Showing papers by "Nancy Y. Ip published in 2002"

Integration of Signals from Receptor Tyrosine Kinases and G Protein-Coupled Receptors

Abstract: Activation of G protein-coupled receptors (GPCRs) leads to stimulation of classical G protein signaling pathways. In addition, GPCRs can activate the mitogen-activated protein kinases (MAPKs) such as

Genotyping on a complementary metal oxide semiconductor silicon polymerase chain reaction chip with integrated DNA microarray

Abstract: A novel method for the fast identification of genetic material utilizing a micro-DNA amplification and analysis device (μ-DAAD) consisting of multiple PCR microreactors with integrated DNA microarrays was developed. The device was fabricated in Si-technology and used for the genotyping of Chinese medicinal plants on the basis of differences in the noncoding region of the 5S-rRNA gene. Successful amplification of the genetic material and the consecutive analysis of the fluorescent-labeled amplicons in the μ-DAAD by the integrated oligonucleotide probes were demonstrated. Parallel analysis was performed by loading the four PCR reactors of the μ-DAAD with different samples of 3-μL volume. Temperature sensors and heating elements of the μ-DAAD enable precise temperature control and fast cycling, allowing the rapid completion of a combined amplification and analysis (hybridization) experiment.

Induction of Cdk5 activity in rat skeletal muscle after nerve injury.

Abstract: Cyclin-dependent kinase 5 (Cdk5) was originally identified as a serine/threonine kinase and subsequently demonstrated to play a critical role in the development of CNS. We recently reported the novel function of Cdk5 in the neuregulin signaling pathway during the development of neuromuscular junction (NMJ). Here, we report the regulation of Cdk5 and p35 in rat skeletal muscle after nerve injury. Northern blot analysis revealed that Cdk5 and p35 transcripts were up-regulated in muscle after nerve denervation. The temporal profiles for the regulation of Cdk5 and p35 transcripts were different, suggesting that these changes in gene transcription might be regulated by different mechanism. Our finding on the ability of tetrodotoxin to induce p35 transcript in muscle suggested that electrical activity could regulate p35 expression. In addition to the induction of mRNA expression, the total Cdk5 and p35-associated kinase activity in muscle increased prominently after nerve denervation. Taken together, our findings suggest that Cdk5 and p35 may play important physiological roles in muscle regeneration following nerve injury.

Identification of Candidate Genes Induced by Retinoic Acid in Embryonal Carcinoma Cells

Abstract: Retinoic acid (RA) induced the terminal differentiation of a human embryonal carcinoma cell line (NT2/D1) into several morphologically distinct cell types, including the postmitotic CNS neurons. Although RA has been suggested to play an important role in brain development, little is known about the molecular mechanism by which RA induces neuronal differentiation. In the present study, RNA fingerprinting by arbitrarily primed PCR (RAP-PCR) was used to identify the transcripts in NT2/D1 cells that were differentially regulated by RA. Northern blot analysis of the differentially amplified PCR fragments revealed 11 genes that were regulated by RA. Of these, seven were up-regulated and four were down-regulated along the course of RA treatment. More importantly, four of the RA-regulated genes that were identified in the present study are novel. Our findings suggested that there are a number of RA-regulated genes that have yet to be identified. RAP-PCR provides a useful tool for studying the patterns of transcript expression during the course of RA treatment and allows the cloning of novel genes involved in the process of neuronal differentiation. Furthermore, it provides a basis for the selection of genes that are involved in the RA-induced signaling pathway in the human CNS.

Cloning of the α Component of the Chick Ciliary Neurotrophic Factor Receptor: Developmental Expression and Down-Regulation in Denervated Skeletal Muscle

Abstract: A full-length cDNA clone encoding for the chick CNTFR alpha (alpha component of the ciliary neurotrophic factor receptor) was isolated by screening an embryonic day 13 chick brain cDNA library with a rat CNTFR alpha probe. The isolated cDNA clone contained a approximately 2-kb insert with an open reading frame of 362 amino acids. The identification of this clone as chick CNTFR alpha was based on the homology in amino acid sequence (approximately 70%) with the rat and human CNTFR alpha. Hydropathy analysis revealed that the chick CNTFR alpha contains a hydrophobic region at the amino terminus that is typical of secretory signal peptides, as well as a hydrophobic region at the carboxyl terminus that is characteristic of glycosylphosphatidylinositol-linked proteins. The expression of chick CNTFR alpha was developmentally regulated and was widely distributed in neural tissues, such as brain and spinal cord. In the periphery, chick CNTFR alpha transcript was expressed at high levels in the skeletal muscle and was only barely detectable in the liver. Unexpectedly, the expression of chick CNTFR alpha mRNA in skeletal muscle was decreased by approximately 10-fold at 1.5 days after denervation. This is in sharp contrast to the result previously obtained with CNTFR alpha in denervated rat muscle.

Expression of Cdk5 and its activators in NT2 cells during neuronal differentiation.

Abstract: We have recently developed a rapid protocol involving NT2 cell aggregation and treatment with retinoic acid (RA) to produce terminally differentiated CNS neurons. As a first step to explore the functional roles of cell-cycle regulatory proteins in the process of neuronal differentiation, the expression profiles of cyclin-dependent kinases (Cdks) and their regulators were examined in NT2 cells following treatment with RA. One of the Cdks, Cdk5, has been demonstrated to affect the process of neuronal differentiation and suggested to play an important role in development of the nervous system. We found that the expression of Cdk5 was gradually increased, while its activators (p35 and p39) as well as Cdk5 kinase activity were induced in NT2 cells during the process of neuronal differentiation. Moreover, both p35 and p39 were localized along the axons and varicosity-like structures of differentiated NT2 neurons. Taken together, our results demonstrated that NT2 cells provide a good in vitro model system to examine signaling pathways involved in the regulation of Cdk5 activators and to elucidate the functional roles of Cdk5 in neuronal differentiation.

Differential expression of ciliary neurotrophic factor receptor in skeletal muscle of chick and rat after nerve injury

Abstract: The activities of ciliary neurotrophic factor (CNTF) were initially thought to be restricted to cells in the nervous system. However, the recent identification of its receptor specificity-conferring alpha component (CNTFR alpha) in skeletal muscle has provided the clue to the unexpected actions of CNTF in the periphery. In the present study, we demonstrated that the mRNA expression of CNTFR alpha in chick skeletal muscle was decreased by approximately 10-fold after nerve transection; this finding is in sharp contrast to the dramatic up-regulation observed in denervated rat muscle. As a first step toward investigating the differential regulation of CNTFR alpha in chick and rat, we examined the mRNA expression of CNTFR alpha in different types of muscle following nerve injury in young and adult animals. Our findings demonstrated that the differential expression of CNTFR alpha observed in denervated skeletal muscle of the chick and rat was not dependent on age or muscle type. The temporal profile of the changes in CNTFR alpha expression was, however, dependent on the age of the chick as well as the types of muscles. Furthermore, the low level of CNTFR alpha expression observed in denervated chick muscle recovered to almost control levels in regenerating skeletal muscle. Taken together, our findings provided the first extensive analysis on the mRNA expression of CNTFR alpha and the alpha subunit of the acetylcholine receptor in various skeletal muscles of the chick following nerve injury and regeneration.

Agrin-Deficient Myotube Retains Its Acetylcholine Receptor Aggregation Ability when Challenged with Agrin

Abstract: Agrin is a synapse-organizing molecule that mediates the nerve-induced aggregation of acetylcholine receptors (AChRs) and other postsynaptic components at the developing and regenerating vertebrate neuromuscular junctions. At the neuromuscular junction, three different cell types can express agrin, i.e., neuron, muscle, and Schwann cell. Several lines of evidence suggested that neuron-derived agrin is the AChR-aggregating factor, but the possible roles of muscle-derived agrin in the formation of AChR aggregate are not known. By using the recombinant DNA method, a clonal stable C2C12 cell line transfected with antisense agrin cDNA was created. RNA dot blot and western blot analysis indicated that the expression of agrin in the transfected cell was abolished by DNA transfection. When the agrin-deficient C2C12 cells were induced to form myotubes and subsequently cocultured with agrin cDNA transfected fibroblasts, AChR aggregates were formed in the cocultures. In addition, acetylcholinesterase (AChE) aggregates in agrin-deficient myotubes were also induced by exogenous agrin and the AChE aggregates were colocalized with the AChR aggregates. The agrin-deficient myotubes could also respond to neuron-induced AChR aggregation after coculturing with neuroblastoma cells. Thus, the agrin-deficient myotubes retain their ability to exhibit the agrin- or neuron-induced AChR aggregation. This result suggests that the formation of postsynaptic specializations during development and regeneration is mediated by neuron-derived agrin but not the agrin from muscle.