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1.
Microcirculation ; 13(6): 467-76, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16864413

RESUMO

OBJECTIVES: Normal muscle growth is accompanied by capillary proliferation, which usually lags behind the increase in muscle size, causing a decline in mean capillary density (CD). It is not known, however, how the capillary distribution is affected and what impact it has on the oxygenation of the muscle. METHODS: The capillarization of soleus muscles of rats (64-425 g) was determined with the method of capillary domains. As well as quantifying CD, capillary to fiber ratio (C:F), and fiber size, this method provides a measure of the heterogeneity of capillary spacing. Capillary locations were used to mathematically model oxygenation levels within the muscle. RESULTS: The increase in muscle mass was largely attributable to 5-fold increase in fiber size, accompanied by a more than 3-fold rise in C:F. The mismatch between rates of angiogenesis and muscle growth resulted in a decrease in CD. However, the heterogeneity of capillary spacing was unaffected (heterogeneity index logRSD: 0.091 +/- 0.013; mean +/- SD) as was muscle PO2, with modal values between 4 and 60 mmHg (0.5 and 8 kPa). CONCLUSIONS: Angiogenesis during normal muscle growth does not maintain CD, but with similar heterogeneity of capillary spacing it preserves the potential for adequate intramuscular oxygenation.


Assuntos
Modelos Cardiovasculares , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/crescimento & desenvolvimento , Neovascularização Fisiológica , Consumo de Oxigênio/fisiologia , Animais , Masculino , Microcirculação/fisiologia , Músculo Esquelético/metabolismo , Ratos , Ratos Wistar
2.
Adv Exp Med Biol ; 530: 509-17, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-14562746

RESUMO

Non-steady state measurements of oxygen diffusion through various model layers can be performed using a diffusion chamber that was described earlier [1, 2]. A closer analysis of these measurements showed that they not only yield the oxygen diffusion coefficient (DO2) of the diffusion layer, but also the oxygen permeability (PO2). In this study DO2 and PO2 have been determined in solutions of metmyoglobin (metMb) with concentrations varying between 5 and 40 g/dL at 25 degrees C. Both DO2 and PO2 decreased with increasing metMb concentration. This decrease was comparable to the values reported for DO2 in protein solutions by Kreuzer and Hoofd [3]. Using this diffusion chamber for non-steady state measurements, oxygen diffusion coefficients, oxygen permeability and oxygen solubility (aO2 = PO2/DO2) of various model layers could be determined.


Assuntos
Metamioglobina/metabolismo , Oxigênio/metabolismo , Difusão
4.
Drug Discov Today ; 8(3): 100-1, 2003 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-12568772

RESUMO

Gene-mapping studies that look for complex traits among human populations have deepened our understanding of disease causes, but do they hold promise for identifying drug targets?


Assuntos
Bases de Dados de Ácidos Nucleicos , Tecnologia Farmacêutica/métodos , Bases de Dados de Ácidos Nucleicos/tendências , Humanos , Farmacogenética/métodos , Farmacogenética/tendências , Tecnologia Farmacêutica/tendências
5.
Hear Res ; 159(1-2): 125-31, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11520640

RESUMO

Immunophilin FK binding protein-12 (FKBP-12), the soluble receptor for the immunosuppressant drug FK506, is involved in a number of neuronal activities including increased nerve regeneration in the peripheral nervous system and enhanced recovery in animal models of neurodegenerative diseases. In addition, FKBP-12 is tightly bound to the calcium release channel ryanodine receptor and physiologically interacts with the inositol 1,4,5-trisphosphate receptor. In nearly all cell types, release of intracellular Ca(2+) and subsequent second messenger signaling involves activation of these ion channels. We determined the distribution of FKBP-12 within the mammalian cochlea and dorsal cochlear nucleus (DCN) in order to gain insight into Ca(2+) regulation within the cochlea and to possibly identify potential cellular targets for neuroimmunophilin ligands that may prove useful in protection and recovery following ototoxic insult. FKBP-12 protein and mRNA were found to be abundant throughout rat and guinea pig cochlea and DCN.


Assuntos
Cóclea/metabolismo , Núcleo Coclear/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína 1A de Ligação a Tacrolimo/genética , Proteína 1A de Ligação a Tacrolimo/metabolismo , Animais , Western Blotting , Cobaias , Imuno-Histoquímica , Hibridização In Situ , Masculino , Regeneração Nervosa/genética , Regeneração Nervosa/fisiologia , Ratos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Distribuição Tecidual
7.
J Biol Chem ; 275(48): 37712-7, 2000 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-10956649

RESUMO

The novel transmembrane aspartic protease BACE (for Beta-site APP Cleaving Enzyme) is the beta-secretase that cleaves amyloid precursor protein to initiate beta-amyloid formation. As such, BACE is a prime therapeutic target for the treatment of Alzheimer's disease. BACE, like other aspartic proteases, has a propeptide domain that is removed to form the mature enzyme. BACE propeptide cleavage occurs at the sequence RLPR downward arrowE, a potential furin recognition motif. Here, we explore the role of furin in BACE propeptide domain processing. BACE propeptide cleavage in cells does not appear to be autocatalytic, since an inactive D93A mutant of BACE is still cleaved appropriately. BACE and furin co-localize within the Golgi apparatus, and propeptide cleavage is inhibited by brefeldin A and monensin, drugs that disrupt trafficking through the Golgi. Treatment of cells with the calcium ionophore, leading to inhibition of calcium-dependent proteases including furin, or transfection with the alpha(1)-antitrypsin variant alpha(1)-PDX, a potent furin inhibitor, dramatically reduces cleavage of the BACE propeptide. Moreover, the BACE propeptide is not processed in the furin-deficient LoVo cell line; however, processing is restored upon furin transfection. Finally, in vitro digestion of recombinant soluble BACE with recombinant furin results in complete cleavage only at the established E46 site. Taken together, our results strongly suggest that furin, or a furin-like proprotein convertase, is responsible for cleaving the BACE propeptide domain to form the mature enzyme.


Assuntos
Doença de Alzheimer/enzimologia , Ácido Aspártico Endopeptidases/metabolismo , Subtilisinas/metabolismo , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide , Ácido Aspártico Endopeptidases/química , Sequência de Bases , Catálise , Linhagem Celular , Primers do DNA , Endopeptidases , Furina , Complexo de Golgi/enzimologia , Humanos , Hidrólise , Dados de Sequência Molecular , Proteínas Recombinantes/metabolismo
8.
J Biol Chem ; 275(44): 34574-9, 2000 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-10942772

RESUMO

Parkinson's disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies. Recently, two point mutations in alpha-synuclein were found to be associated with familial PD, but as of yet no mutations have been described in the homologous genes beta- and gamma-synuclein. alpha-Synuclein forms the major fibrillar component of Lewy bodies, but these do not stain for beta- or gamma-synuclein. This result is very surprising, given the extent of sequence conservation and the high similarity in expression and subcellular localization, in particular between alpha- and beta-synuclein. Here we compare in vitro fibrillogenesis of all three purified synucleins. We show that fresh solutions of alpha-, beta-, and gamma- synuclein show the same natively unfolded structure. While over time alpha-synuclein forms the previously described fibrils, no fibrils could be detected for beta- and gamma-synuclein under the same conditions. Most importantly, beta- and gamma-synuclein could not be cross-seeded with alpha-synuclein fibrils. However, under conditions that drastically accelerate aggregation, gamma-synuclein can form fibrils with a lag phase roughly three times longer than alpha-synuclein. These results indicate that beta- and gamma-synuclein are intrinsically less fibrillogenic than alpha-synuclein and cannot form mixed fibrils with alpha-synuclein, which may explain why they do not appear in the pathological hallmarks of PD, although they are closely related to alpha-synuclein and are also abundant in brain.


Assuntos
Proteínas do Tecido Nervoso/química , Doença de Parkinson/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Dicroísmo Circular , Primers do DNA , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Dobramento de Proteína , Homologia de Sequência de Aminoácidos , Análise Espectral/métodos , Sinucleínas , alfa-Sinucleína , beta-Sinucleína , gama-Sinucleína
9.
J Biol Chem ; 275(27): 20647-51, 2000 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-10749877

RESUMO

Beta-site amyloid precursor protein cleaving enzyme (BACE) is a novel transmembrane aspartic protease that possesses all the known characteristics of the beta-secretase involved in Alzheimer's disease (Vassar, R., Bennett, B. D., Babu-Khan, S., Kahn, S., Mendiaz, E. A., Denis, P., Teplow, D. B., Ross, S., Amarante, P., Loeloff, R., Luo, Y., Fisher, S., Fuller, J., Edenson, S., Lile, J., Jarosinski, M. A., Biere, A. L., Curran, E., Burgess, T., Louis, J. -C., Collins, F., Treanor, J., Rogers, G., and Citron, M. (1999) Science 286, 735-741). We have analyzed the sequence and expression pattern of a BACE homolog termed BACE2. BACE and BACE2 are unique among aspartic proteases in that they possess a carboxyl-terminal extension with a predicted transmembrane region and together they define a new family. Northern analysis reveals that BACE2 mRNA is expressed at low levels in most human peripheral tissues and at higher levels in colon, kidney, pancreas, placenta, prostate, stomach, and trachea. Human adult and fetal whole brain and most adult brain subregions express very low or undetectable levels of BACE2 mRNA. In addition, in situ hybridization of adult rat brain shows that BACE2 mRNA is expressed at very low levels in most brain regions. The very low or undetectable levels of BACE2 mRNA in the brain are not consistent with the expression pattern predicted for beta-secretase.


Assuntos
Encéfalo/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Glicoproteínas/genética , Proteínas de Membrana/genética , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide , Animais , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/metabolismo , Encéfalo/anatomia & histologia , Endopeptidases/química , Glicoproteínas/metabolismo , Humanos , Hibridização In Situ , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Ratos , Alinhamento de Sequência
10.
Science ; 286(5440): 735-41, 1999 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-10531052

RESUMO

Cerebral deposition of amyloid beta peptide (Abeta) is an early and critical feature of Alzheimer's disease. Abeta generation depends on proteolytic cleavage of the amyloid precursor protein (APP) by two unknown proteases: beta-secretase and gamma-secretase. These proteases are prime therapeutic targets. A transmembrane aspartic protease with all the known characteristics of beta-secretase was cloned and characterized. Overexpression of this protease, termed BACE (for beta-site APP-cleaving enzyme) increased the amount of beta-secretase cleavage products, and these were cleaved exactly and only at known beta-secretase positions. Antisense inhibition of endogenous BACE messenger RNA decreased the amount of beta-secretase cleavage products, and purified BACE protein cleaved APP-derived substrates with the same sequence specificity as beta-secretase. Finally, the expression pattern and subcellular localization of BACE were consistent with that expected for beta-secretase. Future development of BACE inhibitors may prove beneficial for the treatment of Alzheimer's disease.


Assuntos
Doença de Alzheimer/enzimologia , Peptídeos beta-Amiloides/biossíntese , Precursor de Proteína beta-Amiloide/metabolismo , Ácido Aspártico Endopeptidases/isolamento & purificação , Ácido Aspártico Endopeptidases/metabolismo , Doença de Alzheimer/tratamento farmacológico , Motivos de Aminoácidos , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide , Animais , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/genética , Sítios de Ligação , Encéfalo/enzimologia , Encéfalo/metabolismo , Linhagem Celular , Clonagem Molecular , Endopeptidases , Endossomos/enzimologia , Expressão Gênica , Biblioteca Gênica , Complexo de Golgi/enzimologia , Humanos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/farmacologia , Peptídeos/metabolismo , Inibidores de Proteases/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transfecção
11.
J Biol Chem ; 274(28): 19509-12, 1999 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-10391881

RESUMO

Parkinson's disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies, the major components of which are filaments consisting of alpha-synuclein. Two recently identified point mutations in alpha-synuclein are the only known genetic causes of PD. alpha-Synuclein fibrils similar to the Lewy body filaments can be formed in vitro, and we have shown recently that both PD-linked mutations accelerate their formation. This study addresses the mechanism of alpha-synuclein aggregation: we show that (i) it is a nucleation-dependent process that can be seeded by aggregated alpha-synuclein functioning as nuclei, (ii) this fibril growth follows first-order kinetics with respect to alpha-synuclein concentration, and (iii) mutant alpha-synuclein can seed the aggregation of wild type alpha-synuclein, which leads us to predict that the Lewy bodies of familial PD patients with alpha-synuclein mutations will contain both, the mutant and the wild type protein. Finally (iv), we show that wild type and mutant forms of alpha-synuclein do not differ in their critical concentrations. These results suggest that differences in aggregation kinetics of alpha-synucleins cannot be explained by differences in solubility but are due to different nucleation rates. Consequently, alpha-synuclein nucleation may be the rate-limiting step for the formation of Lewy body alpha-synuclein fibrils in Parkinson's disease.


Assuntos
Corpos de Lewy/química , Proteínas do Tecido Nervoso/genética , Doença de Parkinson/genética , Escherichia coli/genética , Humanos , Cinética , Mutação , Proteínas do Tecido Nervoso/química , Doença de Parkinson/patologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Solubilidade , Sinucleínas , alfa-Sinucleína
12.
J Biol Chem ; 274(14): 9843-6, 1999 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-10092675

RESUMO

Parkinson's disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies, the major component of which are filaments consisting of alpha-synuclein. Two recently identified point mutations in alpha-synuclein are the only known genetic causes of PD, but their pathogenic mechanism is not understood. Here we show that both wild type and mutant alpha-synuclein form insoluble fibrillar aggregates with antiparallel beta-sheet structure upon incubation at physiological temperature in vitro. Importantly, aggregate formation is accelerated by both PD-linked mutations. Under the experimental conditions, the lag time for the formation of precipitable aggregates is about 280 h for the wild type protein, 180 h for the A30P mutant, and only 100 h for the A53T mutant protein. These data suggest that the formation of alpha-synuclein aggregates could be a critical step in PD pathogenesis, which is accelerated by the PD-linked mutations.


Assuntos
Mutação , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Doença de Parkinson/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Linhagem Celular , Dicroísmo Circular , Clonagem Molecular , Humanos , Conformação Proteica , Estrutura Secundária de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier , Sinucleínas , alfa-Sinucleína
13.
Ann N Y Acad Sci ; 884: 270-91, 1999 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-10842600

RESUMO

Sensorineural hearing loss results from the degeneration of hair cells and/or auditory neurons in the cochlea of the inner ear. BDNF and NT-3 were shown to support survival of auditory neurons both in vitro and in vivo. Cochlea from P3-P4 rats were cultured as floating explants and hair cells in the organ of Corti were identified by phalloidin-FITC immunostaining. Treatment with cisplatin (35 micrograms/mL) or neomycin (0.6 mM) resulted in 21.2 +/- 6.0% and 7.4 +/- 4.7% surviving hair cells, respectively, after 3 days in culture. GDNF, added together with the ototoxins, increased their number to 46.7% and 37.4%, respectively. In cultures of dissociated cochlea from 4-week-old rat, cisplatin (5 mg/mL) added 24 h after seeding resulted in only 6.1 +/- 1.2% surviving neurons. However, when cisplatin was added together with GDNF (10 ng/mL), 32.8 +/- 1.0% of the neurons survived. The efficacy of GDNF in animal models of ototoxicity was tested next. Guinea pigs were pretreated with GDNF in one ear, delivered either by infusion into the inner ear (scala tympani) with Alzet minipumps (50 ng/mL at a 0.5 microL/h), or injected into the middle ear (120 microL at 1 mg/mL) through the tympanic membrane. The ear that did not receive GDNF always served as control. Ototoxicity was induced systemically either by intraperitoneal cisplatin injections (1 mg/kg/day for 15 days or two injections of 7.5 mg/kg at a 5-day interval or by a combination of kanamycin (200-300 mg/kg, administered subcutaneously) and ethacrinic acid (40 mg/kg, intravenous). It was found that the number of surviving hair cells in GDNF-treated ears was about twice that of control ears in animals exposed to the ototoxins. The transducing GDNF receptor (ret) is expressed in the inner ear.


Assuntos
Células Ciliadas Auditivas Externas/efeitos dos fármacos , Fatores de Crescimento Neural , Proteínas do Tecido Nervoso/farmacologia , Fármacos Neuroprotetores/farmacologia , Rampa do Tímpano/efeitos dos fármacos , Animais , Antibacterianos/efeitos adversos , Antineoplásicos/efeitos adversos , Células Cultivadas , Cisplatino/efeitos adversos , Inibidores Enzimáticos/efeitos adversos , Ácido Etacrínico/efeitos adversos , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Cobaias , Humanos , Canamicina/efeitos adversos , Ratos , Ratos Long-Evans , Ratos Wistar
14.
J Neurochem ; 71(5): 1912-9, 1998 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-9798915

RESUMO

Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for midbrain dopaminergic neurons. To begin to understand the intracellular signaling pathways used by GDNF, we investigated the role of phosphatidylinositol 3-kinase activity in GDNF-stimulated cellular function and differentiation of dopaminergic neurons. We found that treatment of dopaminergic neuron cultures with 10 ng/ml GDNF induced maximal levels of Ret phosphorylation and produced a profound increase in phosphatidylinositol 3-kinase activity, as measured by western blot analysis and lipid kinase assays. Treatment with 1 microM 2-(4-morpholinyl)-8-phenylchromone (LY294002) or 100 nM wortmannin, two distinct and potent inhibitors of phosphatidylinositol 3-kinase activity, completely inhibited GDNF-induced phosphatidylinositol 3-kinase activation, but did not affect Ret phosphorylation. Furthermore, we examined specific biological functions of dopaminergic neurons: dopamine uptake activity and morphological differentiation of tyrosine hydroxylase-immunoreactive neurons. GDNF significantly increased dopamine uptake activity and promoted robust morphological differentiation. Treatment with LY294002 completely abolished the GDNF-induced increases of dopamine uptake and morphological differentiation of tyrosine hydroxylase-immunoreactive neurons. Our findings show that GDNF-induced differentiation of dopaminergic neurons requires phosphatidylinositol 3-kinase activation.


Assuntos
Dopamina/metabolismo , Proteínas de Drosophila , Fatores de Crescimento Neural , Proteínas do Tecido Nervoso/farmacologia , Neurônios/efeitos dos fármacos , Inibidores de Fosfoinositídeo-3 Quinase , Androstadienos/farmacologia , Animais , Diferenciação Celular/fisiologia , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Morfolinas/farmacologia , Neurônios/citologia , Neurônios/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-ret , Ratos/embriologia , Ratos Sprague-Dawley , Receptores Proteína Tirosina Quinases/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Wortmanina
15.
Eur J Neurosci ; 10(4): 1270-81, 1998 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9749781

RESUMO

We have investigated in vitro the influence of pituitary intermediate lobe melanotrophs on the differentiation of their afferent hypothalamic dopaminergic neurons. The presence of melanotrophs in primary cultures of foetal hypothalamic neurons induces an increase of the number of dopaminergic neurons (while the total neuronal population remains unchanged) and induces a stimulation of their neuritic outgrowth. These effects are mediated by diffusible factors since they are reproduced by application of conditioned medium issued from co-cultures with intermediate lobe cells from newborn rats. Moreover, by immunoneutralization of alpha-melanocyte-stimulating hormone (alphaMSH) in the co-culture or conditioned medium, or by application of the peptide itself, we demonstrate that the neuritotrophic effect on dopaminergic neurons is mediated by alphaMSH, the main secretory product of melanotrophs, whereas the inductive effect on the number of dopaminergic neurons is attributable to another diffusible neurotrophic factor(s) present in foetal, but not adult, adenohypophysis. Similar effects are observed on cultures of newborn hypothalamic neurons. However, at this stage of neuronal development, alphaMSH also increases the number of dopaminergic neurons, which could be due to a change of neuronal receptivity. We show that the neuritotrophic influence of alphaMSH is restricted to the dopaminergic neurons connected to the melanotrophs, and that in addition, these neurons systematically co-express the tyrosine hydroxylase and glutamate decarboxylase as the neurons innervating the melanotrophs in situ. These findings indicate that the differentiation of dopaminergic hypothalamic neurons is influenced by the target cells, melanotrophs, and that this trophic influence implicates alphaMSH.


Assuntos
Dopamina/fisiologia , Hipotálamo/citologia , Neurônios/fisiologia , Hipófise/metabolismo , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Diferenciação Celular/fisiologia , Técnicas de Cocultura , Desenvolvimento Embrionário e Fetal/fisiologia , Hipotálamo/embriologia , Hipotálamo/crescimento & desenvolvimento , Dados de Sequência Molecular , Hipófise/citologia , Ratos , Ratos Wistar , Estimulação Química , alfa-MSH/análise
16.
Neuroreport ; 9(10): 2183-7, 1998 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-9694197

RESUMO

Glial-derived neurotrophic factor (GDNF) was tested for its ability to prevent hearing and sensory cell loss in guinea pigs exposed to acoustic trauma. Hearing was measured prior to any treatment. Animals were exposed to damaging levels of noise either before or after local application of GDNF to one ear. Four weeks later, hearing and sensory cell loss was greater in the control ear than in the ear receiving GDNF before acoustic trauma or 2 h after trauma, but not 4 or 6 h after trauma. The results indicate that GDNF treatment in vivo can prevent cochlear sensory cell damage and hearing loss if present during or shortly after acoustic trauma.


Assuntos
Cóclea/lesões , Perda Auditiva Provocada por Ruído/prevenção & controle , Fatores de Crescimento Neural , Proteínas do Tecido Nervoso/farmacologia , Fármacos Neuroprotetores/farmacologia , Animais , Limiar Auditivo/efeitos dos fármacos , Limiar Auditivo/fisiologia , Cóclea/patologia , Feminino , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Cobaias , Células Ciliadas Vestibulares/efeitos dos fármacos , Perda Auditiva Provocada por Ruído/patologia , Perda Auditiva Provocada por Ruído/fisiopatologia , Degeneração Neural/patologia , Degeneração Neural/prevenção & controle , Fatores de Tempo
17.
J Neurochem ; 70(4): 1383-93, 1998 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9523554

RESUMO

Here we report the generation and characterization of two distinct monoclonal antibodies, G-90 and B-1531, specific to glial cell line-derived neurotrophic factor (GDNF). ELISA results confirmed that G-90 and B-1531 both recognize GDNF. Western blots showed that G-90 recognized only the GDNF dimer, whereas B-1531 recognized both the monomer and dimer. Peptide competition ELISA (PCE) and BIAcore data suggested that G-90 and B-1531 recognize different epitopes: PCE confirmed that B-1531 binds to NH2-terminal peptides between amino acids 18 and 37, whereas G-90 does not; BIAcore data showed that B-1531 binds to the NH2 terminus of GDNF, whereas G-90 does not. G-90, in a concentration-dependent manner, completely neutralized the GDNF-induced increases of choline acetyltransferase in cultured motoneuron and of dopamine uptake and morphological differentiation in dopaminergic neuron cultures. B-1531 had no neutralizing effects. GDNF-induced Ret autophosphorylation in NGR-38 cells was completely neutralized by G-90, whereas B-1531 had a moderate effect. These data show that G-90 and B-1531 are specific antibodies to GDNF. The data also suggest that the NH2 terminus of GDNF is not critical for activity. Partial inhibition of Ret phosphorylation is insufficient to down-regulate GDNF-induced biological activity.


Assuntos
Anticorpos Monoclonais/imunologia , Proteínas de Drosophila , Fatores de Crescimento Neural , Proteínas do Tecido Nervoso/imunologia , Animais , Especificidade de Anticorpos , Técnicas Biossensoriais , Western Blotting , Colina O-Acetiltransferase/farmacocinética , Dopamina/farmacocinética , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas do Tecido Nervoso/farmacologia , Fosforilação , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-ret , Ratos , Ratos Sprague-Dawley , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Recombinantes
18.
J Neurochem ; 69(3): 986-94, 1997 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9282920

RESUMO

The c-ret protooncogene encodes Ret, the functional tyrosine kinase receptor for glial cell line-derived neurotrophic factor (GDNF). K-252b, a known protein tyrosine kinase inhibitor, has been shown earlier to inhibit the trophic activity of brain-derived neurotrophic factor on dopaminergic (DAergic) neurons and nerve growth factor on basal forebrain cholinergic neurons while potentiating neurotrophin-3 activity on central cholinergic and peripheral sensory neurons and PC12 cells. We tested whether K-252b would modulate GDNF-induced differentiation in DAergic neuron cultures. Exposure to 1 ng/ml GDNF increased dopamine (DA) uptake 80% above control, whereas treatment with 5 microM K-252b decreased the efficacy of GDNF by 60%. Concentrations of GDNF of <100 pg/ml were completely inhibited, whereas concentrations of >100 pg/ml were moderately active, between 10 and 20% above control. In addition, K-252b shifted the ED50 from 20 to 200 pg/ml. GDNF treatment increased soma size and neurite outgrowth in tyrosine hydroxylase-immunoreactive neurons. K-252b inhibited differentiation of these morphological parameters induced by GDNF. Furthermore, GDNF stimulated Ret autophosphorylation at maximal levels, whereas the inhibition of DA uptake and morphological differentiation by K-252b correlated with a significantly decreased level of Ret autophosphorylation. Therefore, K-252b is able to inhibit intracellular activities induced by GDNF on mesencephalic DAergic neurons.


Assuntos
Carbazóis/farmacologia , Dopamina/metabolismo , Proteínas de Drosophila , Inibidores Enzimáticos/farmacologia , Mesencéfalo/metabolismo , Fatores de Crescimento Neural , Proteínas do Tecido Nervoso/farmacologia , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Diferenciação Celular , Células Cultivadas , Feto , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Humanos , Alcaloides Indólicos , Cinética , Proteínas do Tecido Nervoso/antagonistas & inibidores , Neurônios/citologia , Neurônios/efeitos dos fármacos , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-ret , Ratos , Ratos Sprague-Dawley , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/farmacologia , Tirosina 3-Mono-Oxigenase/análise
19.
Cell ; 85(7): 1113-24, 1996 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-8674117

RESUMO

We report the expression cloning and characterization of GDNFR-alpha, a novel glycosylphosphatidylinositol-linked cell surface receptor for glial cell line-derived neurotrophic factor (GDNF). GDNFR-alpha binds GDNF specifically and mediates activation of the Ret protein-tyrosine kinase (PTK). Treatment of Neuro-2a cells expressing GDNFR-alpha with GDNF rapidly stimulates Ret autophosphorylation. Ret is also activated by treatment with a combination of GDNF and soluble GDNFR-alpha in cells lacking GDNFR-alpha, and this effect is blocked by a soluble Ret-Fc fusion protein. Ret activation by GDNF was also observed in cultured embryonic rat spinal cord motor neurons, a cell type that responds to GDNF in vivo. A model for the stepwise formation of a GDNF signal-transducing complex including GDNF, GDNFR-alpha, and the Ret PTK is proposed.


Assuntos
Proteínas de Drosophila , Proteínas do Tecido Nervoso/genética , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Receptores de Fator de Crescimento Neural/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas/efeitos dos fármacos , Clonagem Molecular , Relação Dose-Resposta a Droga , Feto/citologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Glicosilfosfatidilinositóis/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Neurônios Motores/metabolismo , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/farmacologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/farmacologia , Fosforilação , Proteínas Proto-Oncogênicas c-ret , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/metabolismo , Retina/citologia , Medula Espinal/citologia , Tirosina/metabolismo
20.
J Biol Chem ; 269(45): 27833-9, 1994 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-7961712

RESUMO

Three neurotrophic factors, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and nerve growth factor (NGF) form noncovalent homodimers in solution. Since they are highly homologous proteins, it seemed probable that two monomers of these proteins might associate together to form a heterodimer. This was tested by denaturing the two different proteins together in 6 M guanidine HCl and refolding them in phosphate-buffered saline. When the refolded mixture of BDNF and NT-3 was subjected to Mono S cation exchange chromatography, a new peak was observed eluting between NT-3 and BDNF, which accounted for about 30% of the protein used. This new protein species migrated as a single band upon native gel electrophoresis with mobility between that of the NT-3 homodimer and the BDNF homodimer, indicating that a complex had been formed. Sedimentation equilibrium data show that the dissociation constant of this heterodimer is < 3 x 10(-10) M. The heterodimer was stable upon incubation at 37 degrees C in phosphate-buffered saline over 11 days. Having determined that the heterodimer is highly stable, it was subjected to various biological assays. Autophosphorylation assay using TrkB receptor showed that the heterodimer is indistinguishable from the BDNF or NT-3 homodimer in the ability to induce phosphorylation of the receptor. It was also indistinguishable from the homodimers in the neurotrophic activity using chick dorsal root ganglion explant. In the sympathetic neuron survival assay, the heterodimer behaved more similarly to NT-3, whereas in the dopamine uptake assay, it was intermediate between the two homodimers. In addition, the heterodimer was shown to be retrogradely transported in the dorsal root ganglion neurons. A heterodimer between NGF and BDNF is formed but much less effectively than the NT-3.BDNF heterodimer, and it is not stable even at 4 degrees C. These results indicate that BDNF and NT-3 have an intersubunit contact surface for dimerization resembling each other's but different from the contact surface of NGF.


Assuntos
Fatores de Crescimento Neural/química , Proteínas do Tecido Nervoso/química , Conformação Proteica , Dobramento de Proteína , Células 3T3 , Animais , Bioensaio , Fator Neurotrófico Derivado do Encéfalo , Células CHO , Galinhas , Cromatografia por Troca Iônica , Dicroísmo Circular , Cricetinae , Eletroforese em Gel de Poliacrilamida , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/fisiologia , Guanidina , Guanidinas , Humanos , Substâncias Macromoleculares , Camundongos , Fatores de Crescimento Neural/biossíntese , Fatores de Crescimento Neural/isolamento & purificação , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/isolamento & purificação , Neurotrofina 3 , Fosforilação , Desnaturação Proteica , Receptor do Fator Neutrófico Ciliar , Receptores de Fator de Crescimento Neural/biossíntese , Receptores de Fator de Crescimento Neural/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Transfecção
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