RESUMO
OBJECTIVE: To explore the mechanism of Xianglian Huazhuo formula (, XLHZ) blocking the development of chronic atrophic gastritis (CAG) to gastric cancer (GC) through bioinformatics analysis and in vitro. METHODS: Pathological morphology of gastric mucosa of rats were observed. High-throughput sequencing was used to analyze the miRNA expression profile of gastric mucosa. The miRanda, miRDB and miRWalk databases were used to predict the differential target genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed for differential target genes. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to verify the differentially expressed miRNAs and target genes. Western blot, EdU, wound healing and flow cytometry were used to observe the effect of XLHZ on epithelial-mesenchymal transition (EMT) markers, proliferation, migration, apoptosis and cell cycle of CAG cells in vitro. RESULTS: A total of five differentially expressed miRNAs and four differential target genes were screened in this study. GO analysis showed that the target genes were enriched in regulation of neuron development, regulation of transcription factor activity and regulation of RNA polymerase. KEGG pathways database differences in gene enrichment of target genes in the Wnt signaling pathway, Phospholipase D signaling pathway and mitogen-activated protein kinase signaling pathway. qRT-PCR confirmed that miRNAs and its target genes were consistent with the screening results. In vitro, our study revealed that XLHZ could increase the expression of E-cadherin, decrease the expression of transforming growth factor ß1, vimentin and ß-catenin, inhibite the proliferation and migration of CAG cells, cause cell cycle arrest at G0/G1 and G2/M phase, induce the apoptosis of CAG cells, and prevent the progression of CAG to GC. CONCLUSION: This study provided a new idea for the mechanism of blocking the progression of CAG to GC by XLHZ, which may be related to the expression of miR-20a-3p, miR-320-3p, miR-34b-5p, miR-483-3p and miR-883-3p and their target genes transferrin receptor, nuclear receptor subfamily 4 member 2, delta like canonical Notch ligand 1 and a kinase anchor protein 12 in CAG. In the future, we will continue to investigate the linkage between the active ingredients of XLHZ and the relevant miRNAs and their target genes, so as to provide more sufficient experimental basis for clinically effective prevention of CAG to GC.
Assuntos
Medicamentos de Ervas Chinesas , Gastrite Atrófica , Sequenciamento de Nucleotídeos em Larga Escala , MicroRNAs , Neoplasias Gástricas , Gastrite Atrófica/genética , Gastrite Atrófica/metabolismo , Ratos , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Animais , Humanos , Masculino , Medicamentos de Ervas Chinesas/farmacologia , Proliferação de Células , Apoptose/genética , Transição Epitelial-Mesenquimal/genética , Ratos Sprague-Dawley , Mucosa Gástrica/metabolismo , Movimento CelularRESUMO
Successful peripheral nerve regeneration and functional recovery require the reestablishment of the neuron-Schwann cell relationship in the regenerating rat sciatic nerve, neurons differentially regulate Schwann cell genes. The message for the low-affinity NGF receptor, p75NGFR, is induced in Schwann cells distal to the injury and is repressed as regenerating axons make contact with these cells. The inverse is true for mRNA of the myelin gene P0; expression decreases distal to injury and increases as new axons contact Schwann cells and a program of myelination is initiated. Using an in vitro co-culture paradigm in which primary neurons and adult Schwann cells are separated by a microporous membrane, we show that axon contact is not an absolute requirement for neuronal regulation of Schwann cell genes. In this system neurons but not other cell types, repress the expression of Schwann cell p75NGFR while inducing the expression of the POU domain transcription factor, suppressed cAMP inducible POU, and myelin P0. These results demonstrate that regenerating axons can direct the Schwann cell genetic program from a distance through diffusible molecules.
Assuntos
Comunicação Celular , Regulação da Expressão Gênica , Substâncias de Crescimento/farmacologia , Neurônios/fisiologia , Receptores de Fator de Crescimento Neural/genética , Células de Schwann/fisiologia , Animais , Células Cultivadas , Embrião de Galinha , Meios de Cultura , Técnicas de Cultura/métodos , Difusão , Gânglios Espinais/fisiologia , Gânglios Espinais/ultraestrutura , Masculino , Proteínas da Mielina/genética , Proteínas da Mielina/metabolismo , Regeneração Nervosa/fisiologia , Neurônios/ultraestrutura , Fator 6 de Transcrição de Octâmero , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Receptores de Fator de Crescimento Neural/metabolismo , Células de Schwann/ultraestrutura , Fatores de Transcrição/genéticaRESUMO
OBJECTIVE: This study was designed to determine concussion incidence, risk, and relative risk among middle and high school athletes participating in various sports. METHOD: Data were retrospectively obtained from 10,334 athletes of 12 different sports in Hawaii. In addition to determining the overall concussion incidence, comparisons of incidence, risk, and relative risk were made according to age, sex, concussion history, sport, and football position. RESULTS: The overall incidence of concussion among youth athletes was 1,250 (12.1%). The relative risk for a concussion was almost two times greater in 18-year olds than in 13-year-old athletes. In comparable sports, girls had a 1.5 times higher concussion risk than boys. Athletes with a prior concussion had 3-5 times greater risk to sustain a concussion than those with no history of a concussion. Among varied sports, wrestling and martial arts had the highest relative risk of a concussion, followed by cheerleading, football, and track and field. No differences in concussion risks were found among the football players in different positions. CONCLUSIONS: Older youths, females, those with a history of concussion, and those participating in high contact sports were found to have higher risks of sustaining a concussion. The findings increase awareness of concussion patterns in young athletes and raise concerns regarding protective strategies and concussion management in youth sports.
Assuntos
Atletas , Traumatismos em Atletas/epidemiologia , Concussão Encefálica/epidemiologia , Esportes , Adolescente , Fatores Etários , Traumatismos em Atletas/complicações , Concussão Encefálica/etiologia , Feminino , Futebol Americano , Humanos , Incidência , Masculino , Testes Neuropsicológicos , Estudos Retrospectivos , Risco , Fatores SexuaisRESUMO
The Ras/Raf/MEK/MAP kinase cascade transmits signals from activated cell-surface receptors to transcription factors in the nucleus and is an essential component of metazoan intracellular signaling pathways (see, for example, [1-6]). In the mouse, the Raf protein kinase family is comprised of three homologous genes, Raf-1, A-Raf and B-Raf [5] which are ubiquitously expressed in the developing embryo [7]. We have introduced into the mouse germ line a loss-of-function mutation in the X-chromosomal A-Raf gene, by homologous recombination in embryonic stem cells. On a predominantly C57 Bl/6 genetic background, A-Raf-deficient mice displayed neurological and intestinal abnormalities and died between 7 and 21 days post-partum. When the mutated allele was maintained on a predominantly 129/OLA background, by contrast, A-Raf-deficient animals survived to adulthood, did not display obvious intestinal abnormalities, were fertile, but did have a subset of the neurological defects.
Assuntos
Anormalidades do Sistema Digestório , Genes Letais , Sistema Nervoso/fisiopatologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Animais , Sistema Digestório/fisiopatologia , Feminino , Mutação em Linhagem Germinativa , Heterozigoto , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas c-raf , Recombinação Genética , Cromossomo XRESUMO
We examined the potential for the C-C chemokine RANTES to stimulate dorsal root ganglia (DRG) cell migration. Embryonic day 12 (E12.5) mouse DRG cells migrated in response to RANTES, in vitro, differentiating to the nociceptive phenotype within 18 h. In addition, RANTES stimulated intracellular calcium mobilization in DRG cells. RANTES expression was demonstrated by polymerase chain reaction analysis to be present in E10.5 limb bud, E12.5 DRG, Schwann cells, spinal cord and skin. RANTES protein was detected immunohistochemically in E12.5 DRG and the cutaneous layers of the developing hind limb. Thus, RANTES expression is spatially and temporally consistent with an effector molecule in sensory neuropoiesis, potentially expanding the role of this chemokine to include neurotropism.
Assuntos
Quimiocina CCL5/farmacologia , Quimiotaxia/efeitos dos fármacos , Neurônios Aferentes/efeitos dos fármacos , Animais , Southern Blotting , Cálcio/metabolismo , Células Cultivadas , Quimiocina CCL5/biossíntese , Extremidades , Gânglios Espinais/citologia , Gânglios Espinais/embriologia , Técnicas Imunoenzimáticas , Transporte de Íons/efeitos dos fármacos , Camundongos , Crista Neural/citologia , Especificidade de Órgãos , Reação em Cadeia da Polimerase , Células de Schwann/metabolismo , Pele/metabolismo , Medula Espinal/metabolismoRESUMO
We describe monoclonal antibodies (MAB) reactive with subsets of mouse and human thymic epithelial cells. Rat MAb CDR1 reacts with mouse but not human cortical epithelial cells. Immunologic staining of thymic nurse cells in suspension indicates the CDR1 antigen is located on the cell surface. Mouse MAb CDR2 reacts with human but not mouse cortical thymic epithelial cells. Rat MAb MD1 and MD2 detect different determinants expressed by most medullary epithelial cells in mouse thymus but fewer such cells in human thymus. In addition, MD1 detects flattened subcapsular cells rarely in mouse thymus but frequently in human thymus. Two-color stains using an anti-keratin antiserum demonstrate the epithelial nature of the cells reactive with these antibodies. The antigens detected by CDR1 and MD1 first appear during the neonatal period, achieving adult distribution by postnatal days 14 and 4, respectively. The extra-thymic staining of these MAb is described. On the basis of their intra- and extra-thymic reactivities, these MAb differ from those previously reported and may permit dissection of the thymic microenvironment.
Assuntos
Anticorpos Monoclonais/imunologia , Timo/imunologia , Envelhecimento/imunologia , Animais , Animais Recém-Nascidos/imunologia , Antígenos de Superfície/análise , Antígenos de Superfície/imunologia , Epitélio/imunologia , Histocitoquímica , Humanos , Técnicas Imunoenzimáticas , Queratinas/análise , Masculino , Camundongos , Camundongos Endogâmicos AKR , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Ratos , Timo/crescimento & desenvolvimentoRESUMO
We report here that S-100 beta, a protein with neurotrophic activity on central nervous system neurons, stimulates neuritic outgrowth from cultures of dorsal root ganglia (DRG). S-100 beta elicited neurites from explant and dissociated cell cultures of embryonic chick DRG, and the extent of the response varied with the age of the embryo. Specificity was demonstrated by the observation that incubation of S-100 beta with antibodies directed against S-100 beta reduced the neurite outgrowth, whereas incubation of S-100 beta with normal rabbit serum had little effect. S-100 beta also stimulated the area of neuritic outgrowth from organotypic cultures of fetal rat DRG, showing that the activity of the protein is not restricted to a particular species or culture condition. A mutant S-100 beta lacking neurotrophic activity on cerebral cortex neurons was unable to effectively stimulate neurite outgrowth from DRG cultures. These studies suggest that S-100 beta may play a role in neuronal growth and/or maintenance in the peripheral nervous system.
Assuntos
Gânglios Espinais/efeitos dos fármacos , Proteínas S100/farmacologia , Animais , Axônios/fisiologia , Embrião de Galinha , Técnicas de Cultura , Desenvolvimento Embrionário e Fetal , Gânglios Espinais/ultraestrutura , Imunoglobulina G , Ratos/embriologia , Proteínas S100/imunologiaRESUMO
Interactions between migratory granule neurons and the developing molecular layer of the mouse cerebellum were examined using an in situ binding assay. Single cell suspensions of postnatal granule neurons specifically adhere to unfixed frozen cerebellar tissue sections. We investigated the influence of postnatal age of granule neurons and of tissue on this interaction. Granule neurons from P10 (the time of peak migratory activity) bind preferentially to the molecular layer. Premigratory granule neurons, P5, do not bind age-matched cerebellar tissue. Postmigratory granule neurons, P14 and older, adhere to the molecular and internal granular layers of age-matched and older cerebellar tissue but not to younger tissue. These binding patterns are most simply explained as a single receptor-ligand system in which both elements exhibit independent developmental regulation. Although granule neurons lose the ability to bind with increasing age, the molecular layer ligand retains its capacity for this interaction into adulthood, long after normal migration has ceased.
Assuntos
Envelhecimento/fisiologia , Adesão Celular , Cerebelo/crescimento & desenvolvimento , Animais , Diferenciação Celular , Cerebelo/citologia , Cerebelo/fisiologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Family violence is ubiquitous in our society and, thus, is encountered in all medical practices. The purpose of this study was to ascertain whether physicians wearing buttons with an anti-abuse message have more conversations about violence compared with physicians not wearing such buttons. Six of 11 family practice residents wore Minnesota Medical Association buttons that invited conversations about abuse. For four weeks, all 11 residents recorded daily the number of conversations about violence that occurred in the medical setting. Analyses comparing the two groups showed that the physicians wearing buttons had significantly more conversations than those not wearing buttons (c2 = 9.040, p < 0.005). Physicians wearing buttons had a higher percentage of days with conversations about domestic violence than physicians without buttons (c2 = 7.695, p < 0.01). From the significant p-values documented, we conclude that wearing the buttons increases conversations about family violence and makes physicians more consistent in talking about violence with patients.
Assuntos
Violência Doméstica/prevenção & controle , Relações Médico-Paciente , Currículo , Medicina de Família e Comunidade/educação , Feminino , Humanos , Internato e Residência , MasculinoRESUMO
IL-6 is a major immunomodulatory cytokine with neuroprotective activity. The absence of interleukin-6 (IL-6) results in increased vulnerability of dopaminergic neurons to the neurotoxicant, MPTP, and a compromised reactive microgliosis. To determine how astrogliosis may contribute to nigrostriatal degeneration in IL-6 (-/-) mice, the inflammatory profiles of astrocytes of IL-6 genotype were compared. Fourteen cytokines and four chemokines were simultaneously assayed in the supernatants of LPS-stimulated primary astrocyte cultures. In a time course of 6, 18 and 48 h and LPS stimulations of 0, 0.1, 1, 10 and 100 ng/ml, IL-6 (-/-) astrocytes secreted significantly greater amounts of the pro-inflammatory cytokines IL-1alpha, IL-1beta and TNFalpha than did IL-6 (+/+) cells. Elevated levels of IL-10 and IL-12p40 were only detected at 48 h post-stimulation with greater IL-10 in IL-6 (-/-) supernatants and greater IL-12p40 in IL-6 (+/+) supernatants. IL-6 (+/+) astrocytes produced more G-CSF and GM-CSF when compared with IL-6 (-/-) astrocytes. Chemokine levels were greater in supernatants of IL-6 (+/+) astrocytes than IL-6 (-/-) cells prior to 48 h post-stimulation. At that time, higher levels of MIP-1alpha were maintained in IL-6 (+/+) supernatant, while similar levels of MCP-1 in supernatants of both IL-6 (+/+) and IL-6 (-/-) cells were measured. Additionally, LPS (100 ng/ml) resulted in greater levels of KC and Rantes in IL-6 (-/-) astrocyte supernatants compared with IL-6 (+/+) supernatants at that time. These results suggest that the autocrine modulatory activities of IL-6 affect multiple cytokine secretory pathways, which could participate in neurodegenerative processes.
Assuntos
Astrócitos/imunologia , Quimiocinas/metabolismo , Citocinas/metabolismo , Inflamação/metabolismo , Interleucina-6/genética , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Células Cultivadas , Quimiocina CCL5/metabolismo , Citocinas/imunologia , Relação Dose-Resposta Imunológica , Feminino , Inflamação/patologia , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Interleucina-6/imunologia , Interleucina-6/metabolismo , Lipopolissacarídeos/toxicidade , Intoxicação por MPTP/imunologia , Masculino , CamundongosRESUMO
OBJECTIVE: To evaluate the gastrointestinal safety and efficacy of the COX inhibiting nitric oxide donator AZD3582 in patients with hip or knee osteoarthritis. METHODS: 970 patients were randomised (7:7:2) to AZD3582 750 mg twice daily, naproxen 500 mg twice daily, or placebo twice daily in a double blind study. The primary end point was the six week incidence of endoscopic gastroduodenal ulcers (diameter > or =3 mm). Overall damage measured on the Lanza scale was a secondary end point. Safety and tolerability assessments included endoscopic upper gastrointestinal erosions and the gastrointestinal symptom rating scale (GSRS). Efficacy was primarily assessed by WOMAC. RESULTS: The incidence of ulcers with AZD3582 was 9.7% and with naproxen 13.7% (p = 0.07, NS), v 0% on placebo. The incidence of Lanza scores >2 was higher with naproxen (43.7%) than with AZD3582 (32.2%) (p<0.001). Compared with baseline, significantly fewer ulcers and erosions developed in stomach and stomach/duodenum combined, and fewer erosions developed in stomach, duodenum, and both combined on AZD3582 than on naproxen. GSRS reflux and abdominal pain subscale scores were lower for AZD3582 than for naproxen but there was no difference for indigestion, constipation, and diarrhoea. AZD3582 was as effective as naproxen at improving WOMAC scores. Both agents were well tolerated, with no significant effects on blood pressure. CONCLUSIONS: At doses with similar efficacy in relieving osteoarthritis symptoms, the primary end point of six week endoscopic gastroduodenal ulcer incidence was not significantly different between AZD3582 and naproxen. Most secondary endoscopic gastrointestinal end points favoured AZD3582.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Naftalenos/uso terapêutico , Naproxeno/uso terapêutico , Osteoartrite do Quadril/tratamento farmacológico , Osteoartrite do Joelho/tratamento farmacológico , Adulto , Idoso , Anti-Inflamatórios não Esteroides/efeitos adversos , Pressão Sanguínea/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase/efeitos adversos , Inibidores de Ciclo-Oxigenase/uso terapêutico , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Naftalenos/efeitos adversos , Naproxeno/efeitos adversos , Úlcera Péptica/induzido quimicamente , Qualidade de Vida , Resultado do TratamentoRESUMO
Thy-1 is a cell membrane differentiation antigen with a restricted distribution in murine tissues. In both mice and rats the antigen is widely expressed in the CNS, while in the neonatal cerebellum it is expressed at very low levels. We have devised a protocol of immersion fixation by freeze-substitution that preserves both antigenicity and tissue morphology. We have stained freeze-substituted tissue sections of developing mouse cerebella with monoclonal anti-Thy-1. Thy-1 is faintly detectable at birth in Purkinje cells and in the molecular layer. The intensity in these two sites increases to a maximum at day 9; this subsequently decreases in the Purkinje cell cytoplasm until most are negative by day 21, but persists in the molecular layer into adulthood. Thy-1 is not detectable in the external granular layer and is only detectable in the glomeruli of the internal granular layer. Ascending fibre tracts are positive from day 5 onwards. The chronologic and anatomic expressions of Thy-1 are compatible with a role of Thy-1 in the generation and maintenance of synapses.
Assuntos
Antígenos de Superfície/análise , Córtex Cerebelar/imunologia , Envelhecimento , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Animais Recém-Nascidos/imunologia , Anticorpos Monoclonais , Córtex Cerebelar/citologia , Córtex Cerebelar/crescimento & desenvolvimento , Dendritos/imunologia , Liofilização , Técnicas Imunoenzimáticas , Camundongos , Antígenos Thy-1RESUMO
Schwann cells support and facilitate axonal growth during development and successful regeneration in the peripheral nerve. In the regenerating rat sciatic nerve, Schwann cells provide a trophic milieu for primary sensory, sympathetic, and motoneurons. We have characterized a neurotrophic activity produced by adult rat sciatic nerve Schwann cells and a spontaneously immortal Schwann cell clone (iSC). This activity elicits neurite outgrowth from chick embryo explants of both CNS and PNS. The iSC activity has been concentrated by cation-exchange chromatography and compared to known neurotrophins in bioassay. Pooled bound fractions elicit neurite outgrowth from sympathetic, ciliary and motoneurons. In collagen matrix cocultures of iSC and E4 ventral horn (before motor axon extension to muscle targets), the iSC activity can direct the initial axonal extension from motoneurons. The data presented suggest that Schwann cell-produced activity may mediate motoneuron axonal extension before contact with their peripheral source of neurotrophin.
Assuntos
Axônios , Substâncias de Crescimento/isolamento & purificação , Interleucina-6 , Neurônios Motores/efeitos dos fármacos , Células de Schwann/fisiologia , Animais , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Embrião de Galinha , Cromatografia por Troca Iônica , Colágeno , Gânglios Parassimpáticos/citologia , Gânglios Parassimpáticos/embriologia , Gânglios Simpáticos/citologia , Gânglios Simpáticos/embriologia , Inibidores do Crescimento/farmacologia , Substâncias de Crescimento/farmacologia , Fator Inibidor de Leucemia , Linfocinas/farmacologia , Neurônios Motores/ultraestrutura , Fatores de Crescimento Neural , Proteínas do Tecido Nervoso/farmacologia , Ratos , Células de Schwann/química , Medula Espinal/citologiaRESUMO
Successful mammalian peripheral nerve regeneration is dependent on activated Schwann cells. Schwann cells facilitate neuronal regrowth through the production of tropic cell membrane molecules, neurotrophins, and extracellular matrix components. To better understand Schwann cell function in the regenerating nerve, we have designed a method of isolating proliferating adult Schwann cells from the injured rat sciatic nerve. Relying on the mitotic signal that is present after a crush injury, we can obtain sufficient numbers of dividing Schwann cells within one week of initial culture. A spontaneously immortal Schwann cell clone (iSC) was observed in and isolated from one of these primary cultures. These cells were transformed at a time of maximal Schwann cell activation in response to injury. Both the primary Schwann cells and the iSC have been characterized as Schwann cells by morphology, immunohistochemistry and gene expression.
Assuntos
Regeneração Nervosa , Células de Schwann/citologia , Nervo Isquiático/fisiologia , Animais , Northern Blotting , Separação Celular/métodos , Células Clonais , Masculino , Compressão Nervosa , Proteínas do Tecido Nervoso/análise , Células PC12 , RNA/análise , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Fator de Crescimento Neural/genética , Proteínas S100/análise , Células de Schwann/fisiologiaRESUMO
The 7S NGF complex from the male mouse submaxillary gland consists of the alpha, gamma, and beta subunits in the ratio alpha 2 gamma 2 beta. The beta (NGF) subunit contains all the known biolocial activity of 7S NGF. The alpha and gamma subunits are both members of glandular kallikrein gene family, yet only gamma subunit has protease activity. The gamma subunit plays a role in the processing of preproNGF to its mature form, while the role of the alpha subunit is not yet understood. Despite the fact that 7S NGF has been extensively characterized, no other NGF complex has been characterized, nor have the alpha or gamma subunits been observed in tissues which express NGF. We have therefore purified and characterized the NGF complex from the submaxillary glands of the rat Mastomys natalensis in order to more fully understand the roles of the alpha and gamma subunits. The NGF complex from M. natalensis contains subunits similar to those found in mouse 7S NGF. Although similar, there are significant differences between mouse and M. natalensis NGF complexes, especially in the degree of post-translational modification of the gamma and NGF subunits, the expression of esterase activity and the ease with which the complexes dissociate. Evidence is presented that suggests that the NGF complex from M. natalensis may consist of subunits in the ratio alpha 2 gamma beta. The amino acid sequence of the M. natalensis NGF suggests some, but not all, ways in which these differences arise.
Assuntos
Muridae/metabolismo , Fatores de Crescimento Neural/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Bioensaio , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Focalização Isoelétrica , Masculino , Camundongos , Dados de Sequência Molecular , Fatores de Crescimento Neural/antagonistas & inibidores , Fatores de Crescimento Neural/isolamento & purificaçãoRESUMO
A rat monoclonal antibody (OZ42), raised against immature mouse granule cells, recognizes a region of the external granular layer of postnatally developing cerebellar cortex. This region, about three cells thick, is adjacent to the developing molecular layer and contains postmitotic, premigratory granule cells. The OZ42 reactivity commenced near postnatal day 3 (P3), the deep external granular layer was strongly reactive by P10 and this level was maintained while granule cells remained in the external granular layer (approximately P15). Isolated immature granule cells in cytospin preparations specifically reacted with OZ42. Reactivity was extranuclear and was substantially reduced when cells were prepared by trypsinization, suggesting that at least some of the antigen is associated with the outer surface of the plasma membrane. Other postnatal reactivity to OZ42 (P0 to P3) was found in a band of cells in the deep cortical layers overlying the corpus callosum through the entorhinal cortex, terminating adjacent to the hippocampus. Reactivity in some regions of the corpus callosum and anterior commissure was seen from P0 to P5. No reactivity of non-neural tissues was observed at any stage. In the embryo there was extensive staining of the CNS and PNS at E10 and E14, which was largely gone by E16. Weaver mutant mice examined for reactivity to OZ42 showed that the granule cell death and cerebellar disorganization in P10 homozygous mutants was associated with a substantial decrease in OZ42 reactivity in the external granular layer. At P14 and P20, OZ42 reactivity in the weaver external granular layer was restricted to single cells, rather than an entire layer of cells, further indicating that the OZ42 antigen is present on granule cells rather than the substratum. By Western analysis of non-reducing SDS-PAGE gels, OZ42 recognized a single band with the molecular weight between 120 and 145 kD in P10, but not adult cerebellum and BALB/c mice. An OZ42-specific band at 60-70 kD was also seen under reducing conditions and occasionally in non-reducing conditions. These bands were not recognized by antibodies against NCAM, L1 and AMOG. Immunoprecipitation and cross-blocking with antiserum to TAG-1 suggested that OZ42 recognized the same molecule in the mouse cerebellum that has been described in embryonic rat and mouse spinal cord. The developmentally regulated expression of the neural-specific molecule recognized by OZ42 in the postnatal cerebellum suggests it my be involved with the early stages of granule cell axon elongation.
Assuntos
Cerebelo/análise , Neurônios/análise , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Animais Recém-Nascidos/metabolismo , Anticorpos Monoclonais/biossíntese , Western Blotting , Movimento Celular , Cerebelo/citologia , Cerebelo/embriologia , Cerebelo/crescimento & desenvolvimento , Feminino , Técnicas Imunoenzimáticas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Mutantes Neurológicos , Testes de Precipitina , Ratos , Ratos Endogâmicos , Fatores de TempoRESUMO
Interleukin-6 (IL-6) was produced by the spontaneously immortal Schwann cell clone, iSC, when cocultured with PC12 cells. The iSC cell-derived IL-6 in coculture conditioned media caused the neuronal differentiation of naive PC12 cells and this bioactivity was neutralized by preincubation of conditioned media with antisera to IL-6. Cocultured iSC transcribe IL-6 message as confirmed by northern analysis. Stimuli that induce IL-6 production in the hematopoietic lineage induced transcription and production of IL-6 by iSC cells. Lipopolysaccharide, tumor necrosis factor-alpha, IL-1 alpha, IL-6, and serum withdrawal induced iSC cell IL-6 mRNA. The kinetics of IL-6 production was confirmed in the mouse IL-6-dependent B9 bioassay and that activity could be neutralized with antisera to IL-6. Expression of both the IL-6 receptor and the gp130 signal transduction component by iSC as determined by northern analysis suggests an autocrine regulatory mechanism. The observed iSC production of IL-6 in vitro led to an investigation of the sciatic nerve crush model of Schwann cell activation in vivo. In the initial 12 h after crush injury, IL-6 message is induced. IL-6 mRNA expression was highest distal to the crush injury. Our in vitro data demonstrate that iSC cells produce IL-6 in response to PC12 cell coculture and to stimuli that induce IL-6 production in the hematopoietic lineage. The induction of IL-6 message distal to a crush injury suggests another mechanism by which Schwann cells facilitate peripheral nerve regeneration.
Assuntos
Antígenos CD , Interleucina-6/biossíntese , Células de Schwann/metabolismo , Nervo Isquiático/lesões , Ferimentos não Penetrantes/metabolismo , Animais , Células Cultivadas , Receptor gp130 de Citocina , Citocinas/farmacologia , Técnicas Citológicas , Glicoproteínas de Membrana/genética , Compressão Nervosa , Células PC12 , RNA Mensageiro/metabolismo , Ratos , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Receptores de Interleucina-6 , Nervo Isquiático/metabolismo , Transdução de SinaisRESUMO
A specific mutation (A53T) in the encoding region for alpha-synuclein has been identified in a large multigenerational family with an autosomal dominant parkinsonism known as the Contursi kindred. In this study, we used a monoclonal antibody directed against alpha-synuclein in order to identify novel proteins in the brain of an affected member of this kindred who had come to autopsy. Homogenates from the frontal cortex and caudate nucleus were examined using Western blot techniques and compared to matched autopsy specimens from control subjects and patients with various forms of parkinsonism. Western blots, using a 15-min exposure time, revealed the expected 19-kDa band representing alpha-synuclein in all brain samples examined. However, a novel band in the 36-kDa range was also present in the Contursi brain which was not seen in cortex or caudate from control brains or in frontal cortex from 14 cases of typical Parkinson's disease. With a 24-h exposure time, this band was faintly seen in the caudate nucleus of three of the Parkinson's disease cases. Surprisingly, the 36-kDa band (as well as other high-molecular-weight bands) was also present in frontal cortex and caudate nucleus in 3 additional cases that met diagnostic criteria for both Parkinson's disease and Alzheimer's disease. A preliminary analysis of samples from the frontal cortex of 10 Alzheimer's disease cases revealed a 36-kDa band in only one instance. The identification of novel alpha-synuclein-immunoreactive bands in these various forms of parkinsonism may open new research avenues for exploring the relationship between abnormal protein deposition in the brain and one or more neurodegenerative disorders, including the Contursi form of familial parkinsonism.
Assuntos
Doença de Alzheimer/genética , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/genética , Doença de Parkinson/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Especificidade de Anticorpos , Western Blotting , Química Encefálica/genética , Análise Mutacional de DNA , Saúde da Família , Feminino , Lobo Frontal/química , Lobo Frontal/patologia , Humanos , Corpos de Lewy/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Degeneração Neural/genética , Degeneração Neural/patologia , Proteínas do Tecido Nervoso/imunologia , Fosfoproteínas/análise , Fosfoproteínas/imunologia , Substância Negra/química , Substância Negra/patologia , Sinucleínas , alfa-SinucleínaRESUMO
The hnmp-1 (hematopoietic neural membrane protein) gene encodes a protein with striking similarity to the tetra-transmembrane-spanning protein encoded by pmp22. hnmp-1 was cloned from an elutriated human monocyte library and is expressed in various human hematopoietic and lymphoid lineages as well as adult mouse spleen and thymus. In the mouse nervous system, HNMP-1 mRNA is temporally expressed by Schwann cells during sciatic nerve myelination. Dorsal root ganglia sensory and spinal cord alpha-motoneurons acquire HNMP-1 protein selectively throughout development. In the fiber tracts of the spinal cord and in sciatic nerve, HNMP-1 protein is axon-associated. Additionally a rapid and sustained level of HNMP-1 expression is observed in response to acute PNS injury. HNMP-1 is constituitively induced in sciatic nerve of Trembler J mice, which are mutant for pmp22 and have a demyelinating/hypomyelinating phenotype. The expression pattern of HNMP-1 suggests a possible role for this molecule during active myelination.