RESUMO
Galectin-2 (Gal-2) is an animal lectin with specificity for ß-galactosides. It is predominantly expressed and suggested to play a protective function in the gastrointestinal tract; therefore, it can be used as a protein drug. Recombinant proteins have been expressed using Escherichia coli and used to study the function of Gal-2. The recombinant human Gal-2 (hGal-2) protein purified via affinity chromatography after being expressed in E. coli was not completely homogeneous. Mass spectrometry confirmed that some recombinant Gal-2 were phosphogluconoylated. In contrast, the recombinant mouse Gal-2 (mGal-2) protein purified using affinity chromatography after being expressed in E. coli contained a different form of Gal-2 with a larger molecular weight. This was due to mistranslating the original mGal-2 stop codon TGA to tryptophan (TGG). In this report, to obtain a homogeneous Gal-2 protein for further studies, we attempted the following methods: for hGal-2, 1) replacement of the lysine (Lys) residues, which was easily phosphogluconoylated with arginine (Arg) residues, and 2) addition of histidine (His)-tag on the N-terminus of the recombinant protein and cleavage with protease after expression; for mGal-2, 3) changing the stop codon from TGA to TAA, which is commonly used in E. coli. We obtained an almost homogeneous recombinant Gal-2 protein (human and mouse). These results have important implications for using Gal-2 as a protein drug.
Assuntos
Escherichia coli , Galectina 2 , Camundongos , Animais , Humanos , Galectina 2/química , Escherichia coli/genética , Escherichia coli/metabolismo , Códon de Terminação/metabolismo , Proteínas Recombinantes/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
Osteoclasts are the only bone-resorbing cells in organisms and understanding their differentiation mechanism is crucial for the treatment of osteoporosis. In the present study, we investigated the effect of Thiamet G, an O-GlcNAcase specific inhibitor, on osteoclastogenic differentiation. Thiamet G treatment increased global O-GlcNAcylation in murine RAW264 cells and suppressed receptor activator of nuclear factor-κB ligand (RANKL)-dependent formation in tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells, thereby suppressing the upregulation of osteoclast specific genes. Meanwhile, knockdown of O-linked N-acetylglucosamine (O-GlcNAc) transferase promoted the formation TRAP-positive multinuclear cells. Thiamet G treatment also suppressed RANKL and macrophage colony-stimulating factor (M-CSF) dependent osteoclast formation and bone-resorbing activity in mouse primary bone marrow cells and human peripheral blood mononuclear cells. These results indicate that the promotion of O-GlcNAc modification specifically suppresses osteoclast formation and its activity and suggest that chemicals affecting O-GlcNAc modification might potentially be useful in the prevention or treatment of osteoporosis in future.
Assuntos
Diferenciação Celular/efeitos dos fármacos , N-Acetilglucosaminiltransferases/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Piranos/farmacologia , Tiazóis/farmacologia , Animais , Diferenciação Celular/fisiologia , Feminino , Glicosilação/efeitos dos fármacos , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Masculino , Camundongos , Células RAW 264.7RESUMO
Galectins are a group of animal lectins characterized by their specificity for ß-galactosides. Of these, galectin-2 (Gal-2) is predominantly expressed in the gastrointestinal tract. In the current study, we used a mouse gastric mucous fraction to investigate whether Gal-2 is secreted from epithelial cells and identify its potential ligands in gastric mucus. Gal-2 was detected in the mouse gastric mucous fraction and could be eluted from it by the addition of lactose. Affinity chromatography using recombinant mouse galectin-2 (mGal-2)-immobilized adsorbent and subsequent LC-MS/MS identified MUC5AC, one of the major gastric mucin glycoproteins, as a potential ligand of mGal-2. Furthermore, MUC5AC was detected in the mouse gastric mucous fraction by Western blotting, and recombinant mGal-2 was adsorbed to this fraction in a carbohydrate-dependent manner. These results suggested that Gal-2 and MUC5AC in mouse gastric mucus interact in a ß-galactoside-dependent manner, resulting in a stronger barrier structure protecting the mucosal surface.
Assuntos
Galectina 2/química , Trato Gastrointestinal/química , Mucina-5AC/química , Animais , Humanos , Lactose , Camundongos , Muco , EstômagoRESUMO
Helicobacter pylori is associated with the onset of gastritis, peptic ulcers, and gastric cancer. Galectins are a family of ß-galactoside-binding proteins involved in diverse biological phenomena. Galectin-2 (Gal-2), a member of the galectin family, is predominantly expressed in the gastrointestinal tract. Although some galectin family proteins are involved in immunoreaction, the role of Gal-2 against H. pylori infection remains unclear. In this study, the effects of Gal-2 on H. pylori morphology and survival were examined. Gal-2 induced H. pylori aggregation depending on ß-galactoside and demonstrated a bactericidal effect. Immunohistochemical staining of the gastric tissue indicated that Gal-2 existed in the gastric mucus, as well as mucosa. These results suggested that Gal-2 plays a role in innate immunity against H. pylori infection in gastric mucus.
Assuntos
Galactosídeos/farmacologia , Galectina 2/farmacologia , Helicobacter pylori/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Animais , Infecções por Helicobacter , Helicobacter pylori/crescimento & desenvolvimento , Humanos , Masculino , CamundongosRESUMO
Galactoseß1-4Fucose (GalFuc) is a unique disaccharide found in invertebrates including nematodes. A fungal galectin CGL2 suppresses nematode development by recognizing the galactoseß1-4fucose epitope. The Caenorhabditis elegans galectin LEC-6 recognizes it as an endogenous ligand and the Glu67 residue of LEC-6 is responsible for this interaction. We found that mammalian galectin-2 (Gal-2) also has a comparable glutamate residue, Glu52. In the present study, we investigated the potential nematode-suppressing activity of Gal-2 using C. elegans as a model and focusing on Gal-2 binding to the GalFuc epitope. Gal-2 suppressed C. elegans development whereas its E52D mutant (Glu52 substituted by Asp), galectin-1 and galectin-3 had little effect on C. elegans growth. Lectin-staining using fluorescently-labeled Gal-2 revealed that, like CGL2, it specifically binds to the C. elegans intestine. Natural C. elegans glycoconjugates were specifically bound by immobilized Gal-2. Western blotting with anti-GalFuc antibody showed that the bound glycoconjugates had the GalFuc epitope. Frontal affinity chromatography with pyridylamine-labeled C. elegans N-glycans disclosed that Gal-2 (but not its E52D mutant) recognizes the GalFuc epitope. Gal-2 also binds to the GalFuc-bearing glycoconjugates of Ascaris and the GalFuc epitope is present in the parasitic nematodes Nippostrongylus brasiliensis and Brugia pahangi. These results indicate that Gal-2 suppresses C. elegans development by binding to its GalFuc epitope. The findings also imply that Gal-2 may prevent infestations of various parasitic nematodes bearing the GalFuc epitope.
Assuntos
Caenorhabditis elegans/crescimento & desenvolvimento , Dissacarídeos/química , Epitopos/química , Galectina 2/metabolismo , Animais , Ascaris suum/crescimento & desenvolvimento , Ascaris suum/metabolismo , Sítios de Ligação , Biomphalaria , Caenorhabditis elegans/metabolismo , Dissacarídeos/metabolismo , Epitopos/metabolismo , Galectina 2/química , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos ICRRESUMO
Phthalates, known as reproductive toxicants and endocrine disruptors, are widely used as plasticizers in polyvinyl chloride products. The present study was conducted for risk identification of dermal exposure to phthalates. When dibutyl phthalate was applied to the skin of hairless rats and humans, only monobutyl phthalate appeared through the skin, and the permeability of the skin was higher than that after the application of the monoester directly. The inhibition of skin esterases made the skin impermeable to the metabolite following dermal exposure to dibutyl ester, whereas removal of the stratum corneum from the skin did not change the skin permeation behavior. Similar phenomena were observed for benzyl butyl phthalate. The skin permeability of monobenzyl phthalate was higher than that of monobutyl phthalate in humans, although the reverse was observed in rats. Species difference in skin permeation profile corresponded to the esterase activity of the skin homogenate. Di(2-ethylhexyl) phthalate, which was not metabolized by esterases in the skin, was not transported across the skin. These results suggest that highly lipophilic phthalates may be transported easily across the stratum corneum lipids. The water-rich viable layer may become permeable to these phthalates by their metabolism into monoesters, which are relatively hydrophilic. Skin metabolism is essential to the percutaneous absorption of phthalates. Because esterase activity has large inter-individual differences, further study will be needed for individual risk identification of dermal exposure to phthalates.
Assuntos
Poluentes Ambientais/toxicidade , Ácidos Ftálicos/toxicidade , Absorção Cutânea , Animais , Dibutilftalato , Dietilexilftalato/administração & dosagem , Dietilexilftalato/farmacocinética , Dietilexilftalato/toxicidade , Exposição Ambiental , Poluentes Ambientais/farmacocinética , Esterases/antagonistas & inibidores , Feminino , Humanos , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Ácidos Ftálicos/administração & dosagem , Ácidos Ftálicos/farmacocinética , Plastificantes/administração & dosagem , Plastificantes/farmacocinética , Plastificantes/toxicidade , Ratos , Ratos Pelados , Medição de Risco , Pele/enzimologia , Especificidade da EspécieRESUMO
Galectins comprise a group of animal lectins characterized by their specificity for ß-galactosides. Galectin-2 (Gal-2) is predominantly expressed in the gastrointestinal tract and has been identified as one of the main gastric mucosal proteins that are proposed to have a protective role in the stomach. As Gal-2 is known to form homodimers in solution, this may result in crosslinking of macromolecules with the sugar structures recognized by Gal-2. In this study, we report that Gal-2 could interact with mucin, an important component of gastric mucosa, in a ß-galactoside-dependent manner. Furthermore, Gal-2 and mucin could form an insoluble precipitate, potentially through the crosslinking of mucins via Gal-2 and the formation of a lattice, resulting in a large insoluble complex. Therefore, we suggest that Gal-2 plays a role in the gastric mucosa by strengthening the barrier structure through crosslinking the mucins on the mucosal surface.
Assuntos
Galectina 2/química , Galectina 2/metabolismo , Mucinas/química , Mucinas/metabolismo , Animais , Células Epiteliais/metabolismo , Galectina 2/genética , Mucosa Gástrica/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Lactose/química , Lactose/metabolismo , Peso Molecular , Plasmídeos , Multimerização Proteica , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SuínosRESUMO
Polycationic compounds, such as poly-L-arginine and poly-L-ornithine (PLO), enhance the nasal absorption of hydrophilic macromolecular drugs. However, the bio availability corresponding to the dose of these enhancers has not been obtained in an open system study, where an administered solution is transferred to the pharynx because they do not exhibit mucoadhesion/retention in the nasal cavity. In this study, we prepared PEGylated-poly-L-ornithine (PEG-PLO) and investigated the effects of PEGylation on in vitro adhesion/retention properties, permeation enhancement efficiency, and cytotoxicity. PEG-PLO bearing 3-4 polyethylene glycol (PEG) chains per PLO molecule was more retentive than unmodified PLO on an inclined plate. The permeability of a model drug, FD-4, across Caco-2 cell sheets was enhanced by PEG-PLO as well as by PLO. PLO showed cytotoxicity at high concentrations, whereas PEG-PLO did not decrease cell viability, even above the concentration giving a sufficient enhancement effect. These findings suggest that PEGylation of polycationic absorption enhancers improves their adhesion/retention and decreases their cytotoxicity, which may lead to enhancers with greater utility.
Assuntos
Absorção Gastrointestinal/fisiologia , Peptídeos/metabolismo , Polietilenoglicóis/metabolismo , Tensoativos/metabolismo , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Avaliação Pré-Clínica de Medicamentos/métodos , Absorção Gastrointestinal/efeitos dos fármacos , Humanos , Peptídeos/síntese química , Peptídeos/farmacologia , Polietilenoglicóis/síntese química , Polietilenoglicóis/farmacologia , Tensoativos/síntese química , Tensoativos/farmacologiaRESUMO
A novel system for delivering recombinant human growth hormone (rhGH) that is noninvasive and has a simple method of administration is strongly desired to improve the compliance of children. The aim of this study was to investigate the potential for the intranasal (i.n.) co-administration of rhGH with poly-L-arginine (PLA) as a novel delivery system by evaluating the effects of the concentration and molecular weight of PLA on the nasal absorption of rhGH. The influence of the formation of insoluble aggregates and a soluble complex in the dosage formulation on nasal rhGH absorption was also evaluated by size-exclusion chromatography and ultrafiltration. PLA enhanced the nasal absorption of rhGH at each concentration and molecular weight examined. Nasal rhGH absorption increased dramatically when the PLA concentration was 1.0 % (w/v) due to the improved solubility of rhGH in the formulation. A delay in rhGH absorption was observed when the molecular weight of PLA was increased. This appeared to be because the increase in molecular weight caused the formation of a soluble complex. It seems that the PLA concentration affects the absorption-enhancing effect on rhGH, while the molecular weight of PLA affects the time when the maximum plasma rhGH concentration was reached (Tmax) of rhGH after i.n. administration, mainly because of the interactions among rhGH, PLA, and additives. Therefore, the transnasal rhGH delivery system using PLA is considered to be a promising alternative to subcutaneous (s.c.) injection if these interactions are sufficiently controlled.
Assuntos
Hormônio do Crescimento Humano/administração & dosagem , Hormônio do Crescimento Humano/farmacocinética , Absorção Nasal/efeitos dos fármacos , Peptídeos , Administração Intranasal , Animais , Hormônio do Crescimento Humano/sangue , Injeções Intravenosas , Injeções Subcutâneas , Masculino , Peso Molecular , Mucosa Nasal/metabolismo , Peptídeos/administração & dosagem , Peptídeos/química , Peptídeos/farmacologia , Ratos Wistar , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/sangue , Proteínas Recombinantes/farmacocinéticaRESUMO
PURPOSE: To develop an in silico method based on Fick's law of diffusion to estimate the skin concentration following dermal exposure to chemicals with a wide range of lipophilicity. METHODS: Permeation experiments of various chemicals were performed through rat and porcine skin. Permeation parameters, namely, permeability coefficient and partition coefficient, were obtained by the fitting of data to two-layered and one-layered diffusion models for whole and stripped skin. The mean skin concentration of chemicals during steady-state permeation was calculated using the permeation parameters and compared with the observed values. RESULTS: All permeation profiles could be described by the diffusion models. The estimated skin concentrations of chemicals using permeation parameters were close to the observed levels and most data fell within the 95% confidence interval for complete prediction. The permeability coefficient and partition coefficient for stripped skin were almost constant, being independent of the permeant's lipophilicity. CONCLUSIONS: Skin concentration following dermal exposure to various chemicals can be accurately estimated based on Fick's law of diffusion. This method should become a useful tool to assess the efficacy of topically applied drugs and cosmetic ingredients, as well as the risk of chemicals likely to cause skin disorders and diseases.
Assuntos
Simulação por Computador , Modelos Biológicos , Compostos Orgânicos/farmacocinética , Absorção Cutânea , Pele/metabolismo , Administração Cutânea , Animais , Difusão , Feminino , Masculino , Permeabilidade , Ratos , Ratos Pelados , SuínosRESUMO
Chitosan, a natural cationic polysaccharide derived from crustaceans and shellfish shells, is known for its advantageous biological properties, including biodegradability, biocompatibility, and antibacterial activity. Chitosan and its composite materials are studied for their potential for bone tissue repair. However, the effects of chitosan degradation products, glucosamine (GlcN) and chitosan oligosaccharide (COS), on osteoclasts remain unclear. If these chitosan degradation products promote osteoclastic differentiation, careful consideration is required for the use of chitosan and related materials in bone repair applications. Here, we assessed the effects of high (500 µg/mL) and low (0.5 µg/mL) concentrations of GlcN and COS on osteoclastic differentiation in human peripheral blood mononuclear cells (PBMCs) and murine macrophage-like RAW264 cells. A tartrate-resistant acid phosphatase (TRAP) enzyme activity assay, TRAP staining, and actin staining were used to assess osteoclastic differentiation. High concentrations of GlcN and COS, but not low concentrations, suppressed macrophage colony-stimulating factor (M-CSF)- and RANKL-dependent increases in TRAP enzyme activity, TRAP-positive multinuclear osteoclast formation, and actin ring formation in PBMCs without cytotoxicity. Similar effects were observed in the RANKL-dependent osteoclastic differentiation of RAW264 cells. In conclusion, chitosan degradation products do not possess osteoclast-inducing properties, suggesting that chitosan and its composite materials can be safely used for bone tissue repair.
RESUMO
Galectin-1 (Gal-1) is an evolutionarily conserved sugar-binding protein found in intra- and extracellular spaces. Extracellularly, it binds to glycoconjugates with ß-galactoside(s) and functions in various biological phenomena, including immunity, cancer, and differentiation. Under extracellular oxidative conditions, Gal-1 undergoes oxidative inactivation, losing its sugar-binding ability, although it exhibits sugar-independent functions. An age-related decrease in serum Gal-1 levels correlates with decreasing bone mass, and Gal-1 knockout promotes osteoclastic bone resorption and suppresses bone formation. However, the effect of extracellular Gal-1 on osteoclast differentiation remains unclear. Herein, we investigated the effects of extracellular Gal-1 on osteoclastogenesis in human peripheral blood mononuclear cells (PBMCs) and mouse macrophage RAW264 cells. Recombinant Gal-1 suppressed the macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand-dependent osteoclast formation, actin ring formation, and bone-resorption activity of human PBMCs. Similar results were obtained for RAW264 cells. Gal-1 knockdown increased osteoclast-like cell formation, suggesting that it affected differentiation in an autocrine-like manner. Oxidized Gal-1 slightly affected differentiation, and in the presence of lactose, the differentiation inhibitory effect of galectin-1 was not observed. These findings suggest that extracellular Gal-1 inhibits osteoclast differentiation in a ß-galactoside-dependent manner, and an age-related decrease in serum Gal-1 levels may contribute to reduced osteoclast activity and decreasing bone mass.
Assuntos
Reabsorção Óssea , Galectina 1 , Tirosina , Animais , Humanos , Camundongos , Reabsorção Óssea/metabolismo , Diferenciação Celular , Galectina 1/metabolismo , Galectina 1/farmacologia , Leucócitos Mononucleares , Açúcares , Tirosina/análogos & derivados , Células RAW 264.7/metabolismoRESUMO
Corticotropin-releasing factor (CRF) is mainly secreted from the hypothalamic paraventricular nuclei and plays a crucial role in stress-related responses. Recent studies have reported that CRF is a neuromodulator in the central nervous system. In the cerebellum, CRF is essential for the induction of long-term depression (LTD) at the parallel fiber-Purkinje cell synapses. Given that LTD is thought to be one of the fundamental mechanisms of motor learning, CRF may affect motor learning. However, the role of CRF in motor learning in vivo remains unclear. In this study, we aimed to examine the role of CRF in motor learning. This was achieved through a series of behavioral experiments involving the in vivo administration of CRF and its antagonists. Rats injected with CRF directly into the cerebellum exhibited superior performance on the rotarod test, especially during initial training phases, compared to control subjects. Conversely, rats receiving a CRF receptor antagonist demonstrated reduced endurance on the rotating rod compared to controls. Notably, CRF mRNA expression levels in the cerebellum did not show significant variance between the CRF-injected and control groups. These findings imply a critical role of endogenous CRF in cerebellar motor learning and suggest that exogenous CRF can augment this process. (199 words).
Assuntos
Cerebelo , Hormônio Liberador da Corticotropina , Aprendizagem , Animais , Hormônio Liberador da Corticotropina/metabolismo , Masculino , Ratos , Aprendizagem/fisiologia , Aprendizagem/efeitos dos fármacos , Cerebelo/metabolismo , Cerebelo/efeitos dos fármacos , Cerebelo/fisiologia , Atividade Motora/efeitos dos fármacos , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Ratos Sprague-DawleyRESUMO
OBJECTIVE: Galectins are sugar-binding proteins that participate in many biological processes, such as immunity, by regulating host immune cells and their direct interaction with pathogens. They are involved in mediating infection by Schistosoma mansoni, a parasitic trematode that causes schistosomiasis. However, their direct effects on schistosomes have not been investigated. RESULTS: We found that galectin-2 recognizes S. mansoni glycoconjugates and investigated whether galectin-1, 2, and 3 can directly affect S. mansoni in vitro. Adult S. mansoni were treated with recombinant galectin-1, 2, and 3 proteins or praziquantel, a positive control. Treatment with galectin-1, 2, and 3 had no significant effect on S. mansoni motility, and no other differences were observed under a stereoscopic microscope. Hence, galectin-1, 2, and 3 may have a little direct effect on S. mansoni. However, they might play a role in the infection in vivo via the modulation of the host immune response or secretory molecules from S. mansoni. To the best of our knowledge, this is the first study to investigate the direct effect of galectins on S. mansoni and helps in understanding the roles of galectins in S. mansoni infection in vivo.
Assuntos
Galectinas , Schistosoma mansoni , Esquistossomose mansoni , Animais , Galectina 1/farmacologia , Galectinas/farmacologia , Praziquantel/farmacologia , Schistosoma mansoni/efeitos dos fármacos , Schistosoma mansoni/fisiologia , Esquistossomose mansoni/tratamento farmacológicoRESUMO
Xeroderma pigmentosum (XP) is a rare autosomal recessive hereditary disorder. As patients with XP are deficient in nucleotide excision repair, they show severe photosensitivity symptoms. Although skin protection from ultraviolet (UV) radiation is essential to improve the life expectancy of such patients, the optimal protective effect is not achieved even with sunscreen application, owing to the low usability of the preparations. Nanosheets are two-dimensional nanostructures with a thickness in the nanometer range. The extremely large aspect ratios of the nanosheets result in high transparency, flexibility, and adhesiveness. Moreover, their high moisture permeability enables their application to any area of the skin for a long time. We fabricated preparations containing avobenzone (BMDBM) based on freestanding poly (L-lactic acid) (PLLA) nanosheets through a spin-coating process. Although monolayered PLLA nanosheets did not contain enough BMDBM to protect against UV radiation, the layered nanosheets, consisting of five discrete BMDBM nanosheets, showed high UV absorbance without lowering the adhesive strength against skin. Inflammatory reactions in XPA-deficient mice after UV radiation were completely suppressed by the application of BMDBM-layered nanosheets to the skin. Thus, the BMDBM layered nanosheet could serve as a potential sunscreen preparation to improve the quality of life of patients with XP.
RESUMO
BACKGROUND: Alopecia areata (AA) is considered a highly heritable, T-cell-mediated autoimmune disease of the hair follicle. However, no convincing susceptibility gene has yet been pinpointed in the major histocompatibility complex (MHC), a genome region known to be associated with AA as compared to other regions. METHODS: We engineered mice carrying AA risk allele identified by haplotype sequencing for the MHC region using allele-specific genome editing with the CRISPR/Cas9 system. Finally, we performed functional evaluations in the mice and AA patients with and without the risk allele. FINDINGS: We identified a variant (rs142986308, p.Arg587Trp) in the coiled-coil alpha-helical rod protein 1 (CCHCR1) gene as the only non-synonymous variant in the AA risk haplotype. Furthermore, mice engineered to carry the risk allele displayed a hair loss phenotype. Transcriptomics further identified CCHCR1 as a novel component interacting with hair cortex keratin in hair shafts. Both, these alopecic mice and AA patients with the risk allele displayed morphologically impaired hair and comparable differential expression of hair-related genes, including hair keratin and keratin-associated proteins (KRTAPs). INTERPRETATION: Our results implicate CCHCR1 with the risk allele in a previously unidentified subtype of AA based on aberrant keratinization in addition to autoimmune events. FUNDING: This work was supported by JSPS KAKENHI (JP16K10177) and the NIHR UCLH Biomedical Research center (BRC84/CN/SB/5984).
Assuntos
Alopecia em Áreas/genética , Proteínas de Transporte/genética , Predisposição Genética para Doença , Peptídeos e Proteínas de Sinalização Intracelular/genética , Complexo Principal de Histocompatibilidade/genética , Alelos , Alopecia em Áreas/imunologia , Alopecia em Áreas/patologia , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Modelos Animais de Doenças , Genoma/genética , Cabelo/crescimento & desenvolvimento , Cabelo/imunologia , Cabelo/patologia , Folículo Piloso/imunologia , Folículo Piloso/metabolismo , Folículo Piloso/patologia , Haplótipos/genética , Humanos , Queratinas , Queratinas Específicas do Cabelo/genética , Queratinas Específicas do Cabelo/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Camundongos , Linfócitos T/metabolismo , Linfócitos T/patologiaRESUMO
The aim of the present study was to assess the potential of biocompatible polymeric nanosheets as topical and transdermal drug-delivery devices. Nanosheets are two-dimensional nanostructures with a thickness in the nanometer order, and their extremely large aspect ratios result in unique properties, including high transparency, flexibility, and adhesiveness. Nanosheet formulations containing betamethasone valerate (BV) as a model drug and consisting of poly (L-lactic acid) or poly (lactic-co-glycolic) acid were fabricated through a spin-coating-assisted layer-by-layer method using a water-soluble sacrificial membrane. The fabricated formulations could incorporate and release higher amounts of BV compared with a commercial ointment, and the amounts could be controlled by the polymers used, the amount of BV added, and the use of controlled-release membranes. The presence of BV had a minimal effect on thickness, transparency, adhesiveness, and moisture permeability of nanosheets, permitting their application to any area of skin for a long period of time. Therefore, this biocompatible polymeric nanosheet formulation represents a novel and promising topical and transdermal drug delivery device, which has potential to deliver drugs regardless of the area of skin.
Assuntos
Sistemas de Liberação de Medicamentos , Nanoestruturas/administração & dosagem , Poliésteres/administração & dosagem , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/administração & dosagem , Administração Tópica , Adulto , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/química , Valerato de Betametasona/administração & dosagem , Valerato de Betametasona/química , Liberação Controlada de Fármacos , Feminino , Glucocorticoides/administração & dosagem , Glucocorticoides/química , Humanos , Masculino , Nanoestruturas/química , Poliésteres/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Ratos Pelados , Pele/metabolismo , Suínos , Adulto JovemRESUMO
Galectin-2 (Gal-2) is a lectin thought to play protective roles in the gastrointestinal tract. Oxidation of mouse Gal-2 (mGal-2) by hydrogen peroxide (H2 O2 ) results in the loss of sugar-binding activity, whereas S-nitrosylation of mGal-2, which does not change its sugar-binding profile, has been shown to protect the protein from H2 O2 -induced inactivation. One of the two cysteine residues, C57, has been identified as being responsible for controlling H2 O2 -induced inactivation; however, the underlying molecular mechanism has not been elucidated. We performed structural analyses of mGal-2 using nuclear magnetic resonance (NMR) and found that residues near C57 experienced significant chemical shift changes following S-nitrosylation, and that S-nitrosylation slowed the H2 O2 -induced aggregation of mGal-2. We also revealed that S-nitrosylation improves the thermal stability of mGal-2 and that the solvent accessibility and/or local dynamics of residues near C57 and the local dynamics of the core-forming residues in mGal-2 are reduced by S-nitrosylation. Structural models of Gal-2 indicated that C57 is located in a hydrophobic pocket that can be plugged by S-nitrosylation, which was supported by the NMR experiments. Based on these results, we propose two structural mechanisms by which S-nitrosylation protects mGal-2 from H2 O2 -induced aggregation without changing its sugar-binding profile: (a) stabilization of the hydrophobic pocket around C57 that prevents oxidation-induced destabilization of the pocket, and (b) prevention of oxidation of C57 during the transiently unfolded state of the protein, in which the residue is exposed to H2 O2 . DATABASE: Nuclear magnetic resonance assignments for non-S-nitrosylated mGal-2 and S-nitrosylated mGal-2 have been deposited in the BioMagResBank (http://www.bmrb.wisc.edu/) under ID code 27237 for non-S-nitrosylated mGal-2 and ID code 27238 for S-nitrosylated mGal-2.
Assuntos
Galectina 2/química , Espectroscopia de Ressonância Magnética/métodos , Óxido Nítrico/química , S-Nitrosotióis/química , Animais , Cisteína/química , Cisteína/metabolismo , Galectina 2/metabolismo , Peróxido de Hidrogênio/farmacologia , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Modelos Moleculares , Óxido Nítrico/metabolismo , Oxidantes/farmacologia , Oxirredução/efeitos dos fármacos , Agregados Proteicos/efeitos dos fármacos , Conformação Proteica , S-Nitrosotióis/metabolismoRESUMO
OBJECTIVE: To develop a bioadhesive phosphorescent particle that can be used as a marker in video-oculography to assess eye movements in the dark without drug treatment. METHODS: The marker was prepared by spray-coating a Sr4Al14O25: Eu2+, Dy3+ phosphor with a carboxyvinyl polymer. The morphologic, luminescent and adhesive properties were assessed. The dynamic properties of VOR measured by the marker were compared with those obtained by tracking the pupil under miotic treatment. RESULTS: Non-aggregated and non-fused particles having diameters of about 5µm could be prepared by polymeric coating of the phosphor, resulting in particles small enough not to restrict eye movement. Although the phosphorescent of the particles decreased with increasing thickness of the coating layer, the coated particles were detectable in the dark for at least 60 min. The thicker the coating layer was, the higher the adhesiveness of the particles obtained. The particles having the thickest coating layer were retained on the corneal surface during VOR measurement and thus performed well as a marker in video-oculography. The dynamic properties of VOR measured by the marker were essentially identical to those obtained by tracking the pupil under miotic treatment. CONCLUSION: Our marker will contribute to understanding the mechanisms underlying motor learning.
Assuntos
Técnicas de Diagnóstico Oftalmológico , Movimentos Oculares/fisiologia , Medições Luminescentes/métodos , Gravação em Vídeo/métodos , Adesividade , Animais , Biomarcadores/análise , Córnea , Escuridão , Camundongos Endogâmicos C57BL , Tamanho da Partícula , PolimerizaçãoRESUMO
Skin concentrations of topically administered compounds need to be considered in order to evaluate their efficacies and toxicities. This study investigated the relationship between the skin permeation and concentrations of compounds, and also predicted the skin concentrations of these compounds using their permeation parameters. Full-thickness skin or stripped skin from pig ears was set on a vertical-type diffusion cell, and lidocaine (LID) solution was applied to the stratum corneum (SC) in order to determine in vitro skin permeability. Permeation parameters were obtained based on Fick's second law of diffusion. LID concentrations at each depth of the SC were measured using tape-stripping. Concentration-depth profiles were obtained from viable epidermis and dermis (VED) by analyzing horizontal sections. The corresponding skin concentration at each depth was calculated based on Fick's law using permeation parameters and then compared with the observed value. The steady state LID concentrations decreased linearly as the site became deeper in SC or VED. The calculated concentration-depth profiles of the SC and VED were almost identical to the observed profiles. The compound concentration at each depth could be easily predicted in the skin using diffusion equations and skin permeation data. Thus, this method was considered to be useful for promoting the efficient preparation of topically applied drugs and cosmetics.