RESUMO
BACKGROUND: Vitrification is commonly used for cryopreservation of gametes. OBJECTIVE: The study aimed at evaluating viability and developmental competence of bovine oocytes vitrified by Rapid-I method. MATERIALS AND METHODS: Oocytes after collection (group 1) and IVM (group 2) were vitrified using medium containing 18% Ficoll, 40% ethylene glycol, 0.3 M sucrose. To evaluate viability, oocytes after warming were stained with fluorescein diacetate and ethidium bromide. In experiment 2, oocytes after IVM and vitrification were activated by 0.5 µM ionomycin in TCM 199 combined with 2 mM 6-DMAP in TCM 199 with 10% fetal bovine serum. RESULTS: Survival rate in group 1 was 58%, and 88% in the control. In group 2, 63% viable oocytes were found, compared to 82% in the control group. After parthenogenetic activation 27.2% morulas were observed. This percentage was lower than in the non-vitrified group (31%). CONCLUSION: Maturity stage of bovine oocytes has no effect on their survival after vitrification.
Assuntos
Criopreservação/veterinária , Oócitos , Vitrificação , Animais , Bovinos , Sobrevivência Celular , Etilenoglicol , Feminino , Fertilização in vitroRESUMO
BACKGROUND: Vitrification by Rapid-I method could be essential for felid rescue programs to protect wild felid in the future. OBJECTIVE: This study was aimed at adapting the Rapid I method and evaluating the viability of serval and Pallas cat oocytes compared to oocytes of the domestic cat. MATERIALS AND METHODS: Oocytes after collection and in vitro maturation were vitrified using Cryotech medium (Cryotech, Japan) and a Rapid-I device (Vitrolife, Sweden). To evaluate viability, oocytes after warming were stained with fluorescein diacetate and ethidium bromide. RESULTS: Survival rate in the control group (domestic cat) was 75 %. In the experimental group, 70% (serval) and 60% (pallas cat) viable oocytes were found. CONCLUSION: The Rapid-I method can be applied successfully for the vitrification of wild felid oocytes.
Assuntos
Sobrevivência Celular , Criopreservação/veterinária , Felidae , Felis , Oócitos/citologia , Animais , Criopreservação/métodos , Crioprotetores , Feminino , VitrificaçãoRESUMO
Although the phylogeography of European mammals has been extensively investigated since the 1990s, many studies were limited in terms of sampling distribution, the number of molecular markers used and the analytical techniques employed, frequently leading to incomplete postglacial recolonisation scenarios. The broad-scale genetic structure of the European badger (Meles meles) is of interest as it may result from historic restriction to glacial refugia and/or recent anthropogenic impact. However, previous studies were based mostly on samples from western Europe, making it difficult to draw robust conclusions about the location of refugia, patterns of postglacial expansion and recent demography. In the present study, continent-wide sampling and analyses with multiple markers provided evidence for two glacial refugia (Iberia and southeast Europe) that contributed to the genetic variation observed in badgers in Europe today. Approximate Bayesian computation provided support for a colonisation of Scandinavia from both Iberian and southeastern refugia. In the whole of Europe, we observed a decline in genetic diversity with increasing latitude, suggesting that the reduced diversity in the peripheral populations resulted from a postglacial expansion processes. Although MSVAR v.1.3 also provided evidence for recent genetic bottlenecks in some of these peripheral populations, the simulations performed to estimate the method's power to correctly infer the past demography of our empirical populations suggested that the timing and severity of bottlenecks could not be established with certainty. We urge caution against trying to relate demographic declines inferred using MSVAR with particular historic or climatological events.
Assuntos
Evolução Molecular , Variação Genética , Genética Populacional , Mustelidae/genética , Animais , Teorema de Bayes , DNA Mitocondrial/genética , Europa (Continente) , Haplótipos , Repetições de Microssatélites , Modelos Genéticos , Filogeografia , Dinâmica PopulacionalRESUMO
This study describes, for the first time, the relationship between morphology and ploidy in domestic cat embryos. Blastocyst morphology and quality were assessed using time-lapse recordings, while ploidy was analyzed using fluorescence in situ hybridization. Out of 54 blastocysts, clear fluorescence signals for all the molecular probes used were observed in 24 (44.4%) blastocysts, while in another 14 (25.9%) blastocysts, fluorescence signals only allowed for sex assessment. No clear signals were observed in the remaining 16 blastocysts (29.7%). Of the 24 blastocysts with clear signals, normal ploidy was detected in 10 (41.4%), 7 (29.2%) were diagnosed as haploid, and the remaining 7 blastocysts (29.2%) were mosaics. Additionally, results showed the distribution of diploid, haploid, and mosaic blastocysts in relation to the occurrence of morphological disorders and to embryo quality. The presence of abnormal embryo morphology and karyotype disorders may affect further development and the pregnancy rate. Due to the comparable proportion of good and poor quality blastocysts with disturbed ploidy, it is important to implement new methods of embryo assessment, especially when techniques used in humans, such as pronuclear observation, cannot be used.
Assuntos
Blastocisto , Ploidias , Animais , Gatos , Feminino , Hibridização in Situ Fluorescente/veterinária , Gravidez , Taxa de GravidezRESUMO
Plasmodium falciparum, the most lethal of the malarial parasites that infect humans, undergoes three cycles of development in its vertebrate host and elicits stage-specific immune responses. This stage specificity of the immune response has made it difficult to isolate antigens that would be useful in developing a vaccine against malaria. A complementary DNA clone for a glycophorin-binding protein of Plasmodium falciparum merozoites has been isolated and characterized. The protein interacts with glycophorin, the erythrocyte receptor, during invasion of the host cell by the parasite. Antigenic determinants of this protein expressed in Escherichia coli have been used to produce antibodies to a glycophorin-binding protein. The antibodies show schizont-specific immunofluorescence and react with the merozoite protein. The primary sequence of these determinants reveals a 150-nucleotide tandem-repeating sequence coding for a 50-amino-acid repeat. The characterization of the Plasmodium falciparum glycophorin-binding protein represents one approach toward designing serologic agents to block the parasite's development in the vertebrate host.
Assuntos
Eritrócitos/parasitologia , Plasmodium falciparum/genética , Animais , DNA/genética , DNA Recombinante/metabolismo , Glicoforinas/metabolismo , Soros Imunes/imunologia , Camundongos/imunologia , Plasmodium falciparum/fisiologiaRESUMO
A phosphatidylinositol-glycan-specific phospholipase D (PI-G PLD) that specifically hydrolyzes the inositol phosphate linkage in proteins anchored by phosphatidylinositol-glycans (PI-Gs) has recently been purified from human and bovine sera. The primary structure of bovine PI-G PLD has now been determined and the functional activity of the enzyme has been studied. Expression of PI-G PLD complementary DNA in COS cells produced a protein that specifically hydrolyzed the inositol phosphate linkage of the PI-G anchor. Cotransfection of PI-G PLD with a PI-G-anchored protein resulted in the secretion of the PI-G-anchored protein. The results suggest that the expression of PI-G PLD may influence the expression and location of PI-G-anchored proteins.
Assuntos
Fosfolipase D/química , Sequência de Aminoácidos , Animais , Bovinos , Linhagem Celular , Clonagem Molecular , DNA/genética , Expressão Gênica , Glicosilfosfatidilinositóis , Humanos , Hidrólise , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fosfatidilinositóis/metabolismo , Fosfolipase D/genética , Fosfolipase D/metabolismo , Polissacarídeos/metabolismo , Homologia de Sequência do Ácido Nucleico , Transfecção , TripsinaRESUMO
Binding of antibodies to effector cells by way of receptors to their constant regions (Fc receptors) is central to the pathway that leads to clearance of antigens by the immune system. The structure and function of this important class of receptors on immune cells is addressed through the molecular characterization of Fc receptors (FcR) specific for the murine immunoglobulin G isotype. Structural diversity is encoded by two genes that by alternative splicing result in expression of molecules with highly conserved extracellular domains and different transmembrane and intracytoplasmic domains. The proteins encoded by these genes are members of the immunoglobulin supergene family, most homologous to the major histocompatibility complex molecule E beta. Functional reconstitution of ligand binding by transfection of individual FcR genes demonstrates that the requirements for ligand binding are encoded in a single gene. These studies demonstrate the molecular basis for the functional heterogeneity of FcR's, accounting for the possible transduction of different signals in response to a single ligand.
Assuntos
Linfócitos/fisiologia , Macrófagos/fisiologia , Receptores Fc , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA/genética , Regulação da Expressão Gênica , Antígenos de Histocompatibilidade Classe II/genética , Imunoglobulina G , Proteínas de Membrana , Camundongos , Conformação Proteica , Receptores Fc/genética , Receptores de IgG , Transcrição Gênica , TransfecçãoRESUMO
Essentials DS-1040 inhibits the activated form of thrombin-activatable fibrinolysis inhibitor (TAFIa). Infusion of DS-1040 was safe and well tolerated in healthy young and elderly subjects. DS-1040 substantially decreased TAFIa activity but had no impact on bleeding time. DS-1040 may provide an option of safer thrombolytic therapy. SUMMARY: Background Current treatments for acute ischemic stroke and venous thromboembolism, such as recombinant tissue-type plasminogen activator and thrombectomy, are limited by a narrow time window and the risk of bleeding. DS-1040 is a novel low molecular weight compound that inhibits the activated form of thrombin-activatable fibrinolysis inhibitor (TAFIa), and was developed as a fibrinolysis enhancer for the treatment of thromboembolic diseases. Objectives This first-in-human, randomized, placebo-controlled, three-part, phase 1 study was conducted to evaluate the safety, pharmacokinetics and pharmacodynamics of DS-1040 in healthy subjects. Subjects/Methods Young (18-45 years) or elderly (65-75 years) subjects (N = 103) were randomized to receive single ascending doses of DS-1040 ranging from 0.1 mg to 40 mg, or placebo, administered either as a 0.5-h intravenous infusion or as a 24-h continuous infusion. Results All doses of DS-1040 were tolerated, and no serious adverse events (AEs) or discontinuations resulting from AEs occurred during the study. Bleeding time remained within the normal range for all doses tested in all subjects. Plasma exposure of DS-1040 increased proportionally with increase in dose. Elderly subjects had higher exposures to DS-1040 and prolonged elimination times, probably because of decreased renal clearance. DS-1040 caused a substantial dose-dependent and time-dependent decrease in TAFIa activity and in 50% clot lysis time. The levels of D-dimer, indicative of endogenous fibrinolysis, increased in some individuals following DS-1040 treatment. No effects of DS-1040 on coagulation parameters or platelet aggregation were observed. Conclusions The novel fibrinolysis-enhancing agent DS-1040 has favorable pharmacokinetic/pharmacodynamic properties and a favorable safety profile, warranting further clinical development.
Assuntos
Carboxipeptidase B2/antagonistas & inibidores , Fibrinólise/efeitos dos fármacos , Fibrinolíticos/administração & dosagem , Inibidores de Proteases/administração & dosagem , Adolescente , Adulto , Fatores Etários , Idoso , Testes de Coagulação Sanguínea , Carboxipeptidase B2/metabolismo , Relação Dose-Resposta a Droga , Feminino , Fibrinolíticos/efeitos adversos , Fibrinolíticos/farmacocinética , Voluntários Saudáveis , Hemorragia/induzido quimicamente , Humanos , Infusões Intravenosas , Masculino , Pessoa de Meia-Idade , Inibidores de Proteases/efeitos adversos , Inibidores de Proteases/farmacocinética , Fatores de Risco , Adulto JovemRESUMO
The morphogenesis of bacteriophage lambda proheads is under the control of the four phage genes B, C, Nu3 and E, and the two Escherichia coli genes groEL and groES . It has been shown previously that extracts prepared from cells infected with a lambda C-E- mutant accumulate a gpB polymer, which behaves as a biologically active intermediate in prohead assembly. This gpB activity has been called a preconnector , as it is probably a precursor to the head-tail connector. We now report the partial purification of biologically active preconnectors and the characterization of its structure. In the electron microscope, preconnectors appear as donut -like structures composed of several subunits displaying radial symmetry. Optical filtration of periodic arrays of preconnectors showed that the structure has 12-fold rotational symmetry. Side views of the preconnector reveal that it resembles an asymmetrical dumbell . This information has been used to construct a three-dimensional model of the preconnector . The implications of this structure for prohead shape and function, and for DNA packaging are discussed.
Assuntos
Bacteriófago lambda/análise , Proteínas Virais/isolamento & purificação , Bacteriófago lambda/ultraestrutura , Eletroforese em Gel de Poliacrilamida , Filtração , Aumento da Imagem , Microscopia Eletrônica , Modelos MolecularesRESUMO
The in vitro development of human mast cells from fetal liver cells with recombinant human stem cell factor in serum-containing RPMI was compared to that in AIM-V media with and without serum. Compared to serum-containing media, AIM-V medium caused mast cells to develop earlier and in greater numbers. By 2 weeks, about 60% of cells in serum-free AIM-V medium were phenotypic mast cells, approximately 2 times the percentages in serum-containing media. By 6 weeks the percentages of mast cells were > or =80% under all conditions, but the number of mast cells was 3-4-fold greater in serum-free AIM-V medium than in serum-supplemented media. Mast cells obtained in serum-free AIM-V medium exhibited rounded nuclei, like tissue-derived mast cells; mast cells obtained in serum-supplemented media had segmented nuclei. By 10-12 weeks of culture about 40% of the AIM-V-derived cells showed strong chymase immunocytochemical staining, a pattern observed for only 14% of the cells in serum-containing media. AIM-V medium is a suitable medium for the development of human mast cells in vitro, and permits an earlier, more selective and greater expansion of mast cells than serum-containing media.
Assuntos
Técnicas de Cultura/métodos , Feto/citologia , Fígado/citologia , Mastócitos/citologia , Contagem de Células , Diferenciação Celular , Separação Celular , Meios de Cultura Livres de Soro , HumanosRESUMO
Reduced leptin (Ob protein) signaling is proposed to be a stimulus for the activation of neuropeptide Y (NPY) gene activity and increased expression of mRNA for the long form of the leptin receptor (Ob-Rb) in the hypothalamic arcuate nucleus. To determine if Ob-Rb protein is expressed in arcuate nucleus NPY neurons, we developed an affinity-purified polyclonal antibody against amino acids 956-1102 of human Ob-Rb. This antibody specifically recognizes the cytoplasmic tail of Ob-Rb and does not react with shorter leptin-receptor variants. Western immunoblots of Ob-Rb-transfected COS cells showed a single 150-kD band, and immunofluorescence revealed intense perinuclear staining in the cytoplasm. A 150-kD band was also present in Western immunoblots of hypothalamus. Immunocytochemical staining of brain slices revealed immunoreactive Ob-Rb protein concentrated in many neuronal cell bodies in the same regions of the forebrain that also express Ob-Rb mRNA. In the hypothalamus, Ob-Rb-positive cell bodies were abundant in the arcuate nucleus and ventromedial nucleus, with lesser numbers in the dorsomedial nucleus and paraventricular nucleus. Immunostaining was also detected in cell bodies of pyramidal cell neurons of the pyriform cortex and cerebral cortex, in neurons of the thalamus, and on the surface of ependymal cells lining the third ventricle. The choroid plexus, which expresses the short Ob-Ra form, was negative. Combined immunocytochemistry for Ob-Rb protein and fluorescence in situ hybridization for NPY mRNA identified arcuate nucleus neurons containing both NPY mRNA and Ob-Rb protein. The present finding of Ob-Rb protein in neurons that express NPY mRNA supports the hypothesis that arcuate nucleus NPY neurons are direct targets of leptin and play an important role in regulation of food intake and body weight.
Assuntos
Núcleo Arqueado do Hipotálamo/metabolismo , Proteínas de Transporte/biossíntese , Neurônios/metabolismo , Neuropeptídeo Y/genética , Receptores de Superfície Celular , Processamento Alternativo , Animais , Anticorpos/metabolismo , Western Blotting , Células COS , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Hipotálamo/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Fígado/metabolismo , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores para LeptinaRESUMO
A Plasmodium falciparum glycophorin binding protein (GBP-130) has been implicated in protective immunity to malaria. The gene for GBP-130 encodes a protein containing 11 tandemly repetitive 50 amino acid units. We report an immunization trial in Aotus monkeys using a recombinant DNA protein containing three of these 50 amino acid repeats. When administered with aluminum hydroxide, this antigen induced low levels of antibodies that reacted with the recombinant protein by ELISA and with parasite antigens in immunoblot and immunofluorescence assays, but not by immunoprecipitation. When administered with Freund's complete adjuvant, this antigen induced high levels of antibodies that reacted in ELISA, immunoblot, immunofluorescence, and immunoprecipitation assays. Serum from immunized monkeys did not inhibit parasite growth, and protection from intravenous challenge with P. falciparum-infected erythrocytes was not observed in any experimental group. These results suggest that the repetitive region of GBP-130 is not a useful vaccine candidate.
Assuntos
Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/uso terapêutico , Proteínas Recombinantes/imunologia , Sequência de Aminoácidos , Animais , Aotidae/imunologia , Aotidae/parasitologia , Epitopos/imunologia , Imunização , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas de Protozoários/imunologia , Coelhos , Sequências Repetitivas de Ácido NucleicoRESUMO
PURPOSE: To determine whether color Doppler sonography can be a sensitive alternative to screening arteriography for identifying arterial injury in patients with penetrating traumatic neck injuries. METHODS: Fifty-two patients admitted to our trauma center with penetrating neck injuries (gunshot wounds and lacerations) were examined prospectively with color Doppler sonography, and findings were compared with the results of angiography (n = 44), with findings at surgery (n = 4), and with clinical status (n = 4). RESULTS: Color Doppler sonography correctly detected all serious injuries of the carotid arteries (n = 6; 5 diagnosed at angiography and 1 at surgery) and all injuries of the vertebral arteries (n = 4; all diagnosed at angiography). Sonography missed 1 instance of reversible narrowing of the internal and external carotid arteries and did not show 2 normal vertebral arteries. CONCLUSION: Color Doppler sonography was as accurate as angiography in screening clinically stable patients with zone II or III injuries and no signs of active bleeding. Our initial results suggest that in the future, sonography may be used as a screening examination for arterial lesions in patients with penetrating neck injuries.
Assuntos
Lesões do Pescoço , Ultrassonografia Doppler em Cores , Ferimentos Penetrantes/diagnóstico por imagem , Adolescente , Adulto , Angiografia , Artérias/diagnóstico por imagem , Artérias/lesões , Artérias/cirurgia , Artérias Carótidas/diagnóstico por imagem , Artérias Carótidas/cirurgia , Lesões das Artérias Carótidas , Artéria Carótida Externa/diagnóstico por imagem , Artéria Carótida Interna/diagnóstico por imagem , Estenose das Carótidas/diagnóstico por imagem , Feminino , Hemorragia/diagnóstico por imagem , Humanos , Masculino , Pessoa de Meia-Idade , Pescoço/irrigação sanguínea , Pescoço/diagnóstico por imagem , Estudos Prospectivos , Artéria Vertebral/diagnóstico por imagem , Artéria Vertebral/lesões , Artéria Vertebral/cirurgia , Ferimentos por Arma de Fogo/diagnóstico por imagem , Ferimentos por Arma de Fogo/cirurgia , Ferimentos Penetrantes/cirurgiaRESUMO
PURPOSE: To show that postgadolinium three-dimensional time-of-flight MR angiography shows abnormal intradural vessels associated with spinal dural arteriovenous fistula better than routine MR imaging and provides screening information useful for subsequent diagnostic conventional angiography and/or posttreatment evaluation. METHODS: Precontrast and postcontrast MR imaging and MR angiograms, as well as subsequent digital subtraction angiograms, were obtained for eight patients with dural arteriovenous fistulas, diagnosed with digital subtraction angiography and verified with surgery. In four patients, MR studies also were obtained after surgery. RESULTS: All patients had cord hyperintensity of T2-weighted images and postgadolinium enhancement on T1-weighted images. Five had vessellike signal abnormalities in the subarachnoid space on MR. Abnormal intradural vessels were detected in all eight patients with MR angiography. Comparison with digital subtraction angiography revealed these vessels to be primarily enlarged veins of the coronal venous plexus on the cord surface. In six patients, the medullary vein draining the fistula was demonstrated, indicating the level of the fistula, later identified by digital subtraction angiography. After surgical obliteration of the fistula, the draining medullary vein and most or all of the abnormal coronal veins were no longer demonstrated, with decrease or resolution of cord hyperintensity on T2-weighted images. CONCLUSION: Postgadolinium, spinal MR angiography in cases of suspected dural arteriovenous fistula provides information about intradural veins that supplements the diagnostic value of the MR imaging results, facilitates the subsequent digital subtraction angiography study, and, in treated cases, reflects the success of surgery and/or embolization.
Assuntos
Fístula Arteriovenosa/diagnóstico , Dura-Máter/irrigação sanguínea , Angiografia por Ressonância Magnética , Medula Espinal/irrigação sanguínea , Idoso , Angiografia Digital , Artérias/patologia , Artérias/cirurgia , Fístula Arteriovenosa/patologia , Fístula Arteriovenosa/cirurgia , Meios de Contraste , Diagnóstico Diferencial , Combinação de Medicamentos , Feminino , Gadolínio DTPA , Humanos , Processamento de Imagem Assistida por Computador , Masculino , Meglumina , Pessoa de Meia-Idade , Exame Neurológico , Compostos Organometálicos , Ácido Pentético/análogos & derivados , Veias/patologia , Veias/cirurgiaRESUMO
This article discusses the utility and technique of magnetic resonance imaging and intraoperative spinal sonography in the evaluation of cystic and cavitary lesions of the spinal cord and canal. The pathophysiology and fluid dynamics of these lesions are also discussed.
Assuntos
Cistos/diagnóstico , Imageamento por Ressonância Magnética , Canal Medular/patologia , Doenças da Medula Espinal/diagnóstico , Medula Espinal/anormalidades , Cistos/congênito , Cistos/diagnóstico por imagem , Humanos , Canal Medular/diagnóstico por imagem , Medula Espinal/diagnóstico por imagem , Medula Espinal/patologia , Doenças da Medula Espinal/congênito , Doenças da Medula Espinal/diagnóstico por imagem , Neoplasias da Medula Espinal/diagnóstico , Neoplasias da Medula Espinal/diagnóstico por imagem , Tomografia Computadorizada por Raios X , UltrassonografiaRESUMO
A significant amount of progress has been achieved on characterizing the interaction of the IgE Fc molecule with the Fc epsilon RI alpha. However, there is yet no definitive structural information which precisely defines the nature of this interaction. It is clear that this information will only be provided by the resolution of the X-ray crystallographic structures of the IgE Fc molecule, the Fc epsilon RI alpha subunit extracellular domain, and the IgE Fc-Fc epsilon RI alpha complex. It is anticipated that these structures will be determined in the near future, and that they may provide some insight into the development of potential therapeutics effective in the management of IgE-mediated allergic diseases.
Assuntos
Imunoglobulina E/metabolismo , Receptores de IgE/metabolismo , Animais , Basófilos/imunologia , Sítios de Ligação , Células CHO , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Humanos , Mastócitos/imunologia , Camundongos , Modelos Moleculares , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Ratos , Receptores de IgE/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Using conventional anatomical methods, renal arteries of 140 human fetuses were studied. It was found (21.1%) that the accessory renal arteries occurred in a three-fold manner: 1. as single arteries (19.2%), 2. as double arteries (2.1%) and 3. as triplex arteries (0.7%). More often they originated from the right part of the circumference of the abdominal aorta, mainly in the female fetuses. These arteries penetrated the following segments of the kidney: the inferior (12.9%), the superior (2.3%), the anterior inferior (2.8%), the posterior (2.1%) and the anterior superior (1.5%). They crossed the renal pelvis more often in front (12.2%) than from behind of it (5%). The frequency of the occurrence of the accessory arteries depends not from the age of the fetus.
Assuntos
Artéria Renal/embriologia , Aorta Abdominal/anatomia & histologia , Aorta Abdominal/embriologia , Feminino , Feto , Idade Gestacional , Humanos , Masculino , Artéria Renal/anatomia & histologiaRESUMO
Protein-protein interactions are often dependent on the post-translational modification of one component of a complex. To facilitate the study of these interactions in signal transduction, we have developed the yeast tribrid system, a modification of the yeast two-hybrid system. We demonstrate that the interactions are dependent upon the presence of a tyrosine kinase, an SH2 domain and a tyrosine containing substrate. Using the gamma subunit of the high-affinity IgE receptor, Fc epsilon RI, this approach has been used to isolate a novel SH2-containing family member. The mRNA encoding this novel protein is differentially expressed in rat tissues. The yeast tribrid system can be readily adapted for the characterization of novel tyrosine kinases or substrates, as well as the study of protein-protein interactions which involve other post-translational modifications.
Assuntos
Fosfotirosina/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores de IgE/metabolismo , Saccharomyces cerevisiae/genética , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Sequência de Bases , Proteínas de Transporte/genética , Clonagem Molecular , DNA Complementar , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Galactose/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , Fosforilação , Plasmídeos/genética , Regiões Promotoras Genéticas , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Ratos , Receptores de IgE/genética , Proteínas Recombinantes de Fusão , Serina Endopeptidases/genética , Transdução de Sinais , Quinase Syk , beta-Galactosidase/genéticaRESUMO
Phosphatidylinositol-glycan-specific phospholipase D (PI-G PLD) specifically hydrolyzes the inositol-phosphate linkage in phosphatidylinositol-glycan (PI-G) anchored proteins. We recently deduced the primary structure of this enzyme and demonstrated specific enzymatic activity in transfected cells. Co-transfection of PI-G PLD with a natural PI-G anchored protein resulted in the secretion of the PI-G anchored protein via a PI-G PLD specific mechanism. We have taken advantage of these observations to develop an alternative system that may be useful for expressing and secreting proteins not amenable to secretion by conventional methods. Chimeric PI-G anchored proteins were constructed by transferring the COOH-terminal signal peptide for PI-G anchor attachment from placental alkaline phosphatase or from the low affinity IgG receptor, FcGRIIIB, to proteins that are not normally PI-G anchored. This process facilitates the cell surface expression of several proteins including the high affinity IgE receptor alpha subunit, FcERI alpha, which otherwise requires at least one other subunit for surface expression. Co-expression of these chimeric PI-G anchored proteins with PI-G PLD resulted in their secretion via a PI-G PLD specific mechanism.