Background and aims: In focal areas of advanced human atherosclerotic lesions, the intimal fluid ... more Background and aims: In focal areas of advanced human atherosclerotic lesions, the intimal fluid is acidic. An acidic medium impairs the ABCA1-mediated cholesterol efflux from macrophages, so tending to increase their content of free cholesterol, which is then available for esterification by the macrophage enzyme ACAT1. Here we investigated whether low extracellular pH would affect the activity of ACAT1. Methods:-Human monocyte-derived macrophages were first incubated with acetyl-LDL at neutral and acidic conditions (pH 7.5, 6.5, and 5.5) to generate foam cells, and then the foam cells were incubated with [ 3 H]oleate-BSA complexes, and the formation of [ 3 H]oleate-labeled cholesteryl esters was measured. ACAT1 activity was also measured in cell-free macrophage extracts. Results:-In acidic media, ACAT1-dependent cholesteryl [ 3 H]oleate generation became compromised in the developing foam cells and their content of free cholesterol increased. In line with this finding, ACAT1 activity in the soluble cell-free fraction derived from macrophage foam cells peaked at pH 7, and gradually decreased under acidic pH with a rapid drop below pH 6.5. Incubation of macrophages under progressively more acidic conditions (until pH 5.5) lowered the cytosolic pH of macrophages (down to pH 6.0). Such intracellular acidification did not affect macrophage gene expression of ACAT1 or the neutral CEH. Conclusions: Exposure of human macrophage foam cells to acidic conditions lowers their intracellular pH with simultaneous decrease in ACAT1 activity. This reduces cholesterol esterification and thus leads to accumulation of potentially toxic levels of free cholesterol, a contributing factor to macrophage foam cell death.
A genetic fits ion between streptavidin of Streptomyces avidinii and luciferase of Pyrophorus pla... more A genetic fits ion between streptavidin of Streptomyces avidinii and luciferase of Pyrophorus plagiophthalamus was constructed. The fits ion protein was produced in the Sf9 insect cell line using the baculovirus expression vector system (REVS). Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the proteinsjrom cells infected with the recombinant virus, VLl393-LucGR-StreptAv, revealed that the fusion protein migrated with an apparent molecular weight of 75 kDa. Light emission measurements showed that the infected cells produced about 255 mg of the chimeric protein per liter of cell culture (127.5 J.1g/1 x 10 6 cells). Precipitation of the LucGR-StreptAv fusion protein with biotinylated acrylic beads as well as immunoblot analyses using biotinylated immunoglobulins indicated that both fusion moieties of the chimeric protein product were fitnctional with respect to their physical and enzymatic activities.
Cerebellar granule neurons cultured with serum develop a mature neuronal phenotype, including sti... more Cerebellar granule neurons cultured with serum develop a mature neuronal phenotype, including stimulus-coupled release of glutamate, and depend on elevated potassium for survival. We find that cells cultured with serum undergo two phases of cell death. By 6 d in vitro, 30-50% of the cells present are dead; after this time the remaining cells die. Elevated potassium prevents only this later phase of death, whereas neurotrophins protect these cells against the early phase of death. Factors that bind p75 NTR or TNF-R, members of the same receptor family, exhibit voltage-sensitive calcium channel-dependent protection, whereas ligands of expressed Trk receptors show additional calcium channel-independent protection. The cells express TrkB protein and show elevated c-Fos and c-Jun levels in response to BDNF. No TrkA is detected, although p75 NTR protein is expressed and NGF induces depolarizationdependent elevation of c-Jun levels. In the presence of the protein kinase C inhibitor bisindolylmaleimide, BDNF-induced survival promotion is reduced partially, whereas NGF-induced death is unmasked. Basal survival mechanisms are insensitive to inhibition of PK-C or PI-3 kinase. We conclude that BDNF promotes survival in part via its TrkB receptor, whereas there is an additional pathway promoting survival and elevating c-Jun evoked by both NGF and BDNF via a non-Trk receptor.
Radial glial cells play an essential role through their function as guides for neuronal migration... more Radial glial cells play an essential role through their function as guides for neuronal migration during development. Disruption of metabotropic glutamate receptor 5 (mGluR5) function retards the growth of radial glial processes in vitro. Neuregulins (NRG) are activated by proteolytic cleavage and regulate (radial) glial maintenance via ErbB3/ErbB4 receptors. We show here that blocking ErbB4 disrupts radial process extension. Soluble NRG acting on ErbB4 receptors is able to promote radial process extension in particular where process elongation has been impeded by blockade of mGluR5, the nonselective cation channel canonical transient receptor potential 3 (TRPC3), or matrix metalloproteases (MMP). NRG does not restore retarded process growth caused by ErbB4 blockade. Stimulation of muscarinic receptors restores process elongation due to mGluR5 blockade but not that caused by TRPC3, MMP or ErbB4 blockade suggesting that muscarinic receptors can replace mGluR5 with respect to radial process extension. Additionally, NRG/ ErbB4 causes Ca 21 mobilization in a population of cells through cooperation with ErbB1 receptors. Our results indicate that mGluR5 promotes radial process growth via NRG activation by a mechanism involving TRPC3 channels and MMPs. Thus neurotransmitters acting on G-protein coupled receptors could play a central role in the maintenance of the radial glial scaffold through activation of NRG/ErbB4 signaling.
1. The effect of acute morphine administration on the rotational behavior of unilaterally brain-l... more 1. The effect of acute morphine administration on the rotational behavior of unilaterally brain-lesioned mice and on the amphetamine-induced rotational behavior of unilaterally brain-lesioned rats was studied. 2. The effect of repeated morphine administration on the rotational behavior of mice and rats was also investigated. 3. Acute administration of 40 mg/kg of morphine induced strong ipsilateral rotation in unilaterally brain-lesioned mice. 4. One day after withdrawal, mice given morphine repeatedly for 5 days and treated acutely with 40 mg/kg of morphine rotated significantly less ipsilaterally than mice that had received the same dose of morphine for the first time. 5. Rats given 2 mg/kg of morphine 1 hr before administration of 5 mg/kg of amphetamine tended to rotate slightly more in the ipsilateral direction than the similarly lesioned control rats that received amphetamine alone. 6. After 1 day of withdrawal from 5 days of repeated morphine administration, rats given morphine before amphetamine tended to rotate less ipsilaterally than those given morphine before amphetamine for the first time. 7. Thus, repeated administration of morphine appears to induce tolerance to the effect of morphine on circling behavior in unilaterally brain-lesioned mice and rats.
The guidance of developing neurons to the right position in the central nervous system is of cent... more The guidance of developing neurons to the right position in the central nervous system is of central importance in brain development. Canonical transient receptor potential (TRPC) channels are thought to mediate turning responses of growth cones to guidance cues through fine control of calcium transients. Proliferating and 1-to 5day-differentiated neural progenitor cells (NPCs) showed expression of Trpc1 and Trpc3 mRNA, while Trpc4-7 was not clearly detected. Time-lapse imaging showed that the motility pattern of neuronal cells was phasic with bursts of rapid movement (> 60 mm/h), changes in direction, and intermittent slow phases or stallings (< 40 mm/ h), which frequently occurred in close contact with radial glial processes. Genetic interference with the TRPC3 and TRPC1 channel enhanced the motility of NPCs (burst frequency/stalling frequency). TRPC3-deficient cells or cells treated with the TRPC3 blocker pyr3 infrequently changed direction and seldom contacted radial glial processes. TRPC channels are also activated by group I metabotropic glutamate receptors (mGluR1 and mGluR5). As shown here, pyr3 blocked the calcium response mediated through mGluR5 in radial glial processes. Furthermore, 2-methyl-6-(phenylethynyl)pyridine, a blocker of mGluR5, affected the motility pattern in a similar way as TRPC3/6 double knockout or pyr3. The results suggest that radial glial cells exert attractant signals to migrating neuronal cells, which alter their motility pattern. Our results suggest that mGluR5 acting through TRPC3 is of central importance in radial glial-mediated neuronal guidance.
Gia (?) Gitt, Ca^^ Gia To test whether py subunits would be involved in the stimulation of cAMP a... more Gia (?) Gitt, Ca^^ Gia To test whether py subunits would be involved in the stimulation of cAMP accumulation via the human a2B-AR in Sf9 cells the effects of Gpy and PARK (which functions as a Gpy scavenger) on forskolin-stimulated cAMP accumulation was tested (Fig. 1). While pARK enhanced forskolin-stimulated cAMP accumulation the GPy coexpression considerably reduced cAMP accumulation. This means that the adenylyl cyclase of Sf9 cells is inhibited by Py rather than stimulated. The a2B-AR-stimulated AMP accumulation in Sf9 cells can thus not be due to Py. The basal and forskolin
Five recombinant baculoviruses, each containing a different insect luciferase gene encoding a pro... more Five recombinant baculoviruses, each containing a different insect luciferase gene encoding a protein with characteristic light emission properties, namely, luc GR (546 nm), luc FF (556 nm), luc YG (560 nm), luc YE (578 nm), and luc OR (593 nm) were constructed. All genes were inserted under the transcriptional control of the polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV) and expressed in Spodoptera frugiperda insect cells during viral infection. The biological activity of the different luciferases was characterized by using intact recombinant baculovirus infected cells. Addition of the substrate, D-luciferin, immediately prior to the analysis allowed monitoring of light emission by flow cytometry. Also, the kinetics of the light emission of lucGR was analyzed with the flow cytometer. The emission peaks of the infected cells were clearly separated by wavelength scanning. Especially, the firefly luciferase (lucFF) had a broad peak and transient luminescence. The highest maximal intensity values in vivo were recorded for luc GR and luc YG. SDS-PAGE analysis showed that the major protein expressed had a molecular weight similar to authentic luciferase. Flow cytometry and insect luciferases with clearly separated emission spectra appear to be of value for sensitive in vivo analysis of gene promoter activity.
O uso da terapia antirretroviral (TARV) tem aumentado à sobrevida das pessoas vivendo com HIV/AID... more O uso da terapia antirretroviral (TARV) tem aumentado à sobrevida das pessoas vivendo com HIV/AIDS (PVHA) alterando consideravelmente a história natural da infecção. Entretanto, em alguns casos, o uso destes medicamentos tem sido associado à distúrbios nutricionais e metabólicos. O objetivo deste estudo foi descrever os distúrbios nutricionais e metabólicos ocasionados pelo uso da TARV e o respectivo papel terapêutico da abordagem nutricional em PVHA. Trata-se de uma revisão narrativa, realizada durante o segundo semestre do ano de 2012, utilizando os seguintes descritores: AIDS, HIV, PVHA, TARV, estado nutricional, acompanhamento nutricional. O principal distúrbio nutricional encontrado nesta revisão, relacionado ao uso de TARV, foi a síndrome lipodistrófica associada à dislipidemias e hiperglicemias, bem como, alterações ósseas e aumento de gordura corporal. Neste sentido, o acompanhamento nutricional é extremamente importante para a melhora da qualidade de vida e prevenção de demais complicações metabólicas em PVHA.
A single G-protein-coupled receptor might activate multiple G-protein species. This multiplex cou... more A single G-protein-coupled receptor might activate multiple G-protein species. This multiplex coupling ability can be used by tissues to regulate signalling; for the pharmacologist, such multiplex coupling might cause difficulties in the interpretation of experimental data. In this article, we present mathematical models for the activation of two separate G-protein species by a single receptor. Issues addressed concern mutual antagonism between the G proteins and the availability of an already activated receptor for interaction with a new G protein (receptor-G-protein-effector complexing versus free diffusion of G proteins) in addition to receptor-G-protein precoupling at different G-protein and receptor expression levels. The output from the receptor models uses, as readout, a new model for adenylyl cyclase regulation by two allosteric regulators (i.e. G(s) and G(i)).
By studying the influence of two toxins from the black mamba Dendroaspis polylepis on the kinetic... more By studying the influence of two toxins from the black mamba Dendroaspis polylepis on the kinetics of [3H]-N-methylscopolamine binding to muscarinic acetylcholine receptors from rat cerebral cortex, it was revealed that these toxins, MT alpha and MT beta, interact with the receptors via kinetically distinct mechanisms. MT beta bound to receptors in a one-step, readily reversible process with the dissociation constant K(d)=5.3 microM. The binding mechanism of MTalpha was more complex, involving at least two consecutive steps. A fast receptor-toxin complex formation (K(T)=3.8 microM) was followed by a slow process of isomerisation of this complex (k(i)=1.8 x 10(-2) s(-1), half-time 39 s). A similar two-step interaction mechanism has been established for a related toxin, MT2 from the green mamba D. angusticeps (K(T)=1.4 microM, k(i)=8.3 x 10(-4) s(-1), half-time 840 s). The slow isomerisation process delays the effect of MT alpha and MT2, but increases their apparent potency compared to toxins unable to induce the isomerisation process.
We studied the cellular response to orexin type 1 receptor (OX 1 R) stimulation in differentiated... more We studied the cellular response to orexin type 1 receptor (OX 1 R) stimulation in differentiated IMR-32 neuroblastoma cells. In vitro differentiation of IMR-32 cells with 5-bromo-2Ј-deoxyuridine leads to a neuronal phenotype with long neurite extensions and an upregulation of mainly N-type voltage-gated calcium channels. Transduction of differentiated IMR-32 cells with baculovirus harboring an OX 1 R-green fluorescent protein cDNA fusion construct resulted in appearance of fluorescence that was confined mainly to the plasma membrane in the cell body and to neurites. Application of orexin-A to fluorescent cells led to an increase in intracellular free Ca 2ϩ concentration, [Ca 2ϩ ] i. At low nanomolar concentrations of orexin-A, the response was reversibly attenuated by removal of extracellular Ca 2ϩ , by application of a high concentration (10 mM) of Mg 2ϩ , and by the pharmacological channel blocker dextromethorphan. A diacylglycerol, dioctanoylglycerol, but not thapsigargin or depolarization with potassium, mimicked the OX 1 R response with regard to Mg 2ϩ sensitivity. A reverse transcription-PCR screening identified mRNAs for all transient receptor potential canonical (TRPC) channels, including TRPC3, TRPC6, and TRPC7, which are known to be activated by diacylglycerol. Expression of a dominant-negative TRPC6 channel subunit blunted the responses to both dioctanoylglycerol and OX 1 R stimulation. The results suggest that the OX 1 R activates a Ca 2ϩ entry pathway that involves diacylglycerol-activated TRPC channels in neuronal cells.
The gene encoding the gamma-chain of the human Interleukin-2 receptor was expressed in lepidopter... more The gene encoding the gamma-chain of the human Interleukin-2 receptor was expressed in lepidopteran insect cells using the baculovirus expression vector system. The corresponding gene was inserted under the polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus and expressed in the Spodoptera frugiperda insect cell line Sf9 during viral infection. The recombinant receptor protein was identified by immunoblotting in cell lysates, prepared from insect cells infected with the recombinant virus. At 40 h post infection the corresponding protein was detected as two major bands with apparent molecular weights of 50-60 kDa using a rabbit anti-human IL-2R gamma-receptor specific antiserum. Metabolic labelling with [35S]-methionine and SDS-PAGE analysis of the recombinant baculovirus infected insect cells verified the immunoblotting data. The expressed IL-2R gamma- protein could also be determined on the surface of infected insect cells by flow cytometer analysis.
Proceedings of the National Academy of Sciences, 2005
Fragile X syndrome, a common form of inherited mental retardation, is caused by the absence of th... more Fragile X syndrome, a common form of inherited mental retardation, is caused by the absence of the fragile X mental retardation protein (FMRP) due to a mutation in the FMR1 gene. We investigated the differentiation of neural stem cells generated from the brains of fmr1- knockout (KO) mice and from postmortem tissue of a fragile X fetus. Mouse and human FMRP-deficient neurospheres generated more TuJ1-positive cells (3-fold and 5-fold, respectively) than the control neurospheres generated from normal mouse and human brains, and these cells showed morphological alterations with fewer and shorter neurites and a smaller cell body volume. The number of cells expressing glial fibrillary acidic protein and generated by these neurospheres was reduced because of increased apoptotic cell death. Furthermore, there was an increase in a population of cells with intense oscillatory Ca 2+ responses to neurotransmitters in differentiated cells lacking FMRP. In addition, the number of cells in a coho...
Pfl�gers Archiv European Journal of Physiology, 2002
The different roles of glycolytic and mitochondrial pathways in glucose-induced metabolic activat... more The different roles of glycolytic and mitochondrial pathways in glucose-induced metabolic activation and insulin secretion were studied in islets of Langerhans. Single islets were perifused with 3 mM glucose together with agents affecting the production or consumption of ATP. Subsequently, glucose was raised to 11 mM and the effects of the agents on metabolic and secretory responses were evaluated. Metabolism was monitored continuously with an oxygen-sensitive microelectrode inserted into the islet. Insulin secretion was determined by assaying insulin in perifusate with ELISA. Inhibitors of mitochondrial ATP production reduced the metabolic and secretory response to glucose. When glycolytic ATP production was reduced, initial but not sustained glucose-stimulated insulin release was observed. Inhibition of mitochondrial pyruvate transport reduced the glucose-induced decline in pO 2. Although mitochondrial metabolism was eventually similar to normal, insulin release was only 20% of normal. Increased energy expenditure also changed the kinetics of the glucose-induced decline in pO 2 and decreased the insulin release by 50%. In conclusion, glucose-induced enhancement of insulin release was only seen when the rise of the sugar concentration triggered a rapid and sustained increase of mitochondrial metabolism. This activation of mitochondrial metabolism required a good metabolic state prior to the glucose challenge.
Background and aims: In focal areas of advanced human atherosclerotic lesions, the intimal fluid ... more Background and aims: In focal areas of advanced human atherosclerotic lesions, the intimal fluid is acidic. An acidic medium impairs the ABCA1-mediated cholesterol efflux from macrophages, so tending to increase their content of free cholesterol, which is then available for esterification by the macrophage enzyme ACAT1. Here we investigated whether low extracellular pH would affect the activity of ACAT1. Methods:-Human monocyte-derived macrophages were first incubated with acetyl-LDL at neutral and acidic conditions (pH 7.5, 6.5, and 5.5) to generate foam cells, and then the foam cells were incubated with [ 3 H]oleate-BSA complexes, and the formation of [ 3 H]oleate-labeled cholesteryl esters was measured. ACAT1 activity was also measured in cell-free macrophage extracts. Results:-In acidic media, ACAT1-dependent cholesteryl [ 3 H]oleate generation became compromised in the developing foam cells and their content of free cholesterol increased. In line with this finding, ACAT1 activity in the soluble cell-free fraction derived from macrophage foam cells peaked at pH 7, and gradually decreased under acidic pH with a rapid drop below pH 6.5. Incubation of macrophages under progressively more acidic conditions (until pH 5.5) lowered the cytosolic pH of macrophages (down to pH 6.0). Such intracellular acidification did not affect macrophage gene expression of ACAT1 or the neutral CEH. Conclusions: Exposure of human macrophage foam cells to acidic conditions lowers their intracellular pH with simultaneous decrease in ACAT1 activity. This reduces cholesterol esterification and thus leads to accumulation of potentially toxic levels of free cholesterol, a contributing factor to macrophage foam cell death.
A genetic fits ion between streptavidin of Streptomyces avidinii and luciferase of Pyrophorus pla... more A genetic fits ion between streptavidin of Streptomyces avidinii and luciferase of Pyrophorus plagiophthalamus was constructed. The fits ion protein was produced in the Sf9 insect cell line using the baculovirus expression vector system (REVS). Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the proteinsjrom cells infected with the recombinant virus, VLl393-LucGR-StreptAv, revealed that the fusion protein migrated with an apparent molecular weight of 75 kDa. Light emission measurements showed that the infected cells produced about 255 mg of the chimeric protein per liter of cell culture (127.5 J.1g/1 x 10 6 cells). Precipitation of the LucGR-StreptAv fusion protein with biotinylated acrylic beads as well as immunoblot analyses using biotinylated immunoglobulins indicated that both fusion moieties of the chimeric protein product were fitnctional with respect to their physical and enzymatic activities.
Cerebellar granule neurons cultured with serum develop a mature neuronal phenotype, including sti... more Cerebellar granule neurons cultured with serum develop a mature neuronal phenotype, including stimulus-coupled release of glutamate, and depend on elevated potassium for survival. We find that cells cultured with serum undergo two phases of cell death. By 6 d in vitro, 30-50% of the cells present are dead; after this time the remaining cells die. Elevated potassium prevents only this later phase of death, whereas neurotrophins protect these cells against the early phase of death. Factors that bind p75 NTR or TNF-R, members of the same receptor family, exhibit voltage-sensitive calcium channel-dependent protection, whereas ligands of expressed Trk receptors show additional calcium channel-independent protection. The cells express TrkB protein and show elevated c-Fos and c-Jun levels in response to BDNF. No TrkA is detected, although p75 NTR protein is expressed and NGF induces depolarizationdependent elevation of c-Jun levels. In the presence of the protein kinase C inhibitor bisindolylmaleimide, BDNF-induced survival promotion is reduced partially, whereas NGF-induced death is unmasked. Basal survival mechanisms are insensitive to inhibition of PK-C or PI-3 kinase. We conclude that BDNF promotes survival in part via its TrkB receptor, whereas there is an additional pathway promoting survival and elevating c-Jun evoked by both NGF and BDNF via a non-Trk receptor.
Radial glial cells play an essential role through their function as guides for neuronal migration... more Radial glial cells play an essential role through their function as guides for neuronal migration during development. Disruption of metabotropic glutamate receptor 5 (mGluR5) function retards the growth of radial glial processes in vitro. Neuregulins (NRG) are activated by proteolytic cleavage and regulate (radial) glial maintenance via ErbB3/ErbB4 receptors. We show here that blocking ErbB4 disrupts radial process extension. Soluble NRG acting on ErbB4 receptors is able to promote radial process extension in particular where process elongation has been impeded by blockade of mGluR5, the nonselective cation channel canonical transient receptor potential 3 (TRPC3), or matrix metalloproteases (MMP). NRG does not restore retarded process growth caused by ErbB4 blockade. Stimulation of muscarinic receptors restores process elongation due to mGluR5 blockade but not that caused by TRPC3, MMP or ErbB4 blockade suggesting that muscarinic receptors can replace mGluR5 with respect to radial process extension. Additionally, NRG/ ErbB4 causes Ca 21 mobilization in a population of cells through cooperation with ErbB1 receptors. Our results indicate that mGluR5 promotes radial process growth via NRG activation by a mechanism involving TRPC3 channels and MMPs. Thus neurotransmitters acting on G-protein coupled receptors could play a central role in the maintenance of the radial glial scaffold through activation of NRG/ErbB4 signaling.
1. The effect of acute morphine administration on the rotational behavior of unilaterally brain-l... more 1. The effect of acute morphine administration on the rotational behavior of unilaterally brain-lesioned mice and on the amphetamine-induced rotational behavior of unilaterally brain-lesioned rats was studied. 2. The effect of repeated morphine administration on the rotational behavior of mice and rats was also investigated. 3. Acute administration of 40 mg/kg of morphine induced strong ipsilateral rotation in unilaterally brain-lesioned mice. 4. One day after withdrawal, mice given morphine repeatedly for 5 days and treated acutely with 40 mg/kg of morphine rotated significantly less ipsilaterally than mice that had received the same dose of morphine for the first time. 5. Rats given 2 mg/kg of morphine 1 hr before administration of 5 mg/kg of amphetamine tended to rotate slightly more in the ipsilateral direction than the similarly lesioned control rats that received amphetamine alone. 6. After 1 day of withdrawal from 5 days of repeated morphine administration, rats given morphine before amphetamine tended to rotate less ipsilaterally than those given morphine before amphetamine for the first time. 7. Thus, repeated administration of morphine appears to induce tolerance to the effect of morphine on circling behavior in unilaterally brain-lesioned mice and rats.
The guidance of developing neurons to the right position in the central nervous system is of cent... more The guidance of developing neurons to the right position in the central nervous system is of central importance in brain development. Canonical transient receptor potential (TRPC) channels are thought to mediate turning responses of growth cones to guidance cues through fine control of calcium transients. Proliferating and 1-to 5day-differentiated neural progenitor cells (NPCs) showed expression of Trpc1 and Trpc3 mRNA, while Trpc4-7 was not clearly detected. Time-lapse imaging showed that the motility pattern of neuronal cells was phasic with bursts of rapid movement (> 60 mm/h), changes in direction, and intermittent slow phases or stallings (< 40 mm/ h), which frequently occurred in close contact with radial glial processes. Genetic interference with the TRPC3 and TRPC1 channel enhanced the motility of NPCs (burst frequency/stalling frequency). TRPC3-deficient cells or cells treated with the TRPC3 blocker pyr3 infrequently changed direction and seldom contacted radial glial processes. TRPC channels are also activated by group I metabotropic glutamate receptors (mGluR1 and mGluR5). As shown here, pyr3 blocked the calcium response mediated through mGluR5 in radial glial processes. Furthermore, 2-methyl-6-(phenylethynyl)pyridine, a blocker of mGluR5, affected the motility pattern in a similar way as TRPC3/6 double knockout or pyr3. The results suggest that radial glial cells exert attractant signals to migrating neuronal cells, which alter their motility pattern. Our results suggest that mGluR5 acting through TRPC3 is of central importance in radial glial-mediated neuronal guidance.
Gia (?) Gitt, Ca^^ Gia To test whether py subunits would be involved in the stimulation of cAMP a... more Gia (?) Gitt, Ca^^ Gia To test whether py subunits would be involved in the stimulation of cAMP accumulation via the human a2B-AR in Sf9 cells the effects of Gpy and PARK (which functions as a Gpy scavenger) on forskolin-stimulated cAMP accumulation was tested (Fig. 1). While pARK enhanced forskolin-stimulated cAMP accumulation the GPy coexpression considerably reduced cAMP accumulation. This means that the adenylyl cyclase of Sf9 cells is inhibited by Py rather than stimulated. The a2B-AR-stimulated AMP accumulation in Sf9 cells can thus not be due to Py. The basal and forskolin
Five recombinant baculoviruses, each containing a different insect luciferase gene encoding a pro... more Five recombinant baculoviruses, each containing a different insect luciferase gene encoding a protein with characteristic light emission properties, namely, luc GR (546 nm), luc FF (556 nm), luc YG (560 nm), luc YE (578 nm), and luc OR (593 nm) were constructed. All genes were inserted under the transcriptional control of the polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV) and expressed in Spodoptera frugiperda insect cells during viral infection. The biological activity of the different luciferases was characterized by using intact recombinant baculovirus infected cells. Addition of the substrate, D-luciferin, immediately prior to the analysis allowed monitoring of light emission by flow cytometry. Also, the kinetics of the light emission of lucGR was analyzed with the flow cytometer. The emission peaks of the infected cells were clearly separated by wavelength scanning. Especially, the firefly luciferase (lucFF) had a broad peak and transient luminescence. The highest maximal intensity values in vivo were recorded for luc GR and luc YG. SDS-PAGE analysis showed that the major protein expressed had a molecular weight similar to authentic luciferase. Flow cytometry and insect luciferases with clearly separated emission spectra appear to be of value for sensitive in vivo analysis of gene promoter activity.
O uso da terapia antirretroviral (TARV) tem aumentado à sobrevida das pessoas vivendo com HIV/AID... more O uso da terapia antirretroviral (TARV) tem aumentado à sobrevida das pessoas vivendo com HIV/AIDS (PVHA) alterando consideravelmente a história natural da infecção. Entretanto, em alguns casos, o uso destes medicamentos tem sido associado à distúrbios nutricionais e metabólicos. O objetivo deste estudo foi descrever os distúrbios nutricionais e metabólicos ocasionados pelo uso da TARV e o respectivo papel terapêutico da abordagem nutricional em PVHA. Trata-se de uma revisão narrativa, realizada durante o segundo semestre do ano de 2012, utilizando os seguintes descritores: AIDS, HIV, PVHA, TARV, estado nutricional, acompanhamento nutricional. O principal distúrbio nutricional encontrado nesta revisão, relacionado ao uso de TARV, foi a síndrome lipodistrófica associada à dislipidemias e hiperglicemias, bem como, alterações ósseas e aumento de gordura corporal. Neste sentido, o acompanhamento nutricional é extremamente importante para a melhora da qualidade de vida e prevenção de demais complicações metabólicas em PVHA.
A single G-protein-coupled receptor might activate multiple G-protein species. This multiplex cou... more A single G-protein-coupled receptor might activate multiple G-protein species. This multiplex coupling ability can be used by tissues to regulate signalling; for the pharmacologist, such multiplex coupling might cause difficulties in the interpretation of experimental data. In this article, we present mathematical models for the activation of two separate G-protein species by a single receptor. Issues addressed concern mutual antagonism between the G proteins and the availability of an already activated receptor for interaction with a new G protein (receptor-G-protein-effector complexing versus free diffusion of G proteins) in addition to receptor-G-protein precoupling at different G-protein and receptor expression levels. The output from the receptor models uses, as readout, a new model for adenylyl cyclase regulation by two allosteric regulators (i.e. G(s) and G(i)).
By studying the influence of two toxins from the black mamba Dendroaspis polylepis on the kinetic... more By studying the influence of two toxins from the black mamba Dendroaspis polylepis on the kinetics of [3H]-N-methylscopolamine binding to muscarinic acetylcholine receptors from rat cerebral cortex, it was revealed that these toxins, MT alpha and MT beta, interact with the receptors via kinetically distinct mechanisms. MT beta bound to receptors in a one-step, readily reversible process with the dissociation constant K(d)=5.3 microM. The binding mechanism of MTalpha was more complex, involving at least two consecutive steps. A fast receptor-toxin complex formation (K(T)=3.8 microM) was followed by a slow process of isomerisation of this complex (k(i)=1.8 x 10(-2) s(-1), half-time 39 s). A similar two-step interaction mechanism has been established for a related toxin, MT2 from the green mamba D. angusticeps (K(T)=1.4 microM, k(i)=8.3 x 10(-4) s(-1), half-time 840 s). The slow isomerisation process delays the effect of MT alpha and MT2, but increases their apparent potency compared to toxins unable to induce the isomerisation process.
We studied the cellular response to orexin type 1 receptor (OX 1 R) stimulation in differentiated... more We studied the cellular response to orexin type 1 receptor (OX 1 R) stimulation in differentiated IMR-32 neuroblastoma cells. In vitro differentiation of IMR-32 cells with 5-bromo-2Ј-deoxyuridine leads to a neuronal phenotype with long neurite extensions and an upregulation of mainly N-type voltage-gated calcium channels. Transduction of differentiated IMR-32 cells with baculovirus harboring an OX 1 R-green fluorescent protein cDNA fusion construct resulted in appearance of fluorescence that was confined mainly to the plasma membrane in the cell body and to neurites. Application of orexin-A to fluorescent cells led to an increase in intracellular free Ca 2ϩ concentration, [Ca 2ϩ ] i. At low nanomolar concentrations of orexin-A, the response was reversibly attenuated by removal of extracellular Ca 2ϩ , by application of a high concentration (10 mM) of Mg 2ϩ , and by the pharmacological channel blocker dextromethorphan. A diacylglycerol, dioctanoylglycerol, but not thapsigargin or depolarization with potassium, mimicked the OX 1 R response with regard to Mg 2ϩ sensitivity. A reverse transcription-PCR screening identified mRNAs for all transient receptor potential canonical (TRPC) channels, including TRPC3, TRPC6, and TRPC7, which are known to be activated by diacylglycerol. Expression of a dominant-negative TRPC6 channel subunit blunted the responses to both dioctanoylglycerol and OX 1 R stimulation. The results suggest that the OX 1 R activates a Ca 2ϩ entry pathway that involves diacylglycerol-activated TRPC channels in neuronal cells.
The gene encoding the gamma-chain of the human Interleukin-2 receptor was expressed in lepidopter... more The gene encoding the gamma-chain of the human Interleukin-2 receptor was expressed in lepidopteran insect cells using the baculovirus expression vector system. The corresponding gene was inserted under the polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus and expressed in the Spodoptera frugiperda insect cell line Sf9 during viral infection. The recombinant receptor protein was identified by immunoblotting in cell lysates, prepared from insect cells infected with the recombinant virus. At 40 h post infection the corresponding protein was detected as two major bands with apparent molecular weights of 50-60 kDa using a rabbit anti-human IL-2R gamma-receptor specific antiserum. Metabolic labelling with [35S]-methionine and SDS-PAGE analysis of the recombinant baculovirus infected insect cells verified the immunoblotting data. The expressed IL-2R gamma- protein could also be determined on the surface of infected insect cells by flow cytometer analysis.
Proceedings of the National Academy of Sciences, 2005
Fragile X syndrome, a common form of inherited mental retardation, is caused by the absence of th... more Fragile X syndrome, a common form of inherited mental retardation, is caused by the absence of the fragile X mental retardation protein (FMRP) due to a mutation in the FMR1 gene. We investigated the differentiation of neural stem cells generated from the brains of fmr1- knockout (KO) mice and from postmortem tissue of a fragile X fetus. Mouse and human FMRP-deficient neurospheres generated more TuJ1-positive cells (3-fold and 5-fold, respectively) than the control neurospheres generated from normal mouse and human brains, and these cells showed morphological alterations with fewer and shorter neurites and a smaller cell body volume. The number of cells expressing glial fibrillary acidic protein and generated by these neurospheres was reduced because of increased apoptotic cell death. Furthermore, there was an increase in a population of cells with intense oscillatory Ca 2+ responses to neurotransmitters in differentiated cells lacking FMRP. In addition, the number of cells in a coho...
Pfl�gers Archiv European Journal of Physiology, 2002
The different roles of glycolytic and mitochondrial pathways in glucose-induced metabolic activat... more The different roles of glycolytic and mitochondrial pathways in glucose-induced metabolic activation and insulin secretion were studied in islets of Langerhans. Single islets were perifused with 3 mM glucose together with agents affecting the production or consumption of ATP. Subsequently, glucose was raised to 11 mM and the effects of the agents on metabolic and secretory responses were evaluated. Metabolism was monitored continuously with an oxygen-sensitive microelectrode inserted into the islet. Insulin secretion was determined by assaying insulin in perifusate with ELISA. Inhibitors of mitochondrial ATP production reduced the metabolic and secretory response to glucose. When glycolytic ATP production was reduced, initial but not sustained glucose-stimulated insulin release was observed. Inhibition of mitochondrial pyruvate transport reduced the glucose-induced decline in pO 2. Although mitochondrial metabolism was eventually similar to normal, insulin release was only 20% of normal. Increased energy expenditure also changed the kinetics of the glucose-induced decline in pO 2 and decreased the insulin release by 50%. In conclusion, glucose-induced enhancement of insulin release was only seen when the rise of the sugar concentration triggered a rapid and sustained increase of mitochondrial metabolism. This activation of mitochondrial metabolism required a good metabolic state prior to the glucose challenge.
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Papers by Karl Åkerman