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In the clinical investigation of a family with debilitating centrofacial pruritus by exome sequencing, we have observed a clear segregation of the TRPM3 variant outlined, which is highly suggestive of a causal relationship.
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Dermatoses Faciais/genética , Prurido/genética , Canais de Cátion TRPM/genética , Feminino , Genes Dominantes , Variação Genética , Humanos , Pessoa de Meia-Idade , Linhagem , Sequenciamento do ExomaRESUMO
Autoimmune progesterone dermatitis (APD) is a rare skin disorder with varying presentations, resulting from hypersensitivity to endogenous progesterone during the luteal phase of the menstrual cycle. The diagnosis has been traditionally confirmed with intradermal progesterone testing (IPT) or intramuscular challenge with progesterone or its derivatives. We present a case of a 31-year-old woman with suspected APD who underwent IPT to progesterone. The patient's cyclical symptoms, positive skin reaction and symptoms following IPT were sufficient to make a diagnosis of APD. However, we also tested 10 healthy female controls without symptoms of APD, and found that 9 of these also developed positive skin reactions to intradermal progesterone at 15 min, 24 and 48 h, albeit to a lesser extent. Therefore, these results raise doubts about the validity of using IPT to make a diagnosis of APD. Further research on appropriate testing is needed.
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Doenças Autoimunes/diagnóstico , Dermatite/diagnóstico , Testes Intradérmicos/métodos , Progesterona/efeitos adversos , Dermatopatias/patologia , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Hipersensibilidade/etiologia , Hipersensibilidade/imunologia , Fase Luteal/imunologia , Ciclo Menstrual/imunologia , Avaliação de Resultados em Cuidados de Saúde , Progesterona/imunologia , Dermatopatias/imunologiaRESUMO
STUDY QUESTION: What is the likelihood of identifying genetic or endocrine abnormalities in a group of boys with 46, XY who present to a specialist clinic with a suspected disorder of sex development (DSD)? SUMMARY ANSWER: An endocrine abnormality of the gonadal axis may be present in a quarter of cases and copy number variants (CNVs) or single gene variants may be present in about half of the cases. WHAT IS KNOWN ALREADY: Evaluation of 46, XY DSD requires a combination of endocrine and genetic tests but the prevalence of these abnormalities in a sufficiently large group of boys presenting to one specialist multidisciplinary service is unclear. STUDY, DESIGN, SIZE, DURATION: This study was a retrospective review of investigations performed on 122 boys. PARTICIPANTS/MATERIALS, SETTING, METHODS: All boys who attended the Glasgow DSD clinic, between 2010 and 2015 were included in the study. The median external masculinization score (EMS) of this group was 9 (range 1-11). Details of phenotype, endocrine and genetic investigations were obtained from case records. MAIN RESULTS AND THE ROLE OF CHANCE: An endocrine abnormality of gonadal function was present in 28 (23%) with a median EMS of 8.3 (1-10.5) whilst the median EMS of boys with normal endocrine investigations was 9 (1.5-11) (P = 0.03). Endocrine abnormalities included a disorder of gonadal development in 19 (16%), LH deficiency in 5 (4%) and a disorder of androgen synthesis in 4 (3%) boys. Of 43 cases who had array-comparative genomic hybridization (array-CGH), CNVs were reported in 13 (30%) with a median EMS of 8.5 (1.5-11). Candidate gene analysis using a limited seven-gene panel in 64 boys identified variants in 9 (14%) with a median EMS of 8 (1-9). Of the 21 boys with a genetic abnormality, 11 (52%) had normal endocrine investigations. LIMITATIONS, REASONS FOR CAUTION: A selection bias for performing array-CGH in cases with multiple congenital malformations may have led to a high yield of CNVs. It is also possible that the yield of single gene variants may have been higher than reported if the investigators had used a more extended gene panel. WIDER IMPLICATIONS OF THE FINDINGS: The lack of a clear association between the extent of under-masculinization and presence of endocrine and genetic abnormalities suggests a role for parallel endocrine and genetic investigations in cases of suspected XY DSD. STUDY FUNDING/COMPETING INTEREST(S): RN was supported by the James Paterson Bursary and the Glasgow Children's Hospital Charity Summer Scholarship. SFA, RM and EST are supported by a Scottish Executive Health Department grant 74250/1 for the Scottish Genomes Partnership. EST is also supported by MRC/EPSRC Molecular Pathology Node and Wellcome Trust ISSF funding. There are no conflicts of interest. TRIAL REGISTRATION NUMBER: None.
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Transtorno 46,XY do Desenvolvimento Sexual/diagnóstico , Testes Genéticos/métodos , Hormônios Esteroides Gonadais/sangue , Biomarcadores/sangue , Criança , Pré-Escolar , Hibridização Genômica Comparativa , Transtorno 46,XY do Desenvolvimento Sexual/sangue , Transtorno 46,XY do Desenvolvimento Sexual/epidemiologia , Transtorno 46,XY do Desenvolvimento Sexual/genética , Genótipo , Humanos , Lactente , Masculino , Fenótipo , Prevalência , Estudos RetrospectivosRESUMO
ß-Amyloid precursor protein (APP) and its cleaved products are strongly implicated in Alzheimer's disease (AD). Endosomes are highly active APP processing sites, and endosome anomalies associated with upregulated expression of early endosomal regulator, rab5, are the earliest known disease-specific neuronal response in AD. Here, we show that the rab5 effector APPL1 (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif) mediates rab5 overactivation in Down syndrome (DS) and AD, which is caused by elevated levels of the ß-cleaved carboxy-terminal fragment of APP (ßCTF). ßCTF recruits APPL1 to rab5 endosomes, where it stabilizes active GTP-rab5, leading to pathologically accelerated endocytosis, endosome swelling and selectively impaired axonal transport of rab5 endosomes. In DS fibroblasts, APPL1 knockdown corrects these endosomal anomalies. ßCTF levels are also elevated in AD brain, which is accompanied by abnormally high recruitment of APPL1 to rab5 endosomes as seen in DS fibroblasts. These studies indicate that persistent rab5 overactivation through ßCTF-APPL1 interactions constitutes a novel APP-dependent pathogenic pathway in AD.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Síndrome de Down/metabolismo , Endossomos/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Idoso , Precursor de Proteína beta-Amiloide/genética , Animais , Encéfalo/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Fibroblastos/metabolismo , Técnicas de Silenciamento de Genes , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Neurônios/metabolismoRESUMO
Synaptic roles for neurofilament (NF) proteins have rarely been considered. Here, we establish all four NF subunits as integral resident proteins of synapses. Compared with the population in axons, NF subunits isolated from synapses have distinctive stoichiometry and phosphorylation state, and respond differently to perturbations in vivo. Completely eliminating NF proteins from brain by genetically deleting three subunits (α-internexin, NFH and NFL) markedly depresses hippocampal long-term potentiation induction without detectably altering synapse morphology. Deletion of NFM in mice, but not the deletion of any other NF subunit, amplifies dopamine D1-receptor-mediated motor responses to cocaine while redistributing postsynaptic D1-receptors from endosomes to plasma membrane, consistent with a specific modulatory role of NFM in D1-receptor recycling. These results identify a distinct pool of synaptic NF subunits and establish their key role in neurotransmission in vivo, suggesting potential novel influences of NF proteins in psychiatric as well as neurological states.
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Encéfalo/fisiologia , Atividade Motora/fisiologia , Proteínas de Neurofilamentos/metabolismo , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Animais , Axônios/efeitos dos fármacos , Axônios/fisiologia , Encéfalo/efeitos dos fármacos , Cocaína/farmacologia , Inibidores da Captação de Dopamina/farmacologia , Potenciação de Longa Duração/fisiologia , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Proteínas de Neurofilamentos/genética , Receptores de Dopamina D1/metabolismo , Sinapses/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacosRESUMO
AIMS: To investigate the pattern of changes in HbA1c in people with Type 1 diabetes managed by long-term Continuous subcutaneous insulin infusion. METHODS: We studied HbA1c changes using computerized clinic records in 35 adult people with Type 1 diabetes and an elevated HbA1c (≥ 64 mmol/mol, 8.0%) on multiple daily insulin injections, who were then switched to continuous subcutaneous insulin infusion for at least 5 years. RESULTS: We identified three subgroups with similar baseline HbA1c but different long-term responses to pump therapy: group A--those with improvement followed by deterioration (57%); group B--those with improvement that was sustained throughout the 5 years (31%); and group C-those where HbA1c did not change significantly from baseline (12%). The patients in group C had a higher BMI: 31.0 ± 5.2 vs. 25.9 ± 3.3 vs. 25.2 ± 3.1 kg/m² (group C vs. group A and group B; P = 0.02). CONCLUSIONS: Improved glycaemic control with continuous subcutaneous insulin infusion was maintained over 5 years by 88% of people with Type 1 diabetes in this study, but there were variations in the long-term efficacy, with some people improving and worsening, others maintaining strict control and a few subcutaneous insulin infusion 'non-responders'.
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Diabetes Mellitus Tipo 1/tratamento farmacológico , Hiperglicemia/prevenção & controle , Hipoglicemia/prevenção & controle , Hipoglicemiantes/administração & dosagem , Sistemas de Infusão de Insulina , Insulina/administração & dosagem , Qualidade da Assistência à Saúde , Adulto , Índice de Massa Corporal , Estudos de Coortes , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/complicações , Feminino , Seguimentos , Hemoglobinas Glicadas/análise , Humanos , Hipoglicemiantes/efeitos adversos , Hipoglicemiantes/uso terapêutico , Infusões Subcutâneas , Insulina/efeitos adversos , Insulina/uso terapêutico , Sistemas de Infusão de Insulina/efeitos adversos , Resistência à Insulina , Londres , Masculino , Adesão à Medicação , Pessoa de Meia-Idade , Ambulatório Hospitalar , Sobrepeso/complicaçõesRESUMO
Interfacial sliding speed and contact pressure between the sub-units of particulate soft matter assemblies can vary dramatically across systems and with dynamic conditions. By extension, frictional interactions between particles may play a key role in their assembly, global configuration, collective motion, and bulk material properties. For example, in tightly packed assemblies of microgels - colloidal microspheres made of hydrogel - particle stiffness controls the fragility of the glassy state formed by the particles. The interplay between particle stiffness and shear stress is likely mediated by particle-particle normal forces, highlighting the potential role of hydrogel-hydrogel friction. Here we study friction at a twinned "Gemini" interface between hydrogels. We construct a lubrication curve that spans four orders of magnitude in sliding speed, and find qualitatively different behaviour from traditional lubrication of engineering material surfaces; fundamentally different types of lubrication occur at the hydrogel Gemini interface. We also explore the role played by polymer solubility and hydrogel-hydrogel adhesion in hydrogel friction. We find that polymer network elasticity, mesh size, and single-chain relaxation times can describe friction at the gel-gel interface, including a transition between lubrication regimes with varying sliding speed.
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BACKGROUND: There is emerging literature regarding the efficacy of trauma-focussed writing therapies (TF-WTs) for posttraumatic stress. Such therapies have the potential to reduce posttraumatic stress symptoms (PTSS) in a brief time frame and can be delivered remotely. There remains a need for further research assessing the efficacy of different types of TF-WTs, as well comparing them to alternative control conditions not previously assessed. The present study assessed two TF-WTs that had differing writing instructions in comparison to an intervention that involved writing about positive experiences. METHODS: Adult community participants (n = 83) with subthreshold or clinical PTSD symptoms were randomized to one of three conditions (two of which involved trauma-focussed writing, and the other involved writing about positive experiences). All conditions involved three weekly telehealth-delivered writing appointments. Outcomes were measured using the PTSD Checklist (PCL-5) and the Depression, Anxiety and Stress Scales (DASS-21), and were evaluated at baseline, one-week post-intervention, and five-weeks post-intervention. This trial was registered with the Australian and New Zealand Clinical Trials Registry (ANZCTR Protocol 12620001065987). RESULTS: There was no evidence that the two TF-WTs were more efficacious in reducing PTSS or producing clinically meaningful change in comparison to positive experiences writing. Instead, a significant reduction from baseline to follow-up in PTSS, depression, anxiety and stress was observed in all three conditions. LIMITATIONS: The results should be interpreted with consideration of the modest sample size and absence of longer-term follow-up. CONCLUSIONS: Three-session trauma-focussed writing delivered via telehealth may not be superior to writing about positive experiences.
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Transtornos de Estresse Pós-Traumáticos , Redação , Humanos , Transtornos de Estresse Pós-Traumáticos/terapia , Transtornos de Estresse Pós-Traumáticos/psicologia , Feminino , Masculino , Adulto , Pessoa de Meia-Idade , Resultado do Tratamento , Psicoterapia Breve/métodos , Telemedicina , Adulto Jovem , Escalas de Graduação PsiquiátricaAssuntos
Higienizadores de Mão , Autorrelato , Estudos Transversais , Eczema , Pessoal de Saúde , HumanosRESUMO
New studies provide further evidence that the neuronal cytoskeleton is the product of a dynamic interplay between axonal transport processes and locally regulated assembly mechanisms. These data confirm that the axonal cytoskeleton in mammalian systems is largely stationary and is maintained by a smaller pool of moving subunits or polymers. Slow axonal transport in certain lower species, however, may exhibit quite different features.
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Transporte Axonal/fisiologia , Axônios/fisiologia , Actinas/fisiologia , Animais , Transporte Biológico , Citoesqueleto/fisiologia , Filamentos Intermediários/fisiologia , Proteínas dos Microtúbulos/fisiologia , Proteínas de Neurofilamentos/fisiologia , FosforilaçãoRESUMO
Once presumed to be relatively uniform, the axonal cytoskeleton can vary markedly in size and composition along its length. New studies emphasize the interactiveness of neurofilaments and identify a family of cytoskeletal proteins that may cross-link the various cytoskeletal polymers of the axon, and anchor this network to the membrane skeleton. These and other findings support a model of the axonal cytoskeleton as a stationary but dynamic structure. Current evidence continues to support the possibility that axonally transported polymers/oligomers and/or monomers may serve as precursors to the cytoskeleton in different situations. Although the motors for slow transport of cytoskeletal proteins remain elusive, possible candidates are emerging.
Assuntos
Transporte Axonal/fisiologia , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Humanos , Microtúbulos/metabolismoRESUMO
OBJECTIVES: Hand sanitisers are urgently needed in the time of COVID-19, and as a result of shortages, some people have resorted to making their own formulations, including the repurposing of distilleries. We wish to highlight the importance of those producing hand sanitisers to avoid methylated spirits containing methanol and to follow WHO recommended formulations. METHODS: We explore and discuss reports of methanol toxicity through ingestion and transdermal absorption. We discuss the WHO formulations and explain the rationale behind the chosen ingredients. SHORT CONCLUSION: We advise those producing hand sanitisers to follow WHO recommended formulations, and advise those producing hand sanitisers using methylated spirits, to avoid formulations which contain methanol.
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Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/prevenção & controle , Desinfetantes/farmacologia , Etanol/farmacologia , Metanol/farmacologia , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Betacoronavirus/fisiologia , COVID-19 , Infecções por Coronavirus/virologia , Desinfetantes/química , Desinfetantes/normas , Desinfetantes/toxicidade , Composição de Medicamentos , Etanol/química , Desinfecção das Mãos/instrumentação , Humanos , Metanol/química , Metanol/toxicidade , Pneumonia Viral/virologia , SARS-CoV-2 , Organização Mundial da SaúdeRESUMO
The 200-kD subunit of neurofilaments (NF-H) functions as a cross-bridge between neurofilaments and the neuronal cytoskeleton. In this study, four phosphorylated NF-H variants were identified as major constituents of axons from a single neuron type, the retinal ganglion cell, and were shown to have characteristics with different functional implications. We resolved four major Coomassie Blue-stained proteins with apparent molecular masses of 197, 200, 205, and 210 kD on high resolution one-dimensional SDS-polyacrylamide gels of mouse optic axons (optic nerve and optic tract). Proteins with the same electrophoretic mobilities were radiolabeled within retinal ganglion cells in vivo after injecting mice intravitreally with [35S]methionine or [3H]proline. Extraction of the radiolabeled protein fraction with 1% Triton X-100 distinguished four insoluble polypeptides (P197, P200, P205, P210) with expected characteristics of NF-H from two soluble neuronal polypeptides (S197, S200) with few properties of neurofilament proteins. The four Triton-insoluble polypeptides displayed greater than 90% structural homology by two-dimensional alpha-chymotryptic iodopeptide map analysis and cross-reacted with four different monoclonal and polyclonal antibodies to NF-H by immunoblot analysis. Each of these four polypeptides advanced along axons primarily in the Group V (SCa) phase of axoplasmic transport. By contrast, the two Triton-soluble polypeptides displayed only a minor degree of alpha-chymotryptic peptide homology with the Triton-insoluble NF-H forms, did not cross-react with NF-H antibodies, and moved primarily in the Group IV (SCb) wave of axoplasmic transport. The four NF-H variants were generated by phosphorylation of a single polypeptide. Each of these polypeptides incorporated 32P when retinal ganglion cells were radiolabeled in vivo with [32P]orthophosphate and each cross-reacted with monoclonal antibodies specifically directed against phosphorylated epitopes on NF-H. When dephosphorylated in vitro with alkaline phosphatase, the four variants disappeared, giving rise to a single polypeptide with the same apparent molecular mass (160 kD) as newly synthesized, unmodified NF-H. The NF-H variants distributed differently along optic axons. P197 predominated at proximal axonal levels; P200 displayed a relatively uniform distribution; and P205 and P210 became increasingly prominent at more distal axonal levels, paralleling the distribution of the stationary neurofilament network.(ABSTRACT TRUNCATED AT 400 WORDS)
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Axônios/metabolismo , Citoesqueleto/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Filamentos Intermediários/metabolismo , Neurônios/metabolismo , Retina/metabolismo , Células Ganglionares da Retina/metabolismo , Animais , Autorradiografia , Axônios/ultraestrutura , Densitometria , Eletroforese em Gel de Poliacrilamida , Feminino , Immunoblotting , Proteínas de Filamentos Intermediários/análise , Filamentos Intermediários/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peso Molecular , Proteínas de Neurofilamentos , Neurônios/ultraestrutura , Mapeamento de Peptídeos , Fosforilação , Células Ganglionares da Retina/ultraestruturaRESUMO
We have studied the fate of neurofilament proteins (NFPs) in mouse retinal ganglion cell (RGC) neurons from 1 to 180 d after synthesis and examined the proximal-to-distal distribution of the newly synthesized 70-, 140-, and 200-kD subunits along RGC axons relative to the distribution of neurofilaments. Improved methodology for intravitreal delivery of [3H]proline enabled us to quantitate changes in the accumulation and subsequent decline of radiolabeled NFP subunits at various postinjection intervals and, for the first time, to estimate the steady state levels of NFPs in different pools within axons. Two pools of newly synthesized triplet NFPs were distinguished based on their kinetics of disappearance from a 9-mm "axonal window" comprising the optic nerve and tract and their temporal-spatial distribution pattern along axons. The first pool disappeared exponentially between 17 and 45 d after injection with a half-life of 20 d. Its radiolabeled wavefront advanced along axons at 0.5-0.7 mm/d before reaching the distal end of the axonal window at 17 d, indicating that this loss represented the exit of neurofilament proteins composing the slowest phase of axoplasmic transport (SCa or group V) from axons. About 32% of the total pool of radiolabeled neurofilament proteins, however, remained in axons after 45 d and disappeared exponentially at a much slower rate (t 1/2 = 55 d). This second NFP pool assumed a nonuniform distribution along axons that was characterized proximally to distally by a 2.5-fold gradient of increasing radioactivity. This distribution pattern did not change between 45 and 180 d indicating that neurofilament proteins in the second pool constitute a relatively stationary structure in axons. Based on the relative radioactivities and residence time (or turnover) of each neurofilament pool in axons, we estimate that, in the steady state, more neurofilament proteins in mouse RGC axons may be stationary than are undergoing continuous slow axoplasmic transport. This conclusion was supported by biochemical analyses of total NFP content and by electron microscopic morphometric studies of neurofilament distribution along RGC axons. The 70-, 140-, and 200-kD subunits displayed a 2.5-fold proximal to distal gradient of increasing content along RGC axons. Neurofilaments were more numerous at distal axonal levels, paralleling the increased content of NFP.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Axônios/ultraestrutura , Citoesqueleto/ultraestrutura , Proteínas de Filamentos Intermediários/biossíntese , Filamentos Intermediários/ultraestrutura , Animais , Transporte Axonal , Feminino , Masculino , Camundongos , Microtúbulos/ultraestrutura , Células Ganglionares da Retina/ultraestrutura , Fatores de Tempo , Vias Visuais/ultraestruturaRESUMO
Microtubule-associated proteins (MAPs) in neurons establish functional associations with microtubules, sometimes at considerable distances from their site of synthesis. In this study we identified MAP 1A in mouse retinal ganglion cells and characterized for the first time its in vivo dynamics in relation to axonally transported tubulin. A soluble 340-kD polypeptide was strongly radiolabeled in ganglion cells after intravitreal injection of [35S]methionine or [3H]proline. This polypeptide was identified as MAP 1A on the basis of its co-migration on SDS gels with MAP 1A from brain microtubules; its co-assembly with microtubules in the presence of taxol or during cycles of assembly-disassembly; and its cross-reaction with well-characterized antibodies against MAP 1A in immunoblotting and immunoprecipitation assays. Glial cells of the optic nerve synthesized considerably less MAP 1A than neurons. The axoplasmic transport of MAP 1A differed from that of tubulin. Using two separate methods, we observed that MAP 1A advanced along optic axons at a rate of 1.0-1.2 mm/d, a rate typical of the Group IV (SCb) phase of transport, while tubulin moved 0.1-0.2 mm/d, a group V (SCa) transport rate. At least 13% of the newly synthesized MAP 1A entering optic axons was incorporated uniformly along axons into stationary axonal structures. The half-residence time of stationary MAP 1A in axons (55-60 d) was 4.6 times longer than that of MAP 1A moving in Group IV, indicating that at least 44% of the total MAP 1A in axons is stationary. These results demonstrate that cytoskeletal proteins that become functionally associated with each other in axons may be delivered to these sites at different transport rates. Stable associations between axonal constituents moving at different velocities could develop when these elements leave the transport vector and incorporate into the stationary cytoskeleton.
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Proteínas Associadas aos Microtúbulos/metabolismo , Retina/metabolismo , Células Ganglionares da Retina/metabolismo , Animais , Transporte Axonal/fisiologia , Axônios/metabolismo , Axônios/fisiologia , Transporte Biológico , Citoesqueleto/metabolismo , Citoesqueleto/fisiologia , Imuno-Histoquímica , Camundongos , Proteínas Associadas aos Microtúbulos/farmacocinética , Proteínas Associadas aos Microtúbulos/fisiologia , Microtúbulos/metabolismo , Microtúbulos/fisiologia , Células Ganglionares da Retina/fisiologia , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/farmacocinética , Tubulina (Proteína)/fisiologiaRESUMO
The possibility that proteins are modified during axoplasmic transport in central nervous system axons was examined by analyzing neurofilament proteins (200,000, 140,000, and 70,000 mol wt) along the mouse primary optic pathway (optic nerve and optic tract). The major neurofilament proteins (NFPs) exhibited considerable microheterogeneity. At least three forms of the " 140,000" neurofilament protein differing in molecular weight by SDS PAGE (140,000-145,000 mol wt) were identified. The "140,000" proteins, and their counterparts in purified neurofilament preparations, displayed similar isoelectric points and the same peptide maps. The "140,000" NFPs exhibited regional heterogeneity when consecutive segments of the optic pathway were separately examined on polyacrylamide gels. Two major species (145,000 and 140,000 mol wt) were present along the entire length of the optic pathway. The third protein (143,000 mol wt) was absent proximally but became increasingly prominent in distal segments. After intravitreal injection of [(3)H]proline, newly synthesized radiolabeled proteins in the "140,000" mol wt region entered proximal mouse retinal ganglion cell (RGC) axons as two major species corresponding to the 145,000 and 14,000 mol wt NFPs observed on stained gels. When transported NFPs reached more distal axonal regions (30 d postinjection or longer), a 143,000 mol wt protein appeared that was similar in isoelectric point and peptide map to the 145,000 and 140,000 mol wt species. The results suggest that (a) the composition of CNS neurofilaments, particularly the "140,000" component, is more complex than previously recognized, that (b) retinal ganglion cell axons display regional differentiation with respect to these cytoskeletal proteins, and that (c) structural heterogeneity of "140,000" NFPs arises, at least in part, from posttranslational modification during axoplasmic transport. When excised but intact optic pathways were incubated in vitro at pH 7.4, a 143,000 NFP was rapidly formed by a calcium-dependent enzymatic process active at endogenous calcium levels. Changes in major proteins other than those in the 145,000-140,000 mol wt region were minimal. In optic pathways from mice injected intravitreally with L-[(3)H]proline, tritiated 143,000 mol wt NFP formed rapidly in vitro if radioactively labeled NFPs were present in distal RGC axonal regions (31 d postinjection). By contrast, no 143,000 mol wt NFP was generated if radioactively labeled NFPs were present proximally in RGC axons (6 d postinjection). The enzymatic process that generates 143,000 mol wt NFP in vitro, therefore, appears to have a nonuniform distribution along the RGC axons. The foregoing results and other observations, including the accompanying report (J. Cell Biol., 1982, 94:159-164), imply that CNS axons may be regionally specialized with respect to structure and function.
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Transporte Axonal , Citoesqueleto/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Axônios/metabolismo , Cálcio/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peso Molecular , Vias Neurais/metabolismo , Nervo Óptico/metabolismo , Fragmentos de Peptídeos/análise , Biossíntese de Proteínas , Retina/metabolismoRESUMO
Tubulin proteins in mouse retinal ganglion cell (RGC) neurons were analyzed to determine whether they undergo posttranslational processing during axoplasmic transport. Alpha- and beta-tubulin comprised heterogeneous proteins in the primary optic pathway (optic nerve and optic tract) when examined by two-dimensional (2D) PAGE. In addition, however, alpha-tubulin exhibited regional heterogeneity when consecutive 1.1-mm segments of the optic pathway were analyzed separately. In proximal segments, alpha-tubulin consisted of two predominant proteins separable by isoelectric point and several less abundant species. In more distal segments, these predominant proteins decreased progressively and the alpha-tubulin region of the gel was represented by less abundant multiple forms only; beta-tubulin region of the gel was represented by less abundant multiple forms only; beta-tubulin was the same in all segments. After intravitreal injection of [3H]proline to mice, radiolabeled alpha- and beta-tubulin heteroproteins were conveyed together at a rate of 0.1-0.2 mm/d in the slowest phase of axoplasmic transport. At 45 d postinjection, the distribution of radiolabeled heterogeneous forms a alpha- and beta-tubulin in consecutive segments of optic pathway resembled the distribution of unlabeled proteins by 2D PAGE, indicating that regional heterogeneity of tubulin arises during axonal transport. Peptide mapping studies demonstrated that the progressive alteration of alpha-tubulin revealed by PAGE analysis cannot be explained by contamination of the alpha-tubulin region by other proteins on gels. The results are consistent with the posttranslational processing of alpha-tubulin during axoplasmic transport. These observations, along with the accompanying report (J. Cell Biol., 1982, 94:150-158), provide additional evidence that CNS axons may be regionally specialized.