RESUMO
It is generally admitted that actin filaments are anchored to a membrane by membranar actin-binding-proteins. However, we found that actin may also interact directly with membrane phospholipids. The actin-phospholipid complex has been investigated at the air-water interface using a film balance technique. In order to probe the effect of the phospholipid headgroup on the actin-phospholipid interaction, we focus mainly on phospholipids that have the same acyl chain length but different headgroups. For all the phospholipids, the apparent area per molecule (the total surface divided by the number of lipid molecules) increases after the injection of the protein into the subphase, which suggests an intercalation of actin between the phospholipid molecules. This effect seems to be more important for DMPE and DMPS than for DMPG, suggesting that the headgroup plays an important role in this intercalation. The critical surface pressure associated to the liquid expanded-liquid condensed (LE-LC) phospholipid transition increases with the concentration of G-actin and thus suggests that G-actin acts as an impurity, simply competing as a surfactant at the air-water interface. On the other hand, F-actin affects the LE to LC transition of phospholipids differently. In this case, the LE to LC transition is broader and F-actin slightly decreases the critical surface pressure, which suggests that electrostatic interactions are involved.
Assuntos
Citoesqueleto de Actina/química , Actinas/química , Bicamadas Lipídicas/química , Fosfolipídeos/química , Adsorção , Ar , Fosfatidiletanolaminas/química , Fosfatidilgliceróis/química , Eletricidade Estática , Estresse Mecânico , Propriedades de Superfície , Termodinâmica , Unitiol/química , ÁguaRESUMO
The vasopressin precursor is composed of 3 domains in line, namely vasopressin, MSEL-neurophysin and a glycopeptide referred to as copeptin, which are separated during the processing. In guinea pig neurohypophysis, the precursor is partially processed so that a two-domain fragment, MSEL-neurophysin--copeptin, can be found along with free MSEL-neurophysin adn copeptin. Guinea pig copeptin has been sequenced. It is a glycopeptide composed of 38 amino acid residues rather than the 39 found in other mammalian copeptins. Compared with other copeptins, that from guinea pig shows a few substitutions and the deletion of one acidic residue, probably in position 32. This deletion might be responsible for incomplete cleavage by the trypsin-like processing enzyme.
Assuntos
Glicopeptídeos/análise , Neuro-Hipófise/análise , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Carboidratos/análise , Quimotripsina , Cobaias , Pepsina A , Fragmentos de Peptídeos/análise , Especificidade da Espécie , TripsinaRESUMO
The guinea pig two-domain precursor of MSEL-neurophysin and copeptin has been passed through a trypsin-Sepharose column in order to mimic the enzyme processing by a membrane-bound endopeptidase. Only two cleavages were observed located in the inter-domain sequence (at Arg-94 and Arg-98), in contrast to several additional cleavages found when free neurophysin or copeptin is subjected to soluble trypsin. Because the physiological maturation involves a single cleavage at Arg-94, both local accessibility in the precursor and narrow specificity of the enzyme are implied in the processing.
Assuntos
Arginina Vasopressina/metabolismo , Glicopeptídeos/metabolismo , Neurofisinas/metabolismo , Ocitocina , Precursores de Proteínas/metabolismo , Tripsina/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia de Afinidade , Cromatografia em Agarose , Cobaias , Hidrólise , Processamento de Proteína Pós-TraducionalRESUMO
The vasopressin precursor is composed of 3 domains, namely vasopressin, MSEL-neurophysin and a glycopeptide. Processing occurs during axonal transport from hypothalamus to neurohypophysis from which the 3 fragments can be isolated. The glycopeptide fragment of the rat vasopressin precursor has been purified and sequenced. Despite the fact that rat MSEL-neurophysin is shortened (93 residues instead of 95 for other mammals), rat glycopeptide has 39 residues, as do the other mammalian glycopeptides, suggesting a similar processing. Fifteen substitutions are however observed when compared to ox glycopeptide. The C-terminal part of MSEL-neurophysin (residues 77-93) and the glycopeptide are encoded by the same exon and the homologies when compared with their bovine counterparts are 58% and 62% respectively. In contrast, the central part of rat MSEL-neurophysin (residues 10-76), which is encoded by a separate exon, displays 96% of homology; vasopressin and the N-terminal part of MSEL-neurophysin (residues 1-9), encoded by a third exon, are nearly invariant.
Assuntos
Glicopeptídeos/isolamento & purificação , Precursores de Proteínas/análise , Vasopressinas/análise , Sequência de Aminoácidos , Animais , Bovinos , Humanos , Ratos , Especificidade da EspécieRESUMO
Hydrin 2 (vasotocinyl-Gly), a hydroosmotic peptide resulting from differential processing of provasotocin and recently identified in frog neurohypophysis, has been looked for in the pituitary gland of an exotic toad (Bufo marinus) and of a reptile (Vipera aspis). Hydrin 2 has been found in the amphibian but not in the reptile. This result confirms the evolutionary specificity of hydrin 2 that has been identified only in frogs and toads but not in birds and reptiles. Occurrence of hydrin 2 is explained by its regulatory function on the water permeability of the skin of anurans.
Assuntos
Evolução Biológica , Neuropeptídeos/análise , Hipófise/análise , Vasotocina/análogos & derivados , Animais , Bufo marinus , Cromatografia Líquida de Alta Pressão , Ocitocina/análogos & derivados , Ocitocina/isolamento & purificação , Serpentes , Especificidade da Espécie , Vasotocina/análise , Vasotocina/isolamento & purificaçãoRESUMO
Guinea pig neurohypophysial hormones have been purified by two procedures, one involving molecular sieving and paper chromatoelectrophoresis, the other high-pressure reverse-phase liquid chromatography. Arginine vasopressin and oxytocin have been identified by their amino acid compositions and their retention times in HPLC determined through their biological properties. No partially processed precursor, including a neurohormone and a neurophysin, has been detected. Because the cleavage of the three-domain vasopressin-neurophysin-copeptin precursor is apparently complete between the first two domains, whereas it is not between the second and the third, it is supposed that two distinct enzymic systems are involved in the processing.
Assuntos
Hormônios Neuro-Hipofisários/isolamento & purificação , Vasopressinas/genética , Sequência de Aminoácidos , Animais , Cromatografia em Gel/métodos , Cromatografia Líquida de Alta Pressão/métodos , Eletroforese em Papel/métodos , Cobaias , Neuro-Hipófise/análise , Hormônios Neuro-Hipofisários/genéticaRESUMO
A 'big' frog (Rana esculenta) neurophysin, encompassing sequences homologous to mammalian MSEL-neurophysin and copeptin, has been passed through a trypsin-Sepharose column in order to compare its conformation with that of the two-domain intermediate precursor isolated from guinea pig. Whereas the polypeptide possesses 8 arginine residues, only two cleavages were observed located in a putative inter-domain sequence (at Arg-94 and Arg-114). Because free vasotocin has been isolated from the frog, it is assumed that pro-vasotocin has a three-domain conformation similar to that of pro-vasopressin but processing in amphibians involves only one step rather than two steps as in mammals.
Assuntos
Arginina Vasopressina , Glicopeptídeos , Neurofisinas , Ocitocina , Precursores de Proteínas , Sequência de Aminoácidos , Animais , Evolução Biológica , Bovinos , Dados de Sequência Molecular , Conformação Proteica , Rana esculenta , Homologia de Sequência do Ácido Nucleico , VasopressinasRESUMO
The neurohypophysial hormones of the 1-month-old bovine fetus have been identified by their positions in ion-exchange chromatography and their retention times in high-pressure reverse-phase partition chromatography. Arginine vasopressin and oxytocin have been recognized. The molar ratio vasopressin/oxytocin in neurohypophysis is about 6 in the 1-month-old fetus compared with 4 in the 3-month-old fetus, 2.7 in the 7-month-old fetus and 1 in the adult. Vasotocin is virtually absent even in the early fetus (less than 0.1% of arginine vasopressin). The occurrence of a vasotocin gene expressed in the fetus but silent in the adult appears unlikely.
Assuntos
Neuro-Hipófise/embriologia , Hormônios Neuro-Hipofisários/análise , Vasotocina/análise , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Desenvolvimento Embrionário e FetalRESUMO
alpha- and beta-melanocyte-stimulating hormones (alpha-MSH and beta-MSH) have been isolated from Xenopus laevis neurointermediate pituitary and microsequenced. Intracellular alpha-MSH is not N-acetylated after proteolytic processing of pro-opiomelanocortin in contrast to mammalian alpha-MSHs. There is a high preservation of the melanotropic amino acid sequence common to all MSHs although in Xenopus beta-MSH a histidine residue replaces the glutamic acid residue found in position 8 of mammalian beta-MSHs.
Assuntos
Hormônios Estimuladores de Melanócitos/análise , Hipófise/análise , Pró-Opiomelanocortina/metabolismo , Xenopus laevis/metabolismo , alfa-MSH/análise , Acetilação , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Glutamatos , Ácido Glutâmico , Histidina , Hormônios Estimuladores de Melanócitos/metabolismo , Dados de Sequência Molecular , Peptídeo Hidrolases/metabolismo , Hipófise/metabolismo , Homologia de Sequência do Ácido Nucleico , alfa-MSH/metabolismoRESUMO
Neurohypophysial hormones and neurophysins are derived from common precursors processed during the axonal transport from the hypothalamus to the neurohypophysis. Two neurohormones, an oxytocin-like and a vasopressin-like, on one hand, two neurophysins, termed VLDV-and MSEL-neurophysins according to residues in positions 2, 3, 6 and 7, on the other, are usually found in vertebrate species. In contrast to placental mammals that have oxytocin and arginine vasopressin, marsupials have undergone a peculiar evolution. Two pressor peptides, lysipressin and vasopressin for American species, lysipressin and phenylpressin for Australian macropods, have been identified in individual glands and it is assumed that the primordial vasopressin gene has been duplicated in these lineages. On the other hand, the reptilian mesotocin is still present in Australian species instead of the mammalian oxytocin, while the North American opossum has both hormones and South American opossums have only oxytocin. The neurophysin domain of each precursor is encoded by 3 exons and different evolutionary rates have been found for the 3 corresponding parts of the protein. The central parts, encoded by the central exons, are evolutionarily very stable and nearly identical in the 2 neurophysins of a given species. Recurrent gene conversions have apparently linked the evolutions of the 2 precursor lineages. In mammals, the 3-domain precursor of vasopressin is processed in 2 stages: a first cleavage splitting off vasopressin and a second cleavage separating MSEL-neurophysin from copeptin. Two distinct enzymatic systems seem to be involved in these cleavages. Processing is usually complete at the level of the neurohypophysis, but an intermediate precursor encompassing MSEL -neurophysin and copeptin linked by an arginine residue has been characterized in guinea pig. In vitro processing of this intermediate through trypsin--Sepharose reveals cleavages only in the interdomain region. In non-mammalian tetrapods, such as birds and amphibians, mesotocin and vasotocin are associated with neurophysins in precursors similar to those found in mammals. However, processing of the vasotocin precursor seems to be different from the processing of the vasopressin precursor, with a single cleavage leading to the hormone release.
Assuntos
Neurofisinas/metabolismo , Hormônios Neuro-Hipofisários/metabolismo , Precursores de Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Evolução Biológica , Dados de Sequência Molecular , Neurofisinas/genética , Hormônios Neuro-Hipofisários/genética , Precursores de Proteínas/genética , Processamento Pós-Transcricional do RNARESUMO
A quantitative image analysis technique was developed to assess the cytokine content of immunocytochemically stained cytokine producing cells. Peripheral blood mononuclear cells were stimulated to induce cytokine production with the superantigen streptococcal pyrogenic exotoxin-A. We have developed a method based on indirect immunocytochemistry which identifies IFN-gamma producing cells by a characteristic morphology generated by the accumulation of IFN-gamma in the Golgi organelle. An image analysis technique permitted discrimination between these producer cells and IFN-gamma binding target cells, which showed a different appearance, with staining restricted to the cell surface membrane. A semi-automated routine programme allowed the signal from a video camera to be processed by computerised image analysis methodology. This enabled us to measure the number of cytokine producing cells, the cytokine staining intensity in individual cells and the cell size expressed in actual cell area. The incidence of IFN-gamma producing cells determined by image analysis measurement was compared to results obtained using manual microscopy. Cell size was assessed by the image analysis system as well as by flow cytometry. Administration of pooled human IgG for intravenous use (IVIg) to the superantigen stimulated cells significantly down-regulated IFN-gamma production, both in terms of the numbers of producer cells and in terms of cytokine staining intensity in individual cells. In addition blast transformation of cells was substantially reduced. These effects, mediated by IVIg, were also evident following delayed IVIg administration 24 h after the initial cell stimulation.
Assuntos
Imunoglobulina G/imunologia , Interferon gama/biossíntese , Superantígenos/imunologia , Adulto , Tamanho Celular , Células Cultivadas , Citometria de Fluxo , Humanos , Processamento de Imagem Assistida por Computador , Interferon gama/imunologia , Ativação Linfocitária/imunologia , Linfócitos/imunologiaRESUMO
From guinea pig posterior pituitaries, a MSEL-type neurophysin (neurophysin containing methionine-2, serine-3, glutamic acid-6 and leucine-7), a glycopeptide referred to as copeptin and their common precursor have been purified to homogeneity and sequenced. The performed acid-oxidized precursor, subjected to trypsin hydrolysis, has given 9 peptides, 6 of which (T1-T6) identical to those given by oxidized MSEL-neurophysin except that T6 has an additional C-terminal arginine residue when compared to its homologue. The other 3 tryptic peptides (T7-T9) are identical to those given by copeptin. The 132-residue precursor therefore comprises a MSEL-type neurophysin (93 residues) and copeptin (38 residues) linked by an arginine residue. The molar proportion of this bound form compared with the free polypeptides is approximately 20%. It is believed that this precursor is a part of the vasopressin-MSEL-neurophysin-copeptin precursor incompletely processed during the transport from hypothalamus to neurohypophysis.
Assuntos
Arginina Vasopressina/isolamento & purificação , Glicopeptídeos/isolamento & purificação , Neurofisinas/isolamento & purificação , Ocitocina , Precursores de Proteínas/isolamento & purificação , Sequência de Aminoácidos , Animais , Cobaias , Peso Molecular , Fragmentos de Peptídeos/isolamento & purificação , Neuro-Hipófise/análiseRESUMO
This report presents six cases (among 43 surgical cases) of left ventricular EMF presenting as pure mitral insufficiency without any echocardiographic or hemodynamic signs of left ventricular EMF (except apical diverticulum in two cases). Five of these cases were diagnosed at operation, and the sixth was diagnosed on the basis of a characteristic right ventricular angiogram. At operation a fibrotic lesion localized to the level of the anteropapillary muscle, with severe mitral insufficiency, was found. Five patients underwent successful mitral valve replacement and one a successful conservative mitral valve procedure. Postoperative angiograms, done in two patients, showed a normal contour of the left ventricle. A review of the literature did not reveal any previous descriptions of this type of limited left ventricular EMF, mimicking rheumatic mitral insufficiency. In our surgical experience, we have encountered three types of left ventricular EMF: obliterative, extensive, and limited. We emphasize EMF as a possible cause of mitral insufficiency in African children.
Assuntos
Fibrose Endomiocárdica/diagnóstico , Insuficiência da Valva Mitral/diagnóstico , Cardiopatia Reumática/diagnóstico , Adolescente , Criança , Diagnóstico Diferencial , Fibrose Endomiocárdica/diagnóstico por imagem , Fibrose Endomiocárdica/cirurgia , Feminino , Humanos , Masculino , Insuficiência da Valva Mitral/diagnóstico por imagem , Insuficiência da Valva Mitral/cirurgia , Radiografia , Cardiopatia Reumática/diagnóstico por imagem , Cardiopatia Reumática/cirurgiaRESUMO
The purpose of this study was to evaluate prospectively the occurrence of cardiac arrhythmias during the combined therapy with intravenous aminophylline and terbutaline in 29 consecutive patients with status asthmaticus. The 24-hour Holter recordings were performed during continuous intravenous infusions of aminophylline (0.56 +/- 0.20 mg/kg/h) and terbutaline (0.034 +/- 0.014 microgram/kg/min). Serum theophylline concentration was 12.1 +/- 3.8 micrograms/ml and never reached the toxic level (greater than 20 micrograms/ml). Premature ventricular contractions (PVCs) were absent in 35 percent of patients and 48 percent had rare unifocal PVCs (less than 10/h). Only 17 percent of patients (five of 29) exhibited severe ventricular arrhythmias: PVCs greater than 10/h (n = 3), multifocal PVCs (n = 1); and a short run of ventricular tachycardia (n = 1). Serious supraventricular arrhythmias occurred in only 7 percent of patients (two of 29) who developed sustained runs of atrial tachycardia. These arrhythmias were always clinically well tolerated and spontaneously resolved without any antiarrhythmic treatment. We conclude that severe arrhythmias are rarely observed during combined therapy with aminophylline and terbutaline in status asthmaticus.
Assuntos
Aminofilina/efeitos adversos , Arritmias Cardíacas/induzido quimicamente , Asma/tratamento farmacológico , Estado Asmático/tratamento farmacológico , Terbutalina/efeitos adversos , Adulto , Idoso , Aminofilina/administração & dosagem , Arritmias Cardíacas/sangue , Arritmias Cardíacas/diagnóstico , Quimioterapia Combinada , Eletrocardiografia , Feminino , Humanos , Infusões Intravenosas , Masculino , Pessoa de Meia-Idade , Monitorização Fisiológica , Potássio/sangue , Estudos Prospectivos , Terbutalina/administração & dosagem , Teofilina/sangueRESUMO
Twenty patients with endomyocardial fibrosis (EMF), the largest series reported to date, were operated upon between June, 1978, and June, 1980. Eleven were male and nine female, ranging in age from 6 to 23 years (mean 13.3 years). There were seven cases of right ventricular (RVEMF), six of left ventricular (LVEMF), and seven cases of bilateral EMF. All patients underwent endocardiectomy and atrioventricular valve replacement with a xenograft. Four patients had an additional valvular annuloplasty. There were four postoperative deaths (all bilateral EMF): two from low cardiac output and one each from hepatic failure and cerebral malaria. There was one late death from serum hepatitis. The other patients had a relatively difficult postoperative course, but none of the 20 patients atrioventricular block. The longest follow-up of the 15 survivors is 28 months (mean 16.7 months). All patients are symptom free. Three take digitalis and/or diuretics. Ten have been recatheterized from 6 months to 1 year after operation. Intracardiac pressures, the ventricular cineangiogram, liver, and heart size returned to normal in patients with LVEMF; in RVEMF, despite clinical improvement, most of these parameters remained abnormal. Of special interest were (1) our recognitions of an early type of LVEMF and (2) our surgical preservation of a thin juxta-annular rim of fibrosis in the right ventricle to avoid atrioventricular block. Operation is indicated in all patients with LVEMF, despite greater risk. Early intervention is advised in RVEMF to avoid irreversible liver damage and cardiac enlargement.
Assuntos
Fibrose Endomiocárdica/cirurgia , Adolescente , Adulto , Angiocardiografia , Criança , Fibrose Endomiocárdica/diagnóstico , Fibrose Endomiocárdica/diagnóstico por imagem , Feminino , Humanos , Masculino , Métodos , Mortalidade , Complicações Pós-OperatóriasRESUMO
Abstract Hydrin 2 (vasotocinyl-Gly), found along with vasotocin in the neurohypophysis of frogs and toads but not of other vasotocin-bearers such as birds and reptiles, is believed to act on water permeability of frog skin, whereas vasotocin mainly fulfils the antidiuretic function on the kidney. In order to understand the peculiar regulation of provasotocin differential processing in amphibians, conversion of hydrin 2 into vasotocin has been attempted using bovine pituitary granule alpha-amidating enzyme. Generated vasotocin has pharmacological properties and Chromatographic behaviour in high-performance liquid chromatography identical to those of synthetic vasotocin. However, the low yield of conversion (5% to 10%) suggests that additional factor(s) might be involved in the physiological processing.
RESUMO
Human MSEL-neurophysin has been dissected into two halves by endopeptidase Lys-C, taking advantage of a peculiar Lys59-Ala60 bond. Two sub-domains, N-terminal (1-59) and C-terminal (60-93), have been separated. These sub-domains have been purified by reverse-phase high pressure liquid chromatography and identified by their N-terminal sequences. The N-terminal fragment comprises two chains 1-18 and 19-59, because of the presence of a second lysine residue in position 18, whereas the C-terminal fragment (60-93) is a single chain. Hormone-binding experiments have been carried out using vasopressin or vasopressinyl-Gly-Lys-Arg and testing the ability of the hormone-neurophysin complex to precipitate at pH 3.9 with 10% NaCl. The N-terminal sub-domain precipitates in presence of vasopressin in the same way as native neurophysin whereas the C-terminal sub-domain does not. It can be concluded that the hormone-binding site is located in the 1-59 region of neurophysin.
Assuntos
Arginina Vasopressina/metabolismo , Endopeptidases/metabolismo , Metaloendopeptidases , Neurofisinas/metabolismo , Ocitocina , Precursores de Proteínas/metabolismo , Vasopressinas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismoRESUMO
A Ca(2+)-dependent endopeptidase cleaving at the carboxyl side of the paired Lys-Arg residues has been found in the neurosecretory granules of the rat neurointermediate pituitary. The specificity pattern on synthetic fluorogenic substrates, the inhibitor profile, the pH optimum of 5.0 and the Ca(2+)-dependence are compatible with an involvement of this enzyme in the prooxytocin and the provasopressin processing within the granules. The enzymatic features of the neurohypophysial granule endopeptidase resemble those of the insulinoma granule type II endopeptidase and suggest that the same Ca(2+)-dependent protease or closely related enzymes could be involved in processing Lys-Arg-containing prohormones in neuroendocrine cells.
Assuntos
Cálcio/farmacologia , Grânulos Citoplasmáticos/enzimologia , Endopeptidases/metabolismo , Ocitocina/metabolismo , Neuro-Hipófise/enzimologia , Precursores de Proteínas/metabolismo , Vasopressinas/metabolismo , Sequência de Aminoácidos , Animais , Arginina/metabolismo , Concentração de Íons de Hidrogênio , Lisina/metabolismo , Dados de Sequência Molecular , Neuro-Hipófise/ultraestrutura , Ratos , Especificidade por SubstratoRESUMO
The biological properties of vasotocin, hydrin 1 (vasotocinyl-Gly-Lys-Arg) and hydrin 2 (vasotocinyl-Gly), in particular the hydro-osmotic activities on the frog skin, the frog urinary bladder and the frog kidney, have been compared. Hydrins are as active or more active than vasotocin on the first two organs but they are virtually devoid of antidiuretic activity in the rat and the frog, in contrast to vasotocin. It appears that where the oxytocin ring (residues 1-6), present in the three peptides, is necessary for the action on the three organs, the C-terminal amidated group of vasotocin is necessary for the renal receptor but not for the skin and bladder receptors. It is known that amphibians have two types of vasotocin receptors, V1 and V2, homologous to the vascular/hepatic V1 and the renal V2 vasopressin receptors of mammals, respectively. We suggest that adaptation has led to specialization of (at least) two subtypes of hydro-osmotic V2 receptors, the renal subtype on which vasotocin is mainly active for the reabsorption of tubular water, and the skin/bladder subtype on which hydrin 2 is specifically involved in ensuring the rehydration of the animal. Cooperative evolution might have created in anuran Amphibia, on the one hand, two hydro-osmotic peptides, vasotocin and hydrin 2, derived from a single precursor through differential processing; and on the other hand, two corresponding receptors in kidney and skin for internal and external water recovery.