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Biomedical subjects

Y Fan

Publications and source records attributed to Y Fan.

At least 19 recordsLinked to original sources
PubMed2003

Nondestructive evaluation of explosively welded clad rods by resonance acoustic spectroscopy.

A resonance acoustic spectroscopy technique is assessed for nondestructive evaluation of explosively welded clad rods. Each rod is modeled as a two-layered cylinder with a spring-mass system to represent a thin interfacial layer containing the weld. A range of interfacial profiles is generated in a set of experimental samples by varying the speed of the explosion that drives the copper cladding into the aluminum core. Excellent agreement is achieved between measured and calculated values of the resonant frequencies of the system, through appropriate adjustment of the interfacial mass and spring constants used in the wave scattering calculations. Destructive analysis of the interface in the experimental specimens confirms that key features of the interfacial profile may be inferred from resonance acoustic spectroscopy analysis applied to ultrasonic measurements.

Acoustics↗
PubMed2001

Preparing a human membrane and secreted protein-enriched cDNA library using PCR primers derived from a genomic database.

We describe here a strategy for preparing a human membrane and secreted protein (MSP)-enriched cDNA library based on human MSP- and non-MSP-encoding cDNA sequences in the databases. The signal peptide parts of the MSP-encoding cDNA sequences, which currently comprise about half of the estimated total number in humans, were analyzed for common patterns. These patterns form a 'minimal' set of polymerase chain reaction primer candidates of length varying from 9 to 21 nt. The products stemming from each primer candidate were determined and the results allowed us to obtain an 'optimal' mixed-length primer set. Ninety-six percent of the primers in this set were predicted to yield </=10% undesired products, and the desired MSP-cDNA products could be easily separated by gel electrophoresis. The present analysis establishes a methodology for preparing a cDNA library that enables the analysis of individual MSPs. This methodology may also help identify new MSPs. As many cell regulatory processes are mediated by secreted proteins and their membrane-bound receptors, the preparation of a MSP-enriched cDNA library should benefit research on MSPs.

Amino Acid Sequence↗
PubMed2001

Quantitative structure-antitumor activity relationships of camptothecin analogues: cluster analysis and genetic algorithm-based studies.

Topoisomerase 1 (top1) inhibitors are proving useful against a range of refractory tumors, and there is considerable interest in the development of additional top1 agents. Despite crystallographic studies, the binding site and ligand properties that lead to activity are poorly understood. Here we report a unique approach to quantitative structure-activity relationship (QSAR) analysis based on the National Cancer Institute's (NCI) drug databases. In 1990, the NCI established a drug discovery program in which compounds are tested for their ability to inhibit the growth of 60 different human cancer cell lines in culture. More than 70 000 compounds have been screened, and patterns of activity against the 60 cell lines have been found to encode rich information on mechanisms of drug action and drug resistance. Here, we use hierarchical clustering to define antitumor activity patterns in a data set of 167 tested camptothecins (CPTs) in the NCI drug database. The average pairwise Pearson correlation coefficient between activity patterns for the CPT set was 0.70. Coherence between chemical structures and their activity patterns was observed. QSAR studies were carried out using the mean 50% growth inhibitory concentrations (GI(50)) for 60 cell lines as the dependent variables. Different statistical methods, including stepwise linear regression, principal component regression (PCR), partial least-squares regression (PLS), and fully cross-validated genetic function approximation (GFA) were applied to construct quantitative structure-antitumor relationship models. For our data set, the GFA method performed better in terms of correlation coefficients and cross-validation analysis. A number of molecular descriptors were identified as being correlated with antitumor activity. Included were partial atomic charges and three interatomic distances that define the relative spatial dispositions of three significant atoms (the hydroxyl hydrogen of the E-ring, the lactone carbonyl oxygen of the E-ring, and the carbonyl oxygen of the D-ring). The cross-validated r(2) for the final GFA model was 0.783, indicating a predictive QSAR model.

Algorithms↗
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

Enhanced apoptosis under low serum conditions in human glioblastoma cells by connexin 43 (Cx43).

Connexin 43 (Cx43), a structural component of gap junctions, is believed to function as a tumor suppressor gene. Previously, we showed that expression of Cx43 suppresses cell proliferation and tumorigenicity of human glioblastoma cells [Huang et al., Cancer Res 58:5089-5096, 1998] and enhances apoptosis in response to chemotherapeutic agents [Huang et al., Int J Cancer 92:130-138, 2001]. In the present study, we demonstrated that expression of Cx43 in human glioblastoma cells potentiated an apoptotic program under low-serum conditions. The Cx43-mediated effect was coupled with a decreased expression of the specific apoptosis-inhibitor bcl-2. Overexpression of bcl-2 in Cx43-transfected cells conferred resistance to apoptosis induced under low-serum conditions, suggesting that the Cx43-mediated apoptosis under low-serum conditions is regulated, in part, through the downregulation of bcl-2 expression. Furthermore, application of the phosphatidylinositol-3'-OH kinase inhibitor LY294002 specifically induced apoptosis in Cx43-transfected cells. Our results demonstrate a new role of Cx43 in the mediation of apoptosis under low serum conditions.

Apoptosis↗
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

Investigation of skeletal muscle denervation and reinnervation using magnetic resonance spectroscopy.

OBJECTIVE: To determine changes in skeletal muscle metabolism after nerve transection and repair and to correlate metabolic changes with functional recovery. STUDY DESIGN AND SETTING: Male Wistar rats were divided into 6 experimental groups plus a control group. The posterior tibial nerve was transected and reapproximated. At varying times after surgery (1, 2, 4, 6, or 8 weeks) animals were sacrificed, the gastrocnemius muscle was harvested, and proton nuclear magnetic resonance (NMR) spectroscopy was performed. Functional recovery was measured using the sciatic function index. RESULTS: Animals undergoing nerve repair all showed functional recovery whereas the nonrepaired nerve group did not. Concentration of glucose and lactate increased after denervation and then returned toward normal. Choline concentration decreased and then returned toward normal. In animals not undergoing nerve repair, the metabolic abnormalities persisted and showed no sign of recovery over the 8-week observation period. CONCLUSIONS: 1H NMR spectroscopy is a potentially useful tool to study changes in skeletal muscle metabolism after motor nerve injury. SIGNIFICANCE: NMR spectroscopy is rapidly developing into a clinically useful tool. High-field magnets have improved resolution and data acquisition. Basic experiments, such as those described here, will help guide the use of NMR spectroscopy in clinical medicine and will also lead to a better understanding of basic mechanisms of nerve injury and repair.

Action Potentials↗
PubMed2001

Individual somatic H1 subtypes are dispensable for mouse development even in mice lacking the H1(0) replacement subtype.

H1 linker histones are involved in facilitating the folding of chromatin into a 30-nm fiber. Mice contain eight H1 subtypes that differ in amino acid sequence and expression during development. Previous work showed that mice lacking H1(0), the most divergent subtype, develop normally. Examination of chromatin in H1(0-/-) mice showed that other H1s, especially H1c, H1d, and H1e, compensate for the loss of H1(0) to maintain a normal H1-to-nucleosome stoichiometry, even in tissues that normally contain abundant amounts of H1(0) (A. M. Sirotkin et al., Proc. Natl. Acad. Sci. USA 92:6434-6438, 1995). To further investigate the in vivo role of individual mammalian H1s in development, we generated mice lacking H1c, H1d, or H1e by homologous recombination in mouse embryonic stem cells. Mice lacking any one of these H1 subtypes grew and reproduced normally and did not exhibit any obvious phenotype. To determine whether one of these H1s, in particular, was responsible for the compensation present in H1(0-/-) mice, each of the three H1 knockout mouse lines was bred with H1(0) knockout mice to generate H1c/H1(0), H1d/H1(0), or H1e/H1(0) double-knockout mice. Each of these doubly H1-deficient mice also was fertile and exhibited no anatomic or histological abnormalities. Chromatin from the three double-knockout strains showed no significant change in the ratio of total H1 to nucleosomes. These results suggest that any individual H1 subtype is dispensable for mouse development and that loss of even two subtypes is tolerated if a normal H1-to-nucleosome stoichiometry is maintained. Multiple compound H1 knockouts will probably be needed to disrupt the compensation within this multigene family.

Animals↗
PubMed2001

Association of caries activity with the composition of dental plaque fluid.

This study tests the hypothesis that caries activity is associated with lower degrees of saturation with respect to enamel mineral in dental plaque fluid following sucrose exposure. Plaque fluids were obtained from caries-free, caries-positive, and caries-active subjects. Samples were collected before and at 3 and 7 min after a sucrose rinse on consecutive weeks and analyzed for organic acids, inorganic ions, pH, calcium activity, and, in selected samples, total protein. After sucrose, pH values were significantly lower in the caries-active group in comparison with the caries-free and caries-positive groups. Total and free calcium concentrations increased with decreasing pH, with free calcium being about one-third of total calcium. The caries-active group exhibited significantly lower degrees of saturation with respect to enamel mineral, after sucrose, and had significantly higher mutans streptococci levels in plaque than did the caries-free samples. Thus, saturation levels in post-sucrose plaque fluids reflect the cariogenic potential of dental plaque.

Adolescent↗
PubMed2001

An isoform of eIF4E is a component of germ granules and is required for spermatogenesis in C. elegans.

Control of gene expression at the translational level is crucial for many developmental processes. The mRNA cap-binding protein, eIF4E, is a key player in regulation of translation initiation; appropriate levels of eIF4E are essential for normal cell-cycle regulation and tissue differentiation. The observation that eIF4E levels are elevated during gametogenesis in several organisms suggests that eIF4E might have a specific role in gamete formation as well. We show that one of the five isoforms of C. elegans eIF4E, IFE-1, is enriched in the germline and is a component of germ granules (P granules). The association of IFE-1 with P granules requires the P-granule protein PGL-1. In vitro PGL-1 interacts directly with IFE-1, but not with the other four isoforms of eIF4E. Analysis of animals depleted of IFE-1 by RNAi shows that IFE-1 is required for spermatogenesis, specifically for efficient progression through the meiotic divisions and for the production of functional sperm, in both hermaphrodites and males. The requirement for IFE-1 is highly sensitive to temperature. IFE-1 is not required for oogenesis, as ife-1(RNAi) hermaphrodites produce viable progeny when normal sperm are supplied. Consistent with a primary role in spermatogenesis, ife-1 mRNA levels are highest in regions of the gonad undergoing spermatogenesis. Our results suggest that C. elegans spermatogenesis requires either this specific isoform of eIF4E or an elevated level of eIF4E.

Animals↗