Showing papers by "Nancy Y. Ip published in 2001"

Cdk5 is involved in neuregulin-induced AChR expression at the neuromuscular junction.

Abstract: Here we describe an important involvement of Cdk5/p35 in regulating the gene expression of acetylcholine receptor (AChR) at the neuromuscular synapse. Cdk5 and p35 were prominently expressed in embryonic muscle, and concentrated at the neuromuscular junction in adulthood. Neuregulin increased the p35-associated Cdk5 kinase activity in the membrane fraction of cultured C2C12 myotubes. Co-immunoprecipitation studies revealed the association between Cdk5, p35 and ErbB receptors in muscle and cultured myotubes. Inhibition of Cdk5 activity not only blocked the NRG-induced AChR transcription, but also attenuated ErbB activation in cultured myotubes. In light of our finding that overexpression of p35 alone led to an increase in AChR promoter activity in muscle, Cdk5 activation is sufficient to mediate the up-regulation of AChR gene expression. Taken together, these results reveal the unexpected involvement of Cdk5/p35 in neuregulin signaling at the neuromuscular synapse.

Surface Characterization of a Silicon-Chip-Based DNA Microarray

Abstract: The immobilization of DNA (deoxyribonucleic acid) on solid supports is a crucial step for any application in the field of DNA microarrays. It determines the efficacy of the hybridization and influences the signal strength for the detection. We used solid supports made from silicon wafers as an alternative substrate to the commonly used microscope glass slides. The covalent immobilization of thiol-terminated DNA oligonucleotides on self-assembled layers of (3-mercaptopropyl)trimethoxysilane (MPTS) by disulfide bond formation was investigated. Contact angle measurement, variable angle spectral ellipsometry (VASE), X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM) were used to characterize the changing properties of the surface during the DNA array fabrication. During wafer processing the contact angle changed from 3° for the hydroxylated surface to 48.5° after deposition of MPTS. XPS data demonstrated that all sulfur in the MPTS layer was present in the form of reduced SH or S−S grou...

Expression of the P2Y1 nucleotide receptor in chick muscle: its functional role in the regulation of acetylcholinesterase and acetylcholine receptor.

Abstract: In vertebrate neuromuscular junctions, ATP is stored at the motor nerve terminals and is co-released with acetylcholine during neural stimulation. Here, we provide several lines of evidence that the synaptic ATP can act as a synapse-organizing factor to induce the expression of acetylcholinesterase (AChE) and acetylcholine receptor (AChR) in muscles, mediated by a metabotropic ATP receptor subtype, the P2Y1 receptor. The activation of the P2Y1receptor by adenine nucleotides stimulated the accumulation of inositol phosphates and intracellular Ca2+ mobilization in cultured chick myotubes. P2Y1 receptor mRNA in chicken muscle is very abundant before hatching and again increases in the adult. The P2Y1 receptor protein is shown to be restricted to the neuromuscular junctions and colocalized with AChRs in adult muscle (chicken, Xenopus , and rat) but not in the chick embryo. In chicks after hatching, this P2Y1 localization develops over ∼3 weeks. Denervation or crush of the motor nerve (in chicken or rat) caused up to 90% decrease in the muscle P2Y1 transcript, which was restored on regeneration, whereas the AChR mRNA greatly increased. Last, mRNAs encoding the AChE catalytic subunit and the AChR α-subunit were induced when the P2Y1 receptors were activated by specific agonists or by overexpression of P2Y1 receptors in cultured myotubes; those agonists likewise induced the activity in the myotubes of promoter–reporter gene constructs for those subunits, actions that were blocked by a P2Y1-specific antagonist. These results provide evidence for a novel function of ATP in regulating the gene expression of those two postsynaptic effectors.

Chips and Qi: microcomponent-based analysis in traditional Chinese medicine

Abstract: Over the last 50 years or so Traditional Chinese medicine (TCM) has been subject to intensive basic and clinical research. Although the effectiveness and remarkable safety of TCM have been documented after controlled clinical studies, there are several herbal and animal parts that are toxic or difficult to identify. DNA polymorphism-based assays have recently been developed for the identification of herbal medicines. In this approach, small amounts of DNA are amplified by the polymerase chain reaction and the reactions products are analyzed by gel electrophoresis, sequencing, or hybridization with species-specific probes. With the DNA based identification of TCM materials as an example, chip-based analytical micro devices were developed with the goal of fabricating an integrated device that will enable sample preparation, amplification, and analysis on a single microchip-based device ("lab-on-a-chip"). The application of a silicon-based polymerase chain reaction microreactor and a DNA microarray for the DNA sequence-based identification of toxic medicinal plants is reported here.

N-methyl-d-aspartate receptor antagonists

Abstract: The present invention is based in part on the surprising discovery that Tanshinones from Salvia miltiorrhiza act as allosteric high-potency N-methyl-D-aspartate receptor antagonists. Pharmacological blockade of excessive activation of N-methyl-D-aspartate receptors (NMDARs) greatly reduces ischemic injury of neurons in cell culture and animal models. Tanshinones thus represent a novel class of compounds with NMDA receptor blocking activities with potential for the development of safe neuroprotective drugs for therapy of stroke and other neurodegenerative and neuropsychiatric disorders.

A reporter gene assay for the detection of phytoestrogens in traditional Chinese medicine.

Abstract: Bupleurum & Peony Formula (Jia Wei Xiao Yao San) is a herbal formula which possesses a clinical history for the treatment of menopausal syndrome and menstrual irregularity. The present investigation reports the ability to monitor the formula's phytoestrogen content that will allow for the implementation of a standardization protocol that is based on a quantifiable biological response. Utilizing an oestrogen-sensitive chimeric receptor/reporter gene element which has been stably transfected into HeLa cells, the botanical formula was shown to induce the expression of the reporter gene, luciferase, in a dose dependent manner. Pretreatment of the HeLa cells with the botanical formula produced a 5-fold increase in bioluminescence compared with the control. Additionally, our studies showed that the response of the cells, when challenged by the botanical formula, was oestrogen specific. Pretreatment of the cells with tamoxifen effectively blocked the activation of the chimeric oestrogen receptor by the botanical formula. The cell line provides a sensitive assay that can easily detect the presence of phytoestrogens in complex botanical formulas.

Expression of Retinoid Receptors During the Retinoic Acid-Induced Neuronal Differentiation of Human Embryonal Carcinoma Cells

Abstract: Retinoic acid (RA), a derivative of vitamin A, is essential for normal patterning and neurogenesis during development. Until recently, studies have been focused on the physiological roles of RA receptors (RARs), one of the two types of nuclear receptors, whereas the functions of the other nuclear receptors, retinoid X receptors (RXRs), have not been explored. Accumulating evidence now suggests that RXRalpha is a critical receptor component mediating the effects of RA during embryonic development. In this study, we have examined the expression profiles of RXRalpha and RARs during the RA-induced neuronal differentiation in a human embryonal carcinoma cell line, NT2. Distinct expression profiles of RXRalpha, RARalpha, RARbeta, and RARgamma were observed following treatment with RA. In particular, we found that RA treatment resulted in a biphasic up-regulation of RXRalpha expression in NT2 cells. The induced RXRalpha was found to bind specifically to the retinoid X response element based on gel mobility retardation assays. Furthermore, immunocytochemical analysis revealed that RXRalpha expression could be localized to the somatoaxonal regions of the NT2 neurons, including the tyrosine hydroxylase- and vasoactive intestinal peptide-positive neurons. Taken together, our findings provide the first demonstration of the cellular localization and regulation of RXRalpha expression in NT2 cells and suggest that RXRalpha might play a crucial role in the cellular functions of human CNS neurons.

Optical response of photodiode in integrated DNA detection system

Abstract: This paper describes the fabrication and optical response of photodiodes when they are used in an integrated detection system for DNA analysis. Compared with the traditional approach of external CCD camera based detection systems, the proposed approach can produce a more highly integrated system with higher density. By the use of the natural spectral sensitivity of the photodiode, the need for color filters is eliminated in the integration. Experimental results show that matched and unmatched DNA samples with optical labels can be distinguished by the measured photodiode current when properly excited by optical illumination.

Surface characterization of DNA microarray on silicon dioxide and compatible silicon materials in the immobilization process

Abstract: In this work, the surface properties of a DNA microarray formed on silicon based solid support are studied at different stages during the hybridization process. A modified immobilization process using the covalent immobilization of thiol-terminated DNA oligonucleotides on self-assembled layers of (3-mercaptopropyl) trimethoxysilane (MPTS) by disulfide bond formation is used to selectively attach DNA probes onto the surface of silicon dioxide. Contact angle measurement is used to monitor the bonding of MPTS on the surface. Atomic force microscopy (AFM) shows an increase in particle size before and after the growth of the MPTS layer. Fluorescence microscopy reveals the success of hybridization of complementary oligonucleotides labeled by FAM to the probe. The effects of modified immobilization process on other common material in silicon processing are also studied. As a result of the corrosive chemical used in the process, common metals used in micro-fabrication processes like aluminum are etched away. Silicon nitride is not affected by the immobilization and hybridization process, and thus can be used as a passivation and isolation material to conform the DNA to a specific area for DNA microarray to reduce cross-talk. The fluorescence image from the scanner indicates silicon nitride can effectively be used as an isolation material with linewidth down to 1 μm.

Characterization of immobilized DNA probes on the surface of silicon compatible materials

Abstract: In this paper, the objectives and methodologies of deoxyribonucleic acid (DNA) analysis based on micro arrays produced by microelectronic fabrication technology is reviewed. The important issue of material compatibility between DNA material and silicon based material is addressed. With a special surface treatment, DNA probes have been successfully attached (or immobilized) on the surface of silicon compatible materials (mainly silicon dioxide). Through the use of optical labels that emit optical fluorescence under the illumination of a UV light source, the number of DNA probes immobilized on the silicon dioxide surface can be quantified. The results are further analyzed with atomic force microscopy to visualize the surface conditions. The successful immobilization of DNA probes on silicon wafers is very important for the fabrication of DNA chips for DNA analysis.