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Biomedical subjects

Jun Xing

Publications and source records attributed to Jun Xing.

At least 19 recordsLinked to original sources
PubMed2006

Structural transition of the inhibitory region of troponin I within the regulated cardiac thin filament.

Contraction and relaxation of cardiac muscle are regulated by the inhibitory and regulatory regions of troponin I (cTnI). Our previous FRET studies showed that the inhibitory region of cTnI in isolated troponin experiences a structural transition from a beta-turn/coil motif to an extended conformation upon Ca(2+) activation. During the relaxation process, the kinetics of the reversal of this conformation is coupled to the closing of the Ca(2+)-induced open conformation of the N-domain of troponin C (cTnC) and an interaction between cTnC and cTnI in their interface. We have since extended the structural kinetic study of the inhibitory region to fully regulated thin filament. Single-tryptophan and single-cysteine mutant cTnI(L129W/S151C) was labeled with 1,5-IAEDANS at Cys151, and the tryptophan-AEDANS pair served as a donor-acceptor pair. Labeled cTnI mutant was used to prepare regulated thin filaments. Ca(2+)-induced conformational changes in the segment of Trp129-Cys151 of cTnI were monitored by FRET sensitized acceptor (AEDANS) emission in Ca(2+) titration and stopped-flow measurements. Control experiments suggested energy transfer from endogenous tryptophan residues of actin and myosin S1 to AEDANS attached to Cys151 of cTnI was very small and Ca(2+) independent. The present results show that the rate of Ca(2+)-induced structural transition and Ca(2+) sensitivity of the inhibitory region of cTnI were modified by (1) thin filament formation, (2) the presence of strongly bound S1, and (3) PKA phosphorylation of the N-terminus of cTnI. Ca(2+) sensitivity was not significantly changed by the presence of cTm and actin. However, the cTn-cTm interaction decreased the cooperativity and kinetics of the structural transition within cTnI, while actin filaments elicited opposite effects. The strongly bound S1 significantly increased the Ca(2+) sensitivity and slowed down the kinetics of structural transition. In contrast, PKA phosphorylation of cTnI decreased the Ca(2+) sensitivity and accelerated the structural transition rate of the inhibitory region of cTnI on thin filaments. These results support the idea of a feedback mechanism by strong cross-bridge interaction with actin and provide insights on the molecular basis for the fine tuning of cardiac function by beta-adrenergic stimulation.

Binding Sites↗
PubMed2006

Comparison of headspace and direct single-drop microextraction and headspace solid-phase microextraction for the measurement of volatile sulfur compounds in beer and beverage by gas chromatography with flame photometric detection.

Three approaches based on headspace single-drop microextraction (HS-SDME), direct single-drop microextraction (Direct-SDME), and headspace solid-phase microextraction (HS-SPME), have been compared for analyzing volatile sulphur compounds (VSCs) in beer and beverage. Procedures and performance of the three methods have been contrasted through the determination of extraction efficiencies, precision, linearity and limits of detection. The overall process of HS-SDME and HS-SPME was applied to GC-FPD determination of five VSCs in beer and beverage.

Beer↗
PubMed2006

Defective expression of Galpha12 in the testes of azoospermia patients and in the spermatozoa with low motility.

Antibody to the Galpha12-subunit of guanine nucleotide regulatory proteins was used to determine whether the Galpha12 is present in adult human spermatogenic cells and to determine its role in dyszoospermia. Immunoblots from testes and spermatozoa demonstrated the presence of Galpha12 in the samples. Immunohistochemical analyses of testes found that Galpha12 was expressed in the cytoplasm of Leydig cells and was expressed in spermatids from the elongating Sb phase to mature sperm. Indirect immunofluorescence of human spermatozoa revealed the presence of Galpha12 in the neck region and the midpiece of the sperm. Galpha12 in spermatids and spermatozoa partially co-localized with F-actin and alpha-tubulin. Immunohistochemical analyses of tissues from three patients with non-obstructive azoospermia showed abnormal expression of Galpha12 in more than 45% of spermatids. Furthermore, Western blots and indirect immunofluorescence found defective expression of Galpha12 in low-motility spermatozoa with midpieces that were bent on themselves. Therefore, it suggests that Galpha12 plays a role in polarity and tail formation as spermatids mature. Furthermore, Galpha12 may be a candidate protein responsible for azoospermia caused by spermatogenic disturbance or midpiece deformities.

Actins↗
PubMed2006

Structural studies and mechanism of Saccharomyces cerevisiae dolichyl-phosphate-mannose synthase: insights into the initial step of synthesis of dolichyl-phosphate-linked oligosaccharide chains in membranes of endoplasmic reticulum.

Dolichyl-phosphate-mannose (Dol-P-Man) synthase catalyzes the reversible formation of a key intermediate that is involved as a mannosyl donor in at least three different pathways for the synthesis of glycoconjugates important for eukaryotic development and viability. The enzyme is found associated with membranes of the endoplasmic reticulum (ER), where it transfers mannose from the water soluble cytoplasmic donor, guanosine 5'-diphosphate (GDP)-Man, to the membrane-bound, extremely hydrophobic, and long-chain polyisoprenoid acceptor, dolichyl-phosphate (Dol-P). The enzyme from Saccharomyces cerevisiae has been utilized to investigate the structure and activity of the protein and interactions of the enzyme with Dol-P and synthetic Dol-P analogs containing fluorescent probes. These interactions have been explored utilizing fluorescence resonance energy transfer (FRET) to establish intramolecular distances within the protein molecule as well as intermolecular distances to determine the localization of the active site and the hydrophobic substrate on the enzyme's surface. A three-dimensional (3D) model of the enzyme was produced with bound substrates, Dol-P, GDP-Man, and divalent cations to delineate the binding sites for these substrates as well as the catalytic site. The FRET analysis was used to characterize the functional properties of the enzyme and to evaluate its modeled structure. The data allowed for proposing a molecular mechanism of catalysis as an inverting mechanism of mannosyl residue transfer.

Amino Acid Sequence↗
PubMed2006

A pathway of structural changes produced by monastrol binding to Eg5.

Monastrol is a small molecule inhibitor that is specific for Eg5, a member of the kinesin 5 family of mitotic motors. Crystallographic models of Eg5 in the presence and absence of monastrol revealed that drug binding produces a variety of structural changes in the motor, including in loop L5 and the neck linker. What is not clear from static crystallographic models, however, is the sequence of structural changes produced by drug binding. Furthermore, because crystallographic structures can be influenced by the packing forces in the crystal, it also remains unclear whether these drug-induced changes occur in solution, at physiologically active concentrations of monastrol or of other drugs that target this site. We have addressed these issues by using a series of spectroscopic probes to monitor the structural consequences of drug binding. Our results demonstrated that the crystallographic model of an Eg5-ADP-monastrol ternary complex is consistent with several solution-based spectroscopic probes. Furthermore, the kinetics of these spectroscopic signal changes allowed us to determine the temporal sequence of drug-induced structural transitions. These results suggested that L5 may be an element in the pathway that links the state of the nucleotide-binding site to the neck linker in kinesin motors.

Anisotropy↗
PubMed2006

[Screening and analysis of coding SNPs of HLA-DQA1 gene involved in susceptibility for cervical cancer].

BACKGROUND & OBJECTIVE: Polymorphisms of human leukocyte antigen (HLA) gene play an important role in the development of cervical cancer. This study was to screen single nucleotide polymorphisms (SNPs) of HLA-DQA1 gene involved in susceptibility of cervical cancer by a bioinformatics approach, and analyze their correlations to abnormal gene functions. METHODS: SNPs of HLA-DQA1 were screened from a public database dbSNP by SNPper software, and relevant FASTA subsequences were also obtained from dbSNP. PARSESNP software was used to analyze cSNPs. RESULTS: Two SNPs, rs9272693 and rs9272703, which may induce mis-sense mutation, were identified in codon region of HLA-DQA1 gene. A PSSM difference>10 was used to predict deleterious mutation. CONCLUSIONS: SNPper software in combination with PARSESNP software could be used to analyze SNPs of HLA-DQA1 gene and select the variants in a conserved region, and it provides an evaluation criterion. But the results need to be verified in cervical cancer patients and control populations.

Databases as Topic↗
PubMed2005

Quantitative determination of compounds in tobacco essential oils by comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry.

A quantitative analysis of the individual compounds in tobacco essential oils is performed by comprehensive two-dimensional gas chromatography (GC x GC) combined with flame ionization detector (FID). A time-of-flight mass spectrometer (TOF/MS) was coupled to GC x GC for the identification of the resolved peaks. The response of a flame ionization detector to different compound classes was calibrated using multiple internal standards. In total, 172 compounds were identified with good match and 61 compounds with high probability value were reliably quantified. For comparative purposes, the essential oil sample was also quantified by one-dimensional gas chromatography-mass spectrometry (GC/MS) with multiple internal standards method. The results showed that there was close agreement between the two analysis methods when the peak purity and match quality in one-dimensional GC/MS are high enough.

Gas Chromatography-Mass Spectrometry↗
PubMed2005

Docking and rolling, a model of how the mitotic motor Eg5 works.

Whereas kinesin I is designed to transport cargoes long distances in isolation, a closely related kinesin motor, Eg5, is designed to generate a sustained opposing force necessary for proper mitotic spindle formation. Do the very different roles for these evolutionarily related motors translate into differences in how they generate movement? We have addressed this question by examining when in the ATPase cycle the Eg5 motor domain and neck linker move through the use of a series of novel spectroscopic probes utilizing fluorescence resonance energy transfer, and we have compared our results to kinesin I. Our results are consistent with a model in which movement in Eg5 occurs in two sequential steps, an ATP-dependent docking of the neck linker, followed by a rotation or "rolling" of the entire motor domain on the microtubule surface that occurs with ATP hydrolysis. These two forms of movement are consistent with the functions of a motor designed to generate sustained opposing force, and hence, our findings support the argument that the mechanochemical features of a molecular motor are shaped more by the demands placed on it than by its particular family of origin.

Adenosine Diphosphate↗
PubMed2005

[Physiological and ecological effects of inter- and mixed cropping rape with milk vetch].

The investigation on the growth, yield and benefit of rape inter- and mixed cropped with milk vetch showed that comparing with sowing rape under zero tillage, the physiological and ecological characters of transplanted rape, including its height, leaf size, root diameter, opening degree and yield were obviously improved. Under zero tillage, mixed cropping had an obvious advantage than inter- and single cropping. After tillage, the yield of transplanted rape under mixed cropping was 11.9% more than that under single cropping with zero tillage. The benefit of transplanting rape under mixed cropping with tillage was the biggest, followed by single cropping rape with zero tillage. It could be concluded that the patterns of transplanting rape under mixed cropping after tillage and zero tillage had the best physiological and ecological effects, not only increasing rape yield and income, but also improving soil fertility.

Astragalus Plant↗
PubMed2004

Switching of troponin I: Ca(2+) and myosin-induced activation of heart muscle.

The principal task of the Ca(2+) activation of striated muscle is the release of the troponin I (TnI) inhibitory region (TnI-I) from actin. TnI-I release facilitates the repositioning of tropomyosin across the actin surface and the formation of strong, force generating, actin-myosin cross-bridges. Full activation of the Ca(2+) regulatory switch (CRS) requires two switching steps in cTnI: binding of the TnI regulatory region to hydrophobic sites in the N-domain of Ca(2+)-bound troponin C and release of the adjacent TnI-I from actin. Using Förster resonance energy transfer, we have examined the requirements for full activation of the cardiac CRS. In the presence of actin, both Ca(2+) and strong cross-bridges are required for full activation. Actin desensitizes the CRS to Ca(2+) and produces cooperativity in the Ca(2+) activation of the CRS. Strong cross-bridges eliminate cooperativity and re-sensitize the CRS to Ca(2+). We propose a kinetic scheme and a structural model to account for these findings.

Animals↗
PubMed2004

Development of new solid-phase microextraction fibers by sol-gel technology for the determination of organophosphorus pesticide multiresidues in food.

Allyloxy bisbenzo 16-crown-5 trimethoxysilane was first used as precursor to prepare the sol-gel-derived bisbenzo crown ether/hydroxyl-terminated silicone oil (OH-TSO) SPME coating. The coating procedure involving sol solution composition and conditioning process was presented. Compared with commercial SPME stationary phases, the new coatings showed higher extraction efficiency and therefore could provide higher sensitivity for organphosphorous pesticides (OPs). Limits of detection (LODs) were in the range of 0.003-1.0 ng/g for these OPs in food samples (honey, juice, orange and pakchoi). The optimal extraction conditions of the new coatings to OPs in these samples were investigated by adjusting extraction time, salt addition, extraction temperature, and dilution ratios of samples with distilled water by using SPME coupled with gas chromatography (GC)-flame photometric detection (FPD). The method was applied to determine the concentrations of OPs in real samples.

Chromatography, Gas↗
PubMed2004

Nucleic acid sequence-based amplification methods to detect avian influenza virus.

Infection of poultry with highly pathogenic avian influenza virus (AIV) can be devastating in terms of flock morbidity and mortality, economic loss, and social disruption. The causative agent is confined to certain isolates of influenza A virus subtypes H5 and H7. Due to the potential of direct transfer of avian influenza to humans, continued research into rapid diagnostic tests for influenza is therefore necessary. A nucleic acid sequence-based amplification (NASBA) method was developed to detect a portion of the haemagglutinin gene of avian influenza A virus subtypes H5 and H7 irrespective of lineage. A further NASBA assay, based on the matrix gene, was able to detect examples of all known subtypes (H1-H15) of avian influenza virus. The entire nucleic acid isolation, amplification, and detection procedure was completed within 6h. The dynamic range of the three AIV assays was five to seven orders of magnitude. The assays were sensitive and highly specific, with no cross-reactivity to phylogenetically or clinically relevant viruses. The results of the three AIV NASBA assays correlated with those obtained by viral culture in embryonated fowl's eggs.

Animals↗
PubMed2004

Solid-phase microextraction and gas chromatography-electron capture detection analysis of trace organochlorine pesticides in water using novel benzo-15-crown-5 sol-gel coating.

A novel dihydroxy-terminated benzo-15-crown-5 is synthesized and applied to prepare the solid-phase microextraction (SPME) fiber coating with sol-gel technology. Headspace SPME, as a simple, solvent-free method, is applied to the analysis of 16 organochlorine pesticides (OCPs) present at trace levels in a water sample. A homemade crown ether fiber coated with 80- micro m thickness was used for extraction. Analyses are performed using gas chromatography-electroncapture detection. The optimization of the extraction process is studied. Compared with commercially available SPME fibers, polydimethylsiloxane, the new phases show better selectivity and sensitivity toward OCPs. The linear concentrations range from 1 to 1000 ng/L, the detection limits are in the range of 0.01-0.5 ng/L, the recoveries are over 85%, and relative standard deviations are below 7.2% for these OCPs.

Chromatography, Gas↗
PubMed2004

[Differential expression of motility-related protein-1/CD9 gene in gastric cancers and premalignant lesions].

OBJECTIVE: To clone and identify gastric cancer-related genes and explore the possible pathogenic mechanism of gastric cancer. METHODS: The differentially expressed cDNA bands were isolated by fluorescent mRNA differential display in gastric cancer specimens, matched normal gastric mucosa and premalignant lesions. The motility-related protein-1 (MRP-1/CD9) gene was one of the down-regulated genes. MRP-1/CD9 gene expression in different kinds of gastric tissue was analyzed by Northern blot and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: MRP-1/CD9 expression was down-regulated in all gastric cancer tissues. Northern blot analysis confirmed this differential expression. RT-PCR analysis showed that the MRP-1/CD9 gene expression was much lower in gastric cancers(0.31+/-0.18) than in the matched normal gastric tissue (0.49+/-0.24) and in the premalignant lesions (0.47+/-0.18) (P<0.05), and its expression in intestinal type gastric cancer (0.38+/-0.16) was higher than that in diffuse type gastric cancer (0.22+/-0.17) (P<0.05). CONCLUSION: The MRP-1/CD9 gene expression was down-regulated in gastric cancer, its expression was probably related to the carcinogenesis and histology types of gastric cancer.

Antigens, CD↗
PubMed2003

Application of polyphenylmethylsiloxane coated fiber for solid-phase microextraction combined with microwave-assisted extraction for the determination of organochlorine pesticides in Chinese teas.

Polyphenylmethylsiloxane (PPMS) as a novel coating for solid-phase microextraction (SPME) combined with microwave-assisted extraction (MAE) has been applied to determine the concentrations of organochlorine pesticides (OCPs) in Chinese teas. The characteristics of PPMS fiber, the extraction modes of SPME, the extraction time, temperature, and salt effects were investigated. Microwave irradiation time and power were also studied. Compared with commercial polydimethylsiloxane (PDMS) fiber and homemade sol-gel polymethylsiloxane (PMS) fiber, the novel porous sol-gel PPMS fiber exhibited high sensitivity and selectivity for OCPs compounds, higher thermal stability (to 350 degrees C) and long service life (more than 150 times). The recoveries of MAE is compared with that of ultrasonic extraction (USE), MAE-SPME-gas chromatography (GC)/electron-capture detection (ECD) methods showed better results for Chinese teas. Linear ranges of OCPs in the blank green tea was 0.1-10(3) ng/l. Detection limits of this method are below 0.081 ng/l. Recoveries of this method are between 39.05 and 94.35%. The repeatability of the technique was less than 16% relative standard deviation (R.S.D.). The tested pesticides in three Chinese teas were at the ng/g level.

Chromatography, Gas↗
PubMed2003

Kinetics of conformational transitions in cardiac troponin induced by Ca2+ dissociation determined by Förster resonance energy transfer.

Upon Ca2+ activation of cardiac muscle, several structural changes occur in the troponin subunits. These changes include the opening of the cardiac troponin C (cTnC) N-domain, the change of secondary structure of the inhibitory region of cardiac troponin I (cTnI), and the change in the separation between these two proteins in the cTnC-cTnI interface. We have used Förster resonance energy transfer in Ca2+ titration and stopped-flow experiments to delineate these transitions using a reconstituted cardiac troponin. Energy transfer results were quantified to yield time-dependent profiles of changes in intersite distances during Ca2+ dissociation. The closing of the cTnC N-domain induced by release of regulatory Ca2+ from cTnC occurs in one step (t1/2 approximately 5 ms), and this transition is not affected by Ca2+ release from the C-domain. The other two transitions triggered by Ca2+ dissociation are biphasic with the fast phase (t1/2 approximately 5 ms) correlated with Ca2+ release from the cTnC N-domain. These transitions are slower than the release of bound regulatory Ca2+ (t1/2 3.6 ms) and are coupled to one another in a cooperative manner in restoring their conformations in the deactivated state. The kinetic results define the magnitudes of structural changes relevant in Ca2+ switching between activation and deactivation of cardiac muscle contraction.

Animals↗
PubMed2003

Novel benzo-15-crown-5 sol-gel coating for solid-phase microextraction.

A novel dihydroxy-terminated benzo-15-crown-5 was synthesized and applied to prepare a solid-phase microextraction (SPME) fiber coating with sol-gel technology. The optimization of the sol-gel process was studied. The coating method with sol-gel was improved and completed in one run, which economized materials and allowed easier control of the fiber thickness. The repeatability of coating fiber to fiber was better than 4.94% (RSD). The surface of the fiber coating was well-distributed and an electron microscopy experiment suggested a porous structure for crown ether coating, providing high surface areas and allowing for high extraction efficiency. The coating has a high thermal stability (350 degrees C), long lifetime and can stand solvent (organic and inorganic) rinsing due to the chemical binding between the coating and the fiber surface. Non-polar benzene, toluene, ethylbenzene, xylenes, chlorobenzenes, polar phenolic compounds and arylamines were used to evaluate the character of the fiber coating by headspace SPME-gas chromatography technology. For phenols, the linear concentrations ranged from 5 to 1000 microg/l, the detection limits were between 0.05 and 1 microg/l, and the RSD was less than 5%. The addition of benzo-crown ether not only increases the thermal stability of the fiber coating, but also enhances the selectivity of the fiber coating. Compared with commercially available SPME fibers poly(dimethylsiloxane) and polyacrylate, the few phases showed better selectivity and sensitivity towards non-polar and polar aromatic compounds.

Amines↗