Biomedical subjects

Y Xiao

Publications and source records attributed to Y Xiao.

At least 19 recordsLinked to original sources

PubMed1999

Membrane topography of the renal phosphate carrier NaPi-2: limited proteolysis studies.

The rat sodium/phosphate cotransporter NaPi-2 is a 70 kDa polypeptide (p70) for which eight transmembrane segments have been predicted. We have shown that p70 exists predominantly as p45 and p40 fragments which are linked by disulfide bonds. In this work, the p40 fragment, corresponding to the C-terminus of NaPi-2, was purified from renal brush-border membranes using non-reducing and then reducing column electrophoresis followed by enzymatic deglycosylation and SDS-PAGE. The N-terminal sequence obtained for this fragment, VEAIG, indicates that the formation of p45 and p40 arises from the cleavage of p70 between arginine-319 and valine-320. In order to determine the membrane topography of NaPi-2, brush-border membrane vesicles were digested with various proteases and the transporter-derived proteolytic peptides were subsequently identified by Western blotting using N- and C-terminal-directed antibodies. Our results lead us to propose an alternative topographical model in which p45 and p40 possess three transmembrane domains each and indicate that the processing site of p70 for the generation of p45 and p40 is localized in a large protein core facing the extracellular milieu. This localization of the cleavage site indicated that NaPi-2 could either be processed intracellularly by vesicular proteases or extracellularly by secretory proteases or by brush-border membrane ectoenzymes.

Animals↗

PubMed1999

Liquid chromatographic studies with immobilized neuronal nicotinic acetylcholine receptor stationary phases: effects of receptor subtypes, pH and ionic strength on drug-receptor interactions.

Nicotinic acetylcholine receptor (nAChR) alpha3-subunits, beta4-subunits, alpha3/beta4-subunit combination and alpha4/beta2-subunit combination were immobilized on chromatographic stationary phases and the binding affinities of the different nAChR subtypes were chromatographically evaluated. The observed relative binding affinities of epibatidine were alpha4/beta2>alpha3/beta4 and epibatidine did not bind at alpha3-subunits and beta4-subunits. No significant difference in binding affinities was observed on the alpha4/beta2 nAChRs immobilized in immobilized artificial membrane (IAM) particles and those sterically immobilized on Superdex 200 beads. The effects of mobile phase pH and ionic strength on the binding affinities of the alpha3/beta4 nAChRs support were also investigated. The results are consistent with the proposed ligand-nAChR binding model in which a cationic center exists at the binding site.

Animals↗

PubMed1999

Delineation of a membrane-proximal beta-rich domain in the GABAA receptor by progressive deletions.

The type A gamma-aminobutyric acid (GABAA) receptor plays a major inhibitory role in the central nervous system. Structural elucidation of the GABAA receptor has been impeded by the large size of the receptor. We present here the delineation of a minimal structural domain as the first step of dissecting the receptor structure. This was achieved through prediction-assisted progressive deletions: the prediction of a candidate structural domain rich in beta-strands with no close similarity to known structures was tested by deleting putative secondary structure elements from the ends of the proposed domain, as well as mutations within the terminal secondary structures. Such progressive deletions revealed the limits of an integral domain, spanning Cys180 to Met293 (numbering of human alpha1 subunit). Below these limits the intact domain structure, as indicated by its circular dichroism, collapses. Based on its putative position, this domain is provisionally designated the membrane-proximal beta-rich domain of GABAA receptor. The inclusion of sequences from the first two out of four previously suggested transmembrane segments and one of the two conserved Cys residues in this domain defines important constraints to the receptor structure.

Amino Acid Sequence↗

PubMed1999

Epistatic interactions of deletion mutants in the genes encoding the F1-ATPase in yeast Saccharomyces cerevisiae.

The F1-ATPase is a multimeric enzyme (alpha3 beta3 gamma delta epsilon) primarily responsible for the synthesis of ATP under aerobic conditions. The entire coding region of each of the genes was deleted separately in yeast, providing five null mutant strains. Strains with a deletion in the genes encoding alpha-, beta-, gamma- or delta-subunits were unable to grow, while the strain with a null mutation in epsilon was able to grow slowly on medium containing glycerol as the carbon source. In addition, strains with a null mutation in gamma or delta became 100% rho0/rho- and the strain with the null mutation in gamma grew much more slowly on medium containing glucose. These additional phenotypes were not observed in strains with the double mutations: Delta alpha Delta gamma, Delta beta Delta gamma, Deltaatp11 Delta gamma, Delta alpha Delta delta, Delta beta Delta delta or Deltaatp11 Delta delta. These results indicate that epsilon is not an essential component of the ATP synthase and that mutations in the genes encoding the alpha- and beta-subunits and in ATP11 are epistatic to null mutations in the genes encoding the gamma- and delta-subunits. These data suggest that the propensity to form rho0/rho- mutations in the gamma and delta null deletion mutant stains and the slow growing phenotypes of the null gamma mutant strain are due to the assembly of F1 deficient in the corresponding subunit. These results have profound implications for the physiology of normal cells.

Epistasis, Genetic↗

PubMed1999

Maturational changes in gene expression for adenine nucleotide translocator isoforms and betaF1-ATPase in rabbit heart.

Maturational changes in myocardial respiratory control have been related to postnatal accumulation of adenine nucleotide translocator (ANT) in the inner mitochondrial membrane. Alternatively alterations in relative isoform distribution for this nuclear-encoded gene during myocardial maturation might be responsible for changing the kinetics of respiratory control. Rabbit hearts were analyzed for adenine nucleotide translocator isoform (ANT1, ANT2, ANT3) gene expression and distribution at four ages (fetal, 29/31 days of gestation; 1 h postnatal; 9 days postnatal; and 3-4 months postnatal). Transcript levels for the coordinately expressed betaF1-ATPase were also examined in these hearts. These studies demonstrated that mRNA expression for ANT1 in coordination with betaF1-ATPase increased substantially after 9 days of age in rabbit hearts. Expression of the minor isoform ANT3 parallels ANT1, though no change in expression of the kidney-specific isoform ANT2 occurs in heart during this developmental period. Previous work has demonstrated that ANT protein accumulation is closely coordinated with mRNA expression for ANT1. These results support previous studies, which indicate that the operational mode of myocardial respiratory control depends on adenine nucleotide mRNA expression. Changes in relative adenine nucleotide translocator isoform distribution do occur during fetal to mature transition and may contribute to observed changes in the mode of respiratory control.

Aging↗

PubMed1999

Heterogeneity of Chinese hamster X chromosomes in X-ray-induced chromosomal aberrations.

PURPOSE: To study the frequencies and distribution of X-ray-induced chromosomal aberrations in different arms as well as different regions of the long arms of Chinese hamster X chromosomes by fluorescence in situ hybridization (FISH). MATERIAL AND METHODS: Female embryonic primary cells of Chinese hamster were exposed to 1 and 4 Gy X-rays during the G1 stage and metaphases were collected 20 h later with colcemid-blocking. Induced aberrations involving different arms as well as different regions of the long arm of the X chromosomes were analysed by three-colour FISH using arm-specific painting probes. Distributions of aberrations in the arms were statistically analysed by chi2-test, with the hypothesis that aberrations are proportionally distributed on the basis of their relative lengths of the arms. RESULTS: The long arms of the X chromosomes were frequently involved in breaks than was the short arm. The result of the chi2-test indicates a non-proportional distribution of breaks between the arms, while exchanges (dicentrics and translocations) involving the arms were proportionally distributed. Differential involvement of regions of the long arm, i.e. Xq1 and Xq2, in breaks was also observed. Xq21, a known common fragile site in the X chromosome, was often involved in terminal deletions. CONCLUSION: Arm-specific probes of Chinese hamster chromosomes are useful for the detailed study of X-ray-induced aberrations in the X chromosome. The heterogeneity of the Chinese hamster X chromosome in response to X-ray-induced aberrations exists not only between the short (euchromatin) and the long (heterochromatin) arms, but also between different heterochromatic regions of the long arm of the X chromosome.

Animals↗

PubMed1999

Comparing two methods for calculating phantom scatter.

Two methods are compared for calculating the field-size dependence of the phantom scatter component of dose for x-ray beams. One model sums three Gaussian distributions; the other model is a two-parameter function. With a measurement of the beam quality as input to determine parameters, both models accurately reproduce the relative phantom scatter. However, there are important differences between the models. For all beam energies, the two-parameter model characterizes the absolute phantom scatter as a function of depth and field size, while, also for all beam energies, the six-parameter Gaussian model characterizes the relative phantom scatter at a single depth of 10 cm. For small field sizes, the phantom scatter calculated from the two-parameter model agrees with Monte Carlo calculations better than the Gaussian model. In the Gaussian model, the parameters can be obtained for beam energies between 60Co and 25 MV by linear interpolation based on the measured beam quality. In the two-parameter model, and for energies above 4 MV, the parameters can be obtained using linear functions of the dose-weighted average linear attenuation coefficient, which is related to beam quality.

Calibration↗

PubMed1999

Equivalent fields and scatter integration for photon fields.

Equivalent fields are often used in radiation oncology for calculation of dose. This avoids the need to make a scatter integration but has limited applicability and some inaccuracy. We have evaluated the alternative of explicit integration of phantom-scatter dose using a functional representation, sigma, of the ratio between the scatter dose and the primary dose. The independent parameters are depth and the side of the square field. The function chosen has the advantage that the integral for a right triangle is available in closed form, which simplifies the determination of the dose from phantom-scattered photons for irregular fields by summation over such triangles. This approach accounts for the influence of depth and beam quality, which the commonly used equivalent-field tables and the area-over-perimeter relation ignore. The accuracy of this procedure is determined by the accuracy of the function sigma. This has about a 1% error of total dose for high-energy x-rays. We conclude that the tables and rules can be replaced by a computer-implemented integration of the phantom-scatter dose represented by this function sigma and using sectors or right triangles. Summing the closed-form contributions from component right triangles reduces the calculation time, which is particularly desirable when many fields are employed, as for intensity-modulated techniques and inverse planning. Measurements performed on irregular MLC-shaped fields compared well with the result from calculations.

Models, Statistical↗

PubMed1999

Activation and Ca2+ permeation of stably transfected alpha3/beta4 neuronal nicotinic acetylcholine receptor.

The alpha3/beta4 rat neuronal nicotinic acetylcholine receptor, stably transfected in human embryonic kidney cells, was examined using the whole-cell-clamp technique and 2-dimensional confocal imaging. Application of agonists (nicotine, cytisine, epibatidine) activated a large (100-200 pA/pF) inwardly rectifying monovalent current, with little current at voltages between 0 and +40 mV. Rapid application of nicotine and cytisine indicated EC50 values of congruent with22 and congruent with64 microM, respectively, and suggested second order binding kinetics (Hill coefficient approximately 2). The time constant of desensitization (decay) of nicotine-activated current was concentration-dependent (typically approximately 10 s at 30 microM versus approximately 1.0 s at 100-1000 microM), but not voltage-dependent and was significantly smaller than the approximately 200 s reported for the alpha3/beta4 receptor expressed in Xenopus oocytes. Nicotine-activated current was rapidly and reversibly blocked by coapplication of mecamylamine and d-tubocurarine. At -80 mV holding potentials, the current was also suppressed by approximately 25% either upon complete removal or elevation of Ca2+ to 10 mM. Total replacement of Na+ by Ca2+ also completely blocked the current. On the other hand, evidence for permeation of Ca2+ was indicated by increased inward current at -40 mV upon elevation of Ca2+ from 2 to 10 mM, as well as a rise in the cytosolic Ca2+ proportional to the current carried by the receptor. These findings are consistent with the idea that Ca2+, in addition to its channel-permeating properties, may also regulate the receptor from an extracellular site. Our results suggest that the alpha3/beta4 neuronal nicotinic acetylcholine receptor, when stably expressed in human embryonic kidney 293 cells, has desensitization kinetics and Ca2+ regulatory mechanisms somewhat different from those described for the receptor expressed in Xenopus oocytes.

Animals↗

PubMed1998

Immobilized nicotinic receptor stationary phase for on-line liquid chromatographic determination of drug-receptor affinities.

Nicotinic acetylcholine receptors (nAChR) are ligand-gated ion channels which mediate nicotinic cholinergic transmission in the nervous system. A nAChR subtype composed of alpha3 and beta4 subunits (alpha3/beta4 subtype), prepared from a stably transfected KXalpha3beta4R2 cell line, has been immobilized in the phospholipid monolayer of an immobilized artificial membrane (IAM) liquid chromatography (LC) stationary phase. Approximately 60 mg of protein was immobilized per gram IAM particles. The resulting phase was used for the rapid on-line chromatographic determination of drug binding affinities to nAChRs. Relative binding affinities were determined by frontal chromatography for (+/-)-epibatidine (Kd: 0.27 +/- 0.05 nM) > A85380 (Kd: 17.2 +/- 0.5 nM) > (-)-nicotine (Kd: 88 +/- 33 nM) > carbachol (Kd: 1280 +/- 30 nM) > atropine (Kd: 14,570 +/- 2600 nM). These results are consistent with the affinity rank order obtained from binding assays using membrane homogenates. The immobilized receptor LC stationary phase was stable and reproducible. This approach opens up the possibility of the production of a variety of immobilized receptor LC stationary phases which may be used for direct determination of drug-receptor binding interactions and for the rapid on-line screening of combinatorial pools for drug candidates.

Animals↗

PubMed1998

Demonstration of a tandem pair of complement protein modules in GABA(B) receptor 1a.

We have subcloned and expressed the N-terminal portion of the recently sequenced metabotropic GABA receptor, GABA(B)R1a. This region of the receptor contains a complement protein-like amino acid sequence. The purified 140-residue recombinant protein fragment was soluble and stable. Mass spectrometry indicated formation of four disulfide bonds, as expected if two complement protein modules (CPs, also known as SCRs, Sushi domains) are formed. The circular dichroism spectrum was unusual and characteristic of CPs. Differential scanning calorimetry demonstrated a melting point (64 degrees C), and total enthalpy commensurate with two fully folded domains. We thus conclude that the 1a subtype of the GABA(B) receptor, but not the 1b subtype, contains a pair of CPs and we present a three-dimensional model of this region.

Amino Acid Sequence↗

PubMed1998

[Detection of minimal residual disease in acute lymphoblastic leukemia using PCR-molecule hybridization of RNA transcripts].

OBJECTIVE: To detect acute lymphoblastic leukemia's(ALL's) minimal residual disease(MRD) rapidly and effectively. METHODS: In this assay, the gamma T-cell receptor gene rearrangements serve as marker genes. The gene rearrangements are amplified from the diagnostic specimens using a consensus V segment primer and a consensus J segment primer to which the promoter T7 RNA polymerase has been appended. The PCR product from this amplification is transcribed into a radiolabeled RNA probe. The opposite DNA strand is transcribed into test RNA from the PCR product of different staged specimens. The test RNA is hybridized with the probe, and later the digestion with RNase A, Polyacrylamide gel electrophoresis and autoradiography are in progress. RESULTS: According to the mechanism, the perfectly matched RNA duplex can prevent the digestion of RNase A, and the presence of the leukemia cells in the test specimen can be determined. Logarithmical dilution experiments with DNA of a cell line from ALL have shown that this assay's sensitivity is at the 10(-5) level. Minimal residual disease was successfully detected in a case of ALL during its complete remission stage. But if the probe and test RNA are not from the same individual, the results of this kind of cross hybridization are negative. CONCLUSION: The above results suggest that this assay can become an effective measure in the detection of ALL-MRD clinically.

Child↗

PubMed1998

[Sequencing analysis of human K-ras oncogene exon 1 in Chinese people].

OBJECTIVE: To investigate the mutation of human K-ras oncogene in the blood of patients with lung cancer. METHODS: PCR and sequencing methods were used to analyse the exon 1. Twelve patients'blood samples and 31 normals' blood samples were detected. RESULTS: In all 43 samples, the K-ras codon 11 was CCT (proline), and the opposite strand sequenced was AGG. It was different from the standard GCT(Alanine) sequenced by foreign authors. CONCLUSION: The 11th codon of the human K-ras oncogene in Chinese is CCT (Proline). As the K-ras codon 12 is a hot spot, this finding may be of importance to clinical genetic diagnosis.

Asian People↗

PubMed1998

Temperature threshold and modulation of energy metabolism in the cardioplegic arrested rabbit heart.

Hypothermia protects ischemic tissues by reducing ATP utilization and accumulation of harmful metabolites. However, it also reduces ATP production, which might cause deterioration in the energy supply/demand ratio. Modulation of energy supply/demand according to temperature has not been previously studied in detail. In this study, isolated, perfused rabbit hearts (n = 60) were used to determine the effects of various temperatures on myocardial energy metabolism and function during cardioplegic arrest. Ischemia was induced by crystalloid cardioplegic solution at 4, 18, 30, and 34 degrees C for 120 min, respectively. At each temperature, the hearts were divided into a glucose-treated group which contained 22 mM glucose in cardioplegic solution as the only substrate and a control group which contained 22 mM mannitol to keep same osmolarity. Following 15 min reperfusion, recovery of left ventricular developed pressure (DP), +/- dP/dtmax, and the product of heart rate and DP were significantly higher in 30, 18, and 4 degrees C groups than those in 34 degrees C control group. The functional recovery was also significantly higher in the 34 degrees C glucose-treated group than that in the 34 degrees C control group, but there was no difference between those groups at 30 degrees C and the temperature below 30 degrees C. Myocardial ATP concentration was significantly lower in 34 degrees C control group than those in other groups. There is a close relationship between myocardial ATP concentration and functional recovery (R2 = 0.90). The accumulations of lactate and CO2 were significantly higher at 34 degrees C in glucose-treated group than those in the control group. However, there was no significant difference between these two groups at 30 degrees C and the temperature below 30 degrees C. These results indicate that under these study conditions: (1) a marked decrease in energy supply/demand occurs above 30 degrees C, implying that a temperature threshold exists; and (2) this can be ameliorated by provision of glucose as substrate in cardioplegia solution.

Adenosine Triphosphate↗

PubMed1998

Temperature threshold and preservation of signaling for mitochondrial membrane proteins during ischemia in rabbit heart.

Temperature modulates both myocardial energy requirements and production. We have previously demonstrated that myocardial protection induced by hypothermic adaptation preserves expression of genes regulating heat shock protein and the nuclear-encoded mitochondrial proteins, the adenine nucleotide translocator isoform 1 (ANT1), and the beta subunit of F1-ATPase (beta F1-ATPase). This preservation is associated with a reduction in ATP depletion similar to that noted in cardioplegic arrested hearts preserved at a critical temperature (30 degrees C) or below. We tested the hypothesis that expression of these genes may also be subject to this temperature threshold phenomenon. Isolated perfused rabbit hearts were subjected to ischemic cardioplegic arrest at 4, 30, or 34 degrees C for 120 min. Cardiac function indices and steady-state mRNA levels for ANT1, beta F1-ATPase, and HSP70-1 were measured prior to ischemia (B) and after 45 min of reperfusion. Cardiac function was significantly depressed in the 34 degrees C group. Ischemia at 34 degrees C reduced steady-state mRNA levels for ANT1 and beta F1-ATPase from B, but these levels were similarly preserved at 4 and 30 degrees C. HSP70-1 levels were mildly elevated (fourfold) above B to similar levels at all three temperatures. These results indicate that mRNA expression for ANT1 and beta F1-ATPase is specifically preserved in a pattern consistent with the temperature threshold phenomenon. HSP70-1 expression is not influenced by ischemic temperature. Preservation of gene expression for these mitochondrial proteins implies that signaling for mitochondrial biogenesis or resynthesis is maintained after ischemic insult.

Animals↗

PubMed1998

Aging and diethylstilbestrol-induced aneuploidy in male germ cells: a transgenic mouse model.

The incidence of aneuploidy in male germ cells was evaluated by analyzing extra marker chromosome(s) signal(s) in round and/or hook spermatids of transgenic mice. Two types of transgenic mice were used as models. The inserted foreign DNA (lambda-gt10LacZ shuttle vector and/or pSVc-myc plasmid) was located at the middle of the long arms of chromosome 2 (lambda DNA) and/or chromosome 8 (c-myc). The number of marker chromosomes present could easily be detected after fluorescence in situ hybridization (FISH) in testicular cells. The frequency of spontaneous aneuploidy of chromosome 2 was similar in round spermatids of lambda and lambda-myc mice. Differential involvement of chromosomes 2 and 8 was observed in both round and hook spermatids. The frequency of spontaneous aneuploidy in round spermatids was higher than that in hook spermatids. The frequency of aneuploidy of marker chromosomes was significantly higher in older mice (2 years old) than in younger ones. Diethylstilbestrol (DES)-induced aneuploidy was dose dependent, and was not influenced by the stage at which germ cells were treated with DES. These results demonstrate the usefulness of a transgenic mouse model for the study of aneuploidy in germ cells.

Aging↗

PubMed1998

Antigenic characterization of HIV-1 gp41 binding proteins.

The envelope protein HIV-1 gp41 has been shown to exert various effects on human T-cells, B-cells and monocytes like inhibition of cell proliferation, modulation of MHC expression and cytokine production. In contrast to gpl20, where several receptor molecules have been identified, the receptor for gp41 is still unknown. Using a sepharose column, coupled with recombinant soluble gp41, (rsgp41; Env amino acids 539-684), five gp41-binding proteins of 37, 45, 50, 62 and 100 kDa had been isolated from lysates of the B-cell line Raji. Two mouse antiserums were generated against the proteins P45 and P62 and were tested against the binding specificity of both antiserums. In Western blot analysis the antiserums recognized two protein bands of 45 and 62 kDa in complete Raji cell lysates, as well as the purified proteins P45 and P62, respectively, but did not show any cross-reaction, indicating that the two proteins do not share any immunological epitopes. Besides, the polyclonal antiserums did not recognize the other gp41-binding proteins P37, P50 and P100. Using the P62 antiserum proteins of the same size as in Raji cell lysates were stained in the lysates of the monocytic cell line U937 and the T-cell line H9, demonstrating distribution of P62 in different blood cells. P45 seems not to be identical to HLA-C which had been shown to bind to gp41. These results indicate, that P45 and P62 are two separate gp41-binding proteins without homology to each other or to the other gp41-binding proteins.

Animals↗

PubMed1998

Search for human T-lymphotropic virus type I carriers among northeastern Chinese.

Human T-lymphotropic virus type I (HTLV-I) is thought to be the causative agent of adult T-cell leukemia/ lymphoma (ATL). This virus infection is endemic in southwestern Japan, parts of Africa and the Caribbean Islands. We examined sera of 1645 subjects of Liaoning province, northeastern China to detect HTLV-I carriers in an effort to reveal the migratory route taken by the early Japanese (Jomon people). As a result, all sera were found to be negative as tested by particle-agglutination (PA), immunofluorescence (IF), enzyme-linked immunoabsorbent (ELISA) and Western blotting methods. This suggests that the Jomon people, who are thought to have brought HTLV-I to the Japan archipelago tens of thousands of years ago, did not come from northeast China.

Adult↗