RESUMO
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.
Assuntos
Ceratopogonidae/classificação , Gado/parasitologia , Filogenia , Animais , Ceratopogonidae/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Feminino , Países Baixos , Homologia de Sequência , Especificidade da Espécie , Asas de Animais/anatomia & histologiaRESUMO
We are using transgenic mice to study the regulation of the bovine vasopressin (VP) and oxytocin (OT) genes. Prompted by the observation that mice bearing a bovine OT transgene express bovine OT RNA in their testes, we investigated the expression of the VP-OT locus in normal mice and cattle. Normal wild-type mice do not have detectable levels of either VP or OT RNA in their testes. Normal cattle are also devoid of detectable VP transcripts, but have relatively high levels of testicular OT RNA. Additionally, OT, but not VP, peptide is detectable by HPLC. In situ hybridization to RNA in bovine testicular tissue sections localized OT transcripts to seminiferous tubules, with a distribution similar to that of alpha-inhibin, suggesting expression in Sertoli cells. Interestingly, the bovine OT RNAs in the transgenic mouse testes were also shown by in situ hybridization to have the same distribution. These data suggest that the cis-acting regulatory sequences responsible for expression of the OT gene in bovine Sertoli testis reside within the limits of the transgene used in this study. Further, the trans-acting factors present in murine testicular cells are able to recognize these elements, although they do not express the endogenous mouse OT gene in this tissue.
Assuntos
Regulação da Expressão Gênica , Ocitocina/genética , Túbulos Seminíferos/metabolismo , Testículo/metabolismo , Animais , Bovinos , Sondas de DNA , Hipotálamo/química , Masculino , Camundongos , Camundongos Transgênicos , Hibridização de Ácido Nucleico , Ocitocina/análise , Hipófise/química , RNA/análise , Sondas RNA , Túbulos Seminíferos/química , Testículo/química , Distribuição Tecidual , Vasopressinas/genéticaRESUMO
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.
Assuntos
Vasopressinas/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Camundongos , Precursores de Proteínas/metabolismoRESUMO
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.
Assuntos
Antioxidantes/química , Modelos Químicos , Picratos , Trimetazidina/análogos & derivados , Trimetazidina/química , Bepridil/análogos & derivados , Bepridil/metabolismo , Compostos de Bifenilo , Simulação por Computador , Cresóis/química , Sequestradores de Radicais Livres/química , Radicais Livres/metabolismo , Hidroquinonas/química , Fenóis/química , Relação Estrutura-Atividade , Vitamina E/químicaRESUMO
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.
Assuntos
Expressão Gênica , Hipotálamo/metabolismo , Hipófise/metabolismo , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Vasopressinas/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Primers do DNA , Glicopeptídeos/análise , Glicopeptídeos/biossíntese , Masculino , Dados de Sequência Molecular , Neuroblastoma , Neurofisinas/análise , Neurofisinas/biossíntese , Reação em Cadeia da Polimerase , Precursores de Proteínas/biossíntese , Radioimunoensaio , Ratos , Ratos Wistar , Células Tumorais Cultivadas , Vasopressinas/análise , Vasopressinas/biossínteseRESUMO
Previous studies in highly sensitized patients waiting for a renal transplant showed a lack of correlation between the formation of alloantibodies and the alloantigen specific cytotoxic T cell precursor frequency. The frequencies of CTLp against HLA class I antigens, toward which patients had formed antibodies (not acceptable mismatches, NAM), were similar to those against HLA antigens, toward which no antibodies were present (acceptable mismatches, AM). In more recent studies we tested whether the immunological triggering leading to antibody formation might have resulted in a different population of cytotoxic T cells. Limiting dilution assays performed in the absence or presence of antibodies against CD8 showed that CTL directed against NAM were significantly less inhibited by anti-CD8 compared with those directed against AM. Those "primed" CTL could also be distinguished from the more "naive" CTL on the basis of their resistance to cyclosporine. In contrast to CsA, therapeutic concentrations of FK506 were able to inhibit both "naive" and "primed" CTLs. The clinical relevance of these data is currently being investigated.
Assuntos
Ciclosporina/farmacologia , Transplante de Rim/imunologia , Linfócitos T Citotóxicos/imunologia , Tacrolimo/farmacologia , Ciclosporina/uso terapêutico , Feminino , Antígenos de Histocompatibilidade Classe I/imunologia , Teste de Histocompatibilidade , Humanos , Imunofenotipagem , Técnicas In Vitro , Linfócitos T Citotóxicos/efeitos dos fármacos , Tacrolimo/uso terapêuticoRESUMO
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.
Assuntos
Sistema Imunitário/química , Oligopeptídeos/análise , Adulto , Animais , Cromatografia Líquida de Alta Pressão , Reações Cruzadas , Feminino , Humanos , Soros Imunes/imunologia , Masculino , Pessoa de Meia-Idade , Radioimunoensaio/métodos , Ratos , Ratos Wistar , Baço/química , Timo/química , Extratos de Tecidos/químicaRESUMO
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.
Assuntos
Antígenos HLA/imunologia , Imunidade Materno-Adquirida/imunologia , Linfócitos T/imunologia , Adulto , Criança , Epitopos , Pai , Feminino , Haplótipos , Humanos , Interleucina-2/farmacologia , Masculino , Células-Tronco/efeitos dos fármacos , Células-Tronco/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
The volumes of distribution of many acidic drugs have been shown to be close to that of their binding protein, i.e. serum albumin. The distribution of basic drugs mainly bound to alpha 1-acid glycoprotein (AAG) can be questioned with respect to its dependency upon the distribution of this plasma protein. So, a pharmacokinetic study was performed in 7 subjects with human 125I-labelled alpha 1-acid glycoprotein. The steady-state volume of distribution was found to be 5.37 +/- 0.82L. The central volume was 3.23 +/- 0.33L, close to that of plasma volume and the peripheral volume was 2.14 +/- 0.63L. These data allowed the establishment of an equation giving access to the volume of distribution of a basic drug by relating its unbound fraction to physiological distribution of alpha 1-acid glycoprotein. The values yielded by this equation show that the actual and calculated volumes of distribution of basic drugs mainly bound to AAG are discrepant. This protein is thus not the main factor controlling the distribution of basic drugs within the body.
Assuntos
Orosomucoide/metabolismo , Adulto , Humanos , Injeções Intravenosas , Cinética , Pessoa de Meia-Idade , Orosomucoide/sangue , Ligação ProteicaRESUMO
We report on the use of europium (Eu) as a suitable nonradioactive alternative for target cell labeling in limiting dilution analysis (LDA) assays set up to determine cytotoxic T-lymphocyte precursor (CTLp) frequencies. A nonradioactive alternative to the commonly used chromium-51 (51Cr) release assay seems desirable because working with radioisotopes has some major disadvantages concerning possible health risks, environmental load, costs of facilities necessary for working with radioisotopes, and shelf life. Some groups have successfully applied the Eu release assay based on detection by time-resolved fluorometry, to tests in which NK- or LAK-cell activity or cytotoxic T-lymphocyte reactions were measured. This led to the investigation whether this method could also be applicable to the more specific determination of CTLp frequencies in LDA assays. After optimal labeling conditions had been established, the sensitivity of the Eu release assay was determined by performing several LDA assays in which the target cells were labeled with either Eu or radioactive 51Cr. When CTLp frequencies were compared, it was shown that the Eu release assay is at least as sensitive and specific as the 51Cr release assay. Moreover, although the labeling procedure takes longer, sample processing is much faster: only 1 second per sample. The fact that the Eu release assay is not radioactive enables the assay to be performed at any laboratory and even--because the frequency of CTLps may have implications for organ graft survival and for donor selection in bone marrow transplantation--to do so on a routine basis.
Assuntos
Testes Imunológicos de Citotoxicidade/métodos , Európio , Células-Tronco Hematopoéticas , Linfócitos T Citotóxicos , Contagem de Células , Radioisótopos de Cromo , Humanos , Fatores de TempoRESUMO
The binding of nimesulide within human serum to isolated proteins and to erythrocytes was studied by equilibrium dialysis. Within the range of therapeutic concentrations, nimesulide was 99% bound to serum involving a nonsaturated process (NKA = 91). This binding was almost identical to binding of nimesulide to serum albumin (NKA = 95). Physiological concentrations of free fatty acids did not affect binding of nimesulide to serum albumin. The retention of nimesulide by erythrocytes suspended in buffer was moderate (67%), although in whole blood no erythrocyte binding was observed because of the greater affinity of this drug for serum. Over the range of therapeutic concentrations (2.5 to 63 mumol/L), the free fraction of nimesulide in serum remains constant. Serum binding was decreased in samples obtained from patients with renal failure or hepatic cirrhosis associated with hypoalbuminaemia and hyperbilirubinaemia, respectively. At therapeutic concentrations, the binding of nimesulide was unaffected by warfarin, cefoperazone, furosemide (frusemide), glibenclamide, tamoxifen or digitoxin. However, valproic acid and fenofibrate (80 mumol/L) may displace nimesulide. 4-Hydroxy-nimesulide, the principal metabolite, significantly increased the free fraction of nimesulide. Although methotrexate had no effect on the free fraction of nimesulide, the free fraction of methotrexate was significantly increased in the presence of nimesulide. The present study also demonstrated 2 distinct nimesulide binding sites (site I and site II) on serum albumin (10 mumol/L) with different affinities: site II KA = 3.57 x 10(5) L/mol and site I KA = 1.24 x 10(5) L/mol. Interaction studies using markers that bind specifically to site I (warfarin and azapropazone) and site II (diazepam and ibuprofen) indicated that nimesulide binds to site II with higher affinity and to a lesser extent to site I.
Assuntos
Anti-Inflamatórios não Esteroides/metabolismo , Proteínas Sanguíneas/metabolismo , Sulfonamidas/metabolismo , Humanos , Nefropatias/metabolismo , Hepatopatias/metabolismo , Ligação ProteicaRESUMO
The characteristics of beta adrenergic receptors of human erythrocyte membranes were investigated using (-)125iodocyanopindolol as a radioligand. Inhibition of (-)125iodocyanopindolol specific binding was checked using either atenolol and metoprolol as beta 1 selective antagonists or ICI 118551 and IPS 339 as beta 2 selective antagonists. The results showed non linear Hofstee's plots suggesting that both beta 1 and beta 2 adrenergic receptors are present. Analysis of the data yielded a beta 1/beta 2 adrenergic receptor ratio of approximately 33/67. Thus it is concluded that beta 2 subtype is predominant on human erythrocyte membranes.
Assuntos
Eritrócitos/análise , Receptores Adrenérgicos beta/análise , Agonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Humanos , Iodocianopindolol , Cinética , Pindolol/análogos & derivados , Pindolol/metabolismo , Ensaio RadioliganteRESUMO
Numerous data have suggested that beta-adrenoceptor-mediated responses were decreased in uremia and that parathormone could be implicated in this phenomenon. In a previous paper we have shown that the beta2 receptor density of mononuclear cells of uremic patients is significantly increased despite a significant increase in plasma epinephrine, suggesting that an endogenous substance could interfere and disregulate the beta 2 receptor density. In order to further evaluate this phenomenon we have firstly studied the influence of one non uremic and five uremic plasma ultrafiltrates on the binding of (-)-[125I]iodocyanopindolol using rat lung beta adrenoceptors. The results show that uremic plasma ultrafiltrates induce a decrease in the Bmax value without any variation on the Kd value. In a second step we have assessed the ability of human synthetic 1-34 and 53-84 parathormone to interact directly with beta-adrenoceptors. No variation in the (-)-[125I]iodocyanopindolol binding parameters was observed. These results suggest that an uremic endogenous substance might interfere on the beta adrenergic receptors and that the alteration in the beta-adrenergic response in uremia is probably not due to a direct action of parathormone on the beta-adrenoceptors.
Assuntos
Pindolol/análogos & derivados , Receptores Adrenérgicos beta/metabolismo , Uremia/sangue , Animais , Humanos , Técnicas In Vitro , Radioisótopos do Iodo , Iodocianopindolol , Cinética , Pulmão/metabolismo , Masculino , Hormônio Paratireóideo/sangue , Pindolol/metabolismo , Propranolol/farmacologia , Ratos , Ratos Endogâmicos , UltrafiltraçãoRESUMO
Isoxicam binding to HSA was studied using equilibrium dialysis and fluorescence methods. It was shown that this drug binds to or near site I (warfarin or azapropazone site) and to site II (the diazepam site) as a secondary site, although it is generally considered that their respective drug structural requirements are often exclusive. The binding parameters were calculated with different mathematical models; a site oriented model with or without fixing the number of binding sites as integer values and a stoichiometric model. The relevant results are in good agreement under the selected experimental conditions. The stoichiometric method indicates that no positive cooperativity occurred during the binding process but other interactions between the two sites cannot be excluded.
Assuntos
Anti-Inflamatórios não Esteroides/metabolismo , Piroxicam/análogos & derivados , Albumina Sérica/metabolismo , Sítios de Ligação , Fluorescência , Humanos , Piroxicam/metabolismo , Ligação ProteicaRESUMO
Previous investigations have suggested that beta-adrenoceptor-mediated responses were decreased in uremia. To evaluate this phenomenon further, beta 2-receptor density in mononuclear cells, plasma catecholamines and plasma parathyroid hormone were studied in two groups of normotensive patients: group U, twenty-five chronic uremic patients with end-stage renal failure; group C, twenty-eight control subjects. Each group was divided into three age and sex-matched subgroups. Beta 2-receptor density was determined using (-)125 iodocyanopindolol. Despite a significant increase in plasma epinephrine in the group of uremic patients, there was a significant increase in beta 2-adrenoceptor density. On the other hand the uremic state did not influence (-)125 iodocyanopindolol binding affinity and plasma norepinephrine. Parathyroid hormone, as expected, was significantly elevated in all the uremic subgroups. It can be concluded that the uremic state is associated with an unexpected upregulation of beta 2-receptor density in mononuclear cells. The role of an endogenous beta-blocking substance is suggested.
Assuntos
Falência Renal Crônica/metabolismo , Linfócitos/análise , Monócitos/análise , Receptores Adrenérgicos beta/análise , Adulto , Fatores Etários , Idoso , Catecolaminas/sangue , Feminino , Humanos , Técnicas In Vitro , Iodocianopindolol , Masculino , Pessoa de Meia-Idade , Hormônio Paratireóideo/sangue , Pindolol/análogos & derivados , Pindolol/metabolismoRESUMO
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.
Assuntos
Ocitocina/metabolismo , Precursores de Proteínas/metabolismo , Vasopressinas/metabolismo , Sequência de Aminoácidos/genética , Animais , Humanos , Neurofisinas/química , Neurofisinas/fisiologia , Ocitocina/química , Ocitocina/genética , Dobramento de Proteína , Precursores de Proteínas/química , Precursores de Proteínas/genética , Vasopressinas/química , Vasopressinas/genéticaRESUMO
Abstract In vitro assays using crude synaptosomal membrane preparations from the cortex and cytosolic fraction of hippocampus have shown that in the brain, steroids can bind to the intracellular corticosteroid receptors as well as to the membrane-bound GABA-receptor complex. Corticosteroid, deoxycorticosterone and spironolactone bound with higher affinity to the mineralocorticoid (relative binding affinity (IC(50) in nM) 1.2, 3.9 and 4.9, respectively) than to the glucocorticoid receptors (IC(50) 5.2, 14.0 and 88.0 nM, respectively) in hippocampal cytosol. They enhanced significantly the binding of [(35)S]t-butylbicyclophosphorothionate to cortical membrane. Steroids such as 3alpha,5alpha-tetrahydroxydeoxycorticosterone and 3a-hydroxy-5alpha-dihydroprogesterone displaced the binding of [(35)S]t-butylbicyclophosphorothionate with IC(50) (in nM) of 236.7 and 315.0, respectively. In the presence of 10 to 12.5muM added GABA, they bound with higher affinity (IC(50) 18.0 and 20.5 nM, respectively). Pentobarbital also bound to this site with IC(50) of 430,000 and 240,000 nM, respectively, in the absence and presence of GABA. These compounds also enhanced the binding of [(3)H]flunitrazepam which was not affected by the presence of added GABA. They showed no affinity for mineralocorticoid or glucocorticoid receptors. Data from this study showed that steroids which preferentially bound to the mineralocorticoid receptor sites (corticosterone, deoxycorticosterone and spironolactone) also enhanced the binding of [(35)S]t-butylbicyclophosphorothionate to their recognition sites. Steroids which did not interact with the intracellular receptors (3alpha,5alpha-tetrahydroxydeoxycorticosterone and 3a-hydroxy-5alpha-dihydroprogesterone) displaced [(35)S]t-butylbicyclophosphorothionate binding and enhanced [(3)H]flunitrazepam binding.
RESUMO
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.
Assuntos
Transporte Proteico/fisiologia , Vasopressinas/genética , Vasopressinas/metabolismo , Animais , Linhagem Celular Tumoral , Diabetes Insípido/metabolismo , Diabetes Insípido/fisiopatologia , Retículo Endoplasmático/metabolismo , Expressão Gênica , Mutagênese , Neuroblastoma , Neurofisinas/química , Neurofisinas/genética , Dobramento de Proteína , Estrutura Terciária de Proteína , Ratos , Vesículas Secretórias/metabolismo , Vasopressinas/químicaRESUMO
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.
Assuntos
Mutação da Fase de Leitura , Precursores de Proteínas/genética , Processamento de Proteína Pós-Traducional , Vasopressinas/genética , Animais , Transporte Biológico , Diabetes Insípido/genética , Retículo Endoplasmático/metabolismo , Deleção de Genes , Camundongos , Precursores de Proteínas/biossíntese , Precursores de Proteínas/metabolismo , Ratos , Ratos Brattleboro , Distribuição Tecidual , Células Tumorais Cultivadas , Vasopressinas/biossíntese , Vasopressinas/metabolismoRESUMO
Previously it has been shown that vasopressin (VP) and oxytocin are converted by aminopeptidase activity in brain membranes into fragments with potent CNS activities. This report concerns the properties of this enzyme activity, addressed as VP-converting aminopeptidase (VP-AP) activity, in membranes of the rat brain. The VP-AP activity had a pH optimum at pH 7.0 and had a Km of 17 microM for its action on VP. Amastatin was the most potent aminopeptidase inhibitor. Enzyme activity was inhibited by relatively low concentrations of metal chelators. Treatment of brain membranes by EDTA resulted in loss of enzyme activity that was completely reversed by 10 microM Zn2+, indicating that VP-AP activity is a metallopeptidase. Several VP analogues and fragments, in particular VP(1-8), inhibited the action of enzyme activity on VP. Among peptides unrelated to VP, angiotension I, somatostatin, and porcine ACTH(1-39) markedly inhibited enzyme activity. Solubilization of VP-AP activity from brain membranes and gel filtration on Sephadex G200 showed two peaks of activity, one eluting with an apparent mass of about 140 kDa, the other in the void volume. Gel filtration fractions were able to convert [3H][Phe3]VP in a step-wise fashion. The VP-AP-like activity was found in many tissues outside the brain. Highest activity was present in lung, kidney, parts of the gastrointestinal tract, ovary, and uterus. The results indicate that VP-AP activity is a widely distributed enzyme with probably multiple functions, one of which involves the metabolism of vasopressin in the brain.