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Biomedical subjects

Y Xu

Publications and source records attributed to Y Xu.

At least 307 records · Page 17Linked to original sources
PubMed2000

Enhanced mitochondrial DNA repair and cellular survival after oxidative stress by targeting the human 8-oxoguanine glycosylase repair enzyme to mitochondria.

Oxidative damage to mitochondrial DNA (mtDNA) has been implicated as a causative factor in many disease processes and in aging. We have recently discovered that different cell types vary in their capacity to repair this damage, and this variability correlates with their ability to withstand oxidative stress. To explore strategies to enhance repair of oxidative lesions in mtDNA, we have constructed a vector containing a mitochondrial transport sequence upstream of the sequence for human 8-oxoguanine DNA glycosylase. This enzyme is the glycosylase/AP lyase that participates in repair of purine lesions, such as 8-oxoguanine. Western blot analysis confirmed that this recombinant protein was targeted to mitochondria. Enzyme activity assays showed that mitochondrial extracts from cells transfected with the construct had increased enzyme activity compared with cells transfected with vector only, whereas nuclear enzyme activity was not changed. Repair assays showed that there was enhanced repair of oxidative lesions in mtDNA. Additional studies revealed that this augmented repair led to enhanced cellular viability as determined by reduction of the tetrazolium compound to formazan, trypan blue dye exclusion, and clonogenic assays. Therefore, targeting of DNA repair enzymes to mitochondria may be a viable approach for the protection of cells against some of the deleterious effects of oxidative stress.

Base Sequence↗
PubMed2000

Disruption of a single copy of the SERCA2 gene results in altered Ca2+ homeostasis and cardiomyocyte function.

A mouse model carrying a null mutation in one copy of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase isoform 2 (SERCA2) gene, in which SERCA2 protein levels are reduced by approximately 35%, was used to investigate the effects of decreased SERCA2 level on intracellular Ca(2+) homeostasis and contractile properties in isolated cardiomyocytes. When compared with wild-type controls, SR Ca(2+) stores and Ca(2+) release in myocytes of SERCA2 heterozygous mice were decreased by approximately 40-60% and approximately 30-40%, respectively, and the rate of myocyte shortening and relengthening were each decreased by approximately 40%. However, the rate of Ca(2+) transient decline (tau) was not altered significantly, suggesting that compensation was occurring in the removal of Ca(2+) from the cytosol. Phospholamban, which inhibits SERCA2, was decreased by approximately 40% in heterozygous hearts, and basal phosphorylation of Ser-16 and Thr-17, which relieves the inhibition, was increased approximately 2- and 2.1-fold. These results indicate that reduced expression and increased phosphorylation of phospholamban provides compensation for decreased SERCA2 protein levels in heterozygous heart. Furthermore, both expression and current density of the sarcolemmal Na(+)-Ca(2+) exchanger were up-regulated. These results demonstrate that a decrease in SERCA2 levels can directly modify intracellular Ca(2+) homeostasis and myocyte contractility. However, the resulting deficit is partially compensated by alterations in phospholamban/SERCA2 interactions and by up-regulation of the Na(+)-Ca(2+) exchanger.

Animals↗
PubMed2000

Polymorphisms of the DNA repair gene XRCC1 and risk of gastric cancer in a Chinese population.

Gastric cancer remains the leading cause of cancer death in China and other countries in eastern Asia. Studies of gastric cancer have revealed that it is a disease of complex etiology involving dietary, infectious, environmental, occupational and genetic factors. DNA repair capacity has been suggested as a genetic factor contributing to variation in susceptibility to cancer. In the present study, we described an association between 2 polymorphisms of the DNA repair gene XRCC1 and risk of gastric cancer in a Chinese population. We used a polymerase chain reaction-based assay to detect Pvu II and Nci I restriction fragment length polymorphisms (XRCC1 26304 C-->T and XRCC1 28152 G-->A, respectively) in 188 patients with gastric cancer and 166 healthy controls. The XRCC1 26304 T allele (194Trp) frequency (34.6%) was higher and the XRCC1 28152 A allele (399Gln) frequency (25.6%) was lower in healthy Chinese controls than previously reported healthy U.S. Caucasian controls (7.2% and 34.1%, respectively). Multivariate logistic regression analysis revealed that the putative high-risk genotypes XRCC1 26304 CC and XRCC1 28152 GA/AA were associated with a non-significant increased risk for gastric cancer (adjusted odds ratio [OR]=1.45, 95% confidence interval [CI]= 0.93-2.25 and OR=1.53, 95% CI= 0.98-2.39, respectively) compared with other genotypes. However, the XRCC1 26304 CC genotype was associated with a significantly increased risk for gastric cardia cancer (adjusted OR=1.86, 95% CI=1.09-3.20). Individuals with both putative high-risk genotypes (CC and GA/AA) had a significantly higher risk (adjusted OR=1.73, 95% CI=1.12-2.69), particularly for gastric cardia cancer (adjusted OR=2.18, 95% CI=1.21-3.94) than individuals with other genotypes. These findings support the hypothesis that these 2 XRCC1 variants may contribute to the risk of developing gastric cancer, particularly gastric cardia cancer.

Adult↗
PubMed2000

Mac-1-negative B-1b phenotype of natural antibody-producing cells, including those responding to Gal alpha 1,3Gal epitopes in alpha 1,3-galactosyltransferase-deficient mice.

Human natural Abs against Galalpha1-3Galbeta1-4GlcNAc (Gal) epitopes are a major barrier to xenotransplantation. Studies in this report, which use combined multiparameter flow cytometric sorting and enzyme-linked immunospot assay, demonstrate that anti-Gal IgM-producing cells are found exclusively in a small B cell subpopulation (i.e., CD21(-/low) IgM(high) B220(low) CD5(-) Mac-1(-) 493(-) cells) in the spleens of alpha1, 3-galactosyltransferase-deficient mice. All IgM-producing cells were detected in a similar splenic subpopulation of alpha1, 3-galactosyltransferase-deficient and wild-type mice. A higher frequency of B cells with anti-Gal surface IgM receptors was observed in the peritoneal cavity than in the spleen, but these did not actively secrete Abs, and showed phenotypic properties of B-1b cells (CD21(-/low) IgM(high) CD5(-) CD43(+) Mac-1(+)). However, these became Mac-1(-) and developed anti-Gal Ab-producing activity after in vitro culture with LPS. The splenic B cells with anti-Gal receptors consisted of both Mac-1(+) B-1b cells and Mac-1(-) B-1b-like cells. The latter comprised most anti-Gal IgM-producing cells. Our studies indicate that anti-Gal natural IgM Abs are produced by a B1b-like, Mac-1(-) splenic B cell population and not by plasma cells or B-1a cells. They are consistent with a model whereby B-1b cells lose Mac-1 expression upon Ag exposure and that these, rather than plasma cells, become the major IgM Ab-producing cell population.

Animals↗
PubMed2000

Solution structure of BmP02, a new potassium channel blocker from the venom of the Chinese scorpion Buthus martensi Karsch.

BmP02 is a 28-amino acid residue peptide purified from the venom of the Chinese scorpion Buthus martensi Karsch, which had been demonstrated to be a weak blocker of apamin-sensitive calcium-activated potassium channels. Two-dimensional NMR spectroscopy techniques were used to determine the solution structure of BmP02. The results show that BmP02 formed a alpha/beta scorpion fold, the typical three-dimensional structure adopted by most short chain scorpion toxins whose structures have been determined. However, in BmP02 this alpha/beta fold was largely distorted. The alpha-helix was shortened to only one turn, and the loop connecting the helix to the first beta-strand exhibited conformational heterogeneity. The instability of BmP02 could be attributed to a proline at position 17, which is usually a glycine. Because the residue at this position makes intense contact with the alpha-helix, it was supposed that the bulky side chain of proline had pushed the helix away from the beta-sheet. This had a significant influence on the structure and function of BmP02. The alpha-helix rotated by about 40 degrees to avoid Pro17 while forming two disulfides with the second beta-strand. The rotation further caused both ends of the helix to be unwound due to covalent restrictions. According to its structure, BmP02 was supposed to interact with its target via the side chains of Lys11 and Lys13.

Amino Acid Sequence↗
PubMed2000

Chimeras of Dictyostelium myosin II head and neck domains with Acanthamoeba or chicken smooth muscle myosin II tail domain have greatly increased and unregulated actin-dependent MgATPase activity.

Phosphorylation of the regulatory light chain of Dictyostelium myosin II increases V(max) of its actin-dependent MgATPase activity about 5-fold under normal assay conditions. Under these assay conditions, unphosphorylated chimeric myosins in which the tail domain of the Dictyostelium myosin II heavy chain is replaced by either the tail domain of chicken gizzard smooth muscle or Acanthamoeba myosin II are 20 times more active because of a 10- to 15-fold increase in V(max) and a 2- to 7-fold decrease in apparent K(ATPase) and are only slightly activated by regulatory light chain phosphorylation. Actin-dependent MgATPase activity of the Dictyostelium/Acanthamoeba chimera is not affected by phosphorylation of serine residues in the tail whose phosphorylation completely inactivates wild-type Acanthamoeba myosin II. These results indicate that the actin-dependent MgATPase activity of these myosins involves specific, tightly coupled, interactions between head and tail domains.

Acanthamoeba↗
PubMed2000

Structural basis for signal transduction by the Toll/interleukin-1 receptor domains.

Toll-like receptors (TLRs) and the interleukin-1 receptor superfamily (IL-1Rs) are integral to both innate and adaptive immunity for host defence. These receptors share a conserved cytoplasmic domain, known as the TIR domain. A single-point mutation in the TIR domain of murine TLR4 (Pro712His, the Lps(d) mutation) abolishes the host immune response to lipopolysaccharide (LPS), and mutation of the equivalent residue in TLR2, Pro681His, disrupts signal transduction in response to stimulation by yeast and gram-positive bacteria. Here we report the crystal structures of the TIR domains of human TLR1 and TLR2 and of the Pro681His mutant of TLR2. The structures have a large conserved surface patch that also contains the site of the Lps(d) mutation. Mutagenesis and functional studies confirm that residues in this surface patch are crucial for receptor signalling. The Lps(d) mutation does not disturb the structure of the TIR domain itself. Instead, structural and functional studies indicate that the conserved surface patch may mediate interactions with the down-stream MyD88 adapter molecule, and that the Lps(d) mutation may abolish receptor signalling by disrupting this recruitment.

Amino Acid Sequence↗
PubMed2000

Abnormal contractile function in transgenic mice expressing a familial hypertrophic cardiomyopathy-linked troponin T (I79N) mutation.

This study characterizes a transgenic animal model for the troponin T (TnT) mutation (I79N) associated with familial hypertrophic cardiomyopathy. To study the functional consequences of this mutation, we examined a wild type and two I79N-transgenic mouse lines of human cardiac TnT driven by a murine alpha-myosin heavy chain promoter. Extensive characterization of the transgenic I79N lines compared with wild type and/or nontransgenic mice demonstrated: 1) normal survival and no cardiac hypertrophy even with chronic exercise; 2) large increases in Ca(2+) sensitivity of ATPase activity and force in skinned fibers; 3) a substantial increase in the rate of force activation and an increase in the rate of force relaxation; 4) lower maximal force/cross-sectional area and ATPase activity; 5) loss of sensitivity to pH-induced shifts in the Ca(2+) dependence of force; and 6) computer simulations that reproduced experimental observations and suggested that the I79N mutation decreases the apparent off rate of Ca(2+) from troponin C and increases cross-bridge detachment rate g. Simulations for intact living fibers predict a higher basal contractility, a faster rate of force development, slower relaxation, and increased resting tension in transgenic I79N myocardium compared with transgenic wild type. These mechanisms may contribute to mortality in humans, especially in stimulated contractile states.

Animals↗
PubMed2000

Phosphorylation of murine p53 at ser-18 regulates the p53 responses to DNA damage.

Ser-15 of human p53 (corresponding to Ser-18 of mouse p53) is phosphorylated by ataxia-telangiectasia mutated (ATM) family kinases in response to ionizing radiation (IR) and UV light. To determine the effects of phosphorylation of endogenous murine p53 at Ser-18 on biological responses to DNA damage, we introduced a missense mutation (Ser-18 to Ala) by homologous recombination into both alleles of the endogenous p53 gene in mouse embryonic stem (ES) cells. Our analyses showed that phosphorylation of murine p53 at Ser-18 in response to IR or UV radiation was required for a full p53-mediated response to these DNA damage-inducing agents. In contrast, phosphorylation of p53 at Ser-18 was not required for ATM-dependent cellular resistance after exposure to IR. Additionally, efficient acetylation of the C terminus of p53 in response to DNA damage did not require phosphorylation of murine p53 at Ser-18.

Acetylation↗
PubMed2000

Adenosine: A partial agonist of the growth hormone secretagogue receptor.

The growth hormone secretagogue receptor (GHS-R) is involved in the regulation of pulsatile GH release. However, until recently, natural endogenous ligands for the receptor were unknown. We fractionated porcine hypothalamic extracts and assayed fractions for activity on HEK293 cells expressing GHS-R and aequorin. A partial agonist was isolated and identified using microspray tandem mass spectrometry as adenosine. GHS-R activation by adenosine and synthetic adenosine agonists is inhibited by the GHS-R selective antagonists L-765,867, D-Lys(3)-GHRP-6, and by theophylline and XAC. Cross desensitization of the GHS-R occurs with both MK-0677 and adenosine. Ligand binding and site directed mutagenesis studies show that adenosine binds to a binding site that is distinct from the previously characterized MK-0677 and GHRP-6 binding pocket. We propose, that adenosine is a physiologically important endogenous GHS-R ligand and speculate that GHS-R ligands modulate dopamine release from hypothalamic neurons.

Adenosine↗
PubMed2000

p53 transcriptional activity is essential for p53-dependent apoptosis following DNA damage.

p53-mediated transcription activity is essential for cell cycle arrest, but its importance for apoptosis remains controversial. To address this question, we employed homologous recombination and LoxP/Cre-mediated deletion to produce mutant murine embryonic stem (ES) cells that express p53 with Gln and Ser in place of Leu25 and Trp26, respectively. p53(Gln25Ser26) was stable but did not accumulate after DNA damage; the expression of p21/Waf1 and PERP was not induced, and p53-dependent repression of MAP4 expression was abolished. Therefore, p53(Gln25Ser26) is completely deficient in transcriptional activation and repression activities. After DNA damage by UV radiation, p53(Gln25Ser26) was phosphorylated at Ser18 but was not acetylated at C-terminal sites, and its DNA binding activity did not increase, further supporting a role for p53 acetylation in the activation of sequence-specific DNA binding activity. Most importantly, p53(Gln25Ser26) mouse thymocytes and ES cells, like p53(-/-) cells, did not undergo DNA damage-induced apoptosis. We conclude that the transcriptional activities of p53 are required for p53-dependent apoptosis.

Animals↗
PubMed2000

Functional analyses of positions across the 5' splice site of the trypanosomatid spliced leader RNA. Implications for base-pair interaction with U5 and U6 snRNAs.

In this study, we have used a genetic compensatory approach to examine the functional significance of the previously proposed interaction of spliced leader (SL) RNA with U5 small nuclear RNA (snRNA) (Dungan, J. D., Watkins, K. P., and Agabian, N. (1996) EMBO J. 15, 4016-4029; Xu, Y.-X., Ben Shlomo, H., and Michaeli, S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8473-8478) and the interaction of the SL RNA intron with U6 snRNA analogous to cis-splicing. Mutations were introduced at positions -4, -1, +1, +4, +5, and +7/+8 relative to the SL RNA 5' splice site that were proposed to interact with U5 and U6 snRNAs. All mutants exhibited altered splicing phenotypes compared with the parental strain, showing the importance of these intron and exon positions for trans-splicing. Surprisingly, mutation at invariant +1 position did not abolish splicing completely, unlike cis-splicing, but position +2 had the most severe effect on trans-splicing. Compensatory mutations were introduced in U5 and U6 snRNAs to examine whether the defects resulted from failure to interact with these snRNAs by base pairing. Suppression was observed only for positions +5 and +7/+8 with U5 compensatory mutations and for position +5 with a U6 compensatory mutation, supporting the existence of a base pair interaction of U5 and U6 with the SL RNA intron region. The failure to suppress the other SL RNA mutants by the U5 compensatory mutations suggests that another factor(s) interacts with these key SL RNA positions.

5' Untranslated Regions↗
PubMed2000

Laminin expression in adult and developing retinae: evidence of two novel CNS laminins.

Components of the extracellular matrix exert myriad effects on tissues throughout the body. In particular, the laminins, a family of heterotrimeric extracellular glycoproteins, have been shown to affect tissue development and integrity in such diverse organs as the kidney, lung, skin, and nervous system. Of these, we have focused on the roles that laminins play in the differentiation and maintenance of the nervous system. Here, we examine the expression of all known laminin chains within one component of the CNS, the retina. We find seven laminin chains-alpha3, alpha4, alpha5, beta2, beta3, gamma2, and gamma3-outside the retinal basement membranes. Anatomically, these chains are coexpressed in one or both of two locations: the matrix surrounding photoreceptors and the first synaptic layer where photoreceptors synapse with retinal interneurons. Biochemically, four of these chains are coisolated from retinal extracts in two independent complexes, confirming that two novel heterotrimers-alpha4beta2gamma3 and alpha5beta2gamma3-are present in the retinal matrix. During development, all four of these chains, along with components of laminin 5 (the alpha3, beta3, and gamma2 chains) are also expressed at sites at which they could exert important effects on photoreceptor development. Together, these data suggest the existence of two novel laminin heterotrimers in the CNS, which we term here laminin 14 (composed of the alpha4, beta2, and gamma3 chains) and laminin 15 (composed of the alpha5, beta2, and gamma3 chains), and lead us to hypothesize that these laminins, along with laminin 5, may play roles in photoreceptor production, stability, and synaptic organization.

Adult↗
PubMed2000

Microsatellite instability in inflammatory bowel disease-associated neoplastic lesions is associated with hypermethylation and diminished expression of the DNA mismatch repair gene, hMLH1.

Twelve to 15% of sporadic colorectal cancers display defective DNA mismatch repair (MMR), manifested as microsatellite instability (MSI). In this group of cancers, promoter hypermethylation of the MMR gene hMLH1 is strongly associated with, and believed to be the cause of, MSI. A subset of colorectal neoplastic lesions arising in inflammatory bowel disease (IBD) is also characterized by MSI. We wished to determine whether hMLH1 hypermethylation was associated with diminished hMLH1 protein expression and MSI in IBD neoplasms. We studied 148 patients with IBD neoplasms, defined as carcinoma or dysplasia occurring in patients with ulcerative colitis or Crohn's disease. MSI was evaluated using multiplex fluorescent PCR to amplify loci D2S123, BAT-25, BAT-26, D5S346, and D17S250 in all cases. Lesions were characterized as high-frequency MSI (MSI-H) if they manifested instability at two or more loci, low-frequency MSI (MSI-L) if unstable at only one locus, or MS-stable (MSS) if showing no instability at any loci. Methylation-specific PCR was performed to determine the methylation status of the hMLH1 promoter region. hMLH1 protein expression was also evaluated by immunohistochemistry. Thirteen (9%) of 148 neoplasms arising in IBD were MSI-H, comprising 11 carcinomas and 2 dysplastic lesions. Sixteen additional lesions (11%) were MSI-L, comprising 11 carcinomas and 5 dysplastic lesions. The remaining 118 neoplasms (80%) were MSS. Six (46%) of 13 MSI-H, 1 (6%) of 16 MSI-L, and 4 (15%) of 27 MSS lesions showed hMLH1 hypermethylation (P = 0.013). Diminished hMLH1 protein expression in neoplastic cell nuclei relative to surrounding normal cell nuclei was demonstrated immunohistochemically in four of four (100%) hypermethylated lesions tested. In IBD neoplasia, hMLH1 promoter hypermethylation occurs frequently in the setting of MSI, particularly MSI-H. Furthermore, hMLH1 hypermethylation and MSI are strongly associated with diminished hMLH1 protein expression in IBD neoplasms. These findings suggest that hMLH1 hypermethylation causes defective DNA MMR in at least a subset of IBD neoplasms.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Rates of hippocampal atrophy correlate with change in clinical status in aging and AD.

BACKGROUND: The cognitive continuum in the elderly population can be conceptually divided into those who are functioning normally (control subjects), those with a mild cognitive impairment (MCI), and those with probable AD. OBJECTIVES: To test the hypothesis that the annualized rates of hippocampal atrophy differ as a function of both baseline and change in clinical group membership (control, MCI, or AD). METHODS: The authors identified 129 subjects from the Mayo Clinic AD Research Center/AD Patient Registry who met established criteria for normal control subjects, MCI, or probable AD, both at entry and at the time of a subsequent clinical follow-up evaluation 3 +/- 1 years later. Each subject underwent an MRI examination of the head at the time of the initial assessment and at follow-up clinical assessment; the annualized percentage change in hippocampal volume was computed. Subjects who were classified as controls or patients with MCI at baseline could either remain cognitively stable or could decline to a lower functioning group over the period of observation. RESULTS: The annualized rates of hippocampal volume loss for each of the three initial clinical groups decreased progressively in the following order: AD > MC > control. Within the control and MCI groups, those who declined had a significantly greater rate of volume loss than those who remained clinically stable. The mean annualized rates of hippocampal atrophy by follow-up clinical group were: control-stable 1.73%, control-decliner 2.81%, MCI-stable 2.55%, MCI-decliner 3.69%, AD 3. 5%. CONCLUSION: Rates of hippocampal atrophy match both baseline cognitive status and the change in cognitive status over time in elderly persons who lie along the cognitive continuum from normal to MCI to AD.

Aged↗
PubMed2000

Protein threading using PROSPECT: design and evaluation.

The computer system PROSPECT for the protein fold recognition using the threading method is described and evaluated in this article. For a given target protein sequence and a template structure, PROSPECT guarantees to find a globally optimal threading alignment between the two. The scoring function for a threading alignment employed in PROSPECT consists of four additive terms: i) a mutation term, ii) a singleton fitness term, iii) a pairwise-contact potential term, and iv) alignment gap penalties. The current version of PROSPECT considers pair contacts only between core (alpha-helix or beta-strand) residues and alignment gaps only in loop regions. PROSPECT finds a globally optimal threading efficiently when pairwise contacts are considered only between residues that are spatially close (7 A or less between the C(beta) atoms in the current implementation). On a test set consisting of 137 pairs of target-template proteins, each pair being from the same superfamily and having sequence identity </= 30%, PROSPECT recognizes 69% of the templates correctly and aligns 66% of the structurally alignable residues correctly. These numbers may be compared with the 55% fold recognition and 64% alignment accuracy for the same test set using only scoring terms i), ii), and (iv), indicating the significant contribution from the contact term. The fold recognition and alignment accuracy are further improved to 72% and 74%, respectively, when the secondary structure information predicted by the PHD program is used in scoring. PROSPECT also allows a user to incorporate constraints about a target protein, e.g., disulfide bonds, active sites, and NOE distance restraints, into the threading process. The system rigorously finds a globally optimal threading under the specified constraints. Test results have shown that the constraints can further improve the performance of PROSPECT. Proteins 2000;40:343-354.

Algorithms↗
PubMed2000

Human monoclonal antibody 98-6 reacts with the fusogenic form of gp41.

A mixture of two peptides from gp41 (N36 and C34) forms an alpha-helical structure that is thought to represent the fusogenic form of gp41. A human anti-gp41 monoclonal antibody (mAb 98-6), generated from the cells of an infected individual, reacted poorly with C34, but binding was strongly enhanced when N36 was added, indicating that the mAb reacts with a conformational epitope present in the fusogenic structure formed by the interaction of peptides N36 and C34. The epitope recognized by mAb 98-6 was found in lysates of virions on oligomeric forms of gp41 (dimers, trimers, and tetramers). On infected cells, the epitope was present as oligomers of gp41, as monomers of gp41, and as part of the envelope polyprotein gp160, obtained after biotinylation of intact cells, which were then lysed and immunoprecipitated with various mAbs. In lysates of infected cells, the epitope was present as part of both monomeric gp41 and gp160. These studies demonstrate that infected humans can respond to the fusogenic form of gp41 and that the anti-gp41 mAb studied here recognizes a conformational epitope formed by the interaction of two regions of gp41, which forms an alpha-helical bundle. This epitope is found on several forms of gp41 as it occurs in virions, on the surface of infected cells, and in infected cells.

Antibodies, Monoclonal↗