lymphoid organs express this chemokine. We show that DC-CK1, in contrast to RANTES, MIP-1alpha and interleukin-8, preferentially attracts naive T cells (CD45RA+). The specific expression of DC-CK1 by dendritic cells at the site of initiation of an immune response, combined with its chemotactic activity for naive T cells, suggests that DC-CK1 has an important rule in the induction of immune responses.
Surface loops adjacent to the cation-binding site of the complement factor B von Willebrand factor type A module determine C3b binding specificity.
The interaction of factor B with C3b deposited on the surface of pathogens is the first step in the activation of the alternative complement pathway. The role of the von Willebrand factor type A (VWFA) module of factor B in this interaction has been investigated by generating three chimeras, Ch1-Ch3, in which surface loops of the VWFA module flanking the cation-binding residues were replaced by the corresponding sequences of C2, a factor B-like molecule which does not bind C3b. The location of the three loops was inferred from a homology model based on the structure of the integrin alphaM VWFA module [Ch1, betaA-alpha1 loop: Ch2, alpha3-alpha4 loop; and Ch3, betaD-alpha5 loop; Lee, J.-O., et al. (1995b) Cell 80, 631-638]. The function of the chimeras was studied by means of hemolytic assays and assays of the individual steps of the alternative complement pathway, i.e., binding to the C3b analogue cobra venom factor and factor D cleavage. These experiments showed that Ch1 and Ch3 define regions that are involved in C3b binding whereas Ch2 does not appear to be involved in binding specificity. The inability of Ch1 to register the enhancement of cobra venom factor binding normally seen after factor D cleavage suggested that the betaA-alpha1 loop mediates the conformational regulation of ligand binding affinity. Homology modeling of the chimeras has been used to visualize the surface structures which potentially define the C3b binding site.
Ataxia telangiectasia mutant protein activates c-Abl tyrosine kinase in response to ionizing radiation.
Ataxia telangiectasia (AT) is a rare human autosomal recessive disorder with pleiotropic phenotypes, including neuronal degeneration, immune dysfunction, premature ageing and increased cancer risk. The gene mutated in AT, ATM, encodes a putative lipid or protein kinase. Most of the human AT patient phenotypes are recapitulated in Atm-deficient mice. Cells derived from Atm-/- mice, like those from AT patients, exhibit abnormal response to ionizing radiation. One of the known responses to ionizing radiation is the activation of a nuclear tyrosine kinase encoded by the c-abl proto-oncogene. Ionizing radiation does not activate c-Abl in cells from AT patients or in thymocytes or fibroblasts from the Atm-deficient mice. Ectopic expression of a functional ATM kinase domain corrects this defect, as it phosphorylates the c-Abl tyrosine kinase in vitro at Ser 465, leading to the activation of c-Abl. A mutant c-Abl with Ser 465 changed to Ala 465 is not activated by ionizing radiation or ATM kinase in vivo. These findings identify the c-Abl tyrosine kinase as a downstream target of phosphorylation and activation by the ATM kinase in the cellular response to ionizing radiation.
Interaction of the adaptor protein Shc and the adhesion molecule cadherin.
In mitogenic signaling pathways, Shc participates in the growth factor activation of Ras by interacting with activated receptors and/or the Grb-2.Sos complex. Using several experimental approaches we demonstrate that Shc, through its SH2 domain, forms a complex with the cytoplasmic domain of cadherin, a transmembrane protein involved in the Ca2+-dependent regulation of cell-cell adhesion. This interaction is demonstrated in a yeast two-hybrid assay, by co-precipitation from mammalian cells, and by direct biochemical analysis in vitro. The Shc-cadherin association is phosphotyrosine-dependent and is abrogated by addition of epidermal growth factor to A-431 cells maintained in Ca2+-free medium, a condition that promotes changes in cell shape. Shc may therefore participate in the control of cell-cell adhesion as well as mitogenic signaling through Ras.
Biochemical and mutational studies of the 5'-3' exonuclease of DNA polymerase I of Escherichia coli.
In order to improve our understanding of the 5'-3' exonuclease reaction catalyzed by Escherichia coli DNA polymerase I, we have constructed expression plasmids and developed purification methods for whole DNA polymerase I and its 5'-3' exonuclease domain that allow the production of large quantities of highly purified material suitable for biophysical and other studies. We have studied the enzymatic properties of the 5'-3' exonuclease, both as an isolated domain and in the context of the whole polymerase, using a variety of model oligonucleotides to explore the enzyme-substrate interaction. The 5'-3' exonuclease is known to be a structure-specific nuclease that cleaves a 5' displaced strand at the junction between single-stranded and duplex regions. Since the isolated domain shows the same structure specificity as the whole polymerase, the correct geometry of substrate binding is achieved without the assistance of the polymerase domain. The 5'-3' exonuclease reaction has a strict requirement for a free 5' end on the displaced strand; however, the upstream template and primer strands are dispensable. Site-directed mutagenesis of the ten carboxylate residues that are highly conserved among bacterial and bacteriophage 5'-3' exonucleases indicates that nine of them are important in the reaction. This finding is discussed in relation to structural and mutational data for related 5' nucleases.
Defects of the mismatch repair gene MSH2 are implicated in the development of murine and human lymphoblastic lymphomas and are associated with the aberrant expression of rhombotin-2 (Lmo-2) and Tal-1 (SCL).
Mutations in the DNA mismatch repair (MMR) gene hMSH2 underlie a novel pathway of tumorigenesis for some cancers of epithelial origin. Mice deficient in MSH2 are susceptible to lymphomas but defects in this gene have not been identified in human lymphoid tumors. To determine if the lymphomas these mice develop are related to a particular subtype of human lymphoma we evaluated 20 clinically ill homozygous MSH2-/- mice ranging in age from 2 to 13 months. The murine tumors comprised a single histopathologic entity representing the malignant counterpart of precursor thymic T cells and closely resembled human precursor T-cell lymphoblastic lymphoma (LBL). Evaluation of the expression of three T-cell malignancy associated genes showed that Rhombotin-2 (RBTN-2 also known as Lmo-2), TAL-1 (also known as SCL), and HOX-11 were expressed in 100%, 40%, and 0% of the murine tumors, respectively. The MSH2-/- murine model of precursor T-cell LBL was substantiated by the finding of a nearly identical expression profile of RBTN-2, TAL-1, and HOX-11 in 10 well-characterized cases of human LBL. Direct evidence for MSH2 abnormalities in human LBL was established by sequence analysis of exon 13 of hMSH2, which revealed coding region mutations in 2 of 10 cases. Our findings implicate defects in the MMR system with the aberrant expression of T-cell specific proto-oncogenes and define a new pathway of human lymphomagenesis.
Kinetic mechanism of human hypoxanthine-guanine phosphoribosyltransferase: rapid phosphoribosyl transfer chemistry.
Hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) is the locus of Lesch-Nyhan syndrome, the activator of the prodrugs 6-mercaptopurine and allopurinol, and a target for antiparasitic chemotherapy. The three-dimensional structure of the recombinant human enzyme in complex with GMP has recently been solved [Eads, J., Scapin, G., Xu, Y., Grubmeyer, C., & Sacchettini, J. C. (1994) Cell 78, 325-334]. Here, ligand binding, pre-steady state kinetics, isotope trapping, and isotope exchange experiments are presented which detail the sequential kinetic mechanism of the enzyme. In the forward reaction, in which a base (hypoxanthine or guanine) reacts with PRPP to form nucleoside monophosphate and PPi, binding of PRPP precedes that of the base, and in the reverse direction, IMP binds first. Compared to k(cat), phosphoribosyl group transfer is rapid in both the forward (131 vs 6.0 s(-1)) and reverse (9 vs 0.17 s(-1)) directions. In the forward direction, product pyrophosphate dissociates rapidly (> 12 s(-1)) followed by release of IMP (6.0 s(-1)). In the reverse direction, Hx dissociates rapidly (9.5 s(-1)) and PRPP dissociates slowly (0.24 s(-1)). The more rapid rate of utilization of guanine than hypoxanthine in the forward reaction is the result of the faster release of product GMP rather than the result of differences in the rate of the chemical step. The kinetic mechanism, with rapid chemistry and slow product dissociation, accounts for the previously observed ability of the alternative product guanine to stimulate, rather than inhibit, the pyrophosphorolysis of IMP. The overall equilibrium for the hypoxanthine phosphoribosyl transfer reaction lies far toward nucleotide product (Keq approximately 1.6 x 10(5)), at the high end for PRPP-linked nucleotide formation. The three-dimensional structure of the HGPRTase x IMP complex has been solved to 2.4 A resolution and is isomorphous with the GMP complex. The results of the ligand binding and kinetic studies are discussed in light of the structural data.
Chromosomal regions associated with segregation distortion of molecular markers in F2, backcross, doubled haploid, and recombinant inbred populations in rice (Oryza sativa L.).
Chromosomal regions associated with marker segregation distortion in rice were compared based on six molecular linkage maps. Mapping populations were derived from one interspecific backcross and five inter-subspecific (indica/japonica) crosses, including two F2 populations, two doubled haploid (DH) populations, and one recombinant inbred (RI) population. Mapping data for each population consisted of 129-629 markers. Segregation distortion was determined based on chi-square analysis (P < 0.01) and was observed at 6.8-31.8%, of the mapped marker loci. Marker loci associated with skewed allele frequencies were distributed on all 12 chromosomes. Distortion in eight chromosomal regions bracketed previously identified gametophyte (ga) or sterility genes (S). Distortion in three other chromosomal regions was found only in DH populations, where japonica alleles were over-represented, suggesting that loci in these regions may be associated with preferential regeneration of japonica genotypes during anther culture. Three additional clusters of skewed markers were observed in more than one population in regions where no gametophytic or sterility loci have previously been reported. A total of 17 segregation distortion loci may be postulated based on this study and their locations in the rice genome were estimated.
Identification and purification of cellular proteins that specifically interact with the RNA constitutive transport elements from retrovirus D.
Human immunodeficiency virus (HIV) encodes a transacting protein, Rev, which interacts with an RNA element (RRE) to mediate nuclear export of unspliced viral mRNA. Recently, the RNA constitutive transport elements (CTE) from Mason-Pfizer monkey virus (MPMV) and simian retrovirus type I (SRV-1) were shown to render Rev-independent expression of gag, pol, or env genes in subgenomic constructs of HIV-1 and to support replication of HIV-1 mutants lacking RRE and Rev. Since CTEs act in cis, in the absence of any viral regulatory proteins, it is widely believed that they interact directly with the cellular export machinery by means of RNA-protein interaction. In this report, Electrophoretic mobility shift and UV-crosslinking assays were carried out to identify nuclear proteins that interact specifically with CTEs from both MPMV and SRV-1. Two of the four proteins (65 and 40 kDa, respectively) that bound CTE RNA did not interact in the same assays with RNA of a nonfunctional CTE mutant generated by site-directed mutagenesis. Both proteins have been partially purified. The nature of these proteins and their roles in RNA intracellular trafficking are currently under investigation.
Lymphotactin is produced by NK cells and attracts both NK cells and T cells in vivo.
Injection of lymphotactin (Lptn) into the peritoneum caused an influx of lymphocytes at 24 h. Phenotypic analysis of the cellular influx showed that a large proportion of these cells were T lymphocytes; however, a large number of NK cells was also present. This effect of murine Lptn (mLptn) was specific since the cellular influx was blocked with a mLptn-specific mAb. Similar results were observed when Lptn was injected s.c. and the tissue was analyzed by immunohistochemistry using an anti-CD3epsilon mAb. Microchemotaxis assays confirmed that murine NK cells respond to mLptn, and also showed human NK clones to be similarly responsive to recombinant human Lptn (rhLptn). Immunohistochemical analysis of IL-2-activated murine NK cells and Northern analysis of human NK clones revealed that these cells also produce Lptn, suggesting that a self-regulatory migration mechanism exists in NK cells. Together these data confirm, in vivo, the lymphocyte specificity of Lptn previously observed in vitro and extend its chemotactic effects to the NK cell lineage. We also investigated the functional consequences of truncating the carboxyl terminus of hLptn. This truncated molecule (which is missing the carboxyl-terminal 22 amino acids of hLptn) had no detectable activity on human PBLs. In addition, while hLptn was found to attract murine splenocytes in vitro, the carboxyl-terminal truncated hLptn was again inactive on murine splenocytes. This observation indicates the presence of structural features in the carboxyl terminus of Lptn that are necessary for its biologic activity.
Identification of a novel gene encoding the yeast mitochondrial dicarboxylate transport protein via overexpression, purification, and characterization of its protein product.
A gene encoding the mitochondrial dicarboxylate transport protein (DTP) has been identified for the first time from any organism. Our strategy involved overexpression of putative mitochondrial transporter genes, selected based on analysis of the yeast genome, followed by purification and functional reconstitution of the resulting protein products. The DTP gene from the yeast Saccharomyces cerevisiae encodes a 298-residue basic protein which, in common with other mitochondrial anion transporters of known sequence and function, displays the mitochondrial transporter signature motif, three homologous 100-amino acid sequence domains, and six predicted membrane-spanning regions. The product of this gene has been abundantly expressed in Escherichia coli where it accumulates in inclusion bodies. Upon solubilization of the overexpressed DTP from isolated inclusion bodies with Sarkosyl, 28 mg of DTP was obtained per liter of E. coli culture at a purity of 75%. The purified, overexpressed DTP was then reconstituted in phospholipid vesicles where both its kinetic properties (i.e. Km = 1. 55 mM and Vmax = 3.0 micro;mol/min/mg protein) and its substrate specificity were determined. The intraliposomal substrates malonate, malate, succinate, and phosphate effectively supported [14C]malonate uptake, whereas other anions tested did not. External substrate competition studies revealed a similar specificity profile. Inhibitor studies indicated that the reconstituted transporter was sensitive to inhibition by n-butylmalonate, p-chloromercuribenzoate, mersalyl, and to a lesser extent pyridoxal 5'-phosphate but was insensitive to N-ethylmaleimide and selective inhibitors of other mitochondrial anion transporters. In combination, the above findings indicate that the identified gene encodes a mitochondrial transport protein which upon overexpression and reconstitution displays functional properties that are virtually identical to those of the native mitochondrial dicarboxylate transport system. In conclusion, the present investigation has resulted in identification of a gene encoding the mitochondrial DTP and thus eliminates a major impediment to molecular studies with this metabolically important transporter. Based on both structural and functional considerations, the yeast DTP is assignable to the mitochondrial carrier family. Additionally, the development of a procedure that enables the expression and isolation of large quantities of functional DTP provides the foundation for comprehensive investigations into the structure/function relationships within this transporter via site-directed mutagenesis, as well as for the initiation of crystallization trials.
Microstructure-Confined Mechanical and Electric Properties of the Electrorheological Fluids under the Oscillatory Mechanical Field
The dynamic properties of two kinds of aluminosilicate-based electrorheological (ER) fluids were investigated under on-state and off-state electric fields. The dynamic responsive (frequency sweep spectrum and strain sweep spectrum) differences between these two ER suspensions were found under the off-state electric field, whereas similarities were observed once an external electric field was applied. Their dc conductances under an oscillatory mechanical field and their mechanical strengths under an oscillatory electric field were also examined. The periodically varied dc current in an oscillatory mechanical field and the decreased shear stress in an ac field were both found in these ER suspensions. The different in situ microstructures of these two ER fluids suggested on the basis of the percolation model, as well as their different response times measured experimentally, were presented to understand the experimental observations. Our results have a direct implication concerning the practical application of ER fluids.
Amphiphilic sites for general anesthetic action? Evidence from 129Xe-[1H] intermolecular nuclear Overhauser effects.
Because a strong correlation exists between the potency of general anesthetics and their ability to dissolve in oil, a lipophilic site of action is often assumed. We show here that a lipophilic molecule may preferentially target less lipophilic sites after interaction with a membrane takes place. Xenon, a chemically inert and structureless general anesthetic, was chosen as an unbiased molecular probe for assessment of its dynamic distribution. Site-selective intermolecular 129Xe-[1H] nuclear Overhauser effects were used to measure the specific interaction between xenon and protons in different regions in a phosphatidylcholine lipid membrane. It was evident that xenon-membrane interaction was directed toward the amphiphilic head region, with significant involvement of interfacial water, despite xenon's apolar and highly lipophilic nature in the gas phase. This result may suggest the importance of amphiphilicity in association with anesthetic action.
Stress among package truck drivers.
In 1992, a cross-sectional questionnaire study of package truck drivers in one company was conducted at four widely scattered sites throughout the US; 317 drivers participated, representing 82% of those eligible. The package truck drivers scored significantly above the US working population comparison norm on all summary and individual scales derived from the SCL 90-R, indicating a substantial increase in psychologic distress for this group. The Global Severity Index, the best single summary measure of psychological distress in the SCL 90-R, revealed a mean T score for the drivers of 64.20, 91st percentile of the normative population. The group perceived significantly more daily stressful events than the average working adult, and their sensitivity to these events was also increased. Role overload, a component of the Occupational Stress Inventory, was the most consistent factor associated with symptoms of psychological distress on multiple regression analysis. This study suggests that job stress is a psychological health hazard for these drivers.
c-myc-Dependent hepatoma cell apoptosis results from oxidative stress and not a deficiency of growth factors.
Expression of c-myc regulates apoptotic cell death in the human hepatoma cell line HuH-7 during culture in serum-free medium (SFM) plus zinc. To understand the mechanism of this c-myc effect, the ability of various serum-contained factors to prevent apoptosis was determined. Apoptosis was not inhibited by growth factors and was even accelerated by supplementation with insulin-like growth factor I or insulin. Cell death was prevented by SFM supplementation with the amino acid glutamine but not serine or asparagine. Improved cell survival with glutamine was associated with increased levels of glutathione (GSH). In HuH-7 cells cultured in SFM plus zinc, c-myc expression led to decreased levels of GSH, and elevated intracellular levels of hydrogen peroxide (H2O2). Cell death induced by c-myc expression was inhibited by the addition of catalase or dimethyl sulfoxide, a hydroxyl radical scavenger, or by increased intracellular expression of catalase. In contrast to findings in fibroblasts, c-myc-dependent apoptosis during serum deprivation in HuH-7 hepatoma cells was unrelated to a loss of growth factors. Apoptosis resulted from H2O2-mediated oxidative stress with associated glutamine dependent intracellular GSH depletion.
Strategy for identification of sequence variants in COL7A1 and a novel 2-bp deletion mutation in recessive dystrophic epidermolysis bullosa.
The diagnostic hallmark of the dystrophic forms of epidermolysis bullosa (DEB), a group of heritable blistering skin diseases, is abnormalities in the anchoring fibrils at the dermal-epidermal basement membrane zone. Since type VII collagen is the major, if not the exclusive, component of the anchoring fibrils, the corresponding gene (COL7A1) is the candidate gene in DEB. Recent cloning of the type VII collagen cDNA and elucidation of the exon-intron organization of the gene have provided the basis for us to develop a novel strategy for identification of sequence variants in COL7A1. Optimization of 72 balanced primer pairs corresponding to flanking intronic sequences allowed PCR amplification of all 118 exons directly from genomic DNA. The PCR products were examined by heteroduplex analysis followed by comparative nucleotide sequencing. More than 100 sequence variants have been identified thus far in COL7A1 using this method, some of which are single base pair polymorphisms and many of which are pathogenetic mutations contributing to the blistering phenotype in DEB. The comprehensive method described is useful for rapid, reliable, and sensitive detection of sequence variants in COL7A1. We demonstrate the utility of this novel strategy in mutation detection and prenatal exclusion of RDEB in a consanguineous family at risk for recurrence.