RESUMO
A generalization of the hybrid scheme for multireference methods as recently put forward by Saitow and Yanai [J. Chem. Phys. 152, 114 111 (2020)] is presented. The hybrid methods are constructed by defining internal and external excitation spaces and evaluating these two subsets of excitations at different levels of theory. New hybrids that use the mix of internally contracted multireference coupled-cluster, unshifted multireference coupled electron pair, and multireference perturbation methods are derived and benchmarked. A new separation of the excitation space, which combines all singles and doubles excitations to the virtual orbitals into the external space, is also presented and tested. In general, the hybrid methods improve upon their non-hybrid parent method and offer a good compromise between computational complexity and numerical accuracy.
RESUMO
Gain-of-function missense mutations in voltage-gated sodium channel Nav1.7 have been linked to small-fiber neuropathy, which is characterized by burning pain, dysautonomia and a loss of intraepidermal nerve fibers. However, the mechanistic cascades linking Nav1.7 mutations to axonal degeneration are incompletely understood. The G856D mutation in Nav1.7 produces robust changes in channel biophysical properties, including hyperpolarized activation, depolarized inactivation, and enhanced ramp and persistent currents, which contribute to the hyperexcitability exhibited by neurons containing Nav1.8. We report here that cell bodies and neurites of dorsal root ganglion (DRG) neurons transfected with G856D display increased levels of intracellular Na(+) concentration ([Na(+)]) and intracellular [Ca(2+)] following stimulation with high [K(+)] compared with wild-type (WT) Nav1.7-expressing neurons. Blockade of reverse mode of the sodium/calcium exchanger (NCX) or of sodium channels attenuates [Ca(2+)] transients evoked by high [K(+)] in G856D-expressing DRG cell bodies and neurites. We also show that treatment of WT or G856D-expressing neurites with high [K(+)] or 2-deoxyglucose (2-DG) does not elicit degeneration of these neurites, but that high [K(+)] and 2-DG in combination evokes degeneration of G856D neurites but not WT neurites. Our results also demonstrate that 0 Ca(2+) or blockade of reverse mode of NCX protects G856D-expressing neurites from degeneration when exposed to high [K(+)] and 2-DG. These results point to [Na(+)] overload in DRG neurons expressing mutant G856D Nav1.7, which triggers reverse mode of NCX and contributes to Ca(2+) toxicity, and suggest subtype-specific blockade of Nav1.7 or inhibition of reverse NCX as strategies that might slow or prevent axon degeneration in small-fiber neuropathy.
Assuntos
Cálcio/metabolismo , Eritromelalgia/metabolismo , Gânglios Espinais/metabolismo , Mutação de Sentido Incorreto , Canal de Sódio Disparado por Voltagem NAV1.3/metabolismo , Neuritos/metabolismo , Canais de Sódio/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Animais , Cálcio/toxicidade , Células Cultivadas , Gânglios Espinais/citologia , Humanos , Canal de Sódio Disparado por Voltagem NAV1.3/genética , Neuritos/patologia , Potássio/metabolismo , Ratos , Ratos Sprague-Dawley , Sódio/metabolismo , Canais de Sódio/genética , Trocador de Sódio e Cálcio/antagonistas & inibidoresRESUMO
AIMS: Little is known about the long-term effects of intensive multifactorial treatment early in the diabetes disease trajectory. In the absence of long-term data on hard outcomes, we described change in 10-year modelled cardiovascular risk in the 5 years following diagnosis, and quantified the impact of intensive treatment on 10-year modelled cardiovascular risk at 5 years. METHODS: In a pragmatic, cluster-randomized, parallel-group trial in Denmark, the Netherlands and the UK, 3057 people with screen-detected Type 2 diabetes were randomized by general practice to receive (1) routine care of diabetes according to national guidelines (1379 patients) or (2) intensive multifactorial target-driven management (1678 patients). Ten-year modelled cardiovascular disease risk was calculated at baseline and 5 years using the UK Prospective Diabetes Study Risk Engine (version 3ß). RESULTS: Among 2101 individuals with complete data at follow up (73.4%), 10-year modelled cardiovascular disease risk was 27.3% (sd 13.9) at baseline and 21.3% (sd 13.8) at 5-year follow-up (intensive treatment group difference -6.9, sd 9.0; routine care group difference -5.0, sd 12.2). Modelled 10-year cardiovascular disease risk was lower in the intensive treatment group compared with the routine care group at 5 years, after adjustment for baseline cardiovascular disease risk and clustering (-2.0; 95% CI -3.1 to -0.9). CONCLUSIONS: Despite increasing age and diabetes duration, there was a decline in modelled cardiovascular disease risk in the 5 years following diagnosis. Compared with routine care, 10-year modelled cardiovascular disease risk was lower in the intensive treatment group at 5 years. Our results suggest that patients benefit from intensive treatment early in the diabetes disease trajectory, where the rate of cardiovascular disease risk progression may be slowed.
Assuntos
Diabetes Mellitus Tipo 2/prevenção & controle , Angiopatias Diabéticas/prevenção & controle , Adulto , Idoso , Terapia Combinada/estatística & dados numéricos , Diagnóstico Precoce , Métodos Epidemiológicos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Cardiovasculares , Resultado do TratamentoRESUMO
A comparison of four different methods for the extraction of spore DNA from Stachybotrys chartarum was conducted. Spore DNA was extracted and purified using either one of three different commercial kits or water. All preparations utilized bead milling. Genomic DNA extracted from 10(1) to 10(7) spores was assessed by both agarose gel electrophoresis and real-time quantitative polymerase chain reaction (qPCR) performed against multi-copy (rRNA) and single-(tubulin) gene targets. The spore isolation technique we employed was verified to be pure by light microscopy. Although all preparatory methods led to successful detection by qPCR, S. chartarum spore DNA prepared using the Qiagen Plant kit was notably better over the extraction range.
Assuntos
DNA Fúngico/análise , DNA Fúngico/isolamento & purificação , Biologia Molecular/métodos , Esporos Fúngicos/química , Stachybotrys/química , DNA Fúngico/genética , DNA Ribossômico/análise , DNA Ribossômico/genética , Eletroforese em Gel de Ágar , Genoma Fúngico , Reação em Cadeia da Polimerase , Esporos Fúngicos/genética , Stachybotrys/genética , Tubulina (Proteína)/genéticaRESUMO
The myelinated axon provides a model in which it is possible to examine how various types of ion channels are incorporated into a membrane to form an excitable neuronal process. The available evidence now indicates that mammalian myelinated fibers contain a repertoire of physiologically active membrane molecules including at least four types of ion channels and an electrogenic Na+,K(+)-pump. Physiological properties of myelinated fibers reflect the distribution of these various types of channels and pumps, as well as interactions with myelinating Schwann cells in the PNS or oligodendrocytes in the CNS. A growing body of data also suggests a role for astrocytes and Schwann cells at nodes of Ranvier. This article reviews the current understanding of the ion channel organization of the mammalian myelinated fiber.
Assuntos
Fibras Nervosas Mielinizadas/fisiologia , Condução Nervosa , Canais de Potássio/fisiologia , Canais de Sódio/fisiologia , Potenciais de AçãoRESUMO
Glial cells are nervous-system cells that have classically been considered to be inexcitable. Despite their lack of electrical excitability, they can express voltage-activated Na+ channels with properties similar to the Na+ channels used by excitable cells to generate action potentials. The functional role that these voltage-activated Na+ channels play in glia is unclear. Three functions have been proposed: (1) glial cells might synthesize Na+ channels and donate them to adjacent neurons, thereby reducing the biosynthetic load of neurons; (2) Na+ channels might endow glial cells with the ability to sense electric activity of neighboring neurons, and might thus play a role in neuro-glial communication; and (3) Na+ influx through voltage-gated Na+ channels could be important to fuel the glial (Na+,K+)-ATPase, thereby facilitating and possibly modulating K+ uptake from the extracellular space.
Assuntos
Ativação do Canal Iônico/fisiologia , Neuroglia/fisiologia , Canais de Sódio/fisiologia , Animais , Eletrofisiologia , Humanos , Neuroglia/enzimologia , Neuroglia/metabolismoRESUMO
It is well known that non-viable mold contaminants such as macrocyclic trichothecene mycotoxins of Stachybotrys chartarum are highly toxinigenic to humans. However, the method of recovering native mycotoxin has been without consensus. Inconsistencies occur in the methods of isolation, suspension, preparation, and quantitation of the mycotoxin from the spores. The purpose of this study was to provide quantitatively comparative data on three concurrent preparations of 10(6)S. chartarum spores. The experiments were designed to specifically evaluate a novel method of mycotoxin extraction, solubilization, and the subsequent inhibitory effect in an established in vitro luminescence protein translation assay from 30 day-old spores. The mycotoxin-containing spores swabbed from wallboard cultures were milled with and without glass beads in 100% methanol, 95% ethanol, or water. Milled spore lysates were cleared of cell debris by filter centrifugation followed by a second centrifugation through a 5000 MWCO filter to remove interfering proteins and RNases. Cleared lysate was concentrated by centrivap and suspended in either alcohol or water as described. The suspensions were used immediately in the in vitro luminescence protein translation assay with the trichothecene, T-2 toxin, as a control. Although, mycotoxin is reported to be alcohol soluble, the level of translation inhibition was not reliably satisfactory for either the methanol or ethanol preparations. In fact, the methanol and ethanol control reactions were not significantly different than the alcohol prepared spore samples. In addition, we observed that increasing amounts of either alcohol inhibited the reaction in a dose dependent manner. This suggests that although alcohol isolation of mycotoxin is desirable in terms of time and labor, the presence of alcohol in the luminescence protein translation reaction was not acceptable. Conversely, water extraction of mycotoxin demonstrated a dose dependent response, and there was significant difference between the water controls and the water extracted mycotoxin reactions. In our hands, water was the best extraction agent for mycotoxin when using this specific luminescence protein translation assay kit.
Assuntos
Micotoxinas/química , Stachybotrys/química , Tricotecenos/química , Etanol/química , Luciferases/química , Luminescência , Metanol/química , Micotoxinas/análise , Micotoxinas/isolamento & purificação , Micotoxinas/toxicidade , Solubilidade , Solventes , Esporos Bacterianos/química , Tricotecenos/análise , Tricotecenos/isolamento & purificação , Tricotecenos/toxicidade , Água/químicaRESUMO
A hollow-core waveguide structure for on-chip atomic spectroscopy is presented. The devices are based on Anti-Resonant Reflecting Optical Waveguides and may be used for a wide variety of applications which rely on the interaction of light with gases and vapors. The designs presented here feature short delivery paths of the atomic vapor into the hollow waveguide. They also have excellent environmental stability by incorporating buried solid-core waveguides to deliver light to the hollow cores. Completed chips were packaged with an Rb source and the F = 3 ≥ F' = 2, 3, 4 transitions of the D2 line in 85Rb were monitored for optical absorption. Maximum absorption peak depths of 9% were measured.
RESUMO
Dorsal root ganglion (DRG) neurons produce multiple sodium currents, including several different TTX-sensitive (TTX-S) currents and TTX-resistant (TTX-R) currents, which are produced by distinct sodium channels. We previously demonstrated that, after sciatic nerve transection, the levels of SNS and NaN sodium channel alpha-subunit transcripts and protein in small (18-30 micrometer diameter) DRG neurons are reduced, as are the amplitudes and densities of the slowly inactivating and persistent TTX-R currents produced by these two channels. In this study, we asked whether glial-derived neurotrophic factor (GDNF), which has been shown to prevent some axotomy-induced changes such as the loss of somatostatin expression in DRG neurons, can ameliorate the axotomy-induced downregulation of SNS and NaN TTX-R sodium channels. We show here that exposure to GDNF can significantly increase both slowly inactivating and persistent TTX-R sodium currents, which are paralleled by increases in SNS and NaN mRNA and protein levels, in axotomized DRG neurons in vitro. We also show that intrathecally administered GDNF increases the amplitudes of the slowly inactivating and persistent TTX-R currents, and SNS and NaN protein levels, in peripherally axotomized DRG neurons in vivo. Finally, we demonstrate that GDNF upregulates the persistent TTX-R current in SNS-null mice, thus demonstrating that the upregulated persistent sodium current is not produced by SNS. Because TTX-R sodium channels have been shown to be important in nociception, the effects of GDNF on axotomized DRG neurons may have important implications for the regulation of nociceptive signaling by these cells.
Assuntos
Gânglios Espinais/metabolismo , Fatores de Crescimento Neural , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Canais de Sódio/metabolismo , Animais , Axotomia , Células Cultivadas , Feminino , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Imuno-Histoquímica , Injeções Espinhais , Masculino , Camundongos , Camundongos Knockout , Canal de Sódio Disparado por Voltagem NAV1.8 , Canal de Sódio Disparado por Voltagem NAV1.9 , Proteínas do Tecido Nervoso/administração & dosagem , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neuropeptídeos/genética , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/cirurgia , Sódio/metabolismo , Canais de Sódio/genética , Tetrodotoxina/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Two TTX-resistant sodium channels, SNS and NaN, are preferentially expressed in c-type dorsal root ganglion (DRG) neurons and have been shown recently to have distinct electrophysiological signatures, SNS producing a slowly inactivating and NaN producing a persistent sodium current with a relatively hyperpolarized voltage-dependence. An attenuation of SNS and NaN transcripts has been demonstrated in small DRG neurons after transection of the sciatic nerve. However, it is not known whether changes in the currents associated with SNS and NaN or in the expression of SNS and NaN channel protein occur after axotomy of the peripheral projections of DRG neurons or whether similar changes occur after transection of the central (dorsal root) projections of DRG neurons. Peripheral and central projections of L4/5 DRG neurons in adult rats were axotomized by transection of the sciatic nerve and the L4 and L5 dorsal roots, respectively. DRG neurons were examined using immunocytochemical and patch-clamp methods 9-12 d after sciatic nerve or dorsal root lesion. Levels of SNS and NaN protein in the two types of injuries were paralleled by their respective TTX-resistant currents. There was a significant decrease in SNS and NaN signal intensity in small DRG neurons after peripheral, but not central, axotomy compared with control neurons. Likewise, there was a significant reduction in slowly inactivating and persistent TTX-resistant currents in these neurons after peripheral, but not central, axotomy compared with control neurons. These results indicate that peripheral, but not central, axotomy results in a reduction in expression of functional SNS and NaN channels in c-type DRG neurons and suggest a basis for the altered electrical properties that are observed after peripheral nerve injury.
Assuntos
Gânglios Espinais/metabolismo , Neurônios/metabolismo , Nervo Isquiático/metabolismo , Canais de Sódio/metabolismo , Tetrodotoxina , Animais , Axotomia , Células Cultivadas , Feminino , Gânglios Espinais/citologia , Canal de Sódio Disparado por Voltagem NAV1.8 , Canal de Sódio Disparado por Voltagem NAV1.9 , Neurônios/citologia , Neuropeptídeos/metabolismo , Ratos , Ratos Sprague-Dawley , Rizotomia , Nervo Isquiático/citologia , Nervo Isquiático/fisiologia , Sódio/metabolismo , Canais de Sódio/efeitos dos fármacos , Canais de Sódio/genética , Tetrodotoxina/farmacologiaRESUMO
TTX-resistant (TTX-R) sodium currents are preferentially expressed in small C-type dorsal root ganglion (DRG) neurons, which include nociceptive neurons. Two mRNAs that are predicted to encode TTX-R sodium channels, SNS and NaN, are preferentially expressed in C-type DRG cells. To determine whether there are multiple TTX-R currents in these cells, we used patch-clamp recordings to study sodium currents in SNS-null mice and found a novel persistent voltage-dependent sodium current in small DRG neurons of both SNS-null and wild-type mice. Like SNS currents, this current is highly resistant to TTX (Ki = 39+/-9 microM). In contrast to SNS currents, the threshold for activation of this current is near 70 mV, the midpoint of steady-state inactivation is -44 +/- 1 mV, and the time constant for inactivation is 43+/-4 msec at 20 mV. The presence of this current in SNS-null and wild-type mice demonstrates that a distinct sodium channel isoform, which we suggest to be NaN, underlies this persistent TTX-R current. Importantly, the hyperpolarized voltage-dependence of this current, the substantial overlap of its activation and steady-state inactivation curves and its persistent nature suggest that this current is active near resting potential, where it may play an important role in regulating excitability of primary sensory neurons.
Assuntos
Gânglios Espinais/metabolismo , Gânglios Espinais/fisiologia , Neurônios Aferentes/metabolismo , Neurônios Aferentes/fisiologia , Canais de Sódio/metabolismo , Canais de Sódio/fisiologia , Tetrodotoxina/farmacologia , Animais , Cinética , Camundongos , Técnicas de Patch-Clamp , Canais de Sódio/efeitos dos fármacosRESUMO
The storage lesion which limits the shelf life of human blood in blood banking is associated with a metabolic loss of 2,3-diphosphoglycerate and ATP. This metabolic loss is driven by intracellular ATPase which are usually considered to include the ion pumps and the reactions which maintain the discoid shape of the human erythrocyte. Under the acidic conditions of blood storage, the energy-yielding reactions of the glycolytic pathway are restricted at the hexokinase and phosphofructokinase steps. We show here that under such circumstances the enzyme of the diphosphoglycerate shunt, diphosphoglycerate mutase/phosphatase and the glycolytic enzyme phosphoglycerate kinase can form a futile cycle with ATPase activity. This ATPase activity responds to 2-phosphoglycolate which is known to activate both diphosphoglycerate mutase and diphosphoglycerate phosphatase reactions. When the enzymes of the futile cycle are combined with the enzymes of the lower glycolytic pathway in a reconstitution experiment designed to represent conditions within the stored erythrocyte, the futile cycle does provide an ATPase activity which results in the metabolic loss of 2,3-diphosphoglycerate. An isotope incorporation experiment demonstrates that the futile cycle is active in glucose-depleted erythrocytes.
Assuntos
Eritrócitos/metabolismo , Glicólise , 2,3-Difosfoglicerato , Trifosfato de Adenosina/metabolismo , Ácidos Difosfoglicéricos/metabolismo , Humanos , NAD/metabolismo , Fosfoglicerato Quinase/metabolismoRESUMO
We have examined the hypothesis that the human erythrocyte isozyme of pyruvate kinase (EC 2.7.1.40) is a hybrid of the two isozymes present in liver. Rabbit antiserum against purified human erythrocyte pyruvate kinase inactivates the erythrocyte isozyme and the major liver isozyme from human tissue but does not inactivate the minor liver isozyme. The electrophoretic mobilities of the erythrocyte and major liver isozymes are altered by anti-erythrocyte enzyme antibody while the mobility of the minor liver isozyme is unaffected. Gel diffusion analysis indicates cross-reactivity between the erythrocyte and major liver isozyme but no cross-reactivity with the minor liver isozyme. The hybrid hypothesis would predict cross-reactivity including changes in activity and mobility of all isozymes and we conclude, therefore that the hypothesis is incorrect.
Assuntos
Fígado/enzimologia , Animais , Reações Cruzadas , Epitopos , Eritrócitos/enzimologia , Humanos , Isoenzimas/imunologia , Piruvato Quinase/sangue , Piruvato Quinase/imunologia , RatosRESUMO
AIMS: Establishing a balance between the benefits and harms of treatment is important among individuals with screen-detected diabetes, for whom the burden of treatment might be higher than the burden of the disease. We described the association between cardio-protective medication and health-related quality of life (HRQoL) among individuals with screen-detected diabetes. METHODS: 867 participants with screen-detected diabetes underwent clinical measurements at diagnosis, one and five years. General HRQoL (EQ5D) was measured at baseline, one- and five-years, and diabetes-specific HRQoL (ADDQoL-AWI) and health status (SF-36) at one and five years. Multivariable linear regression was used to quantify the association between change in HRQoL and change in cardio-protective medication. RESULTS: The median (IQR) number of prescribed cardio-protective agents was 2 (1 to 3) at diagnosis, 3 (2 to 4) at one year and 4 (3 to 5) at five years. Change in cardio-protective medication was not associated with change in HRQoL from diagnosis to one year. From one year to five years, change in cardio-protective agents was not associated with change in the SF-36 mental health score. One additional agent was associated with an increase in the SF-36 physical health score (2.1; 95%CI 0.4, 3.8) and an increase in the EQ-5D (0.05; 95%CI 0.02, 0.08). Conversely, one additional agent was associated with a decrease in the ADDQoL-AWI (-0.32; 95%CI -0.51, -0.13), compared to no change. CONCLUSIONS: We found little evidence that increases in the number of cardio-protective medications impacted negatively on HRQoL among individuals with screen-detected diabetes over five years.
Assuntos
Cardiotônicos/uso terapêutico , Doenças Cardiovasculares/prevenção & controle , Diabetes Mellitus Tipo 2/tratamento farmacológico , Qualidade de Vida , Adulto , Idoso , Doenças Cardiovasculares/epidemiologia , Estudos de Coortes , Diabetes Mellitus Tipo 2/diagnóstico , Angiopatias Diabéticas/prevenção & controle , Prescrições de Medicamentos/estatística & dados numéricos , Feminino , Nível de Saúde , Humanos , Masculino , Programas de Rastreamento , Saúde Mental , Pessoa de Meia-Idade , Padrões de Prática Médica , Inquéritos e Questionários , Reino Unido/epidemiologiaRESUMO
Axonal membrane structure during acute experimental allergic encephalomyelitis (EAE) was examined with freeze-fracture electron microscopy. Axons without myelin sheaths were prevalent within EAE spinal cords. Often these axons were associated with astrocytic processes, though membrane specializations were not observed at these sites. The demyelinated axons exhibited a highly asymmetrical partitioning of intramembranous particles (IMP), with approximately 2,000 particles/micron2 on P-faces and approximately 150/micron2 on E-faces. This distribution and density of IMP is similar to myelinated internodal membrane. The IMP were generally randomly distributed along the axons. However, in some regions, E-faces of demyelinated axons without paranodal-like membrane specialization in the vicinity displayed a greater than normal (approximately 500/micron2) particle density. Many of the IMP in these regions of increased density were of a large (greater than 10 nm) diameter. Axonal membrane bounded by a single set of paranodal oligodendroglial loops ('heminodal') was also observed, and the axolemma adjacent to the terminal glial loop exhibited a gradient of morphologies. The E-faces of presumed heminodal membrane most often displayed a moderately low density of IMP. However, in several instances, heminodal membrane exhibited a moderately high IMP density (approximately 1,100/micron2), similar to that observed within normal nodal membrane. In all cases, a high percentage of the E-face IMP within heminodal membrane were large. The results demonstrate that acute demyelination is associated with a maintenance of the integrity of certain components of the axolemma and an apparent dedifferentiation in other constituents.
Assuntos
Axônios/ultraestrutura , Encefalomielite Autoimune Experimental/patologia , Membranas Intracelulares/ultraestrutura , Medula Espinal/ultraestrutura , Animais , Doenças Desmielinizantes/patologia , Técnica de Fratura por Congelamento , Cobaias , Substâncias Macromoleculares , Microscopia Eletrônica , Oligodendroglia/ultraestrutura , Ratos , Ratos Endogâmicos LewRESUMO
The macromolecular structure of the axon membrane in 26-28-day-old Jimpy mice and control optic nerve were examined with quantitative freeze-fracture electron microscopy. Premyelinated and myelinated axons were observed in control optic nerves, with axonal diameters of premyelinated axons being generally smaller than that of myelinated axons (approximately 0.2-0.4 micron vs approximately 0.5-1.5 micron, respectively). Axon membrane from control optic nerves exhibited an asymmetrical partitioning of intramembranous particles (IMP). P-faces of internodal membrane displayed nearly twice as many IMP as the premyelinated axolemma (1,731 vs 893 micron-2, respectively). E-faces of internodal and premyelinated axolemma exhibited IMP densities of 124 and 157 micron-2, respectively. Few myelinated axons were apparent in optic nerves from Jimpy mice. The amyelinated axons of Jimpy mice displayed a spectrum of axonal diameters, ranging from approximately 0.2 to 1.5 micron. P-face densities of amyelinated axons, considered as a group, exhibited a wide range (600-2,100 micron-2). However, large diameter (greater than or equal to 0.5 micron) axons exhibited a significantly greater P-face IMP density than that of small caliber (greater than 0.5 micron) axons (1,525 vs 1,032 micron-2, respectively). Aggregations of E-face IMP were not observed along amyelinated axons of Jimpy optic nerves. The results demonstrate that the changes in P-face IMP density that occur during development of normal myelinated axons also occur in developing axons of Jimpy optic nerve, irrespective of a lack of normal glial cell association, and provide further evidence that the primary defect of hypomyelination within Jimpy mice is not attributed to the neuron.
Assuntos
Axônios/ultraestrutura , Membrana Celular/ultraestrutura , Nervo Óptico/ultraestrutura , Animais , Técnica de Fratura por Congelamento , Camundongos , Camundongos Jimpy , Microscopia Eletrônica , Bainha de Mielina/ultraestrutura , Valores de Referência , Especificidade da EspécieRESUMO
The expression of rat brain voltage-sensitive Na+ channel mRNAs in Schwann cells was examined using in situ hybridization cytochemistry and RT-PCR. The mRNAs of rat brain Na+ channel subtype II and III, but not subtype I, were detected in cultured Schwann cells from sciatic nerve and in intact sciatic nerve, which contains Schwann cells but not neuronal cell bodies. These results indicate that rat brain Na+ channel mRNAs, which have been considered as mainly neuronal-type messages, are also expressed in glial cells in vitro and in vivo.
Assuntos
Células de Schwann/fisiologia , Canais de Sódio/fisiologia , Animais , Células Cultivadas , Expressão Gênica , Hibridização In Situ , Técnicas In Vitro , Sondas RNA , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/fisiologiaRESUMO
B104 neuroblastoma cells are excitable, but the ion channels underlying electrogenesis in these cells have not been identified. RT-PCR, restriction enzyme analysis and in situ hybridization were used to study sodium channel mRNAs in B104 cells. High levels of sodium channel alpha-subunit mRNAs III, NaG and Na6 and beta 1-subunit mRNA were detected by RT-PCR in B104 cells. Low levels of types I and II alpha-subunit mRNAs were also present. In situ hybridization with subtype-specific riboprobes detected sodium channel alpha-subunit mRNAs III, NaG and Na6 and beta 1-subunit mRNA in B104 cells; analysis of the percentage of B104 cells expressing each alpha-subunit mRNA subtype suggests that some cells express the mRNAs for several alpha-subunits.
Assuntos
Neuroblastoma/metabolismo , Prosencéfalo/metabolismo , RNA Mensageiro/metabolismo , Canais de Sódio/biossíntese , Actinas/biossíntese , Animais , Sequência de Bases , Linhagem Celular , Sequência Consenso , Primers do DNA , DNA Complementar , Humanos , Hibridização In Situ , Substâncias Macromoleculares , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Ratos , Mapeamento por Restrição , Células Tumorais CultivadasRESUMO
Previous electrophysiological work has demonstrated expression of a voltage-gated Na+ channel (VGSC) specifically in two highly metastatic prostatic epithelial tumour cell lines: MAT-LyLu (rat) and PC-3 (human). However, the identity of the channel(s) present was uncertain. The present study used a combination of molecular biological techniques to demonstrate that full-length skeletal muscle type 1 (SkM1) VGSC mRNA is present in the mRNA pool of the MAT-LyLu cell line. mRNA for this particular channel type was also expressed in the PC-3 cells. In situ hybridisation data suggested that the level and pattern of rSkM1 mRNA expression were different in the Dunning cells of markedly different metastatic potential. Interestingly, the same type of mRNA was also detected in the weakly metastatic counterparts of the cells: AT-2 (rat) and LNCaP (human).
Assuntos
Músculo Esquelético/metabolismo , Neoplasias da Próstata/metabolismo , Canais de Sódio/metabolismo , Animais , Humanos , Ativação do Canal Iônico , Masculino , RNA Mensageiro/metabolismo , Ratos , Canais de Sódio/genética , Células Tumorais CultivadasRESUMO
Two tetrodotoxin-resistant (TTX-R) voltage-gated sodium channels, SNS and NaN, are preferentially expressed in small dorsal root ganglia (DRG) and trigeminal ganglia neurons, most of which are nociceptive, of rat and mouse. We report here the sequence of NaN from human DRG, and demonstrate the presence of two TTX-R currents in human DRG neurons. One current has physiological properties similar to those reported for SNS, while the other displays hyperpolarized voltage-dependence and persistent kinetics; a similar TTX-R current was recently identified in DRG neurons of sns-null mouse. Thus SNS and NaN channels appear to produce different currents in human DRG neurons.