RESUMEN
BACKGROUND: Cathepsin K, which is involved in bone resorption, is a good target for treating osteoporosis, but no clinically approved medicine has been developed. Recently, allosteric inhibitors with high specificity and few side effects have been attracting attention for use in new medicines. METHODS: Cathepsin K inhibitors were isolated from the methanol extract of Chamaecrista nomame (Leguminosae) using cathepsin K inhibition activity-assisted multi-step chromatography. Standard kinetic analysis was employed to examine the mechanism of cathepsin K inhibition when an isolated inhibitor and its derivative were used. The allosteric binding of these cathepsin K inhibitors was supported by a docking study using AutoDock vina. Combinations of allosteric cathepsin K inhibitors expected to bind to different allosteric sites were examined by means of cathepsin K inhibition assay. RESULTS: Two types of cathepsin K inhibitors were identified in the methanol extract of Chamaecrista nomame. One type consisted of cassiaoccidentalin B and torachrysone 8-ß-gentiobioside, and inhibited both cathepsin K and B with similar inhibitory potential, while the other type of inhibitor consisted of pheophytin a, and inhibited cathepsin K but not cathepsin B, suggesting that pheophytin a binds to an allosteric site of cathepsin K. Kinetic analysis of inhibitory activity suggested that pheophytin a and its derivative, pheophorbide b, bind allosterically to cathepsin K. This possibility was supported by a docking study on cathepsin K. The cathepsin K inhibitory activity of pheophytin a and pheophorbide b was enhanced by combining them with the allosteric inhibitors NSC 13345 and NSC94914, which bind to other allosteric sites on cathepsin K. CONCLUSIONS: Different allosteric inhibitors that bind to different sites in combination, as shown in this study, may be useful for designing new allosteric inhibitory drugs with high specificity and few side effects.
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Resorción Ósea , Metanol , Humanos , Catepsina K/metabolismo , Sitio Alostérico , Cinética , Catepsinas/metabolismoRESUMEN
We examined whether the acetylenic fatty acids 6-octadecynoic acid (6-ODA) and 9-octadecynoic acid (9-ODA) perform as ligands for free fatty acid receptors of medium- and long-chain fatty acids FFAR1 and FFAR4, previously called GPR40 and GPR120, respectively. Phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 was increased through FFAR1 but not through FFAR4 expressed in HEK 293â¯cells, suggesting that 6-ODA and 9-ODA function as an FFAR1 ligand, but not as an FFAR4 ligand. Activation of ERK in FFAR1-expressing HEK293â¯cells by 6-ODA and 9-ODA peaked at 10â¯min after stimulation followed by a slow decrease, similar to ERK activation by rosiglitazone, which peaked at 10â¯min after stimulation and lasted longer. Glucose-dependent production of insulin from MIN6 insulinoma cells was induced by 6-ODA and 9-ODA in an FFAR1-dependent manner. In this process, 6-ODA and 9-ODA stimulated the production of insulin not in the first phase that occurred within 10â¯min after stimulation but in the second phase. F-actin-remodeling that reflects insulin granule recruiting to the plasma membrane in the second phase of insulin secretion by 6-ODA and 9-ODA suggested that they have an FFAR1-dependent function in insulin secretion from MIN6 cells.
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Ácidos Grasos/metabolismo , Insulina/metabolismo , Insulinoma/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Actinas/metabolismo , Alquinos/farmacología , Animales , Línea Celular Tumoral , Ácidos Grasos Monoinsaturados/farmacología , Ácidos Grasos Insaturados/farmacología , Glucosa/metabolismo , Células HEK293 , Humanos , RatonesRESUMEN
Covalent agonists of PPARγ cause unique receptor conformational changes and behave as selective PPARγ modulators, whereas there are few covalent agonists other than endogenous unsaturated fatty acids metabolites. Previously, we established a cell-based strategy to identify new PPARγ ligands and synthesized a new-type of covalent agonist that possesses the hybrid structure of a plant-derived cinnamic acid derivative and GW9662, a covalent antagonist. Herein, we report six analogues that differ in how the two fragments are linked together. Compounds with a simplified linker showed potent agonistic activity with improved EC50 values (less than 5 nM), indicating that close proximity between the two fragments improves binding affinity. When the position of cinnamic acid moiety was placed at 4' carbon of aniline ring, PPARγ agonist activity was completely abolished. Docking studies suggested that the activation profile likely depends on interaction with the cavity around helix 3, ß-sheet, and Ω-loop region in the ligand-binding domain. Furthermore, a cell-based assay revealed that agonist-type compounds activate PPARγ transcription in a manner dependent on covalent linkage with the Cys285 residue leading to prolonged transactivation. This activation feature reflects pharmacological benefits of covalent drugs, suggesting that these hybrid compounds may serve as potential leads for a new-class of covalent PPARγ ligands.
Asunto(s)
Anilidas/farmacología , Cinamatos/química , PPAR gamma/agonistas , Cisteína/química , Células Hep G2 , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Reproducibilidad de los Resultados , Activación Transcripcional/efectos de los fármacosRESUMEN
Zinc-requiring ectoenzymes, including both secreted and membrane-bound enzymes, are considered to capture zinc in their active site for their activation in the early secretory pathway. This idea has been confirmed by our studies conducted using tissue-nonspecific alkaline phosphatase (TNAP), which is elaborately activated by means of a two-step mechanism by zinc transporter 5 (ZNT5)-ZNT6 heterodimers and ZNT7 homodimers, through protein stabilization followed by enzyme activation with zinc in the early secretory pathway. However, the molecular basis of the activation process in other zinc-requiring ectoenzymes remains largely unknown. In this study, we investigated this activation process by using three cancer-promoting zinc-requiring ectoenzymes, autotaxin (ATX), matrix metalloproteinase 9 (MMP9), and carbonic anhydrase IX (CAIX), and the chicken DT40 cell mutants that we generated; we specifically focused on clarifying whether the same or a similar activation mechanism operates in these ectoenzymes. ATX activation required ZNT5-ZNT6 heterodimers and ZNT7 homodimers in a manner similar to TNAP activation, although the protein stability of ATX was differently regulated from that of TNAP. MMP9 required ZNT5-ZNT6 heterodimers and ZNT7 homodimers for its activation as well as secretion; MMP9 was not secreted into the spent medium unless both zinc-transport complexes were present. Finally, CAIX activation by zinc was mediated not only by ZNT5-ZNT6 heterodimers and ZNT7 homodimers but also by ZNT4 homodimers; thus, these three zinc-transport complexes redundantly contribute to CAIX activation. Our results provide pivotal insights into the activation processes of zinc-requiring ectoenzymes, and furthermore, they offer novel insights for potential cancer therapy applications given the cancer-promoting potencies of ATX, MMP9, and CAIX.
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Anhidrasa Carbónica IX/metabolismo , Proteínas de Transporte de Catión/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Neoplasias/enzimología , Hidrolasas Diéster Fosfóricas/metabolismo , Zinc/metabolismo , Animales , Proteínas de Transporte de Catión/química , Línea Celular , Pollos , Dimerización , Activación EnzimáticaRESUMEN
Recent findings indicate that mRNA splicing inhibitors can be potential anticancer candidates. We have previously established a screening system which monitors mRNA processing in order to identify mRNA processing inhibitors. Among a number of dietary resources, isoflavone fractions showed an inhibitory effect of mRNA processing. These findings demonstrate that a variety of dietary sources have an impact on mRNA biogenesis.
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Evaluación Preclínica de Medicamentos/métodos , Glycine max/química , Isoflavonas/farmacología , ARN Mensajero/metabolismo , Línea Celular , Células HeLa/efectos de los fármacos , Humanos , Hibridación Fluorescente in Situ , Luciferasas de Renilla/genética , Procesamiento Postranscripcional del ARN , Empalme del ARNRESUMEN
Secretory and membrane-bound zinc-requiring enzymes are thought to be activated by binding zinc in the early secretory pathway. One such enzyme, tissue-non-specific alkaline phosphatase (TNAP), is activated through a two-step mechanism, via protein stabilization and subsequent enzyme activation through metalation, by ZnT5-ZnT6 heterodimers or ZnT7 homodimers. However, little is known about the molecular basis underlying the activation process. In the present study, we found that the di-proline motif (PP-motif) in luminal loop 2 of ZnT5 and ZnT7 is important for TNAP activation. TNAP activity was significantly reduced in cells lacking ZnT5-ZnT6 heterodimers and ZnT7 homodimers [triple knockout (TKO) cells]. The decreased TNAP activity was restored by expressing hZnT5 with hZnT6 or hZnT7, but significantly less so (almost 90% less) by expressing mutants thereof in which the PP-motif was mutated to alanine (PP-AA). In TKO cells, overexpressed hTNAP was not completely activated, and it was converted less efficiently into the holo form by expressing a PP-AA mutant of hZnT5 with hZnT6, whose defects were not restored by zinc supplementation. The zinc transport activity of hZnT7 was not significantly impaired by the PP-AA mutation, indicating that the PP-motif is involved in the TNAP maturation process, although it does not control zinc transport activity. The PP-motif is highly conserved in ZnT5 and ZnT7 orthologues, and its importance for TNAP activation is conserved in the Caenorhabditis elegans hZnT5 orthologue CDF5. These results provide novel molecular insights into the TNAP activation process in the early secretory pathway.
Asunto(s)
Proteínas Portadoras/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Línea Celular , PollosRESUMEN
Macroautophagy, or autophagy, is a cellular response in which unnecessary cytoplasmic components, including lipids and organelles, are self-degraded. Recent studies closely related autophagy to activation of hepatic stellate cells (HSCs), a process critical in the pathogenesis of liver fibrosis. During HSC activation, cytoplasmic lipid droplets (LDs) are degraded as autophagic cargo, and then cells express fibrogenic genes. Thus, inhibition of autophagy in HSCs is a potential therapeutic approach for attenuating liver fibrosis. We found that tetrandrine, a bisbenzylisoquinoline alkaloid isolated from Stephania tetrandra, induced lipid accumulation, a phenotype associated with quiescent HSCs, through blockade of autophagy in the rat-derived HSC line HSC-T6. Tetrandrine inhibited autophagic flux without affecting lysosomal function. A phenotypic comparison using siRNA knockdown suggested that tetrandrine may target regulators, involved in fusion between autophagosomes and lysosomes (e.g., syntaxin 17). Moreover, perilipin 1, an LD-coated protein, co-localized specifically with LC3, a marker protein for autophagosomes, in tetrandrine-treated HSC-T6 cells. This suggests a potential role for perilipin 1 in autophagy-mediated LD degradation in HSCs. Our results identified tetrandrine as a potential tool for prevention and treatment of HSC activation.
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Autofagia/efectos de los fármacos , Bencilisoquinolinas/farmacología , Células Estrelladas Hepáticas/efectos de los fármacos , Metabolismo de los Lípidos/efectos de los fármacos , Línea Celular , HumanosRESUMEN
Dietary zinc deficiency puts human health at risk, so we explored strategies for enhancing zinc absorption. In the small intestine, the zinc transporter ZIP4 functions as an essential component of zinc absorption. Overexpression of ZIP4 protein increases zinc uptake and thereby cellular zinc levels, suggesting that food components with the ability to increase ZIP4 could potentially enhance zinc absorption via the intestine. In the present study, we used mouse Hepa cells, which regulate mouse Zip4 (mZip4) in a manner indistinguishable from that in intestinal enterocytes, to screen for suitable food components that can increase the abundance of ZIP4. Using this ZIP4-targeting strategy, two such soybean extracts were identified that were specifically able to decrease mZip4 endocytosis in response to zinc. These soybean extracts also effectively increased the abundance of apically localized mZip4 in transfected polarized Caco2 and Madin-Darby canine kidney cells and, moreover, two apically localized mZip4 acrodermatitis enteropathica mutants. Soybean components were purified from one extract and soyasaponin Bb was identified as an active component that increased both mZip4 protein abundance and zinc levels in Hepa cells. Finally, we confirmed that soyasaponin Bb is capable of enhancing cell surface endogenous human ZIP4 in human cells. Our results suggest that ZIP4 targeting may represent a new strategy to improve zinc absorption in humans.
Asunto(s)
Proteínas de Transporte de Catión/agonistas , Enterocitos/metabolismo , Fármacos Gastrointestinales/metabolismo , Glycine max/química , Absorción Intestinal , Extractos Vegetales/metabolismo , Zinc/metabolismo , Animales , Células CACO-2 , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Línea Celular , Membrana Celular/metabolismo , Enfermedades Carenciales/metabolismo , Enfermedades Carenciales/prevención & control , Suplementos Dietéticos , Perros , Endocitosis , Enterocitos/citología , Fármacos Gastrointestinales/análisis , Fármacos Gastrointestinales/química , Fármacos Gastrointestinales/uso terapéutico , Regulación de la Expresión Génica , Humanos , Ratones , Extractos Vegetales/química , Extractos Vegetales/uso terapéutico , Estabilidad Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Saponinas/análisis , Saponinas/metabolismo , Semillas/química , Zinc/deficienciaRESUMEN
6-Octadecynoic acid (6-ODA), a fatty acid with a triple bond, was identified in the methanol extract of Marrubium vulgare L. as an agonist of peroxisome proliferator-activated receptor γ (PPARγ). Fibrogenesis caused by hepatic stellate cells is inhibited by PPARγ whose ligands are clinically used for the treatment of diabetes. Plant extracts of Marrubium vulgare L., were screened for activity to inhibit fibrosis in the hepatic stellate cell line HSC-T6 using Oil Red-O staining, which detects lipids that typically accumulate in quiescent hepatic stellate cells. A methanol extract with activity to stimulate accumulation of lipids was obtained. This extract was found to have PPARγ agonist activity using a luciferase reporter assay. After purification using several chromatographic methods, 6-ODA, a fatty acid with a triple bond, was identified as a candidate of PPARγ agonist. Synthesized 6-ODA and its derivative 9-octadecynoic acid (9-ODA), which both have a triple bond but in different positions, activated PPARγ in a luciferase reporter assay and increased lipid accumulation in 3T3-L1 adipocytes in a PPARγ-dependent manner. There is little information about the biological activity of fatty acids with a triple bond, and to our knowledge, this is the first report that 6-ODA and 9-ODA function as PPARγ agonists.
Asunto(s)
Ácidos Grasos Monoinsaturados/farmacología , Células Estrelladas Hepáticas/efectos de los fármacos , PPAR gamma/agonistas , Extractos Vegetales/farmacología , Células 3T3-L1 , Alquinos/farmacología , Animales , Ácidos Grasos Insaturados/farmacología , Humanos , Marrubium/química , RatonesRESUMEN
A number of enzymes become functional by binding to zinc during their journey through the early secretory pathway. The zinc transporters (ZnTs) located there play important roles in this step. We have previously shown that two zinc transport complexes, ZnT5/ZnT6 heterodimers and ZnT7 homo-oligomers, are required for the activation of alkaline phosphatases, by converting them from the apo- to the holo-form. Here, we investigated the molecular mechanisms of this activation. ZnT1 and ZnT4 expressed in chicken DT40 cells did not contribute to the activation of tissue nonspecific alkaline phosphatase (TNAP). The reduced activity of TNAP in DT40 cells deficient in both ZnT complexes was not restored by zinc supplementation nor by exogenous expression of other ZnTs that increase the zinc content in the secretory pathway. Moreover, we showed that expression of ZnT5/ZnT6 heterodimers reconstituted with zinc transport-incompetent ZnT5 mutant failed to restore TNAP activity but could stabilize the TNAP protein as the apo-form, regardless of zinc status. These findings demonstrate that TNAP is activated not simply by passive zinc binding but by an elaborate two-step mechanism via protein stabilization followed by enzyme conversion from the apo- to the holo-form with zinc loaded by ZnT complexes in the early secretory pathway.
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Fosfatasa Alcalina/metabolismo , Proteínas de Transporte de Catión/metabolismo , Multimerización de Proteína , Zinc/metabolismo , Fosfatasa Alcalina/genética , Animales , Apoenzimas/genética , Apoenzimas/metabolismo , Proteínas de Transporte de Catión/genética , Línea Celular Transformada , Pollos , Activación Enzimática/genética , Estabilidad de Enzimas/genética , Humanos , Mutación , Unión ProteicaRESUMEN
We report the tumor cell-selective prodrugs based on the arsonic acid-presenting iron oxide nanoparticles. We synthesized the well-dispersed nanoparticles having arsonoacetic acid which is composed of the low toxic As(V) form. From the analyses of the reaction products, it is suggested that the reduction by dithiothreitol with arsonoacetic acid and the modified nanoparticles could generate the highly-toxic As(III) species. In the MTT assays, it was found that the cell viabilities of HeLaS3 and especially HepG2 were reduced in the presence of the modified nanoparticles. In contrast, a slight effect on viability was observed with primary mouse hepatocytes. The viabilities showed good agreements with the amounts of intracellular reduced glutathione concentrations. Furthermore, the valid concentrations of the modified nanoparticles for tumor-specific cytotoxicity were similar level in MRI measurements. These results indicate that arsonic acid-presenting nanoparticles should be a good platform for developing highly-sensitive tumor-specific prodrugs.
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Antineoplásicos/farmacología , Arsenicales/farmacología , Compuestos Férricos/farmacología , Nanopartículas/química , Profármacos/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Arsenicales/química , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Compuestos Férricos/síntesis química , Compuestos Férricos/química , Células HeLa , Células Hep G2 , Hepatocitos/efectos de los fármacos , Humanos , Ratones , Profármacos/síntesis química , Profármacos/química , Relación Estructura-ActividadRESUMEN
A number of proteins complete mRNA processing in the nucleus, thus, inhibitor of mRNA processing is worth finding to analyze the mechanism of mRNA maturation in detail. Here, we established a monitoring system for mRNA processing using a test compound, spliceostatin A (SSA), which inhibits mRNA splicing. This system should serve to facilitate the discovery of novel compounds from natural resources that inhibit mRNA processing.
Asunto(s)
Bioensayo , Núcleo Celular/efectos de los fármacos , Piranos/farmacología , Empalme del ARN/efectos de los fármacos , ARN Mensajero/antagonistas & inhibidores , Compuestos de Espiro/farmacología , Núcleo Celular/genética , Núcleo Celular/metabolismo , Relación Dosis-Respuesta a Droga , Expresión Génica/efectos de los fármacos , Genes Reporteros , Células HeLa , Humanos , Luciferasas de Renilla , Precursores del ARN/antagonistas & inhibidores , Precursores del ARN/genética , Empalme del ARN/genética , ARN Mensajero/genética , Empalmosomas/efectos de los fármacos , Empalmosomas/genéticaRESUMEN
The extract of Salvia officinalis (Common Sage) exhibited inhibitory activity of STAT3 signal after screening of several plants extracts using the STAT3-responsive reporter system. Cirsiliol, luteolin, and carnosol were identified from the methanol extract of Silvia officinalis as inhibitors of STAT3 signaling and the effects of these three compounds on STAT3 protein or growth inhibition on cancer cells was compared. Luteolin at the dose of 90 µM clearly suppressed the phosphorylation of STAT3 induced by IL-6, while carnosol was prone to decrease total STAT3 proteins at high doses (>90 µM). Cirsiliol had almost no effect. Since the three compounds exhibited similar concentration-dependent suppression patterns in the reporter assay except for cirsiliol became plateau beyond 30 µM, these compounds appeared to function as STAT3 inhibitory factors in different ways. The direct anti-proliferative activity of three compounds was examined with or without the anti-cancer drug gefitinib using HepG2 and A549 cells. The anti-proliferative effect of the three compounds was additively enhanced by gefitinib. At the doses of 3.6 µM, statistically significant suppression of proliferation was observed in HepG2 cells only by cirsiliol among the three compounds in the absence of gefitinib but all three compounds were prone to suppress the proliferation of HepG2 cells and A549 cells dose-dependently although cirsiliol showed a modest dose-dependency and this suppression of proliferation was enhanced by the addition of gefitinib. Cirsiliol, a dimethyoxylated flavone, activated the natural killer activity of KHYG-1 cells against erythroleukemia K562 cells like a hexamethoxylated flavone, nobiletin, suggesting that it may also have an indirect anti-cancer potential through activation of NK cells. These results shed light on the putative anti-cancer potential of Salvia officinalis.
RESUMEN
The majority of CDF/ZnT zinc transporters form homo-oligomers. However, ZnT5, ZnT6, and their orthologues form hetero-oligomers in the early secretory pathway where they load zinc onto zinc-requiring enzymes and maintain secretory pathway functions. The details of this hetero-oligomerization remain to be elucidated, and much more is known about homo-oligomerization that occurs in other CDF/ZnT family proteins. Here, we addressed this issue using co-immunoprecipitation experiments, mutagenesis, and chimera studies of hZnT5 and hZnT6 in chicken DT40 cells deficient in ZnT5, ZnT6, and ZnT7 proteins. We found that hZnT5 and hZnT6 combine to form heterodimers but do not form complexes larger than heterodimers. Mutagenesis of hZnT6 indicated that the sites present in transmembrane domains II and V in which many CDF/ZnT proteins have conserved hydrophilic amino acid residues are not involved in zinc binding of hZnT6, although they are required for zinc transport in other CDF/ZnT family homo-oligomers. We also found that the long N-terminal half of hZnT5 is not necessary for its functional interaction with hZnT6, whereas the cytosolic C-terminal tail of hZnT5 is important in determining hZnT6 as a partner molecule for heterodimer formation. In DT40 cells, cZnT5 variant lacking the N-terminal half was endogenously induced during periods of endoplasmic reticulum stress and so seemed to function to supply zinc to zinc-requiring enzymes under these conditions. The results outlined here provide new information about the mechanism of action through heterodimerization of CDF/ZnT proteins that function in the early secretory pathway.
Asunto(s)
Proteínas de Transporte de Catión/química , Vías Secretoras , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Línea Celular , Pollos , Dimerización , Humanos , Datos de Secuencia Molecular , Unión Proteica , Alineación de SecuenciaRESUMEN
Screening of mRNA export factors in Saccharomyces cerevisiae and Drosophila melanogaster has identified a number of mRNA processing factors involved in multiple mRNA processing steps. However, only limited information is available on human cells. Here we established a screening system searching for mRNA processing factors in human cells by combining the luciferase reporter system and fluorescence in situ hybridization, which evaluates the nuclear/cytoplasmic distribution of bulk poly(A)+ RNA. This system makes it possible to search for the compounds affecting mRNA processing from the various resources.
Asunto(s)
Genes Reporteros/genética , Espacio Intracelular/metabolismo , Luciferasas/genética , Poli A/metabolismo , Transporte de ARN , Células HeLa , Humanos , ARN Mensajero/metabolismoRESUMEN
The SLC39A family of zinc transporters can be divided into four subfamilies (I, II, LIV-1, and gufA) in vertebrates, but studies of their functions have been restricted exclusively to members of subfamilies II and LIV-1. In this study, we characterized SLC39A9 (ZIP9), the only member of subfamily I in vertebrates. Confocal microscopy demonstrated that transiently expressed, HA-tagged human ZIP9 (hZIP9-HA) was localized to the trans-Golgi network regardless of zinc status. Disruption of the ZIP9 gene in DT40 cells did not change the growth rate, sensitivity to high zinc and manganese concentrations during long-term culture, or cellular zinc status after short-term incubation with zinc. The alkaline phosphatase activity of ZIP9(-/-) cells did not change in cells cultured in medium containing normal zinc levels. In contrast, the activity of this enzyme decreased in wild-type cells cultured in zinc deficient medium but less so in ZIP9(-/-) cells under these conditions. Stable over-expression of hZIP9-HA moderately decreased alkaline phophatase activity. These results suggest that ZIP9 functions to regulate zinc homeostasis in the secretory pathway without significantly altering cytosolic zinc homeostasis.
Asunto(s)
Proteínas de Transporte de Catión/metabolismo , Homeostasis , Vías Secretoras , Zinc/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Proteínas de Transporte de Catión/deficiencia , Proteínas de Transporte de Catión/genética , Células HeLa , Humanos , Datos de Secuencia Molecular , Red trans-Golgi/metabolismoRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0197165.].
RESUMEN
The biological and molecular properties of tetrodotoxin (TTX)-sensitive voltage-gated Na(+) currents (I(Na)) in murine vas deferens myocytes were investigated using patch-clamp techniques and molecular biological analyses. In whole-cell configuration, a fast, transient inward current was evoked in the presence of Cd(2+), and was abolished by TTX (K(d) = 11.2 nM), mibefradil (K(d) = 3.3 microM), and external replacement of Na(+) with monovalent cations (TEA(+), Tris(+), and NMDG(+)). The fast transient inward current was enhanced by veratridine, an activator of voltage-gated Na(+) channels, suggesting that the fast transient inward current was a TTX-sensitive I(Na). The values for half-maximal (V(half)) inactivation and activation of I(Na) were -46.3 mV and -26.0 mV, respectively. RT-PCR analysis revealed the expression of Scn1a, 2a, and 8a transcripts. The Scn8a transcript and the alpha-subunit protein of Na(V)1.6 were detected in smooth muscle layers. Using Na(V)1.6-null mice (Na(V)1.6(-/-)) lacking the expression of the Na(+) channel gene, Scn8a, I(Na) were not detected in dispersed smooth muscle cells from the vas deferens, while TTX-sensitive I(Na) were recorded in their wild-type (Na(V)1.6(+/+)) littermates. This study demonstrates that the molecular identity of the voltage-gated Na(+) channels responsible for the TTX-sensitive I(Na) in murine vas deferens myocytes is primarily Na(V)1.6.
Asunto(s)
Potenciales de Acción/fisiología , Activación del Canal Iónico/fisiología , Potenciales de la Membrana/fisiología , Miocitos del Músculo Liso/fisiología , Canales de Sodio/fisiología , Sodio/metabolismo , Conducto Deferente/fisiología , Animales , Células Cultivadas , Masculino , Ratones , Ratones Endogámicos BALB CRESUMEN
BACKGROUND: Although vascular endothelial growth factor (VEGF) is a primary mediator of retinal angiogenesis, VEGF inhibition alone is insufficient to prevent retinal neovascularization. Hence, it is postulated that there are other potent ischemia-induced angiogenic factors. Erythropoietin possesses angiogenic activity, but its potential role in ocular angiogenesis is not established. METHODS: We measured both erythropoietin and VEGF levels in the vitreous fluid of 144 patients with the use of radioimmunoassay and enzyme-linked immunosorbent assay. Vitreous proliferative potential was measured according to the growth of retinal endothelial cells in vitro and with soluble erythropoietin receptor. In addition, a murine model of ischemia-induced retinal neovascularization was used to evaluate erythropoietin expression and regulation in vivo. RESULTS: The median vitreous erythropoietin level in 73 patients with proliferative diabetic retinopathy was significantly higher than that in 71 patients without diabetes (464.0 vs. 36.5 mIU per milliliter, P<0.001). The median VEGF level in patients with retinopathy was also significantly higher than that in patients without diabetes (345.0 vs. 3.9 pg per milliliter, P<0.001). Multivariate logistic-regression analyses indicated that erythropoietin and VEGF were independently associated with proliferative diabetic retinopathy and that erythropoietin was more strongly associated with the presence of proliferative diabetic retinopathy than was VEGF. Erythropoietin and VEGF gene-expression levels are up-regulated in the murine ischemic retina, and the blockade of erythropoietin inhibits retinal neovascularization in vivo and endothelial-cell proliferation in the vitreous of patients with diabetic retinopathy in vitro. CONCLUSIONS: Our data suggest that erythropoietin is a potent ischemia-induced angiogenic factor that acts independently of VEGF during retinal angiogenesis in proliferative diabetic retinopathy.
Asunto(s)
Retinopatía Diabética/patología , Eritropoyetina/metabolismo , Retina/citología , Neovascularización Retiniana/patología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Cuerpo Vítreo/química , Animales , Estudios de Casos y Controles , Bovinos , Proliferación Celular , Células Cultivadas , Retinopatía Diabética/tratamiento farmacológico , Retinopatía Diabética/fisiopatología , Relación Dosis-Respuesta a Droga , Eritropoyetina/análisis , Eritropoyetina/antagonistas & inhibidores , Proteínas de la Matriz Extracelular , Femenino , Humanos , Modelos Logísticos , Masculino , Ratones , Persona de Mediana Edad , Cadenas Pesadas de Miosina , Miosina Tipo IIB no Muscular , Proteínas/farmacología , ARN Mensajero/metabolismo , Receptores de Eritropoyetina/fisiología , Retina/efectos de los fármacos , Retina/metabolismo , Neovascularización Retiniana/fisiopatología , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/análisis , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
The chemo-enzymatic synthesis of an artificially N-glycosylated derivative of glucagon, a peptide hormone that regulates the blood sugar level, is described. We synthesized the glycosylated glucagon by chemical synthesis of an N-acetylglucosaminyl peptide and enzymatic transfer of an oligosaccharide using the transglycosylation activity of the glycosynthase-like mutant of Mucor hiemalis endo-ß-N-acetylglucosaminidase (Endo-M) and sialo-oligosaccharide oxazoline as a donor substrate. The sialo-oligosaccharide-attached glucagon synthesized showed high resistance against protease degradation and stimulated the release of glucose from mouse hepatocytes when added to cells. The synthetic glucagon showed slightly higher activity than native glucagon and has potential as a therapeutic agent for treating diabetic patients.