RESUMO
OBJECTIVE: To compare in a randomized, non-inferiority trial the embryo transfer (ET) technique based on uterine length measurement before transfer (ULMbET) with transabdominal ultrasound-guided embryo transfer (TA-UGET) in a large population of patients undergoing in-vitro fertilization (IVF). METHODS: Patients undergoing IVF with ET with cleaving fresh embryos were randomized to receive ULMbET or TA-UGET. The transfer of one to three embryos on day 2-3 of culture was performed using a soft catheter either under transabdominal ultrasound guidance (TA-UGET group) or after measurement of the uterine cavity by transvaginal ultrasound and calculation of the discharge site (ULMbET group). The primary outcome measures were clinical pregnancy rate, ongoing pregnancy rate and implantation rate, and secondary outcomes included patient discomfort during ET. RESULTS: A total of 1648 patients undergoing IVF were randomized to receive ULMbET (n = 828) or TA-UGET (n = 820) and were included in the analysis. Comparable clinical pregnancy rate (38.2% vs 38.9%), implantation rate (24.8% vs 25.2%) and ongoing pregnancy rate (33.1% vs 34.8%) were observed in ULMbET and TA-UGET groups. The discomfort intensity score and the proportion of patients with moderate-to-severe discomfort during ET were significantly higher in the TA-UGET group (2.6 vs 1.5 visual analog scale points and 19.8% vs 1.2%; P = 0.045 and P = 0.003, respectively). CONCLUSION: The ULMbET technique leads to IVF results comparable to those obtained with UGET, but is better tolerated than is TA-UGET and is technically easier to perform for a single operator. Copyright © 2016 ISUOG. Published by John Wiley & Sons Ltd.
Assuntos
Transferência Embrionária/métodos , Fertilização in vitro/métodos , Ultrassonografia de Intervenção , Útero/diagnóstico por imagem , Adulto , Implantação do Embrião , Feminino , Humanos , Gravidez , Resultado do Tratamento , Útero/patologiaRESUMO
BACKGROUND: Epidemiological studies have suggested an increased risk of cardiovascular events (CVE) during acute stressful and/or frightful moments. A possible explanation for this could be an effect of acute stress on hemostasis. A recent study demonstrated an increase in factor VIII after watching a horror movie. Primary hemostasis, however, is thought to play a more prominent role in the etiology of CVE. The objective of this study was therefore to assess the influence of viewing a 'bloodcurdling' horror movie on platelet reactivity in healthy volunteers. METHODS: We performed a randomized cross-over study in healthy adults. Subjects were allocated to two movies in random sequence: a horror and a control movie. Blood was drawn at baseline and after 24â¯min of viewing time. The primary endpoint was the change in Platelet Function Analyzer® Closure Time (Δ PFA-CT) after watching the movie. RESULTS: In total, 20 participants, aged 18-30â¯years, completed the study protocol. The delta PFA-CT was statistically significantly shorter with a mean in the delta difference of -9.7â¯s (SEM 4.0, 95% C.I. -18.0 to -1.3) during the horror movie versus the control movie. The Light Transmission Aggregometry endpoints were in line with the PFA-CT, albeit only the highest level of Arachidonic Acid agonist demonstrated a statistically significant mean difference in the delta of aggregation of 13.15% (SEM 7.0, 95% C.I. 1.6-27.9). CONCLUSION: A 'blood curdling' horror movie increases platelet reactivity. These data are supportive of a role of platelet reactivity in acute stress induced cardiovascular event risk.
Assuntos
Filmes Cinematográficos , Ativação Plaquetária , Angústia Psicológica , Adulto , Plaquetas/citologia , Doenças Cardiovasculares/etiologia , Estudos Cross-Over , Feminino , Voluntários Saudáveis , Humanos , Masculino , Agregação Plaquetária , Adulto JovemRESUMO
The site of action potential initiation in substantia nigra neurons was investigated by using simultaneous somatic and dendritic whole-cell recording in brain slices. In many dopamine neurons, action potentials were observed first at the dendritic recording site. Anatomical reconstruction showed that in these neurons, the axon emerged from the dendrite from which the recording had been made. Action potentials showed little attention in the dendritic tree, which in dopamine neurons was shown to be due to recruitment of dendritic sodium channels and may be related to the dendritic release of dopamine. We conclude that in substantia nigra neurons, the site of action potential initiation, and thus the final site of synaptic integration, is in the axon. As the axon can originate from a dendrite, up to 240 microns away from the soma, synaptic input to the axon-bearing dendrite may be privileged with respect to its ability to influence action potential initiation.
Assuntos
Axônios/fisiologia , Dendritos/fisiologia , Neurônios/ultraestrutura , Substância Negra/ultraestrutura , Potenciais de Ação , Animais , Axônios/ultraestrutura , Dendritos/ultraestrutura , Dopamina/metabolismo , Potenciais da Membrana , Neurônios/metabolismo , Ratos , Ratos Wistar , Tetrodotoxina/farmacologia , Ácido gama-Aminobutírico/metabolismoRESUMO
Fast excitatory synaptic transmission in the CNS is mediated by AMPA-type glutamate receptor (GluR) channels. Heterologous expression suggested that the Ca2+ permeability of these receptors critically depends on the subunit composition. Using patch-clamp techniques in brain slices, we found that the Ca2+ permeability of native AMPA-type GluRs was markedly higher in nonpyramidal (PCa/PK approximately 0.63) than in pyramidal (PCa/PK approximately 0.05) neurons of rat neocortex. Analysis of mRNA in single cells indicated that the relative abundance of GluR-B-specific mRNA was significantly lower in nonpyramidal (GluR-B/GluR-non-B approximately 0.3) than in pyramidal (GluR-B/GluR-non-B approximately 3) cells. This suggests that differences in relative abundance of GluR-B-specific mRNA generate functional diversity of AMPA-type GluRs in neurons with respect to Ca2+ permeability.
Assuntos
Canais de Cálcio/fisiologia , Cálcio/metabolismo , Córtex Cerebral/fisiologia , Expressão Gênica , Neurônios/fisiologia , Células Piramidais/fisiologia , Receptores de AMPA/fisiologia , Receptores de Glutamato/biossíntese , Córtex Visual/fisiologia , Animais , Sequência de Bases , Primers do DNA , Técnicas In Vitro , Substâncias Macromoleculares , Potenciais da Membrana/fisiologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Ratos , Ratos WistarRESUMO
Glutamate receptors activated by NMDA (NMDARs) or AMPA (AMPARs) are clustered on dendritic spines of pyramidal cells. Both the AMPAR-mediated postsynaptic responses and the synaptic AMPAR immunoreactivity show a large intersynapse variability. Postsynaptic responses mediated by NMDARs show less variability. To assess the variability in NMDAR content and the extent of their coexistence with AMPARs in Schaffer collateral-commissural synapses of adult rat CA1 pyramidal cells, electron microscopic immunogold localization of receptors has been used. Immunoreactivity of NMDARs was detected in virtually all synapses on spines, but AMPARs were undetectable, on average, in 12% of synapses. A proportion of synapses had a very high AMPAR content relative to the mean content, resulting in a distribution more skewed toward larger values than that of NMDARs. The variability of synaptic NMDAR content [coefficient of variation (CV), 0.64-0.70] was much lower than that of the AMPAR content (CV, 1.17-1.45). Unlike the AMPAR content, the NMDAR content showed only a weak correlation with synapse size. As reported previously for AMPARs, the immunoreactivity of NMDARs was also associated with the spine apparatus within spines. The results demonstrate that the majority of the synapses made by CA3 pyramidal cells onto spines of CA1 pyramids express both NMDARs and AMPARs, but with variable ratios. A less-variable NMDAR content is accompanied by a wide variability of AMPAR content, indicating that the regulation of expression of the two receptors is not closely linked. These findings support reports that fast excitatory transmission at some of these synapses is mediated by activation mainly of NMDARs.
Assuntos
Células Piramidais/química , Receptores de AMPA/análise , Receptores de N-Metil-D-Aspartato/análise , Sinapses/química , Animais , Linhagem Celular , Dendritos/química , Humanos , Imuno-Histoquímica , Ratos , Ratos WistarRESUMO
The regulation of AMPA receptors at the postsynaptic membrane is a fundamental component of synaptic plasticity. In the hippocampus, the induction of long-term potentiation increases the delivery of GluR1, a major AMPA receptor subunit in hippocampal pyramidal neurons, to the synaptic plasma membrane through a mechanism that requires the PDZ binding domain of GluR1. Synapse-associated protein 97 (SAP97), a member of the membrane-associated guanylate kinase family, is believed to associate with AMPA receptors (AMPARs) containing the GluR1 subunit, but the functional significance of these interactions is unclear. We investigated the interaction of GluR1 with SAP97, the only PDZ protein known to interact with GluR1. We find that interactions involving SAP97 and GluR1 occur early in the secretory pathway, while the receptors are in the endoplasmic reticulum or cis-Golgi. In contrast, few synaptic receptors associate with SAP97, suggesting that SAP97 dissociates from the receptor complex at the plasma membrane. We also show that internalization of GluR1, as triggered by NMDAR activation, does not require SAP97. These results implicate GluR1-SAP97 interactions in mechanisms underlying AMPA receptor targeting.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Receptores de AMPA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Biotinilação , Membrana Celular/química , Membrana Celular/metabolismo , Células Cultivadas , Córtex Cerebral/química , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Hipocampo/química , Hipocampo/citologia , Hipocampo/metabolismo , Imuno-Histoquímica , Proteínas de Membrana , Modelos Neurológicos , Neurônios/metabolismo , Neurônios/ultraestrutura , Testes de Precipitina , Ratos , Ratos Sprague-Dawley , Frações Subcelulares/metabolismo , Sinapses/metabolismo , Sinapses/ultraestruturaRESUMO
We have used the whole cell technique to microinject human fibroblasts with either 1,4,5-inositol trisphosphate (InsP3) or 'caged' InsP3, in order to study the mechanisms of transmembrane signalling related to mitogenic stimulations. Cytosolic Ca2+ elevations in response to 1,4,5 InsP3 diffusing from the patch pipette were difficult to detect, while 1,4,5 InsP3, photoreleased after loading the cell with its inactive precursor, was capable of generating not only a single cytosolic Ca2+ rise but sometimes triggered an oscillatory calcium response, similar to that often observed under mitogenic stimulation. We estimated that less than 100 nM InsP3 was sufficient to generate Ca2+ responses. The Ca2+ rise produced by the photoreleased InsP3 could fully activate the K+ channels present in the plasma membrane of human fibroblasts.
Assuntos
Cálcio/metabolismo , Membrana Celular/metabolismo , Citosol/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Células Cultivadas , Fibroblastos , Humanos , Inositol 1,4,5-Trifosfato/análogos & derivados , Inositol 1,4,5-Trifosfato/farmacologia , FotóliseRESUMO
Ca2+ transients (measured with Fluo-3) were induced in single mouse ovarian oocytes by photolytic liberation of InsP3. The time course of cytosolic Ca2+ changes induced in this way is composed of distinct phases: upstroke, fast decline, slow declining plateau and fast decline to rest level. All the phases reflect mainly intracellular redistributions of the ion and not influx, since they are not strongly dependent on external Ca2+ or on changes in transmembrane potential. Often sustained Ca2+ oscillations followed the first InsP3-induced Ca2+ transient. These persisted for several minutes in the absence of external Ca2+. The initial rate of Ca2+ rise and the delay between the InsP3 stimulus and Ca2+ upstroke are correlated with the amount of liberated InsP3. A second InsP3 stimulation, applied during the plateau, causes only small Ca2+ elevations, lacking the upstroke phase. A second, full sized, transient could be elicited only after a complete return to the basal level. Vanadate, applied intracellularly, appeared to inhibit the re-uptake phase into the stores, stabilizing the plateau level. The present observations suggest that in mouse oocytes the InsP3-sensitive stores provide only a small and graded Ca2+ release which may then act as a trigger for a more substantial Ca(2+)-induced Ca2+ release (CICR) process.
Assuntos
Cálcio/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Oócitos/metabolismo , Animais , Feminino , Cinética , Potenciais da Membrana , Camundongos , Ovário/metabolismo , Fotólise , Vanadatos/farmacologiaRESUMO
Bradykinin (BK) and platelet-derived growth factor (PDGF) act as mitogens and stimulate phosphatidylinositol (PI) turnover in human fibroblasts. By coupling whole-cell electrophysiological measurements with cytosolic Ca2+ determinations using fura-2 microfluorimetry, we have studied the changes in cytosolic calcium and in membrane conductance in single cells following stimulation with BK or PDGF. Both agonists produce variable patterns of response which include: single transient, sustained pulsations, damped oscillations, no response. In all cases, there is a very good temporal correlation between increases in intracellular Ca2+ and membrane current. The cytosolic calcium elevation appears to be insensitive to membrane potential changes, indicating that Ca2+ is released from an intracellular source. The Ca2(+)-activated current is not blocked by 1 microM apamin or by 0.5 mM (+)-tubocurarine; it is instead strongly reduced by 5 mM tetraethylammonium (TEA). We can conclude that BK and PDGF induce very similar early responses in human fibroblasts, and that the variable pattern of response does not depend on the particular mitogen used. The membrane currents are due to a kind of Ca2(+)-activated K+ channels which, according to their voltage-dependence and specific blockers, belong to the "maxi K+" class.
Assuntos
Bradicinina/farmacologia , Cálcio/metabolismo , Membrana Celular/fisiologia , Citosol/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Calcimicina/farmacologia , Células Cultivadas , Condutividade Elétrica , Fibroblastos , Humanos , Potenciais da Membrana , Fosfatidilinositóis/metabolismo , Potássio/metabolismo , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/metabolismo , Tubocurarina/farmacologiaRESUMO
The regional and subcellular localizations of glycine receptor complex messenger RNAs were determined in the adult rat central nervous system using non-radioactive in situ hybridization. The present investigation focused on glycine receptors alpha1 and alpha2 subunit messenger RNAs. Within the central nervous system we observed that the glycine receptor alpha1 and alpha2 subunit messenger RNAs are widely expressed. At the subcellular level, these messenger RNAs are present either in neuronal somata and dendrites or somata only. Furthermore, among different regions as well as within the same region the subcellular localizations of both alpha subunit messenger RNAs are cell type-dependent. In contrast, the regional distributions of beta subunit and gephyrin messenger RNAs are essentially as previously described [Fujita M. (1991) Brain Res. 560, 23-37; Malosio M.-L. et al. (1991) Eur. molec. Biol. Org. J. 9, 2401-2409; Kirsch J. et al. (1993) Eur. J. Neurosci. 5, 1109-1117] and their messenger RNAs are confined predominantly within the somata of neurons [Kirsch J. et al. (1993); Racca et al. (1997) J. Neurosci. 17, 1691-1700]. These results demonstrate that the glycine receptor complex messenger RNAs are broadly expressed in the central nervous system and that the glycine receptor alpha1 and alpha2 subunit messenger RNAs differ in their subcellular localization depending on the neuronal population. The latter finding suggests that different mechanisms for the localization of glycine receptor alpha1 and alpha2 subunit messenger RNAs are used by distinct populations of neurons.
Assuntos
Dendritos/metabolismo , RNA Mensageiro/biossíntese , Receptores de Glicina/biossíntese , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Dendritos/ultraestrutura , Hibridização In Situ , Sondas de Oligonucleotídeos , Ratos , Ratos Sprague-Dawley , Medula Espinal/citologia , Medula Espinal/metabolismo , Medula Espinal/ultraestrutura , Frações Subcelulares/metabolismo , Frações Subcelulares/ultraestruturaRESUMO
Proton-proton small angle correlations have been measured in neon-nucleus collisions, using the 4 pi detector Diogene, at 400 and 800 MeV per nucleon incident energies. Values of the size of the emitting region are obtained by comparison with the Koonin formula, taking into account the biases of the apparatus. The dependence of the density on target mass and incident energy is also analysed.
Assuntos
Transferência de Energia , Neônio , Física Nuclear , Prótons , Carbono , Partículas Elementares , Chumbo , Modelos Teóricos , Nióbio , Aceleradores de Partículas , Fluoreto de SódioRESUMO
Mean multiplicities of pi+ and pi- in 4He collisions with C, Cu, and Pb at 200, 600, and 800 MeV/u, and with C and Pb at 400 MeV/u have been measured using the large solid angle detector Diogene. The independence of pion multiplicity on projectile incident energy, target mass and proton multiplicity is studied in comparison with intra-nuclear cascade predictions. The discrepancy between experimental results and theory is pointed out and discussed.
Assuntos
Hélio , Mésons , Modelos Teóricos , Prótons , Radiometria , Carbono , Cobre , Transferência de Energia , Íons , Chumbo , Método de Monte Carlo , Física NuclearRESUMO
BACKGROUND: In 2004, a law regulating assisted reproduction techniques (ART) was passed in Italy. The new rules allow for the formation and transfer of a maximum of three embryos at one time, whereas embryo selection and embryo storage are prohibited. The aim of this study is to evaluate the impact of these restrictions on ICSI outcome in couples affected by severe male factor infertility. METHODS: Thirteen Italian ART Units were involved in this study. Data were collected on ICSI cycles performed during 2 years before (control group) and 2 years after (study group) the enforcement of the law. Only cases of obstructive azoospermia (OA), non-obstructive azoospermia (NOA) and severe oligoastenoteratozoospermia (OAT) (sperm count Assuntos
Azoospermia
, Legislação Médica
, Taxa de Gravidez/tendências
, Técnicas de Reprodução Assistida/legislação & jurisprudência
, Injeções de Esperma Intracitoplásmicas
, Adulto
, Transferência Embrionária
, Feminino
, Humanos
, Itália
, Masculino
, Gravidez
RESUMO
BRCA1 germline mutations predispose women to early onset, familial breast and ovarian cancer. BRCA1 has been recently implicated in the cellular response to agents that disrupt the mitotic spindle. In this report, we studied BRCA1 contribution to paclitaxel response in MCF-7 breast cancer cells. We show that MCF-7 cells transfected with BRCA1 siRNA display a significant increase in resistance to paclitaxel compared with the control cells. We next demonstrate that downregulation of BRCA1 reduces the mitotic index and triggers premature cyclin B1 degradation and decrease in Cdk1 activity following paclitaxel treatment, suggesting that BRCA1 downregulation results in precocious inactivation of the spindle checkpoint. These findings were confirmed by showing that BRCA1 downregulation induces premature sister-chromatids separation in MCF-7 cells following spindle damage. Furthermore, we show that BRCA1 up-regulates the expression of the protein kinase BubR1, essential component of the functional spindle checkpoint, whose downregulation is known to result in paclitaxel resistance in MCF-7 cells. Altogether, our findings support the notion that downregulation of BRCA1 expression mediates paclitaxel resistance through premature inactivation of spindle checkpoint in MCF-7 breast cancer cells. They link BRCA1 to the mitotic checkpoint that plays an essential role in the maintenance of chromosomal stability.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Proteína BRCA1/genética , Neoplasias da Mama/metabolismo , Paclitaxel/farmacologia , Fuso Acromático/efeitos dos fármacos , Proteína BRCA1/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Instabilidade Cromossômica , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , RNA Interferente Pequeno/metabolismo , Fuso Acromático/ultraestrutura , Regulação para CimaRESUMO
The level of arousal in mammals is correlated with metabolic state and specific patterns of cortical neuronal responsivity. In particular, rhythmic transitions between periods of high activity (up phases) and low activity (down phases) vary between wakefulness and deep sleep/anesthesia. Current opinion about changes in cortical response state between sleep and wakefulness is split between neuronal network-mediated mechanisms and neuronal metabolism-related mechanisms. Here, we demonstrate that slow oscillations in network state are a consequence of interactions between both mechanisms. Specifically, recurrent networks of excitatory neurons, whose membrane potential is partly governed by ATP-modulated potassium (K(ATP)) channels, mediate response-state oscillations via the interaction between excitatory network activity involving slow, kainate receptor-mediated events and the resulting activation of ATP-dependent homeostatic mechanisms. These findings suggest that K(ATP) channels function as an interface between neuronal metabolic state and network responsivity in mammalian cortex.
Assuntos
Córtex Cerebral/fisiologia , Rede Nervosa , Neurônios/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Canais de Potássio/metabolismo , RatosRESUMO
Some synaptic neurotransmitter receptors, such as those for glycine, have somato-dendritic distributions. Although the machinery for protein synthesis and several mRNAs are present in dendrites and close to synapses in central neurons, so far the mRNAs for neurotransmitter receptors have not been found unequivocally in dendrites. The glycine receptor (GlyR), a ligand-gated channel mediating a chloride-dependent inhibition, is composed of transmembrane alpha and beta subunits. GlyRs are only present at glycinergic postsynaptic differentiation, where they are stabilized by the associated protein gephyrin. With light nonradioactive in situ hybridization (ISH), we observe that GlyR alpha subunit mRNAs are present in both somata and dendrites of most neurons of the ventral horn of rat spinal cord, whereas the beta subunit and gephyrin mRNAs are predominantly in somata. Interestingly, within dendrites GlyR alpha subunit mRNAs form aggregates that are mostly localized peripherally to the dendritic axial core. Electron microscopic ISH shows that GlyR alpha subunit mRNAs are associated with postsynaptic differentiations. At these sites, the GlyR alpha subunit mRNAs are detected in close association with subsynaptic cisternae. This targeting of alpha subunit mRNAs to postsynaptic domains could provide a means of dynamically modulating synaptic efficacy by changing the composition and the density of receptors at glycinergic synapses.
Assuntos
Dendritos/química , Proteínas do Tecido Nervoso/genética , RNA Mensageiro/análise , Receptores de Glicina/genética , Sinapses/química , Animais , Proteínas de Transporte/metabolismo , Canais de Cloreto/metabolismo , Hibridização In Situ , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/biossíntese , Ratos , Ratos Sprague-Dawley , Medula Espinal/citologiaRESUMO
There is a growing body of evidence that local protein synthesis beneath synapses may provide a novel mechanism underlying plastic phenomena. In vivo and in vitro biochemical data show that dendrites can perform translation and glycosylation. Using antibodies directed against the eukaryotic protein synthetic machinery, we sought to identify the structures implicated in nonperinuclear translation, namely dendritic and postsynaptic protein synthesis. We performed a morphological and immunocytochemical analysis of ventromedial horn rat spinal cord neurons using both light and electron microscopy. We show at the cellular level that, in vivo, protein synthesis macrocomplexes (ribosomes and eIF-2) as well as the endomembranous system implicated in cotranslational and posttranslational modifications (endoplasmic reticulum and Golgi cisternae) penetrated some dendrites. Membrane-limited organelles of different shape and size are present close to the postsynaptic differentiations of most synapses, independently of their localization on the neuronal surface. We demonstrate (1) that some cisternae are immunoreactive for antibodies against ribosomal proteins and eIF-2, and (2) that markers of endoplasmic reticulum (BiP), intermediate compartment, and Golgi complex (rab1, CTR433, TGN38) label subsets of these subsynaptic organelles. Therefore, these findings indicate that synapses are equipped with the essential elements required for the synthesis and insertion of a well folded and glycosylated transmembrane protein.
Assuntos
Dendritos/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Sinapses/metabolismo , Animais , Feminino , Glicosilação , Imuno-Histoquímica , Microscopia Eletrônica , Organelas/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
The physiological properties of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptor (GluR) channels are determined by their subunit composition. In particular, the expression of the GluR-B subunit determines the divalent ion selectivity of the channel. We studied the distribution of GluR-B mRNA and protein in adult rat hippocampal GABAergic neurons combining non-radioactive in situ hybridization with immunocytochemistry. The majority of GABAergic hippocampal neurons were GluR-B mRNA-positive, but overall the levels of GluR-B transcript were lower than in pyramidal and granule cells, which showed the highest hybridization signal. The different GluR-B mRNA expression levels in GABAergic versus principal neurons were also observed at the protein level. There was a paucity of GluR-B/C immunoreactivity in the vast majority of somata of the GABAergic neurons studied, which contrasted with the strong expression of GluR-B/C proteins in the hippocampal principal neurons. Our results demonstrate the general low expression of the GluR-B subunit mRNA and GluR-B/C proteins in GABAergic hippocampal neurons. Considering the dominant role of the GluR-B subunit in determining the divalent ion permeability of the receptor, it is likely that most GABAergic neurons express AMPA receptor channels with different calcium permeabilities.
Assuntos
Hipocampo/química , Interneurônios/química , Neurônios/química , Fragmentos de Peptídeos/análise , Receptores de AMPA/química , Ácido gama-Aminobutírico/fisiologia , Animais , Glutamato Descarboxilase/análise , Glutamato Descarboxilase/genética , Hipocampo/citologia , Imuno-Histoquímica , Masculino , RNA Mensageiro/análise , Ratos , Ratos Sprague-DawleyRESUMO
The ability of purinergic agonists to induce Ca2+ responses has been tested in two lines of murine fibroblasts: normal NIH 3T3 fibroblasts and NIH 115.14, a clone expressing high levels [1] of the c-ras protooncogene. Both kinds of cells are responsive to ATP in the range 1 microM-1 mM; ADP and ATP gamma S are almost as potent as ATP, while AMP is unable to elicit a response. Ca2+ measurements performed in single cells by image analysis show great variability among cells but in each individual responding cell the Ca2+ rise occurs in an all-or-none fashion. The transient Ca2+ response does not depend on influx from the extracellular medium. Electrophysiological experiments reveal the activation of an outward current (at -50 mV) by ATP, probably due to Ca(2+)-activated K+ channels, confirming the absence of a substantial Ca2+ influx. Finally, stimulation by ATP produces a small but significant increase in the production of inositol phosphates. These results indicate that these cell lines possess purinergic receptors which are not integral membrane channels and which are coupled to InsP3 formation and may be therefore classified as P2Y.
Assuntos
Genes ras , Receptores Purinérgicos/metabolismo , Transdução de Sinais , Células 3T3 , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular Transformada , Cinética , Potenciais da Membrana , Camundongos , Fosfatidilinositóis/metabolismoRESUMO
Triple-differential cross sections of charged pions were measured for collisions of Ne projectiles at E/A = 800 MeV with NaF, Nb, and Pb targets. The reaction plane was estimated event by event from the light-baryon momentum distribution. For heavy targets, preferential emission of charged pions away from the interaction zone towards the projectile side was observed in the transverse direction. Such a preferential emission, which is not predicted by cascade calculations, may be attributed to a stronger pion absorption by the heavier spectator remnant.