RESUMEN
The use of nanovectors in several medicinal treatments has reached a great importance in the last decade. Some drugs need to be protected to increase their lifetimes in the blood flow, to avoid degradation, to be delivered into target cells or to decrease their side effects. The goal of this work was to design and prepare nanovectors formed by novel surfactants derived from the [Ru(bpy)3]2+ complex. These amphiphilic molecules are assembled to form metallomicelles which can act as pharmaceutical agents and, at the same time, as nanovectors for several drugs. TEM images showed a structural transition from spherical to elongated micelles when the surfactant concentration increased. Fluorescence microscopy confirmed the internalization of these metallomicelles into diverse cell lines and cytotoxicity assays demonstrated specificity for some human cancer cells. The encapsulation of various antibiotics was carried out as well as a thorough study about the DNA condensation by the metallomicelles. To the best of our knowledge, applications of these metallomicelles have not been shown in the literature yet.
Asunto(s)
Complejos de Coordinación/química , Micelas , Nanopartículas/química , Rutenio/química , Células A549 , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Complejos de Coordinación/farmacología , Composición de Medicamentos , Células Hep G2 , Humanos , Células MCF-7 , Microscopía de Fuerza Atómica , Microscopía Confocal , Microscopía Electrónica de Transmisión , Nanopartículas/ultraestructura , Rutenio/farmacologíaRESUMEN
With the idea of improving and advancing the design and preparation of new reagents based on cationic surfactants for gene therapy, two luminescent metallosurfactants derived from the [Ru(2,2'-bpy)3](2+) complex were synthesized. Their interaction with DNA and the effect they exert on the conformation of the polynucleotide were studied by using different techniques. The equilibrium binding constants, Kb, of the two surfactants to DNA were obtained at different molar ratios X=[surfactant]/[DNA]. The observed sigmoidal dependence of Kb on X confirms the cooperative character of the binding. After the addition of a determined surfactant concentration, the condensation of the polymer was observed. The amount of surfactant needed to produce this conformational change is lower for the double stranded surfactant than for the single chain surfactant due to a stronger hydrophobic interaction. The addition of α-cyclodextrin molecules to the metallosurfactant/DNA solutions results in polynucleotide decompaction, which confirms the importance of the hydrophobic interactions in the condensation of the polynucleotide. Results also show the importance of choosing both a proper system to study and the most seeming measuring technique to use. It is demonstrated that, in some cases, the use of several techniques is desirable to obtain reliable and accurate results.
Asunto(s)
ADN/química , Metales/química , Rubidio/química , Tensoactivos/química , Espectrometría de FluorescenciaRESUMEN
A study of a dinitro-diester calix[4]arene (5,17-(3-nitrobenzylideneamino)-11,23-di-tert-butyl-25,27-diethoxycarbonyl methyleneoxy-26,28-dihydroxycalix[4]arene) interaction with calf-thymus DNA was carried out using several techniques. The measurements were done at various molar ratios X=[calixarene]/[DNA]. Results show diverse changes in the DNA conformation depending on the X value. Thus, at low macrocycle concentrations, the calixarene binds to the polynucleotide. This interaction, mainly in groove mode, weakens the hydrogen bonds between base pairs of the helix inducing denaturation of the double strands, as well as condensation of the macromolecule, from an extended coil state to a globular state. An opposite effect is observed at X molar ratios higher than 0.07. The de-condensation of DNA happens, that is, the transition from a compact state to a more extended conformation, probably due to the stacking of calixarene molecules in the solution. Results also show the importance of making a proper choice of the system under consideration.
Asunto(s)
Calixarenos/metabolismo , ADN/metabolismo , Desnaturalización de Ácido Nucleico , Animales , Bovinos , Dicroismo Circular , Etidio/química , Microscopía de Fuerza Atómica , Espectrometría de Fluorescencia , ViscosidadRESUMEN
Two new surfactants derived from the tris(2,2'-bipyridine) ruthenium(II) complex, [Ru(2,2'-bpy)(3)](2+), were synthesized and characterized: the double-tailed [Ru(2,2'-bipy)2(4,4'-(C(11)H(23))(2)-2,2'-bipy)](2+) surfactant (RuC11) and the mono-tailed [Ru(2,2'-bipy)(2)(4-(CH(3))-4'-(C(13)H(27))-2,2'-bipy)](2+) surfactant (RuC13). The main characteristic of these species is the presence of an inorganic complex as the polar head of the surfactant with interesting luminescence properties, which were used to study the interaction of these cationic surfactants with α-, ß- and γ-cyclodextrins (CD). The results showed the formation of [2]- and [3]-pseudorotaxanes. The binding constant values as well as the stoichiometry of the complexes formed were obtained; the results were confirmed, from a qualitative point of view, with NMR spectra.
RESUMEN
Although there is a WHO guidance for a limit on residual DNA for parenterally administered vaccines produced on continuous cell lines, there is no corresponding guidance for oral vaccines. To help determine an oral limit, we performed a study of Vero cell DNA uptake in rats, in which the relative uptake and persistence of Vero cell DNA administered orally was compared to its uptake when delivered intramuscularly (IM). The results of this study allowed the generation of an empirically derived IM versus oral factor (10(6)) representing the relative inefficiency of DNA uptake by oral administration. This factor was then applied to the WHO recommended parenteral limit of 10 ng/dose to determine a corresponding upper limit on the level of residual Vero cell DNA for an oral vaccine of 10 mg. As a conservative approach, this empirically determined limit was reduced 100-fold to 100 microg. Thus, the results of this animal study, together with additional evidence in the literature, support a residual DNA safety limit of 100 microg per dose for an oral vaccine produced on a continuous cell line.
Asunto(s)
ADN/administración & dosificación , ADN/efectos adversos , Vacunas/normas , Administración Oral , Animales , Línea Celular , Chlorocebus aethiops , ADN/farmacocinética , Desoxirribonucleasas , Endocitosis , Endosomas/fisiología , Femenino , Humanos , Masculino , Guías de Práctica Clínica como Asunto , Vacunas/administración & dosificación , Células Vero , Organización Mundial de la SaludRESUMEN
HFE is related to major histocompatibility complex (MHC) class I proteins and is mutated in the iron-overload disease hereditary haemochromatosis. HFE binds to the transferrin receptor (TfR), a receptor by which cells acquire iron-loaded transferrin. The 2.8 A crystal structure of a complex between the extracellular portions of HFE and TfR shows two HFE molecules which grasp each side of a twofold symmetric TfR dimer. On a cell membrane containing both proteins, HFE would 'lie down' parallel to the membrane, such that the HFE helices that delineate the counterpart of the MHC peptide-binding groove make extensive contacts with helices in the TfR dimerization domain. The structures of TfR alone and complexed with HFE differ in their domain arrangement and dimer interfaces, providing a mechanism for communicating binding events between TfR chains. The HFE-TfR complex suggests a binding site for transferrin on TfR and sheds light upon the function of HFE in regulating iron homeostasis.
Asunto(s)
Antígenos HLA/química , Hemocromatosis , Antígenos de Histocompatibilidad Clase I/química , Proteínas de la Membrana , Receptores de Transferrina/química , Cristalografía por Rayos X , Antígenos HLA/genética , Antígenos HLA/metabolismo , Hemocromatosis/genética , Proteína de la Hemocromatosis , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Hierro/metabolismo , Sustancias Macromoleculares , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica , Receptores de Transferrina/metabolismo , Transferrina/metabolismoRESUMEN
HFE, the protein that is mutated in hereditary haemochromatosis, binds to the transferrin receptor (TfR). Here we show that wild-type HFE and TfR localize in endosomes and at the basolateral membrane of a polarized duodenal epithelial cell line, whereas the primary haemochromatosis HFE mutant, and another mutant with impaired TfR-binding ability accumulate in the ER/Golgi and at the basolateral membrane, respectively. Levels of the iron-storage protein ferritin are greatly reduced and those of TfR are slightly increased in cells expressing wild-type HFE, but not in cells expressing either mutant. Addition of an endosomal-targeting sequence derived from the human low-density lipoprotein receptor (LDLR) to the TfR-binding-impaired mutant restores its endosomal localization but not ferritin reduction or TfR elevation. Thus, binding to TfR is required for transport of HFE to endosomes and regulation of intracellular iron homeostasis, but not for basolateral surface expression of HFE.
Asunto(s)
Antígenos HLA/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Hierro/metabolismo , Proteínas de la Membrana , Receptores de Transferrina/metabolismo , Transporte Biológico Activo , Línea Celular , Polaridad Celular , Endocitosis , Endosomas/metabolismo , Células Epiteliales/metabolismo , Proteínas Fluorescentes Verdes , Antígenos HLA/genética , Hemocromatosis/genética , Hemocromatosis/inmunología , Hemocromatosis/metabolismo , Proteína de la Hemocromatosis , Antígenos de Histocompatibilidad Clase I/genética , Homeostasis , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Modelos Biológicos , Mutación , Unión Proteica , Receptores de LDL/genética , Receptores de LDL/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
HFE is a class I major histocompatibility complex (MHC)-related protein that is mutated in patients with the iron overload disease hereditary hemochromatosis. HFE binds to transferrin receptor (TfR), the receptor used by cells to obtain iron in the form of diferric transferrin (Fe-Tf). Previous studies demonstrated that HFE and Fe-Tf can bind simultaneously to TfR to form a ternary complex, and that membrane-bound or soluble HFE binding to cell surface TfR results in a reduction in the affinity of TfR for Fe-Tf. We studied the inhibition by soluble HFE of the interaction between soluble TfR and Fe-Tf using radioactivity-based and biosensor-based assays. The results demonstrate that HFE inhibits the TfR:Fe-Tf interaction by binding at or near the Fe-Tf binding site on TfR, and that the Fe-Tf:TfR:HFE ternary complex consists of one Fe-Tf and one HFE bound to a TfR homodimer.
Asunto(s)
Compuestos Férricos/metabolismo , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Proteínas de la Membrana , Receptores de Transferrina/metabolismo , Transferrina/metabolismo , Unión Competitiva , Técnicas Biosensibles , Antígenos HLA/genética , Hemocromatosis/metabolismo , Proteína de la Hemocromatosis , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Unión Proteica , Proteínas Recombinantes/metabolismoRESUMEN
HFE is a class I major histocompatibility complex (MHC)-related protein that is mutated in patients with the iron storage disease hereditary hemochromatosis. HFE binds tightly to transferrin receptor (TfR), the receptor that mediates uptake of iron-loaded transferrin. The binding affinities for TfR of HFE mutants, designed using the HFE crystal structure, were measured using biosensor assays. The results allow localization of the TfR binding site on HFE to the C-terminal portion of the alpha1 domain helix and an adjacent loop, a region distinct from the ligand binding sites on class I MHC and related proteins. A biosensor-derived pH-dependent affinity profile for the HFE-TfR interaction is discussed in terms of HFE's hypothesized role in intracellular trafficking.
Asunto(s)
Antígenos HLA/metabolismo , Hemocromatosis/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Proteínas de la Membrana , Receptores de Transferrina/metabolismo , Animales , Sitios de Unión , Células CHO , Cricetinae , Antígenos HLA/química , Antígenos HLA/genética , Hemocromatosis/genética , Proteína de la Hemocromatosis , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Concentración de Iones de Hidrógeno , Ligandos , Mutagénesis Sitio-Dirigida , Conformación ProteicaRESUMEN
Generating monoclonal antibodies (mAbs) against one polypeptide chain of a heterodimeric protein can be difficult when the other chain is more immunogenic. To influence the immune response in favor of the less immunogenic protein, we rendered adult mice tolerant to the immunodominant protein using a procedure based on the phenomenon of high zone tolerance. We then immunized the tolerized mice with a heterodimeric protein containing the immunogenic protein and produced hybridomas in the usual way. Screening the hybridomas for reactivity against the immunodominant protein and against the heterodimer revealed that this tolerization procedure can result in an increase of hybridomas producing mAbs against the protein of interest by up to 90-fold. This method should be of general utility for the production of mAbs against weakly antigenic proteins in mixtures of antigens.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Tolerancia Inmunológica , Epítopos Inmunodominantes/inmunología , Epítopos Inmunodominantes/farmacología , Lectinas Tipo C , Proteínas de la Membrana , Animales , Anticuerpos Monoclonales/inmunología , Antígenos CD/inmunología , Antígenos CD/farmacología , Células CHO/metabolismo , Cricetinae , Dimerización , Femenino , Antígenos HLA/inmunología , Antígenos HLA/metabolismo , Antígenos HLA/farmacología , Proteína de la Hemocromatosis , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Antígenos de Histocompatibilidad Clase I/farmacología , Hibridomas , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/farmacología , Ratones , Ratones Endogámicos BALB C , Subfamília D de Receptores Similares a Lectina de las Células NK , Microglobulina beta-2/inmunologíaRESUMEN
HFE is an MHC-related protein that is mutated in the iron-overload disease hereditary hemochromatosis. HFE binds to transferrin receptor (TfR) and reduces its affinity for iron-loaded transferrin, implicating HFE in iron metabolism. The 2.6 A crystal structure of HFE reveals the locations of hemochromatosis mutations and a patch of histidines that could be involved in pH-dependent interactions. We also demonstrate that soluble TfR and HFE bind tightly at the basic pH of the cell surface, but not at the acidic pH of intracellular vesicles. TfR:HFE stoichiometry (2:1) differs from TfR:transferrin stoichiometry (2:2), implying a different mode of binding for HFE and transferrin to TfR, consistent with our demonstration that HFE, transferrin, and TfR form a ternary complex.
Asunto(s)
Antígenos HLA/química , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/metabolismo , Proteínas de la Membrana , Estructura Secundaria de Proteína , Receptores de Transferrina/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Membrana Celular/metabolismo , Cristalografía por Rayos X/métodos , Antígenos HLA/genética , Hemocromatosis/genética , Proteína de la Hemocromatosis , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Receptores de Transferrina/químicaRESUMEN
We recently reported the positional cloning of a candidate gene for hereditary hemochromatosis called HFE. The gene product, a member of the major histocompatibility complex class I-like family, was found to have a mutation, Cys-282 --> Tyr (C282Y), in 85% of patient chromosomes. This mutation eliminates the ability of HFE to associate with beta2-microglobulin (beta2m) and prevents cell-surface expression. A second mutation that has no effect on beta2m association, H63D, was found in eight out of nine patients heterozygous for the C282Y mutant. In this report, we demonstrate in cultured 293 cells overexpressing wild-type or mutant HFE proteins that both the wild-type and H63D HFE proteins form stable complexes with the transferrin receptor (TfR). The C282Y mutation nearly completely prevents the association of the mutant HFE protein with the TfR. Studies on cell-associated transferrin at 37 degrees C suggest that the overexpressed wild-type HFE protein decreases the affinity of the TfR for transferrin. The overexpressed H63D protein does not have this effect, providing the first direct evidence for a functional consequence of the H63D mutation. Addition of soluble wild-type HFE/beta2m heterodimers to cultured cells also decreased the apparent affinity of the TfR for its ligand under steady-state conditions, both in 293 cells and in HeLa cells. Furthermore, at 4 degrees C, the added soluble complex of HFE/beta2m inhibited binding of transferrin to HeLa cell TfR in a concentration-dependent manner. Scatchard plots of these data indicate that the added heterodimer substantially reduced the affinity of TfR for transferrin. These results establish a molecular link between HFE and a key protein involved in iron transport, the TfR, and raise the possibility that alterations in this regulatory mechanism may play a role in the pathogenesis of hereditary hemochromatosis.
Asunto(s)
Antígenos HLA/genética , Hemocromatosis/genética , Antígenos de Histocompatibilidad Clase I/genética , Proteínas de la Membrana , Receptores de Transferrina/metabolismo , Secuencia de Aminoácidos , Dimerización , Proteína de la Hemocromatosis , Humanos , Ligandos , Mutación Puntual , Pruebas de Precipitina , Unión Proteica , Relación Estructura-Actividad , Transfección , Transferrina/metabolismo , Microglobulina beta-2/metabolismoRESUMEN
Recognition of self peptides bound to the class I major histocompatibility complex molecule HLA-B27 is thought to trigger proliferation of autoreactive T cells and result in autoimmune arthritic diseases. Previous work from other laboratories established that a predominant feature of endogenous peptides eluted from purified B27 is an arginine at position 2. We studied the binding of peptides containing both natural and unnatural amino acids by the subtype HLA-B*2702, with the goal of gaining insight into peptide binding by this B27 subtype that is associated with susceptibility to arthritic disease. A soluble from of B*2702 was depleted of endogenous peptides. We tested the binding of peptides substituted with cysteine, homocysteine, or an alpha-amino-epsilon-mercapto hexanoic acid side chain (Amh) instead of the naturally occurring arginine at position 2, to determine whether the peptide sulfhydryl residue could be covalently linked to cysteine 67 in the B*2702 binding cleft. Although none of the altered peptide sequences bound covalently to B*2702, the affinities of the homocysteine- and Amh-substituted peptides were close to that of the native peptide sequence. Substitutions at position 2 with other side chains, such as glutamine and methionine, also resulted in peptides that bound with only slightly reduced affinity. These results demonstrate that peptide side chains other than arginine at position 2 can be accomodated within the B*2702 peptide binding site with only minor reductions in affinity. This extended repertoire of permissible B27-binding peptides should be taken into account for a consideration of disease-associated peptide sequences.