JoVE   
You do not have subscription access to articles in this section. Learn more about access.

  JoVE General

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Neuroscience

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Immunology and Infection

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Clinical and Translational Medicine

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Bioengineering

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Applied Physics

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Chemistry

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Behavior

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Environment

|   

JoVE Science Education

General Laboratory Techniques

You do not have subscription access to videos in this collection. Learn more about access.

Basic Methods in Cellular and Molecular Biology

You do not have subscription access to videos in this collection. Learn more about access.

Model Organisms I

You do not have subscription access to videos in this collection. Learn more about access.

Model Organisms II

You have trial access to videos in this collection until May 31, 2014.

In JoVE (1)

Other Publications (14)

Articles by Ping Chi in JoVE

 JoVE General

Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice

1Department of Medicine, Memorial Sloan Kettering Cancer Center, 2Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, 3Developmental Biology Program, Memorial Sloan Kettering Cancer Center, 4Howard Hughes, Medical Institute, 5Laboratory of Chromatin Biology and Epigenetics, The Rockefeller University


JoVE 2802

The interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. They form complex networks between smooth muscle cells and post-ganglionic neuronal fibers to regulate GI contractility. Here, we present immunofluorescence methods cross-sectional and whole-mount visualization of murine ICC networks.

Other articles by Ping Chi on PubMed

Roscovitine: a Novel Regulator of P/Q-type Calcium Channels and Transmitter Release in Central Neurons

Roscovitine is widely used for inhibition of cdk5, a cyclin-dependent kinase expressed predominantly in the brain. A novel function of roscovitine, i.e. an effect on Ca(2+) channels and transmitter release in central neurons, was studied by whole-cell voltage-clamp recordings and time-lapse fluorescence imaging techniques. Extracellular application of roscovitine markedly enhanced the tail calcium current following repolarization from depolarized voltages. This effect was rapid, reversible and dose dependent. Roscovitine dramatically slowed the deactivation kinetics of calcium channels. The deactivation time constant was increased 3- to 6-fold, suggesting that roscovitine could prolong the channel open state and increase the calcium influx. The potentiation of tail calcium currents caused by roscovitine and by the L-channel activator Bay K 8644 was not occluded but additive. Roscovitine-induced potentiation of tail calcium currents was significantly blocked by the P/Q-channel blocker CgTx-MVIIC, indicating that the major target of roscovitine is the P/Q-type calcium channel. In mutant mice with targeted deletion of p35, a neuronal specific activator of cdk5, roscovitine regulated calcium currents in a manner similar to that observed in wild-type mice. Moreover, intracellular perfusion of roscovitine failed to modulate calcium currents. These results suggest that roscovitine acts on extracellular site(s) of calcium channels via a cdk5-independent mechanism. Roscovitine potentiated glutamate release at presynaptic terminals of cultured hippocampal neurons detected with the vesicle trafficking dye FM1-43, consistent with the positive effect of roscovitine on the P/Q-type calcium channel, the major mediator of action potential-evoked transmitter release in the mammalian CNS.

Regulation of Neurotransmitter Release by Synapsin III

Synapsin III is the most recently identified member of the synapsin family, a group of synaptic vesicle proteins that play essential roles in neurotransmitter release and neurite outgrowth. Here, through the generation and analysis of synapsin III knock-out mice, we demonstrate that synapsin III regulates neurotransmitter release in a manner that is distinct from that of synapsin I or synapsin II. In mice lacking synapsin III, the size of the recycling pool of synaptic vesicles was increased, and synaptic depression was reduced. The number of vesicles that fuse per action potential was similar between synapsin III knock-out and wild-type mice, and there was no change in the quantal content of EPSCs; however, IPSCs were greatly reduced in synapsin III-deficient neurons. The density and distribution of synaptic vesicles in presynaptic terminals did not appear to be different in synapsin III knock-out mice in comparison to wild-type littermates. In addition to the changes in neurotransmitter release, we observed a specific delay in axon outgrowth in cultured hippocampal neurons from synapsin III knock-out mice. Our data indicate that synapsin III plays unique roles both in early axon outgrowth and in the regulation of synaptic vesicle trafficking.

Synaptic Vesicle Mobilization is Regulated by Distinct Synapsin I Phosphorylation Pathways at Different Frequencies

During action potential firing, the rate of synapsin dissociation from synaptic vesicles and dispersion into axons controls the rate of vesicle availability for exocytosis at the plasma membrane. Here we show that synapsin Ia's dispersion rate tracks the synaptic vesicle pool turnover rate linearly over the range 5-20 Hz and that the molecular basis for this lies in regulation at both the calcium-calmodulin-dependent kinase (CaM kinase) and the mitogen-activated protein (MAP) kinase/calcineurin sites. Our results show that CaM kinase sites control vesicle mobilization at low stimulus frequency, while MAP kinase/calcineurin sites are critical at both lower and higher stimulus frequencies. Thus, multiple signaling pathways serve to allow synapsin's control of vesicle mobilization over different stimulus frequencies.

Genomic Analysis of Glucocorticoid-regulated Promoters in Murine T-lymphoma Cells

We have undertaken a high-throughput analysis to identify targets of glucocorticoid regulation in P1798 murine T-lymphoma cells. G1/S-arrested cultures were treated for 8 hours with 0.1 microM dexamethasone (dex) in the presence and absence of 1 microg/ml cycloheximide. Untreated cultures and cultures exposed to cycloheximide alone were prepared as controls. RNA was isolated and gene expression analyzed using Affymetrix MG-U74A oligonucleotide arrays (Gene Chips). Three independent experiments were performed. The data were analyzed using a variety of statistical and analytical approaches in order to identify primary transcriptional targets of the glucocorticoid receptor. We identified 44 genes that increase by > 2-fold in both dex-treated and dex + cycloheximide-treated cultures (relative to control and cycloheximide-treated cultures) in three replicate experiments. Statistical analysis of control data indicate that the probability that a given probeset would, as a result of random error, increase > 2-fold both in the presence and absence of cycloheximide in two independent experiments is approximately 7 x 10(-9). We have retrieved from the Celera mouse genomic sequence 8 kb of promoter sequence, spanning 4 kb either side of the 5'-end of the cDNA from eight of the induced genes. These sequences were analyzed for potential glucocorticoid receptor binding sites. Five of these genes contain the sequence ACAnnnTGTnCT within 4 kb of the presumptive transcriptional start site. Eight control genes were selected at random and analyzed for the sequence ACAnnnTGTnCT. Two control genes had such sequences within 4 kb of the transcriptional start site.

Chromatin Remodeling and Cancer, Part I: Covalent Histone Modifications

Dynamic chromatin remodeling underlies many, if not all, DNA-templated biological processes, including gene transcription; DNA replication and repair; chromosome condensation; and segregation and apoptosis. Disruption of these processes has been linked to the development and progression of cancer. The mechanisms of dynamic chromatin remodeling include the use of covalent histone modifications, histone variants, ATP-dependent complexes and DNA methylation. Together, these mechanisms impart variation into the chromatin fiber, and this variation gives rise to an 'epigenetic landscape' that extends the biological output of DNA alone. Here, we review recent advances in chromatin remodeling, and pay particular attention to mechanisms that appear to be linked to human cancer. Where possible, we discuss the implications of these advances for disease-management strategies.

Chromatin Remodeling and Cancer, Part II: ATP-dependent Chromatin Remodeling

Connections between perturbations that lie outside of our genome, that is, epigenetic alternations, and tumorigenesis have become increasingly apparent. Dynamic chromatin remodeling of the fundamental nucleosomal structure (covered in this review) or the covalent marks residing in the histone proteins that make up this structure (covered previously in part I) underlie many fundamental cellular processes, including transcriptional regulation and DNA-damage repair. Dysregulation of these processes has been linked to cancer development. Mechanisms of chromatin remodeling include dynamic interplay between ATP-dependent complexes, covalent histone modifications, utilization of histone variants and DNA methylation. In part II of this series, we focus on connections between ATP-dependent chromatin-remodeling complexes and oncogenesis and discuss the potential clinical implications of chromatin remodeling and cancer.

Synthesis and Properties of Chitosan/polypeptide Bioconjugate Composite

Adhesive polypeptide containing the key component of mussel adhesive protein was synthesized by ring-opening polymerization, and a hybrid material of the adhesive polypeptide and chitosan was prepared through the solution method. Some strong hydrogen bond interaction existed, but without the chemical bond between chitosan and polypeptide molecular in the composites, which was demonstrated by IR and XRD. Tensile strength and elongation-at-break of the composite increased with the increase of the polypeptide content. However, the mechanical properties decreased when the content of polypeptide was more than 2% in the composite; all in all, the mechanical properties of the composite were better than the pure chitosan. Furthermore, the introduction of polypeptide was beneficial in improving the hydrophilicity and cell affinity of the composite. The results indicated that the novel chitosan/polypeptide composite has excellent biocompatibility, which could be a scaffold material for cell culture in tissue engineering.

In Situ Preparation of Glycoconjugate Hollow Microspheres Mimics the Extracellular Matrix Via Interfacial Polymerization

A novel chitin-graft-polyleucine microsphere with hollow construction was prepared via ring-opening polymerization initiated with chitin based on interfacial protocol. The FT-IR and (1)H NMR analysis demonstrated the conjugation with regulated graft length. The study provided a facile one-step route to obtain microsphere with glycoconjugation structure. This hybrid polysaccharide-polypeptide microsphere may give promising application in drug delivery and tissue engineering.

One-step Preparation of Glycopeptide Microspheres Based on Alpha-amino Acid-N-carboxyanhydride Polymerization Using Interfacial Protocols

A type of polysaccharide-polypeptide hybrid material, chitin derivative with polypeptide side chains was prepared by the graft copolymerization of L-leucine N-carboxyanhydride triggered by water-soluble chitin (WSC). The studies on surface tension and aggregation demonstrated surface activity of WSC. Using this extraordinary property, the microspheres were synchronously obtained via interfacial polymerization. The method employed here to form the microspheres was in direct contrast to previous syntheses that used either templating method or oil-in-water emulsion. The study provided a facile approach for synthesizing microspheres with a variety of distinct polypeptide and regulated graft length, which had mimetic structure of glycoconjugation in extracellular matrix. Furthermore, the swelling capability of the microsphere in both acidic aqueous and organic solvents would give promising application in drug delivery.

Emergent Right Lobe Adult-to-adult Living-donor Liver Transplantation for High Model for End-stage Liver Disease Score Severe Hepatitis

The aim of this study was to explore the feasibility of emergency right lobe adult-to-adult living-donor liver transplantation (LDLT) for high model for end-stage liver disease (MELD) score severe hepatitis. Consecutive 10 high MELD score severe hepatitis patients underwent emergency right lobe adult-to-adult LDLT in our hospital from April to December 2007. The MELD score was 34.50 +/- 2.088. The outcomes of these recipients were retrospectively analyzed. Among them, eight cases of ABO blood group were identical and two cases compatible, one case was Rh negative. Two recipients died and the rest of the recipients and all donors are safe; perioperative and 2-year survival rate was 80%. The mean graft-recipient weight ratio (GRWR) was 1.27% +/- 0.25%, and graft volume to recipient standard liver volume ratio (GV/ESLVR) was 56.7% +/- 6.75%. Of the 10 patients, three received right lobe grafts with middle hepatic vein (MHV), four without MHV, three without MHV but followed by V and VIII hepatic vein outflow reconstruction. An encouraging outcome was achieved in this group: elevated serum creatinine, serum endotoxin, decreased serum prothrombin activity, and Tbil returned to normal on postoperative days 3, 7, 14, and 28, respectively. One-year survival rate was 80%. Outcomes of emergency right lobe adult-to-adult LDLT for high MELD score severe hepatitis were fairly encouraging and acceptable. Emergency right lobe adult-to-adult LDLT is an effective and life-saving modality for high MELD score acute liver failure patients following severe hepatitis.

Hormone Activity of Hydroxylated Polybrominated Diphenyl Ethers on Human Thyroid Receptor-beta: in Vitro and in Silico Investigations

Hydroxylated polybrominated diphenyl ethers (HO-PBDEs) may disrupt thyroid hormone status because of their structural similarity to thyroid hormone. However, the molecular mechanisms of interactions with thyroid hormone receptors (TRs) are not fully understood.

Covalent Histone Modifications--miswritten, Misinterpreted and Mis-erased in Human Cancers

Post-translational modification of histones provides an important regulatory platform for processes such as gene transcription and DNA damage repair. It has become increasingly apparent that the misregulation of histone modification, which is caused by the deregulation of factors that mediate the modification installation, removal and/or interpretation, actively contributes to human cancer. In this Review, we summarize recent advances in understanding the interpretation of certain histone methylations by plant homeodomain finger-containing proteins, and how misreading, miswriting and mis-erasing of histone methylation marks can be associated with oncogenesis and progression. These observations provide us with a greater mechanistic understanding of epigenetic alterations in human cancers and might also help direct new therapeutic interventions in the future.

Estrogenic Activity of Anthraquinone Derivatives: in Vitro and in Silico Studies

Comprehension of the ligand-receptor interactions is a prerequisite for constructing mechanism based quantitative structure-activity relationship (QSAR) models on xenoestrogenic activity. Molecular docking was performed to simulate the interactions between anthraquinone derivative (AQs) molecules and the estrogen receptor alpha (ERalpha). Hydrogen bonding, hydrophobic, and pi-pi interactions were found to be the dominant interactions between AQs and the receptor, which implied the estrogenic activities of the compounds. The recombinant yeast-based assay was employed to determine the estrogenic activities of 20 AQs. On the basis of the observed interactions between the AQs and ERalpha, appropriate molecular structural parameters were computed to develop a QSAR model. The polarizability term, the binding energy, the average molecular polarizability, the most negative formal charge in the molecule, and the average of the negative potentials on the molecular surface were significant parameters explaining the estrogenicity. The developed QSAR model had good robustness, predictive ability, and mechanism interpretability. The interactions between the AQs and ERalpha and the partition ability of the AQs into the biophase are main factors governing the estrogenic activities. Moreover, the applicability domain of the model was described.

ETV1 is a Lineage Survival Factor That Cooperates with KIT in Gastrointestinal Stromal Tumours

Gastrointestinal stromal tumour (GIST) is the most common human sarcoma and is primarily defined by activating mutations in the KIT or PDGFRA receptor tyrosine kinases. KIT is highly expressed in interstitial cells of Cajal (ICCs)-the presumed cell of origin for GIST-as well as in haematopoietic stem cells, melanocytes, mast cells and germ cells. Yet, families harbouring germline activating KIT mutations and mice with knock-in Kit mutations almost exclusively develop ICC hyperplasia and GIST, suggesting that the cellular context is important for KIT to mediate oncogenesis. Here we show that the ETS family member ETV1 is highly expressed in the subtypes of ICCs sensitive to oncogenic KIT mediated transformation, and is required for their development. In addition, ETV1 is universally highly expressed in GISTs and is required for growth of imatinib-sensitive and resistant GIST cell lines. Transcriptome profiling and global analyses of ETV1-binding sites suggest that ETV1 is a master regulator of an ICC-GIST-specific transcription network mainly through enhancer binding. The ETV1 transcriptional program is further regulated by activated KIT, which prolongs ETV1 protein stability and cooperates with ETV1 to promote tumorigenesis. We propose that GIST arises from ICCs with high levels of endogenous ETV1 expression that, when coupled with an activating KIT mutation, drives an oncogenic ETS transcriptional program. This differs from other ETS-dependent tumours such as prostate cancer, melanoma and Ewing sarcoma where genomic translocation or amplification drives aberrant ETS expression. It also represents a novel mechanism of oncogenic transcription factor activation.

Waiting
simple hit counter