RESUMO
INTRODUCTION: Celiac disease (CD) is common, affecting approximately 1% of the population. The cornerstone of management is a gluten-free diet, with dietetic advice being the key to aiding implementation. The aim of the study was to assess group clinics in comparison with traditional individual appointments. METHODS: Patients with a new diagnosis of CD, confirmed histologically, were prospectively recruited over 18 months in Sheffield, United Kingdom. Patients received either a group clinic or traditional one-to-one appointment, led by a dietitian. Quality-of-life questionnaires were completed at baseline, as well as biochemical parameters being recorded. Patients were followed up at 3 months, where adherence scores were assessed as well as biochemical parameters and quality of life questionnaires being completed. RESULTS: Sixty patients with CD were prospectively recruited and received either an individual (n = 30) or group clinic (n = 30). A statistically significant reduction in tissue transglutaminase was noted following group clinics (mean 58.5, SD 43.4 U/mL vs mean 13.2, SD 5.7 U/mL, P < 0.01). No significant differences in baseline and follow-up biochemical parameters between one-to-one and group clinics were noted. At follow-up, there was no statistically significant difference between mean gluten-free diet adherence scores (mean 3.1, SD 0.4 vs mean 3.1, SD 0.7, P = 0.66) between one-to-one and group clinics. DISCUSSION: This first study assessing group clinics in CD demonstrates they are as effective as traditional one-to-one clinics, with the added benefits of peer support and greater efficiency, with an estimated 54% reduction of dietetic resources.
Assuntos
Doença Celíaca/dietoterapia , Nutricionistas , Grupo Associado , Consultas Médicas Compartilhadas , Apoio Social , Cooperação e Adesão ao Tratamento/estatística & dados numéricos , Adulto , Idoso , Ansiedade/psicologia , Doença Celíaca/imunologia , Doença Celíaca/fisiopatologia , Doença Celíaca/psicologia , Depressão/psicologia , Feminino , Proteínas de Ligação ao GTP/imunologia , Humanos , Imunoglobulina A/imunologia , Masculino , Pessoa de Meia-Idade , Proteína 2 Glutamina gama-Glutamiltransferase , Qualidade de Vida , Transglutaminases/imunologia , Resultado do Tratamento , Reino UnidoAssuntos
Doença Celíaca/diagnóstico , Erros de Diagnóstico/prevenção & controle , Proteínas de Ligação ao GTP/sangue , Hemólise , Imunoensaio/normas , Imunoglobulina G/sangue , Transglutaminases/sangue , Anticorpos Antiproteína Citrulinada/sangue , Doença Celíaca/sangue , Doença Celíaca/imunologia , Erros de Diagnóstico/estatística & dados numéricos , Emulsões , Reações Falso-Negativas , Proteínas de Ligação ao GTP/imunologia , Gliadina/sangue , Gliadina/imunologia , Humanos , Hiperlipidemias/sangue , Hiperlipidemias/imunologia , Imunoglobulina A/sangue , Fosfolipídeos/sangue , Proteína 2 Glutamina gama-Glutamiltransferase , Óleo de Soja/sangue , Transglutaminases/imunologiaRESUMO
High-mobility group box 1 (HMGB1) is a chromatin-associated protein that, in response to stress or injury, translocates from the nucleus to the extracellular milieu, where it functions as an alarmin. HMGB1's function is in part determined by the complexes (HMGB1c) it forms with other molecules. However, structural modifications in the HMGB1 polypeptide that may regulate HMGB1c formation have not been previously described. In this report, we observed high-molecular weight, denaturing-resistant HMGB1c in the plasma and peripheral blood mononuclear cells of individuals with systemic lupus erythematosus (SLE) and, to a much lesser extent, in healthy subjects. Differential HMGB1c levels were also detected in mouse tissues and cultured cells, in which these complexes were induced by endotoxin or the immunological adjuvant alum. Of note, we found that HMGB1c formation is catalyzed by the protein-cross-linking enzyme transglutaminase-2 (TG2). Cross-link site mapping and MS analysis revealed that HMGB1 can be cross-linked to TG2 as well as a number of additional proteins, including human autoantigens. These findings have significant functional implications for studies of cellular stress responses and innate immunity in SLE and other autoimmune disease.
Assuntos
Autoantígenos/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteína HMGB1/metabolismo , Leucócitos Mononucleares/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Transglutaminases/metabolismo , Autoantígenos/imunologia , Células Cultivadas , Proteínas de Ligação ao GTP/imunologia , Proteína HMGB1/imunologia , Humanos , Leucócitos Mononucleares/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/patologia , Peso Molecular , Proteína 2 Glutamina gama-Glutamiltransferase , Especificidade por Substrato , Transglutaminases/imunologiaRESUMO
The total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase activity, respiratory bursts (release of superoxide anion), superoxide dismutase activity, and phagocytic activity and clearance efficiency to the pathogen Vibrio alginolyticus were measured when white shrimp, Litopenaeus vannamei, (7.5 ± 0.5 g) were individually injected with diethyl pyrocarbonate-water (DEPC-H2O) or different dsRNA at 3 days of injection. In addition, haemolymph glucose and lactate, and haemocytes crustacean hyperglycemic hormone (CHH), transglutaminase I (TGI), transglutaminase II (TGII) and clottable protein (CP) mRNA expression were determined for the shrimp that received DEPC-H2O and different dsRNA after 3 days, and then transferred to 22 and 28 °C from 28 °C. Results showed that respiratory burst, phagocytic activity and clearance efficiency significantly decreased, but hyaline cells significantly increased in the shrimp received LvTGII dsRNA after 3 days. In hypothermal stress studies, LvTGI and CHH were significantly up-regulated in LvTGII-depleted shrimp following exposure to 28 and 22 °C, and haemolymph glucose and lactate were significantly enhanced in LvTGII-depleted shrimp. The injection of LvTGII dsRNA also significantly increased the mortality of L. vannamei challenged with the pathogen V. alginolyticus. These results suggest that LvTGII is an important component on the immune resistance of shrimp, and is involved in the regulation of some immune parameters and carbohydrate metabolites, as well as has a complementary effect with LvTGI in immunological and physiological response of shrimp.
Assuntos
Proteínas de Ligação ao GTP/imunologia , Penaeidae/enzimologia , Penaeidae/imunologia , Transglutaminases/imunologia , Vibrio alginolyticus/imunologia , Análise de Variância , Animais , Contagem de Células Sanguíneas , Primers do DNA/genética , Hemócitos/fisiologia , Monofenol Mono-Oxigenase/metabolismo , Penaeidae/microbiologia , Fagocitose/imunologia , Proteína 2 Glutamina gama-Glutamiltransferase , Explosão Respiratória/imunologia , Superóxido Dismutase/metabolismoRESUMO
AIM: To examine the possible ameliorative effect of breastfeeding and the uptake of human colostrum against coeliac disease in autistic rats. METHODS: Female rats were fed a standard diet and received a single intraperitoneal injection of 600 mg/kg sodium valproate on day 12.5 after conception. In study 1, neonatal rats were randomly subjected to blood tests to investigate autism. In study 2, the 1(st) group was fed by the mother after an injection of interferon-γ (IFN-γ) and administration of gliadin. The pups in the 2(nd) group were prevented from accessing maternal milk, injected IFN-γ, administered gliadin, and hand-fed human colostrum. The normal littermates fed by the table mothers were injected with physiological saline and served as normal controls in this study. RESULTS: The protein concentration was higher in group 2 than in group 1 in the duodenum (161.6 ± 9 and 135.4 ± 7 mg/g of tissue, respectively, P < 0.01). A significant increase (P < 0.001) in body weight was detected in human colostrum-treated pups on post natal day (PND) 7 and 21 vs suckling pups in group 1. A delay in eye opening was noticed in the treated rats in group 1 on PND 13 compared with the control group and group 2. Administration of a single intraperitoneal injection of 600 mg/kg sodium valproate on day 12.5 after conception resulted in significantly reduced calcium and vitamin D levels in study 1 compared with the control groups (P < 0.001). However, human colostrum uptake inhibited increases in the level of transglutaminase antibody in autistic pups with coeliac disease. CONCLUSION: The effects of early-life nutrition and human colostrum on the functional maturation of the duodenal villi in autistic rats with coeliac disease that might limit or prevent the coeliac risk with autism.
Assuntos
Fenômenos Fisiológicos da Nutrição Animal , Transtorno Autístico/complicações , Alimentação com Mamadeira , Doença Celíaca/prevenção & controle , Colostro/metabolismo , Duodeno/metabolismo , Animais , Animais Lactentes , Transtorno Autístico/sangue , Transtorno Autístico/induzido quimicamente , Autoanticorpos/sangue , Biomarcadores/sangue , Cálcio/sangue , Doença Celíaca/sangue , Doença Celíaca/induzido quimicamente , Doença Celíaca/complicações , Doença Celíaca/imunologia , Doença Celíaca/patologia , Modelos Animais de Doenças , Duodeno/ultraestrutura , Feminino , Proteínas de Ligação ao GTP/imunologia , Idade Gestacional , Gliadina , Humanos , Interferon gama , Absorção Intestinal , Masculino , Estado Nutricional , Gravidez , Proteína 2 Glutamina gama-Glutamiltransferase , Ratos , Ratos Wistar , Transglutaminases/imunologia , Ácido Valproico , Vitamina D/análogos & derivados , Vitamina D/sangue , Aumento de PesoRESUMO
Interferon beta (IFNß) reduces disease burden in relapsing-remitting multiple sclerosis (MS) patients. In this study, IFNß-1b-treated MS patient gene expression profiles and biological knowledgebases were integrated to study IFNß's pleiotropic mechanisms of action. Genes involved in immune regulation, mitochondrial fatty acid metabolism and antioxidant activity were discovered. Plausible mediators of neuronal preservation included NRF2, downregulation of OLA1, an antioxidant suppressor, and the antioxidant gene ND6, implicated in optic neuropathy and MS-like lesions. Network analysis highlighted IKBKE, which likely has a role in both viral response and energy metabolism. A comparative analysis of therapy-naive MS- and IFNß-associated gene expression suggests an IFNß insufficiency in MS. We observed more gene expression changes in long-term treatment than during acute dosing. These distinct short- and long-term effects were driven by different transcription factors. Multi-gene biomarker signatures of IFNß treatment effects were developed and subsequently confirmed in independent IFNß-1b-treated MS studies, but not in glatiramer acetate-treated patients.
Assuntos
Adjuvantes Imunológicos/uso terapêutico , Interferon beta/uso terapêutico , Esclerose Múltipla/tratamento farmacológico , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/imunologia , Adenosina Trifosfatases/metabolismo , Adulto , Antioxidantes/metabolismo , Biomarcadores Farmacológicos/metabolismo , Regulação para Baixo , Ácidos Graxos/genética , Ácidos Graxos/imunologia , Ácidos Graxos/metabolismo , Feminino , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/imunologia , Proteínas de Ligação ao GTP/metabolismo , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/imunologia , Quinase I-kappa B/metabolismo , Interferon beta-1b , Interferon beta/imunologia , Masculino , Pessoa de Meia-Idade , Mitocôndrias/genética , Mitocôndrias/imunologia , Mitocôndrias/metabolismo , Esclerose Múltipla/genética , Esclerose Múltipla/imunologia , NADH Desidrogenase/genética , NADH Desidrogenase/imunologia , NADH Desidrogenase/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/imunologia , Fator 2 Relacionado a NF-E2/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismo , TranscriptomaRESUMO
BACKGROUND: Due to the restrictive nature of a gluten-free diet, celiac patients are looking for alternative therapies. While drug-development programs include gluten challenges, knowledge regarding the duration of gluten challenge and gluten dosage is insufficient.We challenged adult celiac patients with gluten with a view to assessing the amount needed to cause some small-bowel mucosal deterioration. METHODS: Twenty-five celiac disease adults were challenged with low (1-3 g) or moderate (3-5g) doses of gluten daily for 12 weeks. Symptoms, small-bowel morphology, densities of CD3+ intraepithelial lymphocytes (IELs) and celiac serology were determined. RESULTS: Both moderate and low amounts of gluten induced small-bowel morphological damage in 67% of celiac patients. Moderate gluten doses also triggered mucosal inflammation and more gastrointestinal symptoms leading to premature withdrawals in seven cases. In 22% of those who developed significant small- intestinal damage, symptoms remained absent. Celiac antibodies seroconverted in 43% of the patients. CONCLUSIONS: Low amounts of gluten can also cause significant mucosal deterioration in the majority of the patients. As there are always some celiac disease patients who will not respond within these conditions, sample sizes must be sufficiently large to attain to statistical power in analysis.
Assuntos
Doença Celíaca/patologia , Glutens/efeitos adversos , Mucosa Intestinal/patologia , Intestino Delgado/patologia , Adulto , Idoso , Anticorpos/sangue , Formação de Anticorpos , Autoanticorpos/análise , Biópsia , Doença Celíaca/complicações , Doença Celíaca/imunologia , Estudos de Coortes , Enterite/patologia , Ensaio de Imunoadsorção Enzimática , Feminino , Proteínas de Ligação ao GTP/imunologia , Glutens/administração & dosagem , Humanos , Imunoglobulina A/sangue , Imuno-Histoquímica , Mucosa Intestinal/imunologia , Intestino Delgado/imunologia , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Proteína 2 Glutamina gama-Glutamiltransferase , Transglutaminases/imunologia , Adulto JovemRESUMO
We tested two post-exposure prophylaxes (PEPs) for rabies in laboratory animals; one was a traditional antirabies vaccine for humans via intramuscular route (IM), and the other was a DNA vaccine administered by intranasal route (IN). In contrast to The World Health Organization's recommended five-dose PEP, we gave only four doses without hyper-immune antirabies sera, making the PEP more rigorous. All animals were challenged with challenge virus strain (CVS); 16h later, PEP was applied. All animals that received the PEP with DNA/IN survived, and 87% of the rabbits and 80% of the mice that received the PEP with traditional antirabies vaccine/IM survived. Negative controls succumbed to infection. The expression of G protein was detected in the NALT, cerebellum, cerebral cortex (neocortex), cerebellum and hippocampus, mainly in the glial cells (microglia) and microvessels. On the other hand, plasmid construct was detected in brain and its mRNA expression in medium and posterior encephalon. The efficiency of this DNA/IN PEP is probably due to the early expression of the antigen in the brain stimulating the immune system locally.
Assuntos
Vacina Antirrábica/uso terapêutico , Raiva/prevenção & controle , Administração Intranasal , Animais , Encéfalo/imunologia , Encéfalo/patologia , Encéfalo/virologia , Linhagem Celular , Corantes , Cricetinae , DNA Complementar/biossíntese , DNA Complementar/genética , Amarelo de Eosina-(YS) , Feminino , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/imunologia , Hematoxilina , Esquemas de Imunização , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Plasmídeos/genética , RNA Viral/genética , RNA Viral/imunologia , Coelhos , Vacina Antirrábica/administração & dosagem , Vacina Antirrábica/imunologia , Vírus da Raiva/genética , Vírus da Raiva/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Vacinas de DNA/uso terapêuticoRESUMO
Allogeneic mixed lymphocyte tumor cell cultures (MLTCs) were established using lymphocytes from non-small-cell lung cancer (NSCLC) patient UKY-53 and HLA-A2+ NSCLC tumor cells (UKY-29). The tumor cells expressed the lymphocyte costimulatory molecule CD80 (UKY29.7). Cytolytic activity showed the cytotoxic T-lymphocytes (CTL) lysed UKY-29, but not K562 or Daudi. The CTL also lysed: HLA-A2+ and -A24+ tumor cell lines from a number of tumor histologies. The CTL also lysed Epstein Barr virus transformed (EBV) B-cells, UKY-29EBV, autologous to the stimulating cell line, UKY29TC. These data suggested the presence of both tumor-specific and allogeneic reactivities in the bulk CTL population. Subsequent cDNA cloning analysis and sequencing demonstrated that the bulk CTL population was recognizing: (i) allogeneic target HLA-CW3, and two minor histocompatibility antigens; (ii) guanine nucleotide-binding protein, G(S) (GNAS), and (iii) inositol myophosphatase (IMPA). All three antigens, we believe, were restricted by HLA-A2. Whereas the system described was initially intended to identify tumor-associated antigens recognized by CTL, the nature of the allogeneic system provides a unique opportunity for the identification of epitopes that confer both allo and minor antigen recognition.
Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP/imunologia , Proteínas de Ligação ao GTP/imunologia , Biblioteca Gênica , Antígenos HLA-C/imunologia , Monoéster Fosfórico Hidrolases/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Cromograninas , DNA Complementar/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/genética , Antígeno HLA-A2/imunologia , Antígenos HLA-C/genética , Humanos , Teste de Cultura Mista de Linfócitos , Dados de Sequência Molecular , Monoéster Fosfórico Hidrolases/genética , RNA Mensageiro/genética , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Electroacupuncture (EA) has been used to treat Major Depressive Disorder (MDD). However, its efficacy is inconclusive and the mechanism is still unclear. Thus, the objective of this study is to investigate the therapeutic effect of EA on GTP-binding-protein (G protein) in platelet membrane using fluoxetine as a comparison. METHODS: A randomized controlled trial (RCT) was performed on 90 MDD patients, who were divided into three groups treated with fluoxetine, EA and sham EA respectively. Antibodies were utilized to quantify the levels of G protein alpha subtypes in the platelet membrane before and after 6-week anti-depressive treatment. Thirty age and sex-matched normal individuals were used as controls. RESULT: All the treatments had the same therapeutic effects in treating moderate depression. Both levels of Galphai and Galphaq in depression patients were significantly higher than those in controls and were not reduced by treatments, although the severity was considerably relieved. LIMITATIONS: The duration of treatment was limited to six weeks only. CONCLUSION: EA might be served as an alternative treatment for moderate depression and we further demonstrate that the abnormal levels of Galpha protein in platelet membrane might be a potential risk factor for MDD.
Assuntos
Transtorno Depressivo Maior/sangue , Transtorno Depressivo Maior/terapia , Eletroacupuntura/métodos , Fluoxetina/uso terapêutico , Proteínas de Ligação ao GTP/sangue , Glicoproteínas da Membrana de Plaquetas/metabolismo , Inibidores Seletivos de Recaptação de Serotonina/uso terapêutico , Adulto , Anticorpos/imunologia , Terapia Combinada , Transtorno Depressivo Maior/tratamento farmacológico , Feminino , Proteínas de Ligação ao GTP/imunologia , Proteínas de Ligação ao GTP/metabolismo , Humanos , Masculino , Glicoproteínas da Membrana de Plaquetas/imunologia , Índice de Gravidade de DoençaRESUMO
The nucleotide sequence of the full length HSD-3.8 cDNA (accession number AF311312), encoding a human sperm component, was determined to consist of 3818 bp with a reading frame of 2778 bp encoding a deduced polypeptide composed of 926 amino acids. A 0.7 kb fragment containing three immunological epitopes of HSD-3.8 cDNA was prepared and used to construct recombinant expression vectors. The constructs were transformed into E.coli BL-21, and the fusion proteins were expressed, isolated and purified. Using the polyclonal antibodies raised against the purified expressed fusion proteins, positive immunostaining occurred over the surface of the postacrosomal zone of human spermatozoa and of germ cells within the seminiferous epithelium of human testis. Intense staining of large pachytene primary spermatocytes occurred. The capacity of the recombinant protein to reduce fertility as an immunogen in adult female rats was assessed. Immunized animals were infertile or exhibited marked reduction in their fertility. Analysis of the deduced HSD-3.8 polypeptide revealed the presence of a tetratricopeptide repeat (TPR) motif, a P-loop sequence that acts as a binding site for ATP/GTP and phosphorylation sites for PKC, CK2 and cAMP/cGMP-dependent protein kinases. A blot overlay assay with [alpha-(32)P]GTP showed that the polypeptide encoded by the 0.7 kb fragment of HSD-3.8 is a GTP binding protein. It was also shown to possess GTPase activity and to be phosphorylated by PKC in vitro. In conclusion, HSD-3.8 is a GTP binding protein and its activity may be regulated by phosphorylation.
Assuntos
Proteínas de Ligação ao GTP/biossíntese , Proteínas de Ligação ao GTP/genética , Testículo/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Antígenos de Superfície , Ligação Competitiva , Western Blotting , DNA Complementar/isolamento & purificação , Feminino , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/imunologia , Proteínas de Ligação ao GTP/fisiologia , Guanosina Trifosfato/metabolismo , Humanos , Masculino , Dados de Sequência Molecular , Fosforilação , Coelhos , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Espermatozoides/química , Espermatozoides/fisiologia , SinaptofisinaRESUMO
To analyze the selectivity of delta receptor subtypes to regulate different classes of G proteins, the expression of the alpha-subunits of Gi2, Gi3, Go1, Go2, Gq and G11 transducer proteins was reduced by administration of oligodeoxynucleotides (ODNs) complementary to sequences in their respective mRNAs. Mice receiving antisense ODNs to Gi2 alpha, Gi3 alpha, Go2 alpha and G11 alpha subunits showed an impaired antinociceptive response to all the delta agonists evaluated. An ODN to Go1 alpha specifically blocked the antinociceptive effect of the agonist of delta-1 receptors, [D-Pen2,5]enkephalin (DPDPE), without altering the activity of [D-Ala2]deltorphin II or [D-Ser2]-Leu-enkephalin-Thr (DSLET). In mice treated with an ODN to Gq alpha, the effects of the agonists of delta-2-opioid receptors were reduced, but not those of DPDPE. Thus, Go1 proteins are selectively linked to delta-1-mediated analgesia, and Gq proteins are related to delta-2-evoked antinociception. After impairing the synthesis of Go1 alpha subunits, DPDPE exhibited an antagonistic activity on the antinociception produced by [D-Ala2]deltorphin II. After treatment with ODNs complementary to sequences in Gq alpha or PLC-beta 1 mRNAs, the analgesic capacity of [D-Ala2]deltorphin II was diminished. However, the delta-2-agonist did not alter the antinociceptive activity of DPDPE. An ODN complementary to nucleotides 7 to 26 of the murine delta receptor reduced the analgesic potency of [D-Ala2]deltorphin II, but not that observed for DPDPE. In these mice, [D-Ala2]deltorphin II did not antagonize the effect of DPDPE. These results suggest the existence of different molecular forms of the delta opioid receptor, and the involvement of inositol-signaling pathways in the supraspinal antinociceptive effects of delta agonists.
Assuntos
Analgesia , Proteínas de Ligação ao GTP/análise , Inositol/metabolismo , Receptores Opioides delta/fisiologia , Analgésicos Opioides/farmacologia , Animais , D-Penicilina (2,5)-Encefalina , Encefalinas/farmacologia , Proteínas de Ligação ao GTP/imunologia , Masculino , Camundongos , Oligonucleotídeos Antissenso/farmacologia , Receptores Opioides delta/classificação , Fosfolipases Tipo C/fisiologiaRESUMO
We have isolated from an American lobster (Homarus americanus) olfactory organ cDNA library a clone, hG alpha(q), with >80% identity to mammalian and arthropod G alpha(q) sequences. In brain and olfactory organ, hG alpha(q) mRNA was expressed predominantly in neurons, including virtually all the neuronal cell body clusters of the brain. G alpha(q) protein was also expressed broadly, appearing on western blots as a single band of 46 kDa in brain, eyestalk, pereiopod, dactyl, tail muscle, olfactory organ, and aesthetasc hairs. These results suggest that hG alpha(q) plays a role in a wide variety of signal transduction events. Its presence in the olfactory aesthetasc hairs, which are almost pure preparations of the outer dendrites of the olfactory receptor neurons, the expression of a single hG alpha(q) mRNA species (6 kb) in the olfactory organ, and the localization of hG alpha(q) mRNA predominantly in the olfactory receptor neurons of the olfactory organ strongly suggest that one function of hG alpha(q) is to mediate olfactory transduction.
Assuntos
Proteínas de Ligação ao GTP/genética , Nephropidae/genética , Neurônios Receptores Olfatórios/química , Animais , Especificidade de Anticorpos , Northern Blotting , Western Blotting , Química Encefálica/fisiologia , Clonagem Molecular , DNA Complementar , Proteínas de Ligação ao GTP/análise , Proteínas de Ligação ao GTP/imunologia , Gânglios dos Invertebrados/química , Gânglios dos Invertebrados/citologia , Expressão Gênica/fisiologia , Hibridização In Situ , Mecanorreceptores/química , Dados de Sequência Molecular , Sistema Nervoso/química , Sistema Nervoso/citologia , RNA Mensageiro/análise , Homologia de Sequência de Aminoácidos , Transdução de Sinais/fisiologia , Olfato/fisiologiaRESUMO
In several cell systems histamine has been shown to stimulate both adenylyl cyclase and phospholipase C through activation of a G protein-coupled H2 receptor. To analyze the bifurcating signal emanating from the activated H2 receptor and to identify the G proteins involved, H1 and H2 histamine receptors were functionally expressed in baculovirus-infected insect cells. Histamine challenge lead to concentration-dependent cAMP formation and Ca2+ mobilization in Sf9 cells infected with a virus encoding the H2 receptor, whereas H1 receptor stimulation only resulted in pronounced phospholipase C activation. To analyze the G protein coupling pattern of histamine receptors, activated G proteins were labeled with [alpha-32P]GTP azidoanilide and identified by selective immunoprecipitation. In insect cell membranes expressing H1 histamine receptors, histamine led to incorporation of the label into alpha q-like proteins, whereas activation of the H2 receptor resulted in labeling of alpha q- and alpha s-like G protein alpha-subunits. In COS cells transfected with H2 receptor complementary DNA, histamine caused concentration-dependent accumulation of cAMP and inositol phosphates; the latter effect was insensitive to pertussis toxin treatment. Histamine stimulation led to a pronounced increase in inositol phosphate production when complementary DNAs coding for alpha q, alpha 11, alpha 14, or alpha 15 G protein alpha-subunits were cotransfected. This increase was specific for Gq family members, as overexpression of alpha 12 or alpha s did not enhance histamine-stimulated phospholipase C activation. In membranes of guinea pig heart, addition of [alpha-32P]GTP azidoanilide resulted in labeling of alpha q and alpha 11 via the activated H1 and also via H2 receptors. These data demonstrate that dual signaling of the activated H2 histamine receptor is mediated by coupling of the receptor to Gs and Gq family members.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Receptores Histamínicos H2/metabolismo , Fosfolipases Tipo C/metabolismo , Toxina Adenilato Ciclase , Adenilil Ciclases/metabolismo , Animais , Baculoviridae/genética , Ligação Competitiva , Células COS/efeitos dos fármacos , Células COS/metabolismo , Membrana Celular/metabolismo , AMP Cíclico/biossíntese , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/imunologia , Guanidinas/metabolismo , Guanidinas/farmacologia , Cobaias , Histamina/farmacologia , Antagonistas dos Receptores H2 da Histamina/metabolismo , Antagonistas dos Receptores H2 da Histamina/farmacologia , Fosfatos de Inositol/biossíntese , Insetos/metabolismo , Insetos/virologia , Masculino , Toxina Pertussis , Piperidinas/metabolismo , Piperidinas/farmacologia , Pirilamina/metabolismo , Pirilamina/farmacologia , Receptores Histamínicos H1/genética , Receptores Histamínicos H1/metabolismo , Receptores Histamínicos H2/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Virulência de Bordetella/farmacologiaAssuntos
Anticorpos/farmacologia , Proteínas de Ligação ao GTP/biossíntese , Expressão Gênica/fisiologia , Neurônios/fisiologia , Oligonucleotídeos Antissenso/farmacologia , Animais , Especificidade de Anticorpos , Cálcio/metabolismo , Células Cultivadas , Proteínas de Ligação ao GTP/imunologia , Expressão Gênica/efeitos dos fármacos , Hipotálamo/fisiologia , Hibridização In Situ , Substâncias Macromoleculares , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Reação em Cadeia da Polimerase , Ratos , Ratos Wistar , Gânglio Cervical Superior/fisiologiaRESUMO
In Chinese hamster ovary cells expressing the substance P (SP) receptor clone (CHO-SPR cells), we examined SP-stimulated [Ca2+]i changes by microscopic fluorescence analysis and electrophysiological recordings. In fura-2-loaded cells, SP (1 microM) induced a prolonged elevation of [Ca2+]i, which comprised a rapid and transient Ca2+ mobilization and a prolonged phase of Ca2+ entry, but thrombin (1 unit/ml) induced only transient elevation of [Ca2+]i. The formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) was stimulated to 230% above the control by SP but 10% by thrombin 10 s after stimulation. In whole cell clamp recordings, SP induced a long lasting inward current, whereas thrombin did not evoke an inward current. Gq alpha antibody applied intracellularly blocked the SP-induced current, but GS alpha antibody did not block it. Furthermore, decavanadate and heparin, inhibitors of Ins(1,4,5)P3 binding to its receptor, suppressed the SP-induced current. In cell-attached patch, bath-applied SP activated channel currents carried by Ba2+, Ca2+, or Na+. In inside-out patches, Ins(1,4,5)P3, but neither inositol 1,3,4-trisphosphate nor inositol 1,3,4,5-tetrakisphosphate, activated channel currents carried by Ba2+, Ca2+, or Na+. The channel activity induced by Ins(1,4,5)P3 was abolished by heparin. These results demonstrate that SP induces Ca2+ entry through activation of cation channels and suggest that Ins(1,4,5)P3 may regulate both SP-induced Ca2+ mobilization and Ca2+ entry in CHO-SPR cells.
Assuntos
Canais de Cálcio/fisiologia , Cálcio/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Receptores da Neurocinina-1/fisiologia , Substância P/farmacologia , Sequência de Aminoácidos , Animais , Anticorpos/farmacologia , Bário/farmacologia , Células CHO , Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Células Clonais , Cricetinae , DNA Complementar/metabolismo , Proteínas de Ligação ao GTP/imunologia , Proteínas de Ligação ao GTP/metabolismo , Heparina/farmacologia , Inositol 1,4,5-Trifosfato/farmacologia , Fosfatos de Inositol/farmacologia , Cinética , Potenciais da Membrana/efeitos dos fármacos , Dados de Sequência Molecular , Fosfatidilinositóis/metabolismo , Receptores da Neurocinina-1/biossíntese , Receptores da Neurocinina-1/efeitos dos fármacos , Sódio/farmacologia , Trombina/farmacologia , Fatores de Tempo , Transfecção , Vanadatos/farmacologiaRESUMO
We have recently localized a small GTP-binding protein (Rab6p) thought to be involved in vesicular membrane transport, to the medial and trans-cisternae of the Golgi apparatus in NRK (normal rat kidney) cells. Here, we have localized and quantified Rab6p during the development in culture of embryonic neurons, up to synapse formation, and compared its subcellular distribution and level of expression to that of synaptophysin, a major integral membrane protein of small synaptic vesicles. Using immunocytochemistry (laser scanning confocal microscopy, immunoelectron microscopy), fractionation and immunoisolation methods, we show that during the early phase of synaptogenesis, Rab6p is associated with synaptophysin-containing membranes of a trans-Golgi subcompartment, post-Golgi vesicles and small synaptic vesicles or their precursors. Concomitantly, Rab6p undergoes translocation from cytosol to membranes and its level of expression increases. However, at late stages, the association of Rab6p to small synaptic vesicles sharply decreases and its level of expression plateaus. These findings suggest a role for Rab6p in the post-Golgi transport of synaptophysin, at an early step of the biogenesis of small synaptic vesicles.