RESUMO
BACKGROUND: Many parallels exist between growth and development of the placenta and that of cancer. One parallel is shared expression of antigens that may have functional importance and may be recognized by the immune system. Here, we characterize expression and regulation of one such antigen, Trophoblast glycoprotein (TPGB; also called 5T4), in the placenta across gestation, in placentas of preeclamptic (PE) pregnancies, and in purified microvesicles and exosomes. METHODS: Trophoblast glycoprotein expression was analyzed by real-time reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunohistochemistry. Regulation of 5T4 in cytotrophoblast cells was examined under either differentiating conditions of epidermal growth factor or under varying oxygen conditions. Microvesicles and exosomes were purified from supernatant of cultured and perfused placentas. RESULTS: Trophoblast glycoprotein expression was prominent at the microvillus surface of syncytiotrophoblast and on the extravillous trophoblast cells, with minimal expression in undifferentiated cytotrophoblasts and normal tissues. Trophoblast glycoprotein expression was elevated in malignant tumors. In cytotrophoblasts, 5T4 was induced by in vitro differentiation, and its messenger RNA (mRNA) was increased under conditions of low oxygen. PE placentas expressed higher 5T4 mRNA than matched control placentas. Trophoblast glycoprotein was prominent within shed placental microvesicles and exosomes. CONCLUSION: Given the potential functional and known immunological importance of 5T4 in cancer, these studies reveal a class of proteins that may influence placental development and/or sensitize the maternal immune system. In extravillous trophoblasts, 5T4 may function in epithelial-to-mesenchymal transition during placentation. The role of syncytiotrophoblast 5T4 is unknown, but its abundance in shed syncytial vesicles may signify route of sensitization of the maternal immune system.
Assuntos
Exossomos/metabolismo , Vesículas Extracelulares/metabolismo , Glicoproteínas de Membrana/metabolismo , Placenta/metabolismo , Trofoblastos/metabolismo , Diferenciação Celular , Feminino , Humanos , Glicoproteínas de Membrana/genética , Placentação/fisiologia , Gravidez , Primeiro Trimestre da Gravidez/metabolismo , Segundo Trimestre da Gravidez/metabolismoRESUMO
BACKGROUND: Composite tissue allotransplantation (CTA) may restore a variety of tissue defects, but carries the potential risks of graft failure and/or immunosuppression-related complications. Ischemia-reperfusion injury has been documented in CTA is known to contribute to acute rejection of solid organ grafts. This study describes the influence of subcritical ischemic time (ie, ischemia sufficient to generate reversible cell damage) on signs of rejection of musculocutaneous allograft components of subcritical ischemic time, namely, ischemia sufficient to generate reversible cell injury. Although skin is considered the most antigenic component of a composite allograft and is currently used for rejection surveillance, muscle and adipose are more susceptible to ischemia-related injury. METHODS: Vascularized epigastric flaps were transplanted from WKY to Fisher 344 rats after 1 or 3 hours of ischemia. Biopsies taken on postoperative day 6 were graded for signs of acute rejection according to criteria modified from previously published grading systems for CTA rejection. RESULTS: Skin and muscle exposed to 3 hours of ischemia showed significantly higher rejection scores than after 1 hour of ischemia, as evidenced by a more aggressive diffuse lymphocytic infiltration with disruption of tissue architecture. The rejection score in skin with 3-hour ischemia was 5.0 +/- 0.1 versus 3.7 +/- 0.2 with 1-hour (Mann-Whitney U test; P < .05). The rejection score in muscle exposed to 3-hour ischemia was 3.6 +/- 0.3 versus 2.5 +/- 0.1 with 1-hour (P < .05). CONCLUSIONS: Muscle and skin demonstrated increased acute rejection of allotransplants with increased subcritical ischemic time. This study supports the use of aggressive methods to reduce subcritical ischemic injury during allotransplantation of composite tissue and inclusion of muscle in postoperative biopsies in this early investigational period of CTA.
Assuntos
Rejeição de Enxerto/patologia , Músculo Esquelético/transplante , Transplante de Pele/patologia , Transplante de Tecidos/patologia , Transplante Homólogo/patologia , Tecido Adiposo/patologia , Tecido Adiposo/transplante , Animais , Isquemia/patologia , Masculino , Modelos Animais , Músculo Esquelético/patologia , Ratos , Ratos Endogâmicos F344 , Ratos Endogâmicos WKY , Traumatismo por Reperfusão/patologia , Pele/patologiaRESUMO
The G12 subfamily of heterotrimeric G proteins, comprised of the alpha-subunits Galpha12 and Galpha13, has been implicated as a signaling component in cellular processes ranging from cytoskeletal changes to cell growth and oncogenesis. In an attempt to elucidate specific roles of this subfamily in cell regulation, we sought to identify molecular targets of Galpha12. Here we show a specific interaction between the G12 subfamily and the cytoplasmic tails of several members of the cadherin family of cell-surface adhesion proteins. Galpha12 or Galpha13 binding causes dissociation of the transcriptional activator beta-catenin from cadherins. Furthermore, in cells lacking the adenomatous polyposis coli protein required for beta-catenin degradation, expression of mutationally activated Galpha12 or Galpha13 causes an increase in beta-catenin-mediated transcriptional activation. These findings provide a potential molecular mechanism for the previously reported cellular transforming ability of the G12 subfamily and reveal a link between heterotrimeric G proteins and cellular processes controlling growth and differentiation.
Assuntos
Caderinas/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Transdução de Sinais/fisiologia , Transativadores , Adenocarcinoma/genética , Adenocarcinoma/patologia , Proteína da Polipose Adenomatosa do Colo , Caderinas/química , Adesão Celular , Linhagem Celular , Núcleo Celular/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Cisteína Endopeptidases/metabolismo , Citoplasma/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP , Genes APC , Proteínas Heterotriméricas de Ligação ao GTP/genética , Humanos , Rim , Complexos Multienzimáticos/metabolismo , Complexo de Endopeptidases do Proteassoma , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Transcrição Gênica , Células Tumorais Cultivadas , Ubiquitinas/metabolismo , beta CateninaRESUMO
Monitoring physiological changes in the brain parenchyma has important applications in the care of neurosurgical patients. A technique is described for measuring extracellular neurochemicals by cerebral microdialysis with simultaneous recording of electroencephalographic (EEG) and single-unit (neuron) activity in selected targets in the human brain. Forty-two patients with medically intractable epilepsy underwent stereotactically guided implantation of a total of 423 intracranial depth electrodes to delineate potentially resectable seizure foci. The electrodes had platinum alloy contacts for EEG recordings and four to nine 40-microm microwires for recording single-unit neuron activity. Eighty-six electrodes also included microdialysis probes introduced via the electrode lumens. During monitoring on the neurosurgical ward, electrophysiological recording and cerebral microdialysis sampling were performed during seizures, cognitive tasks, and sleep-waking cycles. The technique described here could be used in developing novel approaches for evaluation and treatment in a variety of neurological conditions such as head injury, subarachnoid hemorrhage, epilepsy, and movement disorders.
Assuntos
Encéfalo/fisiopatologia , Eletroencefalografia , Epilepsia/fisiopatologia , Epilepsia/cirurgia , Neurônios/fisiologia , Adolescente , Adulto , Aminoácidos/metabolismo , Encéfalo/metabolismo , Criança , Cognição , Eletrodos Implantados/efeitos adversos , Epilepsia/metabolismo , Feminino , Humanos , Masculino , Microdiálise , Pessoa de Meia-Idade , Técnicas EstereotáxicasRESUMO
Gz is a member of the Gi family of trimeric G proteins whose precise signalling function has not been defined. It can be distinguished from other members of the family by several interesting biochemical properties of its alpha subunit, Gzalpha. One particularly intriguing property is its extremely slow GTPase activity; its kcat for GTP hydrolysis is as much as 200-fold less than other Galpha's. Since there is evidence that cellular factors can accelerate the GTPase activities of Galpha subunits, we have suspected that cells expressing Gzalpha may contain a GTPase-activating protein, or GAP, that would enhance its hydrolytic ability. Using purified Gzalpha-GTP as a substrate, we have identified and characterized such a GAP that acts on Gzalpha, which we have termed Gz-GAP. The protein responsible for this activity is specific for Gzalpha and is found in the membrane fraction of bovine brain and in several other tissues that express Gz. Since G protein effectors are in many cases capable of stimulating the GTPases rate of Galpha subunits, we speculate that a novel effector for Gz is responsible for the activity.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP , Proteínas de Ligação ao GTP/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP , Proteínas/metabolismo , Animais , Bovinos , Linhagem Celular , Proteínas Ativadoras de GTPase , Guanosina Trifosfato/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , SpodopteraRESUMO
Pertussis toxin (PTX) has been widely used as a reagent to characterize the involvement of heterotrimeric G-proteins in signalling. This toxin catalyses the ADP-ribosylation of specific G-protein alpha subunits of the Gi family, and this modification prevents the occurrence of the receptor-G-protein interaction. This review focuses on the biochemical properties and signalling of those G-proteins historically classified as 'PTX-resistant' due to the inability of the toxin to influence signalling through them. These G-proteins include members of the Gq and G12 families and one Gi family member, i.e. Gz. Signalling pathways controlled by these G-proteins are well characterized only for Gq family members, which activate specific isoforms of phospholipase C, resulting in increases in intracellular calcium and activation of protein kinase C (PKC), among other responses. While members of the G12 family have been implicated in processes that regulate cell growth, and Gz has been shown to inhibit adenylate cyclase, the specific downstream targets to these G-proteins in vivo have not been clearly established. Since two of these proteins, G12 alpha and Gz alpha, are excellent substrates for PKC, there is the potential for cross-talk between their signalling and Gq-dependent processes leading to activation of PKC. In tissues that express these G-proteins, a number of guanine-nucleotide-dependent, PTX-resistant, signalling pathways have been defined for which the G-protein involved has not been identified. This review summarizes these pathways and discusses the evidence both for the participation of specific PTX-resistant G-proteins in them and for the regulation of these processes by PKC.
Assuntos
Toxina Adenilato Ciclase , Proteínas de Ligação ao GTP/metabolismo , Toxina Pertussis , Transdução de Sinais/fisiologia , Fatores de Virulência de Bordetella/farmacologia , Animais , Proteínas de Ligação ao GTP/química , Mamíferos/metabolismo , Modelos Biológicos , Neurônios/metabolismo , Receptores de Superfície Celular/metabolismo , Alinhamento de SequênciaRESUMO
Polypeptides that define a protein family termed RGS (for regulators of G-protein signalling) are encoded by the SST2 gene of the yeast Saccharomyces cerevisiae, the EGL-10 gene of the nematode Caenorhabdatis elegans, and several related mammalian genes. Genetic studies in invertebrates and mammalian cell-transfection experiments indicate that RGS proteins negatively regulate signalling pathways involving seven transmembrane receptors and heterotrimeric G proteins. However, the biochemical mechanism by which RGS proteins control these pathways is unknown. Here we report the characterization of human RGS10, a member of this protein family. Co-immunoprecipitation studies demonstrate that RGS10 associates specifically with the activated forms of two related G-protein subunits, G alphai3, and G alphaz, but fails to interact with the structurally and functionally distinct G alphas subunit. In vitro assays with purified proteins indicate that RGS10 increases potently and selectively the GTP hydrolytic activity of several members of the G alphai family, including G alphai3, G alphaz, and G alpha0. These results demonstrate that RGS proteins can attenuate signalling pathways involving heterotrimeric G proteins by serving as GTPase-activating proteins for specific types of G alpha subunits.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas/metabolismo , Proteínas RGS , Sequência de Aminoácidos , Animais , Clonagem Molecular , Ativação Enzimática , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Hidrólise , Dados de Sequência Molecular , Testes de Precipitina , Ratos , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
Gz alpha is a G protein alpha subunit with biochemical properties that distinguish it from other members of the G protein alpha subunit family. One such property is its ability to be stoichiometrically phosphorylated by protein kinase C (PKC), both in vitro and in intact cells. The site of this phosphorylation has been mapped to a region near the N terminus of Gz alpha, but no functional significance of the modification has been established. To investigate this question, we have developed a baculovirus/Sf9 cell expression system to produce Gz alpha. The protein purified from Sf9 cells is functional as assessed by its ability both to bind guanine nucleotide in a Mg(2+)-sensitive fashion and to serve as a substrate for phosphorylation by PKC. Furthermore, addition of the G protein beta gamma complex purified from bovine brain inhibits phosphorylation of Gz alpha in a dose-dependent manner. Conversely, phosphorylation of Gz alpha inhibits its ability to interact with beta gamma subunits. These results establish a functional consequence for PKC-catalyzed phosphorylation of Gz alpha and suggest a mechanism for regulation of signaling through Gz by preventing reassociation of its subunits.
Assuntos
Encéfalo/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteína Quinase C/metabolismo , Animais , Baculoviridae , Bovinos , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/isolamento & purificação , Cinética , Substâncias Macromoleculares , Fosforilação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Spodoptera , TransfecçãoRESUMO
Azidoanilido-GTP (AA-GTP), a hydrolysis-resistant, photoreactive GTP analog, is becoming an increasingly popular tool for identifying activation of specific G proteins by receptors within native plasma membranes. Despite the use of AA-GTP as an affinity probe, surprisingly little is known regarding the ability of various G protein alpha subunits to bind this analog. To directly address this issue, we compared the ability of four purified G protein alpha subunits (Go, Gi2, Gs, and Gz) to bind AA-GTP with their ability to bind GTP gamma S, a GTP analog commonly used to characterize the GTP-binding properties of G proteins. All four G alpha subunits tested bound AA-GTP in a manner distinct from their binding of GTP gamma S. One of these proteins, Gs alpha, required millimolar levels of free Mg2+ for significant binding of AA-GTP, while Go alpha and Gi alpha 2 displayed peak AA-GTP binding at approximately 100 microM free Mg2+. The fourth G alpha subunit, Gz, bound AA-GTP very poorly relative to GTP gamma S regardless of the magnesium concentration. These results indicate that individual G protein alpha subunits differ markedly in their ability to bind AA-GTP. Use of AA-GTP to identify specific G protein-receptor interactions must therefore take into account the varied abilities of G alpha subunits to bind this analog.
Assuntos
Marcadores de Afinidade/metabolismo , Azidas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/análogos & derivados , Marcadores de Afinidade/síntese química , Animais , Azidas/síntese química , Bovinos , Guanosina Trifosfato/síntese química , Guanosina Trifosfato/metabolismo , Técnicas In Vitro , Magnésio/fisiologia , Fragmentos de Peptídeos/metabolismo , Fotoquímica , Ligação Proteica , Proteínas Recombinantes/metabolismoRESUMO
Plasma membranes from Chinese hamster ovary (CHO) cells transfected with the serotonin 5-HT1A receptor have been incubated with full or partial receptor agonists and the photoreactive GTP analog, 4-azidoanilido-[alpha-32P]-GTP ([32P]-AA-GTP), to characterize the resulting receptor-G-protein interactions. Subsequent solubilization and immunoprecipitation of the membranes with anti-G(i)alpha-2 or anti-G(i)alpha-3 immunoglobulins revealed that full and partial agonists produce concentration-dependent labeling of the respective G-proteins with [32P]-AA-GTP. Full agonists of the 5-HT1A receptor [serotonin 5-hydroxytryptamine (5-HT) and 8-hydroxy-2-(di-n-propylamino)tetraline (8-OH-DPAT)] produced a 7-12-fold increase in the labeling of G(i)alpha-2 and G(i)alpha-3, whereas partial agonists (rauwolscine and ipsapirone) produced a smaller incorporation (2-5-fold) of [32P]-AA-GTP by the same G-proteins. The concentration of agonist producing half-maximal binding of [32P]-AA-GTP by G(i)alpha-3 [5-HT, 48 +/- 1 nM; 8-OH-DPAT, 28 +/- 1 nM; ipsapirone, 22 +/- 6 nM] compared to G(i)alpha-2 [5-HT, 124 +/- 38 nM; 8-OH-DPAT, 40 +/- 1 nM, ipsapirone, 82 +/- 7 nM] was lower with all agonists except rauwolscine, where the EC50's were similar (G(i)alpha-2, 604 +/- 145 nM; Gi alpha-3, 708 +/- 130 nM).(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Agonistas do Receptor de Serotonina/farmacologia , Inibidores de Adenilil Ciclases , Marcadores de Afinidade , Animais , Azidas , Células CHO , Cricetinae , Guanosina Trifosfato/análogos & derivados , Células HeLa , Humanos , Fragmentos de Peptídeos/metabolismo , Radioisótopos de Fósforo , Testes de Precipitina , Pirimidinas/farmacologia , Ioimbina/farmacologiaRESUMO
A lambda gt11 expression library containing cDNA from total chick embryo was screened with S103L, a rat monoclonal antibody which reacts specifically with the core protein of the chick cartilage chondroitin sulfate proteoglycan. One clone was identified which produced a 220-kDa beta-galactosidase/S103L-binding fusion protein. Sequencing the entire 1.5-kilobase cDNA insert showed that it contained a single open reading frame, which encoded a portion of the proteoglycan core protein from the chondroitin sulfate domain. This was confirmed by comparison with amino acid sequence data from peptide CS-B, which was derived from the chondroitin sulfate domain (Krueger, R.C., Jr., Fields, T. A., Hildreth, J., IV, and Schwartz, N.B. (1990) J. Biol. Chem. 265, 12075-12087). Furthermore, the 3' end of the insert overlapped with 23 bases at the 5' end of the published sequence for the C-terminal globular domain (Sai, S., Tanaka, T., Kosher, R. A., and Tanzer, M. L. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 5081-5085), which oriented this clone, as well as the CS peptide, along the protein core. The cDNA insert hybridized with a 9-kilobase mRNA from sternal chondrocytes as well as a similar sized message in brain but did not hybridize to any message from rat chondrosarcoma or from undifferentiated limb bud mesenchyme. In further studies, the fusion protein as well as a cyanogen bromide fragment (70 kDa) derived from it were isolated and shown to react with S103L, indicating that cleavage at methionine residues does not disrupt the antibody recognition site. Purification and N-terminal sequencing of the antigenic CNBr fragment derived from the fusion protein revealed that its N terminus is preceded by a methionine in the fusion protein and overlaps with the N terminus of peptide CS-B. As peptide CS-B is not recognized by S103L and the C terminus of peptide CS-B lies beyond the proteoglycan portion of the antigenic CNBr fragment, the S103L epitope is either contained within the 11 amino acids preceding the N terminus of peptide CS-B or it spans the clostripain cleavage site at the origin of the N terminus of peptide CS-B.
Assuntos
Cartilagem/metabolismo , Epitopos/genética , Proteínas da Matriz Extracelular , Glicoproteínas/genética , Proteoglicanas , Agrecanas , Sequência de Aminoácidos , Animais , Sequência de Bases , Embrião de Galinha , Galinhas , Proteoglicanas de Sulfatos de Condroitina/genética , Clonagem Molecular , Brometo de Cianogênio , DNA/genética , Epitopos/análise , Biblioteca Gênica , Glicoproteínas/imunologia , Lectinas Tipo C , Modelos Estruturais , Dados de Sequência Molecular , Mapeamento de Peptídeos , Proteínas Recombinantes de Fusão/imunologia , Mapeamento por Restrição , Homologia de Sequência do Ácido NucleicoRESUMO
Peptides were derived from the large chondroitin sulfate proteoglycan from chick cartilage by clostripain digestion. Using differential chondroitinase ABC and keratanase treatment and direct carbohydrate analysis, three major peptides of 86, 75, and 27 kDa were shown to bear only chondroitin sulfate chains. Another major peptide of 65 kDa was shown to contain both chondroitin sulfate and keratan sulfate chains, allowing it to be separated from the peptides derived from the chondroitin sulfate domain by DEAE-cellulose chromatography. An additional new peptide (100 kDa) containing keratan sulfate chains was found only in clostripain digests of proteoglycan-hyaluronate-link protein aggregates. Unlike any of the other peptides derived from clostripain digestion of proteoglycan monomer or aggregate, this peptide had the properties of a functional hyaluronate binding region. All of these peptides were purified to apparent homogeneity by preparative electroelution from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and deglycosylated with anhydrous hydrogen fluoride. Automated Edman degradation of the two largest chondroitin sulfate peptides revealed that they had unique N termini and several unrecognized residues, which were all subsequently revealed to be modified serine residues following deglycosylation. The keratan sulfate-bearing peptide also had a unique N terminus, which contained a single unrecognized residue, even after HF deglycosylation. Finally, the N terminus of the hyaluronate binding region was blocked. These studies allow estimates of core peptide masses in the absence of carbohydrate as well as provide primary amino acid sequence for O-xylosylated serine residues in the multiply substituted proteoglycans.
Assuntos
Cartilagem/metabolismo , Proteínas da Matriz Extracelular , Glicoproteínas/metabolismo , Glicosaminoglicanos/metabolismo , Proteoglicanas , Agrecanas , Sequência de Aminoácidos , Animais , Sítios de Ligação , Embrião de Galinha , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Lectinas Tipo C , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos/isolamento & purificação , Conformação ProteicaRESUMO
Expressive language samples were obtained from normal and language-disordered children through standard interview procedures and through wireless radiotelemetry. Comparisons of these samples using Developmental Sentence Scoring (DSS), mean length of utterance (MLU), and inflectional scoring indicated significant differences between the groups of children, and among the interview-telemetry samples. Telemetry samples yielded different overall scores from those obtained with the interview samples, and there were significant differences in the types of language performance within the samples. Reliable telemetry scores across days were obtained. Difficulties in using the equipment are described and the procedures are compared.
Assuntos
Transtornos do Desenvolvimento da Linguagem/diagnóstico , Transtornos da Linguagem/diagnóstico , Telemetria/métodos , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Rádio , Fatores de Tempo , Comportamento VerbalRESUMO
To evaluate the effect of elicitation stimuli and situations, expressive language samples were obtained from 10 normal children and 10 language-disordered children in four location-stimulus combinations and from wireless radio telemetry. These language samples were analyzed using the procedures of Developmental Sentence Scoring, Mean Length of Utterance, and inflectional scoring. No differences existed between the locations of home and clinic, but significant differences were noted among samples elicited using pictures as oppossed to open-ended questions as compared to telemetry samples. Normal and language-disordered children responded differently to these elicitation variables. Open-ended questions and telemetry yielded the highest performances on Developmental Sentence Scoring and inflectional scoring. Telemetry samples yielded the shortest response lengths. The two groups of children also responded differently in the specific scoring categories.