RESUMO
One of the major functions of human skin is to provide protection from the environment. Although we cannot entirely avoid, for example, sun exposure, it is likely that exposure to other environmental factors could affect cutaneous function. A number of studies have identified smoking as one such factor that leads to both facial wrinkle formation and a decline in skin function. In addition to the direct physical effects of tobacco smoke on skin, its inhalation has additional profound systemic effects for the smoker. The adverse effects on the respiratory and cardiovascular systems from smoking are well known. Central to the pathological changes associated with smoking is the elastic fibre, a key component of the extracellular matrices of lungs. In this study we examined the systemic effect of chronic smoking (>40 cigarettes/day; >5 years) on the histology of the cutaneous elastic fibre system, the nanostructure and mechanics of one of its key components, the fibrillin-rich microfibril, and the micromechanical stiffness of the dermis and epidermis. We show that photoprotected skin of chronic smokers exhibits significant remodelling of the elastic fibre network (both elastin and fibrillin-rich microfibrils) as compared to the skin of age- and sex-matched non-smokers. This remodelling is not associated with increased gelatinase activity (as identified by in situ zymography). Histological remodelling is accompanied by significant ultrastructural changes to extracted fibrillin-rich microfibrils. Finally, using scanning acoustic microscopy, we demonstrated that chronic smoking significantly increases the stiffness of both the dermis and the epidermis. Taken together, these data suggest an unappreciated systemic effect of chronic inhalation of tobacco smoke on the cutaneous elastic fibre network. Such changes may in part underlie the skin wrinkling and loss of skin elasticity associated with smoking. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
Assuntos
Fibrilinas/efeitos dos fármacos , Envelhecimento da Pele/efeitos dos fármacos , Fumar Tabaco/efeitos adversos , Adulto , Biópsia , Derme/efeitos dos fármacos , Derme/ultraestrutura , Elasticidade/efeitos dos fármacos , Elastina/efeitos dos fármacos , Elastina/ultraestrutura , Epiderme/efeitos dos fármacos , Epiderme/ultraestrutura , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/ultraestrutura , Feminino , Humanos , Imuno-Histoquímica , Masculino , Microfibrilas/efeitos dos fármacos , Microfibrilas/ultraestrutura , Pessoa de Meia-Idade , Pele/efeitos dos fármacos , Pele/ultraestruturaRESUMO
Fibrillins form multifunctional microfibrils in most connective tissues. Deficiencies in fibrillin assembly can result in fibrillinopathies, such as Marfan syndrome. We demonstrate the presence of heparin/heparan sulfate binding sites in fibrillin-2 and -3. Multimerization of all three fibrillins drastically increased the apparent affinity of their interaction with heparin/heparan sulfate. Surprisingly, contrary to other reports heparin/heparan sulfate strongly inhibited homo- and heterotypic N-to-C-terminal fibrillin interactions. These data suggest that heparin/heparan sulfate controls the formation of microfibrils at the bead interaction stage.
Assuntos
Heparina/farmacologia , Heparitina Sulfato/farmacologia , Microfibrilas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fibrilina-1 , Fibrilina-2 , Fibrilinas , Heparina/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Microfibrilas/efeitos dos fármacos , Proteínas dos Microfilamentos/química , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Estrutura Quaternária de ProteínaRESUMO
BACKGROUND: Approximately 50% of patients with fibrillary glomerulonephritis (GN) progress to end-stage renal disease (ESRD) within 2 years of diagnosis, and no standard therapy exists. The data on rituximab therapy for fibrillary GN are limited and have inconsistent outcomes. Here, we report the largest case series to date using rituximab for fibrillary GN. METHODS: Retrospective chart reviews were conducted on 12 patients with fibrillary GN who were treated with rituximab (1 g i.v. × 2 doses or 375 mg/m(2) × 4 doses) at the Center for Glomerular Diseases at Columbia University Medical Center. Non-progression of disease was defined as stable/improved serum creatinine (SCr) with a minimum of 1 year of follow-up. RESULTS: The median SCr was 2.1 (range 0.7-2.7) mg/dL, median estimated glomerular filtration rate (eGFR) 39 (range 21-98) mL/min/1.73 m(2) and median proteinuria 4497 (range 210-7542) mg/day at the time of rituximab initiation. Four patients had received immunosuppression before rituximab, and nine received immunosuppression after rituximab, with four receiving a second rituximab course. Four of 12 patients were non-progressors, 3 of 12 had progressive renal dysfunction without reaching ESRD, and 5 patients reached ESRD. The median follow-up for patients who did not reach ESRD was 38 (range 14-76) months after rituximab treatment. Non-progressors had lower SCr values, higher eGFRs and shorter median duration from diagnosis to treatment than progressors. No serious adverse events were noted. CONCLUSIONS: Rituximab therapy was associated with non-progression of renal disease in 4 of 12 patients. At the time of treatment, these non-progressors had better renal function and shorter time from diagnosis to treatment than progressors.
Assuntos
Anticorpos Monoclonais Murinos/uso terapêutico , Antineoplásicos/uso terapêutico , Glomerulonefrite/tratamento farmacológico , Microfibrilas/efeitos dos fármacos , Adulto , Idoso , Progressão da Doença , Feminino , Seguimentos , Taxa de Filtração Glomerular , Glomerulonefrite/patologia , Humanos , Masculino , Microfibrilas/patologia , Pessoa de Meia-Idade , Estudos Retrospectivos , RituximabRESUMO
Versican G1 domain-containing fragments (VG1Fs) have been identified in extracts from the dermis in which hyaluronan (HA)-versican-fibrillin complexes are found. However, the molecular assembly of VG1Fs in the HA-versican-microfibril macrocomplex has not yet been elucidated. Here, we clarify the role of VG1Fs in the extracellular macrocomplex, specifically in mediating the recruitment of HA to microfibrils. Sequential extraction studies suggested that the VG1Fs were not associated with dermal elements through HA binding properties alone. Overlay analyses of dermal tissue sections using the recombinant versican G1 domain, rVN, showed that rVN deposited onto the elastic fiber network. In solid-phase binding assays, rVN bound to isolated nondegraded microfibrils. rVN specifically bound to authentic versican core protein produced by dermal fibroblasts. Furthermore, rVN bound to VG1Fs extracted from the dermis and to nondenatured versican but not to fibrillin-1. Homotypic binding of rVN was also seen. Consistent with these binding properties, macroaggregates containing VG1Fs were detected in high molecular weight fractions of sieved dermal extracts and visualized by electron microscopy, which revealed localization to microfibrils at the microscopic level. Importantly, exogenous rVN enhanced HA recruitment both to isolated microfibrils and to microfibrils in tissue sections in a dose-dependent manner. From these data, we propose that cleaved VG1Fs can be recaptured by microfibrils through VG1F homotypical interactions to enhance HA recruitment to microfibrils.
Assuntos
Ácido Hialurônico/metabolismo , Microfibrilas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Versicanas/química , Versicanas/metabolismo , Adulto , Idoso , Anticorpos/farmacologia , Derme/citologia , Derme/metabolismo , Derme/ultraestrutura , Elasticidade/efeitos dos fármacos , Fibrilina-1 , Fibrilinas , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Ligantes , Masculino , Microfibrilas/efeitos dos fármacos , Modelos Biológicos , Peptídeos/farmacologia , Ligação Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/farmacologia , Relação Estrutura-Atividade , Extratos de Tecidos , Versicanas/ultraestruturaRESUMO
This study quantitatively examined short-term effects of 0.02% Mitomycin C (MMC) treatment on the nanostructural changes in human scleral collagen fibrils. Histologic analysis and non-contact mode atomic force microscopy (AFM) were employed to assess the ultrastructural changes in the morphological characteristics of human sclera before and after 0.02% MMC application for 1 and 3 min. The scleral collagen fibrils treated with 0.02% MMC for 1 min showed no significant change in the morphology of collagen fibrils, and a significant change (p < 0.05) in the thickness of scleral tissues and collagen density, compared to the controls. 0.02% MMC application for 3 min led to a significant increase (p < 0.001) in the mean fibril diameter (185.43 +/- 22.64 nm vs. 140.72 +/- 18.06 nm), thickness (0.81 +/- 0.03 mm vs. 0.54 +/- 0.05 mm) and collagen density (1.16 times), compared to the controls This study examined the nanostructural changes in the scleral collagen fibrils before and after MMC application by AFM technique combined with conventional histological analysis (Hematoxylin-eosin and Masson's trichrome). This result indirectly suggests that long-term MMC application might increase the incidence of complications like a scleromalcia.
Assuntos
Alquilantes/administração & dosagem , Mitomicina/administração & dosagem , Esclera/efeitos dos fármacos , Adulto , Alquilantes/efeitos adversos , Colágeno/efeitos dos fármacos , Colágeno/metabolismo , Colágeno/ultraestrutura , Humanos , Masculino , Microfibrilas/efeitos dos fármacos , Microfibrilas/metabolismo , Microfibrilas/ultraestrutura , Microscopia de Força Atômica , Mitomicina/efeitos adversos , Tamanho do Órgão/efeitos dos fármacos , Concentração Osmolar , Esclera/metabolismo , Esclera/ultraestruturaRESUMO
The mechanisms underlying the biosynthesis of cellulose in plants are complex and still poorly understood. A central question concerns the mechanism of microfibril structure and how this is linked to the catalytic polymerization action of cellulose synthase (CESA). Furthermore, it remains unclear whether modification of cellulose microfibril structure can be achieved genetically, which could be transformative in a bio-based economy. To explore these processes in planta, we developed a chemical genetic toolbox of pharmacological inhibitors and corresponding resistance-conferring point mutations in the C-terminal transmembrane domain region of CESA1(A903V) and CESA3(T942I) in Arabidopsis thaliana. Using (13)C solid-state nuclear magnetic resonance spectroscopy and X-ray diffraction, we show that the cellulose microfibrils displayed reduced width and an additional cellulose C4 peak indicative of a degree of crystallinity that is intermediate between the surface and interior glucans of wild type, suggesting a difference in glucan chain association during microfibril formation. Consistent with measurements of lower microfibril crystallinity, cellulose extracts from mutated CESA1(A903V) and CESA3(T942I) displayed greater saccharification efficiency than wild type. Using live-cell imaging to track fluorescently labeled CESA, we found that these mutants show increased CESA velocities in the plasma membrane, an indication of increased polymerization rate. Collectively, these data suggest that CESA1(A903V) and CESA3(T942I) have modified microfibril structure in terms of crystallinity and suggest that in plants, as in bacteria, crystallization biophysically limits polymerization.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Celulose/química , Glucosiltransferases/química , Glucosiltransferases/genética , Microfibrilas/química , Mutação/genética , Alelos , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Arabidopsis/efeitos dos fármacos , Arabidopsis/enzimologia , Proteínas de Arabidopsis/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Celulose/biossíntese , Cristalização , Resistência a Medicamentos/efeitos dos fármacos , Genes Dominantes/genética , Glucosiltransferases/metabolismo , Espectroscopia de Ressonância Magnética , Microfibrilas/efeitos dos fármacos , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Transporte Proteico/efeitos dos fármacos , Quinolinas/química , Quinolinas/farmacologia , Relação Estrutura-AtividadeRESUMO
We employed second-harmonic generation (SHG) imaging and the zebrafish model to investigate the myopathy caused by statin in vivo with emphasis on the altered microstructures of the muscle sarcomere, the fundamental contractile element of muscles. This approach derives an advantage of SHG imaging to observe the striated skeletal muscle of living zebrafish based on signals produced mainly from the thick myosin filament of sarcomeres without employing exogenous labels, and eliminates concern about the distortion of muscle structures caused by sample preparation in conventional histological examination. The treatment with statin caused a significantly shortened sarcomere relative to an untreated control (1.73±0.09 µm vs 1.91±0.08 µm, P<0.05) while the morphological integrity of the muscle fibers remained largely intact. Mechanistic tests indicated that this microstructural disorder was associated with the biosynthetic pathway of cholesterol, or, specifically, with the impaired production of mevalonate by statins. This microstructural disorder exhibited a strong dependence on both the dosage and the duration of treatment, indicating a possibility to assess the severity of muscle injury according to the altered length of the sarcomeres. In contrast to a conventional assessment of muscle injury using clinical biomarkers in blood, such as creatine kinase that is released from only disrupted myocytes, the ability to determine microstructural modification of sarcomeres allows diagnosis of muscle injury before an onset of conventional clinical symptoms. In light of the increasing prevalence of the incidence of muscle injuries caused by new therapies, our work consolidates the combined use of the zebrafish and SHG imaging as an effective and sensitive means to evaluate the safety profile of new therapeutic targets in vivo.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases/toxicidade , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Doenças Musculares/fisiopatologia , Sarcômeros/fisiologia , Peixe-Zebra/fisiologia , Animais , Colesterol/metabolismo , Relação Dose-Resposta a Droga , Feminino , Processamento de Imagem Assistida por Computador/métodos , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Larva/fisiologia , Masculino , Ácido Mevalônico/metabolismo , Microfibrilas/efeitos dos fármacos , Microfibrilas/fisiologia , Contração Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatologia , Doenças Musculares/induzido quimicamente , Doenças Musculares/metabolismo , Fatores de Tempo , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Aniline blue staining and callose immunolabeling revealed the deposition of significant callose quantities, in the form of fibrils, in the periclinal walls of guard cells (GCs) of stomata of the fern Asplenium nidus. The stomata that were at an early stage of differentiation displayed short callose fibrils at the junctions of the periclinal walls with the dorsal ones, which converged on the site of the future stomatal pore. In stomata being at an advanced stage of differentiation, callose fibrils were radially arranged around the stomatal pore, while in mature closed ones they were focused on the margins of the wall thickenings lining the stomatal pore. The pattern of the callose fibril organization resembled that of cellulose microfibrils in the same walls. Like the cellulose microfibrils, callose fibrils appeared coaligned with the underlying radial arrays of cortical microtubules (MTs). Moreover, the stomata treated with cellulose synthesis inhibitors (coumarin or dichlobenil) and those recovering from treatments with callose synthesis inhibitors (2-deoxy-D-glucose or tunicamycin) exhibited distinct radial callose fibril arrays. Cytochalasin B did not affect the organization of the radial callose fibril arrays. In contrast, oryzalin completely disturbed the pattern of callose deposition in the affected GCs. Therefore, the fibrillar callose orientation in the periclinal GC walls is probably controlled by MTs but not by actin filaments. The MTs seem to orient callose synthases in the plasmalemma, thus determining the fibrillar nature of callose deposits and their radial mode of arrangement. The cellulose microfibrils are not involved in the callose fibril alignment.
Assuntos
Gleiquênias/metabolismo , Glucanos/metabolismo , Microtúbulos/fisiologia , Estômatos de Plantas/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/fisiologia , Cumarínicos/farmacologia , Citocalasina B/farmacologia , Desoxiglucose/farmacologia , Dinitrobenzenos/farmacologia , Gleiquênias/efeitos dos fármacos , Gleiquênias/ultraestrutura , Herbicidas/farmacologia , Microfibrilas/efeitos dos fármacos , Microfibrilas/fisiologia , Microtúbulos/efeitos dos fármacos , Nitrilas/farmacologia , Estômatos de Plantas/efeitos dos fármacos , Estômatos de Plantas/ultraestrutura , Sulfanilamidas/farmacologia , Moduladores de Tubulina/farmacologia , Tunicamicina/farmacologiaRESUMO
Newly deposited microfibrils strongly colocalise with fibronectin in primary fibroblasts. Microfibril formation is grossly inhibited by fibronectin depletion, but rescued by supplementation with exogenous cellular fibronectin. As integrin receptors are key determinants of fibronectin assembly, we investigated whether they also influenced microfibril deposition. Analysis of beta1-integrin-receptor-null fibroblasts, blockage of cell surface integrin receptors that regulate fibronectin assembly and disruption of Rho kinase all result in suppressed deposition of both fibronectin and microfibrils. Antibody activation of beta1 integrins in fibronectin-depleted cultures is insufficient to rescue microfibril assembly. In fibronectin(RGE/RGE) mutant mouse fibroblast cultures, which do not engage alpha5beta1 integrin, extracellular assembly of both fibronectin and microfibrils is markedly reduced. Thus, pericellular microfibril assembly is regulated by fibronectin fibrillogenesis.
Assuntos
Fibronectinas/metabolismo , Fibronectinas/fisiologia , Microfibrilas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Animais , Células Cultivadas , Fibrilina-1 , Fibrilinas , Fibroblastos/metabolismo , Fibronectinas/antagonistas & inibidores , Humanos , Integrina alfa5beta1/antagonistas & inibidores , Integrina alfa5beta1/fisiologia , Integrina beta1/genética , Integrina beta1/metabolismo , Camundongos , Microfibrilas/efeitos dos fármacos , Modelos Biológicos , Polímeros/metabolismo , Ligação Proteica/efeitos dos fármacos , RNA Interferente Pequeno/farmacologiaRESUMO
One of the major pathological features of Alzheimer's disease (AD) is the appearance of senile plaques characterized by extracellular aggregation of amyloid beta-peptide (Abeta) fibrils. Inhibition of Abeta fibril aggregation is therefore viewed as one possible method to halt the progression of AD. Salvianolic acid B (Sal B) is an active ingredient isolated from Salvia miltiorrhiza, a Chinese herbal medicine commonly used for the treatment of cardiovascular and cerebrovascular disorders. Recent findings show that Sal B prevents Abeta-induced cytotoxicity in a rat neural cell line. To understand the mechanism of Sal B-mediated neuroprotection, its effects on the inhibition of Abeta1-40 fibril formation and destabilization of the preformed Abeta1-40 fibrils were studied. The results were obtained using Thioflavin T fluorescence assay and Abeta aggregating immunoassay. We found that Sal B can inhibit fibril aggregation (IC(50): 1.54-5.37 microM) as well as destabilize preformed Abeta fibril (IC(50): 5.00-5.19 microM) in a dose- and time-dependent manner. Sal B is a better aggregation inhibitor than ferulic acid but less active than curcumin in the inhibition of Abeta1-40 aggregation. In electron microscope study, Sal B-treated Abeta1-40 fibrils are seen in various stages of shortening or wrinkling with numerous deformed aggregates of amorphous structure. Circular dichroism data indicate that Sal B dose dependently prevents the formation of beta-structured aggregates of Abeta1-40. Addition of preincubated Sal B with Abeta1-42 significantly reduces its cytotoxic effects on human neuroblastoma SH-SY5Y cells. These results suggest that Sal B has therapeutic potential in the treatment of AD, and warrant its study in animal models.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/toxicidade , Antioxidantes/farmacologia , Benzofuranos/farmacologia , Microfibrilas/efeitos dos fármacos , Peptídeos beta-Amiloides/biossíntese , Benzotiazóis , Agregação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Dicroísmo Circular , Interpretação Estatística de Dados , Ensaio de Imunoadsorção Enzimática , Humanos , Microfibrilas/ultraestrutura , Microscopia Eletrônica , Sais de Tetrazólio , Tiazóis/farmacologiaRESUMO
It is a well-known hypothesis that cortical microtubules control the direction of cellulose microfibril deposition, and that the parallel cellulose microfibrils determine anisotropic cell expansion and plant cell morphogenesis. However, the molecular mechanism by which cortical microtubules regulate the orientation of cellulose microfibrils is still unclear. To investigate this mechanism, chemical genetic screening was performed. From this screening, 'SS compounds' were identified that induced a spherical swelling phenotype in tobacco BY-2 cells. The SS compounds could be categorized into three classes: those that disrupted the cortical microtubules; those that reduced cellulose microfibril content; and thirdly those that had neither of these effects. In the last class, a chemical designated 'cobtorin' was found to induce the spherical swelling phenotype at the lowest concentration, suggesting strong binding activity to the putative target. Examining cellulose microfibril regeneration using taxol-treated protoplasts revealed that the cobtorin compound perturbed the parallel alignment of pre-existing cortical microtubules and nascent cellulose microfibrils. Thus, cobtorin could be a novel inhibitor and an attractive tool for further investigation of the mechanism that enables cortical microtubules to guide the parallel deposition of cellulose microfibrils.
Assuntos
Celulose/metabolismo , Microfibrilas/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Moduladores de Tubulina/farmacologia , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/metabolismo , Linhagem Celular , Parede Celular , Celulose/química , Técnicas de Química Combinatória , Regulação da Expressão Gênica de Plantas , Microfibrilas/metabolismo , Estrutura Molecular , Nicotiana/citologia , Nicotiana/efeitos dos fármacos , Moduladores de Tubulina/químicaRESUMO
The aggregation of alpha-synuclein (alphaS) has been implicated as a critical step in the development of Lewy body diseases (LBD) and multiple system atrophy (MSA). Both retrospective and prospective epidemiological studies have consistently demonstrated an inverse association between cigarette smoking and Parkinson's disease (PD). We used fluorescence spectroscopy with thioflavin S, electron microscopy and atomic force microscopy to examine the effects of nicotine, pyridine, and N-methylpyrrolidine on the formation of alphaS fibrils (f alphaS) from wild-type alphaS (alphaS (WT)) and A53T mutant alphaS (A53T) and on preformed f alpha Ss. Nicotine dose-dependently inhibited the f alphaS formation from both alphaS (WT) and A53T. Moreover, nicotine dose-dependently destabilized preformed f alpha Ss. These effects of nicotine were similar to those of N-methylpyrrolidine. The anti-fibrillogenic activity of nicotine may be exerted not only by the inhibition of f alphaS formation but also by the destabilization of preformed f alphaS. Additionally, this effect may be attributed to N-methylpyrrolidine moieties of nicotine.
Assuntos
Doença por Corpos de Lewy/prevenção & controle , Microfibrilas/efeitos dos fármacos , Microfibrilas/patologia , Nicotina/farmacologia , Nicotina/uso terapêutico , Agonistas Nicotínicos/farmacologia , Agonistas Nicotínicos/uso terapêutico , Benzotiazóis , Humanos , Cinética , Doença por Corpos de Lewy/patologia , Microfibrilas/ultraestrutura , Microscopia de Força Atômica , Microscopia Eletrônica , Mutação , Piridinas/metabolismo , Pirrolidinas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Fluorescência , Tiazóis , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
Versican interacts with hyaluronan (HA) at its N-terminus and with fibrillin-1 at its C terminus. As versican in the dermis connects microfibrils to the HA-rich matrix for viscoelasticity, dermal diseases may involve destruction of these complexes. A recombinant versican protein, rVN, covering the HA binding region (HABR) of human versican and a polyclonal antibody, 6084, against rVN were prepared and characterized. Blotting analyses of skin extracts with 6084 and biotin-conjugated HA revealed that versican was a major HA-binding component in the dermis. Matrix metalloprotease-12, which is expressed in areas of solar elastosis, degraded versican and abrogated its HA-binding ability. Immunohistochemical analyses revealed that the elastic materials in solar elastosis lesions were negative for 6084, but positive for 2B1, an antibody recognizing the C-terminus of versican, indicating loss of the HABR in the aggregated elastic fibers. This loss of the HA-binding ability of versican followed by HA exclusion may be responsible for the pathological and phenotypical changes observed in solar elastosis.
Assuntos
Derme/metabolismo , Ácido Hialurônico/metabolismo , Envelhecimento da Pele/patologia , Envelhecimento da Pele/fisiologia , Versicanas/metabolismo , Anticorpos/imunologia , Sítios de Ligação/imunologia , Tecido Conjuntivo/efeitos dos fármacos , Tecido Conjuntivo/metabolismo , Derme/efeitos dos fármacos , Tecido Elástico/efeitos dos fármacos , Tecido Elástico/metabolismo , Humanos , Metaloproteinase 12 da Matriz/farmacologia , Microfibrilas/efeitos dos fármacos , Microfibrilas/metabolismo , Ligação Proteica/fisiologia , Proteínas Recombinantes/imunologia , Versicanas/efeitos dos fármacos , Versicanas/imunologiaRESUMO
The amyloidoses are the extracellular subset of a group of diseases in which in vivo protein misfolding leads to a pathologic gain of function, i.e., aggregation leading to protein deposition, with subsequent tissue damage. Wild-type and mutant transthyretins (TTR) are the etiologic agents in prototypic systemic amyloidoses. We describe a cell-based assay that measures the cytotoxicity of physiologic concentrations of the amyloidogenic Val30Met TTR variant (V30M TTR) using cells of the same lineage as the in vivo tissue target of amyloid deposition. We have utilized the assay to screen small molecules for their capacity to inhibit the TTR-induced cell damage. We compared the inhibitory activity of each compound with its ability to prevent TTR fibril formation in vitro. Our results emphasize the importance of screening compounds under physiologic conditions. Moreover, if a common conformational intermediate is responsible for cell death in all the amyloid diseases, the cell-based assay has the potential to aid in the discovery of compounds useful in the treatment of amyloidoses caused by other misfolded proteins as well as those caused by TTR.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Pré-Albumina/antagonistas & inibidores , Pré-Albumina/metabolismo , Amiloidose/tratamento farmacológico , Amiloidose/genética , Amiloidose/metabolismo , Linhagem Celular Tumoral , Linhagem da Célula/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Diflunisal/análogos & derivados , Diflunisal/farmacologia , Avaliação Pré-Clínica de Medicamentos , Humanos , Metionina/genética , Microfibrilas/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Pré-Albumina/genética , Pré-Albumina/toxicidade , Resveratrol , Estilbenos/farmacologia , Valina/genéticaRESUMO
The roles of cellulose microfibrils and cortical microtubules in establishing and maintaining the pattern of secondary-cell-wall deposition in tracheary elements were investigated with direct dyes to inhibit cellulose microfibril assembly and amiprophosmethyl to inhibit microtubule polymerization. When direct dyes were added to xylogenic cultures of Zinnia elegans L. mesophyll cells just before the onset of differentiation, the secondary cell wall was initially secreted as bands composed of discrete masses of stained material, consistent with immobilized sites of cellulose synthesis. The masses coalesced, forming truncated, sinuous or smeared thickenings, as secondary cell wall deposition continued. The absence of ordered cellulose microfibrils was confirmed by polarization microscopy and a lack of fluorescence dichroism as determined by laser scanning microscopy. Indirect immunofluorescence showed that cortical microtubules initially subtended the masses of dye-altered secondary cell wall material but soon became disorganized and disappeared. Although most of the secondary cell wall was deposited in the absence of subtending cortical microtubules in dye-treated cells, secretion remained confined to discrete regions of the plasma membrane. Examination of non-dye-treated cultures following application of microtubule inhibitors during various stages of secondary-cell-wall deposition revealed that the pattern became fixed at an early stage such that deposition remained localized in the absence of cortical microtubules. These observations indicate that cortical microtubules are required to establish, but not to maintain, patterned secondary-cell-wall deposition. Furthermore, cellulose microfibrils play a role in maintaining microtubule arrays and the integrity of the secondary-cell-wall bands during deposition.
Assuntos
Parede Celular/ultraestrutura , Celulose/metabolismo , Microfibrilas/ultraestrutura , Microtúbulos/ultraestrutura , Células Vegetais , Asteraceae , Parede Celular/efeitos dos fármacos , Parede Celular/fisiologia , Células Cultivadas , Vermelho Congo/farmacologia , Azul Evans/farmacologia , Imunofluorescência , Microfibrilas/efeitos dos fármacos , Microfibrilas/fisiologia , Microtúbulos/efeitos dos fármacos , Microtúbulos/fisiologia , Modelos Biológicos , Plantas/metabolismoAssuntos
Citoesqueleto/efeitos dos fármacos , Giberelinas/antagonistas & inibidores , Nicotiana/efeitos dos fármacos , Estruturas Vegetais/efeitos dos fármacos , Pirimidinas/toxicidade , Teratogênicos/toxicidade , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Tamanho Celular/efeitos dos fármacos , Tamanho Celular/fisiologia , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Parede Celular/patologia , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Giberelinas/metabolismo , Microfibrilas/efeitos dos fármacos , Microfibrilas/metabolismo , Microfibrilas/patologia , Esteroides/antagonistas & inibidores , Esteroides/metabolismo , Nicotiana/citologiaRESUMO
The lue1 mutant was previously isolated in a bio-imaging screen for Arabidopsis mutants exhibiting inappropriate regulation of an AtGA20ox1 promoter-luciferase reporter fusion. Here we show that lue1 is allelic to fra2, bot1 and erh3, and encodes a truncated katanin-like microtubule-severing protein (AtKSS). Complementation of lue1 with the wild-type AtKSS gene restored both wild-type stature and luciferase reporter levels. Hormonal responses of lue1 to ethylene and gibberellins revealed inappropriate cortical microtubule reorientation during cell growth. Moreover, a fusion between the AtKSS protein and GFP decorated cortical microtubules. A yeast two-hybrid screen with AtKSS as the bait identified proteins related to those involved in microtubule processing, including a katanin p80 subunit and a kinesin ortholog. These results indicate that AtKSS is involved in microtubule dynamics in response to plant hormones.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Microtúbulos/metabolismo , Reguladores de Crescimento de Plantas/farmacologia , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/fisiologia , Sequência de Aminoácidos , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Etilenos/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes Reporter/genética , Giberelinas/farmacologia , Glucuronidase/genética , Glucuronidase/metabolismo , Katanina , Microfibrilas/efeitos dos fármacos , Microfibrilas/metabolismo , Microscopia de Polarização , Microtúbulos/efeitos dos fármacos , Dados de Sequência Molecular , Mutação , Fenótipo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The non-beta-amyloid (Abeta) component of Alzheimer's disease amyloid (NAC) and its precursor alpha-synuclein have been linked to amyloidogenesis in several neurodegenerative diseases. NAC and alpha-synuclein both form beta-sheet structures upon ageing, aggregate to form fibrils, and are neurotoxic. We recently established that a peptide comprising residues 3-18 of NAC retains these properties. To pinpoint the exact region responsible we have carried out assays of toxicity and physicochemical properties on smaller fragments of NAC. Toxicity was measured by the ability of fresh and aged peptides to inhibit the reduction of the redox dye 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) by rat pheochromocytoma PC12 cells and human neuroblastoma SHSY-5Y cells. On immediate dissolution, or after ageing, the fragments NAC(8-18) and NAC(8-16) are toxic, whereas NAC(12-18), NAC(9-16) and NAC(8-15) are not. Circular dichroism indicates that none of the peptides displays beta-sheet structure; rather all remain random coil throughout 24 h. However, in acetonitrile, an organic solvent known to induce beta sheet, fragments NAC(8-18) and NAC(8-16) both form beta-sheet structure. Only NAC(8-18) aggregates, as indicated by concentration of peptide remaining in solution after 3 days, and forms fibrils, as determined by electron microscopy. These findings indicate that residues 8-16 of NAC, equivalent to residues 68-76 in alpha-synuclein, comprise the region crucial for toxicity.
Assuntos
Amiloide/química , Amiloide/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Fragmentos de Peptídeos/toxicidade , Acetonitrilas , Doença de Alzheimer , Sequência de Aminoácidos , Animais , Dicroísmo Circular , Exocitose , Humanos , Microfibrilas/efeitos dos fármacos , Microfibrilas/patologia , Microfibrilas/ultraestrutura , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/fisiologia , Células PC12 , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fosfoproteínas/química , Fosfoproteínas/fisiologia , Conformação Proteica , Ratos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Sinucleínas , alfa-SinucleínaRESUMO
The amyloid beta-peptide (Abeta) is a principal component of insoluble amyloid plaques which are characteristic neuropathological features of Alzheimer's disease. Abeta also exists as a normal soluble protein that undergoes a pathogenic transition to an aggregated, fibrous form. This transition can be affected by extraneous proteinaceous and nonproteinaceous elements, such as zinc ions, which may promote aggregation and/or stabilization of the fibrils. Protein chelation of zinc is typically mediated by histidines, cysteines and carboxylates. Previous studies have demonstrated that the Abeta-Zn2+ binding site is localized within residues 6-28 and that histidines may serve as the principal sites of interaction. To localize key residues within this region, a series of Abeta peptides (residues 1-28) were synthesized that contained systematic His/Ala substitutions. Circular dichroism and electron microscopy were used to monitor the effects of Zn2+ on the peptide beta-sheet conformation and fibril aggregation. Our results indicate that substitution of either His13 or His14 but not His6 eliminates the zinc-mediated effects. These observations indicate a specific zinc binding site within Abeta that involves these central histidine residues.
Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/química , Zinco/metabolismo , Substituição de Aminoácidos , Peptídeos beta-Amiloides/ultraestrutura , Sítios de Ligação , Dicroísmo Circular , Cisteína , Histidina , Microfibrilas/efeitos dos fármacos , Microfibrilas/ultraestrutura , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Estrutura Secundária de Proteína , Zinco/farmacologiaRESUMO
Cultured mesophyll protoplasts of Nicotiana tabacum L. can be hormonally induced into different developmental pathways. In a medium containing auxins (NAA) and cytokinins (BAP) cells divide and eventually give rise to calli. When only auxins are present cells elongate and finally differentiate into very long tubular cells. We focused on the sequence of events leading to elongation. When cultured in a high (1 mg/l) auxin concentration elongating cells seem to pass a certain threshold and increase their nuclear DNA up to about 16C. Cells cultured in a low (0.065 mg/l) auxin concentration only have C-values up to 4C, are unable to pass this threshold and finally fail to elongate. Besides the concentration dependence of the auxin signal, the efflux of auxin seems to be necessary for elongation since addition of TIBA drastically reduces the amount of elongating cells. Concomitant with the changes in nuclear physiology, auxin-induced axiality is seen as sequential rearrangements of microtubules and actin-filaments and of cell wall cellulose microfibrils from 'randomly' arranged in spherical cells to an orientation perpendicular to the long axis of elongating cells.