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Biomedical subjects

X Qian

Publications and source records attributed to X Qian.

At least 19 recordsLinked to original sources
PubMed2001

Evidence for a novel natriuretic peptide receptor that prefers brain natriuretic peptide over atrial natriuretic peptide.

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) exert their physiological actions by binding to natriuretic peptide receptor A (NPRA), a receptor guanylate cyclase (rGC) that synthesizes cGMP in response to both ligands. The family of rGCs is rapidly expanding, and it is plausible that there might be additional, as yet undiscovered, rGCs whose function is to provide alternative signalling pathways for one or both of these peptides, particularly given the low affinity of NPRA for BNP. We have investigated this hypothesis, using a genetically modified (knockout) mouse in which the gene encoding NPRA has been disrupted. Enzyme assays and NPRA-specific Western blots performed on tissues from wild-type mice demonstrate that ANP-activated cGMP synthesis provides a good index of NPRA protein expression, which ranges from maximal in adrenal gland, lung, kidney, and testis to minimal in heart and colon. In contrast, immunoreactive NPRA is not detectable in tissues isolated from NPRA knockout animals and ANP- and BNP-stimulatable GC activities are markedly reduced in all mutant tissues. However, testis and adrenal gland retain statistically significant, high-affinity responses to BNP. This residual response to BNP cannot be accounted for by natriuretic peptide receptor B, or any other known mammalian rGC, suggesting the presence of a novel receptor in these tissues that prefers BNP over ANP.

Adrenal Glands↗
PubMed2001

Dynamic simulations of 13 TATA variants refine kinetic hypotheses of sequence/activity relationships.

The fundamental relationship between DNA sequence/deformability and biological function has attracted numerous experimental and theoretical studies. A classic prototype system used for such studies in eukaryotes is the complex between the TATA element transcriptional regulator and the TATA-box binding protein (TBP). The recent crystallographic study by Burley and co-workers demonstrated the remarkable structural similarity contrasted to different transcriptional activity of 11 TBP/DNA complexes in which the DNAs differed by single base-pairs. By simulating these TATA variants and two other single base-pair variants that were not crystallizable, we uncover sequence-dependent structural, energetic, and flexibility properties that tailor TATA elements to TBP interactions, complementing many previous studies by refining kinetic hypotheses on sequence/activity correlations. The factors that combine to produce favorable elements for TBP activity include overall flexibility; minor groove widening, as well as roll, rise, and shift increases at the ends of the TATA element; untwisting within the TATA element accompanied by large roll at the TATA element ends; and relatively low maximal water densities around the DNA. These features accompany the severe deformation induced by the minor-groove binding protein, which kinks the TATA element at the ends and displaces local water molecules to form stabilizing hydrophobic contacts. Interestingly, the preferred bending direction itself is not a significant predictor of activity disposition, although certain variants (such as wild-type AdMLP, 5'-TATA4G-3', and inactive A29, 5'-TA6G-3') exhibit large preferred bends in directions consistent with their activity or inactivity (major groove and minor groove bends, respectively). These structural, flexibility, and hydration preferences, identified here and connected to a new crystallographic study of a larger group of DNA variants than reported to date, highlight the profound influence of single base-pair DNA variations on DNA motion. Our refined kinetic hypothesis suggests the functional implications of these motions in a kinetic model of TATA/TBP recognition, inviting further theoretical and experimental research.

Base Sequence↗
PubMed2001

Cloning, expression, purification and preliminary X-ray crystallographic studies of yeast Hsp40 Sis1 complexed with Hsp70 Ssa1 C-terminal lid domain.

Heat-shock protein 70 (Hsp70) plays essential roles in a number of cellular processes such as protein folding, assembly and translocation. Heat-shock protein 40 (Hsp40) transiently interacts with Hsp70 and facilitates Hsp70 functions in these processes within cells. Hsp40 recognizes and binds non-native polypeptide and delivers it to Hsp70. Hsp40 can then stimulate the ATPase activity of Hsp70 to refold the polypeptide. To investigate the molecular mechanism by which Hsp40 interacts with Hsp70 to transport the non-native polypeptide, Saccharomyces cerevisiae Hsp40 Sis1 C-terminal peptide-binding fragment complexed with Hsp70 Ssa1 C-terminal lid domain has been produced and crystallized. The complex crystals diffract to 3.3 A and belong to the space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = 112.17, c = 171.31 A. Structure determination by the MAD method is under way.

Adenosine Triphosphatases↗
PubMed2001

A new operation for ventricular septal defect with aortic incompetence.

Two boys, 10 years of age, with ventricular septal defect and severe aortic insufficiency were treated using a new method. An autograft of anterior pulmonary artery wall and the adjacent leaflet and subpulmonary muscle was excised. The excised pulmonary artery segment was used to replace the prolapsed right coronary cusp, and the autograft muscle was used to close the ventricular septal defect. The right ventricular outflow tract and pulmonary valve were repaired using an autologous pericardial patch with an autologous single cusp. The results in both cases were satisfactory.

Aortic Valve Insufficiency↗
PubMed2001

Synthesis and quantitative structure-activity relationships of new 2,5-disubstituted-1,3,4-oxadiazoles.

Fourteen new 2,5-disubstituted-1,3,4-oxadiazoles, namely (i) six 2-[2,2-dimethyl-3-(2,2-dichlorovinyl)-cyclopropyl]-5-substituted-1,3,4-oxadiazoles and (ii) eight 2-substituted-phenoxymethyl-5-substituted-aryl-1,3,4-oxadiazoles, were synthesized and evaluated for their insect growth regulatory activity against second-instar larvae of armyworm (Pseudaletia separata Walker). Two of these compounds (7 and 8) showed good insecticidal activities, with LC(50) values of 14.33 and 15.85 microg/mL, respectively. Steric parameters (e.g., the molecular length d and the ratio of the nonpolar surface area and polar surface area V(1)/V(2)) and semiempirical quantum parameters (e.g., the molecular total energy E(t) and the lowest unoccupied molecular orbital energy E(LUMO) and so on) of these compounds, as well as those of six other 2,5-disubstituted-1,3,4-oxadiazoles reported, were acquired by the molecular modeling method and the PM3-SCF-MO method, respectively. With the help of the synthons' activity contribution method based on the Free-Wilson approach in its Fujita-Ban variant, quantitative structure-activity relationships were studied by regressing half-lethal concentrations against the above parameters.

Animals↗
PubMed2001

In situ hybridization for the identification of yeastlike organisms in tissue section.

The identification of yeast and yeastlike organisms in tissue sections can be very difficult. Biopsy tissues may be limited, with only occasional organisms present. In addition, several common species have overlapping histologic features. Deoxyribonucleic acid probes were designed to detect both the 18S and 28S ribosomal ribonucleic acid sequences of five fungal organisms with a high degree of specificity for each fungus. Each of these organisms--Blastomyces dermatitidis, Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum, and Sporothrix schenckii--can be manifested histologically as round, yeastlike structures, often within a similar size range. Probes were tested against 98 archived, formalin-fixed, paraffin-embedded tissue specimens, each of which had culture-proved involvement by one of these organisms. Assessment of accuracy was based on the presence of yeastlike organisms in consecutive Grocott's methanemine silver (GMS)-stained tissue sections, and agreement with culture results. The results indicated that GMS had a greater overall sensitivity in detecting fungal organisms (95.9%) compared with in situ hybridization (ISH; 82.7%). ISH with oligonucleotide deoxyribonucleic acid probes, however, was more specific, with all species-specific probes yielding 100% specificity (compared with 96.2-100% specificity based on morphology alone). ISH also had a higher positive predictive value (100% in all cases) compared with GMS (83.3-100%). In addition, four cases with rare organisms present (4.1% of cases tested) were detected by ISH but not by GMS staining. These results show that ISH, directed against ribosomal ribonucleic acid, provides a rapid, accurate technique for the identification of yeastlike organisms in histologic tissue sections. Its primary strength lies in the ability to speciate organisms accurately that are too few or atypical to identify based solely on morphologic features.

DNA Probes↗
PubMed2001

Direct detection of Legionella species from bronchoalveolar lavage and open lung biopsy specimens: comparison of LightCycler PCR, in situ hybridization, direct fluorescence antigen detection, and culture.

We developed a rapid thermocycling, real-time detection (also known as real-time PCR) method for the detection of Legionella species directly from clinical specimens. This method uses the LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.) and requires approximately 1 to 2 h to perform. Both a Legionella genus PCR assay and Legionella pneumophila species-specific PCR assay were designed. A total of 43 archived specimens from 35 patients were evaluated, including 19 bronchoalveolar lavage (BAL) specimens and 24 formalin-fixed, paraffin-embedded open lung biopsy specimens. Twenty-five of the specimens were culture-positive for Legionella (9 BAL specimens and 16 tissue specimens). BAL specimens were tested by LightCycler PCR (LC-PCR) methods and by a direct fluorescent antibody (DFA) assay, which detects L. pneumophila serogroups 1 to 6 and several other Legionella species. Tissue sections were tested by the two LC-PCR methods, by DFA, by an in situ hybridization (ISH) assay, specifically designed to detect L. pneumophila, and by Warthin-Starry (WS) staining. The results were compared to the "gold standard" method of bacterial culture. With BAL specimens the following assays yielded the indicated sensitivities and specificities, respectively: Legionella genus detection by Legionella genus LC-PCR, 100 and 100%; Legionella genus detection by DFA assay, 33 and 100%; and L. pneumophila detection by L. pneumophila species-specific LC-PCR, 100 and 100%. With open lung biopsy specimens the following assays yielded the indicated sensitivities and specificities, respectively: Legionella genus detection by LC-PCR 68.8 and 100%; Legionella genus detection by DFA assay, 44 and 100%; Legionella genus detection by WS staining, 63 and 100%; L. pneumophila species-specific detection by LC-PCR, 17 and 100%; and L. pneumophila species-specific detection by ISH, 100 and 100%. The analytical sensitivity of both LC-PCR assays was <10 CFU/reaction. LC-PCR is a reliable method for the direct detection of Legionella species from BAL specimens. The Legionella genus LC-PCR assay could be performed initially; if positive, L. pneumophila species-specific LC-PCR could then be performed (if species differentiation is desired). The speed with which the LC-PCR procedure can be performed offers significant advantages over both culture-based methods and conventional PCR techniques. In contrast, for the methods evaluated, culture was the best for detecting multiple Legionella species in lung tissue. WS staining, Legionella genus LC-PCR, and L. pneumophila species-specific ISH were useful as rapid tests with lung tissue.

Antigens, Bacterial↗
PubMed2001

SH2-B and APS are multimeric adapters that augment TrkA signaling.

Neurotrophins influence growth and survival of sympathetic and sensory neurons through activation of their receptors, Trk receptor tyrosine kinases. Previously, we identified Src homology 2-B (SH2-B) and APS, which are structurally similar adapter proteins, as substrates of Trk kinases. In the present study, we demonstrate that both SH2-B and APS exist in cells as homopentamers and/or heteropentamers, independent of Trk receptor activation. Structure-function analyses revealed that the SH2-B multimerization domain resides within its amino terminus, which is necessary for SH2-B-mediated nerve growth factor (NGF) signaling. Overexpression of SH2-B enhances both the magnitude and duration of TrkA autophosphorylation following exposure of PC12 cells to NGF, and this effect requires the amino-terminal multimerization motif. Moreover, the amino terminus of SH2-B is necessary for TrkA/SH2-B-mediated morphological differentiation of PC12 cells. Together, these results indicate that the multimeric adapters SH2-B and APS influence neurotrophin signaling through direct modulation of Trk receptor autophosphorylation.

Adaptor Proteins, Signal Transducing↗
PubMed2001

In situ hybridization detection of calcitonin mRNA in routinely fixed, paraffin-embedded tissue sections: a comparison of different types of probes combined with tyramide signal amplification.

In situ hybridization (ISH) is a powerful molecular tool used to visualize nucleic acids in tissues and cells. However, its use is limited by the relative lack of sensitivity in detecting low copy numbers of nucleic acids. Several strategies have been developed to improve the threshold levels of in situ detection of nucleic acid by amplification of either target nucleic acid sequences before ISH (such as in situ polymerase chain reaction) or after the hybridization procedure (such as tyramide signal amplification [TSA]). The authors compared the use of different types of probes to detect calcitonin mRNA in 10 cases of formalin-fixed, paraffin-embedded medullary thyroid carcinoma with and without TSA. In addition, dot blot hybridization was used to compare the signal amplification by TSA with oligonucleotide. cDNA, and cRNA probes. These results show that cRNA probes are the most sensitive types of probes for detecting mRNA molecules in formalin-fixed, paraffin-embedded tissue sections and that tyramide amplification can increase the sensitivity for detection of calcitonin mRNA in formalin-fixed, paraffin-embedded tissue sections at least 2- to 4-fold with cRNA probes.

Base Sequence↗
PubMed2000

Molecular characterization of taurine transport in bovine aortic endothelial cells.

Cultured bovine aortic endothelial (BAE) cells expressed a Na(+)/Cl(-)-dependent taurine uptake activity that saturated with an apparent K(0.5) of approximately 4.9 microM for taurine and was inhibited by beta-alanine, guanidinoethane sulfonate, and homotaurine. We isolated a taurine transporter clone from a BAE cell cDNA library that revealed >91% sequence identity at the amino acid level to the previously cloned high-affinity mammalian taurine transporters. The biochemical and pharmacological properties of the bovine taurine transporter cDNA expressed in Xenopus oocyte was similar to those of the high-affinity taurine transporter. Surprisingly, F(-) blocked taurine uptake in BAE cells with an IC(50) of approximately 17.5 mM. The endogenous taurine uptake was also inhibited by the protein kinase C activator phorbol 12-myristate 13-acetate, but not by its inactive analog, 4 alpha-phorbol 12,13-didecanoate. The endogenous uptake was stimulated, however, by hypertonic stress and the increase was due to an increase in the V(max) of taurine uptake. Our results provide the first description of a molecular mechanism that may be responsible for maintaining the intracellular taurine content in the endothelial cells.

Amino Acid Sequence↗
PubMed2000

Synthesis and SAR of benzyl and phenoxymethylene oxadiazole benzenesulfonamides as selective beta3 adrenergic receptor agonist antiobesity agents.

Benzyl and phenoxymethylene substituted oxadiazoles are potent and orally bioavailable beta3 adrenergic receptor (AR) agonists. The 4-trifluormethoxy substituted 5-benzyl oxadiazole 5f has an EC50 of 8 nM in the beta3 AR agonist assay with 100-fold selectivity over beta1 and beta2 AR binding inhibition activity. Its oral bioavailability in dogs is 30 +/- 4%, with a half-life of 3.8 +/- 0.4 h. In the anesthetized rhesus, 5f evoked a dose-dependent glycerolemia (ED50Gly = 0.15 mg/kg). Under these conditions a heart rate increase of 15% was observed at a dose level of 10 mg/kg.

Adrenergic beta-3 Receptor Agonists↗
PubMed2000

Locus-specific contig assembly in highly-duplicated genomes, using the BAC-RF method.

Polyploidy, the presence of multiple sets of chromosomes that are similar but not identical, complicates both chromosome walking and assembly of sequence-ready contigs for many plant taxa including a large number of economically-significant crops. Traditional 'dot-blot hybridization' or PCR-based assays for identifying BAC clones corresponding to a mapped DNA landmark usually do not provide sufficient information to distinguish between allelic and non-allelic loci. A restriction fragment matching method using pools of BAC DNA in combination with dot-blots reveals the locus specificity of individual BACs that correspond to multi-locus DNA probes, in a manner that can efficiently be applied on a large scale. This approach also provides an alternative means of mapping DNA loci that exploits many advantages of 'radiation hybrid' mapping in taxa for which such hybrids are not available. The BAC-RF method is a practical and reliable approach for using high-density RFLP maps to anchor sequence-ready BAC contigs in highly-duplicated genomes, provides an alternative to high-density robotic gridding for screening BAC libraries when the necessary equipment is not available, and permits the expedient isolation of individual members of multigene or repetitive DNA families for a wide range of genetic and evolutionary investigations.

Chromosomes, Bacterial↗
PubMed2000

The Sos1 and Sos2 Ras-specific exchange factors: differences in placental expression and signaling properties.

Targeted disruption of both alleles of mouse sos1, which encodes a Ras-specific exchange factor, conferred mid-gestational embryonic lethality that was secondary to impaired placental development and was associated with very low placental ERK activity. The trophoblastic layers of sos1(-/-) embryos were poorly developed, correlating with high sos1 expression in wild-type trophoblasts. A sos1(-/-) cell line, which expressed readily detectable levels of the closely related Sos2 protein, formed complexes between Sos2, epidermal growth factor receptor (EGFR) and Shc efficiently, gave normal Ras.GTP and ERK responses when treated with EGF for < or =10 min and was transformed readily by activated Ras. However, the sos1(-/-) cells were resistant to transformation by v-Src or by overexpressed EGFR and continuous EGF treatment, unlike sos1(+/-) or wild-type cells. This correlated with Sos2 binding less efficiently than Sos1 to EGFR and Shc in cells treated with EGF for > or =90 min or to v-Src and Shc in v-Src-expressing cells, and with less ERK activity. We conclude that Sos1 participates in both short- and long-term signaling, while Sos2-dependent signals are predominantly short-term.

Animals↗
PubMed2000

A three-dimensional model of the delta-opioid pharmacophore: comparative molecular modeling of peptide and nonpeptide ligands.

A comparative molecular modeling study of delta-opioid ligands was performed under the assumption that potent peptide and nonpeptide agonists may have common three-dimensional (3D) arrangement of pharmacophore groups upon binding to the delta-receptor. Low-energy conformations of the agonists 7-spiroindanyloxymorphone (SIOM) and 2-methyl-4a-alpha-(3-hydroxyphenyl)-1,2,3,4,4a,5,12, 12a-alpha-octahydro-quinolino[2,3,3-g]isoquinoline (TAN-67), and a partial agonist oxomorphindole (OMI) were determined by high-temperature molecular dynamics (MD). A good spatial overlap was found for the pharmacophore groups of SIOM, TAN-67, and OMI, including the basic nitrogen, phenol hydroxyl, and two aromatic ring. Based on this overlap we proposed a 3D pharmacophore model for nonpeptide delta-opioid agonists with a distance of 7.0 +/- 1.3 A between the two aromatic rings and of 8.2 +/- 1.0 A between the nitrogen and phenyl ring. The potent and highly delta-opioid receptor selective agonist [(2S,3R)-TMT(1)]DPDPE, which shares global backbone constraints of the 14-membered disulfide cycle and a strong preference for the trans rotamer of the TMT(1) side chain, was chosen as a peptide template of the delta-opioid pharmacophore. Extensive MD simulations at 300 K with the AMBER force field were performed for [(2S,3R)-TMT(1)]DPDPE and the less potent [(2S, 3S)-TMT(1)]DPDPE analogue. Multiple MD trajectories were collected for each peptide starting from the x-ray structures of DPDPE and [L-Ala(3)]DPDPE and from models proposed in the literature. Low-energy MD conformations were filtered by the nonpeptide pharmacophore query and then directly superimposed with SIOM, OMI, and TAN-67. Two conformers of [(2S,3R)-TMT(1)]DPDPE that showed the best overlap with the nonpeptide pharmacophore (rms deviation </= 1. 0 A for N,O atoms and centroids of two aromatic rings) were selected as possible delta-receptor binding conformations. These conformations have similar backbone structures, and trans rotamers of the TMT(1) side-chain group. They are reasonably close to the crystal structure of [L-Ala(3)]DPDPE, and differ significantly from the crystal structure of DPDPE. The conformer with a gauche(-) rotamer of Phe(4) is most consistent with structure-activity relationships of delta-opioid peptides. The proposed 3D models were used for rational design of new nonpeptide delta-receptor ligands.

Analgesics↗
PubMed2000

Timing of CNS cell generation: a programmed sequence of neuron and glial cell production from isolated murine cortical stem cells.

Multipotent stem cells that generate both neurons and glia are widespread components of the early neuroepithelium. During CNS development, neurogenesis largely precedes gliogenesis: how is this timing achieved? Using clonal cell culture combined with long-term time-lapse video microscopy, we show that isolated stem cells from the embryonic mouse cerebral cortex exhibit a distinct order of cell-type production: neuroblasts first and glioblasts later. This is accompanied by changes in their capacity to make neurons versus glia and in their response to the mitogen EGF. Hence, multipotent stem cells alter their properties over time and undergo distinct phases of development that play a key role in scheduling production of diverse CNS cells.

Animals↗