RESUMEN
A wealth of theoretical and empirical arguments have suggested that music triggers emotional responses by resembling the inflections of expressive vocalizations, but have done so using low-level acoustic parameters (pitch, loudness, speed) that, in fact, may not be processed by the listener in reference to human voice. Here, we take the opportunity of the recent availability of computational models that allow the simulation of three specifically vocal emotional behaviours: smiling, vocal tremor and vocal roughness. When applied to musical material, we find that these three acoustic manipulations trigger emotional perceptions that are remarkably similar to those observed on speech and scream sounds, and identical across musician and non-musician listeners. Strikingly, this not only applied to singing voice with and without musical background, but also to purely instrumental material. This article is part of the theme issue 'Voice modulation: from origin and mechanism to social impact (Part I)'.
Asunto(s)
Música , Canto , Voz , Percepción Auditiva/fisiología , Emociones/fisiología , Humanos , Música/psicología , Voz/fisiologíaRESUMEN
Recombinant human thyroid-stimulating hormone (recTSH) has recently been engineered to detect metastatic lesions in patients operated on for thyroid cancer. In this report, we have compared the microheterogeneity, carbohydrate (CHO) content, mitogenic potency and immunoreactivity of the biotechnology product to those of human TSH of pituitary origin (pitTSH). Compositional analysis revealed that recombinant (rec) TSH produced in Chinese hamster ovary cells was overglycosylated compared with the native hormone (21 and 14%, respectively) with a higher amount of sialic acid and lack of N-acetylgalactosamine. Electrofocusing followed by immunoblotting resolved recTSH into six glycoforms with pIs ranging from 6.0 to 8.6, which were converted to a major species of pI 8.9 by sialidase treatment. pitTSH contained five main isoforms of pI 6.5-8.2 distinct from those of recTSH and partially resistant to sialidase. Binding activity of both human TSHs to porcine thyroid membrane receptors was found to be similar, but recTSH appeared to be 20% active compared to pitTSH in eliciting cAMP production and cell growth in rat FRTL-5 cells. Immunoreactivity of the recombinant hormone was investigated using polyclonal and monoclonal antibodies raised against the native hormone or synthetic peptide sequences of its subunits. While rec- and pitTSH were recognized to a similar extent by anti-protein antibodies, they exhibited a different binding pattern to antipeptide antibodies. Serial dilution of anti-alpha 1-25, anti-alpha 26-51, anti-beta 96-112 antisera bound recTSH to a greater extent than pitTSH, while anti-beta 31-51 and anti-beta 53-76 displayed similar recognition toward both preparations.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Tirotropina/química , Animales , Anticuerpos Monoclonales/metabolismo , Unión Competitiva , Células CHO/metabolismo , Metabolismo de los Hidratos de Carbono , División Celular/efectos de los fármacos , Cricetinae , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Epítopos/metabolismo , Glicosilación , Humanos , Inmunoquímica , Neuraminidasa/farmacología , Hipófisis/metabolismo , Ingeniería de Proteínas , Ratas , Receptores de Tirotropina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Porcinos , Tirotropina/efectos de los fármacos , Tirotropina/genética , Tirotropina/inmunología , Tirotropina/metabolismoRESUMEN
To compare the structural topology of the human TSH to that of the structurally related gonadotropins, 10 peptides covering the entire primary sequence of the alpha- and beta-subunits of TSH were synthesized and used as antigens for the preparation of polyclonal antibodies. The alpha-subunit was synthesized as 4 nonoverlapping peptides (1-25, 26-51, 49-73, 72-92) while the beta-subunit was segmented in 6 overlapping sequences (2-18, 10-38, 31-51, 53-76, 77-96, 92-112). Most of the peptide sequences were predicted to contain a putative antigenic determinant. All antipeptide antisera were found to bind to the corresponding synthetic sequence in an enzyme-linked immunosorbent assay as well as to denatured TSH subunits after Western blotting. The N-terminal half of the alpha-subunit was found differentially accessible in TSH and gonadotropins compared to the free subunit: antipeptide-alpha 1-25 antibodies exhibited variable affinity for the four glycoprotein hormones whereas anti-alpha 26-51 displayed a remarkable recognition of free alpha-subunit. Four peptides proved to be accessible in the TSH beta-subunit: the N-terminal peptide (beta 2-18) elicited antibodies that bound to free TSH-beta and poorly to the dimer while antibodies against the C-terminal sequence (beta 92-112) recognized equally well free beta-subunit and TSH. Antipeptide-beta 31-51 antibodies proved to be specific for TSH while the beta 53-76 contiguous peptide appeared accessible in both TSH and gonadotropins. The current findings therefore demonstrate that most of the sequences predicted to contain antigenic sites in the alpha- or the beta-subunits are indeed accessible at the surface of these proteins. Additionally, both subunits appear to contain amino acid sequences that are differentially expressed in TSH and gonadotropins as well as in free and combined subunits.
Asunto(s)
Anticuerpos/inmunología , Gonadotropinas/química , Mapeo Peptídico/métodos , Péptidos/inmunología , Tirotropina/química , Secuencia de Aminoácidos , Glicoproteínas/química , Hormonas/química , Humanos , Datos de Secuencia Molecular , Péptidos/genéticaRESUMEN
Enzymatic deglycosylation of human thyroid-stimulating hormone (hTSH) was shown to result in a mixture of partially and fully deglycosylated forms of the hormone by gel electrophoresis, silver staining and immunoblotting. Radioiodination of the enzymatic digest, followed by gel filtration and concanavalin A-Sepharose chromatography allowed to separate two different forms of partially deglycosylated [125I]hTSH and a fully deglycosylated hormone. The final recovery was of approx. 60% for [125I]hTSH deglycosylated in its beta-subunit, of 30% for [125I]hTSH missing the oligosaccharide in beta and one in alpha but only of 10% for [125I]hTSH deglycosylated in both the alpha- and beta-subunits. Gel electrophoresis under non-denaturing conditions showed that each form migrated distinctly from free subunits and reverse-phase high performance liquid chromatography after reduction and carboxymethylation identified the presence of the two subunits. Mapping of [125I]hTSH derivatives with polyclonal, monoclonal and anti-peptide antibodies allowed to identify two novel glycosylation-independent epitopes preserved in deglycosylated hTSH while the main immunogenic determinant was lost. When assayed in a bioassay with FRTL-5 cells, the hormone deprived of its beta-linked carbohydrate chain was found to be as effective as the native hormone on cAMP production and cell growth. In contrast, the fully deglycosylated derivative proved to stimulate cAMP release but appeared to be definitely less potent on thyroid cell growth. Our findings thus demonstrate that glycosylation of the alpha-subunit but not that of the beta-subunit is essential to express the domains involved in hTSH immunoreactivity as well as those controlling the post-receptor biological activity of the hormone.
Asunto(s)
Tirotropina/inmunología , Células Cultivadas , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Concanavalina A , AMP Cíclico/biosíntesis , Electroforesis en Gel de Poliacrilamida , Epítopos/inmunología , Glicósido Hidrolasas/metabolismo , Glicosilación , Humanos , Glándula Tiroides/citología , Tirotropina/aislamiento & purificación , Tirotropina/farmacologíaRESUMEN
Isoforms of intrapituitary human TSH were separated by gel isoelectrofocusing, and their immunoreactivity analyzed by subsequent immunoblotting using polyclonal and monoclonal antibodies. Under these conditions, TSH polymorphism could be resolved as seven major isoforms (pI 8.6, 8.3, 8.0, 7.5, 7.0, 6.5, and 6.0) by both silver staining of the gels and binding to anti-TSH polyclonal antibodies. The distribution pattern of these forms appeared totally distinct from that of individual TSH alpha (pI 8.8, 8.4, 8.2, 7.6, 7.4, 6.8, 6.6, 5.8, and 5.4) and TSH beta (pI 8.7, 8.1, 7.2, 6.8, 6.2, and 5.8) subunits. While most anti-TSH polyclonal antibodies recognized neutral and alkaline isoforms of TSH (pI 8.6, 8.3, 8.0, 7.5, 7.0, 6.5, and 6.0) through beta determinants, they displayed a variable potency to bind acidic forms of the hormone (pI 5.8, 5.5, 4.8, and 4.5), in contrast to anti-TSH alpha antisera, which enlighted the broadest spectrum of isoforms. Monoclonal antibodies of various specificities largely reproduced this distribution, indicating that at least five distinct epitopes are coexpressed in the neutral and alkaline forms of TSH, but only two are expressed in the acidic ones. All of the forms were found to induce cAMP production and stimulate growth of FRTL-5 rat thyroid cells, although neutral forms proved to be definitely less potent than the others. We therefore, conclude that TSH isoforms differ in the expression of both their immunoreactive and bioactive domains and that the bioactive/immunoreactive ratio is not an accurate index for the biopotency of the hormone.
Asunto(s)
Tirotropina/fisiología , Anticuerpos/inmunología , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Bioensayo , Humanos , Immunoblotting , Focalización Isoeléctrica , Isomerismo , Radioinmunoensayo , Tirotropina/inmunologíaRESUMEN
The accessibility of the asparagine-linked carbohydrate chains of human thyrotropin (hTSH) and free alpha and beta subunits was investigated by their susceptibility to endoglycosidases H and F as well as to peptide:N-glycosidase F. Iodinated hTSH or subunits were incubated with a commercial enzyme preparation containing both endoglycosidase F and N-glycosidase F activities and further analyzed by sodium dodecyl sulfate gel electrophoresis followed by quantitative autoradiography. We show that, working at the optimum of the N-glycosidase activity, the relative amount of endoglycosidase required for half-deglycosylation was 20-fold higher for native hTSH than for the reduced and dissociated subunits. Under nondenaturing conditions, the 18K beta subunit of hTSH could be readily deglycosylated to a 14K species while the 22K alpha subunit was largely resistant. However, both subunits were converted to an apoprotein of similar apparent molecular weight of 14K following reduction of disulfide bonds. In contrast, the free alpha subunit of human choriogonadotropin appeared fully sensitive to carbohydrate removal under nonreducing conditions despite the presence of a partially deglycosylated 18K intermediate at low concentration of endoglycosidase. Similarly, both hTSH-alpha and hTSH-beta could be completely deglycosylated after acid dissociation of the native hormone. While all three carbohydrate chains of hTSH are sensitive to pure peptide:N-glycosidase F, only one on alpha and the single oligosaccharide present on beta in hTSH appeared to be cleaved by pure endoglycosidase F. Interestingly, one of the two carbohydrate chains present on alpha was also found to be susceptible to endoglycosidase H.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Glicósido Hidrolasas/metabolismo , Tirotropina , Carbohidratos/análisis , Electroforesis en Gel de Poliacrilamida , Humanos , Radioisótopos de Yodo , Cinética , Sustancias Macromoleculares , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa , Peso MolecularRESUMEN
In order to estimate the role played by gaseous diffusion in the mixing disorders of chronic obstructive lung disease (COLD), the effect of breathing a gas mixture lighter than air has been studied. Twenty four patients with severe airflow obstruction have been tested in the following way: in a random order they breathed two different mixtures with the same PO2 : air and helium-oxygen (heliox) for 20 min. Ventilation was monitored during the whole of each run; during the 2 last min arterial blood was sampled. While breathing heliox a slight, non-significant, increase in ventilation has been observed with a slight but statistically significant decrease of PaO2 (p less than 0.01), of PaCO2 (p less than 0.05) and increase of pH (p less than 0.01). These changes suggest a slight increase of distribution disorders with alveolar hyperventilation. For these results to be consistent with stratification, improvement of the diffusion due to low density should have been masked by other phenomena; the possible effects of ternary diffusion, increased viscous resistances and change of transfer factor have been looked at. No conclusive evidence has been found of such counter-effects. Therefore it looks unlikely that stratification be the major factor in distribution impairment in COLD.
Asunto(s)
Helio/farmacología , Enfermedades Pulmonares Obstructivas/fisiopatología , Oxígeno/farmacología , Capacidad de Difusión Pulmonar/efectos de los fármacos , Adulto , Anciano , Dióxido de Carbono/sangre , Humanos , Masculino , Persona de Mediana Edad , Oxígeno/sangre , Alveolos Pulmonares/efectos de los fármacos , Alveolos Pulmonares/fisiopatología , Respiración/efectos de los fármacosRESUMEN
A new analyser for total CO2 in blood--Ericsen E-100--has been tested and its performances compared with those of the manometric Van Slyke apparatus. Ericsen E-100 makes it possible to measure very small samples (50 microliter) in a short time (35 s). It appears that Ericsen E-100 underestimates total CO2; the higher the total CO2 the larger the error. Simple improvements are suggested, especially concerning the way injection is performed in the reaction chamber.
Asunto(s)
Análisis de los Gases de la Sangre/instrumentación , Dióxido de Carbono/sangre , HumanosRESUMEN
A method for measuring dissolved nitrogen in blood is described. Gas-phase chromatography is used. The apparatus is provided with a column of 5 A molecular sieve material and with an extraction chamber. CO2 and O2 are absorbed. As in Lenfant's method, extraction by equilibration is completed by elution. The main features are: the design of the extraction chamber, faciliting gas flow; an auxiliary circuit for freeing the chamber and the reagents from nitrogen, in which the pressure can be equilibrated against the inlet pressure of the column; the care taken to avoid any kind of contamination; the use of a special guide mounted on the blood-transfer syringe, which makes it possible to get reproducible samples at last the checking and measurement of the residual nitrogen which makes it possible to extract and measure the whole of the dissolved nitrogen. At the present stage, the accuracy of the method is better than 1.5% and the coefficient of variation of the reproducibility is 0.57%. These results make the method suitable for measuring (PaN2-PAN2) in cases of chronic respiratory insufficiency.