Biomedical subjects

Y Fan

Publications and source records attributed to Y Fan.

At least 19 recordsLinked to original sources

PubMed2001

Disruption of cytoskeletal integrity impairs Gi-mediated signaling due to displacement of Gi proteins.

beta1 integrins play a crucial role as cytoskeletal anchorage proteins. In this study, the coupling of the cytoskeleton and intracellular signaling pathways was investigated in beta1 integrin deficient (-/-) embryonic stem cells. Muscarinic inhibition of the L-type Ca2+ current (ICa) and activation of the acetylcholine-activated K+ current (IK,ACh) was found to be absent in beta1 integrin-/- cardiomyocytes. Conversely, beta adrenoceptor-mediated modulation of ICa was unaffected by the absence of beta1 integrins. This defect in muscarinic signaling was due to defective G protein coupling. This was supported by deconvolution microscopy, which demonstrated that Gi exhibited an atypical subcellular distribution in the beta1 integrin-/- cardiomyocytes. A critical role of the cytoskeleton was further demonstrated using cytochalasin D, which displaced Gi and impaired muscarinic signaling. We conclude that cytoskeletal integrity is required for correct localization and function of Gi-associated signaling microdomains.

Acetylcholine↗

PubMed2001

Simultaneous detection of multiple cytokines from conditioned media and patient's sera by an antibody-based protein array system.

We have developed a novel technique for high-throughput simultaneous screening of multiple cytokine expression based on a protein array system. Our method has the advantage of showing the specificity of enzyme-linked immunosorbent assays, sensitivity of enhanced chemiluminescence (ECL), and high-throughput of microspot. In this system, the cytokine array membranes were created by spotting capture antibodies onto the membranes. The membranes were then incubated with biological samples such as conditioned media and patient's sera. The bound proteins were then recognized by biotin-conjugated antibodies and detected by horseradish peroxidase-conjugated streptavidin coupled with ECL. Experiments demonstrated that 24 cytokines from conditioned media and patient's sera could be simultaneously detected using this new approach. This methodology should allow us to develop many high-density protein array systems to detect a variety of proteins. To validate and quantitate the expression of key molecules in a wide range of samples, we have developed conditioned medium arrays to evaluate hundreds and even thousands of samples from individual cells and patients in a single microarray. The combinations of protein arrays and conditioned medium arrays or serum arrays will provide a powerful tool to identify the protein expression profiles and rapidly validate their expression in many types and numbers of samples.

Antibodies↗

PubMed2001

Connexin 43 (cx43) enhances chemotherapy-induced apoptosis in human glioblastoma cells.

Stable re-expression of connexin 43 (cx43) in human glioblastoma suppresses transformation and tumorigenicity. The present study was designed to examine the role of cx43 in chemotherapy-induced apoptosis. Expression of cx43 in human glioblastoma cells significantly increased sensitivity to several common chemotherapeutic agents, including etoposide, paclitaxel (Taxol) and doxorubicin, compared with control-transfected cells. The increased sensitivity to chemotherapeutic agents resulted from apoptosis as evidenced by Hoechst dye staining, TUNEL assay and annexin V assay. These cx43-mediated effects were coupled with decreased expression of the specific apoptosis inhibitor bcl-2. Over-expression of bcl-2 in cx43-transfected cells partially confers the resistance to apoptosis induced by etoposide, suggesting that the cx43-mediated apoptosis to chemotherapeutic agents is regulated in part through the down-regulation of bcl-2 expression. Furthermore, the cx43-mediated apoptosis in response to chemotherapeutic drugs may not be linked to increased gap junctional communication in cx43-transfected cells. Our results demonstrate a new role of cx43 in the mediation of apoptosis during chemotherapy.

Antineoplastic Agents↗

PubMed2001

Comparison of human genetic and sequence-based physical maps.

Recombination is the exchange of information between two homologous chromosomes during meiosis. The rate of recombination per nucleotide, which profoundly affects the evolution of chromosomal segments, is calculated by comparing genetic and physical maps. Human physical maps have been constructed using cytogenetics, overlapping DNA clones and radiation hybrids; but the ultimate and by far the most accurate physical map is the actual nucleotide sequence. The completion of the draft human genomic sequence provides us with the best opportunity yet to compare the genetic and physical maps. Here we describe our estimates of female, male and sex-average recombination rates for about 60% of the genome. Recombination rates varied greatly along each chromosome, from 0 to at least 9 centiMorgans per megabase (cM Mb(-1)). Among several sequence and marker parameters tested, only relative marker position along the metacentric chromosomes in males correlated strongly with recombination rate. We identified several chromosomal regions up to 6 Mb in length with particularly low (deserts) or high (jungles) recombination rates. Linkage disequilibrium was much more common and extended for greater distances in the deserts than in the jungles.

Female↗

PubMed2001

IL-12-dependent vascular cell adhesion molecule-1 expression contributes to airway eosinophilic inflammation in a mouse model of asthma-like reaction.

Bronchial-alveolar eosinophilic inflammation is among the characteristic pathological changes in asthma, which has been shown to be correlated with type 2 cytokine and chemokine production. Exogenous IL-12 has been found to be inhibitory for pulmonary eosinophilia in reported studies. Using a murine asthma-like model induced by OVA, we found in the present study that IL-12 gene knockout (KO) mice showed substantially reduced airway recruitment of eosinophils compared with wild-type control mice following OVA sensitization/challenge, although the levels of circulating eosinophils were comparable in these two groups of mice. Cytokine analysis showed Ag-driven Th1 (IFN-gamma) and Th2 (IL-4, IL-5, IL-10, and IL-13) cytokine production by CD4 T cells from local draining lymph nodes and spleen. Similarly, local eotaxin production was comparable in wild-type and IL-12 KO mice. In contrast, immunohistochemical analysis showed that the expression of VCAM-1 on the lung endothelium of IL-12 KO mice was dramatically less than that in wild-type mice. Furthermore, administration of rIL-12 at the stage of sensitization and challenge with OVA restored airway eosinophilia and VCAM-1 expression in IL-12 KO mice. The results suggest that endogenous IL-12 contributes to the recruitment of eosinophils into airways observed in asthma, possibly via enhancement of the expression of VCAM-1 on local vascular endothelial cells.

Administration, Intranasal↗

PubMed2001

Determination of transgenic loci by expression FISH.

DNA targeting by homologous recombination in mouse embryonic stem (ES) cells has become a widely used method for manipulating the mouse genome and for studying the role of specific genes in mammalian development. For certain studies, it is necessary to target two or more DNA sequences residing on a particular chromosome. In these situations, it would be important to distinguish whether two sequential gene targeting events in the ES cells have occurred in cis or in trans. We report here a new application of fluorescence in situ hybridization to RNA molecules present at sites of transcription that allows the identification of cis and trans gene targeting events in ES cells. The method is based on detection of transcripts from commonly used selectable marker genes inserted during homologous recombination. Transcripts are detected in interphase nuclei, making the preparation of mitotic cells unnecessary and obviating the necessity for the more technically demanding DNA detection of genes. The method is applicable to any chromosomal locus, and compared with other methods (e.g., genetic linkage testing in chimeric mice), it will greatly shorten the time required for distinguishing cis and trans gene targeting events in ES cells. The method also may be useful for detecting changes in ploidy of individual chromosomes and loss of heterozygosity of genes in single cells in culture and also in animals, for example, during processes such as tumorigenesis.

Animals↗

PubMed2001

Amplification and cloning of the full-length genome of Japanese encephalitis virus by a novel long RT-PCR protocol in a cosmid vector.

A novel and rapid full-length long RT-PCR technique was established to produce genome-length cDNA from Japanese encephalitis virus. In vitro positive strand RNA transcripts from the full-length RT-PCR amplicon including T7 promoter sequences at the 5' end were proved to be infectious upon transfection. The full-length amplicon without the T7 promoter was cloned into a cosmid vector under the SP6 promoter. This stable clone, designated as pJEV-1, was characterised further and used as a genetic resource for generation of infectious RNA transcripts, gene manipulation and expression. The 'run-off' transcript from pJEV-1 with vector sequences at the either end of the insert was not infectious, but transcripts of the full-length PCR amplicon from pJEV-1 produced infectious virus upon transfection. A transcript with an engineered Xho I site from two ligated PCR fragments amplified from pJEV-1 was also infectious. Furthermore, the coding region for premembrane and envelope proteins (preM-E) from pJEV-1 was subcloned and expressed in the Drosophila Expression System. The expressed protein showed correct molecular size and was immunoreactive with a Japanese encephalitis virus E protein-specific antibody. The derivation of genome-size cDNA from Japanese encephalitis virus and the stable clone will facilitate investigation of this virus and elucidation of its pathogenesis at the molecular level.

Animals↗

PubMed2001

Hepatic lipase gene variation is related to coronary reactivity in healthy young men.

BACKGROUND: Impaired coronary flow reserve (CFR) can be used to indicate vascular dysfunction before the appearance of angiographic lesions. The hepatic lipase (HL) gene has a functional promoter polymorphism at position C-480T, which affects transcription and leads to high activity (C/C) and low activity (C/T, T/T) genotypes. These genotypes modulate HL activity, but their role in coronary artery disease is controversial and the effect on coronary function has not been studied. We investigated whether HL genotypes are associated with coronary artery function in healthy young men. MATERIALS AND METHODS: We studied 49 healthy, mildly hypercholesterolemic men (aged 35 +/- 4 years). Myocardial blood flow was measured at rest and during adenosine induced hyperaemia with positron emission tomography using [15O] H2O. HL genotype was determined by PCR and Nla III enzyme digestion. RESULTS: Resting myocardial blood flow was not statistically different in subjects with high and low activity HL genotypes. However, CFR (the ratio of adenosine flow to resting flow) was 24% higher (4.62 +/- 1.52 vs. 3.73 +/- 1.08 mL g-1 min-1, P = 0.024) in men with the high activity genotype (n = 26) than in those with low activity (n = 23). In multivariate analysis, the HL genotype remained a significant predictor of CFR (P = 0.038) after adjusting for age, body mass index, serum lipids and smoking. CONCLUSIONS: The findings of our preliminary study suggest that the C-480T polymorphism of the HL gene may modify coronary reactivity and reflect differences in the early pathogenesis of coronary dysfunction in these healthy young men. If the association between HL polymorphism and impaired CFR is also present in subjects with other dyslipoproteinemias, the HL polymorphism could be a new risk factor for cardiovascular disease.

Adult↗

PubMed2001

[Design and improvement of stenosis model in cylindrical tubal flow via pulsed Doppler ultrasound in vitro].

A model of disturbed flow downstream of a severe stenosis has been established and improved in vitro, and the relevant method and feasibility for the detection of flow indices are discussed via commercially available Pulsed Doppler Ultrasound (PDU). Detection is done temporally and spatially, upstream and downstream. The methodology is proved to be feasible and has good prosperity of clinical use.

Constriction, Pathologic↗

PubMed2001

[3D visualization and information interaction in biomedical applications].

3D visualization and virtual reality are important trend in the development of modern science and technology, and as well in the studies on biomedical engineering. This paper presents a computer procedure developed for 3D visualization in biomedical applications. The biomedical models are constructed in slice sequences based on polygon cells and information interaction is realized on the basis of OpenGL selection mode in particular consideration of the specialties in this field such as irregularity in geometry and complexity in material etc. The software developed has functions of 3D model construction and visualization, real-time modeling transformation, information interaction and so on. It could serve as useful platform for 3D visualization in biomedical engineering research.

Biomedical Engineering↗

PubMed2001

[Studies on association between lipoprotein lipase gene polymorphisms of Pvu II site and hypertriglyceridemics in Chinese].

OBJECTIVE: To explore whether Pvu II restriction fragment length polymorphisms (RFLP) in the lipoprotein lipase(LPL) gene are associated with hypertriglyceridemics (HTG). METHODS: Pvu II restriction fragment length polymorphisms in the lipoprotein lipase gene on a sample of 135 HTG patients and 193 age-matched healthy individuals in Chengdu area were detected with the method of PCR-RFLP. RESULTS: The P+ P+ genotype frequency and P+ allelic frequency of LPL gene for HTG cases are higher than those for control groups (0.460 vs 0.337,P<0.05; 0.689 vs 0.565,P<0.01). The serum levels of TGapoCII, apoCIII, apoE and TG/HDL-C in P+P+ genotype are higher than those in P-P- genotype(P<0.05). CONCLUSION: The results suggest that P+P+ genotype in the lipoprotein lipase gene is associated with susceptibility to hypertriglyceridemics in Chinese population.

Adult↗

PubMed2001

[Experiment of biodegradation of chlorobenzenes].

Four strains of microorganisms which were able to grow at the presence of 1,4-dichlorobenzene 1,2,4-trichlorobenzene and hexachlorobenzene were isolated from the sludge collected from two different sites after incubating about 2 months. During the growth of the mixed microorganisims on chlorobenzenes, the accumulating consumption of oxygen, the microbial population curve and the concentration of released Cl- were investigated. The result showed that the mixed microorganisms were able to utilize 1,4-dichlorobenzen and 1,2,4-trichlorobenzene as sole carbon source and energy source. The concentration of released Cl- may depend strongly on the activity of the living cells. The order in which the chlorobenzenes were readily degraded was 1,4-dichlorobenzenez[356.7 micrograms/(L.d)] > 1,2,4-trichlorobenzene[110.4 micrograms/(L.d)] > hexachlorobenzene[approximately 6 micrograms/(L.d)]. The number of the substituted chlorine on the chlorobenzenes directly resulted in the resistance to biodegradation.

Bacteria↗

PubMed2000

Improving the performance of MALDI-TOF in oligonucleotide analysis using a new SDIFA technology.

A new technology termed SDIFA is developed to improve the mass resolution of linear matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in oligonucleotide analysis. Unlike the currently used delayed extraction method, SDIFA allows electrical isolation of the sample holder from ion extraction/acceleration and selectively samples part of the desorbed ions, thereby reducing the initial velocity distribution and improving resolution. In addition, a method was introduced to improve the space focusing of TOF. Isotope-limited mass resolution was obtained for oligonucleotides of up to 62 mer, and the true instrumental resolution reaches to 1,800 at 19.2 kDa. It was also demonstrated that excellent resolution was obtained across a large mass range using a single setting of acquisition parameters. This feature allows unambiguous identification of multiple A/T heterozygote samples in a mass range of 5,200-7,800 Da. Moreover, compared with DE, the performance of SDIFA was more stable, reproducible, and less dependent on the experimental conditions including the laser power, sample spots, sample substrates, delayed time, and extraction field strength. This enhanced ease of data acquisition is the key to automated spectrum acquisition.

Oligonucleotides↗

PubMed2000

Effect of diet on colonic-programmed cell death: molecular mechanism of action.

Colon cancer evolves from a progressive inhibition of apoptosis and is influenced strongly by diet. Among dietary factors, butyrate (derived from fermentable fibers) may have utility as a chemopreventive agent because of its ability to promote apoptosis. Because CD95 (APO-1/Fas) transduces signals resulting in apoptosis, we tested the hypothesis that butyrate-dependent colonocyte apoptosis is mediated by this death receptor. Treatment of immortalized mouse colon cells with Fas agonistic antibody induced cell death, indicating that Fas in colonocytes is functional. Antagonism of Fas signaling using a soluble Fas:Fc chimera blocked butyrate induction of apoptosis. Therefore, Fas receptor dependent signal transduction is required for butyrate induction of apoptosis in colonic cells.

Animals↗

PubMed2000

Characterization of the U(L)33 gene product of herpes simplex virus 1.

The U(L)33 protein is one of six genes (including U(L)6, U(L)15, U(L)17, U(L)28, and U(L)32) required for cleavage of viral concatemeric DNA into unit-length genomes and packaging of the virus genomes into preformed capsids. The U(L)25 gene product is dispensable for cleavage of viral DNA but essential for packaging of DNA into capsids. A polyclonal antiserum was produced against an affinity-purified protein containing the full-length U(L)33 gene product of herpes simplex virus 1 fused to glutathione-S-transferase. A protein of approximate M(r) 19,000 that reacted with the antiserum was detected in immunoblots of herpes simplex virus 1-infected cellular lysates. This protein was not detected in lysates of mock-infected cells or cells infected with a mutant virus containing a stop codon in U(L)33, indicating that the 19,000 M(r) protein is the product of the U(L)33 open reading frame. The U(L)33 gene product was not detected in purified virions or capsids. Accumulation of the U(L)33 protein to detectable levels required viral DNA synthesis, indicating that the protein was regulated as a late gene. Indirect immunofluorescence analysis demonstrated that U(L)33 protein accumulated predominantly within replication compartments in the central domains of infected cell nuclei and within the cytoplasm. Localization of the U(L)33 gene product in replication compartments was maintained in cells infected with a variety of cleavage/packaging mutants.

Animals↗

PubMed2000

Enhanced migration and fusion of donor myoblasts in dystrophic and normal host muscle.

Sliced male C57Bl/10Sn (H2-b) donor muscles were grafted into the female histocompatible muscles of untreated, FK506-treated, and T-cell depleted (with or without thymic tolerization) dystrophic (mdx; H2-b) and normal (C57Bl/10Sn; H2-b) hosts, and also into histoincompatible normal (Balb/c; H2-d) hosts. The fate of male donor nuclei was monitored on tissue sections by in situ hybridization with a Y-chromosome specific probe. The results demonstrate that the dystrophic environment is more conducive than normal muscle to donor myoblast migration, with the distance moved being threefold greater at 12 weeks in dystrophic hosts. T-cell depletion was significantly more effective than FK506 treatment at enhancing donor myoblast emigration in both histocompatible and histoincompatible hosts at 3 weeks. Furthermore, the effects of T-cell depletion were sustained in histoincompatible hosts at 12 weeks. These data endorse the use of host T-cell depletion as a promising long-term strategy to improve myoblast transfer therapy (MTT) in the clinical situation.

Animals↗

PubMed2000

IL-10 deficiency prevents IL-5 overproduction and eosinophilic inflammation in a murine model of asthma-like reaction.

Eosinophilic inflammation and bronchial mucus secretion are among the characteristic pathological changes in asthmatic reaction, which is mediated by Th2 type responses. Although it belongs to Th2 cytokines especially in the mouse, IL-10 is often considered an inhibitory cytokine for both Th1 and Th2 cells. In the present study, using a murine asthma model induced by ovalbumin (OVA), we demonstrated that endogenous IL-10 is critical for the development of asthma-like responses. Specifically, in comparison with wild-type controls, IL-10 gene knockout (KO) mice showed significantly reduced IL-5 production, eosinophilic inflammation and mucus production without notable changes in IL-4 and IgE responses following i. p. sensitization and subsequent intranasal challenge with OVA. In addition, Th1-related cytokine (IFN-gamma and IL-12) production in IL-10 KO mice was significantly higher than that in wild-type mice. The results suggest that endogenous IL-10 plays an important role in promoting pulmonary eosinophilic inflammatory reaction and mucus production during asthmatic reaction. The data also argue that IL-10 may be more influential in the development of IL-5-producing Th2 cells which differ from typical Th2 cells producing both IL-4 and IL-5.

Animals↗

PubMed2000

Microdetermination of proteins using m-carboxychlorophosphonazo as detection probe by enhanced resonance light scattering spectroscopy.

Proteins can be determined using a common spectrofluorometer to detect the intensity of resonance light scattering (RLS). Under acidic conditions, the reaction between m-carboxychlorophosphonazo (CPA-mK) and proteins enhances the weak light scattering of CPA-mK drastically. This enhanced intensity is proportional to the concentration of proteins. The linear ranges for human serum albumin are 0.5-35.0 microg/mL, with detection limits of 0.104 microg/mL. The method yields results comparable to those of the calorimetric method using Coomassie Brilliant Blue G-250 (CBB) with relative standard deviations of 0.72-2.10% (n = 10). There is almost no interference by amino acids and most of the metal ions.

Algorithms↗