RESUMEN
In studies on Culicoides attacking livestock in the Netherlands, we chanced upon a species of the Obsoletus complex that we do not recognize, but whose dark wing pattern is distinctive. Nine cytochrome c oxidase (CO1) sequences of our so-called 'dark obsoletus' support its status as a separate species, the sequences differing significantly from those representing Culicoides obsoletus (Meigen) (90-91% homology) and Culicoides scoticus Downes & Kettle (87-88% homology). In the last decade, several research groups in Europe have encountered 'mystery species' related to C. obsoletus and in some instances have made their sequences for various genetic loci available in GenBank. These include a CO1 series submitted from Sweden in 2012 (annotated as 'obsoletus 01, 02, or 03 MA-2012') and of which some share a 99% identity with our sequences for 'dark obsoletus'. Without doubt, the series from the Netherlands, along with a portion of the Swedish submissions, together represent a single species ('dark obsoletus'). Whether this species is referable to the Russian Culicoides gornostaevae Mirzaeva recorded recently from Norway, Sweden and Poland, and based solely upon the external morphology of the male, is not clear. The presence in Western Europe of multiple undescribed species related to C. obsoletus means that the taxonomy of this important vector complex is not fully resolved; consequently, we know little about these cryptic species with regard to seasonality, geographic range and host preference. This is undesirable given that Culicoides-borne arboviruses causing disease in livestock are moving more regularly out of the tropics and spreading north into temperate latitudes.
Asunto(s)
Ceratopogonidae/clasificación , Ganado/parasitología , Filogenia , Animales , Ceratopogonidae/genética , Complejo IV de Transporte de Electrones/genética , Femenino , Países Bajos , Homología de Secuencia , Especificidad de la Especie , Alas de Animales/anatomía & histologíaRESUMEN
It has long been known that under intracellular conditions vasopressin associates tightly to neurophysin, which is present in the same prohormone. As the association has been suggested to play a role during hormone biosynthesis, its role was studied in a cellular context by expressing mutant vasopressin precursors in Neuro2A cells. Mutant vasopressin precursors, in which the association between the vasopressin and neurophysin domains was prevented either by deleting the vasopressin domain from the precursor or by substitution of the essential Tyr2 residue in vasopressin for Gly, were neither processed nor targeted into secretory granules. Rather, both provasopressin mutants were retained in the endoplasmic reticulum. Our results demonstrate that the vasopressin domain is crucial for correct trafficking of the prohormone through the secretory pathway, and suggest that vasopressin-neurophysin association provides correct prohormone folding in the endoplasmic reticulum.
Asunto(s)
Transporte de Proteínas/fisiología , Vasopresinas/genética , Vasopresinas/metabolismo , Animales , Línea Celular Tumoral , Diabetes Insípida/metabolismo , Diabetes Insípida/fisiopatología , Retículo Endoplásmico/metabolismo , Expresión Génica , Mutagénesis , Neuroblastoma , Neurofisinas/química , Neurofisinas/genética , Pliegue de Proteína , Estructura Terciaria de Proteína , Ratas , Vesículas Secretoras/metabolismo , Vasopresinas/químicaRESUMEN
Owing to the heterogeneity of cognitive dysfunctions in patients with Alzheimer's disease (AD), a single drug may not suffice. Combination therapy may be the most appropriate solution, but the cognitive networks in the brain have yet to be unraveled. In the years ahead, more comprehensive and precise knowledge of neurological function will allow scientists to pinpoint ever more sophisticated and effective therapeutic options for AD patients. In this review, the author discusses the currently known advantages and disadvantages of neuropeptides and how they may provide yet another drug target for AD treatment.
RESUMEN
The sorting of soluble proteins into the regulated secretory pathway (RSP) involves aggregation, but whether an additional sorting domain is also required is not clear. By fusing vasopressin prohormone (proVP) fragments to green fluorescent protein (eGFP) we have determined whether a sorting domain can function independently of the aggregative neurophysin domain. Although eGFP itself can be immunolocalised in the RSP of Neuro2A and AtT20 cells, most of the protein enters the constitutive pathway, and is found in the culture medium. In contrast, the N-terminal 27 residues of proVP promote residence in the RSP. Furthermore, only the processed form of this fusion was secreted when stimulated. We suggest a sorting mechanism based on the recognition of a sorting motif, the efficiency of which is enhanced by neurophysin aggregation.
Asunto(s)
Vasopresinas/metabolismo , Animales , Transporte Biológico , Línea Celular , Ratones , Precursores de Proteínas/metabolismoRESUMEN
Biosynthesis of the vasopressin (VP) prohormone in magnocellular neurones of the hypothalamo-neurohypophysial system comprises endoplasmic reticulum (ER) transit, sorting into the regulated secretory pathway and subsequent processing in the individual proteins VP, neurophysin and a glycoprotein. These processes are severely disrupted in the homozygous diabetes insipidus (di/di) Brattleboro rat, which expresses a mutant VP precursor due to a single nucleotide deletion in the neurophysin region of the VP gene resulting in VP deficiency. Previous studies have shown the presence of additional frameshift mutations in VP transcripts, in solitary magnocellular neurones of the di/di rat due to a GA dinucleotide deletion resulting in two different mutant VP precursors with partly restored reading frame. Frameshifted VP precursors are also expressed in several magnocellular neurones in wild-type rats. In this study, we determined if the +1 frameshifted precursors from di/di and wild-type rats can lead to biosynthesis of the hormone VP. Therefore, eukaryotic expression plasmids containing the frameshifted VP cDNAs were transiently expressed in peptidergic tumour cell lines, and cells were analysed by reversed phase high-performance liquid chromatography and specific radioimmunoassays, and by immunofluoresence. Neuro2A neuroblastoma cells expressing the +1 frameshifted precursors of di/di rats retained products in the cell body. Only precursor or insignificant quantities of neurophysin-immunoreactive products were detected. In contrast, in AtT20 cells, frameshifted VP precursors were at least partly processed to yield the VP peptide, indicating that they have access to the regulated secretory pathway. Comparison between the two cell lines showed a very slow ER transit of the wild-type prohormone combined with inefficient processing in Neuro2A cells. The results show that mutant precursors can reach the regulated secretory pathway if ER transport is sufficiently rapid as in the case of AtT20 cells. This suggests that the di/di rat may regain the capacity to biosynthesize authentic VP through these +1 frameshifted precursors in magnocellular neurones.
Asunto(s)
Mutación del Sistema de Lectura , Precursores de Proteínas/genética , Procesamiento Proteico-Postraduccional , Vasopresinas/genética , Animales , Transporte Biológico , Diabetes Insípida/genética , Retículo Endoplásmico/metabolismo , Eliminación de Gen , Ratones , Precursores de Proteínas/biosíntesis , Precursores de Proteínas/metabolismo , Ratas , Ratas Brattleboro , Distribución Tisular , Células Tumorales Cultivadas , Vasopresinas/biosíntesis , Vasopresinas/metabolismoRESUMEN
The trafficking of prohormones and of regulated secretory proteins in general has been studied extensively in the last decades of the last century. Prohormone trafficking starts with correct folding and subsequently efficient sorting into the secretory granule of the regulated secretory pathway. The vasopressin/oxytocin prohormone is particularly interesting for studying protein trafficking, because the physicochemical properties and three-dimensional structure have been largely elucidated. In the case of pro-vasopressin and pro-oxytocin, folding and sorting depend completely on both intramolecular and intermolecular interactions. Proper folding is guided by the hormone-neurophysin association and the sorting event relies on the aggregative properties of the neurophysin domain in the prohormone, as well as a specific sorting signal, which is revealed when the aggregative property of the neurophysin domain is deleted. A comprehensive mechanism for trafficking of the vasopressin/oxytocin prohormone from the endoplasmic reticulum to the secretory granule is proposed.
Asunto(s)
Oxitocina/metabolismo , Precursores de Proteínas/metabolismo , Vasopresinas/metabolismo , Secuencia de Aminoácidos/genética , Animales , Humanos , Neurofisinas/química , Neurofisinas/fisiología , Oxitocina/química , Oxitocina/genética , Pliegue de Proteína , Precursores de Proteínas/química , Precursores de Proteínas/genética , Vasopresinas/química , Vasopresinas/genéticaRESUMEN
Endomorphin (EM)-1 and EM-2 are opioid tetrapeptides, reported within the central nervous system, which have very high specificity and affinity for the mu-opioid receptor. We have used newly developed and well-characterised radioimmunoassays (RIAs) in combination with reversed-phase high-performance liquid chromatography (HPLC) to detect EM-1 and EM-2 immunoreactivity (ir) in rat immune tissues. Endomorphins were detectable in extracts of rat spleen (total EM-1-ir/spleen: 440+/-73 pg, mean+/-SEM, a=group of eight rats; EM-2-ir: 150+/-12 pg) and thymus (EM-1-ir: 152+/-18 pg, mean+/-SEM n=8; EM-2-ir: 156+/-28 pg). EM-2-ir was detectable in extracts of human spleen (338+/-196 pg/g tissue, n=3). Multiple peaks of EM-1-ir and EM-2-ir were observed in rat spleen and thymus extracts, and multiple peaks of EM-2-ir were observed in extracts of human spleen, following reversed-phase HPLC and RIAs. This is the first report of endomorphin immunoreactivity in tissues of the rat and human immune systems.
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Sistema Inmunológico/química , Oligopéptidos/análisis , Adulto , Animales , Cromatografía Líquida de Alta Presión , Reacciones Cruzadas , Femenino , Humanos , Sueros Inmunes/inmunología , Masculino , Persona de Mediana Edad , Radioinmunoensayo/métodos , Ratas , Ratas Wistar , Bazo/química , Timo/química , Extractos de Tejidos/químicaRESUMEN
Insulin-like growth factor-I (IGF-I), a single-chain polypeptide consisting of 70 amino acids and 3 disulfide bridges, is a member of a class of growth factors that are involved in many proliferative and metabolic processes. To assist in solving the crystallographic three-dimensional structure, we have expressed a recombinant fusion protein precursor of IGF-I in a methionine auxotrophic strain of Escherichia coli grown in the presence of selenomethionine. An homogeneous preparation of selenomethionyl-IGF-I was then obtained by chemical cleavage of the fusion protein. The selenomethionine analogue of IGF-I was characterized by electrospray mass spectrometry, peptide mapping, analytical chromatography, and electrophoresis as well as by biological assays. The final preparation of IGF-I was found to incorporate about 90% of selenium and fully retained the functional activity.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/genética , Selenometionina/química , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Factor I del Crecimiento Similar a la Insulina/análogos & derivados , Factor I del Crecimiento Similar a la Insulina/metabolismo , Mapeo Peptídico , Ensayo de Unión Radioligante , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Selenometionina/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Twenty-five compounds (trimetazidine derivatives and other compounds, mostly having a free phenolic group) were examined for their radical scavenging and antioxidant properties. Their reaction with DPPH (2,2-diphenyl-1-picrylhydrazyl) as a measure of radical scavenging capacity was assessed by two parameters, namely EC50 (the concentration of antioxidant decreasing DPPH by 50%), and log Z, a kinetic parameter proposed here and derived from initial second-order rate constants and antioxidant/DPPH ratios. Antioxidant activities were determined by the inhibition of lipid peroxidation and albumin oxidation. The most active compounds were derivatives having a trolox or hydroquinone moiety. Physicochemical and structural properties were determined by molecular modeling as lipophilicity (virtual log P calculations) and H-Surf (solvent-accessible surface of hydroxyl hydrogen) and by quantum mechanical calculations (deltaH(ox) = oxidation enthalpy; deltaH(abs) = enthalpy of hydrogen abstraction). QSAR models were derived to identify molecular mechanisms responsible for the reactivity toward the DPPH radical and for the inhibition of lipid peroxidation. A useful prediction of antioxidant capacity could be achieved from calculated molecular properties and the kinetic parameter developed here.
Asunto(s)
Antioxidantes/química , Modelos Químicos , Picratos , Trimetazidina/análogos & derivados , Trimetazidina/química , Bepridil/análogos & derivados , Bepridil/metabolismo , Compuestos de Bifenilo , Simulación por Computador , Cresoles/química , Depuradores de Radicales Libres/química , Radicales Libres/metabolismo , Hidroquinonas/química , Fenoles/química , Relación Estructura-Actividad , Vitamina E/químicaRESUMEN
1. In this review the structure-function relationships of the different vasopressin prohormone domains are dated and discussed, with special reference to the neurophysin and glycopeptide domains. 2. The primary structures of the currently known neurophysins and glycopeptide sequences are compared and discussed. 3. The hormone-binding and aggregational properties of neurophysin are reviewed and related to a possible function within the regulated secretory pathway. 4. It is proposed, based on the properties reviewed here as well as our own data shown here, that the sorting of the vasopressin prohormone is initiated by hormone binding, which triggers aggregation of the prohormone into the characteristic dense cores of the regulated secretory pathway. 5. This may suggest that prohormone sorting into the regulated secretory pathway is, in general, determined by noncovalent, intramolecular interactions that promote aggregation.
Asunto(s)
Precursores de Proteínas/química , Precursores de Proteínas/fisiología , Vasopresinas/química , Vasopresinas/fisiología , Secuencia de Aminoácidos , Animales , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de ProteínaRESUMEN
PURPOSE: The objective of this work was to assess the influence of binding to plasma proteins and to serum on the brain extraction of four antiinflammatory oxicams. METHODS: The brain extraction of isoxicam, tenoxicam, meloxicam and piroxicam was investigated in rats using the carotid injection technique. Blood protein binding parameters were determined by equilibrium dialysis using human serum, human serum albumin (HSA) and alpha-l-acid glycoprotein (AAG) solutions at various concentrations. RESULTS: All oxicams had low values of brain extraction, between 19% and 39% when dissolved in serum, i.e. under physiological conditions. Brain efflux rate constants calculated from the wash-out curves were the same in the absence or presence of serum. Brain efflux was inversely related to the polarity of the oxicams, such that the higher their H-bonding capacity, the lower their brain efflux. The free dialyzable drug fraction was inversely related to protein concentration. However, rat brain extraction was always higher than expected from in vitro measurements of the dialyzable fraction. CONCLUSIONS: Except for piroxicam whose brain extraction was partially decreased in the presence of proteins, the serum unbound and initially bound fractions of oxicams both seem available for transfer into the brain. Modest affinities for AAG rule out any related effect. More surprising is the apparent lack of effect on brain transfer of the high-affinity binding to HSA and serum. The enhanced brain uptake of meloxicam in the presence of AAG could be a result of interactions between this globular protein and the endothelial wall.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacocinética , Barrera Hematoencefálica/fisiología , Encéfalo/metabolismo , Tiazinas/farmacocinética , Adulto , Animales , Antiinflamatorios no Esteroideos/sangre , Proteínas Sanguíneas/metabolismo , Fenómenos Químicos , Química Física , Humanos , Masculino , Orosomucoide/metabolismo , Unión Proteica , Ratas , Ratas Wistar , Albúmina Sérica/metabolismo , Tiazinas/sangreRESUMEN
We investigated the effects of cyclosporine A, prednisolone and azathioprine on respiration rates, Ca2+-induced swelling and Ca2+ fluxes of rat kidney mitochondria. The three drugs significantly decreased the succinate induced-respiration rates according to concentration-dependent processes. Each drug inhibited about 10% of the respiratory control ratio, with EC50 of 3.7 x 10(-7) M, 5.8 x 10(-9) M for cyclosporine A and azathioprine respectively. Prednisolone was the most effective (19.2%) acting by a two-step process with EC50 of 9.2 x 10(-12) M and 1.9 x 10(-8) M. The combination of the three drugs developed a higher significant decrease of respiratory control ratio than that of each drug but lower than the sum of their respective effects. Inhibitions of swelling and Ca2+ fluxes through mitochondrial membrane due to the combination were not different from those induced by cyclosporine A alone. The action mechanisms of cyclosporine A and prednisolone were total and partial Ca2+ dependent respectively. Azathioprine appears to act by a Ca2+-independent one. It is concluded that azathioprine and prednisolone may worsen cyclosporine A-induced renal mitochondrial alteration of oxidative phosphorylation.
Asunto(s)
Azatioprina/farmacología , Ciclosporina/farmacología , Inmunosupresores/farmacología , Riñón/ultraestructura , Mitocondrias/metabolismo , Fosforilación Oxidativa/efectos de los fármacos , Prednisolona/farmacología , Animales , Calcio/metabolismo , Calcio/farmacología , Masculino , Mitocondrias/efectos de los fármacos , Dilatación Mitocondrial/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Ratas , Ratas Wistar , Succinatos/farmacología , Ácido SuccínicoRESUMEN
The targets of nicotine in the central nervous system (CNS) have been identified as nicotinic acetylcholine (ACh) receptors. Because of their localization in the brain, no data are available about their in vivo modifications. However, nonneuronal nicotine binding sites have been identified on peripheral blood cells. The present study was designed to evaluate the effects of tobacco smoking on granulocyte nicotine binding sites. Binding assays were performed on granulocyte membranes using (-)(3H)nicotine and were analyzed by the Scatchard method in nonsmokers (n=10), smokers (n=10), and ex-smokers (n=10). In nonsmokers, a single-affinity binding site was detected with Kd = 1.08 +/- 0.05 x 10 -9 M. In contrast, in smokers, two different classes of binding sites were identified: one with Kd1 = 2.80 +/- 0.81 x 10 -11 M and another with Kd2 = 3.92 +/- 0.58 x 10 -9 M, similar to the binding site in nonsmokers. Moreover, a twofold increase in nicotine binding-site density was observed in smokers. Ex-smokers recovered a single class of binding sites similar to those observed in nonsmokers when they had stopped smoking for more than 1 yr, whereas recent ex-smokers (1 to 8 mo) displayed a pattern similar to that in smokers, as demonstrated by the persistence of two classes of nicotine binding sites. Chronic nicotine exposure induces modifications of nicotine binding sites on granulocytes. It is noteworthy that the persistence of such alterations corresponds to the very high rate of relapse into smoking observed during the early stages of tobacco cessation. Consequently, monitoring of nicotine binding sites could constitute an interesting approach to understanding the pharmacologic mechanisms involved in tobacco dependence.
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Regulación de la Expresión Génica , Receptores Nicotínicos/genética , Fumar/genética , Adulto , Sitios de Unión , Femenino , Humanos , Masculino , Persona de Mediana Edad , Nicotina/metabolismo , Nicotina/farmacología , Receptores Nicotínicos/metabolismo , Recurrencia , Fumar/metabolismo , Cese del Hábito de Fumar , Factores de Tiempo , TritioRESUMEN
Both aqueous standards and plasmas spiked with standards are used for assays of plasma constituents. In a previous study, high-performance liquid chromatographic (HPLC) assays of plasma norepinephrine, epinephrine, and dopamine performed with spiked plasma reference solutions resulted in lower thresholds and thus gave higher concentrations. To verify the hypothesis that spiked catecholamines bind to plasma proteins (like physiological catecholamines) and thus escape extraction by alumina prior to HPLC, the recovery of standards in human albumin solution was compared with similar investigations using aqueous standards. Our findings confirm that (a) albumin is responsible to a great extent for the drop in recovery and (b) calibration by extraction of aqueous standards is preferable to calibration by spiked plasma for free catecholamine assays.
Asunto(s)
Cromatografía Líquida de Alta Presión/normas , Dopamina/sangre , Epinefrina/sangre , Norepinefrina/sangre , Plasma , Agua , Proteínas Sanguíneas/metabolismo , Calibración , Humanos , Albúmina Sérica/metabolismo , SolucionesRESUMEN
In order to test whether a selective T cell nonresponsiveness to noninherited maternal human leukocyte antigens (NIM) exists, we measured the frequencies of alloreactive T cells of healthy individuals to their NIM HLA antigens and to their noninherited paternal (NIP) HLA antigens by limiting dilution assays. Both the frequencies of cytotoxic T cell precursors (CTLp) and IL-2-producing helper T cell precursors (HTLp) were determined. Similar frequencies were observed for NIM class I-reactive CTLp and NIP class I-reactive CTLp. This was the case when frequencies were determined against total NIM and NIP haplotypes but also when CTLp frequencies against individual NIM and NIP antigens were measured. A positive finding of this study was the confirmation of our earlier observation that CTLp frequencies against individual HLA-A antigens are generally lower than CTLp frequencies against HLA-B. This was found both for the maternal and the paternal HLA-A and -B antigens. Similarly, comparable frequencies of IL-2-producing helper T cell precursors directed against NIM HLA class II antigens and NIP HLA class II antigens were found. When breast-feeding in the neonatal period was considered, no differences in the frequencies of CTLp and HTLp were observed between children who had been breast-fed and children who had not. Therefore the present data do not support the hypothesis that confrontation with noninherited maternal HLA in neonatal life leads to down-regulation of alloreactive T cell responses to the mother.
Asunto(s)
Antígenos HLA/inmunología , Inmunidad Materno-Adquirida/inmunología , Linfocitos T/inmunología , Adulto , Niño , Epítopos , Padre , Femenino , Haplotipos , Humanos , Interleucina-2/farmacología , Masculino , Células Madre/efectos de los fármacos , Células Madre/inmunología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
Eight ligands were used in this study, four basic, three neutral and one acidic. Their binding to serum alpha 1-acid glycoprotein (orosomucoid) was measured at several temperatures, and the data were analysed together by a general model with three unknowns, number of binding sites, delta H0 and delta S0. The partition coefficients of the ligands were measured in octanol/water and heptane/water systems (log Poct. and log Phep.), and their molecular volumes were calculated by molecular modelling techniques. These structural properties allow determination of polarity parameters (delta log Poct.-hep., lambda oct. and lambda hep.) which encode in different proportions the various polar interactions between the solute and the aqueous and organic phases, i.e. hydrogen-bonding capacity and dipolarity/polarizability. This study shows that good correlations exist between delta H0 or delta S0 and polarity parameters, such that the enthalpic contribution to binding increases with increasing polarity of the ligands, mainly hydrogen-bond-donor acidity, whereas their entropic contribution to binding decreases.
Asunto(s)
Orosomucoide/metabolismo , Sitios de Unión , Fenómenos Químicos , Química Física , Enlace de Hidrógeno , Indoles/metabolismo , Isradipino/metabolismo , Ligandos , Nifedipino/análogos & derivados , Nifedipino/metabolismo , Orosomucoide/química , Progesterona/metabolismo , Propanolaminas/metabolismo , Propranolol/metabolismo , Temperatura , Termodinámica , Warfarina/metabolismoRESUMEN
In the oxytocin (OT) gene several regions can be discerned that have a function in regulating its expression. Firstly, in the proximal 5' flanking region regulatory elements have been discovered that are targets for transcription factors of the nuclear hormone receptor family. Through these elements the OT gene of rat and man is responsive to estrogens, thyroid hormones and retinoids. Furthermore, these elements can be employed by the nuclear hormone orphan receptor family for repressive or inductive actions. In the distal 5' flanking region the POU class III proteins Brn-1, Brn-2, Brn-4, that are expressed in magnocellular neurons, and Oct-6 are able to bind, but do not display a significant regulatory activity on the OT gene in heterologous expression systems. Secondly, the OT precursor harbours both the biologically active hormone and the protein neurophysin that is able to associate with the hormone. Heterologous expression of wild-type and mutant vasopressin cDNAs in peptidergic cell lines shows that the highly homologous vasopressin-associated neurophysin domain associates with the hormone domain within the prohormone. This intramolecular interaction between two prohormone domains serves an essential intracellular function, i.e. the proper sorting of the prohormone into the regulated secretory pathway.
Asunto(s)
Oxitocina/genética , Animales , Arginina Vasopresina/biosíntesis , Arginina Vasopresina/genética , Regulación de la Expresión Génica , Genes Reguladores , Humanos , Neurofisinas/metabolismo , Factor 6 de Transcripción de Unión a Octámeros , Oxitocina/biosíntesis , Regiones Promotoras Genéticas , Ratas , Receptores Citoplasmáticos y Nucleares/genética , Factores de Transcripción/metabolismoRESUMEN
The binding interactions of four ligands differing in acid-base properties with human serum albumin (HSA) were examined as a function of temperature. Binding to HSA decreased with increasing temperature for all four ligands. The bound and free ligand concentrations obtained at different temperatures were satisfactorily fitted to a model that incorporates the effect of temperature as an independent covariable and that directly allows the estimation of the enthalpic and entropic components of the ligand-albumin interaction, along with the precision of this estimation. Using this analysis, the binding of acidic ligands could be resolved into two classes of saturable sites, with the determination of the corresponding number of sites, whereas interpretation of binding data at each isolated temperature allowed only the determination of one saturable plus one non-saturable class of site. The thermodynamic constants indicate that binding of ionizable ligands to HSA involves electrostatic plus hydrophobic interactions, whereas only hydrophobic interactions are involved in binding to a second low-affinity class of site when present. Binding of non-ionizable ligands resembles that of the second class of low-affinity sites of ionizable ligands.
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Albúmina Sérica/metabolismo , Benzotiazoles , Sitios de Unión , Humanos , Isradipino/metabolismo , Ligandos , Riluzol , Albúmina Sérica/química , Sulfonamidas/metabolismo , Termodinámica , Tiazoles/metabolismoRESUMEN
To obtain a model for the sorting and processing of preprovasopressin (preproVP), rat VP cDNA was transfected in murine Neuro2A neuroblastoma cells, which do not express VP. The precursor of VP was expressed and processed into the authentic VP gene products VP, neurophysin (NP) and glycopeptide (GP) as determined with reversed phase HPLC and radioimmunoassay. In addition, Neuro2A-specific forms of NP and GP were observed, which may be produced in the constitutive secretory pathway in these cells.