Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 21
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Fitoterapia ; 174: 105859, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38354819

RESUMO

In this study, we examined physiological functions as a key material to develop cosmeceuticals using extracts of Lagerstroemia macrocarpa Wall. Ex Kurz (L. macrocarpa). Initially, the L. macrocarpa extract was treated by different concentration and antioxidant assay (DPPH and ABTS) were performed to measure free radical scavenging ability. In the cytotoxicity experiment, the extract was treated into human epidermal keratinocytes with different concentrations to measure cytotoxicity. We found that the extract induces differentiation markers such as keratin (KRT)1, KRT2, KRT9, KRT10 in keratinocytes. Furthermore, the extract significantly induces involucrin (IVL), loricrin (LOR), claudin1 (CLDN1), and filaggrin (FLG) expression, suggesting that it may enhance skin barrier functions. Especially, the extract restored FLG expression inhibited by interleukin (IL)-4/IL-13 in in vitro atopic dermatitis-like model. Therefore, we expect L. macrocarpa extract will be an effective material to develop the therapeutic and cosmeceutical of atopic dermatitis.


Assuntos
Dermatite Atópica , Lagerstroemia , Humanos , Lagerstroemia/metabolismo , Dermatite Atópica/tratamento farmacológico , Dermatite Atópica/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Proteínas de Filamentos Intermediários/farmacologia , Proteínas de Filamentos Intermediários/uso terapêutico , Estrutura Molecular , Queratinócitos , Extratos Vegetais/metabolismo , Transdução de Sinais , Fator de Transcrição STAT6/metabolismo , Fator de Transcrição STAT6/farmacologia
2.
J Eur Acad Dermatol Venereol ; 33(2): 367-375, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30357932

RESUMO

BACKGROUND: Janus kinase (JAK) inhibition may be a promising new treatment modality for inflammatory (skin) diseases. However, little is known about direct effects of kinase inhibitors on keratinocyte differentiation and function as well as skin barrier formation. OBJECTIVE: Our aim was to address the direct impact of kinase inhibition of the JAK1/3 pathways by tofacitinib on keratinocyte immune function and barrier formation in atopic dermatitis (AD) and psoriasis. METHODS: 3D skin equivalents of both diseases were developed and concurrently pretreated with tofacitinib. To induce AD, 3D skin equivalents were stimulated with recombinant human IL-4 and IL-13. Psoriasis-like conditions were induced by incubation with IL-17A, IL-22 and tumour necrosis factor α (TNFα). The activation of signal transducer and activator of transcription (STAT)1, STAT3 and STAT6 was assessed by Western blot analysis. Microarray analysis and quantitative real-time PCR were used for gene expression analysis. RESULTS: Tofacitinib pretreatment preserved epidermal morphology and reduced STAT3 and STAT6 phosphorylation of AD-like and STAT3 phosphorylation of psoriasis-like culture conditions in 3D skin models compared to sham-controls. Filaggrin expression was fully maintained in the AD-like models, but only partially in psoriasis-like conditions after pretreatment with tofacitinib. In addition, tofacitinib upregulated DSC1, FLG and KRT1. Using gene expression analysis, downregulation of POSTN and IL24 was observed in AD-like conditions, whereas downregulation of IL20 and IL1B was observed in psoriasis-like conditions. CONCLUSION: JAK1/3 inhibition counteracted cytokine-induced AD- and psoriasis-like epidermal morphology and enhanced keratinocyte differentiation in 3D skin models. This effect was more pronounced in the AD-like models compared to the psoriasis-like 3D skin models.


Assuntos
Dermatite Atópica/patologia , Imageamento Tridimensional , Proteínas de Filamentos Intermediários/farmacologia , Janus Quinase 1/efeitos dos fármacos , Piperidinas/farmacologia , Psoríase/patologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Proliferação de Células/efeitos dos fármacos , Simulação por Computador , Dermatite Atópica/tratamento farmacológico , Proteínas Filagrinas , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Psoríase/tratamento farmacológico , Fator de Transcrição STAT6/efeitos dos fármacos , Sensibilidade e Especificidade
3.
Sci Rep ; 8(1): 16158, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30385807

RESUMO

Cationic intrinsically disordered antimicrobial peptides (CIDAMPs) belong to a novel class of epithelial peptide antibiotics with microbicidal activity against various pathogens, including Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Candida albicans. Here we show that treatment of distinct bacteria with different hornerin (HRNR)-derived CIDAMPs cause formation of unique cytoplasmic protein aggregates, suggesting a common intracellular mode of action. We further found that, unlike most amphipathic antimicrobial peptides, HRNR traverses bacterial membranes energy-dependently and accumulates within the cytoplasm. Strikingly, certain structurally different, HRNR-based CIDAMPs were found to bind to an identical panel of distinct bacterial ribosomal proteins, thereby manifesting features of several known classes of antibiotics. This may cause the formation of aberrant proteins and toxic protein aggregates in HRNR-treated pathogens which eventually may induce its death. Our study reveals evidence that structurally distinct CIDAMPs of an abundant body surface protein simultaneously target multiple sites of the bacterial protein synthesis machinery.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Proteínas de Ligação ao Cálcio/química , Proteínas de Filamentos Intermediários/química , Proteínas Intrinsicamente Desordenadas/química , Ribossomos/química , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/farmacologia , Candida albicans/efeitos dos fármacos , Candida albicans/patogenicidade , Membrana Celular/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/patogenicidade , Humanos , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/farmacologia , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/patogenicidade , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Ribossomos/genética , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/patogenicidade
4.
Sci Transl Med ; 8(325): 325ra18, 2016 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-26865566

RESUMO

Atopic dermatitis is a common pruritic skin disease in which barrier dysfunction and cutaneous inflammation contribute to pathogenesis. Mechanisms underlying the associated inflammation are not fully understood, and although Langerhans cells expressing the nonclassical major histocompatibility complex (MHC) family member CD1a are known to be enriched within lesions, their role in clinical disease pathogenesis has not been studied. We observed that house dust mite (HDM) allergen generates neolipid antigens presented by CD1a to T cells in the blood and skin lesions of affected individuals. HDM-responsive CD1a-reactive T cells increased in frequency after birth in individuals with atopic dermatitis and showed rapid effector function, consistent with antigen-driven maturation. In HDM-challenged human skin, we observed phospholipase A2 (PLA2) activity in vivo. CD1a-reactive T cell activation was dependent on HDM-derived PLA2, and such cells infiltrated the skin after allergen challenge. Moreover, we observed that the skin barrier protein filaggrin, insufficiency of which is associated with atopic skin disease, inhibited PLA2 activity and decreased CD1a-reactive PLA2-generated neolipid-specific T cell activity from skin and blood. The most widely used classification schemes of hypersensitivity suggest that nonpeptide stimulants of T cells act as haptens that modify peptides or proteins; however, our results show that HDM proteins may also generate neolipid antigens that directly activate T cells. These data define PLA2 inhibition as a function of filaggrin, supporting PLA2 inhibition as a therapeutic approach.


Assuntos
Antígenos CD1/metabolismo , Fosfolipases A2 do Grupo IV/metabolismo , Proteínas de Filamentos Intermediários/farmacologia , Pyroglyphidae/enzimologia , Adolescente , Adulto , Idoso , Animais , Separação Celular , Citocinas/metabolismo , Dermatite Atópica/sangue , Dermatite Atópica/imunologia , Proteínas Filagrinas , Humanos , Células K562 , Pessoa de Meia-Idade , Pyroglyphidae/efeitos dos fármacos , Pele/imunologia , Pele/patologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Adulto Jovem
6.
J Neurochem ; 119(4): 815-25, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21883224

RESUMO

Abnormalities of α-synuclein (α-syn) and NMDA receptors (NMDARs) are implicated in the pathogenesis of Parkinson's disease. However, how these proteins interact with each other has not been elucidated. Here, the effect of α-syn on NMDARs was investigated by examining the alterations of surface NMDAR NR1 subunits in MES23.5 dopaminergic cells transfected with the human α-syn gene as well as in cells treated with extracellularly added human α-syn. As demonstrated previously that α-syn can enter cells in a non-endocytic manner without being degraded by the cellular proteolytic systems, the extracellularly added α-syn entered the cytoplasm of MES23.5 cells in a concentration-dependent manner. Both the α-syn-transfected cells and α-syn-treated cells exhibited increased intracellular α-syn levels and reduced surface NR1 without altering the total NR1. The α-syn-induced surface NR1 reduction was accompanied by suppression of NMDA-elicited intracellular Ca(2+) elevation and reductions of NMDA-induced caspase 3 activation and cell death, which was abolished by hypotonic shock and K(+) depletion, a procedure that blocks clathrin-mediated endocytosis, and by suppression of RAB5B expression with anti-RAB5B oligonucleotides. The data obtained provide evidence for the first time that α-syn may promote clathrin-mediated NMDAR endocytosis.


Assuntos
Clatrina/metabolismo , Dopamina/metabolismo , Endocitose/efeitos dos fármacos , Agonistas de Aminoácidos Excitatórios/farmacologia , Proteínas de Filamentos Intermediários/metabolismo , N-Metilaspartato/farmacologia , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Transformada , Clatrina/genética , Maleato de Dizocilpina/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Ensaio de Imunoadsorção Enzimática/métodos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Humanos , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/farmacologia , Potássio/metabolismo , Ratos , Receptores de N-Metil-D-Aspartato/genética , Sais de Tetrazólio , Fatores de Tempo , Transfecção/métodos , Regulação para Cima/efeitos dos fármacos , Proteínas rab5 de Ligação ao GTP/metabolismo
7.
J Dermatol Sci ; 62(1): 8-15, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21345654

RESUMO

BACKGROUND: Transient receptor potential vanilloid type 1 (TRPV1) is a cation channel activated by diverse obnoxious stimuli like capsaicin, low pH or heat. Recently, it was revealed that TRPV1 might be deeply associated with skin permeability barrier function, suggesting that modulation of TRPV1 might be beneficial for the skin disorders with barrier damages. OBJECTIVE: We aimed to investigate whether the blockade of TRPV1 activation might accelerate skin barrier recovery and alleviate atopic dermatitis (AD)-like symptoms, employing a novel TRPV1 antagonist, PAC-14028. METHODS: TRPV1 antagonistic effects of PAC-14028 in human keratinocytes and skin were confirmed through capsaicin-evoked calcium influx assay and capsaicin-induced blood perfusion increase. Effects of PAC-14028 on skin barrier recovery were examined in vivo tape-stripping-induced barrier disruption in hairless mice. To determine the effects of PAC-14028 on AD, Dermatophagoides farina (Df)- and oxazolone (OXZ)-induced AD models were employed. RESULTS: PAC-14028 could inhibit capsaicin-evoked calcium influx in keratinocytes at sub-micromolar concentrations. This potent TRPV1 antagonistic activity in keratinocytes was manifested in vivo as the blockade of capsaicin-induced blood perfusion increase, and the accelerated barrier recovery from tape-stripping-induced barrier damages in hairless mice. PAC-14028 could also attenuate dermatitis-associated barrier damages in Df and OXZ models as determined by lower TEWL (trans-epidermal water loss), reformation of neutral lipid layer and reversion of changes in loricrin and filaggrin expression. Importantly, along with accelerated recovery of skin barrier function, PAC-14028 alleviated the general AD-like symptoms, including serum IgE increase, mast cell degranulation, scratching behavior and clinical severity of dermatitis. CONCLUSIONS: These results reflect that the blockade of TRPV1 activation can suppress the atopic dermatitis-like symptoms by accelerating skin barrier recovery.


Assuntos
Acrilamidas/farmacologia , Dermatite Atópica/metabolismo , Piridinas/farmacologia , Pele/metabolismo , Canais de Cátion TRPV/antagonistas & inibidores , Acrilamidas/química , Animais , Cálcio/metabolismo , Capsaicina/farmacologia , Dermatite/metabolismo , Feminino , Proteínas Filagrinas , Humanos , Imunoglobulina E/sangue , Proteínas de Filamentos Intermediários/farmacologia , Lipídeos/química , Mastócitos/metabolismo , Proteínas de Membrana/farmacologia , Camundongos , Oxazolona/farmacologia , Perfusão , Permeabilidade , Piridinas/química
8.
Exp Cell Res ; 316(17): 2779-89, 2010 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-20643125

RESUMO

Platelet-derived growth factor (PDGF) is important in central nervous system (CNS) development, and aberrant expression of PDGF and its receptors has been linked to developmental defects and brain tumorigenesis. We previously found that neural stem and progenitor cells in culture produce PDGF and respond to it by autocrine and/or paracrine signaling. We therefore aimed to examine CNS development after PDGF overexpression in neural stem cells in vivo. Transgenic mice were generated with PDGF-B under control of a minimal nestin enhancer element, which is specific for embryonic expression and will not drive adult expression in mice. The resulting mouse showed increased apoptosis in the developing striatum, which suggests a disturbed regulation of progenitor cells. Later in neurodevelopment, in early postnatal life, mice displayed enlarged lateral ventricles. This enlargement remained into adulthood and it was more pronounced in male mice than in transgenic female mice. Nevertheless, there was an overall normal composition of cell types and numbers in the brain and the transgenic mice were viable and fertile. Adult transgenic males, however, showed behavioral aberrations and locomotor dysfunction. Thus, a tightly regulated expression of PDGF during embryogenesis is required for normal brain development and function in mice.


Assuntos
Comportamento Animal/efeitos dos fármacos , Células-Tronco Embrionárias/citologia , Ventrículos Laterais/patologia , Neurônios/citologia , Proteínas Proto-Oncogênicas c-sis/genética , Animais , Encéfalo/crescimento & desenvolvimento , Desenvolvimento Embrionário , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/farmacologia , Ventrículos Laterais/efeitos dos fármacos , Masculino , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/farmacologia , Nestina , Fatores Sexuais
9.
J Neurochem ; 111(1): 192-203, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19686384

RESUMO

Lewy bodies, alpha-synuclein (alpha-syn) immunopositive intracellular deposits, are the pathological hallmark of Parkinson's disease (PD). Interestingly, Lewybody-like structures have been identified in fetal tissue grafts about one decade after transplantation into the striatum of PD patients. One possible explanation for the accelerated deposition of alpha-syn in the graft is that the aggregation of alpha-syn from the host tissue to the graft is spread by a prion disease-like mechanism. We discuss here an in vitro model which might recapitulate some aspects of disease propagation in PD. We found here that in vitro-generated alpha-syn oligomers induce transmembrane seeding of alpha-syn aggregation in a dose- and time-dependent manner. This effect was observed in primary neuronal cultures as well as in neuronal cell lines. The seeding oligomers were characterized by a distinctive lithium dodecyl sulfate-stable oligomer pattern and could be generated in a dynamic process out of pore-forming oligomers. We propose that alpha-syn oligomers form as a dynamic mixture of oligomer types with different properties and that alpha-syn oligomers can be converted into different types depending on the brain milieu conditions. Our data indicate that extracellular alpha-syn oligomers can induce intracellular alpha-syn aggregation, therefore we hypothesize that a similar mechanism might lead to alpha-syn pathology propagation.


Assuntos
Proteínas de Filamentos Intermediários/química , Proteínas de Filamentos Intermediários/metabolismo , Proteínas de Filamentos Intermediários/farmacologia , Neurônios/efeitos dos fármacos , Animais , Cálcio/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Humanos , Proteínas de Filamentos Intermediários/genética , Camundongos , Mutação/genética , Neuroblastoma/patologia , Neurônios/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/classificação , Estatísticas não Paramétricas , Fatores de Tempo , Transfecção/métodos
10.
Neurotox Res ; 16(3): 238-54, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19526281

RESUMO

Microglia provide immune surveillance for the brain through both the removal of cellular debris and protection against infection by microorganisms and "foreign" molecules. Upon activation, microglia display an altered morphology and increased expression of proinflammatory molecules. Increased numbers of activated microglia have been identified in a number of neurodegenerative diseases including Parkinson's disease (PD). What remains to be determined is whether activated microglia result from ongoing cell death or are involved in disease initiation and progression. To address this question we utilized a transgenic mouse model that expresses a mutated form of a key protein involved in Parkinson's disease, alpha-synuclein. Herein, we report an increase in activated microglia and proinflammatory molecules in 1-month-old transgenic mice well before cell death occurs in this model. Frank microglial activation is resolved by 6 months of age while a subset of proinflammatory molecules remain elevated for 12 months. Both tyrosine hydroxylase mRNA expression and alpha-synuclein protein are decreased in the striatum of older animals evidence of dystrophic neuritic projections. To determine whether mutated alpha-synuclein could directly activate microglia primary microglia-enriched cell cultures were treated with exogenous mutated alpha-synuclein. The data reveal an increase in activated microglia and proinflammatory molecules due to direct interaction with mutated alpha-synuclein. Together, these data demonstrate that mutated alpha-synuclein mediates a proinflammatory response in microglia and this activity may participate in PD pathogenesis.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Citocinas/metabolismo , Regulação da Expressão Gênica/genética , Proteínas de Filamentos Intermediários/genética , Microglia/metabolismo , Mutação/genética , Fatores Etários , Animais , Animais Recém-Nascidos , Antígenos CD36/deficiência , Proteínas de Ligação ao Cálcio/genética , Células Cultivadas , Córtex Cerebral/citologia , Citocinas/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas de Filamentos Intermediários/farmacologia , Intoxicação por MPTP/metabolismo , Intoxicação por MPTP/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos , Microglia/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fatores de Tempo , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismo
11.
J Cosmet Sci ; 55 Suppl: S113-21, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15645087

RESUMO

Fluorescence spectroscopic measurement of the amino acid tryptophan on the hair fibre surface was extended to include in situ fibre irradiation and a novel cyclical wash off, reapplication protocol. When applied to the investigation of a new damage prevention active, it was shown that the active was preferentially degraded in a sacrificial manner and that the underlying fibre surface was maintained in good condition. In addition, tensile strength measurements were performed to assess the mechanical properties of the treated and untreated fibres following UV and sunlight exposure and the results demonstrate the damage prevention effectiveness of the active.


Assuntos
Preparações para Cabelo/farmacologia , Cabelo/efeitos dos fármacos , Cabelo/efeitos da radiação , Proteínas de Filamentos Intermediários/farmacologia , Queratinas/farmacologia , Luz Solar/efeitos adversos , Raios Ultravioleta/efeitos adversos , Cabelo/química , Cabelo/ultraestrutura , Humanos , Microscopia Eletrônica de Varredura , Espectrometria de Fluorescência , Resistência à Tração , Triptofano/química
12.
J Neurochem ; 85(1): 248-56, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12641746

RESUMO

In previous studies, we showed that overexpression of peripherin, a neuronal intermediate filament (IF) protein, in mice deficient for neurofilament light (NF-L) subunits induced a progressive adult-onset degeneration of spinal motor neurons characterized by the presence of IF inclusion bodies reminiscent of axonal spheroids found in amyotrophic lateral sclerosis (ALS). In contrast, the overexpression of human neurofilament heavy (NF-H) proteins provoked the formation of massive perikaryal IF protein accumulations with no loss of motor neurons. To further investigate the toxic properties of IF protein inclusions, we generated NF-L null mice that co-express both peripherin and NF-H transgenes. The axonal count in L5 ventral roots from 6 and 8-month-old transgenic mice showed that NF-H overexpression rescued the peripherin-mediated degeneration of motor neurons. Our analysis suggests that the protective effect of extra NF-H proteins is related to the sequestration of peripherin into the perikaryon of motor neurons, thereby abolishing the development of axonal IF inclusions that might block transport. These findings illustrate the importance of IF protein stoichiometry in formation, localization and toxicity of neuronal inclusion bodies.


Assuntos
Esclerose Lateral Amiotrófica/prevenção & controle , Proteínas de Filamentos Intermediários/biossíntese , Glicoproteínas de Membrana , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Proteínas de Neurofilamentos/biossíntese , Animais , Morte Celular/genética , Quinase 5 Dependente de Ciclina , Quinases Ciclina-Dependentes/metabolismo , Humanos , Corpos de Inclusão/efeitos dos fármacos , Corpos de Inclusão/genética , Corpos de Inclusão/metabolismo , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/farmacologia , Região Lombossacral , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Neurônios Motores/citologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/farmacologia , Proteínas de Neurofilamentos/deficiência , Proteínas de Neurofilamentos/genética , Proteínas de Neurofilamentos/farmacologia , Periferinas , Transporte Proteico/fisiologia , Medula Espinal/citologia , Transgenes
13.
Blood Cells Mol Dis ; 27(3): 705-12, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-11482885

RESUMO

Primitive embryonic stem cells are an ideal starting cell population for studies of gene expression and lineage segregation during development. Despite their potential, it has been difficult to determine culture conditions that cause single-lineage differentiation of these pluripotent cells. Both genetic and epigenetic approaches have been taken to promote neuronal differentiation of embryonic stem cells, including aggregation, exposure to the nonspecific teratogen/morphogen retinoic acid, low-density culture, exposure to growth/differentiation factors, and forced differentiation following expression of lineage-restricted "developmental control" genes. In the current investigation, a hybrid approach involving genetic techniques of "lineage selection" or "forced differentiation" has been employed to develop primitive neural progenitor cell lines. These lines form an important starting point to examine the cascades of gene expression (and inhibition) during neuronal and glial lineage segregation, to study growth factor effects on neural differentiation, and ultimately to provide a source of cells for transplantation to a damaged nervous system.


Assuntos
Embrião de Mamíferos/citologia , Neurônios/citologia , Células-Tronco/citologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem da Célula/efeitos dos fármacos , Vetores Genéticos/genética , Vetores Genéticos/farmacologia , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/farmacologia , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/farmacologia , Nestina , Seleção Genética , Transfecção
14.
J Invest Dermatol ; 114(4): 701-5, 2000 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10733676

RESUMO

Citrulline-containing proteins, mainly originating from keratin K1 and formed by enzymatic deimination of arginine residues, have been identified in the cornified layers of human epidermis. We analyzed the localization and nature of the deiminated proteins in psoriatic epidermis. Immunostaining based on chemical modification of citrulline residues showed that the normal and psoriatic uninvolved epidermis contained deiminated proteins diffusely in the cornified cell layer, whereas the involved epidermis had no detectable or markedly reduced levels of deiminated proteins. Immunolabeling with polyclonal antibodies against a synthetic citrulline-containing peptide corresponding to a deiminated sequence of mouse K1 also suggested markedly decreased deiminated K1 in psoriatic involved lesions. Keratin analyses indicated that deiminated K1 present in normal and psoriatic uninvolved epidermis was not detected in the psoriatic involved epidermis. Double staining with a monoclonal antibody, 34betaB4, and the polyclonal antibodies demonstrated that epidermis with low suprabasal keratin expression was negative for deiminated K1. In contrast, intralesional acrosyringia showing decreased suprabasal keratin immunoreactivity like that of the surrounding psoriatic epidermis showed strong deiminated K1 staining. This suggests that abnormal keratin deimination is restricted to the psoriatic hyperproliferative epidermis, without affecting sweat ductal epithelia.


Assuntos
Queratinas/metabolismo , Psoríase/patologia , Pele/citologia , Pele/patologia , Biópsia/métodos , Divisão Celular , Citrulina/farmacologia , Células Epidérmicas , Epiderme/química , Epiderme/enzimologia , Proteínas Filagrinas , Humanos , Hidrolases/metabolismo , Imuno-Histoquímica , Proteínas de Filamentos Intermediários/farmacologia , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em Proteínas , Pele/química
15.
Biochem Biophys Res Commun ; 255(3): 735-9, 1999 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-10049780

RESUMO

Based on a homology search with endostatin, the C-terminus 185 aa of collagen XVIII, we report the cloning, expression, and antiangiogenic activity of a 22 kDa human collagen XV fragment, that we have named restin. Restin was expressed in the prokaryotic pET expression system. We have shown that restin inhibits the migration of endothelial cells in vitro but has no effect on the proliferation of these cells. A polyclonal antibody raised against endostatin cross-reacted with restin. Systemic administration of restin suppressed the growth of tumors in a xenograft renal carcinoma model.


Assuntos
Colágeno/química , Colágeno/farmacologia , Proteínas de Filamentos Intermediários/genética , Proteínas Associadas aos Microtúbulos , Proteínas de Neoplasias/genética , Neovascularização Fisiológica/efeitos dos fármacos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/farmacologia , Sequência de Aminoácidos , Antineoplásicos/farmacologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Clonagem Molecular , Colágeno Tipo XVIII , Endostatinas , Humanos , Proteínas de Filamentos Intermediários/farmacologia , Dados de Sequência Molecular , Proteínas de Neoplasias/farmacologia , Neoplasias Experimentais/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Homologia de Sequência de Aminoácidos , Transplante Heterólogo/imunologia
16.
Biofactors ; 10(4): 339-45, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10619701

RESUMO

The inhibitory properties of phosphorylated cystatin alpha (P-cystatin alpha) and a conjugated protein of the P-cystatin alpha with filaggrin linker segment peptide (FLSP) against the growth of Staphylococcus bacteria and poliovirus were investigated. Both the P-cystatin alpha and the conjugated protein (P-cystatin alpha-FLSP conjugate) as a model for the cornified envelope of skin inhibited the cysteine protease activity of Staphylococcus aureus V8. The protease activity was inhibited by normal cornified envelope of newborn rat skin, which contains P-cystatin alpha, and P-cystatin alpha in cornified envelope of newborn rat skin also suppressed the growth of S. aureus V8. When P-cystatin alpha or P-cystatin alpha-FLSP conjugate was added to cultured HeLa cells infected with poliovirus, 50-70% of the cell-death due to poliovirus infection was prevented. The poliovirus 3C protease activity in the infected HeLa cells was inhibited by P-cystatin alpha or P-cystatin alpha-FLSP conjugate. As a result, the processing of viral capsid peptides was suppressed. These findings suggest that P-cystatin alpha and P-cystatin alpha-FLSP conjugate could play the role of the barrier against microorganism infections due to inhibition of their cysteine protease activities.


Assuntos
Cistatinas/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Proteínas de Filamentos Intermediários/farmacologia , Poliovirus/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Morte Celular/efeitos dos fármacos , Epiderme/fisiologia , Proteínas Filagrinas , Células HeLa , Humanos , Proteínas de Filamentos Intermediários/química , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Fosforilação , Poliovirus/fisiologia , Ratos , Ratos Sprague-Dawley , Fenômenos Fisiológicos da Pele , Staphylococcus aureus/crescimento & desenvolvimento , Replicação Viral/efeitos dos fármacos
17.
Histochem J ; 23(9): 385-91, 1991 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-1720767

RESUMO

An epidermolytic toxin target was observed in keratohyalin granules of sectioned epidermis by a 'direct' fluorescence procedure using FTC-toxin, but not by an 'indirect' procedure using sequential reaction with toxin, anti-toxin and FTC-secondary antibody. The investigation of the two procedures was extended to keratinocytes. A dispase digestion procedure yielded three fractions which corresponded to basal, spinous and granular cells according to biochemical and morphological criteria. It was shown that the 'direct' and 'indirect' procedures both detected the toxin target in the keratohyalin granules of granular cells, but that the 'indirect' procedure was very insensitive. In control experiments, the profilaggrin of keratohyalin granules was detected readily in cells by a 'direct' procedure using FTC-antiprofilaggrin but only weakly by an 'indirect' double antibody procedure. Insensitivity to 'indirect' procedures thus appears to be a particular property of the keratohyalin granule site. It was shown that the toxin target was readily accessible in permeable (trypsin-isolated) granular cells but inaccessible in impermeable (dispase-isolated) cells.


Assuntos
Epiderme/metabolismo , Exfoliatinas/farmacologia , Proteínas de Filamentos Intermediários/farmacologia , Queratinócitos/metabolismo , Precursores de Proteínas/farmacologia , Receptores Imunológicos/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Anticorpos/imunologia , Sítios de Ligação , Membrana Celular/efeitos dos fármacos , Separação Celular , Endopeptidases , Exfoliatinas/imunologia , Proteínas Filagrinas , Proteínas de Filamentos Intermediários/imunologia , Queratinas/química , Queratinas/imunologia , Camundongos , Microscopia de Fluorescência/métodos , Precursores de Proteínas/imunologia , Receptores Imunológicos/metabolismo
18.
J Comp Neurol ; 308(4): 576-85, 1991 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-1865017

RESUMO

The ability to target specific cytoskeletal components in axons for disruption within intact developing embryos would provide a valuable tool for studying neuronal development. Neurofilaments are an attractive target for such an approach, because they are neuron specific and are expressed late in embryogenesis principally beginning during axon outgrowth. No pharmacological agents are currently available that disrupt neurofilaments without also affecting general development. One approach that has been used successfully to affect proteins in vivo is to inject specific antibodies into living cells. We employed this approach in Xenopus laevis embryos by injecting two antibodies directed against the middle molecular weight neurofilament protein (NF-M) into a single blastomere of a two-cell stage embryo. Injected antibodies could be detected for as long as 3.5 days in cells descended from the injected blastomere. Only cell bodies of neurons descended from anti-NF-M-injected blastomeres contained abnormal accumulations of intermediate filament proteins, and peripheral nerve development was unilaterally retarded in these neurofilament antibody-injected tadpoles. Such accumulations and peripheral nerve defects were not seen in neurons derived from uninjected blastomeres or from blastomeres injected with control antibodies. These data demonstrate the usefulness of specific antibodies to perturb neuronal development in intact frog embryos and, in addition, suggest a role for neurofilaments in axon elongation.


Assuntos
Anticorpos Monoclonais/imunologia , Axônios/fisiologia , Embrião não Mamífero/fisiologia , Proteínas de Filamentos Intermediários/imunologia , Animais , Axônios/ultraestrutura , Embrião não Mamífero/imunologia , Embrião não Mamífero/ultraestrutura , Proteínas de Filamentos Intermediários/farmacologia , Xenopus
19.
Pathol Biol (Paris) ; 37(4): 297-303, 1989 Apr.
Artigo em Francês | MEDLINE | ID: mdl-2660081

RESUMO

Cells in the granular layer of mammalian epidermis contain densely staining bodies called keratohyalin granules. Two types of granules are identified. Larger ones contain phosphorus and consist largely of an unusually histidine rich protein. This protein has a high molecular weight and contains a large fraction of basic aminoacids. However it has a neutral isoelectric point due to extensive phosphorylations. This histidine rich protein undergoes modifications during terminal differentiation, especially when the keratohyalin granules disperse as the granular cells differentiate into the overlying cornified cells. At this point it is dephosphorylated and partially proteolyzed to form lower molecular weight highly basic histidine rich proteins. These cationic molecules aggregate in vitro with keratin intermediate filaments, forming well ordered macrofibrils whose structure resembles the keratin pattern seen in the lower cornified layers. For this reason the name filaggrin is used for these proteins. The high molecular weight precursor is called profilaggrin. Filaggrins are species-distinct products and consist of a number of isoelectric variants. It is established that the filaggrin precursor is composed of tandemly linked multiple copies of filaggrin domains interspersed with short linker peptides. Two functions are proposed for the filaggrin. The first one, based on its interaction with keratin fibres in vitro, is to form the interfilamentous matrix seen in the lower stratum corneum. The second one is to generate a concentrated pool of free aminoacids and derivatives, allowing the stratum corneum to remain hydrated at low environmental humidities. These functions represent sequential rather alternative roles. Epidermal diseases and in vitro cultures illustrate the clear-cut relations between epidermal keratinization and differentiation and the histidine rich proteins pathway.


Assuntos
Proteínas de Filamentos Intermediários , Animais , Fenômenos Químicos , Química , Físico-Química , Epiderme/análise , Proteínas Filagrinas , Cobaias , Humanos , Proteínas de Filamentos Intermediários/metabolismo , Proteínas de Filamentos Intermediários/farmacologia , Camundongos , Precursores de Proteínas , Ratos , Dermatopatias/metabolismo
20.
J Cell Sci ; 87 ( Pt 4): 543-54, 1987 May.
Artigo em Inglês | MEDLINE | ID: mdl-3654791

RESUMO

Sucrose gradient analysis of reaction products obtained from non-epithelial intermediate filament (IF) subunit proteins and a mixture of supercoiled, relaxed and linearized plasmid pBR322 DNA at low ionic strength revealed that limited amounts of these polypeptides interacted exclusively with the supercoiled form of the plasmid DNA. These results were corroborated by electron-microscopic analysis of the reaction products, which showed that only circles of supercoiled pBR322 DNA were completely and smoothly covered with vimentin. IFs reconstituted from pure vimentin reacted with supercoiled pBR322 DNA only through their physical ends. The reaction of an aged preparation of vimentin with supercoiled pBR322 DNA produced large aggregates consisting of a central, axially oriented protein scaffold to which individual loops of DNA were attached at their bases in a halo-like arrangement. The electron-microscopic appearance of such complexes was very reminiscent of that of histone-depleted metaphase chromosomes. Together with the previous observations that non-epithelial IF proteins have high affinities for single-stranded DNA and core histones and that they are structurally and functionally closely related to the nuclear lamins, these results were used to advance a novel hypothesis on the biological role of IF proteins in eukaryotic cells.


Assuntos
DNA Super-Helicoidal/ultraestrutura , Proteínas de Filamentos Intermediários/farmacologia , Plasmídeos/efeitos dos fármacos , Animais , Centrifugação com Gradiente de Concentração , DNA Super-Helicoidal/efeitos dos fármacos , Técnicas In Vitro , Microscopia Eletrônica , Vimentina/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...