Biomedical subjects

W Xiao

Publications and source records attributed to W Xiao.

At least 37 records · Page 2Linked to original sources

PubMed2001

Mutation detection by capillary denaturing high-performance liquid chromatography using monolithic columns.

The high resolving power of the chromatographic separation of single- and double-stranded nucleic acids in 200 microm i.d. monolithic poly(styrene-divinylbenzene) capillary columns was utilized for mutation screening in polymerase chain reaction amplified polymorphic loci. Recognition of mutations is based on the separation of homo- and heteroduplex species by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) under partially denaturing conditions, resulting in characteristic peak patterns both for homozygous and heterozygous samples. Six different single nucleotide substitutions and combinations thereof were confidently identified in 413 bp amplicons from six heterozygous individuals each of which yielded a different unique chromatographic profile. Alternatively, mutations were identified in short, 62 bp PCR products upon their complete on-line denaturation at 75 degrees C taking advantage of the ability of IP-RP-HPLC to resolve single-stranded nucleic acids of identical length that differ in a single nucleotide. Separations in monolithic capillary columns can be readily hyphenated to electrospray ionization mass spectrometry and promise increased sample throughput by operating in arrays similar to those already used in capillary electrophoresis.

Alleles↗

PubMed2001

Denaturing high-performance liquid chromatography: A review.

Denaturing high-performance liquid chromatography (DHPLC) compares two or more chromosomes as a mixture of denatured and reannealed PCR amplicons, revealing the presence of a mutation by the differential retention of homo- and heteroduplex DNA on reversed-phase chromatography supports under partial denaturation. Temperature determines sensitivity, and its optimum can be predicted by computation. Single-nucleotide substitutions, deletions, and insertions have been detected successfully by on-line UV or fluorescence monitoring within 2-3 minutes in unpurified amplicons as large as 1.5 Kb. Sensitivity and specificity of DHPLC consistently exceed 96%. These features and its low cost make DHPLC one of the most powerful tools for the re-sequencing of the human and other genomes. Aside from its application to the mutational analysis of candidate genes, DHPLC has proven instrumental in elucidating human evolution and in the mapping of genes. Employing completely denaturing conditions, the utility of DHPLC has been extended to the genotyping of known polymorphisms by utilizing the ability of poly(styrene-divinylbenzene) to resolve single-stranded DNA molecules of identical size that differ in a single base. Under completely denaturing conditions, it is thus possible to resolve all possible base substitutions with the single exception of C-->G transversions. Improvements in throughput became feasible with the recent introduction of monolithic poly(styrene-divinylbenzene) capillaries that lend themselves to the fabrication of arrays connected to a multi-color laser induced fluorescence scanner or a mass spectrometer.

Base Sequence↗

PubMed2001

Molecular systematics of Xenocyprinae (teleostei: cyprinidae): taxonomy, biogeography, and coevolution of a special group restricted in East Asia.

We surveyed mitochondrial DNA (mtDNA) sequence variation in the subfamily Xenocyprinae from China and used these data to estimate intraspecific, interspecific, and intergeneric phylogeny and assess biogeographic scenarios underlying the geographic structure of lineages. We sequenced 1140 bp of cytochrome b from 30 individuals of Xenocyprinae and one putative outgroup (Myxocypris asiaticus) and also sequenced 297 bp of ND4L, 1380 bp of ND4, 68 bp of tRNA(His), and 69 bp of tRNA(Ser) from 17 individuals of Xenocyprinae and the outgroup (M. asiaticus). We detected high levels of nucleotide variation among populations, species, and genera. The phylogenetic analysis suggested that Distoechodon hupeinensis might be transferred to the genus Xenocypris, the taxonomic status of the genus Plagiognathops might be preserved, and species of Xenocypris and Plagiognathops form a monophyletic group that is sister to the genus Distoechodon and Pseudobrama. The introgressive hybridization might occur among the populations of X. argentea and X. davidi, causing the two species to not be separated by mtDNA patterns according to their species identification, and the process and direction of hybridization are discussed. The spatial distributions of mtDNA lineages among populations of Xenocypris were compatible with the major geographic region, which indicated that the relationship between Hubei + Hunan and Fujian is closer than that between Hubei + Hunan and Sichuan. From a perspective of parasite investigation, our data suggested that the fauna of Hexamita in Xenocyprinae could be used to infer the phylogeny of their hosts.

Animals↗

PubMed2001

Two alternative cell cycle checkpoint pathways differentially control DNA damage-dependent induction of MAG1 and DDI1 expression in yeast.

Eukaryotic cells respond to DNA damage by activating damage checkpoint pathways, which arrest cell cycle progression and induce gene expression. In order to understand how damage checkpoints control the expression of DNA damage-inducible genes, the transcript level of two closely clustered genes, MAG1 and DDI1, was examined in a number of checkpoint mutants. We previously reported that MAG1 induction was abolished in pol2 and rad53 mutants, but not in the mec1-1 mutant. In this study, we found that mec1Delta and dun1Delta null mutants were defective in MAG1 induction, suggesting that MAG1 shares a common regulatory pathway with the RNR1,2,3,4 genes, which are also regulated by the POL2-MEC1-RAD53-DUN1 checkpoint pathway, and that the mec1-1 mutation probably represents a separation-of-function mutation. However, MAG1 is not activated in precisely the same way as the RNR genes, since mutations in CRT1, TUP1 and SSN6, which encode repressors of RNR genes, did not affect basal or induced expression of MAG1. In contrast, the DDI1 transcript level was not affected by any of the above checkpoint mutations. Interestingly, simultaneous inactivation of RAD53 or DUN1 with PDS1, a newly identified checkpoint gene, resulted in severe down-regulation of DDI1 expression, suggesting that DDI1 is controlled by two damage checkpoint pathways, one mediated by POL2-MEC1-RAD53-DUN1 and the other by CHK1-PDS1. On the other hand, deletion of TEL1, a structural homologue of MEC1, did not affect expression of MAG1, DDI1 or RNR3, suggesting that TEL1 plays no role in induction by DNA damage. Based on these and previous studies, we present a model for the role of checkpoint genes in transcriptional regulation in response to DNA damage.

Blotting, Northern↗

PubMed2001

The neuronal t-SNARE complex is a parallel four-helix bundle.

Assembly of the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) complex is an essential step for neurotransmitter release in synapses. The presynaptic plasma membrane associated proteins (t-SNAREs), SNAP-25 (synaptosome-associated protein of 25,000 Da) and syntaxin 1A may form an intermediate complex that later binds to vesicle-associated membrane protein 2 (VAMP2). Using spin labeling electron paramagnetic resonance (EPR), we found that the two t-SNARE proteins assemble into a parallel four-helix bundle that consists of two identical syntaxin 1A components and the N-terminal and C-terminal domains of SNAP-25. Although the structure is generally similar to that of the final SNARE complex, the middle region of the helical bundle appears more flexible in the t-SNARE complex. Such flexibility might facilitate interactions between VAMP2 and the t-SNARE complex.

Electron Spin Resonance Spectroscopy↗

PubMed2001

Crystal structure of the human ubiquitin conjugating enzyme complex, hMms2-hUbc13.

The ubiquitin conjugating enzyme complex Mms2-Ubc13 plays a key role in post-replicative DNA repair in yeast and the NF-kappaB signal transduction pathway in humans. This complex assembles novel polyubiquitin chains onto yet uncharacterized protein targets. Here we report the crystal structure of a complex between hMms2 (Uev1) and hUbc13 at 1.85 A resolution and a structure of free hMms2 at 1.9 A resolution. These structures reveal that the hMms2 monomer undergoes a localized conformational change upon interaction with hUbc13. The nature of the interface provides a physical basis for the preference of Mms2 for Ubc13 as a partner over a variety of other structurally similar ubiquitin-conjugating enzymes. The structure of the hMms2-hUbc13 complex provides the conceptual foundation for understanding the mechanism of Lys 63 multiubiquitin chain assembly and for its interactions with the RING finger proteins Rad5 and Traf6.

Adenosine Triphosphatases↗

PubMed2001

Synchronous fluorescence and absorbance dynamic liquid drop sensor for cr(VI) determination at the femtomole level.

A novel synchronous fluorimetric and absorptiometric technique based on dynamic liquid drops coupled with flow injection is described for the determination of chromium(VI) in aqueous solutions. Drops formed continuously at the end of a quartz capillary tube serve as reactor and optical cell. The reaction between chromium(VI) and 3,3',5,5'-tetramethylbenzidine dichloride (TMB-d) results in a significant decrease in fluorescence and transmitted light intensity, proportional to the logarithm of the concentration of chromium(VI). The transmitted light and fluorescence emission are detected in counter and perpendicular positions of the excitation light by a photomultipler tube and photodiode, respectively. In HCl-NaOAc buffer solution (pH 3.29), the sensor has a wide linear calibration range of 2 x 10(-8)-1 x 10(-4) mol l(-1) chromium(VI) with an absolute mass detection limit of 12.88 fmol.

Calibration↗

PubMed2001

The minimum alveolar concentration of enflurane for laryngeal mask airway extubation in deeply anesthetized children.

UNLABELLED: The end-tidal anesthetic gas concentration required to prevent the anesthetized patient from coughing or moving during or immediately after the laryngeal mask airway (LMA) extubation is not known. We sought to determine the minimum alveolar concentration of enflurane required for the removal of the LMA in children. We studied 21 nonpremedicated children between 4 and 11 yr of age, ASA physical status I, undergoing procedures below the umbilicus. General anesthesia was induced with a mask by using sevoflurane, nitrous oxide, and oxygen, and the LMA was inserted. Anesthesia was maintained with enflurane, nitrous oxide, and oxygen. At the end of surgery, a predetermined end-tidal enflurane concentration was achieved, and the LMA was removed. Each concentration at which the LMA extubation was attempted was predetermined by the up-and-down method (with 0.1% as a step size). When LMA removal was accomplished without coughing, clenching teeth, or gross purposeful muscular movements during or within 1 min after removal, it was considered a successful LMA removal. Removal was considered to be unsuccessful in patients who developed breath holding or laryngospasm during or immediately after LMA removal. The minimum alveolar concentration of enflurane at which 50% of children had a successful LMA removal was found to be 1.02% (95% CL, 0.95%-1.11%), and the 95% effective dose for successful extubation was 1.14% (95% CL, 1.07%-1.66%). In conclusion, the LMA removal may be accomplished without coughing or moving at 1.02% end-tidal enflurane concentration in 50% of anesthetized children aged 4-11 yr. IMPLICATIONS: There may be fewer problems associated with the laryngeal mask airway extubation when patients are deeply anesthetized. The purpose of this study was to determine the minimum concentration of enflurane for successful removal of the laryngeal mask in children.

Anesthesia, Inhalation↗

PubMed2001

Temperature-modulated array high-performance liquid chromatography.

Using novel monolithic poly(styrene-divinylbenzene) capillary columns with an internal diameter of 0.2 mm, we demonstrate for the first time the feasibility of constructing high-performance liquid chromatography arrays for the detection of mutations by heteroduplex analysis under partially denaturing conditions. In one embodiment, such an array can be used to analyze one sample simultaneously at different temperatures to maximize the detection of mutations in DNA fragments containing multiple discrete melting domains. Alternatively, one may inject different samples onto columns kept at the same effective temperature. Further improvements in throughput can be obtained by means of laser-induced fluorescence detection and the differential labeling of samples with up to four different fluorophores. Major advantages of monolithic capillary high-performance liquid chromatographic arrays over their capillary electrophoretic analogs are the chemical inertness of the poly(styrene-divinylbenzene) stationary phase, the physical robustness of the column bed due to its covalent linkage to the inner surface of the fused silica capillary, and the feasibility to modify the stationary phase thereby allowing the separation of compounds not only on the principle of size exclusion, but also adsorption, distribution, and ion exchange. Analyses times are on the order of a few minutes and turnaround time is extremely short as there is no need for the replenishment of the separation matrix between runs.

Chromatography, High Pressure Liquid↗

PubMed2001

Multiplex capillary denaturing high-performance liquid chromatography with laser-induced fluorescence detection.

Denaturing high-performance liquid chromatography (DHPLC) is a sensitive, robust, and operationally inexpensive method for the detection of single-base substitutions and small deletions and insertions. To increase sample throughout, we have developed a multiplexing strategy using fluorophores to distinguish different PCR products. The system combines recent advances in the synthesis of monolithic poly(styrene-divinylbenzene) capillary columns with four-color confocal argon ion laser-induced fluorescence detection. Depending on the change in retention caused by the fluorophores, adjustments in the analysis temperature may be required to ensure the maximum mutation detection sensitivity.

Argon↗

PubMed2001

[The coordinated expression of laminin and its integrin receptor in hepatic sinusoidal capillarization].

OBJECTIVE: To explore the expression of laminin and its integrin receptor alpha 6 in sinusoidal capillaration. METHODS: The rat hepatic fibrosis model was established by injection carbon tetrachloride subcutaneously. Liver sections were examined by scanning electron microscopy and special staining. Then expressions of laminin and integrin alpha 6 subunit were observed by immunohistochemistry and dot immuno-blotting. RESULTS: The pathologic character of hepatic fibrosis-sinusoidal capillaration was observed. Sinusoidal capillaration is characterized by defenestration of sinusoidal endothelial cells (SEC) and basement membrane forming and phenotype transformation. By immunohistochemistry staining. The deposition of laminin surrounding sinusoids according to fibrosis stage was 0, 1.92%, 6.02%, 9.68%, 14.14% respectively, statistically different from each other (P < 0.05). In normal rat no expression of integrin alpha 6 were observed in SEC membranes. When capillaration integrin alpha 6 was detected in a continuous pattern along the sinusoidal in accordance with laminin assembly. The content of integrin alpha 6 in fibrotic liver tissues were significantly higher than in normal liver tissues as measured by dot immuno-blotting (P < 0.05). CONCLUSIONS: During fibrogenesis laminin continuously accumulate in liver tissues and form basement membrane resulting in sinusoidal capillaration, meanwhile are company with induced expression of LN-binding-integrin alpha 6 on SEC membranes. These results suggest that coordinated expression of laminin and laminin-binding-integrin might be important in pathogenesis of sinusoidal capillaration and hepatic fibrosis.

Animals↗

PubMed2001

[Changes of the expression of integrin alpha6beta1 and the activity of focal adhesion kinase in the liver sinusoidal endothelial cell in experimental fibrotic rats].

OBJECTIVE: To investigate the expression of integrin alpha(6)beta(1) and the activity of focal adhesion kinase (FAK) in liver sinusoidal endothelial cells (SECs) from experimental fibrotic rats induced by CCl(4). METHODS: By in situ collagenase perfusion and two-step Percoll gradient centrifugation, SECs were isolated and cultured from normal and CCl(4) -treated Wistar rats. The expression of integrin alpha(6)beta(1) was determined by cell-ELISA, and the activity of FAK was assessed by immunoprecipitation-tyrosine kinase assay. RESULTS: The integrin alpha(6)beta(1) was almost absent in the normal SECs and was up-regulated during the fibrotic process; SECs from experimental fibrotic rats possessed higher expression level of integrin alpha(6)beta(1) than normal SECs (P<0.05). The FAK activity in SECs from experimental fibrotic rats increased significantly as compared with the normal controls (P<0.05). CONCLUSIONS: The expression of integrin alpha(6)beta(1) on SECs and the increase of FAK in SECs may be important in the phenotype and function changes of SECs during hepatic fibrogenesis.

Animals↗

PubMed2001

[The role of YIGSR and RGD peptides in regulating fenestrae of sinusoidal endothelial cells].

OBJECTIVE: To investigate the effects of Tye-Ile-Gly-Ser-Arg (YIGSR) and Arg-Gly-Asp (RGD) polypeptides on the fenestrae of the liver sinusoidal endothelial cells (SECs). METHODS: By in situ collagenase perfusion and two-step percoll gradient centrifugation, SECs were isolated from normal Wistar rats and cultured on collagen type I or laminin-coated coverslips. The population of SECs fenestrae and their diameters were observed using scanning electron microscopical technique; and the synthesis of collagen type IV in SECs was assessed by radio-immunoassay (RIA). RESULTS: The average number of fenestrae and their diameter in the SECs cultured on laminin-coated coverslips were significantly lower than those on collagen type I-coated coverslips. After the treatment with YIGSR pentapeptides (50 micrograms/ml) and RGD tripeptides (50 micrograms/ml) together for 48 hrs, the number and diameters of fenestrae in the SECs that grew on laminin-coated coverslips increased markedly. Meanwhile, the YIGSR and RGD peptides could significantly decrease the synthesis of collagen type IV in SECs that grew on laminin-coated coverslips. CONCLUSION: The results suggested that the YIGSR and RGD polypeptides may have an important role in regulating the morphology and function of SECs.

Animals↗

PubMed2001

[Studies on the genotoxic effects of acrylonitrile].

The effect of acrylonitrile (AN) on the reproduction was investigated in 341 male and 365 female exposed workers, 384 male and 438 female unexposed controls by questionnaires on the process of childbearing and the outcome of child birth. The results showed that AN caused menstrual disorder and dysgenesis in exposed female workers and the wife of exposed male workers. It is concluded that AN is harmful to the reproduction of exposed male and female workers.

Acrylonitrile↗

PubMed2000

Light-induced rotation of a transmembrane alpha-helix in bacteriorhodopsin.

Spin labeling EPR spectroscopy has been used to characterize light-induced conformational changes of bacteriorhodopsin (bR). Pairs of nitroxide spin labels were attached to engineered cysteine residues at strategic positions near the cytoplasmic ends of transmembrane alpha-helices B, F, and G in order to monitor distance changes upon light activation. The EPR analysis of six doubly labeled bR mutants indicates that the cytoplasmic end of helix F not only tilts outwards, but also rotates counter-clockwise during the photocycle. The direction of the rotation of helix F is the opposite of the clockwise rotation previously reported for bovine rhodopsin. The opposite chirality of the F helix rotation in the two systems is perhaps related to the differences in the cis-trans photoisomerization of the retinal in the two proteins. Using time-resolved EPR, we monitored the rotation of helix F also in real time, and found that the signal from the rotation arises concurrently with the reprotonation of the retinal Schiff base.

Animals↗

PubMed2000

A region in domain II of the urokinase receptor required for urokinase binding.

The urokinase receptor is composed of three homologous domains based on disulfide spacing. The contribution of each domain to the binding and activation of single chain urokinase (scuPA) remains poorly understood. In the present paper we examined the role of domain II (DII) in these processes. Repositioning DII to the amino or carboxyl terminus of the molecule abolished binding of scuPA as did deleting the domain entirely. By using alanine-scanning mutagenesis, we identified a 9-amino acid continuous sequence in DII (Arg(137)-Arg(145)) required for both activities. Competition-inhibition and surface plasmon resonance studies demonstrated that mutation of Lys(139) and His(143) to alanine in soluble receptor (suPAR) reduced the affinity for scuPA approximately 5-fold due to an increase in the "off rate." Mutation of Arg(137), Arg(142), and Arg(145), each to alanine, leads to an approximately 100-fold decrease in affinity attributable to a 10-fold decrease in the apparent "on rate" and a 6-fold increase in off rate. These differences were confirmed on cells expressing variant urokinase receptor. suPAR-K139A/H143A displayed a 50% reduction in scuPA-mediated plasminogen activation activity, whereas the 3-arginine variant was unable to stimulate scuPA activity at all. Mutation of the three arginines did not affect binding of a decamer peptide antagonist of scuPA known to interact with DI and DIII. However, this mutation abolished both the binding of soluble DI to DII-III in the presence of scuPA and the synergistic activation of scuPA mediated by DI and wild type DII-DIII. These data show that DII is required for high affinity binding of scuPA and its activation. DII does not serve merely as a spacer function but appears to be required for interdomain cooperativity.

Animals↗

PubMed2000

RNase-L-dependent destabilization of interferon-induced mRNAs. A role for the 2-5A system in attenuation of the interferon response.

The 2-5A system is an interferon-regulated RNA degradation pathway with antiviral, growth-inhibitory, and pro-apoptotic activities. RNase-L mediates the antiviral activity through the degradation of viral RNAs, and the anticellular effects of the 2-5A system are thought to be similarly mediated through the degradation of cellular transcripts. However, specific RNase-L-regulated cellular RNAs have not been identified. To isolate candidate RNase-L substrates, differential display was used to identify mRNAs that exhibited increased expression in RNase-L-deficient N1E-115 cells as compared with RNase-L-transfected cells. A novel interferon-stimulated gene encoding a 43-kDa ubiquitin-specific protease, designated ISG43, was identified in this screen. ISG43 expression is induced by interferon and negatively regulated by RNase-L. ISG43 induction is a primary response to interferon treatment and requires a functional JAK/STAT signaling pathway. The kinetics of ISG43 induction were identical in wild type and RNase-L knock-out fibroblasts; however, the decline in ISG43 mRNA following interferon treatment was markedly attenuated in RNase-L knock-out fibroblasts. The delayed shut-off kinetics of ISG43 mRNA corresponded to an increase in its half-life in RNase-L-deficient cells. ISG15 mRNA also displayed RNase-L-dependent regulation. These findings identify a novel role for the 2-5A system in the attenuation of the interferon response.

Amino Acid Sequence↗

PubMed2000

Route of administration determines induction of T-cell-independent humoral responses to adeno-associated virus vectors.

Vectors based on adeno-associated viruses (AAV) type 2 show promise for treating chronic diseases because transgene expression appears to be stable. This study evaluated the impact of humoral immunity to the capsid proteins on vector uptake by hepatocytes following an intravascular approach. Route of vector administration in mice had a qualitative effect on antivector B cell responses. Administration of vector into the tail vein resulted in T-cell-dependent (TD) B cell responses that were completely inhibited with depleting CD4 antibody. Delivery of vector into the portal circulation via the spleen yielded B cell response that were partially T cell independent (TI) rendering strategies based on T cell inhibition ineffective in allowing vector readministration. The TI B cell response was short lived in comparison to the TD response. Rhesus monkeys produced a B cell memory response to intraportal vector which appeared to be T cell dependent based on Ig isotypes. Gene therapy strategies that require AAV vector readministration should consider vector biodistribution and its impact on B cell activation.

Animals↗