Biomedical subjects

Xiaohong Fang

Publications and source records attributed to Xiaohong Fang.

8 recordsLinked to original sources

PubMed2003

Monitoring molecular beacon DNA probe hybridization at the single-molecule level.

We have monitored the reaction dynamics of the DNA hybridization process on a liquid/solid interface at the single-molecule level by using a hairpin-type molecular beacon DNA probe. Fluorescence images of single DNA probes were recorded by using total internal reflection fluorescence microscopy. The fluorescence signal of single DNA probes during the hybridization to individual complementary DNA probes was monitored over time. Among 400 molecular beacon DNA probes that we tracked, 349 molecular beacons (87.5 %) were hybridized quickly and showed an abrupt fluorescence increase, while 51 probes (12.5 %) reacted slowly, resulting in a gradual fluorescence increase. This ratio stayed about the same when varying the concentrations of cDNA in MB hybridization on the liquid/surface interface. Statistical data of the 51 single-molecule hybridization images showed that there was a multistep hybridization process. Our results also showed that photostability for the dye molecules associated with the double-stranded hybrids was better than that for those with the single-stranded molecular beacon DNA probes. Our results demonstrate the ability to obtain a better understanding of DNA hybridization processes using single-molecule techniques, which will improve biosensor and biochip development where surface-immobilized molecular beacon DNA probes provide unique advantages in signal transduction.

Biosensing Techniques↗

PubMed2003

Integrative nuclear FGFR1 signaling (INFS) as a part of a universal "feed-forward-and-gate" signaling module that controls cell growth and differentiation.

A novel signaling mechanism is described through which extracellular signals and intracellular signaling pathways regulate proliferation, growth, differentiation, and other functions of cells in the nervous system. Upon cell stimulation, fibroblast growth factor receptor-1 (FGFR1), a typically plasma membrane-associated protein, is released from ER membranes into the cytosol and translocates to the cell nucleus by an importin-beta-mediated transport pathway along with its ligand, FGF-2. The nuclear accumulation of FGFR1 is activated by changes in cell contacts and by stimulation of cells with growth factors, neurotransmitters and hormones as well as by a variety of different second messengers and thus was named integrative nuclear FGFR1 signaling (INFS). In the nucleus, FGFR1 localizes specifically within nuclear matrix-attached speckle-domains, which are known to be sites for RNA Pol II-mediated transcription and co-transcriptional pre-mRNA processing. In these domains, nuclear FGFR1 colocalizes with RNA transcription sites, splicing factors, modified histones, phosphorylated RNA Pol II, and signaling kinases. Within the nucleus, FGFR1 serves as a general transcriptional regulator, as indicated by its association with the majority of active nuclear centers of RNA synthesis and processing, by the ability of nuclear FGFR1 to activate structurally distinct genes located on different chromosomes and by its stimulation of multi-gene programs for cell growth and differentiation. We propose that FGFR1 is part of a universal "feed-forward-and-gate" signaling module in which classical signaling cascades initiated by specific membrane receptors transmit signals to sequence specific transcription factors (ssTFs), while INFS elicited by the same stimuli feeds the signal forward to the common coactivator, CREB-binding protein (CBP). Activation of CBP by INFS, along with the activation of ssTFs by classical signaling cascades brings about coordinated responses from structurally different genes located at different genomic loci.

Animals↗

PubMed2003

Synthetic DNA aptamers to detect protein molecular variants in a high-throughput fluorescence quenching assay.

Real-time protein detection in homogeneous solutions is necessary in many biotechnology and biomedical studies. The recent development of molecular aptamers, combined with fluorescence techniques, may provide an easy and efficient approach to protein elucidation. This report describes the development of a fluorescence-based assay with synthetic DNA aptamers that can detect and distinguish molecular variants of proteins in biological samples in a high-throughput process. We used an aptamer with high affinity for the B chain of platelet-derived growth factor (PDGF), labeled it with a fluorophore and a quencher at the two termini, and measured fluorescence quenching by PDGF. The specific quenching can be used to detect PDGF at picomolar concentrations even in the presence of serum and other cell-derived proteins in cell culture media. This is the first successful application of a synthetic aptamer for the detection of tumor-related proteins directly from the tumor cells. We also show that three highly related molecular variants of PDGF (AA, AB, and BB dimers) can be distinguished from one another in this single-step assay, which can be readily adapted to a microtiter plate assay for high-throughput analysis. The use of fluorescence quenching as a measure of binding between the DNA probe and the target protein eliminates potential false signals that may arise in traditional fluorescence enhancement assays as a result of degradation of the DNA aptamer by contaminating nucleases in biological specimens. This assay is applicable to proteins that are not naturally DNA binding. The excellent specificity, ultrahigh sensitivity, and simplicity of this one-step assay addresses a growing need for high-throughput methods that detect changes in the expression of gene products and their variants in cell cultures and biological specimens.

Animals↗

PubMed2003

Specific aptamer-protein interaction studied by atomic force microscopy.

Aptamers are a new class of synthetic DNA/RNA oligonucleotides generated from in vitro selection to selectively bind with various molecules. Due to their molecular recognition capability for proteins, aptamers are becoming promising reagents in protein detection and new drug development. In this study, the specific interaction between the protein immunoglobulin E (IgE) and its 37-nt aptamer has been measured directly by atomic force microscopy. The single-molecule unbinding force between IgE and the aptamer is determined using the Poisson statistical method. The individual unbinding force between IgE and its monoclonal antibody has also been obtained and compared to that between IgE and the aptamer. The results reveal the high affinity of the aptamer to protein, which could match or even surpass that of the antibody to its antigen.

Humans↗

PubMed2002

Molecular aptamer beacons for real-time protein recognition.

One of the most pressing problems facing those attempting to understand the regulation of gene expression and translation is the necessity to monitor protein production in a variety of metabolic states. Thus far, there is no easy solution that will either identify or quantitate proteins in real time. Here we introduce a novel protein probe, molecular aptamer beacon (MAB), for real time protein recognition and quantitative analysis. The MAB combines the signal transduction mechanism of molecular beacons and the molecular recognition specificity of aptamers. An MAB based on a thrombin-binding aptamer was prepared as a model to demonstrate the feasibility. Significant fluorescent signal change was observed when MAB was bound to thrombin, which is attributed to a significant conformational change in MAB from a loose random coil to a compact unimolecular quadruplex. The MAB recognizes its target protein with high specificity and high sensitivity (112 picomolar thrombin concentration) in homogeneous solutions. Ratiometric imaging has been conducted with MAB labeled with two fluorophores, which makes it feasible for protein quantitation in living specimen. The unique properties of the MAB will enable the development of a class of protein probes for real time protein tracing in living specimen and for efficient biomedical diagnosis in homogeneous solutions.

Base Sequence↗

PubMed2002

Integrative nuclear FGFR1 signaling (INFS) pathway mediates activation of the tyrosine hydroxylase gene by angiotensin II, depolarization and protein kinase C.

The integrative nuclear FGFR1 signaling (INFS) pathway functions in association with cellular growth, differentiation, and regulation of gene expression, and is activated by diverse extracellular signals. Here we show that stimulation of angiotensin II (AII) receptors, depolarization, or activation protein kinase C (PKC) or adenylate cyclase all lead to nuclear accumulation of fibroblast growth factor 2 (FGF-2) and FGFR1, association of FGFR1 with splicing factor-rich domains, and activation of the tyrosine hydroxylase (TH) gene promoter in bovine adrenal medullary cells (BAMC). The up-regulation of endogenous TH protein or a transfected TH promoter-luciferase construct by AII, veratridine, or PMA (but not by forskolin) is abolished by transfection with a dominant negative FGFR1TK-mutant which localizes to the nucleus and plasma membrane, but not by extracellularly acting FGFR1 antagonists suramin and inositolhexakisphosphate (IP6). Mechanism of TH gene activation by FGF-2 and FGFR1 was further investigated in BAMC and human TE671 cultures. TH promoter was activated by co-transfected HMW FGF-2 (which is exclusively nuclear) but not by cytoplasmic FGF-1 or extracellular FGFs. Promoter transactivation by HMWFGF-2 was accompanied by an up-regulation of FGFR1 specifically in the cell nucleus and was prevented FGFR1(TK-) but not by IP6 or suramin. The TH promoter was also transactivated by co-transfected wild-type FGFR1, which localizes to both to the nucleus and the plasma membrane, and by an exclusively nuclear, soluble FGFR1(SP-/NLS) mutant with an inserted nuclear localization signal. Activation of the TH promoter by nuclear FGFR1 and FGF-2 was mediated through the cAMP-responsive element (CRE) and was associated with induction of CREB- and CBP/P-300-containing CRE complexes. We propose a new model for gene regulation in which nuclear FGFR1 acts as a mediator of CRE transactivation by AII, cell depolarization, and PKC.

Adrenal Medulla↗

PubMed2002

Molecular beacons: fluorogenic probes for living cell study.

Molecular beacons are a new class of fluorescent probes that can report the presence of specific nucleic acids with high sensitivity and excellent specificity. In addition to their current wide applications in monitoring the progress of polymerase chain reactions, their unique properties make them promising probes for the detection and visualization of target biomolecules in living cells. This article is focused on our recent research in exploring the potential of using molecular beacon for living-cell studies in three important areas: the monitoring of mRNA in living cells, the development of ultrasmall DNA/RNA biosensors, and the novel approach of combining molecular beacon's signal transduction mechanism with aptamer's specificity for real-time protein detection. These applications demonstrate molecular beacon's unique properties in bioanalysis and bioassay development.

Animals↗

PubMedDate not supplied

AFM and STM study of beta-amyloid aggregation on graphite.

Atomic force microscopy (AFM) and scanning tunneling microscopy (STM) have been employed in situ and ex situ to directly study the aggregation of beta-amyloid(1-42) (Abeta42) peptide on hydrophobic graphite. From in situ AFM images, Abeta42 peptides were seen to aggregate into the sheets that preferred to three orientations with characteristic 3-fold symmetry (Proc. Natl. Acad. Sci. USA 96 (1999) 3688). The sheets were formed by parallel narrow lines with a height of 0.8-1.0nm and a width of 12-14nm. The narrow lines looked like beaded chains and have a right-handed axial periodicity. The high-resolution ex situ AFM and STM images showed that some fibrils of beta-amyloid had a characteristic domain texture, indicating they were formed through the association of protofibrils and monomers. The fibril containing lateral associated filaments that exhibited right-handed twist was clearly observed in the STM image. These results provide important clues to study the detailed structure of beta-amyloid aggregates and the mechanism of the Abeta fibrils formation on hydrophobic surface.

Alzheimer Disease↗