intranuclear-microinjection-dna-into-dissociated-adult-mammalian

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to ...

IL-4 is emerging as a candidate cytokine for the treatment of inflammatory and autoimmune diseases. We have reported that IL-4 has anti-angiogenic activity and inhibits the growth of human umbilical vein endothelial cells (HUVEC) in response to vascular endothelial growth factor (VEGF) or fibroblast growth factor-2 (FGF-2). Cell cycle analysis of this effect revealed that IL-4 arrests the growth of FGF-2-stimulated HUVEC in G0 + G1 phases. The absence of subdiploid cells showed that it did not induce apoptosis. Growth arrest was dose-dependent, but the percentage of G0 + G1 phase cells never exceeded 85%. An immunoblot analysis demonstrated that expression of p53 and p21(Waf1) was increased and that of cyclin D1 and cyclin E decreased by IL-4. These results show that IL-4 inhibits endothelial cell growth by altering the expression of cell cycle regulatory molecules.

IL-4 inhibits cell cycle progression of human umbilical vein endothelial cells by affecting p53, p21(Waf1), cyclin D1, and cyclin E expression.

IL-4 hemmt den Ablauf des Zellzyklus von menschlichen Nabelschnur-Endothelzellen durch Einwirkung auf p53, p21 (Waf1), Cyclin D1 und Cyclin E Expression.

The goal of this study was to elucidate the involvement of neuronal and glial calcium-binding proteins in the stimulation of gamma-aminobutyric acid (GABA) transport system by kainate-induced excitotoxicity in the rat retina. We used immunohistochemical method to assess the localization of GABA reuptake and calcium-binding proteins. After systemic administration of kainate, the neuronal GABA transporter does not show an association with calbindin D-28K. However, the localization of the GAT-3 transport system in MÃ¼ller glial cells is closely correlated with the S100 proteins interacting with glial fibrillary acidic protein (GFAP) in response to kainate injury. Furthermore, we demonstrate that kainate-mediated excitotoxicity induced concomitant distribution shift of glial GABA transporter, S100 proteins and GFAP in the distal processes and endfeet of glial cells during the first 48 h.

Concomitant distribution shift of glial GABA transporter and S100 calcium-binding proteins in the rat retina after kainate-induced excitotoxic injury.

Die gleichzeitige Verschiebung der Verteilung glialen GABA-Transporter und S100 Calcium-bindenden Proteinen in der Netzhaut Ratte nach Kainat-induzierten exzitotoxische Verletzungen.

Although nerve injury is known to up- and down-regulate some metabotropic receptors in vagal afferent neurons of the nodose ganglia (NG), the functional significance has not been elucidated. In the present study, thus, we examined whether nerve injury affected receptor-mediated Ca2+ channel modulation in the NG neurons. In this regard, unilateral vagotomy was performed using male Sprague-Dawley rats. One week after vagotomy, Ca2+ currents were recorded using the whole-cell variant of patch-clamp technique in enzymatically dissociated NG neurons. In sham controls, norepinephrine (NE)-induced Ca2+ current inhibition was negligible. Following vagotomy, however, the NE responses were dramatically increased. This phenomenon was in accordance with up-regulation of alpha2A/B-adrenergic receptor mRNAs as quantified using real-time RT-PCR analysis. In addition, neuropeptide Y (NPY) and prostaglandin E2 responses were moderately augmented in vagotomized NG neurons. The altered NPY response appears to be caused by up-regulation of Y2 receptors negatively coupled to Ca2+ channels. In contrast, nerve injury significantly suppressed opioid (tested with DAMGO)-induced Ca2+ current inhibition with down-regulation of micro-receptors. Taken together, these results demonstrated for the first time that the profile of neurotransmitter-induced Ca2+ channel modulation is significantly altered in the NG neurons under pathophysiological state of nerve injury.

Nerve injury alters profile of receptor-mediated Ca2+ channel modulation in vagal afferent neurons of rat nodose ganglia.

Nervenverletzung Profil ver&#xE4;ndert der Rezeptor-vermittelten Ca2 +-Kanal-Modulation in vagalen afferenten Neuronen der Ratte nodosum Ganglien.

Among the autonomic ganglia, major pelvic ganglia (MPG) innervating the urogenital system are unique because both sympathetic and parasympathetic neurons are colocalized within one ganglion capsule. Sympathetic MPG neurons are discriminated from parasympathetic ones by expression of low voltage-activated Ca2+ channels that primarily arise from T-type alpha1H isoform and contribute to the generation of low-threshold spikes. Until now, however, expression profiles of high voltage-activated (HVA) Ca2+ channels in these two populations of MPG neurons remain unknown. Thus, in the present study, we dissected out HVA Ca2+ channels using pharmacological and molecular biological tools. Reverse transcription-polymerase chain reaction analysis showed that MPG neurons contained transcripts encoding all of the known HVA Ca2+ channel isoforms (alpha1B, alpha1C, alpha1D and alpha1E), with the exception of alpha1A. Western blot analysis and pharmacology with omega-agatoxin IVA (1 microM) confirmed that MPG neurons lack the alpha1A Ca2+ channels. Unexpectedly, the expression profile of HVA Ca2+ channel isoforms was identical in the sympathetic and parasympathetic neurons of the MPG. Of the total Ca2+ currents, omega-conotoxin GVIA-sensitive N-type (alpha1B) currents constituted 57 +/- 5% (n = 9) and 60 +/- 3% (n = 6), respectively; nimodipine-sensitive L-type (alpha1C and alpha1D) currents made up 17 +/- 4% and 14 +/- 2%, respectively; and nimodipine-resistant and omega-conotoxin GVIA-resistant R-type currents were 25 +/- 3% and 22 +/- 2%, respectively. The R-type Ca2+ currents were sensitive to NiCl2 (IC50 = 22 +/- 0.1 microM) but not to SNX-482, which was able to potently (IC50 = 76 +/- 0.4 nM) block the recombinant alpha1E/beta2a/alpha2delta Ca2+ currents expressed in human embryonic kidney 293 cells. Taken together, our data suggest that sympathetic and parasympathetic MPG neurons share a similar but unique profile of HVA Ca2+ channel isoforms.

Expression profiles of high voltage-activated calcium channels in sympathetic and parasympathetic pelvic ganglion neurons innervating the urogenital system.

Unter den autonomen Ganglien sind gro&szlig;e Becken Ganglien (MPG) nervenst&auml;rkend Urogenitale System eindeutig, da sowohl sympathische und parasympathische Neuronen innerhalb einer Ganglion Kapsel colocalized sind. Sympathische MPG Neuronen sind von parasympathischen diejenigen durch Ausdruck der niedrigen Spannung aktiviert Ca2 + Kan&auml;le diskriminiert werden, die in erster Linie vom T-Typ-alpha1H-Isoform entstehen und tragen zu der Generation von niedrigschwelligen Spikes. Bis jetzt bleiben jedoch Ausdruck Profile von hoher Spannung aktiviert (HVA) Ca2 + Kan&auml;le in diese zwei Populationen von MPG Neuronen unbekannt. Daher in der vorliegenden Studie seziert wir heraus HVA Ca2 + Kan&auml;le mit pharmakologischen und molekularen biologischen Tools. Reverse Transkription-Polymerase-Kettenreaktion-Analyse zeigte, dass MPG Neuronen enthalten Transkripte Codierung alle der bekannten HVA Ca2 +--Isoformen (alpha1B, alpha1C, alpha1D und alpha1E), mit Ausnahme von alpha1A Kanal. Western-Blot-Analyse und Pharmakologie mit Omega-Agatoxin IVA (1 MicroM) best&auml;tigt, dass MPG Neuronen die alpha1A Ca2 + Kan&auml;le fehlen. Unerwartet war das Profil Ausdruck des HVA Ca2 + Kanal Isoformen in den sympathischen und parasympathischen Neuronen der MPG identisch. Der Gesamt Ca2 + Str&ouml;me, Omega-Conotoxin GVIA-Sensitive N-Typ (alpha1B) Str&ouml;mungen bildeten 57 +/-5 % (n = 9) und 60 +/-3 % (n = 6); Nimodipin-Sensitive L-Typ (alpha1C und alpha1D) Str&ouml;mungen 17 +/-4 % und 14 +/-2 %, gebildet; und Nimodipin-best&auml;ndig und Omega-Conotoxin GVIA s&auml;urefeste R-Typ Str&ouml;mungen waren 25 +/-3 % und 22 +/-2 %, beziehungsweise. Die R-Typ Ca2 +-Str&ouml;me waren sensibel auf NiCl2 (IC50 = 22 +/-0,1 MicroM) aber nicht zu SNX-482, die konnte dabei wirksam (IC50 = 76 +/-0,4 nM) blockieren die rekombinanten alpha1E/beta2a/alpha2delta Ca2 + Str&ouml;mungen in menschlichen embryonalen Nierenzellen 293 ausgedr&uuml;ckt. Zusammen genommen, zufolge unsere Daten sympathische und parasympathische MPG Neuronen eine &auml;hnliche aber eindeutige Profil der HVA-Ca2 +-Kanal-Isoformen teilen.

Ausdruck Profile und hohe Spannung aktiviert Kalzium-Kan&auml;le in sympathische und parasympathische Becken Ganglion Neuronen nervenst&auml;rkend Urogenitale System.

Endocannabinoids (eCB) such as 2-arachidonylglycerol (2-AG) are lipid metabolites that are synthesized in a postsynaptic neurons and act upon CB(1) cannabinoid receptors (CB(1)R) in presynaptic nerve terminals. This retrograde transmission underlies several forms of short and long term synaptic plasticity within the CNS. Here, we constructed a model system based on isolated rat sympathetic neurons, in which an eCB signaling cascade could be studied in a reduced, spatially compact, and genetically malleable system. We constructed a complete eCB production/mobilization pathway by sequential addition of molecular components. Heterologous expression of four components was required for eCB production and detection: metabotropic glutamate receptor 5a (mGluR5a), Homer 2b, diacylglycerol lipase alpha, and CB(1)R. In these neurons, application of l-glutamate produced voltage-dependent modulation of N-type Ca(2+) channels mediated by activation of CB(1)R. Using both molecular dissection and pharmacological agents, we provide evidence that activation of mGluR5a results in rapid enzymatic production of 2-AG followed by activation of CB(1)R. These experiments define the critical elements required to recapitulate retrograde eCB production and signaling in a single peripheral neuron. Moreover, production/mobilization of eCB can be detected on a physiologically relevant time scale using electrophysiological techniques. The system provides a platform for testing candidate molecules underlying facilitation of eCB transport across the plasma membrane.

Molecular reconstruction of mGluR5a-mediated endocannabinoid signaling cascade in single rat sympathetic neurons.

Endocannabinoide (EZB) wie 2-Arachidonylglyzerol (2-AG) sind Lipid Stoffwechselprodukte, die in ein postsynaptisches Neuronen synthetisiert und danach handeln CB1 Cannabinoid-Rezeptoren (CB(1)R) in pr&auml;synaptischen Nervenenden. Diese retrograde &Uuml;bertragung zugrunde liegt, verschiedene Formen von kurzen und langen Begriff synaptische Plastizit&auml;t im CNS. Hier errichteten wir ein Modell-System basierend auf isolierte Ratte sympathischen Neuronen, in denen eine EZB Signalisierung Kaskade in einem reduzierten, r&auml;umlich kompakt und genetisch Klemmverbinder System untersucht werden konnte. Durch fortlaufende Zugabe von molekularen Bestandteilen konstruierten wir einen kompletten EZB Produktion/Mobilisierung Weg. Heterologe Expression von vier Komponenten war bei der EZB-Produktion und -Erkennung: Metabotropic Glutamate Rezeptor 5a (mGluR5a), Homer, 2b, Diacylglycerol Lipase Alpha und CB (1) R. In diese Neuronen produziert Anwendung von l-Glutamat in schnelle enzymatische Produktion von 2-AG, gefolgt von der Aktivierung des CB (1) R. Spannung-abh&auml;ngigen Modulation der Ca(2+) N-Typ-Kan&auml;le vermittelt durch Aktivierung des CB (1) R. mit molekularen Dissektion und pharmakologische Agenten, wir die Aktivierung des mGluR5a Ergebnisse nachweisen Diese Experimente definieren die kritischen Elemente ben&ouml;tigt, um die retrograde EZB Produktions- und signalisieren in ein einzelnes periphere Neuron zu rekapitulieren. Produktion/Mobilisierung der EZB k&ouml;nnen dar&uuml;ber hinaus auf eine physiologisch relevante Zeitskala mit elektrophysiologische Methoden nachgewiesen werden. Das System bietet eine Plattform f&uuml;r die Pr&uuml;fung der Kandidaten-Molek&uuml;le, die Erleichterung der EZB-Transport &uuml;ber die Plasmamembran zugrunde.

Molekulare Rekonstruktion des mGluR5a-vermittelte Endocannabinoid Signalling Kaskade in einzelne Ratte sympathischen Neuronen.

Electrically excitable cells have voltage-dependent ion channels on the plasma membrane that regulate membrane permeability to specific ions. Voltage-gated Ca(2+) channels (VGCCs) are especially important as Ca(2+) serves as both a charge carrier and second messenger. Zebrafish (Danio rerio) are an important model vertebrate for studies of neuronal excitability, circuits, and behavior. However, electrophysiological properties of zebrafish VGCCs remain largely unexplored because a suitable preparation for whole cell voltage-clamp studies is lacking. Rohon-Beard (R-B) sensory neurons represent an attractive candidate for this purpose because of their relatively large somata and functional homology to mammalian dorsal root ganglia (DRG) neurons. Transgenic zebrafish expressing green fluorescent protein in R-B neurons, (Isl2b:EGFP)(ZC7), were used to identify dissociated neurons suitable for whole cell patch-clamp experiments. Based on biophysical and pharmacological properties, zebrafish R-B neurons express both high- and low-voltage-gated Ca(2+) current (HVA- and LVA-I(Ca), respectively). Ni(+)-sensitive LVA-I(Ca) occur in the minority of R-B neurons (30%) and Ï‰-conotoxin GVIA-sensitive Ca(V)2.2 (N-type) Ca(2+) channels underlie the vast majority (90%) of HVA-I(Ca). To identify G protein coupled receptors (GPCRs) that modulate HVA-I(Ca), a panel of neurotransmitters was screened. Application of GABA/baclofen or serotonin produced a voltage-dependent inhibition while application of the mu-opioid agonist DAMGO resulted in a voltage-independent inhibition. Unlike in mammalian neurons, GPCR-mediated voltage-dependent modulation of I(Ca) appears to be transduced primarily via a cholera toxin-sensitive GÎ± subunit. These results provide the basis for using the zebrafish model system to understanding Ca(2+) channel function, and in turn, how Ca(2+) channels contribute to mechanosensory function.

Identification and modulation of voltage-gated Ca2+ currents in zebrafish Rohon-Beard neurons.

Elektrisch erregbar Zellen haben Spannung-abh&auml;ngigen Ionenkan&auml;le auf der Plasmamembran, die Durchl&auml;ssigkeit der Membran f&uuml;r bestimmte Ionen regulieren. Spannung-Gating Ca(2+) Kan&auml;le (VGCCs) sind besonders wichtig, da Ca(2+) Da beide ein kostenlos Carrier und zweite Bote dient. Zebrab&auml;rbling (Danio Rerio) sind ein wichtiges Modell f&uuml;r Studien &uuml;ber neuronale Erregbarkeit, Schaltungen und Verhalten Wirbeltiere. Elektrophysiologische Eigenschaften des Zebrafish VGCCs bleiben jedoch weitgehend unerforscht, weil eine geeignete Aufbereitung f&uuml;r die gesamte Zelle Spannung Klemm-Studien fehlt. Rohon-Bart (R-B) sensorische Neuronen stellen einen attraktiven Kandidaten f&uuml;r diesen Zweck aufgrund ihrer relativ gro&szlig;en Somata und funktionelle Homologie zu S&auml;ugetier-Spinalganglien Ganglien (DRG) Neuronen dar. Transgene Zebrafisch Ausdr&uuml;cken gr&uuml;nes fluoreszierendes Protein in R-B-Neuronen, (Isl2b:EGFP)(ZC7), wurden zur getrennten Neuronen geeignet f&uuml;r ganze Zelle Patch-Clamp-Experimenten zu identifizieren. Basierend auf biophysikalischen und pharmakologische Eigenschaften, Zebrafish R-B Neuronen schnelles beide hoch - und niedrig-Spannung-Gating Ca(2+) Strom (HVA- und LVA-I(Ca), beziehungsweise). NI (+)-sensible LVA-I(Ca) auftreten, in der Minderheit von R-B-Neuronen (30 %) und &omega;-Conotoxin GVIA-Sensitive-Ca (V) 2.2 (N-Typ) Ca(2+) Kan&auml;le zugrunde liegen, die &uuml;berwiegende Mehrheit (90 %) des HVA-I(Ca). Um G-Protein-gekoppelte Rezeptoren (GPCRs) zu identifizieren, die HVA-I(Ca) zu modulieren, wurde ein Gremium von Neurotransmittern gezeigt. Anwendung von GABA/Baclofen oder Serotonin produziert eine Spannung-abh&auml;ngigen Hemmung w&auml;hrend der Anwendung von den Mu-Opioid-Agonisten, die, den DAMGO eine Hemmung der Spannung unabh&auml;ngig gef&uuml;hrt. Im Gegensatz zu scheint GPCR-vermittelte Spannung-abh&auml;ngigen Modulation des I(Ca) in den S&auml;ugetier-Neuronen, in erster Linie &uuml;ber eine Cholera-Toxin-Sensitive G&alpha;-Untereinheit ausgestrahlt werden. Diese Ergebnisse bilden die Grundlage f&uuml;r die Verwendung der Zebrafisch-Modellsystem f&uuml;r das Verst&auml;ndnis Ca(2+)-Kanal-Funktion, und im Gegenzug wie Ca(2+) Kan&auml;le zur Mechanosensory-Funktion.

Identifizierung und Modulation der Spannung-Gating Ca2 + Str&ouml;mungen im Zebrafisch Rohon-Bart Neuronen.

Pelvic ganglia (PG) play critical roles in relaying sympathetic and parasympathetic information from the spinal cord to the penile vasculature and, controlling the penile reflex. Animal studies have shown that androgen deprivation by castration causes erectile dysfunction (ED). Until now, however, neural mechanisms underlying castration-induced ED remain unclear. Therefore, we examined whether androgen deprivation down-regulates nicotinic acetylcholine receptors (nAchRs), which mediate fast excitatory synaptic transmission in the PG. Toward this end, neurogenic ED was demonstrated by measuring the intracavernous pressure in castrated rats. Real-time PCR analysis revealed that the transcripts encoding nAchR Î±3/Î±5/Î²4 subunits were significantly down-regulated in the PG neurons. In addition, down-regulation of the nAchR subunits was reversed by replacement of testosterone. Patch-clamp experiments showed that the nAchR currents were selectively attenuated in the parasympathetic PG neurons innervating the penile vasculature, activation of which elicits penile erection. Taken together, our data suggest that phenotype-specific down-regulation of nAchRs in the PG neurons may contribute to the neurogenic ED in castrated rats.

Phenotype-specific down-regulation of nicotinic acetylcholine receptors in the pelvic ganglia of castrated rats: implications for neurogenic erectile dysfunction.

Becken-Ganglien (PG) spielen wichtige Rollen in Bereitstellung von sympathische und parasympathische Informationen &uuml;ber das R&uuml;ckenmark zum Penis Gef&auml;&szlig;system und Steuerung des Penis Reflexes. Tierversuche haben gezeigt, dass Androgen-Entzug durch Kastration erektilen Dysfunktion (ED) verursacht. Bis jetzt sind Neuronale Mechanismen induziert durch Kastration ED jedoch noch unklar. Daher wir gepr&uuml;ft, ob Androgen-Entzug nach unten-nikotinische Acetylcholin-Rezeptoren (nAchRs), regelt die schnelle erregenden synaptischen &Uuml;bertragung in PG. in Richtung diesem Ende vermitteln, neurogene ED zeigte sich den intracavernous Druck bei kastrierten Ratten gemessen. Echtzeit-PCR-Analyse ergab, dass die Transkripte Codierung nAchR &alpha;3/&alpha;5/&beta;4-Untereinheiten deutlich unten-geregelt in den PG-Neuronen. Dar&uuml;ber hinaus wurde Herunterregulation der nAchR-Untereinheiten durch Ersatz des Testosterons r&uuml;ckg&auml;ngig machen. Patch-Clamp-Experimenten zeigte, dass die nAchR-Str&ouml;me selektiv in den parasympathischen PG-Neuronen, die den Penis Gef&auml;&szlig;system, der Aktivierung, die Erektion des Penis entlockt nervenst&auml;rkend abgeschw&auml;cht wurden. Genommen kann zu die neurogene ED bei kastrierten Ratten zusammen, unsere Daten deuten darauf hin, dass Ph&auml;notyp-spezifische Herunterregulation der nAchRs in den PG-Neuronen beitragen.

Ph&auml;notyp-spezifische Herunterregulation nikotinische Acetylcholin-Rezeptoren in der Beckenh&ouml;hle Ganglien von kastrierten Ratten: Implikationen f&uuml;r neurogene erektile Dysfunktion.

Article	Year
IL-4 inhibits cell cycle progression of human umbilical vein endothelial cells by affecting p53, p21(Waf1), cyclin D1, and cyclin E expression. Molecules and cells\| PubMed ID: 14503851	2003
Concomitant distribution shift of glial GABA transporter and S100 calcium-binding proteins in the rat retina after kainate-induced excitotoxic injury. Neuroscience letters\| PubMed ID: 14642427	2003
Nerve injury alters profile of receptor-mediated Ca2+ channel modulation in vagal afferent neurons of rat nodose ganglia. Neuroscience letters\| PubMed ID: 15196673	2004
Expression profiles of high voltage-activated calcium channels in sympathetic and parasympathetic pelvic ganglion neurons innervating the urogenital system. The Journal of pharmacology and experimental therapeutics\| PubMed ID: 16467454	2006
Molecular reconstruction of mGluR5a-mediated endocannabinoid signaling cascade in single rat sympathetic neurons. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 19864572	2009
Identification and modulation of voltage-gated Ca2+ currents in zebrafish Rohon-Beard neurons. Journal of neurophysiology\| PubMed ID: 20962070	2011
Phenotype-specific down-regulation of nicotinic acetylcholine receptors in the pelvic ganglia of castrated rats: implications for neurogenic erectile dysfunction. Neuroscience letters\| PubMed ID: 21782342	2011

Yu-Jin Won

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