RESUMO
Recent studies suggest that impaired transcription or mitochondrial translation of small RNAs can cause abnormal myelination. A polynucleotide phosphorylase (PNPase) encoded by PNPT1 facilitates the import of small RNAs into mitochondria. PNPT1 mutations have been reported in patients with neurodevelopmental diseases with mitochondrial dysfunction. We report here 2 siblings with PNPT1 mutations who presented delayed myelination as well as mitochondrial dysfunction. We identified compound heterozygous mutations (c.227G>A; p.Gly76Asp and c.574C>T; p.Arg192*) in PNPT1 by quartet whole-exome sequencing. Analyses of skin fibroblasts from the patient showed that PNPase expression was markedly decreased and that import of the small RNA RNaseP into mitochondria was impaired. Exogenous expression of wild-type PNPT1, but not mutants, rescued ATP production in patient skin fibroblasts, suggesting the pathogenicity of the identified mutations. Our cases expand the phenotypic spectrum of PNPT1 mutations that can cause delayed myelination.
Assuntos
Exorribonucleases/genética , Doenças Mitocondriais/genética , Bainha de Mielina/genética , Transtornos do Neurodesenvolvimento/genética , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Encéfalo/patologia , Pré-Escolar , Hibridização Genômica Comparativa , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Doenças Mitocondriais/diagnóstico por imagem , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , Mutação , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Transtornos do Neurodesenvolvimento/diagnóstico por imagem , Transtornos do Neurodesenvolvimento/metabolismo , Transtornos do Neurodesenvolvimento/patologia , RNA/genética , Sequenciamento do ExomaRESUMO
AIMS: To evaluate comprehensively the use of the glycated albumin to HbA1c ratio for estimation of glycaemic control in the previous month. METHODS: A total of 306 children with Type 1 diabetes mellitus underwent ≥10 simultaneous measurements of glycated albumin and HbA1c . Correlation and concordance rates were examined between HbA1c measurements taken 1 month apart (ΔHbA1c ) and glycated albumin/HbA1c ratio fluctuations were calculated as Z-scores from the cohort value at enrolment of this study cohort (method A) or the percent difference from the individual mean over time (method B). RESULTS: Fluctuations in glycated albumin/HbA1c ratio (using both methods) were weakly but significantly correlated with ΔHbA1c , whereas concordance rates were significant for glycaemic deterioration but not for glycaemic improvement. Concordance rates were higher using method B than method A. CONCLUSIONS: The glycated albumin/HbA1c ratio was able to estimate glycaemic deterioration in the previous month, while estimation of glycaemic improvement in the preceding month was limited. Because method B provided a better estimate of recent glycaemic control than method A, the individual mean of several measurements of the glycated albumin/HbA1c ratio over time may also identify individuals with high or low haemoglobin glycation phenotypes in a given population, such as Japanese children with Type 1 diabetes, thereby allowing more effective diabetes management.
Assuntos
Glicemia/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Hemoglobinas Glicadas/metabolismo , Albumina Sérica/metabolismo , Adolescente , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Produtos Finais de Glicação Avançada , Humanos , Japão , Masculino , Adulto Jovem , Albumina Sérica GlicadaRESUMO
AIM: To examine the contribution of PTPN2 coding variants to the risk of childhood-onset Type 1A diabetes. METHODS: PTPN2 mutation analysis was carried out for 169 unrelated Japanese people with childhood-onset Type 1A diabetes. We searched for coding variants that were absent or extremely rare in the general population and were scored as damaging by multiple in silico programs. We performed mRNA analysis and three-dimensional structural prediction of the detected variants, when possible. We also examined possible physical links between these variants and previously reported risk SNPs as well as clinical information from variant-positive children. RESULTS: One frameshift variant (p.Q286Yfs*24) and two probably damaging missense substitutions (p.C232W and p.R350Q) were identified in one child each. Of these, p.Q286Yfs*24 and p.C232W were hitherto unreported, while p.R350Q accounted for 2/121,122 alleles of the exome datasets. The p.Q286Yfs*24 variant did not encode stable mRNA, and p.C232W appeared to affect the structure of the tyrosine-protein phosphatase domain. The three variants were physically unrelated to known risk SNPs. The variant-positive children manifested Type 1A diabetes without additional clinical features and invariably carried risk human leukocyte antigen alleles. CONCLUSIONS: The results provide the first indication that PTPN2 variants contribute to the risk of Type 1A diabetes, independently of known risk SNPs. PTPN2 coding variants possibly induce non-specific Type 1A diabetes phenotypes in individuals with human leukocyte antigen-mediated disease susceptibility. Our findings warrant further validation.
Assuntos
Diabetes Mellitus Tipo 1/genética , Mutação da Fase de Leitura/genética , Mutação de Sentido Incorreto/genética , Proteína Tirosina Fosfatase não Receptora Tipo 2/genética , Adolescente , Criança , Pré-Escolar , Feminino , Predisposição Genética para Doença/genética , Antígenos HLA/genética , Humanos , Lactente , Masculino , Fases de Leitura Aberta/genética , Polimorfismo de Nucleotídeo Único/genética , RNA Mensageiro/genéticaRESUMO
Mitochondrial disorders have the highest incidence among congenital metabolic diseases, and are thought to occur at a rate of 1 in 5000 births. About 25% of the diseases diagnosed as mitochondrial disorders in the field of pediatrics have mitochondrial DNA abnormalities, while the rest occur due to defects in genes encoded in the nucleus. The most important function of the mitochondria is biosynthesis of ATP. Mitochondrial disorders are nearly synonymous with mitochondrial respiratory chain disorder, as respiratory chain complexes serve a central role in ATP biosynthesis. By next-generation sequencing of the exome, we analyzed 104 patients with mitochondrial respiratory chain disorders. The results of analysis to date were 18 patients with novel variants in genes previously reported to be disease-causing, and 27 patients with mutations in genes suggested to be associated in some way with mitochondria, and it is likely that they are new disease-causing genes in mitochondrial disorders. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.
Assuntos
Exoma/genética , Predisposição Genética para Doença , Doenças Mitocondriais/diagnóstico , Doenças Mitocondriais/genética , Núcleo Celular/genética , Estudos de Associação Genética , Humanos , Análise em Microsséries , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNARESUMO
Carnitine palmitoyltransferase II (CPT II) deficiency is an inherited disorder involving beta-oxidation of long-chain fatty acids. CPT II deficiency is a wide-spectrum disorder that includes a lethal neonatal form, an infantile form, and an adult-onset form. However, the ethnic characteristics and the relationship between genotype and clinical manifestation are not well understood. We investigated three non-consanguineous Japanese patients with CPT II deficiency and examined cell lines from 4 unrelated patients and 50 healthy donors. The CPT 2 gene was typed by direct DNA sequencing of polymerase chain reaction-amplified gene products. Case 1 (infantile form) was heterozygous for a phenylalanine to tyrosine substitution at position 383 (p.F383Y) and a novel valine to leucine substitution at 605 (p.V605L). Cases 2, 4, and 5 (infantile form) and case 3 (adult-onset form) were heterozygous for a single mutation at F383Y. Case 6 (adult-onset form) was compound heterozygous at the CPT 2 locus, with deletion of cytosine and thymine at residue 408, resulting in a stop signal at 420 (p.Y408fsX420), and an arginine to cysteine substitution at position 631 (p.R631C). Case 7 (adult-onset form) was homozygous for the p.F383Y mutation. In conclusion, we identified p.F383Y mutations in six of seven patients with CPT II deficiency and two novel variants of the coding gene: p.Y408fsX420 and p.V605L. These mutations differ from those in Caucasian patients, who commonly harbor p.S113L, p.P50H, and p.Q413fsX449 mutations; therefore, our data and those of other Japanese groups suggest that the p.F383Y mutation is significant in Japanese patients with CPT II deficiency.
Assuntos
Carnitina O-Palmitoiltransferase/deficiência , Carnitina O-Palmitoiltransferase/genética , Erros Inatos do Metabolismo Lipídico/genética , Mutação , Adulto , Substituição de Aminoácidos , Povo Asiático , Criança , Pré-Escolar , Feminino , Genótipo , Heterozigoto , Humanos , MasculinoRESUMO
The cDNA sequence of human mitochondrial 3-oxoacyl-CoA thiolase was determined. The nucleotide sequence contains an open reading frame of 1191 base pairs and encodes an amino acid sequence of 397 residues which exhibits 86.6% homology with that of the rat enzyme. Northern blot analysis gave a single mRNA species of 1.6 kb in the human liver, fibroblasts and intercostal muscle.
Assuntos
Acetil-CoA C-Aciltransferase/genética , DNA Complementar/biossíntese , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Complementar/química , Humanos , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Fases de Leitura Aberta , RNA Mensageiro/análise , Homologia de Sequência de AminoácidosRESUMO
Holocarboxylase synthetase (HCS) deficiency is an inherited disease of biotin metabolism characterized by a unique pattern of organic aciduria, metabolic acidosis, and skin lesions. By analysis of five patients in four unrelated families, two mutations were identified: a transition from T to C which causes an amino-acid substitution of proline for leucine at position 237 (L237P) and a single deletion of guanine (delG1067) followed by premature termination. One patient was homozygous for the L237P mutation, three patients in two families were compound heterozygotes of the missense and deletion alleles, and the other patient was heterozygous for the L237P mutation. Inheritance was successfully demonstrated in all of the patients' families by a modified PCR followed by restriction enzyme digestion. The two mutations accounted for seven of eight mutant alleles, while neither mutation was detected in 108 normal healthy Japanese children (216 alleles). Transient expression in cultured fibroblasts from a patient showed that the L237P mutation was responsible for decreased HCS activity. These results suggest that the L237P and delG1067 mutations are frequent disease-causing mutations in Japanese patients with HCS deficiency. This PCR-based technique may therefore be useful for detecting mutations among Japanese patients.
Assuntos
Biotina/metabolismo , Carbono-Nitrogênio Ligases , Ligases/deficiência , Ligases/genética , Erros Inatos do Metabolismo/genética , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Primers do DNA , Feminino , Genótipo , Heterozigoto , Homozigoto , Humanos , Recém-Nascido , Japão , Masculino , Dados de Sequência Molecular , Linhagem , Mutação Puntual , Análise de Sequência , Deleção de Sequência , TransfecçãoRESUMO
OBJECTIVE: We examined the morphological changes induced by repeated endothelial denudation in coronary artery (CA), as well as functional changes in the endothelium-dependent and smooth muscle responses to various vasoactive agents during the process of intimal thickening. METHODS: We observed vascular responses in denuded and non-denuded portions of pig CA while being fed a normal diet (n = 11, N group) or 2% cholesterol diet (n = 25, C group) to intracoronary acetylcholine (ACh), 5-hydroxytryptamine (5-HT), substance P (SP), and isosorbide dinitrate (ISDN) with and without the nitric oxide synthesis inhibitor N omega-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg i.v.) over a period of 8 weeks. Balloon endothelial denudation of the left anterior descending CA was carried out every 2 weeks. RESULTS: In N group, maximum vasoconstriction was obtained with ACh 2 weeks after the first denudation [26 +/- 5% vs. 1 +/- 1% pre-denudation, p < 0.05]. L-NAME did not affect ACh-induced CA diameter changes. Thereafter, the response to ACh was attenuated by repeated denudation in N groups. However, the degree of 5-HT-induced CA narrowing at the denuded portion increased from 7 +/- 4% (0 week) to 88 +/- 8% (8 weeks) (p < 0.05). The changes resulted in severe myocardial ischaemia, and suggested that endothelium-dependent vasodilation was progressively attenuated while hyperreactivity of vascular smooth muscle simultaneously increased. Vasodilation induced by SP was attenuated somewhat, but ISDN-induced vasodilation was preserved. Although mild hypercholesterolaemia was induced in C group, the vascular responses to these vasoactive agents did not differ from those of N group. CONCLUSIONS: Repeated CA endothelial injury and regeneration induce the change of morphology and vascular reactivity in the denuded portion regardless of atherogenic diet. This study strongly suggests that intimal thickening caused by repeated endothelial injury and regeneration induces specific vascular responses to vasoactive agents. Moreover, it is also suggested that during the progression of intimal thickening, increased vascular smooth muscle contraction and decreased endothelium-dependent dilation appear in a stimulus-dependent manner, often leading to severe coronary vasoconstriction accompanied with definitive ECG ST change.
Assuntos
Vasos Coronários/patologia , Endotélio Vascular/lesões , Músculo Liso Vascular/efeitos dos fármacos , Vasoconstritores/farmacologia , Vasodilatadores/farmacologia , Acetilcolina/farmacologia , Animais , Vasos Coronários/fisiopatologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Inibidores Enzimáticos/farmacologia , Hipercolesterolemia/fisiopatologia , Dinitrato de Isossorbida/farmacologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Regeneração , Serotonina/farmacologia , Substância P/farmacologia , SuínosRESUMO
Hyperphosphorylated tau protein is known to be a major component of the paired helical filaments (PHFs) that accumulate in the brain of Alzheimer's patients. The kinase that phosphorylated Ser-208 and Ser-210 in PHF-tau had remained unknown. We used anti-pS208 and anti-pS210 antibodies and Western blots to confirm that the tau-tubulin kinase (TTK) phosphorylates tau at Ser-208 and at Ser-210. Using partial amino acid sequences of purified bovine brain TTK, a mouse cDNA of TTK was isolated and the sequence was determined. Its 963 bp coding region is composed of 320 amino acids and encodes a 36 kDa protein indistinguishable in size from authentic bovine brain TTK. Our immunoblot analysis demonstrated that TTK is ubiquitously distributed in the rat tissues, and that it is developmentally regulated in the rat brain.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Serina/metabolismo , Proteínas tau/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Especificidade de Anticorpos , Sequência de Bases , Western Blotting , Encéfalo/enzimologia , DNA Complementar/análise , Glutationa Transferase/genética , Quinase 3 da Glicogênio Sintase , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
During purification of tau protein kinase I and II from the bovine brain extract, a new tau protein kinase was detected and purified with phosphocellulose, gel filtration, S-Sepharose and AF-Heparin column chromatography. The molecular mass of the enzyme was determined to be 32 kDa by gel filtration and activity staining on SDS-PAGE. The enzyme is a Ser/Thr protein kinase phosphorylating tau, beta-tubulin, MAP2 and alpha-casein. Employing many synthetic peptides, the recognition site of this enzyme appears to be -SR-. The enzyme requires no second messenger and is inhibited with high concentration of heparin, but not by inhibitors of CKI. These results indicate that this enzyme, tau-tubulin kinase is novel and distinct from TPKI, II and CKI, II.
Assuntos
Encéfalo/enzimologia , Proteínas Serina-Treonina Quinases/isolamento & purificação , Proteínas Serina-Treonina Quinases/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas tau/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Caseínas/metabolismo , Bovinos , Cromatografia , Eletroforese em Gel de Poliacrilamida , Heparina/farmacologia , Histonas/metabolismo , Cinética , Proteínas Associadas aos Microtúbulos/metabolismo , Dados de Sequência Molecular , Peso Molecular , Peptídeos/química , Peptídeos/metabolismo , Fosforilação , Cloreto de Potássio/farmacologia , Proteínas Serina-Treonina Quinases/química , Especificidade por SubstratoRESUMO
To study the structure-activity relationships of brain injury-derived neurotrophic peptide (BINP), 12 analogs were synthesized by replacing each amino acid residue with Gly. BINP showed CD spectra typical of an alpha-helical conformation in TFE solution which mimics the membrane environment. In the alpha-helical conformation, BINP showed an amphiphilic profile. Neurotrophic activities of BINP and its analogs were estimated from the effects on supporting septal cholinergic neurons and on rescuing hippocampal neurons from injury caused by glutamate. Both assays showed that the residues on the hydrophobic side of the amphiphilic helix were essential for the neurotrophic activity.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/química , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/farmacologia , Neurônios/efeitos dos fármacos , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Animais , Lesões Encefálicas/metabolismo , Fator Neurotrófico Derivado do Encéfalo/análogos & derivados , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Dicroísmo Circular , Ácido Glutâmico/farmacologia , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Neurônios/citologia , Sistema Nervoso Parassimpático/citologia , Sistema Nervoso Parassimpático/efeitos dos fármacos , RatosRESUMO
In order to visualize the activity of the cAMP-dependent protein kinase (PKA) in living cells, we have constructed a new fluorescence PKA substrate by conjugating a fluorescence probe to a partial amino acid sequence of PKA regulatory domain II which contains a specific autophosphorylation site. The fluorescent peptide was cell-permeable and became phosphorylated when the intracellular cAMP concentration was increased, resulting in a decrease in its fluorescence intensity. In NG108-15 cells, PKA activity was localized to the cytosol around the nucleus. In cultured hippocampal neurons, addition of L-glutamate caused PKA activation associated with increase of the cellular cAMP.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/análise , Corantes Fluorescentes/metabolismo , Neurônios/enzimologia , Sequência de Aminoácidos , Animais , Permeabilidade da Membrana Celular , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citosol/enzimologia , Ativação Enzimática , Ácido Glutâmico/farmacologia , Hipocampo/citologia , Hipocampo/embriologia , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fosforilação , Fosfosserina/metabolismo , Ratos , Células Tumorais CultivadasRESUMO
As replacement of Thr(11) of omega-conotoxin MVIIC with Ala significantly reduced the affinity for both N- and P/Q-type calcium channels, we examined the effect of substitution at this position with other residues. Binding assays using rat cerebellar P2 membranes showed that the affinity is in the order of Leu>Val, aminobutyric acid, Thr>Asn&z.Gt;Ser, Ala, Asp, Phe, Tyr for N-type channels and Thr>Leu, Val, aminobutyric acid, Asn, Ser>Ala&z.Gt;Asp, Phe, Tyr for P/Q-type channels, suggesting that aliphatic amino acids with longer side chains are favorable for block of N-type channels. The effects of substitution were examined electrophysiologically in BHK cells expressing N-type Ca2+ channels. Inhibition of Ba2+ current by the analogs did not completely correlate with binding affinity, although binding to BHK cells was comparable to rat cerebellar membranes.
Assuntos
Alanina/química , Bloqueadores dos Canais de Cálcio/metabolismo , Canais de Cálcio Tipo N/metabolismo , Treonina/química , ômega-Conotoxinas/metabolismo , Sequência de Aminoácidos , Animais , Bário/metabolismo , Bloqueadores dos Canais de Cálcio/química , Canais de Cálcio Tipo N/química , Canais de Cálcio Tipo P/química , Canais de Cálcio Tipo P/metabolismo , Canais de Cálcio Tipo Q/química , Canais de Cálcio Tipo Q/metabolismo , Células Cultivadas , Cricetinae , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Ligação Proteica , Ratos , ômega-Conotoxinas/químicaRESUMO
Despite their high sequence homology, the peptide neurotoxins omega-conotoxin MVIIA and MVIIC selectively block N- and P/Q-type calcium channels, respectively. To study the recognition mechanism of calcium channel subtypes, two chimeric analogs of omega-conotoxin MVIIA and MVIIC were synthesized by exchanging their N- and C-terminal halves. Binding assay for both N- and P/Q-type calcium channels showed that amino acid residues restricted to the N-terminal half are important for the recognition of N-type channels, whereas essential residues for P/Q-type channel recognition are widely spread over the whole omega-conotoxin molecule.
Assuntos
Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/metabolismo , Canais de Cálcio/metabolismo , Fragmentos de Peptídeos/metabolismo , Peptídeos/metabolismo , ômega-Conotoxinas , Sequência de Aminoácidos , Animais , Sítios de Ligação , Membrana Celular/metabolismo , Cerebelo/metabolismo , Dicroísmo Circular , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Peptídeos/química , Conformação Proteica , Dobramento de Proteína , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
omega-Conotoxin MVIIC binds to P/Q-type calcium channels with high affinity and N-type channels with low affinity. To reveal the residues essential for subtype selectivity, we synthesized Ala-scanning analogs of MVIIC. Binding assays using rat cerebellar P(2) membranes suggested that Thr(11), Tyr(13) and Lys(2) are essential for binding to both N- and P/Q-type channels, whereas Lys(4) and Arg(22) are important for binding to P/Q-type channels. These results suggest that MVIIC interacts with P/Q-type channels via a large surface, in good agreement with previous observations using chimeric analogs.
Assuntos
Alanina/química , Bloqueadores dos Canais de Cálcio/metabolismo , Canais de Cálcio Tipo N/metabolismo , Canais de Cálcio Tipo P/metabolismo , Canais de Cálcio Tipo Q/metabolismo , ômega-Conotoxinas/metabolismo , Sequência de Aminoácidos , Animais , Bloqueadores dos Canais de Cálcio/síntese química , Bloqueadores dos Canais de Cálcio/química , Cerebelo/metabolismo , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Dissulfetos/química , Técnicas In Vitro , Membranas Intracelulares/fisiologia , Ativação do Canal Iônico , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Ratos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Relação Estrutura-Atividade , ômega-Conotoxinas/síntese química , ômega-Conotoxinas/químicaRESUMO
Brain injury-derived neurotrophic peptide is the fragmental 13-mer peptide of the novel neurotrophic factor which was extracted and purified from Sponge Gelform made of gelatin implanted at the mechanically-induced injury site in neonatal rat brains. Brain injury-derived neurotrophic peptide supports survival of septal cholinergic and mesencephalic dopaminergic neurons in culture, and rescues hippocampal neurons in culture from glutamate neurotoxicity. Here we studied the binding characteristics of brain injury-derived neurotrophic peptide to synaptosomes from normal adult rat brains and neurons in culture from neonatal rat brains. [125I]Asp-[Tyr11]-brain injury-derived neurotrophic peptide binding to rat brain synaptosomes was specific and saturable. Equilibrium binding studies revealed that [125I]Asp-[Tyr11]-brain injury-derived neurotrophic peptide bound to 1.1 pmol/mg protein with a Kd (dissociation constant) of 0.17 microM in hippocampal synaptosomes and to 2.0 pmol/mg protein with a Kd of 0.38 microM in septal synaptosomes. [125I]Asp-[Tyr11]-brain injury-derived neurotrophic peptide could bind to a subpopulation of hippocampal neurons in culture from embryonic rat brains. Affinity cross-linking with the carboxyl-reactive cross-linking reagent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide-HCl and [125I]Asp-[Tyr11]-brain injury-derived neurotrophic peptide produced radiolabeled bands corresponding to 100,000, 50,000 and 40,000 mol. wt molecules on hippocampal neurons in culture. These results suggest that the 13-mer sequence of brain injury-derived neurotrophic peptide plays a crucial role in expressing the neurotrophic properties of the factor.
Assuntos
Proteínas do Tecido Nervoso/análise , Neurônios/química , Sinaptossomos/química , Animais , Autorradiografia , Sítios de Ligação/fisiologia , Morte Celular/fisiologia , Células Cultivadas , Reagentes de Ligações Cruzadas/metabolismo , Hipocampo/química , Hipocampo/citologia , Radioisótopos do Iodo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Ratos , Núcleos Septais/química , Núcleos Septais/citologia , Sinaptossomos/metabolismoRESUMO
Cell surface glycoconjugates are thought to mediate cell-cell recognition and to play roles in neuronal development and functions. We demonstrated here that exposure of neuronal cells to nanomolar levels of glyco-chains with an N-acetylgalactosamine (GalNAc) residue at the non-reducing termini (GalNAc-S) such as GalNAcbeta4(Neu5Acalpha3)Galbeta4GlcCer (GM2) ganglioside, its oligosaccharide portion, GalNAcbeta4Galbeta4GlcCer (Gg(3)) Cer, GalNAcalpha3GalNAcbeta3Galalpha4Galbeta4GlcCer (Gb(5)) Cer (Forssman hapten) and alpha1-4 linked oligomers of GalNAc, induced a rapid and transient activation of cAMP-dependent protein kinase (PKA) in subplasmalemma. The treatment was accompanied by peripheral actin polymerization and filopodia formation in NG108-15 cells and primary cultured hippocampal neurons, but not in glial cells. A cAMP-dependent protein kinase (PKA) selective inhibitor and an adenylate cyclase inhibitor blocked both PKA activation and the subsequent filopodia formation. A small GTPase cdc42 was a potential downstream target of GalNAc-S-activated PKA. These results suggest that extracellular GalNAc-S serve as potential regulators of the filopodia formation in neuronal cells by triggering the activation of PKA followed by cdc42 up-regulation via a cell surface receptor-like component. Filopodia formation induced by GalNAc-S may have a physiological relevance because long-term exposure to GalNAc-S enhanced F-actin-rich dendrite generation of primary cultured hippocampal neurons, and PKA-dependent dendritic outgrowth and branch formation of primary cultured cerebellar Purkinje neurons, in which actin isoforms were localized to motile structures in dendrites. These findings provide evidence for a novel GalNAc/PKA-signaling cascade in regulating some neuronal maturation.
Assuntos
Acetilgalactosamina/farmacologia , Actinas/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Hipocampo/enzimologia , Neurônios/enzimologia , Animais , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Líquido Extracelular/efeitos dos fármacos , Líquido Extracelular/enzimologia , Hipocampo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Pseudópodes/efeitos dos fármacos , Pseudópodes/enzimologia , RatosRESUMO
We have studied strain-relaxation processes in InAs heteroepitaxy on GaAs(111)A using rocking-curve analysis of reflection high-energy electron diffraction. Strain relaxation in the direction parallel to the surface occurs at approximately 1.5 bilayers (BL) thickness. On the other hand, the lattice constant in the direction normal to the surface remains almost unchanged below approximately 3 BL thickness and is estimated to be approximately 3.3 A. This value, slightly larger than that of bulk GaAs (3.26 A), does not quite reach the value predicted by classical elastic theory, 3.64 A. The present result has been supported by the first-principles total-energy calculations.
RESUMO
Our aim was to visualize the dynamic features of Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity. In order to do so, we synthesized a new reagent by conjugating a fluoroprobe, 6-acryloyl-2-dimethylaminonaphthalene (acrylodan), to syntide 2, a specific peptide substrate for CaMKII. In cell-free conditions, the conjugate was found to be an effective indicator of calmodulin activation by Ca2+ and the subsequent activation of CaMKII. The reagent is cell-permeable and can stain living cells when bath-applied. Using this technique we were able to obtain fluorescence images of stained cells and analyse the dynamic features of CaMKII inside the cells by means of image processing. Regional heterogeneity of CaMKII activation in cultured hippocampal neurones was seen following L-glutamate administration.