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1.
Cells ; 11(16)2022 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-36010656

RESUMO

GM1-gangliosidosis is a catastrophic, neurodegenerative lysosomal storage disease caused by a deficiency of lysosomal ß-galactosidase (ß-Gal). The primary substrate of the enzyme is GM1-ganglioside (GM1), a sialylated glycosphingolipid abundant in nervous tissue. Patients with GM1-gangliosidosis present with massive and progressive accumulation of GM1 in the central nervous system (CNS), which leads to mental and motor decline, progressive neurodegeneration, and early death. No therapy is currently available for this lysosomal storage disease. Here, we describe a proof-of-concept preclinical study toward the development of enzyme replacement therapy (ERT) for GM1-gangliosidosis using a recombinant murine ß-Gal fused to the plant lectin subunit B of ricin (mß-Gal:RTB). We show that long-term, bi-weekly systemic injection of mß-Gal:RTB in the ß-Gal-/- mouse model resulted in widespread internalization of the enzyme by cells of visceral organs, with consequent restoration of enzyme activity. Most importantly, ß-Gal activity was detected in several brain regions. This was accompanied by a reduction of accumulated GM1, reversal of neuroinflammation, and decrease in the apoptotic marker caspase 3. These results indicate that the RTB lectin delivery module enhances both the CNS-biodistribution pattern and the therapeutic efficacy of the ß-Gal ERT, with the potential to translate to a clinical setting for the treatment of GM1-gangliosidosis.


Assuntos
Gangliosídeo G(M1) , Gangliosidose GM1 , Animais , Sistema Nervoso Central/metabolismo , Terapia de Reposição de Enzimas , Gangliosidose GM1/tratamento farmacológico , Gangliosidose GM1/genética , Lectinas/uso terapêutico , Camundongos , Distribuição Tecidual , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
2.
Bull Environ Contam Toxicol ; 105(2): 211-217, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32594201

RESUMO

The objective of this study is to compare the physiological response (content and degradation of photosynthetic pigments, membrane oxidation products and soluble proteins) and multi-element content of Ramalina celastri (lichenized fungi) growing on agricultural fences with no-tillage (associated with transgenic crops and agrochemical application), organic cropping and a non-cultivated area. We found that R. celastri did not differ in its physiological response to agricultural practices, except for the contents of chlorophyll b and phaeophytin a which were high in both cultivated areas. Lichens growing in organic cropping fields have higher arsenic, chromium, uranium and internal transition elements common in the earth's crust, possibly due to the greater resuspension of the material during soil tillage. Lichens that grow on posts close to no-tillage field had higher bromine contents (present in numerous pesticides). We found evidence that R. celastri behaves as a tolerant species to air pollution in agricultural environments.


Assuntos
Poluentes Atmosféricos/análise , Monitoramento Biológico/métodos , Produtos Agrícolas/crescimento & desenvolvimento , Líquens/crescimento & desenvolvimento , Praguicidas/análise , Poluição do Ar/análise , Clorofila/metabolismo , Líquens/química , Líquens/fisiologia , Agricultura Orgânica , Fotossíntese/efeitos dos fármacos , Projetos Piloto , Solo/química
3.
Int J Mol Sci ; 21(3)2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-32024082

RESUMO

The greatest challenges for therapeutic efficacy of many macromolecular drugs that act on intracellular are delivery to key organs and tissues and delivery into cells and subcellular compartments. Transport of drugs into critical cells associated with disease, including those in organs protected by restrictive biological barriers such as central nervous system (CNS), bone, and eye remains a significant hurdle to drug efficacy and impacts commercial risk and incentives for drug development for many diseases. These limitations expose a significant need for the development of novel strategies for macromolecule delivery. RTB lectin is the non-toxic carbohydrate-binding subunit B of ricin toxin with high affinity for galactose/galactosamine-containing glycolipids and glycoproteins common on human cell surfaces. RTB mediates endocytic uptake into mammalian cells by multiple routes exploiting both adsorptive-mediated and receptor-mediated mechanisms. In vivo biodistribution studies in lysosomal storage disease models provide evidence for the theory that the RTB-lectin transports corrective doses of enzymes across the blood-brain barrier to treat CNS pathologies. These results encompass significant implications for protein-based therapeutic approaches to address lysosomal and other diseases having strong CNS involvement.


Assuntos
Barreira Hematoencefálica/metabolismo , Sistema Nervoso Central/metabolismo , Sistemas de Liberação de Medicamentos , Lectinas/química , Substâncias Macromoleculares/metabolismo , Animais , Transporte Biológico , Humanos , Distribuição Tecidual
4.
Dis Model Mech ; 9(7): 769-78, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27482815

RESUMO

Glucocerebrosidase is a lysosomal hydrolase involved in the breakdown of glucosylceramide. Gaucher disease, a recessive lysosomal storage disorder, is caused by mutations in the gene GBA1 Dysfunctional glucocerebrosidase leads to accumulation of glucosylceramide and glycosylsphingosine in various cell types and organs. Mutations in GBA1 are also a common genetic risk factor for Parkinson disease and related synucleinopathies. In recent years, research on the pathophysiology of Gaucher disease, the molecular link between Gaucher and Parkinson disease, and novel therapeutics, have accelerated the need for relevant cell models with GBA1 mutations. Although induced pluripotent stem cells, primary rodent neurons, and transfected neuroblastoma cell lines have been used to study the effect of glucocerebrosidase deficiency on neuronal function, these models have limitations because of challenges in culturing and propagating the cells, low yield, and the introduction of exogenous mutant GBA1 To address some of these difficulties, we established a high yield, easy-to-culture mouse neuronal cell model with nearly complete glucocerebrosidase deficiency representative of Gaucher disease. We successfully immortalized cortical neurons from embryonic null allele gba(-/-) mice and the control littermate (gba(+/+)) by infecting differentiated primary cortical neurons in culture with an EF1α-SV40T lentivirus. Immortalized gba(-/-) neurons lack glucocerebrosidase protein and enzyme activity, and exhibit a dramatic increase in glucosylceramide and glucosylsphingosine accumulation, enlarged lysosomes, and an impaired ATP-dependent calcium-influx response; these phenotypical characteristics were absent in gba(+/+) neurons. This null allele gba(-/-) mouse neuronal model provides a much-needed tool to study the pathophysiology of Gaucher disease and to evaluate new therapies.


Assuntos
Doença de Gaucher/fisiopatologia , Doença de Gaucher/terapia , Glucosilceramidase/deficiência , Modelos Biológicos , Neurônios/enzimologia , Neurônios/patologia , Trifosfato de Adenosina/metabolismo , Animais , Antígenos Transformantes de Poliomavirus/metabolismo , Antígeno CD24/metabolismo , Cálcio/metabolismo , Linhagem Celular Transformada , Células Cultivadas , Doença de Gaucher/enzimologia , Glucosilceramidase/metabolismo , Cariotipagem , Lisossomos/metabolismo , Camundongos Endogâmicos C57BL , Fator 1 de Elongação de Peptídeos/genética , Fator 1 de Elongação de Peptídeos/metabolismo , Regiões Promotoras Genéticas/genética , Especificidade por Substrato
5.
Data Brief ; 6: 1016-22, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26958633

RESUMO

GM1-gangliosidosis is an inherited autosomal recessive disorder caused by mutations in the gene GLB1, which encodes acid ß-galactosidase (ß-gal). The lack of activity in this lysosomal enzyme leads to accumulation of GM1 gangliosides (GM1) in cells. We have developed a high-content-imaging method to assess GM1 levels in fibroblasts that can be used to evaluate substrate reduction in treated GLB1(-/-) cells [1]. This assay allows fluorescent quantification in a multi-well system which generates unbiased and statistically significant data. Fluorescently labeled Cholera Toxin B subunit (CTXB), which specifically binds to GM1 gangliosides, was used to detect in situ GM1 levels in a fixed monolayer of fibroblasts. This sensitive, rapid, and inexpensive method facilitates in vitro drug screening in a format that allows a high number of replicates using low working volumes.

6.
Mol Genet Metab ; 117(2): 199-209, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26766614

RESUMO

New enzyme delivery technologies are required for treatment of lysosomal storage disorders with significant pathologies associated with the so-called "hard-to-treat" tissues and organs. Genetic deficiencies in the GLB1 gene encoding acid ß-galactosidase lead to GM1-gangliosidosis or Morquio B, lysosomal diseases with predominant disease manifestation associated with the central nervous system or skeletal system, respectively. Current lysosomal ERTs are delivered into cells based on receptor-mediated endocytosis and do not effectively address several hard-to-treat organs including those critical for GM1-gangliosidosis patients. Lectins provide alternative cell-uptake mechanisms based on adsorptive-mediated endocytosis and thus may provide unique biodistribution for lysosomal disease therapeutics. In the current study, genetic fusions of the plant galactose/galactosamine-binding lectin, RTB, and the human acid ß-galactosidase enzyme were produced using a plant-based bioproduction platform. ß-gal:RTB and RTB:ß-gal fusion products retained both lectin activity and ß-galactosidase activity. Purified proteins representing both fusion orientations were efficiently taken up into GM1 patient fibroblasts and mediated the reduction of GM1 ganglioside substrate with activities matching mammalian cell-derived ß-galactosidase. In contrast, plant-derived ß-gal alone was enzymatically active but did not mediate uptake or correction indicating the need for either lectin-based (plant product) or mannose-6-phosphate-based (mammalian product) delivery. Native ß-galactosidase undergoes catalytic activation (cleavage within the C-terminal region) in lysosomes and is stabilized by association with protective protein/cathepsin A. Enzymatic activity and lysosomal protein processing of the RTB fusions were assessed following internalization into GM1 fibroblasts. Within 1-4h, both ß-gal:RTB and RTB:ß-gal were processed to the ~64kDa "activated" ß-gal form; the RTB lectin was cleaved and rapidly degraded. The activated ß-gal was still detected at 48h suggesting interactions with protective protein/cathepsin A. Uptake-saturation analyses indicated that the RTB adsorptive-mediated mechanisms of ß-gal:RTB supported significantly greater accumulation of ß-galactose activity in fibroblasts compared to the receptor-mediated mechanisms of the mammalian cell-derived ß-gal. These data demonstrate that plant-made ß-gal:RTB functions as an effective replacement enzyme for GM1-gangliosidosis - delivering enzyme into cells, enabling essential lysosomal processing, and mediating disease substrate clearance at the cellular level. RTB provides novel uptake behaviors and thus may provide new receptor-independent strategies that could broadly impact lysosomal disease treatments.


Assuntos
Gangliosidose GM1/tratamento farmacológico , Proteínas Recombinantes de Fusão/metabolismo , beta-Galactosidase/metabolismo , Células Cultivadas , Terapia de Reposição de Enzimas , Fibroblastos/enzimologia , Humanos , Cinética , Lisossomos/metabolismo , Lectinas de Plantas/química , Lectinas de Plantas/genética , Lectinas de Plantas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Nicotiana , beta-Galactosidase/química , beta-Galactosidase/genética
7.
Sci Rep ; 5: 14144, 2015 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-26382970

RESUMO

Enzyme replacement therapies have revolutionized patient treatment for multiple rare lysosomal storage diseases but show limited effectiveness for addressing pathologies in "hard-to-treat" organs and tissues including brain and bone. Here we investigate the plant lectin RTB as a novel carrier for human lysosomal enzymes. RTB enters mammalian cells by multiple mechanisms including both adsorptive-mediated and receptor-mediated endocytosis, and thus provides access to a broader array of organs and cells. Fusion proteins comprised of RTB and human α-L-iduronidase, the corrective enzyme for Mucopolysaccharidosis type I, were produced using a tobacco-based expression system. Fusion products retained both lectin selectivity and enzyme activity, were efficiently endocytosed into human fibroblasts, and corrected the disease phenotype of mucopolysaccharidosis patient fibroblasts in vitro. RTB-mediated delivery was independent of high-mannose and mannose-6-phosphate receptors, which are exploited for delivery of currently approved lysosomal enzyme therapeutics. Thus, the RTB carrier may support distinct in vivo pharmacodynamics with potential to address hard-to-treat tissues.


Assuntos
Portadores de Fármacos , Sistemas de Liberação de Medicamentos , Ricina , Terapia de Reposição de Enzimas , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Expressão Gênica , Glicosaminoglicanos/metabolismo , Humanos , Iduronidase/administração & dosagem , Iduronidase/genética , Iduronidase/metabolismo , Lectinas Tipo C/metabolismo , Doenças por Armazenamento dos Lisossomos/terapia , Receptor de Manose , Lectinas de Ligação a Manose/metabolismo , Fenótipo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Receptor IGF Tipo 2/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes de Fusão , Ricina/genética , Ricina/metabolismo , Nicotiana/química
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