RESUMEN
Injectable hydrogels are suitable for local cell delivery to the subcutaneous space, but the lack of vasculature remains a limiting factor. Previously we demonstrated that biomaterials containing methacrylic acid promoted vascularization. Here we report the preparation of a semi-interpenetrating polymer network (SIPN), and its evaluation as an injectable carrier to deliver cells and generate blood vessels in a subcutaneous implantation site. The SIPN was prepared by reacting a blend of vinyl sulfone-terminated polyethylene glycol (PEG-VS) and sodium polymethacrylate (PMAA-Na) with dithiothreitol. The swelling of SIPN was sensitive to the PMAA-Na content but only small differences in gelation time, permeability and stiffness were noted. SIPN containing 20 mol% PMAA-Na generated a vascular network in the surrounding tissues, with 2-3 times as many vessels as was obtained with 10 mol% PMAA-Na or PEG alone. Perfusion studies showed that the generated vessels were perfused and connected to the host vasculature as early as seven days after transplantation. Islets embedded in SIPN were viable and responsive to glucose stimulation in vitro. In a proof of concept study in a streptozotocin-induced diabetic mouse model, a progressive return to normoglycemia was observed and the presence of insulin positive islets was confirmed when islets were embedded in SIPN prior to delivery. Our approach proposes a biomaterial-mediated strategy to deliver cells while enhancing vascularization.
Asunto(s)
Células Inmovilizadas/citología , Hidrogeles/química , Trasplante de Islotes Pancreáticos , Islotes Pancreáticos/citología , Polietilenglicoles/química , Ácidos Polimetacrílicos/química , Andamios del Tejido/química , Animales , Materiales Biocompatibles/administración & dosificación , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Recuento de Células , Células Cultivadas , Diabetes Mellitus Experimental/terapia , Ditiotreitol/administración & dosificación , Ditiotreitol/análogos & derivados , Ditiotreitol/farmacología , Hidrogeles/administración & dosificación , Hidrogeles/farmacología , Inyecciones , Trasplante de Islotes Pancreáticos/métodos , Masculino , Ratones , Ratones SCID , Neovascularización Fisiológica/efectos de los fármacos , Polietilenglicoles/administración & dosificación , Polietilenglicoles/farmacología , Ácidos Polimetacrílicos/administración & dosificación , Ácidos Polimetacrílicos/farmacología , RatasRESUMEN
A stimuli-responsive controlled-release delivery system based on carbon nanotubes is demonstrated. Through TEM, FTIR spectroscopic, and thermogravimetric analysis, functional groups have been successfully attached to the open ends of the tubes, thereby enabling functionalized silica spheres to preferentially attach to the ends. This, in essence, plugs the ends of the tube. Controlling release of encapsulated materials within the tubes is illustrated by fluorescein-filled carbon nanotubes. The discharge process can be triggered by exposure to 1,4-dithiothreitol (DTT) or at elevated temperature. Moreover, both triggering systems, DTT and temperature, provide rate of release control through increased DTT concentration or temperature choice, respectively. This delivery system paves the way for the development of a new generation of site-selective, controlled-release, drug-delivery systems, and interactive nanosensor devices.
Asunto(s)
Ditiotreitol/análogos & derivados , Ditiotreitol/química , Sistemas de Liberación de Medicamentos , Nanotubos de Carbono/química , Dióxido de Silicio/química , Fluoresceína/administración & dosificación , Fluoresceína/química , Nanotubos de Carbono/ultraestructura , Dióxido de Silicio/administración & dosificación , Espectroscopía Infrarroja por Transformada de Fourier , TemperaturaRESUMEN
The purpose of this study was to examine whether the expression of two chitin synthase genes, AgCHS1 and AgCHS2, can be repressed by chitosan/AgCHS dsRNA-based nanoparticles through larval feeding in Anopheles gambiae. The AgCHS1 transcript level and chitin content were reduced by 62.8 and 33.8%, respectively, in the larvae fed on chitosan/AgCHS1 dsRNA nanoparticles compared with those of the control larvae fed on chitosan/GFP dsRNA nanoparticles. Our study suggested for the first time that RNA interference (RNAi) in mosquito larvae is systemic, and demonstrated that the larvae fed on the nanoparticles assembled from AgCHS1 and AgCHS2 dsRNA increased larval susceptibilities to diflubenzuron, and calcofluor white (CF) or dithiothreitol, respectively. These results suggest great potential for using such a nanoparticle-based RNAi technology for high-throughput screening of gene functions and for developing novel strategies for pest management.
Asunto(s)
Anopheles/genética , Control de Mosquitos/métodos , Interferencia de ARN , Animales , Anopheles/crecimiento & desarrollo , Anopheles/fisiología , Bencenosulfonatos/toxicidad , Quitina Sintasa/genética , Quitosano/administración & dosificación , Diflubenzurón/toxicidad , Ditiotreitol/análogos & derivados , Ditiotreitol/toxicidad , Conducta Alimentaria , Larva/genética , Nanopartículas/administración & dosificación , ARN Bicatenario/genéticaRESUMEN
Hitherto, the decay mechanisms of nitrosated dithiols as well as formation of related products have not been conclusively elucidated. In this paper, we demonstrate that nitrosated dl-dithiothreitol (DTT) decays via two independent pathways, that is, one producing exclusively nitric oxide and one producing (initially) nitroxyl (HNO/3NO-). The importance of the two decomposition pathways depends on the degree of nitrosation of DTT. Dinitroso-dithiothreitol (NODTTNO) generates quantitatively nitric oxide, whereas mononitroso-dithiothreitol (NODTT) yields initially nitroxyl. Since NODTT and DTT are both targets for nitroxyl, their availability governs the HNO-derived formation of nitric oxide (with NODTT as reactant) or hydroxyl amine and ammonium ion (with DTT as reactant). The formation of NH4+ from the HNO-DTT reaction probably proceeds by a stepwise, NH2OH-independent mechanism, because DTT-derived sulfinamide was identified by N-15 NMR spectrometry as an intermediate. Our data are in line with the assumption that triplet nitroxyl (3NO-) is formed by a unimolecular decay of the deprotonated (thiolate) form of NODTT, because CBS-QB3 calculations predict the existence of a low-lying triplet state of the latter species. The identified pathways are proposed to be of general importance for physiological systems because control experiments showed that the physiological dithiol thioredoxin reacts in a similar manner.
Asunto(s)
Ditiotreitol/análogos & derivados , Óxido Nítrico/química , Compuestos Nitrosos/química , Tolueno/análogos & derivados , Ditiotreitol/química , Cinética , Espectroscopía de Resonancia Magnética , Oxidación-Reducción , Teoría Cuántica , Tolueno/químicaRESUMEN
Interleukin-1 (IL-1) has pleiotropic effects in cartilage. The interferon-induced, double stranded RNA-activated protein kinase PKR that phosphorylates eukaryotic initiation factor 2 (eIF2) alpha has been implicated in cytokine effects in chondrocytes. A compound was recently identified that potently suppresses PKR autophosphorylation (IC50 approximately 200 etaM) and partially restores PKR-inhibited translation in a cell-free system with significant effect in the nanomolar range. The objectives of this study were to exploit this potent PKR inhibitor to assess whether PKR kinase activity is required for catabolic and proinflammatory effects of IL-1alpha in cartilage and to determine whether IL-1alpha causes an increase in eIF2alpha phosphorylation that is antagonized by the PKR inhibitor. Cartilage explants were incubated with the PKR inhibitor and IL-1alpha. Culture media were assessed for sulfated glycosaminoglycan as an indicator of proteoglycan degradation and for prostaglandin E(2). Cartilage extracts were analyzed by Western blot for cyclooxygenase-2 and phosphorylated signaling molecules. Nanomolar concentrations of the PKR inhibitor suppressed proteoglycan degradation and cyclooxygenase-2 accumulation in IL-1alpha-activated cartilage. The PKR inhibitor stimulated or inhibited PGE(2) production with a biphasic dose response relationship. IL-1alpha increased the phosphorylation of both PKR and eIF2alpha, and nanomolar concentrations of PKR inhibitor suppressed the IL-1alpha-induced changes in phosphorylation. The results strongly support PKR involvement in pathways activated by IL-1alpha in chondrocytes.
Asunto(s)
Cartílago/efectos de los fármacos , Condrocitos/efectos de los fármacos , Interleucina-1alfa/farmacología , eIF-2 Quinasa/metabolismo , Animales , Western Blotting , Articulaciones del Carpo/efectos de los fármacos , Articulaciones del Carpo/metabolismo , Cartílago/citología , Cartílago/metabolismo , Bovinos , Condrocitos/citología , Condrocitos/metabolismo , Medios de Cultivo Condicionados/análisis , Medios de Cultivo Condicionados/química , Ciclooxigenasa 2/metabolismo , Ditiotreitol/análogos & derivados , Ditiotreitol/farmacología , Relación Dosis-Respuesta a Droga , Factor 2 Eucariótico de Iniciación/metabolismo , Glicosaminoglicanos/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteoglicanos/metabolismo , Sulfatos/metabolismo , Factores de Tiempo , eIF-2 Quinasa/antagonistas & inhibidoresRESUMEN
The phosphatase of regenerating liver-1, PRL-1, gene was detected in a screen for foveal cone photoreceptor-associated genes. It encodes a small protein tyrosine phosphatase that was previously immunolocalized to the photoreceptors in primate retina. Here we report that in cones and cone-derived cultured cells both PRL-1 activity and PRL-1 gene expression are modulated under oxidative stress. Oxidation reversibly inhibited the phosphatase activity of PRL-1 due to the formation of an intramolecular disulfide bridge between Cys104 within the active site and another conserved Cys, Cys49. This modulation was observed in vitro, in cell culture and in isolated retinas exposed to hydrogen peroxide. The same treatment caused a rapid increase in PRL-1 expression levels in cultured cells which could be blocked by the protein translation inhibitor, cycloheximide. Increased PRL-1 expression was also observed in living rats subjected to constant light exposure inducing photooxidative stress. We further demonstrated that both oxidation and overexpression of PRL-1 upon oxidative stress are greatly enhanced by inhibition of the glutathione system responsible for cellular redox regulation. These findings suggest that PRL-1 is a molecular component of the photoreceptor's response to oxidative stress acting upstream of the glutathione system.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Proteínas de la Membrana/metabolismo , Estrés Oxidativo , Proteínas Tirosina Fosfatasas/metabolismo , Retina/metabolismo , Animales , Dominio Catalítico , Proteínas de Ciclo Celular/análisis , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Línea Celular , Células Cultivadas , Cicloheximida/farmacología , Cisteína/metabolismo , Ditiotreitol/análogos & derivados , Ditiotreitol/farmacología , Relación Dosis-Respuesta a Droga , Glutatión/análisis , Peróxido de Hidrógeno/farmacología , Proteínas Inmediatas-Precoces/análisis , Proteínas Inmediatas-Precoces/química , Proteínas Inmediatas-Precoces/genética , Proteínas de la Membrana/análisis , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Oxidantes/farmacología , Oxidación-Reducción , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Tirosina Fosfatasas/análisis , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/genética , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Sustancias Reductoras/farmacología , Retina/citología , Retina/efectos de los fármacos , Células Fotorreceptoras Retinianas Conos/citología , Células Fotorreceptoras Retinianas Conos/enzimología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , PorcinosRESUMEN
The difference in the heterogeneous binding of Mg(2+), Ca(2+) and Sr(2+) ions by 1-thioglycerol (TG) and 1,4-dithiothreitol (DTT) spontaneously adsorbed monolayers on Au has been studied following the changes in the double layer capacity. A mathematical treatment, based on calculating the electrochemical potential difference at the monolayer-electrolyte interface, has followed our recent work which dealt with the acid-base equilibrium at the interface as a means of calculating the pK of ionizable SAMs and their binding with Cd(2+). Experimentally, spontaneously adsorbed monolayers of TG and DTT were assembled on Au surfaces and studied by impedance spectroscopy and alternating current voltammetry (ACV). The capacity was measured for each of the modified surfaces at increasing concentrations of the divalent metal ions separately. The goal of this study has been to examine the effect of metal ion binding by similar ligands that are differently attached onto the surface. TG and DTT monolayers differ in their flexibility, which is a result of their attachment to the surface through one and two arms, respectively. The general trend of the apparent heterogeneous association constants of the divalent metal ions, which were calculated from the capacity measurements, was substantially different from the classical Irving-Williams series that is applicable to homogeneous systems. This difference could be nicely explained by the reduction of the degree of freedom and flexibility of the attached ligands.
Asunto(s)
Calcio/química , Ditiotreitol/química , Glicerol/análogos & derivados , Magnesio/química , Estroncio/química , Sitios de Unión , Ditiotreitol/análogos & derivados , Glicerol/química , Oro/química , Ligandos , Modelos Químicos , Propiedades de SuperficieRESUMEN
Bone sialoprotein (BSP) is a multifunctional, highly phosphorylated, and glycosylated protein with key roles in biomineralization and tissue remodeling. This work identifies the complete topographical distribution and precise location of both the in vitro and in vivo phosphorylation sites of bovine BSP by a combination of state-of-the-art techniques and approaches. In vitro phosphorylation of native and deglycosylated BSPs by casein kinase II identified seven phosphorylation sites by solid-phase N-terminal peptide sequencing that were within peptides 12-22 (LEDS(P)EENGVFK), 42-62 (FAVQSSSDSS(P)EENGNGDS(P)S(P)EE), 80-91 (EDS(P)DENEDEES(P)E), and 135-145 (EDES(P)DEEEEEE). The in vivo phosphorylation regions and sites were identified by use of a novel thiol reagent, 1-S-mono[(14)C]carboxymethyldithiothreitol. This approach identified all of the phosphopeptides defined by in vitro phosphorylation, but two additional phosphopeptides were defined at residues, 250-264 (DNGYEIYES(P)ENGDPR), and 282-289 (GYDS(P)YDGQ). Furthermore, use of native BSP and matrix-assisted laser desorption ionization time-of-flight mass spectrometry identified several of the above peptides, including an additional phosphopeptide at residues 125-130 (AGAT(P)GK) that was not defined in either of the in vitro and in vivo studies described above. Overall, 7 in vitro and 11 in vivo phosphorylation sites were identified unequivocally, with natural variation in the quantitative extent of phosphorylation at each in vivo phosphorylation site.
Asunto(s)
Sialoglicoproteínas/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Huesos/metabolismo , Quinasa de la Caseína II , Bovinos , Cromatografía Líquida de Alta Presión , Ditiotreitol/análogos & derivados , Ditiotreitol/farmacología , Glicosilación , Sialoproteína de Unión a Integrina , Datos de Secuencia Molecular , Péptidos/química , Fosforilación , Unión Proteica , Conformación Proteica , Proteínas Serina-Treonina Quinasas/química , Estructura Terciaria de Proteína , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Compuestos de Sulfhidrilo/farmacologíaRESUMEN
We used cDNA microarrays in a systematic study of the gene expression responses of HeLa cells and primary human lung fibroblasts to heat shock, endoplasmic reticulum stress, oxidative stress, and crowding. Hierarchical clustering of the data revealed groups of genes with coherent biological themes, including genes that responded to specific stresses and others that responded to multiple types of stress. Fewer genes increased in expression after multiple stresses than in free-living yeasts, which have a large general stress response program. Most of the genes induced by multiple diverse stresses are involved in cell-cell communication and other processes specific to higher organisms. We found substantial differences between the stress responses of HeLa cells and primary fibroblasts. For example, many genes were induced by oxidative stress and dithiothreitol in fibroblasts but not HeLa cells; conversely, a group of transcription factors, including c-fos and c-jun, were induced by heat shock in HeLa cells but not in fibroblasts. The dataset is freely available for search and download at http://microarray-pubs.stanford.edu/human_stress/Home.shtml.
Asunto(s)
Ditiotreitol/análogos & derivados , Regulación de la Expresión Génica/genética , Respuesta al Choque Térmico/genética , Estrés Oxidativo/genética , Adenocarcinoma/genética , Ditiotreitol/farmacología , Retículo Endoplásmico/efectos de los fármacos , Fibroblastos/metabolismo , Células HeLa , Humanos , Neoplasias Pulmonares/genética , Familia de Multigenes/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Presión Osmótica , Tunicamicina/farmacología , Levaduras/genética , Levaduras/metabolismoRESUMEN
A novel radioactive thiol reagent, 1-S-[3H]carboxymethyl-dithiothreitol (DTT-S-C[3H(2)]CO(2)H, [3H]CM-DTT), was designed and synthesized at the micromole level by reaction of dithiothreitol with tritiated iodoacetic acid (I-C[3H(2)].CO(2)H). The reaction progress was followed by reverse-phase high-performance liquid chromatography (RP-HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The usefulness of the synthesized reagent was evaluated in a series of experimental approaches. (i) The synthetic phosphopeptide, NSVS(P)EEGRGDSV, was derivatized by [3H]CM-DTT separated from excess reagent by RP-HPLC. The extent of derivatization was quantitated in terms of the mol of P-Ser/mol of peptide by 3H counting, and the location of the phosphoserine was defined by the N-terminal Edman degradation sequence analysis as being the fourth amino acid residue from the N terminus. (ii) A sample of trypsin-digested alpha-casein was derivatized with [3H]CM-DTT, peptides were separated by RP-HPLC, and aliquots of each fraction were counted for 3H label within the peptide map which rapidly pinpointed the original four phosphoserine-containing peptides. This demonstrated the utility of the synthesized radioactive thiol agent in rapid purification and identification of phosphopeptides from HPLC peptide mapping of proteolytic digests of phosphoproteins. (iii) The [3H]CM-DTT was also used to determine the extent of phosphorylation of phosphoproteins both qualitatively by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography and quantitatively by 3H counting. The synthesized radioactive thiol reagent [3H]CM-DTT proved to be very efficient and sensitive and should be adaptable to a wide range of routinely utilized laboratory approaches in many fields of the biological sciences.
Asunto(s)
Ditiotreitol/síntesis química , Fosfopéptidos/metabolismo , Análisis de Secuencia de Proteína , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Reactivos de Sulfhidrilo/síntesis química , Sitios de Unión , Cromatografía por Intercambio Iónico , Ditiotreitol/análogos & derivados , Ditiotreitol/química , Fosfopéptidos/química , Fosforilación , Fosfoserina/metabolismo , Radiactividad , Reactivos de Sulfhidrilo/química , TritioRESUMEN
Peroxynitrite generated in arteries from superoxide and NO may damage Ca(2+) pumps. Here, we report the effects of peroxynitrite on ATP-dependent azide-insensitive uptake of Ca(2+) into pig coronary artery vesicular membrane fractions F2 [enriched in plasma membrane (PM)] and F3 [enriched in sarcoplasmic reticulum (SR)]. Membranes were pretreated with peroxynitrite and then with DTT to quench this agent. This pretreatment inhibited Ca(2+) uptake in a peroxynitrite concentration-dependent manner, but the effect was more severe in F3 than in F2. The inhibition was thus not overcome by excess DTT used to quench peroxynitrite and was not affected if catalase, SOD, or mannitol was added along with peroxynitrite. Such damage to the pump protein would be difficult to repair if produced during ischemia-reperfusion. The acylphosphates formed with ATP in F3 corresponded mainly to the SR Ca(2+) pump (110 kDa), but in F2 both PM (140 kDa) and 110-kDa bands were observed. Peroxynitrite treatment of F2 inhibited only the 110-kDa band. Inhibition of Ca(2+) uptake and acylphosphate formation from ATP correlated well in peroxynitrite-treated F3 samples. However, inhibition of acylphosphates from orthophosphate (reverse reaction of the pump) was slightly poorer. Peroxynitrite treatment also covalently cross-linked the pump protein, yielding no dimers but only larger oligomers. In contrast, cross-linking of the SR Ca(2+) pump in skeletal and cardiac muscles gives dimers as the first oligomers. Therefore, we speculate that SERCA2 has a different quaternary structure in the coronary artery smooth muscle.
Asunto(s)
ATPasas Transportadoras de Calcio/metabolismo , Vasos Coronarios/metabolismo , Ditiotreitol/análogos & derivados , Músculo Liso Vascular/metabolismo , Ácido Peroxinitroso/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Retículo Sarcoplasmático/metabolismo , Ácido Anhídrido Hidrolasas/efectos de los fármacos , Ácido Anhídrido Hidrolasas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , ATPasas Transportadoras de Calcio/efectos de los fármacos , Proteínas de Transporte de Catión , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Enfermedad de la Arteria Coronaria/metabolismo , Enfermedad de la Arteria Coronaria/fisiopatología , Vasos Coronarios/efectos de los fármacos , Ditiotreitol/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Músculo Liso Vascular/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Ácido Peroxinitroso/farmacología , ATPasas Transportadoras de Calcio de la Membrana Plasmática , Daño por Reperfusión/metabolismo , Daño por Reperfusión/fisiopatología , Retículo Sarcoplasmático/efectos de los fármacos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Fracciones Subcelulares , Sus scrofa , AcilfosfatasaAsunto(s)
Ditiotreitol/análogos & derivados , Proteínas del Ojo/genética , Proteínas de Filamentos Intermediarios/genética , Glicoproteínas de Membrana , Proteínas del Tejido Nervioso/genética , Disco Óptico/fisiología , Animales , Bovinos , Ditiotreitol/farmacología , Fusión de Membrana/fisiología , Mutagénesis Insercional/genética , Mutación/genética , Periferinas , Sustancias Reductoras/farmacología , Células Fotorreceptoras Retinianas Bastones/fisiologíaRESUMEN
Calcineurin is a serine/threonine phosphatase involved in a wide range of cellular responses to calcium mobilizing signals. Previous evidence supports the notion of the existence of a redox regulation of this enzyme, which might be relevant for neurodegenerative processes, where an imbalance between generation and removal of reactive oxygen species could occur. In a recent work, we have observed that calcineurin activity is depressed in two models for familial amyotrophic lateral sclerosis (FALS) associated with mutations of the antioxidant enzyme Cu,Zn superoxide dismutase (SOD1), namely in neuroblastoma cells expressing either SOD1 mutant G93A or mutant H46R and in brain areas from G93A transgenic mice. In this work we report that while wild-type SOD1 has a protective effect, calcineurin is oxidatively inactivated by mutant SOD1s in vitro; this inactivation is mediated by reactive oxygen species and can be reverted by addition of reducing agents. Furthermore, we show that calcineurin is sensitive to oxidation only when it is in an 'open', calcium-activated conformation, and that G93A-SOD1 must have its redox-active copper site available to substrates in order to exert its pro-oxidant properties on calcineurin. These findings demonstrate that both wild-type and mutant SOD1s can interfere directly with calcineurin activity and further support the possibility of a relevant role for calcineurin-regulated biochemical pathways in the pathogenesis of FALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Calcineurina/metabolismo , Ditiotreitol/análogos & derivados , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Aerobiosis , Esclerosis Amiotrófica Lateral/genética , Antioxidantes/farmacología , Ácido Ascórbico/farmacología , Calcio/metabolismo , Cobre/metabolismo , Ditiotreitol/farmacología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Humanos , Degeneración Nerviosa/genética , Degeneración Nerviosa/metabolismo , Oxidación-Reducción , Mutación Puntual , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Large quaternary ammonium (QA) ions block voltage-gated K(+) (Kv) channels by binding with a 1:1 stoichiometry in an aqueous cavity that is exposed to the cytoplasm only when channels are open. S-nitrosodithiothreitol (SNDTT; ONSCH(2)CH(OH)CH(OH)CH(2)SNO) produces qualitatively similar "open-channel block" in Kv channels despite a radically different structure. SNDTT is small, electrically neutral, and not very hydrophobic. In whole-cell voltage-clamped squid giant fiber lobe neurons, bath-applied SNDTT causes reversible time-dependent block of Kv channels, but not Na(+) or Ca(2)+ channels. Inactivation-removed ShakerB (ShBDelta) Kv1 channels expressed in HEK 293 cells are similarly blocked and were used to study further the action of SNDTT. Dose-response data are consistent with a scheme in which two SNDTT molecules bind sequentially to a single channel, with binding of the first being sufficient to produce block. The dissociation constant for the binding of the second SNDTT molecule (K(d2) = 0.14 mM) is lower than that of the first molecule (K(d1) = 0.67 mM), indicating cooperativity. The half-blocking concentration (K(1/2)) is approximately 0.2 mM. Steady-state block by this electrically neutral compound has a voltage dependence (about -0.3 e(0)) similar in magnitude but opposite in directionality to that reported for QA ions. Both nitrosyl groups on SNDTT (one on each sulfur atom) are required for block, but transfer of these reactive groups to channel cysteine residues is not involved. SNDTT undergoes a slow intramolecular reaction (tau approximately 770 s) in which these NO groups are liberated, leading to spontaneous reversal of the SNDTT effect. Competition with internal tetraethylammonium indicates that bath-applied SNDTT crosses the cell membrane to act at an internal site, most likely within the channel cavity. Finally, SNDTT is remarkably selective for Kv1 channels. When individually expressed in HEK 293 cells, rat Kv1.1-1.6 display profound time-dependent block by SNDTT, an effect not seen for Kv2.1, 3.1b, or 4.2.
Asunto(s)
Ditiotreitol/farmacología , Activación del Canal Iónico/fisiología , Compuestos Nitrosos/farmacología , Canales de Potasio/fisiología , Animales , Sitios de Unión/fisiología , Línea Celular , Membrana Celular/fisiología , Citosol/química , Decapodiformes/fisiología , Ditiotreitol/análogos & derivados , Relación Dosis-Respuesta a Droga , Electrofisiología , Cinética , Neuronas/fisiología , Técnicas de Placa-Clamp , Plásmidos , Compuestos de Amonio Cuaternario/farmacología , Canales de Potasio de la Superfamilia Shaker , Spodoptera/genética , Spodoptera/fisiología , TransfecciónRESUMEN
How the ductus arteriosus (DA) closes at birth remains unclear. Inhibition of O2-sensitive K+ channels may initiate the closure but the sensor mechanism is unknown. We hypothesized that changes in endogenous H2O2 could act as this sensor. Using chemiluminescence measurements with luminol (50 [mu]M) or lucigenin (5 [mu]M) we showed significantly higher levels of reactive O2 species in normoxic, compared to hypoxic DA. This increase in chemiluminescence was completely reversed by catalase (1200 U ml-1). Prolonged normoxia caused a significant decrease in K+ current density and depolarization of membrane potential in single fetal DA smooth muscle cells. Removal of endogenous H2O2 with intracellular catalase (200 U ml-1) increased normoxic whole-cell K+ currents (IK) and hyperpolarized membrane potential while intracellular H2O2 (100 nM) and extracellular t-butyl H2O2 (100 [mu]M) decreased IK and depolarized membrane potential. More rapid metabolism of O2- with superoxide dismutase (100 U ml-1) had no significant effect on normoxic K+ currents. N-Mercaptopropionylglycine (NMPG), duroquinone and dithiothreitol all dilated normoxic-constricted DA rings, while the oxidizing agent 5,5'-dithiobis-(2-nitrobenzoic acid) constricted hypoxia-dilated rings. NMPG also increased IK. We conclude that increased H2O2 levels, associated with a cytosolic redox shift at birth, signal K+ channel inhibition and DA constriction.
Asunto(s)
Conducto Arterial/fisiología , Oxígeno/metabolismo , Transducción de Señal/fisiología , Animales , Antioxidantes/farmacología , Benzoquinonas/farmacología , Catalasa/farmacología , Citoplasma/metabolismo , Ácido Ditionitrobenzoico/farmacología , Ditiotreitol/análogos & derivados , Ditiotreitol/farmacología , Transporte de Electrón/fisiología , Peróxido de Hidrógeno/metabolismo , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Oxidación-Reducción , Técnicas de Placa-Clamp , Potasio/metabolismo , Canales de Potasio/fisiología , Conejos , Reactivos de Sulfhidrilo/farmacologíaRESUMEN
Fibroblasts play a critical role in wound repair and in the development of fibrotic diseases, and transforming growth factor-beta (TGF-beta) has been shown to profoundly modulate fibroblast function. However, there is limited information on the TGF-beta receptor types, isoform specificity, and complex formation in skin fibroblasts. Here, we report that normal adult human skin fibroblasts display two isoform-specific, cell surface glycosyl phosphatidylinositol (GPI)-anchored, TGF-beta binding proteins in addition to the type I, II, and III TGF-beta receptors. The identities of these proteins are confirmed on the basis of their affinity for TGF-beta isoforms, immunoprecipitation with specific antireceptor antibodies, and other biochemical analyses. Immunoprecipitation results also indicated oligomeric complex formation between type I and II and between type II and III TGF-beta receptors. Furthermore, by using affinity labeling and two-dimensional electrophoresis, we demonstrate the occurrence of type I and II heterodimers and type I homodimers of TGF-beta receptors on these cells. Because the type I receptor does not bind TGF-beta in the absence of type II receptor, these results indicate that one molecule of TGF-beta induces the formation of a heterooligomeric complex containing more than one molecule each of type I and II TGF-beta receptors on these cells. These cells respond to TGF-beta by markedly down-regulating all five binding proteins and by potently augmenting DNA synthesis. These results allow the expansion of the proposed heteromeric TGF-beta receptor signaling paradigm using mutant cells that are unresponsive to TGF-beta and cell lines that have been transfected to overexpress these receptors, to include normal TGF-beta-responsive cells. In addition, the definition of TGF-beta receptor profiles in human skin fibroblasts provides important information for studying their alterations in these cells in various skin diseases.
Asunto(s)
Receptores de Activinas Tipo I , Glicosilfosfatidilinositoles/análisis , Proteínas Serina-Treonina Quinasas/biosíntesis , Receptores de Factores de Crecimiento Transformadores beta/biosíntesis , Piel/citología , Anticuerpos , Antioxidantes/farmacología , Células Cultivadas , Dimerización , Ditiotreitol/análogos & derivados , Ditiotreitol/farmacología , Electroforesis en Gel Bidimensional , Fibroblastos/química , Fibroblastos/citología , Homeostasis/fisiología , Humanos , Isomerismo , Fosfatidilinositol Diacilglicerol-Liasa , Pruebas de Precipitina , Unión Proteica/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/inmunología , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/química , Receptores de Factores de Crecimiento Transformadores beta/inmunología , Transducción de Señal/fisiología , Piel/química , Transfección , Factor de Crecimiento Transformador beta/química , Factor de Crecimiento Transformador beta/metabolismo , Fosfolipasas de Tipo C/farmacologíaRESUMEN
We describe a conserved yeast gene, ERO1, that is induced by the unfolded protein response and encodes a novel glycoprotein required for oxidative protein folding in the ER. In a temperature-sensitive ero1-1 mutant, newly synthesized carboxypeptidase Y is retained in the ER and lacks disulfide bonds, as shown by thiol modification with AMS. ERO1 apparently determines cellular oxidizing capacity since mutation of ERO1 causes hypersensitivity to the reductant DTT, whereas overexpression of ERO1 confers resistance to DTT. Moreover, the oxidant diamide can restore growth and secretion in ero1 mutants. Genetic tests distinguish the essential function of ERO1 from that of PDI1. We show that glutathione is not required for CPY folding and conclude that Ero1p functions in a novel mechanism that sustains the ER oxidizing potential, supporting net formation of protein disulfide bonds.
Asunto(s)
Secuencia Conservada , Retículo Endoplásmico/química , Glicoproteínas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Compuestos de Sulfhidrilo/metabolismo , Proteínas de Transporte Vesicular , Antioxidantes/farmacología , Transporte Biológico/fisiología , Disulfuros/química , Disulfuros/metabolismo , Ditiotreitol/análogos & derivados , Ditiotreitol/farmacología , Retículo Endoplásmico/enzimología , Inhibidores Enzimáticos/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Regulación Fúngica de la Expresión Génica/fisiología , Genes Fúngicos/fisiología , Glutatión/farmacología , Glicoproteínas/química , Glicoproteínas/metabolismo , Datos de Secuencia Molecular , Mutagénesis/fisiología , Oxidación-Reducción , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro , Pliegue de Proteína , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , TemperaturaRESUMEN
The structure of many proteins entering the secretory pathway is dependent on stabilization by disulfide bonds. To support disulfide-linked folding, the endoplasmic reticulum (ER) must maintain a strongly oxidizing environment compared to the highly reduced environment of the cytosol. We report here the identification and characterization of Ero1p, a novel and essential ER-resident protein. Mutations in Ero1p cause extreme sensitivity to the reducing agent DTT, whereas overexpression confers DTT resistance. Strikingly, compromised Ero1p function results in ER retention of disulfide-stabilized proteins in a reduced, nonnative form, while not affecting structural maturation of a disulfide-free protein. We conclude that there exists a specific cellular redox machinery required for disulfide-linked protein folding in the ER and that Ero1p is an essential component of this machinery.
Asunto(s)
Retículo Endoplásmico/química , Proteínas Fúngicas/metabolismo , Glicoproteínas/genética , Glicoproteínas de Membrana , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Animales , Antioxidantes/farmacología , Secuencia Conservada , Disulfuros/metabolismo , Ditiotreitol/análogos & derivados , Ditiotreitol/farmacología , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/enzimología , Citometría de Flujo , Proteínas Fúngicas/química , Regulación Fúngica de la Expresión Génica/fisiología , Glicoproteínas/química , Glicoproteínas/metabolismo , Glicósido Hidrolasas/química , Glicósido Hidrolasas/metabolismo , Humanos , Masculino , Datos de Secuencia Molecular , Mutagénesis/fisiología , Oxidación-Reducción , Oxidorreductasas , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro , Pliegue de Proteína , Homología de Secuencia de Aminoácido , beta-FructofuranosidasaRESUMEN
Dithiothreitol is known to be effective in vitro to prevent or even revert effects of oxidative stress and radiation in biological systems. However, its use in in vivo conditions has been hampered by its chemical instability and toxicity. Dithiothreitol tetraacetate is stable and less toxic than dithiothreitol. In this work, GLC-MS evidence is presented that dithiothreitol tetraacetate is hydrolyzed in vivo to give dithiothreitol. The activity of a dithiothreitol tetraacetate thiol esterase in blood and in different organs was determined and found to be: small intestine >> liver > pancreas >> testes approximately kidney > or = brain approximately adrenals approximately bone marrow approximately colon approximately lung > plasma approximately heart > total blood. These results suggest that dithiothreitol tetraacetate might be a suitable source of more stable and less toxic dithiothreitol in vivo, and that its production is due to an hydrolytic process requiring a dithiothreitol tetraacetate thiol esterase present in different organs and in blood.
Asunto(s)
Antioxidantes/metabolismo , Ditiotreitol/análogos & derivados , Acetilesterasa/metabolismo , Animales , Ditiotreitol/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Dithiothreitol (DTT) is known to prevent or even reverse several deleterious effects of radiation or of chemical agents operating via free radical and oxidative stress. However, its use has been hampered by its chemical instability and toxic properties. In this work, we synthesized and characterized dithiothreitol tetraacetate (DTT-Ac) which is less toxic and chemically stable, and we provided GLC/MS evidence that it is able to rapidly generate fully deacetylated DTT in liver after its administration to rats. Treatment with DTT-Ac simultaneously with CCl4 or 3 h after the hepatotoxin was able to significantly prevent the CCl4-induced liver necrosis at 24 h after poisoning. DTT-Ac administration was able to significantly reduce the intensity of the covalent binding of CCl4 reactive metabolites to microsomal lipids (CB), but it did not prevent the CCl4-induced initiation of a lipid peroxidation (LP) process as evidenced by diene hyperconjugation of microsomal lipids. Results suggest that DTT-Ac protective effects might be due to its in vivo conversion to DTT which in turn would decrease the intensity of CB via different potential mechanisms to be explored. Protection cannot be attributed to decreases in levels of CCl4 reaching the liver or to chain breaking effects on LP.