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1.
Neuroscience ; 241: 157-69, 2013 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-23531437

RESUMO

Elevated nerve growth factor (NGF) is believed to play a role in many types of pain. An NGF-blocking antibody (muMab 911) has been shown to reduce pain and hyperalgesia in pain models, suggesting a novel therapeutic approach for pain management. Since NGF also plays important roles in peripheral nervous system development and sensory nerve outgrowth, we asked whether anti-NGF antibodies would adversely impact peripheral nerve regeneration. Adult rats underwent a unilateral sciatic nerve crush to transect axons and were subcutaneously dosed weekly for 8weeks with muMab 911 or vehicle beginning 1day prior to injury. Plasma levels of muMab 911 were assessed from blood samples and foot print analysis was used to assess functional recovery. At 8-weeks post-nerve injury, sciatic nerves were prepared for light and electron microscopy. In a separate group, Fluro-Gold was injected subcutaneously at the ankle prior to perfusion, and counts and sizes of retrogradely labeled and unlabeled dorsal root ganglion neurons were obtained. There was no difference in the time course of gait recovery in antibody-treated and vehicle-treated animals. The number of myelinated and nonmyelinated axons was the same in the muMab 911-treated crushed nerves and intact nerves, consistent with observed complete recovery. Treatment with muMab 911 did however result in a small decrease in average cell body size on both the intact and injured sides. These results indicate that muMab 911 did not impair functional recovery or nerve regeneration after nerve injury in adult rats.


Assuntos
Anticorpos Monoclonais/farmacologia , Fator de Crescimento Neural/antagonistas & inibidores , Regeneração Nervosa/fisiologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Nervo Isquiático/fisiologia , Envelhecimento , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Compressão Nervosa , Regeneração Nervosa/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/lesões
2.
Br J Pharmacol ; 155(7): 1093-103, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18776916

RESUMO

BACKGROUND AND PURPOSE: Calcitonin gene-related peptide (CGRP) receptor antagonists effectively abort migraine headache and inhibit neurogenic vasodilatation in humans as well as rat models. Monoclonal antibodies typically have long half-lives, and we investigated whether or not function-blocking CGRP antibodies would inhibit neurogenic vasodilatation with a long duration of action and therefore be a possible approach to preventive therapy of migraine. During chronic treatment with anti-CGRP antibodies, we measured cardiovascular function, which might be a safety concern of CGRP inhibition. EXPERIMENTAL APPROACH: We used two rat blood flow models that measure electrically stimulated vasodilatation in the skin or in the middle meningeal artery (MMA). These vasomotor responses are largely dependent on the neurogenic release of CGRP from sensory afferents. To assess cardiovascular function during chronic systemic anti-CGRP antibody treatment, we measured heart rate and blood pressure in conscious rats. KEY RESULTS: Treatment with anti-CGRP antibodies inhibited skin vasodilatation or the increase in MMA diameter to a similar magnitude as treatment with CGRP receptor antagonists. Although CGRP antibody treatment had a slower onset of action than the CGRP receptor antagonists, the inhibition was still evident 1 week after dosing. Chronic treatment with anti-CGRP antibodies had no detectable effects on heart rate or blood pressure. CONCLUSIONS AND IMPLICATIONS: We showed for the first time that anti-CGRP antibodies exert a long lasting inhibition of neurogenic vasodilatation in two different rat models of arterial blood flow. We have provided strong preclinical evidence that anti-CGRP antibody may be a suitable drug candidate for the preventive treatment of migraine.


Assuntos
Anticorpos Monoclonais/imunologia , Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina , Peptídeo Relacionado com Gene de Calcitonina/antagonistas & inibidores , Vasodilatação/efeitos dos fármacos , Animais , Anticorpos Monoclonais/farmacocinética , Pressão Sanguínea/efeitos dos fármacos , Peptídeo Relacionado com Gene de Calcitonina/imunologia , Modelos Animais de Doenças , Estimulação Elétrica , Meia-Vida , Frequência Cardíaca/efeitos dos fármacos , Masculino , Artérias Meníngeas/efeitos dos fármacos , Artérias Meníngeas/metabolismo , Transtornos de Enxaqueca/fisiopatologia , Transtornos de Enxaqueca/prevenção & controle , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Pele/irrigação sanguínea , Pele/efeitos dos fármacos
3.
J Hand Surg Am ; 26(4): 635-44, 2001 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-11466637

RESUMO

Nerve growth factor (NGF) is thought to play a role in the pathogenesis of neuroma formation as well as in the development of neuropathic pain. In this study we attempted to antagonize NGF by using trkA-IgG, an inhibitor of NGF, consisting of the NGF receptor linked to an immunoglobulin. It was delivered by an implanted osmotic pump directly to the site of a sciatic nerve transection in 16 rats for 30 days. The animals were monitored daily for the first 2 weeks for evidence of auto-cannibalization (autotomy) of the denervated foot (a sign of neuropathic pain). Four (25%) of the 16 rats receiving trkA-IgG exhibited such cannibalization compared with 9 of 15 control rats (60%) that underwent an identical procedure but were not treated with the trkA-IgG solution. One month after surgery the sciatic nerves and representative dorsal root ganglia (DRG) from these rats were evaluated histologically. Six of the 16 experimental rats (38%) demonstrated histological evidence of neuroma formation compared with 12 of the 15 controls (80%). There were no histological differences between the DRG from the two groups. These results support the notion that inhibiting NGF following peripheral nerve injury in the rat can reduce neuroma formation and neuropathic pain without damaging the cell bodies of the transected neurons.


Assuntos
Imunoglobulina G/farmacologia , Fatores de Crescimento Neural/antagonistas & inibidores , Neuroma/fisiopatologia , Nervos Periféricos/fisiopatologia , Receptor trkA/fisiologia , Animais , Canibalismo , Sistemas de Liberação de Medicamentos , Masculino , Neuroma/patologia , Dor/fisiopatologia , Nervos Periféricos/patologia , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/lesões
4.
Hybridoma ; 19(3): 215-27, 2000 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10952410

RESUMO

The binding specificities of a panel of mouse monoclonal antibodies (MAbs) to human nerve growth factor (hNGF) were determined by epitope mapping using chimeric and point mutants of NGF. Subsequently, the MAbs were used to probe NGF structure-function relationships. Six MAbs, which recognize distinct or partially overlapping regions of hNGF, were evaluated for their ability to block the binding of hNGF to the TrkA and p75 NGF receptors in various in vitro assays, which included blocking of TrkA autophosphorylation and blocking of NGF-dependent survival of dorsal root ganglion sensory neurons. Three MAbs (911,912,938) were potent blockers of all activities. Potent blocking of p75 binding occurs only with MAb 909, which recognizes an NGF region identified by mutagenesis as important for NGF-p75 binding. These results are consistent with recently proposed models of binding regions involved in NGF-TrkA and NGF-p75 interactions generated through mutagenic analysis and structure determination of the NGF-TrkA complex. These studies provide insight to the epitope specificities and potency of MAbs that would be useful for physiological NGF blocking studies.


Assuntos
Alanina/genética , Anticorpos Monoclonais/metabolismo , Sítios de Ligação de Anticorpos/genética , Fator de Crescimento Neural/genética , Fator de Crescimento Neural/metabolismo , Animais , Western Blotting , Células CHO , Cricetinae , Humanos , Mutagênese Sítio-Dirigida , Fator de Crescimento Neural/biossíntese , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Mutação Puntual , Estrutura Secundária de Proteína/genética , Ratos , Receptor de Fator de Crescimento Neural/genética , Receptor de Fator de Crescimento Neural/metabolismo , Receptor trkA/antagonistas & inibidores , Receptor trkA/genética , Receptor trkA/imunologia , Receptor trkA/metabolismo , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Transfecção , Células Tumorais Cultivadas
5.
EMBO J ; 19(15): 4046-55, 2000 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-10921885

RESUMO

Bronchoalveolar lavage fluid from mice with experimentally induced allergic pulmonary inflammation contains a novel 9.4 kDa cysteine-rich secreted protein, FIZZ1 (found in inflammatory zone). Murine (m) FIZZ1 is the founding member of a new gene family including two other murine genes expressed, respectively, in intestinal crypt epithelium and white adipose tissue, and two related human genes. In control mice, FIZZ1 mRNA and protein expression occur at low levels in a subset of bronchial epithelial cells and in non-neuronal cells adjacent to neurovascular bundles in the peribronchial stroma, and in the wall of the large and small bowel. During allergic pulmonary inflammation, mFIZZ1 expression markedly increases in hypertrophic, hyperplastic bronchial epithelium and appears in type II alveolar pneumocytes. In vitro, recombinant mFIZZ1 inhibits the nerve growth factor (NGF)-mediated survival of rat embryonic day 14 dorsal root ganglion (DRG) neurons and NGF-induced CGRP gene expression in adult rat DRG neurons. In vivo, FIZZ1 may modulate the function of neurons innervating the bronchial tree, thereby altering the local tissue response to allergic pulmonary inflammation.


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular , Família Multigênica , Proteínas/genética , Hipersensibilidade Respiratória/genética , Sequência de Aminoácidos , Animais , Brônquios/citologia , Líquido da Lavagem Broncoalveolar/química , Sobrevivência Celular , Cisteína , Gânglios Espinais/citologia , Humanos , Imuno-Histoquímica , Hibridização In Situ , Mucosa Intestinal/citologia , Camundongos , Dados de Sequência Molecular , Fator de Crescimento Neural/metabolismo , Proteínas/isolamento & purificação , Proteínas/metabolismo , Ratos , Mucosa Respiratória/citologia , Análise de Sequência de DNA , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Distribuição Tecidual
6.
J Neurosci ; 20(1): 427-37, 2000 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-10627618

RESUMO

Glial cell line-derived neurotrophic factor (GDNF) has potent trophic effects on adult sensory neurons after nerve injury and is one of a family of proteins that includes neurturin, persephin, and artemin. Sensitivity to these factors is conferred by a receptor complex consisting of a ligand binding domain (GFRalpha1-GFRalpha4) and a signal transducing domain RET. We have investigated the normal expression of GDNF family receptor components within sensory neurons and the response to nerve injury. In normal rats, RET and GFRalpha1 were expressed in a subpopulation of both small- and large-diameter afferents projecting through the sciatic nerve [60 and 40% of FluoroGold (FG)-labeled cells, respectively]. GFRalpha2 and GFRalpha3 were both expressed principally within small-diameter DRG cells (30 and 40% of FG-labeled cells, respectively). Two weeks after sciatic axotomy, the expression of GFRalpha2 was markedly reduced (to 12% of sciatic afferents). In contrast, the proportion of sciatic afferents that expressed GFRalpha1 increased (to 66% of sciatic afferents) so that virtually all large-diameter afferents expressed this receptor component, and the expression of GFRalpha3 also increased (to 66% of sciatic afferents) so that almost all of the small-diameter afferents expressed this receptor component after axotomy. There was little change in RET expression. The changes in the proportions of DRG cells expressing different receptor components were mirrored by alterations in the total RNA levels within the DRG. The changes in GFRalpha1 and GFRalpha2 expression after axotomy could be largely reversed by treatment with GDNF.


Assuntos
Proteínas de Drosophila , Fatores de Crescimento Neural , Proteínas do Tecido Nervoso/farmacologia , Células do Corno Posterior/química , Células do Corno Posterior/fisiologia , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Animais , Axotomia , Expressão Gênica/fisiologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Hibridização In Situ , Ligadura , Masculino , Regeneração Nervosa/fisiologia , Proteínas de Neurofilamentos/análise , Proteínas de Neurofilamentos/metabolismo , Sondas de Oligonucleotídeos , Fosforilação , Células do Corno Posterior/efeitos dos fármacos , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas c-ret , RNA Mensageiro/análise , Ratos , Ratos Wistar , Receptores Proteína Tirosina Quinases/análise , Nervo Isquiático/química , Nervo Isquiático/fisiologia , Regulação para Cima/genética
7.
Eur J Neurosci ; 11(5): 1698-704, 1999 May.
Artigo em Inglês | MEDLINE | ID: mdl-10215923

RESUMO

Evidence suggests that nerve growth factor (NGF) is an important mediator in inflammatory pain states: NGF levels increase in inflamed tissue, and neutralization of endogenous NGF prevents the hyperalgesia which normally develops during inflammation of the skin. Here we asked whether NGF contributes to sensitization of primary afferent nociceptors, which are an important component of pain and hyperalgesia in inflamed tissue. An in vitro skin nerve preparation of the rat was used to directly record the receptive properties of thin myelinated (Adelta) and unmyelinated (C) nociceptors innervating normal hairy skin, carrageenan-inflamed skin and carrageenan-inflamed skin where endogenous NGF had been neutralized by application of a trkA-IgG (tyrosine kinase Aimmunoglobulin G) fusion molecule. Following carrageenan inflammation, there was a marked increase in the proportion of nociceptors which displayed ongoing activity (50% of nociceptors developed spontaneous activity compared to 4% of nociceptors innervating normal uninflamed skin), and this was reflected in a significant increase in the average ongoing discharge activity. Spontaneously active fibres were sensitized to heat and displayed a more than twofold increase in their discharge to a standard noxious heat stimulus. Furthermore, the number of nociceptors responding to the algesic mediator bradykinin increased significantly from 28% to 58%. By contrast, the mechanical threshold of nociceptive afferents did not change during inflammation. When the NGF-neutralizing molecule trkA-IgG was coadministered with carrageenan at the onset of the inflammation, primary afferent nociceptors did not sensitize and displayed essentially normal response properties, although the inflammation as evidenced by tissue oedema developed normally. We therefore conclude that NGF is a crucial component for the sensitization of primary afferent nociceptors associated with tissue inflammation.


Assuntos
Fatores de Crescimento Neural/antagonistas & inibidores , Inflamação Neurogênica/imunologia , Nociceptores/fisiologia , Pele/inervação , Animais , Carragenina , Eletrofisiologia , Excipientes , Feminino , Imunoglobulina G/farmacologia , Inflamação Neurogênica/induzido quimicamente , Neurônios Aferentes/química , Neurônios Aferentes/fisiologia , Nociceptores/química , Estimulação Física , Proteínas Proto-Oncogênicas/imunologia , Ratos , Ratos Wistar , Receptores Proteína Tirosina Quinases/imunologia , Receptor trkA , Receptores de Fator de Crescimento Neural/imunologia , Pele/imunologia , Estresse Mecânico
8.
Eur J Neurosci ; 10(4): 1282-91, 1998 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9749782

RESUMO

Nerve growth factor (NGF) has a well characterized role in the development of the nervous system and there is evidence that it interacts with nociceptive primary afferent fibres. Here we applied a synthetic tyrosine kinase A IgG (trkA-IgG) fusion molecule for 10-12 days to the innervation territory of the purely cutaneous saphenous nerve in order to bind, and thereby neutralize endogenous NGF in adult rats. Using neurophysiological analysis of 152 nociceptors we now show that sequestration of NGF results in specific changes of their receptive field properties. The percentage of nociceptors responding to heat dropped significantly from a normal 57% to 32%. This was accompanied by a rightward shift and a reduced slope of the stimulus response function relating the intracutaneous temperature to the neural response. The number of nociceptors responding to application of bradykinin was also significantly reduced from a normal of 28% to 8%. In contrast, the threshold for mechanical stimuli and the response to suprathreshold stimuli remained unaltered, as did the percentage of nociceptors responding to noxious cold. The reduced sensitivity of primary afferent nociceptors was accompanied by a reduction in the innervation density of the epidermis by 44% as assessed with quantitative immunocytochemical analysis of the panaxonal marker PGP 9.5. This demonstrates that endogenous NGF in the adult specifically modulates the terminal arborization of unmyelinated fibres and the sensitivity of primary afferent nociceptors to thermal and chemical stimuli in vivo.


Assuntos
Epiderme/inervação , Fatores de Crescimento Neural/fisiologia , Nociceptores/fisiologia , Receptor trkA , Vias Aferentes/fisiologia , Animais , Proteínas de Transporte/química , Imunoglobulina G/química , Imuno-Histoquímica , Mecanorreceptores/fisiologia , Proteínas de Membrana/química , Fatores de Crescimento Neural/deficiência , Ratos , Proteínas Recombinantes de Fusão/química
9.
Neuron ; 19(1): 63-76, 1997 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-9247264

RESUMO

We have examined the hypothesis that the segregation of LGN axon terminals into ocular dominance (OD) patches in layer 4 of the visual cortex requires neurotrophins, acting as signals to modulate the pattern of synaptic connectivity. Neurotrophin receptor antagonists, composed of the extracellular domain of each member of the trk family of neurotrophin receptors fused to a human Fc domain, were infused directly into visual cortex during the peak phase of OD column formation. Infusion of trkB-IgG, which binds BDNF and NT-4/5, inhibited the formation of OD patches within layer 4, while trkA-IgG and trkC-IgG, which preferentially bind NGF and NT-3, respectively, had no effect. The autoradiographic labeling of LGN terminals in cortical layer 4 was reduced by trkB-IgG, in contrast with the increased labeling observed following NT-4/5 infusion. These data suggest that an endogenous ligand of trkB, normally present in limiting amounts within visual cortex, is necessary for the selective growth and remodeling of LGN axons into eye-specific patches.


Assuntos
Axônios/efeitos dos fármacos , Imunoglobulina G/farmacologia , Proteínas Tirosina Quinases/efeitos dos fármacos , Córtex Visual/efeitos dos fármacos , Humanos , Ligantes , Fatores de Crescimento Neural/farmacologia , Neurotrofina 3
10.
J Neurosci ; 17(1): 470-6, 1997 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-8987771

RESUMO

We have tested the ability of neurotrophins to reverse axotomy-induced changes in adult motor and sensory neurons, using the physiological measure of conduction velocity. Five weeks after axotomy, sensory and motor conduction velocities were greatly reduced. NT-3 at 60 microg/d, pumped directly onto the cut nerve stump, largely prevented the change in sensory fibers. Lower doses were less effective, and NT-4/5 was without effect. In contrast, both NT-3 and NT-4/5 were effective at rescuing motoneurons, with similar dose dependencies. This amelioration of physiological deficits in adult mammalian neurons suggests possible therapeutic application of neurotrophins. We have also studied the physiological effects of neurotrophin deprivation on intact peripheral neurons. After 2 weeks of sequestration of trkB ligands (BDNF and NT-4/5), motor, but not sensory, neuron conduction was significantly slowed. Sequestration of NT-3 was found to affect both motor and sensory fiber velocities but more modestly and only with higher doses of sequestering agent. These data therefore suggest that peripherally produced neurotrophins are necessary for the maintenance of normal functional properties of peripheral neurons.


Assuntos
Neurônios Motores/efeitos dos fármacos , Neurônios Motores/fisiologia , Fatores de Crescimento Neural/farmacologia , Fatores de Crescimento Neural/fisiologia , Neurônios Aferentes/efeitos dos fármacos , Neurônios Aferentes/fisiologia , Animais , Axônios/fisiologia , Denervação , Ligantes , Masculino , Condução Nervosa , Nervos Periféricos/citologia , Nervos Periféricos/efeitos dos fármacos , Nervos Periféricos/fisiologia , Ratos , Ratos Wistar , Fatores de Tempo
11.
Mech Dev ; 61(1-2): 99-111, 1997 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-9076681

RESUMO

Addition of neurons to cultures of non-neuronal cells derived from quail embryonic dorsal root ganglia causes a 2.5-fold increase in the proportion of cells that express the glial marker Schwann cell myelin protein (SMP) when compared to cultures devoid of neurons. This effect is mediated by BDNF because incubation with a trkB immunoadhesin that sequesters BDNF, but not with trkA or trkC immunoadhesins, abolishes this stimulation. This neuronal activity can be mimicked by treatment with soluble BDNF that stimulates specifically the conversion of SMP-negative glial cells into cells that express this phenotype. That BDNF is the endogenous neuron-derived factor affecting glial development is further supported by the observation that BDNF is extensively expressed in developing sensory neurons of the avian ganglia both in vivo and in vitro, but not by the satellite cells. These results show for the first time a paracrine role for neuronal BDNF on differentiation of peripheral glial cells. This effect of BDNF is likely to be mediated by the p75 neurotrophin receptor because: (1) p75 immunoreactive protein is expressed by a subset of satellite cells; (2) neutralization of p75 abolishes the BDNF-induced stimulation; (3) a treatment of non-neuronal cell cultures with equimolar concentrations of either soluble NGF or NT-3 also affects the proportion of cells that become SMP-positive. Whereas NGF stimulates the acquisition of this glial antigen to a similar extent as BDNF, NT-3 inhibits its expression, suggesting that distinct neurotrophins signal differentially through p75. These findings also suggest that the definitive phenotype of peripheral glia is determined by a balance between positive and inhibitory signals arising in adjacent neurons.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/fisiologia , Moléculas de Adesão Celular/metabolismo , Gânglios Espinais/embriologia , Proteínas da Mielina/metabolismo , Animais , Divisão Celular , Células Cultivadas , Coturnix , Indução Embrionária , Fatores de Crescimento Neural/fisiologia , Neuroglia/citologia , Neurônios/citologia , Neurônios/fisiologia , Neurotrofina 3 , Receptor do Fator Neutrófico Ciliar , Receptores de Fator de Crescimento Neural/fisiologia , Células de Schwann/metabolismo
12.
J Neurobiol ; 29(3): 277-92, 1996 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-8907158

RESUMO

We performed a detailed study of the expression of neurotrophin-3 and brain-derived neurotrophic factor transcripts in spinal motoneurons using in situ hybridization of serially sectioned chick embryos aged 3 to 8 days (E3 to E8). Neurotrophin-3 mRNA is detected in motoneuron subsets from E3.5 to E4 only in brachial segments of the neural tube and from E5 in both brachial and lumbar regions. Expression of brain-derived neurotrophic factor mRNA is first evident on E5 in a subset of brachial level motoneurons and from E6 also in motoneurons located in the rostral-most portion of the lateral motor column, as well as in the tail-innervating region of the spinal cord. Analysis along the rostrocaudal extent of the brachial lateral motor column reveals an overlap zone of expression of both neurotrophins of about two segments. In transverse sections of this region, it is observed that neurotrophin-3-positive motoneurons preferentially occupy the lateral part of the column, whereas brain-derived neurotrophic factor-producing motoneurons are localized in a more medial position. These results show that the two factors are synthesized at discrete axial levels of the spinal cord by distinct motoneuron subpopulations. Since brain-derived neurotrophic factor mRNA is expressed within the brachial but not the lumbar lateral motor column, we tested the possibility that brain-derived neurotrophic factor expression is regulated by the type of peripheral target, that is, the wing or the leg. Unilateral transplantation of a wing bud instead of a leg bud and vice versa, prior to the onset of peripheral innervation, failed to alter the original pattern of brain-derived neurotrophic factor mRNA observed in either level of the axis. Thus, the early synthesis of brain-derived neurotrophic factor by subsets of spinal motoneurons is independent of the type of peripheral target and may instead reflect intrinsic differences between motoneuron populations.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Neurônios Motores/fisiologia , Fatores de Crescimento Neural/genética , Animais , Sequência de Bases , Biomarcadores , Morte Celular/genética , Embrião de Galinha , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Hibridização In Situ , Botões de Extremidades/embriologia , Botões de Extremidades/inervação , Botões de Extremidades/fisiologia , Dados de Sequência Molecular , Neurônios Motores/citologia , Neurotrofina 3 , RNA Mensageiro/análise , Medula Espinal/citologia , Medula Espinal/embriologia
13.
Perspect Dev Neurobiol ; 4(1): 101-7, 1996.
Artigo em Inglês | MEDLINE | ID: mdl-9169923

RESUMO

Ciliary neurotrophic factor (CNTF) and growth promoting activity (GPA) are two members of a family of structurally and functionally related cytokines. Although the primary sequences of these proteins are only distantly related, many share striking functional similarities. The question of the potential existence of more, as yet undiscovered, members of this family, especially those most related to CNTF, is discussed. There are several biological systems which exhibit unexplained CNTF-like activities. This has led to speculation that there are indeed other CNTF-like proteins to be found. Because of the poor primary sequence conservation among known members of this family, even those sharing strong functional similarities, it is unlikely that a cloning approach based on sequence homology will find these putative new members of the family. Instead, a more biological approach, based on functional similarities, is more likely to succeed.


Assuntos
Citocinas/fisiologia , Fatores de Crescimento Neural/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Sequência de Aminoácidos , Animais , Galinhas , Fator Neurotrófico Ciliar , Citocinas/química , Humanos , Camundongos , Dados de Sequência Molecular , Fatores de Crescimento Neural/química , Proteínas do Tecido Nervoso/química , Ratos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
14.
J Biol Chem ; 270(39): 23104-10, 1995 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-7559453

RESUMO

Neurotrophins are a family of highly conserved proteins that affect the development and maintenance of distinct neuronal populations. Neurotrophins exist in vivo as homodimers, but we show that neurotrophins can exist as heterodimers in vitro and are pluripotent, being able to bind and to activate different Trk tyrosine kinase receptors as well as promote neuronal differentiation in PC12 cells as effectively as wild type homodimers. These asymmetric neurotrophin dimers allow unique characterization of neurotrophin structure-function relationships with Trk receptors. The chimeric Trk activities of these heterodimers suggest an alternative model of neurotrophin-Trk receptor activation in which the critical Trk-interacting elements may be attributed to a single protomer.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Fatores de Crescimento Neural/farmacologia , Neurônios/citologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Animais , Ligação Competitiva , Cinética , Substâncias Macromoleculares , Camundongos , Modelos Estruturais , Fatores de Crescimento Neural/química , Neurônios/metabolismo , Células PC12 , Fosforilação , Estrutura Secundária de Proteína , Ensaio Radioligante , Ratos , Receptor do Fator Neutrófico Ciliar , Receptor trkA
15.
Nat Med ; 1(8): 774-80, 1995 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-7585179

RESUMO

Evidence suggests that nerve growth factor (NGF) may function as a mediator of some persistent pain states. We have used a synthetic protein, trkA-IgG, to sequester endogenous NGF and block the survival effects of NGF on cultured sensory neurons. We show that administration of trkA-IgG produces a sustained thermal and chemical hypoalgesia and leads to a downregulation of the sensory neuropeptide CGRP (calcitonin gene-related peptide) in treated sensory neurons. Acute administration of the molecule blocks the hyperalgesia that develops with carrageenan-induced inflammation. These data suggest that peripherally produced NGF normally acts to maintain the sensitivity of nociceptive sensory neurons and that, in some inflammatory states, an upregulation of NGF is responsible for alterations in pain-related behaviour. Antagonists of NGF may therefore be of clinical use in treating some chronic pain states.


Assuntos
Fatores de Crescimento Neural/fisiologia , Neurônios Aferentes/fisiologia , Nociceptores/fisiologia , Dor/fisiopatologia , Proteínas Proto-Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Receptores de Fator de Crescimento Neural/fisiologia , Animais , Comportamento Animal/efeitos dos fármacos , Peptídeo Relacionado com Gene de Calcitonina/biossíntese , Capsaicina/farmacologia , Carragenina/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Regulação para Baixo , Imunoglobulina G , Inflamação/fisiopatologia , Neurônios Aferentes/efeitos dos fármacos , Medição da Dor/métodos , Proteínas Proto-Oncogênicas/genética , Ratos , Receptores Proteína Tirosina Quinases/genética , Receptor trkA , Receptores de Fator de Crescimento Neural/genética , Proteínas Recombinantes de Fusão/farmacologia
16.
EMBO J ; 14(12): 2795-805, 1995 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-7796806

RESUMO

The neurotrophins influence survival and maintenance of vertebrate neurons in the embryonic, early post-natal and post-developmental stages of the nervous system. Binding of neurotrophins to receptors encoded by the gene family trk initiates signal transduction into the cell. trkA interacts preferably with nerve growth factor (NGF), trkB with brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5) and trkC with neurotrophin-3 (NT-3). By constructing 17 different chimeras and domain deletions of the human trk receptors and analyzing their binding affinities to the neurotrophins we have shown that an immunoglobulin-like domain located adjacent to the transmembrane domain is the structural element that determines the interaction of neurotrophins with their receptors. Chimeras of trkC where this domain was exchanged for the homologous sequences from trkB or trkA gained high affinity binding to BDNF or NGF respectively, while deletion of this domain in trkC or trkA abolished binding to NT-3 or NGF respectively. This domain alone retained affinities to neurotrophins similar to the full-length receptors and when expressed on NIH 3T3 cells in fusion with the kinase domain showed neurotrophin-dependent activation.


Assuntos
Imunoglobulinas/metabolismo , Fatores de Crescimento Neural/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Sequência de Bases , Ligação Competitiva , Fator Neurotrófico Derivado do Encéfalo , Glicosilação , Humanos , Imunoglobulinas/genética , Camundongos , Dados de Sequência Molecular , Peso Molecular , Proteínas do Tecido Nervoso/metabolismo , Neurotrofina 3 , Fosforilação , Ratos , Receptores de Fator de Crescimento Neural/química , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/isolamento & purificação , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência/fisiologia
17.
Eur J Neurosci ; 7(6): 1403-9, 1995 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-7582115

RESUMO

Localization of mRNA encoding trkB indicates that two truncated isoforms of trkB, T1trkB and T2trkB, are differentially distributed in the rodent nervous system, and that each of these transcripts is co-expressed with catalytic trkB (TK+trkB) in adult motor neurons. In contrast to the prominent expression of T1trkB by non-neuronal cells, T2trkB expression appeared to be restricted to neurons and demonstrated significant overlap with the pattern of TK+trkB distribution. In developing spinal cord ventral horn, an age-related increase in hybridization was observed for truncated isoforms. These findings suggest that truncated trkB may modulate neuronal responses to neurotrophins which act via trkB.


Assuntos
Sistema Nervoso Central/metabolismo , Neurônios/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Envelhecimento/metabolismo , Animais , Autorradiografia , Sequência de Bases , Embrião de Mamíferos/metabolismo , Hibridização In Situ , Isomerismo , Camundongos , Sondas Moleculares/genética , Dados de Sequência Molecular , Neurônios Motores/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor do Fator Neutrófico Ciliar , Receptores de Fator de Crescimento Neural/química , Receptores de Fator de Crescimento Neural/genética
18.
J Biol Chem ; 270(18): 10915-22, 1995 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-7738033

RESUMO

Cardiotrophin-1 (CT-1) is a newly isolated cytokine that was identified based on its ability to induce cardiac myocyte hypertrophy. It is a member of the family of cytokines that includes interleukins-6 and -11, leukemia inhibitory factor (LIF), ciliary neurotrophic factor, and oncostatin M. These cytokines induce a pleiotropic set of growth and differentiation activities via receptors that use a common signaling subunit, gp130. In this work we determine the activity of CT-1 in six in vitro biological assays and examine the composition of its cell surface receptor. We find that CT-1 is inactive in stimulating the growth of the hybridoma cell line, B9 and inhibits the growth of the mouse myeloid leukemia cell line, M1. CT-1 induces a phenotypic switch in rat sympathetic neurons and promotes the survival of rat dopaminergic and chick ciliary neurons. CT-1 also inhibits the differentiation of mouse embryonic stem cells. CT-1 and LIF cross-compete for binding to M1 cells, Kd [CT-1] approximately 0.7 nM, and this binding is inhibited by an anti-gp130 monoclonal antibody. Both ligands can be specifically cross-linked to a protein on M1 cells with the mobility of the LIF receptor (approximately 200 kDa). In addition, CT-1 binds directly to a purified, soluble form of the LIF receptor in solution (Kd approximately 2 nM). These data show that CT-1 has a wide range of hematopoietic, neuronal, and developmental activities and that it can act via the LIF receptor and the gp130 signaling subunit.


Assuntos
Citocinas/metabolismo , Interleucina-6 , Receptores de Citocinas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Ligação Competitiva , Bioensaio , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocinas/farmacologia , Primers do DNA/química , Inibidores do Crescimento/metabolismo , Hematopoese/efeitos dos fármacos , Técnicas In Vitro , Fator Inibidor de Leucemia , Subunidade alfa de Receptor de Fator Inibidor de Leucemia , Ligantes , Linfocinas/metabolismo , Camundongos , Dados de Sequência Molecular , Miocárdio/metabolismo , Neurônios/citologia , Ratos , Receptores de OSM-LIF , Transdução de Sinais
19.
J Neurosci ; 15(1 Pt 2): 477-91, 1995 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-7823156

RESUMO

Using molecular cloning techniques, human homologs of the known members of the trk family of neurotrophin receptors have been cloned and sequenced. Overall, there is a high degree of similarity between the human sequences and those from other mammals; however, there are differences in splicing patterns. There are two spliced forms of the extracellular domain of trkC in the human, a finding that has not been described in other species. In contrast, fewer spliced forms were detected of the intracellular domains of human trkB and trkC than has been described in other mammals. Northern analysis and in situ hybridization experiments indicate that the human trks are expressed in a similar pattern to that described in other mammals. Expression of the trk extracellular domains as fusion proteins with IgG heavy chain yields soluble molecules that mimic intact trks in their binding specificity and affinity. These soluble chimeras block the biological activity of their cognate neurotrophin(s) in vitro.


Assuntos
Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Clonagem Molecular , Espaço Extracelular/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , DNA Complementar/genética , DNA Recombinante , Humanos , Imunoglobulina G/genética , Cadeias Pesadas de Imunoglobulinas/genética , Hibridização In Situ , Sondas Moleculares/genética , Dados de Sequência Molecular , Ratos , Receptores Proteína Tirosina Quinases/genética , Receptor do Fator Neutrófico Ciliar , Receptor trkC , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/metabolismo , Distribuição Tecidual
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