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Cellular and molecular studies on olfactory bulb ensheathing cellsFranceschini, Isabelle A. January 1997 (has links)
No description available.
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Steroidogenesis in cultured mammalian glial cellsSchuliga, Michael, michael.schuliga@deakin.edu.au January 1998 (has links)
A protocol for culturing mammalian type 1 astrocytic cells, using female post-natal rat cerebral cortical tissue, was established and refined for use in steroidogenic metabolic studies incorporating progestin radioisotopes. Cultures were characterised for homogeneity using standard morphological and immunostaining techniques. Qualitative and quantitative studies were conducted to characterise the progesterone (P) metabolic pathways present in astrocytes in vitro. Of particular interest was the formation of the P metabolite, 5á-pregnan-3á-ol-20-one (THP). THP is a GABA(A) receptor agonist, believed to play a vital role in neural functioning and CNS homeostasis. One aim of this study was to observe any modulatory effects selected neuroactive ligands have on the conversion of P into THP, in an attempt to link astrocytic steroidogenesis with neuronal control.
In qualitative studies, chromatographic procedures were used to establish the progestin profile of cerebral cortical astrocytes. Tritiated P, DHP (5á-pregnan-3,20-dione) and THP incurbates were preliminary fractionated by either normal phase (NP) or reverse phase (RP) high performance liquid chromatography (HPLC). The radiometabolites associated with each fraction were further chromatographed, before and/or after chemical derivatistation, by the aforemention HPLC procedures and thin layer chromatography (TLC). Steroid radiometabolites were tentatively identified by comparing their chromatographic mobility with authentic steroids. The identity of the main putative 5á-reduced P metabolities, DHP, THP and 5á-pregnan-3á,20á-diol (20áOH-THP) were further confirmed by isotopic dilution analysis. Their conclusive identification, along with the tentative identification of 20á-hydroxypreg-4-en-3-one (20áOH-P) and 20á-hydroxy-5á-pregnan-3-one (20áOH-DHP), verify the localisation of 5á-reductase, 3á-hydroxy steroif oxidoreductase (HSOR), and 20á-HSOR activity in the cultured astrocytes utilised in this study programme. Other minor metabolites detected were tentatively identified, including 5á-pregnan-3á,21-diol-20-one (THDoc), indicating the presence of 21-hydroxylase enzymatic activity. THDoc, like THP, is a GABA(A) receptor agonist. The chemical and physical characterisation of several yet unidentified progestin metabolites, associated with a highly polar RP HPLC fraction (designated RP peak 1*), indicate the presence of one or more extra hydroxylase enzymes.
Quantitative analysis included a preliminary study. In this study, the percentage yields of radiometabolites formed in cultures incubated with increasing substrate concentrations of (3)H-P for 24 hours were determined. At the lower concentrations examined (ie 0.5 to 50nM), the metabolites associated with the polar RP HPLC fraction (RP peak 1*) collectively have the highest percentage yield. They are subsequently considered metabolic end products of degradative catabolic P pathways. The percentage yield of THP peaks in the medium concentration ranges (ie 5 to 500nM), whereas DHP remains fairly static at a low level with increasing concentration. Both DHP and THP are considered metabolic pathway intermediates. The percentage yield of 20áOH-THP continues to increase with increasing concentration over 5nM, superseding THP approaching the highest concentration examined (5000nM). This indicated the formation of 20áOH-THP does not occur entirely via THP. 20áOH-THP also possibly serves as the direct intermediate in the formation of the main radiometabolites associated with RP peak 1*.
A time/yield study incorporating incubation times from one to 24 hours was also conducted. The full array of radiometabolites (individually or in groups) formed in astrocyte cultures incubated with 50nM tritiated P, DHP of THP, were assayed. Cultures were observed to rapidly convert any DHP into THP, showing astrocytic 3á-HSOR activity is very high. The study also showed 5á-reduction (ie the conversation of P into DHP) is the rate limiting reaction in the two step conversion of P into THP. 5á-Reduction also appears to be a rate limiting step in the formation of 20á-hydroxylated metabolites in astrocytes. Cultures incubated with the tritiated 5á-reduced pregnanes from one to four hours form greater quantities to 20á-hydroxylated radiometabolites compared to cultures incubated with (3)H-P.
The time yield/studies also provided further evidence the unidentified polar radiometabolites associated with RP peak 1* are metabolic end products. For the P and DHP incubates, the collective formation of the aforementioned polar radiometabolites initially lags behind the formation of THP. As the formation of the latter begins to plateau with increasing time between four to 24 hours, the net yield of radiometabolites associated with RP peak 1* continues to rise. The time/yield studies also indicate 5á-reduction and perhaps 3á-hydroxylation are pre-requisite steps in the formation of the polar metabolites. Cultures incubated with the 5á-reduced progestins from one to four hours form higher yields of the radiometabolites associated with RP peak 1* compared to cultures incubated with P as substrate. The net yields of the radiometabolites associated with RP peak 1* for cultures incubated with THP were substantially higher compared to cultures incubated with DHP after equivalent times.
The effect selected neuroligands have on the yield of radiometabolites formed by cultured astrocytes incubated with 50nM (3)H-P was also examined. Dibutyryl cyclic adenosine monophosphate (DBcAMP), not actually a neuroligand per se, but an analog of the intracellular secondary messenger cAMP, was also utilised in these studies. The inhibitory neurotransmitter ã-amino-nbutyric acid (GABA), DBcAMP and isoproterenol (a â-adrenergic receptor agonist) all quickly induce a transient but substantial increase in 20á-HSOR activity in cultured astrocytes. Cultures pretreated with these three compounds (10, 20 and 1µM respectively) form substantially higher yields of 20á-hydroxylated metabolites, including 20áOH-THP (between 200 to 580% greater), when incubated with 50nM (3)H-P for one to four hours. These increases also coincide with increases in the net yield of metabolites formed (by 16 to 48%). The same pre-treated cultures form significantly lower yields of THP, by 25 to 41%, after one hour. This is most likely due to the increased metabolism of any formed THP into 20áOH-THP.
Octopamine (an á-adrenergic agonist) only induces a slight increase in 20á-HSOR activity, having relatively little effect on the yield of 20áOH-THP formed. Pretreatment with octopamine induces a significant increase in the yield of THP for cultures incubated with (3)H-P for four hours (by 24%). The increase in THP formation appears to be due to an increase in 3á-HSOR activity, as judged by the concomitant drop in the yield of the 5á-reduced, 3-keto substrates. An increase in 5á-reductase activity cannot be excluded however. Isoproterenol appears to induce an increase in 5á-reductase activity as isoproterenol appears to induce an increase in 5á-reductase activity as isoproterenol one and four hour incubates have higher yields of DHP. This is in contrast to the other three incubates. After 12 hours, all incubates have higher yields of THP (15-30%).
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The characterization of the olfactory ensheathing cell phenotype by protein analysisSmithson, LAURA 09 October 2008 (has links)
Over the recent years, olfactory ensheathing cells (OECs) have gained world-wide attention due to their reputed potential in promoting spinal cord regeneration and repair. In order to isolate, identify, and characterize OECs in vitro and following implantation, researchers have used three OEC markers: p75NTR, GFAP, and S100. The downfall with using these specific proteins is that Schwann cells, which are located within the olfactory system, as well as migrate into the damaged spinal cord, also express these proteins. It is therefore impossible to distinguish OECs from phenotypically similar Schwann cells using these molecular markers.
Recently proteomic analyses have revealed that OECs (derived from embryonic rat olfactory bulbs), but not Schwann cells (derived from adult rat sciatic nerves) express a variety of proteins. The main aim of this project is to determine if heat shock protein-27 (Hsp27), carbonic anhydrase-III (CA-III), and annexin-A3 (Anx3) markers label OECs but not Schwann cells, both in vivo and in vitro. Additional analyses were also done to determine if smooth muscle α-actin (SMA) and calponin (two smooth muscle-related markers previously shown to label mucosal OECs of adult rats) label bulbar OECs of adult rats and OECs of adult cats.
Using immunohistochemistry we found that SMA labeled olfactory mucosal and bulbar OECs of adult rats and adult cats, Hsp27 labeled olfactory mucosal and bulbar OECs of adult rats and olfactory mucosal OECs of adult cats, while calponin labeled only olfactory mucosal OECs of adult rats. In addition, calponin and SMA did not label Schwann cells (in vivo and in vitro), while Hsp27 labeled this peripheral glial cell. Finally, CA-III did not label OECs of adult rats or adult cats, in vivo or in vitro, and Anx3 did not label OECs in vivo, but showed immunopositive labeling of OECs and Schwann cells in vitro. In conclusion, Hsp27, CA-III, and Anx3 cannot be used as OECs markers either because of their expression in both OECs and Schwann cells or their lack of expression in OECs. Discovering new molecular markers expressed only by OECs is essential in order to determine the properties, fate, and overall potential of OECs in promoting spinal cord regeneration. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2008-09-29 09:50:09.869
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The transcription factors dHAND and eHAND and the growth factor HGF are involved in peripheral nervous system developmentDean, Charlotte Hannah January 2001 (has links)
No description available.
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Properties of rat recombinant K+ channelsAkhtar, Sobia January 2000 (has links)
No description available.
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The impact of reduced neuronal p75NTR expression on sensory neuron phenotype and associated glia2011 October 1900 (has links)
The common neurotrophin receptor, p75NTR, has been implicated in diverse responses of sensory neurons including a role in nociception following nerve injury, suggesting that it may serve a similar role in intact sensory neurons and their satellite glial cells (SGCs). To examine the impact of suppressing neuronal p75NTR expression on known molecular modulators/regulators of the nociceptive state namely, the sodium channels NaV1.8 and NaV1.9, the nerve growth factor receptor TrkA, the potassium channel Kir4.1, glial fibrillary acidic protein (GFAP), SGC p75NTR, connexin 43, we intrathecally infused p75NTR anti-sense oligonucleotides (AS OGN), previously shown by Obata et al. (2006) to effectively suppress p75NTR expression in intact neurons. Male, Wistar rats were divided into three groups, receiving either no treatment (non-infused), seven day intrathecal infusion of p75NTR AS OGN or sense control (SC OGN) via an osmotic pump. Serial L4 and L5 DRG sections were processed for immunohistochemistry to detect alterations in NaV1.8, NaV1.9, TrkA, Kir4.1, p75NTR, GFAP and connexin-43 protein expression. Sciatic nerve sections were also processed for immunohistochemistry to detect NaV1.8, NaV1.9, TrkA and GFAP protein expression.
Infusion of p75NTR AS OGNs resulted in a significant decrease in neuronal p75NTR expression, however no significant change was observed in neuronal NaV1.8, NaV1.9 or TrkA expression relative to SC OGN treated or non-infused controls. On the contrary, SGC expression of phenotypic markers normally associated with the reactive state that is induced in these cells in response to peripheral nerve axotomy was dramatically altered. More specifically, in response to p75NTR AS OGN infusion, there was a significant increase in SGC protein expression of the cytoskeletal protein GFAP and p75NTR, along with a significant decrease in expression of the inward rectifying potassium channel Kir4.1. Preliminary data also revealed this induced reactive state in SGCs to be associated with an increase in the number of SGCs surrounding individual neurons as well as increased SGC expression of the gap junction protein, connexin 43.
In conclusion, reductions in neuronal p75NTR expression and potentially reduced neurotrophin signaling lead to alterations in neuron/glial or axon/glial communication that results in induction of a reactive phenotype in the associated SGCs. With our ever increasing understanding of the role of SGCs modulating pain states, elucidation of the pathways leading to adoption of pathological phenotypes can help in the identification of novel therapeutic targets.
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The role of QKI-5 in CG4 oligodendrocyte differentiation2013 September 1900 (has links)
The Quaking (qk) gene has been implicated in the development of oligodendroglial cells which are the primary source of myelin in the mammalian central nervous system (CNS). Qk encodes three alternatively spliced variants, QKI-5, QKI-6 and QKI-7, all of which are RNA binding proteins. Loss of QKI-6 and QKI-7 results in a dysmyelination phenotype that is present shortly after birth while loss of QKI-5 results in embryonic lethality. CG4 oligodendroglial cells were transfected with either pIRES2-QKI5 to up regulate QKI-5 expression or a QKI-5 specific siRNA to down regulate QKI-5. Cells were cultured for 6d in differentiation medium (DM) following which total RNA and protein was collected from the cell cultures, and coverslips with attached cells were processed for immunofluorescence. Increased QKI-5 expression following transfection with pIRES2-QKI5 resulted in increased Sirt2 and Plp mRNA expression, but did not affect SIRT2 and PLP protein expression. Down regulation of QKI-5 expression had no significant effect on mRNA or protein levels for QKI-6, QKI-7, Plp or Sirt2. Immunocytochemistry revealed that up regulation of QKI-5 resulted in significantly higher percentage of A2B5+ cells and a lower percentage of GalC+ cells, whereas siRNA treatment resulted in an increase in the percentage of GalC+ cells. Our results suggest QKI-5 regulates oligodendrocyte differentiation and modulates the transcription and availability of target mRNAs, such as Sirt2 and Plp, for translation. In order to gain a more complete understanding of the relationship between qk and both Sirt2 and Plp, future studies would include RNA coimmunoprecipitation, miRNA studies, and expanding the list of target genes to include various cell cycle components.
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Suppression of manganese-dependent production of nitric oxide in astrocytes: implications for therapeutic modulation of glial-derived inflammatory mediatorsWright, Tyler T. 15 May 2009 (has links)
Primary cultured astrocytes were treated with Mn in the absence and presence of proinflammatory
cytokines to determine their effect upon stimulation of nitric oxide (NO)
production. Treatments of manganese and cytokines raised NO production to intermediate
levels, whereas combined treatment raised NO creation to much greater levels. Furthermore,
this combined treatment differed from control only in its ability to elevate cellular NO levels
at 24 hours, but not at earlier time points. Combined exposure in astrocytes derived from
mice lacking the nos2 gene prevented any increase in production of NO. Thus, manganese
and cytokines enhance NO production through activation of the nos2 gene. Additionally,
pharmacologic ligands of the peroxisome proliferator-activated receptor gamma (PPARγ)
were used to test the role of this orphan nuclear receptor in modulating Mn-dependent
production of NO. The agonist, 1,1-Bis(3’-indolyl)-1-(p-trifluormethylphenyl) methane
(cDIM1) diminished NO in a dose-dependent manner, whereas addition of the PPARγ
antagonist, GW 9662, amplified cellular NO production, also in a dose-dependent fashion.
Moreover, it was observed that NO production was both attenuated and augmented at similar rates, suggesting the agonist and antagonist work through similar mechanisms. To clarify the
means by which NO levels are manipulated by PPARγ, we measured activation levels of the
transcription factor NF-κB, a primary factor resulting in expression of NOS2. We found that
NF-κB was slightly activated in cells treated solely with manganese or cytokines, whereas
cells treated with both manganese and cytokines showed the highest levels of activation.
Also, we found that these ligands function through an NF-κB dependent mechanism.
Treatment of cDIM1 to astrocytes already treated with manganese and cytokines caused
decreased activation of NF-κB, while addition of GW9662 to similarly treated cells resulted
in increased activation of NF-κB. While these compounds were effective at manipulating
induction of the nos2 gene, they had no effect on induction of guanosine tri-phosphate
cyclohydrolase (GTPCH) the rate limiting enzyme for the production of tetrahydrobiopterin
(BH4), a cofactor essential to the conversion of arginine to NO, Thus, these novel PPARγ
ligands can influence manganese- and cytokine-induced production of NO by an NF-κB
dependent mechanism.
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Characterization of human bitter taste receptor T2R1Upadhyaya, Jasbir Deol 10 September 2010 (has links)
Bitter taste signaling in humans is mediated by a group of 25 bitter receptors (T2Rs) that belong to the G-protein coupled receptor (GPCR) family. Previously, several bitter peptides were isolated and characterized from bitter tasting food protein derived extracts, such as pea protein and soya bean extracts. However, their molecular targets in humans were poorly characterized. In this study, we tested the ability of the bitter tasting tri- and di-peptides to activate the human bitter receptor, T2R1. Using a heterologous expression system, T2R1 gene was transiently expressed in C6-glioma cells and changes in intracellular calcium were measured following addition of the peptides. We found that the bitter tasting tri-peptides are more potent in activating T2R1 than the di-peptides tested. Furthermore, to elucidate the potential ligand binding pocket of T2R1 we used homology molecular modeling. The ligand binding pocket in T2R1 is present on the extracellular surface of the receptor, and is formed by the transmembrane helices 1, 2, 3 and 7 and with extracellular loops 1 and 2.
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Characterization of human bitter taste receptor T2R1Upadhyaya, Jasbir Deol 10 September 2010 (has links)
Bitter taste signaling in humans is mediated by a group of 25 bitter receptors (T2Rs) that belong to the G-protein coupled receptor (GPCR) family. Previously, several bitter peptides were isolated and characterized from bitter tasting food protein derived extracts, such as pea protein and soya bean extracts. However, their molecular targets in humans were poorly characterized. In this study, we tested the ability of the bitter tasting tri- and di-peptides to activate the human bitter receptor, T2R1. Using a heterologous expression system, T2R1 gene was transiently expressed in C6-glioma cells and changes in intracellular calcium were measured following addition of the peptides. We found that the bitter tasting tri-peptides are more potent in activating T2R1 than the di-peptides tested. Furthermore, to elucidate the potential ligand binding pocket of T2R1 we used homology molecular modeling. The ligand binding pocket in T2R1 is present on the extracellular surface of the receptor, and is formed by the transmembrane helices 1, 2, 3 and 7 and with extracellular loops 1 and 2.
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