RESUMO
OBJECTIVES: The purpose of this study was to compare the thickness of the hip capsule in patients with surgical hip disease, either with cam-femoroacetabular impingement (FAI) or non-FAI hip pathology, with that of asymptomatic control hips. METHODS: A total of 56 hips in 55 patients underwent a 3Tesla MRI of the hip. These included 40 patients with 41 hips with arthroscopically proven hip disease (16 with cam-FAI; nine men, seven women; mean age 39 years, 22 to 58) and 25 with non-FAI chondrolabral pathology (four men, 21 women; mean age 40 years, 18 to 63) as well as 15 asymptomatic volunteers, whose hips served as controls (ten men, five women; mean age 62 years, 33 to 77). The maximal capsule thickness was measured anteriorly and superiorly, and compared within and between the three groups with a gender subanalysis using student's t-test. The correlation between alpha angle and capsule thickness was determined using Pearson's correlation coefficient. RESULTS: Superiorly, the hip capsule was significantly greater in cam- (p = 0.028) and non-FAI (p = 0.048) surgical groups compared with the asymptomatic group. Within groups, the superior capsule thickness was significantly greater than the anterior in cam- (p < 0.001) and non-FAI (p < 0.001) surgical groups, but not in the control group. There was no significant correlation between the alpha angle and capsule thickness. There were no gender differences identified in the thickness of the hip capsule. CONCLUSION: The thickness of the capsule does not differ between cam- and non-FAI diseased hips, and thus may not be specific for a particular aetiology of hip disease. The capsule is, however, thicker in diseased surgical hips compared with asymptomatic control hips.Cite this article: K. S. Rakhra, A. A. Bonura, R. Nairn, M. E. Schweitzer, N. M. Kolanko, P. E. Beaule. Is the hip capsule thicker in diseased hips? Bone Joint Res 2016;5:586-593. DOI: 10.1302/2046-3758.511.2000495.
RESUMO
Purified recombinant Parj1 and Parj2 allergens bind an IgE repertoire common to the Parietaria species, allowing their use as marker molecules for diagnosis and therapy of allergic disease induced by the Urticaceae family. Preclinical studies on the in vivo immunogenicity of recombinant Parj1, Parj2 and their isoforms indicated differential capacity to induce IgG1 antibody responses, as indication of potential clinical use. A recombinant hetero-dimeric hybrid derivative (PjED), encompassing the shorter Parj1 isoform (Parj1.0201) and Parj2 allergen, was characterised. In vivo immunisation with PjED induces IgG1 antibodies capable of binding all the isoforms of Parietaria major allergens, overcoming the poor immunogenicity of single monomeric allergens. This feature makes PjED a promising candidate molecule to be further characterised for clinical applications in the treatment of Parietaria allergy.
Assuntos
Formação de Anticorpos/imunologia , Antígenos de Plantas/imunologia , Parietaria/imunologia , Proteínas Recombinantes/imunologia , Animais , Dessensibilização Imunológica/métodos , Feminino , Humanos , Hipersensibilidade/imunologia , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Plantas/imunologia , Pólen/imunologia , Rinite Alérgica Sazonal/imunologiaRESUMO
Parietaria judaica pollen is one of the main sources of allergens in the Mediterranean area. Its allergenic composition has been studied in detail showing the presence of two major allergens (Par j 1 and Par j 2) and two minor allergens belonging to the profilin and calcium binding protein families of allergens (Par j 3 and Par j 4, respectively). Clinical reports support the hypothesis of a limited cross-reactivity between profilin from Parietaria and unrelated sources. We screened a P. judaica cDNA library to identify novel forms of profilins with allergenic activity. This strategy allowed us to isolate a 767 bp cDNA containing the information for a 131 amino acids protein with homology to profilins from unrelated sources greater than that observed with the already published Parietaria profilins. This profilin was expressed in Escherichia coli as a recombinant protein and its immunological prevalence was studied in a population of Parietaria allergic patients from Southern Europe. Immunoblotting analysis showed that the Parietaria profilin was recognized by IgE from 6.5% of the allergic population. Finally, a selected population of profilin allergic patients was enrolled to demonstrate the cross-reactivity of this novel variant with other profilins from grass and date palm. In conclusion, molecular cloning and immunological studies have allowed the isolation, expression and immunological characterization of a novel cross-reactive profilin allergen from P. judaica pollen named Par j 3.0201.
Assuntos
Alérgenos/imunologia , Antígenos de Plantas/imunologia , Parietaria/imunologia , Extratos Vegetais/imunologia , Proteínas de Plantas/imunologia , Profilinas/imunologia , Proteínas Recombinantes/imunologia , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Biblioteca Gênica , Humanos , Masculino , Dados de Sequência Molecular , Profilinas/biossíntese , Profilinas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Alinhamento de SequênciaRESUMO
BACKGROUND: The major allergens in Parietaria pollen, Par j 1 and Par j 2, have been identified as lipid transfer proteins. The family of the Par j 1 allergens is composed of two isoforms, which differ by the presence of a 37 amino acid peptide (Par37) exclusive to the Par j 1.0101 isoform. The goal of this study was to elucidate the biological properties of the Par37 peptide. METHODS: In silico analysis, spectrofluorimetric experiments and in vitro cell culture assays were used to identify the biological properties of Par37. In addition, a mouse model of sensitization was used to study the influence of Par37 in the murine immune response. RESULTS: In silico analysis predicted that Par37 displays characteristics of a host defence peptide. Spectrofluorimetric analysis, real-time PCR and ELISA assays demonstrated that Par37 possesses an LPS-binding activity influencing cell signalling in vitro. In RAW264.7 cells, LPS-induced IL-6 and TNF-α transcription and translation were inhibited after preincubation with Par37. Consistent with these data, inhibition of IFN-γ secretion was observed in murine spleen cells and in human PBMC. Finally, mice immunized with the two Par j 1 isoforms differing in the presence or absence of the Par37 peptide showed different immunological behaviours in vivo. CONCLUSIONS: This study demonstrates that the Par j 1.0101 allergen displays LPS-binding activity due to the presence of a 37 amino acid COOH-terminal region and that this region is capable of influencing cytokine and antibody responses in vitro and in vivo.
Assuntos
Alérgenos/química , Alérgenos/imunologia , Fatores Imunológicos , Parietaria/imunologia , Pólen/imunologia , Alérgenos/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Citocinas/imunologia , Citocinas/metabolismo , Feminino , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/metabolismo , Camundongos , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/imunologia , Peptídeos/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/imunologia , Polimixina B/metabolismo , Ligação Proteica , Alinhamento de Sequência , Baço/imunologiaRESUMO
In this study we wanted to analyse the pattern of the immune response to the Parietaria major allergen Par j 1 in freshly purified peripheral blood mononuclear cell (PBMC) from healthy subjects. We observed that Par j 1 was capable of inducing IFN-γ production by CD3â» and CD16âº/CD56⺠cells exclusively in healthy individuals. Furthermore, a multiparametric analysis allowed us a better definition of two IFN-γ-Par j 1 specific populations (IFN-γ(dim) and IFN-γ(high)) characterized by the presence of different proportions of NKT and NK cells. We also identified the concomitant presence of a subset of IL-10⺠NK cells. Moreover, CFSE staining showed that the Par j 1 preferentially induced the proliferation of CD3â»/CD56âº/CD335⺠cells. Finally, a subset of CD4âº/CD25âº/FoxP3âº/IL-10â» T cells was identified. The result of this pilot study suggest that during a tolerogenic response, the major allergen of the Parietaria pollen works as an activator of both the innate and the adaptive human immune system.
Assuntos
Imunidade Adaptativa , Alérgenos/farmacologia , Imunidade Inata , Células Matadoras Naturais/efeitos dos fármacos , Células T Matadoras Naturais/efeitos dos fármacos , Parietaria/imunologia , Proteínas de Plantas/farmacologia , Adulto , Alérgenos/biossíntese , Alérgenos/genética , Antígenos CD/genética , Antígenos CD/imunologia , Células Cultivadas , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Expressão Gênica/efeitos dos fármacos , Humanos , Interferon gama/genética , Interferon gama/imunologia , Interleucina-10/genética , Interleucina-10/imunologia , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Células T Matadoras Naturais/citologia , Células T Matadoras Naturais/imunologia , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologiaRESUMO
BACKGROUND: Parietaria pollen is one of the major cause of pollinosis in the southern Europe. Specific immunotherapy is the only treatment able to modify the natural outcome of the disease restoring a normal immunity against allergens. METHODS: We designed a recombinant molecule (PjEDloop1) comprised of genetic-engineered variants of the major allergens of the Parietaria pollen (Par j 2/Par j 1). Purity and chemical-physical properties of the derivative were analysed by RP-HPLC chromatography and Photon Correlation Spectroscopy. Immunological activity was evaluated by means of Western blotting, ELISA inhibition and PBMC proliferation assay in 10 Parietaria allergic patients. Basophil activation was studied in six subjects. The immunogenicity of the hybrid was studied looking at the immune responses induced in a mouse model of sensitization. RESULTS: The PjEDloop1 hybrid was produced as a purified recombinant protein with high stability in solution. Western blot, ELISA inhibition and basophil activation test showed that the PjEDloop1 displays a remarkable reduced IgE binding and anaphylactic activity. CD3 reactivity was conserved in all patients. Mice immunization with the rPjEDloop1 induced antibodies and T cell responses comparable to that obtained by the wild type allergens. Such antibodies shared the specificities to rPar j 1 and rPar j 2 with human IgE antibodies. CONCLUSION: Our results demonstrated that a mutant hybrid expressing genetically engineered forms of the major P. judaica allergens displayed reduced allergenicity and retained T cell reactivity for the induction of protective antibodies in vaccination approaches for the treatment of Parietaria pollinosis.
Assuntos
Alérgenos/imunologia , Dessensibilização Imunológica/métodos , Parietaria/imunologia , Proteínas de Plantas/imunologia , Animais , Antígenos de Plantas , Western Blotting , Cromatografia Líquida de Alta Pressão , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Engenharia Genética/métodos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/síntese química , Proteínas Recombinantes/imunologia , Rinite Alérgica Sazonal/terapiaRESUMO
Specific immunotherapy is a well established and clinically proved strategy to cure allergic reactions. The impressive boost of knowledge derived from DNA recombinant technology applied to this field allowed the identification, cloning and expression of several clinically relevant allergens. Recombinant allergens can be easily produced in a pure and reproducible way with immunological properties comparable to natural allergens and matching the requirements of pharmaceutical companies. Parietaria pollinosis is a major health problem in the Mediterranean basin with prolonged symptoms. In this review we will discuss the rational approaches to design hypoallergenic derivatives of the major allergens of this pollen, their immunological properties and possible clinical future implications.
Assuntos
Alérgenos/uso terapêutico , Antígenos de Plantas/uso terapêutico , Dessensibilização Imunológica , Parietaria/imunologia , Rinite Alérgica Sazonal/terapia , Vacinas/uso terapêutico , Alérgenos/química , Alérgenos/genética , Alérgenos/imunologia , Animais , Antígenos de Plantas/química , Antígenos de Plantas/genética , Antígenos de Plantas/imunologia , Clonagem Molecular , Desenho de Fármacos , Humanos , Conformação Proteica , Proteínas Recombinantes/uso terapêutico , Rinite Alérgica Sazonal/imunologia , Relação Estrutura-Atividade , Vacinas/química , Vacinas/imunologiaRESUMO
Amyloidosis, a potentially fatal disease, is characterized by an abnormal deposition of autologous proteins. Heart, liver, kidneys, lung, thyroid, skin and the gastrointestinal tract can be involved; in this last case mucosal alterations or disturbances of the motility leading to pseudo-obstruction, bleeding, diarrhea and malabsorption can be present. However, the data concerning the possible gastrointestinal presentations of amyloidosis are scanty and heterogeneous. We report the case of a patient presenting severe gastrointestinal symptoms caused by a megaduodenum. The patient was thoroughly investigated and lesions appeared limited to the upper gastrointestinal tract in the absence of a systemic disorder. However, at follow up the patient developed cardiac dilatation and bioptic samples revealed the presence of amyloidosis.
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Amiloidose/diagnóstico , Duodenopatias/etiologia , Amiloidose/complicações , Amiloidose/patologia , Dilatação Patológica , Duodenopatias/patologia , Evolução Fatal , Humanos , Masculino , Pessoa de Meia-Idade , Miocárdio/patologiaRESUMO
The diagnosis and therapy of allergic disorders are usually performed with crude extracts which are a heterogeneous mixture of proteins with different allergenic potency. The knowledge of the allergenic composition is a key step for diagnostic and therapeutic options. Parietaria judaica pollen represents one of the main sources of allergens in the Mediterranean area and its major allergens have already been identified (Par j 1 and Par j 2). In addition, inhibition studies performed using a calcium-binding protein (CBP) from grass pollen (Phl p 7) showed the presence of a homologue of this cross-reactive allergen in the Parietaria extract. Screening of a cDNA library allowed us to isolate a 480bp cDNA containing the information for an 87 AA long protein with high level of homology to calcium-binding proteins from other allergenic sources. It was expressed as a recombinant allergen in Escherichia coli and purified by affinity chromatography. Its expression allowed us to study the prevalence of this allergen in a population of allergic patients in southern Europe. Immunoblotting and inhibition studies showed that this allergen shares a pattern of IgE epitopes in common with other 2-EF-hand calcium-binding proteins from botanically non-related species. The immunological properties of the Pj CBP were investigated by CD63 activation assay and CFDA-SE staining. In conclusion, DNA recombinant technology allowed the isolation, expression and immunological characterization of a cross-reactive calcium-binding protein allergen from Parietaria judaica pollen.
Assuntos
Alérgenos/isolamento & purificação , Basófilos/imunologia , Proteínas de Ligação ao Cálcio/imunologia , Proteínas de Ligação ao Cálcio/isolamento & purificação , Imunoglobulina E/imunologia , Parietaria/imunologia , Pólen/imunologia , Alérgenos/genética , Alérgenos/imunologia , Alérgenos/metabolismo , Sequência de Aminoácidos , Antígenos de Plantas/imunologia , Antígenos de Plantas/metabolismo , Sequência de Bases , Basófilos/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proliferação de Células , Clonagem Molecular , Humanos , Imunoglobulina E/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Dados de Sequência Molecular , Pólen/química , Alinhamento de SequênciaAssuntos
Síndromes Compartimentais/diagnóstico , Transtornos Neurológicos da Marcha/diagnóstico , Transtornos Neurológicos da Marcha/etiologia , Neuropatias Fibulares/diagnóstico , Doença Aguda , Síndromes Compartimentais/complicações , Feminino , Humanos , Neuropatias Fibulares/complicações , Adulto JovemRESUMO
BACKGROUND: Allergy is an immunological disorder affecting about 25% of the population living in the industrialized countries. Specific immunotherapy is the only treatment with a long-lasting relief of allergic symptoms and able to reduce the risk of developing new allergic sensitizations and inhibiting the development of clinical asthma in children treated for allergic rhinitis. METHODS: By means of DNA recombinant technology, we were able to design a head to tail dimer expressing disulphide bond variants of the major allergen of the Parietaria pollen. IgE binding activity was studied by Western blot, ELISA inhibition assays and the skin prick test. T cell recognition was studied by peripheral blood mononuclear cell proliferation. The immunogenicity of the hybrid was studied in a mouse model of sensitization. RESULTS: In vitro and in vivo analysis showed that the disruption of specific cysteine residues in both allergens caused a strong reduction in IgE binding activity of the PjEDcys hybrid. In addition,we were able to show that a reduction in the IgE epitope content profoundly reduced the anaphylactic activity of the hybrid (from 100 to 1,000 times less than wild-type allergens) without interfering with the T cell recognition. Sera from BALB/c mice immunized with the hybrid were able to bind the natural Parietaria allergens and to inhibit the binding of human IgE to wild-type Par j 1 and Par j 2 allergens up to 90%. CONCLUSION: Our results demonstrate that hybrid-expressing disulphide bond variants of the major allergens of the Parietaria pollen displayed reduced allergenicity and maintained T cell reactivity for induction of protective antibodies.
Assuntos
Alérgenos/imunologia , Dessensibilização Imunológica , Parietaria/imunologia , Proteínas de Plantas/imunologia , Alérgenos/genética , Animais , Escherichia coli/genética , Feminino , Histamina/imunologia , Humanos , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Leucócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Plantas/genética , Pólen/imunologia , Proteínas Recombinantes/imunologia , Testes Cutâneos , VacinaçãoAssuntos
Antígenos de Plantas/química , Antígenos de Plantas/imunologia , Hipersensibilidade Imediata/diagnóstico , Parietaria/química , Parietaria/imunologia , Alérgenos/imunologia , Proteínas de Ligação ao Cálcio/imunologia , Reações Cruzadas , Proteínas de Plantas/imunologia , Profilinas/imunologiaRESUMO
Leiomyosarcoma of the inferior vena cava is a rare primary tumour. We present a case report of a 67-year-old man with a long history of abdominal pain and gastroesophageal reflux, who was found to have a large retroperitoneal mass confirmed to be a leiomyosarcoma. The clinical and imaging features are outlined, and in addition the treatment and prognosis.
Assuntos
Diagnóstico por Imagem , Leiomiossarcoma/diagnóstico , Neoplasias Vasculares/diagnóstico , Veia Cava Inferior , Idoso , Diagnóstico Diferencial , Humanos , Leiomiossarcoma/cirurgia , Masculino , Prognóstico , Neoplasias Vasculares/cirurgiaRESUMO
BACKGROUND: Par j 1 represents the major allergenic component of Parietaria judaica pollen. Its three-dimensional structure is stabilized by four disulphide bridges. A family of three-dimensional mutants of the recombinant Par j 1 (rPar j 1) allergen, showing reduced allergenicity and retained T cell recognition has been recently developed by site-directed mutagenesis. OBJECTIVE: To develop and characterize a murine model of IgE sensitization to rPar j 1. To evaluate similarities between the murine model and the human IgE response. To investigate in this model the recognition of a hypoallergenic mutant of Par j 1, and to study the immune responses elicited in mice by the mutant itself. METHODS: BALB/c mice were sensitized by two intraperitoneal immunizations with rPar j 1 in alum on days 0 and 21. Allergen-specific serum IgE and IgG responses were studied by direct ELISA and immunoblotting, ELISA inhibition and competitive ELISA. Cell proliferation was evaluated in splenocyte cultures. RESULTS: Sensitization with rPar j 1 induced high levels of IgE and IgG1 vs. low levels of IgG2a. Mouse antibodies specific to rPar j 1 were able to compete with human IgE for recognition of rPar j 1. IgE from mice immunized with rPar j 1 showed a significantly reduced binding activity towards the hypoallergenic variant rPjC, which lacks three disulphide bridges. On the contrary, rPjC was recognized by IgG1 and IgG2a antibodies as well as rPar j 1. The proliferative response to rPjC by splenocytes from mice immunized with rPar j 1 was comparable to that stimulated by rPar j 1. Immunization with rPjC induced low levels of IgE antibodies to the rPjC itself, while IgG and proliferative responses were similar to those induced by rPar j 1. CONCLUSION: Conformational variants of allergens, displaying reduced allergenicity accompanied by retained IgG and T cell recognition, offer a safe, specific and flexible approach to immunotherapy of type I allergy. Our mouse model of IgE sensitization to a recombinant allergen, mimicking the human response to its native counterpart, could provide valuable information for pre-clinical testing of such hypoallergenic molecules.
Assuntos
Alérgenos/imunologia , Dessensibilização Imunológica , Parietaria/imunologia , Proteínas de Plantas/imunologia , Animais , Anticorpos/imunologia , Reações Antígeno-Anticorpo , Divisão Celular , Feminino , Hipersensibilidade/imunologia , Hipersensibilidade/prevenção & controle , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , Pólen , Proteínas Recombinantes/imunologia , Baço/citologiaRESUMO
BACKGROUND: Par j 1 represents a major allergenic component of Parietaria judaica (Pj) pollen, since it is able to induce an immunoglobulin E (IgE) response in 95% of Pj-allergic patients. It belongs to the non-specific lipid transfer protein family, sharing with them a common three-dimensional structure. METHODS: Disulphide bond variants of the recombinant Par j 1 (rPar j 1) allergen were generated by site-directed mutagenesis, and the immunological activity of rPar j 1 and its conformational mutants was compared with the use of the skin prick test (SPT). The ability to bind IgE antibodies was evaluated by Western blot, ELISA and ELISA inhibition. T cell reactivity was measured by peripheral blood mononuclear cell proliferation assay. RESULTS: The disruption of Cys14-Cys29 and Cys30-Cys75 bridging (PjA mutant) caused the loss of the majority of specific IgE-binding activity. Additional disruption of the Cys4-Cys52 bridge (PjC mutant) and the latter Cys50-Cys91 bridge (PjD mutant) led to the abolition of IgE-binding activity. On the SPT, PjB (lacking the Cys4-Cys52 and Cys50-Cys91 bridges) was still capable of triggering a type I hypersensitive reaction in 9 out of 10 patients, and PjA in 3 out of 10 patients, while PjC and PjD did not show any SPT reactivity. All the mutants preserved their T cell reactivity. CONCLUSION: Recombinant hypoallergenic variants of the rPar j 1 allergen described herein may represent a useful tool for improved immunotherapy.
Assuntos
Alérgenos/genética , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Glicoproteínas/genética , Proteínas de Plantas/genética , Animais , Antígenos de Plantas , Sequência de Bases , Proteínas de Transporte/química , DNA de Plantas/genética , Dessensibilização Imunológica , Dissulfetos/química , Variação Genética , Glicoproteínas/química , Humanos , Hipersensibilidade Imediata/imunologia , Hipersensibilidade Imediata/terapia , Imunoglobulina E/metabolismo , Técnicas In Vitro , Ativação Linfocitária , Proteínas de Plantas/química , Coelhos , Linfócitos T/imunologia , Urticaceae/genética , Urticaceae/imunologiaRESUMO
COLL1alpha mRNA is asymmetrically distributed in the Paracentrotus lividus egg. Here we examine the involvement of the cytoskeleton in the localization process of collagen mRNA. The use of drugs such as colchicine and cytochalasin B reveals a perturbation of localization collagen mRNA. Moreover, the presence of specific cis-and trans-acting factors involved in cytoskeleton binding and the localization process was investigated. By Northwestern experiment we found that the 3'UTR of COLL1alpha mRNA is also able to bind two proteins of 54 and 40 kDa in a cellular fraction containing the cytoskeleton. Finally, we found that the protein of 54 kDa is LP54, a protein that binds the 3'UTRs of P. lividus maternal bep messengers and is necessary for their localization.
Assuntos
Colágeno/biossíntese , Colágeno/genética , Colágeno/metabolismo , Citoesqueleto/metabolismo , Óvulo/metabolismo , RNA Mensageiro/metabolismo , Ouriços-do-Mar/embriologia , Ativação Transcricional , Regiões 3' não Traduzidas , Animais , Northern Blotting , Fracionamento Celular , Colchicina/farmacologia , Citocalasina B/farmacologia , DNA Complementar/metabolismo , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Immunoblotting , Imuno-Histoquímica , Hibridização In Situ , Microtúbulos/metabolismo , Óvulo/efeitos dos fármacos , Ligação Proteica , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Bep mRNAs are localized at the animal pole of P. lividus eggs. In the present communication the secondary structures of the 3'UTRs of the bep1, bep3 and bep4 mRNAs are reported. The minimal lengths of these regions required to bind the 54-kDa protein, previously shown to be involved in localization and anchoring of these RNAs, is estimated. Microinjection of the bep3 3'UTR into egg shows that this RNA fragment is also able to become localized to one of the egg poles, as happens for the entire bep3 RNA.
Assuntos
Hormônios de Invertebrado/metabolismo , Proteínas de Membrana/metabolismo , Dobramento de Proteína , Sequência de Aminoácidos , Animais , Hormônios de Invertebrado/química , Hormônios de Invertebrado/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Microinjeções , Dados de Sequência Molecular , Biossíntese de Proteínas , Estrutura Secundária de Proteína , RNA Mensageiro/genética , Ouriços-do-MarRESUMO
Bep mRNAs, i.e., maternal messengers coding for cell surface proteins, are localized in the animal part of Paracertrotus lividus egg and embryos. Here we have examined the involvement of the cytoskeleton in asymmetric distribution of bep3 mRNA. Moreover, in order to understand whether and how cis- and trans-acting factors are necessary for bep3 mRNA localization, we have looked for in vitro-specific interactions between egg proteins and bep3 mRNA. By northwestern assay we have identified a 54-kDa protein that binds to the 3'UTR of bep3 mRNA. This 54-kDa protein also permits association of 3'UTR of bep3 with cytoskeleton elements, indicating its involvement in the localization process. Binding of 54-kDa protein to 3'UTR of bep1 and bep4 has also been demonstrated, suggesting that a binding motif is shared with these other two mRNAs of the same gene family. Northwestern analyses carried out utilizing proteins extracted from different developmental stages indicate that the 54-kDa protein is the only protein able to bind to the 3'UTR of bep3.
Assuntos
Citoesqueleto/química , Proteínas de Membrana/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/análise , Animais , Feminino , Peso Molecular , Óvulo/química , Ouriços-do-MarRESUMO
We have identified and partially characterised two antigens, extracted with 3% butanol, from Paracentrotus lividus embryos dissociated at the blastula stage, and encoded by the cDNA clones previously described as bep1 and bep4 (bep-butanol extracted proteins). The cDNA fragments containing the specific central portions of bep1 and bep4 were expressed as MS2 polymerase fusion proteins in Escherichia coli. These two fusion proteins, called 1C1 (bep1) and 4A1 (bep4), were injected subcutaneously into rabbits and the corresponding polyclonal antibodies generated. Western blot analysis of proteins, extracted with 3% butanol, from sea urchin embryos at the blastula stage (b.e.p.), established that both antibodies recognize two 33 KDa proteins. Reducing and non-reducing electrophoretic conditions show that both antibodies against bep1 and bep4 related proteins react also with a protein band of a molecular weight 66 KDa, indicating that these two antigens probably exist as dimers. Immunolocalization with anti 1C1 and 4A1 antibodies shows the presence of the related antigens also on the cell surface. Fab fragments of the polyclonal antibodies against 1C1 and 4A1 inhibited reaggregation of sea urchin embryonic cells, dissociated from blastula stage embryos. This prevention of reaggregation indicates that these proteins probably play a role in cell interaction during sea urchin embryonic development.