RESUMO
AIM: To examine the responses of mouse odontoblast-lineage cell line (OLC) cultures to xylitol-induced hypertonic stress. METHODOLOGY: OLCs were treated with xylitol, sucrose, sorbitol, mannitol, arabinose and lyxose. Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium assay. The expression of transient receptor potential vanilloids (TRPV) 1, 3 and 4 was detected using a reverse transcriptase-polymerase chain reaction (RT-PCR) assay. The expression of aquaporin (AQP) 2 was detected using immunofluorescence and Western blotting analysis. The expression of interleukin-6 (IL-6) under xylitol-induced hypertonic stress was assessed using an enzyme-linked immunosorbent assay (ELISA). Small interfering ribonucleic acid (siRNA) for AQP-2 was used to inhibition assay. RESULTS: Xylitol-induced hypertonic stress did not decrease OLC viability, unlike the other sugars tested. OLCs expressed TRPV1, 3 and 4 as well as AQP2. Xylitol inhibited lipopolysaccharide (LPS)-induced IL-6 expression after 3 h of hypertonic stress. TRPV1 mRNA expression was upregulated by xylitol. Costimulation with HgCl2 (AQP inhibitor) and Ruthenium red (TRPV1 inhibitor) decreased cell viability with xylitol stimulation. OLCs treated with siRNA against TRPV1 exhibited decreased cell viability with xylitol stimulation. CONCLUSION: OLCs have high-cell viability under xylitol-induced hypertonic stress, which may be associated with TRPV1 and AQP2 expressions.
Assuntos
Aquaporina 2/metabolismo , Odontoblastos/metabolismo , Canais de Cátion TRPV/metabolismo , Xilitol/farmacologia , Animais , Aquaporina 2/antagonistas & inibidores , Aquaporina 2/genética , Soluções Hipertônicas/farmacologia , Interleucina-6/metabolismo , Cinética , Lipopolissacarídeos/farmacologia , Camundongos , Odontoblastos/efeitos dos fármacos , Pressão Osmótica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/genética , Fatores de TempoRESUMO
Because of relevant results that indicated that molecular techniques can provide increased knowledge of animal social systems, they usually complement observational field studies. Despite the great utility of microsatellites, they are not available for all species. Gathering genetic information using microsatellites that were originally designed for other species is a time-saving procedure. The aim of this study was to test the transferability of microsatellites and their usefulness in studies of social behavior of black capuchin monkeys (Sapajus nigritus). We noninvasively sampled adult and subadult black capuchins of three wild groups in southeastern Brazil. Seventeen microsatellites, which were previously designed for and successfully amplified in multiple Neotropical primate species, were tested. Nine of the 17 microsatellite loci tested produced an average of 6.22 alleles (range 2-12) per locus. The allelic richness and the expected heterozygosity for all loci was 5.93 and 0.70, respectively. The combined non-exclusion probability for one candidate parent across all loci was 0.01. The nine microsatellite loci optimized in this study have a great potential for application in studies of social structure and dispersal patterns in S. nigritus populations and in other Neotropical primate species.
Assuntos
Cebus/genética , Técnicas de Transferência de Genes , Repetições de Microssatélites/genética , Comportamento Social , Animais , BrasilRESUMO
BACKGROUND AND PURPOSE: The differences in the characteristics of thymus histology, coexisting autoimmune diseases and related autoantibodies between anti-muscle-specific receptor tyrosine kinase (MuSK)-antibody (Ab)-positive myasthenia gravis (MG) patients, and anti-acetylcholine receptor (AChR)-Ab-positive MG patients are not clearly defined. METHODS: The types of thymus histology, coexisting autoimmune diseases and associated Abs in 83 MuSK-Ab-positive patients nationwide were investigated and were compared with those in AChR-Ab-positive patients followed at our institute (n = 83). As for the autoantibodies associated with thymoma, titin Abs were measured. RESULTS: Thymoma was not present in any of the MuSK-Ab-positive patients but presented in 21 patients (25.3%) amongst the AChR-Ab-positive patients. Titin Abs were absent in MuSK-Ab-positive patients but positive in 25 (30.1%) of the AChR-Ab-positive patients. Concomitant autoimmune diseases were present in eight MuSK-Ab-positive patients (9.6%) amongst whom Hashimoto's thyroiditis and rheumatoid arthritis predominated, whereas 22 AChR-Ab-positive patients (26.5%) had one or more concomitant autoimmune diseases of which Graves' disease predominated. CONCLUSIONS: Differences in frequency of thymoma and thymic hyperplasia, coexisting autoimmune diseases and autoantibody positivity between MuSK-Ab-positive and AChR-Ab-positive MG were indicated, suggesting that, in contrast with AChR-Ab-positive MG, thymus does not seem to be involved in the pathogenic mechanisms of MuSK-Ab-positive MG.
Assuntos
Autoanticorpos/imunologia , Autoantígenos/imunologia , Doenças Autoimunes/complicações , Miastenia Gravis/complicações , Receptores Proteína Tirosina Quinases/imunologia , Receptores Colinérgicos/imunologia , Timo/patologia , Adulto , Povo Asiático , Autoanticorpos/sangue , Autoantígenos/sangue , Conectina/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Miastenia Gravis/imunologia , Miastenia Gravis/patologia , Radioimunoensaio , Timoma/complicações , Timoma/patologia , Hiperplasia do Timo/complicações , Hiperplasia do Timo/patologia , Neoplasias do Timo/complicações , Neoplasias do Timo/patologiaRESUMO
Aquaporin 3 (AQP3) is the predominant water channel protein in human keratinocytes and acts as an inflammatory mediator in some lesions. A chronic, inflammatory process of periodontitis is related with a dramatic change of surrounding fluid homeostasis to plasma extravasation. The exact pattern of aquaporin (AQP) water channel expression and its mechanism in periodontal disease is still unknown. We describe herein an up-regulated AQP3 expression in the epithelial lesion with chronic periodontitis and its functional role. The levels of AQP3 expression in inflamed gingival epithelial tissues were significantly higher than those of healthy subjects. Consistent with these results, AQP3 expression (i.e., levels of mRNA and protein) in cultured rat primary gingival epithelial cells and the human gingival epithelial cell line Ca9-22 were strongly increased in response to TNF-alpha treatment through the 55 kDa TNF-alpha receptor (TNFR I). In this context, small interfering RNA- (siRNA)-mediated "aqp-3 gene silencing," which could reduce AQP3 expression by more than 65%, significantly attenuated selected proinflammatory events of ICAM-1 expression induced by TNF-alpha in Ca9-22. A sixfold increase in leukocyte adherence to TNF-alpha-stimulated epithelial cells was demonstrated by an adherence assay (P < 0.001) and pretreatment with AQP3 siRNA and anti-ICAM-1 antibody reduced leukocyte retention by 85% (P < 0.001). Our study indicates for the first time a novel important mode in the regulation of the inflammatory response through TNF-alpha/TNFR I ligation at the site of epithelial lesions by specialized membrane channel AQP3 and ICAM-1 protein, which is closely implicated in the development of periodontitis mechanisms.
Assuntos
Aquaporina 3/metabolismo , Células Epiteliais/metabolismo , Gengiva/metabolismo , Periodontite/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Aquaporina 3/genética , Adesão Celular , Linhagem Celular , Doença Crônica , Células Epiteliais/imunologia , Feminino , Gengiva/imunologia , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Leucócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Periodontite/imunologia , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Proteínas Recombinantes/metabolismo , Fatores de Tempo , Transfecção , Regulação para CimaRESUMO
BACKGROUND AND OBJECTIVE: Thrombomodulin, a cell transmembrane glycoprotein, binds to thrombin and converts it from a procoagulant protease to an anticoagulant enzyme that activates protein C. Thrombomodulin is very important in regulating the function of thrombin. Elevated soluble thrombomodulin is present in the gingival crevicular fluid of subjects with periodontitis. The objective of the present study was to investigate the mechanisms about the elevated soluble thrombomodulin in gingival crevicular fluid. MATERIAL AND METHODS: Gingival sections from six patients with chronic periodontitis and from three periodontally healthy subjects were immunostained for thrombomodulin detection. Thrombomodulin levels were investigated in the gingival crevicular fluid of 11 subjects with chronic periodontitis. The effects of neutrophil enzymes on thrombomodulin release and on thrombomodulin in the gingival crevicular fluid were examined by an enzyme-linked immunosorbent assay or by Western blotting. RESULTS: The expression of gingival epithelial thrombomodulin was lost or decrease near infiltrating neutrophils. Thrombomodulin was rapidly released from gingival epithelial cells by neutrophil enzymes, and gingival crevicular fluid with periodontitis included the proteolytic cleavage thrombomodulin using immunoblotting analysis. The thrombomodulin release was not caused by rapid cell damage, on lactate dehydrogenase assay. There were significant differences in thrombomodulin content between gingival crevicular fluid samples from healthy and diseased sites, regardless of the degree of probing depth. CONCLUSION: Neutrophil enzymes induced rapid thrombomodulin release from the membrane surface of gingival epithelial cells. This might explain the thrombomodulin increase in gingival crevicular fluid with local diseased gingiva. Elevation of thrombomodulin in gingival crevicular fluid may be a potential marker of epithelial cell membrane injury.
Assuntos
Gengiva/patologia , Líquido do Sulco Gengival/química , Periodontite/patologia , Trombomodulina/análise , Perda do Osso Alveolar/metabolismo , Perda do Osso Alveolar/patologia , Western Blotting , Catepsina G , Catepsinas/análise , Doença Crônica , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/metabolismo , Feminino , Gengiva/metabolismo , Hemorragia Gengival/metabolismo , Hemorragia Gengival/patologia , Humanos , Imuno-Histoquímica , L-Lactato Desidrogenase/análise , Elastase de Leucócito/análise , Masculino , Pessoa de Meia-Idade , Neutrófilos/enzimologia , Bolsa Periodontal/metabolismo , Bolsa Periodontal/patologia , Periodontite/metabolismo , Serina Endopeptidases/análise , Inibidores de Serina Proteinase , Trombomodulina/metabolismo , alfa 1-AntitripsinaRESUMO
We reported a successful mitral valve plasty for a 91-year-old woman with mitral valve prolapse. She has lived healthfully and independently without a big problem. She was admitted to another hospital for acute heart failure. Echo cardiography revealed prolapse of posterior mitral valve leaflet and severe mitral regurgitation. Drug therapy was not enough to control her complaint In spite of her age, the patient was able to support herself, and she and her family desired to have a surgical treatment. Therefore she referred to our hospital and underwent mitral valve plasty. Post operative course was almost uneventful. She discharged the hospital 3 months after the operation. If a selective criteria for individual patients is applied, the nonagenarian can safety undergo cardiac surgery.
Assuntos
Prolapso da Valva Mitral/cirurgia , Valva Mitral/cirurgia , Idoso de 80 Anos ou mais , Procedimentos Cirúrgicos Cardíacos/métodos , Feminino , Humanos , Resultado do TratamentoRESUMO
PURPOSE: Jab1 (Jun activation domain-binding protein 1) has been described as a coactivtor of AP1 transcription factor, and is a subunit of a large protein complex (called the COP9 signalosome). Recent study (K. Tomoda et al., Nature (Lond.), 398: 160-165, 1999) found that Jab1 protein can cause breakdown of p27(kip1) protein in mammalian cells. To investigate whether Jab1 expression is correlated with p27(kip1) protein levels as well as how it might be clinically relevant, we evaluated the expression of Jab1 in a group of epithelial ovarian tumors. EXPERIMENTAL DESIGN: Immunohistochemical analysis was performed in 80 cases of ovarian tumors (33 benign ovarian tumors and 47 ovarian carcinomas). Twenty-six of the 80 cases were evaluated by Western blot analysis. RESULTS: Jab1 overexpression was detected in 68.1% (32 of 47) of malignant tumors and 33.3% (11 of 33) of benign tumors. The positive ratio of Jab1 was increased from benign to malignant ovarian tumors (P = 0.002). A negative correlation between Jab1 and p27(kip1) expression was found in both benign (P = 0.003) and malignant (P = 0.002) ovarian tumors. No significant correlation was observed between Jab1 overexpression and clinicopathological parameters. Kaplan-Meier survival analysis showed that Jab1 overexpression was significantly associated with poor prognosis of patients (P = 0.049). CONCLUSIONS: Jab1 expression is inversely correlated with p27(kip1) expression levels, and Jab1, as a negative regulator of p27(kip1), may be associated with the progression and prognosis of epithelial ovarian tumors.
Assuntos
Carcinoma/patologia , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Neoplasias Ovarianas/patologia , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Complexo do Signalossomo COP9 , Carcinoma/mortalidade , Carcinoma/cirurgia , Proteínas de Ciclo Celular/análise , Inibidor de Quinase Dependente de Ciclina p27 , Proteínas de Ligação a DNA/análise , Inibidores Enzimáticos/análise , Feminino , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular , Metástase Linfática , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/cirurgia , Peptídeo Hidrolases , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida , Fatores de Tempo , Fatores de Transcrição/análise , Proteínas Supressoras de Tumor/análiseRESUMO
Cyclin E plays a pivotal role in the regulation of G1-S transition and relates to malignant transformation of cells. However, the clinical significance of cyclin E in patients with laryngeal squamous cell carcinoma (LSCC) remains unknown. We examined the expression of cyclin E in 102 patients with LSCC and analyzed its relation to clinicopathological parameters, cell proliferation, and clinical outcome. Cyclin E overexpression was observed in 54 cases (52.94%) of LSCC and was significantly correlated with the tumor site (P = 0.012), tumor size (P = 0.006), poor differentiation (P = 0.026), lymph node metastasis (P = 0.012), and advanced stage (P = 0.002). A positive correlation between the cyclin E expression and proliferative activity of tumor cells was found (r = 0.896; P < 0.0001). Kaplan-Meier analysis showed that shorter disease-free and overall survival was significantly associated with proliferating cell nuclear antigen (PCNA) overexpression and cyclin E overexpression. When PCNA and cyclin E are combined, the patients with both PCNA overexpression and cyclin E overexpression had the poorest prognoses when compared with the other cases. Additionally, in early stage (I-II) cases, cyclin E was also revealed to possess a significant prognostic role. By multivariate analysis, lymph node metastasis and cyclin E overexpression were independent prognostic factors for disease-free survival, and tumor size, lymph node metastasis, advanced stage, as well as cyclin E overexpression were independent prognostic factors for overall survival. These findings indicate that cyclin E overexpression is associated with unfavorable clinicopathological parameters and represents an independent marker for cell proliferation and prognosis of LSCC.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Ciclina E/biossíntese , Neoplasias Laríngeas/metabolismo , Adulto , Idoso , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/patologia , Divisão Celular , Ciclina E/análise , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Laríngeas/mortalidade , Neoplasias Laríngeas/patologia , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , Antígeno Nuclear de Célula em Proliferação/análise , Estudos RetrospectivosRESUMO
Experimental conditions are described for simultaneous purification of three forms of lipocortin (lipocortin I, lipocortin II and lipocortin-85) from bovine lung. The procedure yields milligram quantities of all three lipocortins. Using antisera against lipocortin I and lipocortin II, purified proteins show no cross contaminations. All forms of lipocortin exhibit equal potency as in vitro bovine pancreatic phospholipase A2 inhibitors. Protein kinase C catalyzes the in vivo incorporation of about 1.0, 0.7 and 0.4 mole of phosphate per mole of lipocortin I (p35), lipocortin II (p36) and lipocortin-85 (p36 oligomer) respectively. The phosphorylation is specific for protein kinase C and is dependent on the presence of both calcium and phospholipids. While lipocortin I is phosphorylated on threonine residues, lipocortin II and lipocortin-85 are phosphorylated on serine residues.
Assuntos
Glicoproteínas/isolamento & purificação , Pulmão/análise , Fosfolipases A/antagonistas & inibidores , Fosfolipases/antagonistas & inibidores , Animais , Anexinas , Bovinos , Cromatografia DEAE-Celulose , Ácido Egtázico , Eletroforese em Gel de Poliacrilamida , Imunoquímica , Fosfolipases A2 , Fosforilação , Proteína Quinase C/metabolismoRESUMO
Three peaks of calcium binding activity have been identified by the Chelex-100 calcium binding assay of the fractions from DEAE cellulose chromatography of 100,000 X g supernatant of bovine brain. These calcium binding activity peaks have been subjected to extensive purification and three novel calcium binding proteins (Mr 27,000, Mr 48,000 and Mr 63,000) and two previously characterized proteins (calcineurin and calmodulin) have been identified as components of calcium binding activity peaks. Analysis of the calcium binding properties of the novel proteins by equilibrium dialysis suggests these proteins may be intracellular calcium receptors.
Assuntos
Química Encefálica , Proteínas de Ligação ao Cálcio/análise , Proteínas do Tecido Nervoso/análise , Ribonucleoproteínas/análise , Animais , Proteínas de Ligação ao Cálcio/classificação , Calmodulina/análise , Proteínas de Ligação a Calmodulina/análise , Calreticulina , Bovinos , Cromatografia/métodos , Peso Molecular , Ribonucleoproteínas/classificação , UltrafiltraçãoRESUMO
Presenilin-1, mutations of which cause the early-onset of Alzheimer's disease, was shown to be abundantly expressed in the testis as well as the brain. In spite of the high expression level of this protein in the testis, no further analysis has been undertaken. We aimed to study the distribution and developmental changes in presenilin-1 protein, and to provide clues so as to elucidate the role of this protein in the rat testis. To evaluate the specificity of the anti presenilin-1 antibody, rat presenilin-1 protein was expressed in COS-7 cells and the recombinant protein was used for western blot analysis. A positive band of approximately 20 kDa corresponding to the C-terminal fragment of proteolyzed presenilin-1 protein was observed. Using testis and brain tissue samples, a 20 kDa band was detected in both tissues suggesting a similar proteolytic process, but the expression level in the testis was higher than that in the brain. The expression level increased significantly during postnatal testis development. By an immunohistochemical analysis of the rat testis, a strong signal was observed in interstitial cells and further study with cultured TM3 murine Leydig cells revealed an abundant expression of presenilin-1 in Leydig cells. Our study suggests that presenilin-1 expression in Leydig cells may play an important role in Leydig cell function and testis development.
Assuntos
Células Intersticiais do Testículo/metabolismo , Proteínas de Membrana/metabolismo , Testículo/crescimento & desenvolvimento , Animais , Western Blotting , Células COS , Imuno-Histoquímica , Masculino , Proteínas de Membrana/genética , Camundongos , Fragmentos de Peptídeos/metabolismo , Presenilina-1 , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/metabolismoRESUMO
Annexin-I was demonstrated to specifically present in islets and not in exocrine tissues of the rat pancreas and to have a diffuse and homogeneous distribution in all islet cells in our previous study. In the present report, to clarify the functions of annexin-I in rat pancreatic islets, especially in beta-cells, we investigated the role of annexin-I in insulin secretion. Immunoelectron microscopic analysis of pancreatic beta-cells demonstrated that immunogold particles reactive to annexin-I were almost exclusively observed on most of the insulin-containing granules (approximately 90%) and less frequently located in cytosol and other organelles, such as the endoplasmic reticulum and mitochondria. The number of annexin-I gold particles located on insulin granules after oral glucose administration was significantly increased compared with that observed in fasted rats. Moreover, when the isolated islets were stimulated by a high concentration of glucose (20 mM), the phosphorylation of annexin-I was markedly enhanced, and it was synchronized to insulin secretion. This phosphorylation mainly occurred on serine residues. H-7 (100 microM), a potent inhibitor of protein kinase-C, inhibited the phosphorylation to about 90%. These findings suggest that annexin-I might be involved in the regulatory mechanism of glucose-induced insulin secretion in rat pancreatic islets via phosphorylation-dephosphorylation processes.
Assuntos
Anexina A1/metabolismo , Glucose/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina , Animais , Técnicas In Vitro , Secreção de Insulina , Ilhotas Pancreáticas/ultraestrutura , Isoquinolinas/farmacologia , Microscopia Imunoeletrônica , Organelas/metabolismo , Fosforilação , Piperazinas/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Ratos , Transdução de SinaisRESUMO
The ischemic tolerance is known to show protective effects on the neurons and the restricted Ca2+ influx through Ca2+ channels might be involved. In alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor, ribonucleic acid (RNA) editing of the GluR2 subunit determines receptor desensitization and Ca2+ permeability. The authors investigated the effect of ischemic tolerance on the messenger RNA editing of Q/R and R/G sites of GluR2 subunit in hippocampus. It was found that the rate of RNA editing in Q/R site showed no change (100% edited), whereas that in R/G site decreased significantly (83.3% normal editing level to 60.4%) at day 3 (preconditioning period) and returned to normal level at day 14 (after preconditioning period). Further investigation revealed that the decrease of editing rate in ischemic tolerance resulted mainly from the decrease of editing in CA1 area.
Assuntos
Isquemia Encefálica/genética , Hipocampo/metabolismo , Edição de RNA , Receptores de AMPA/genética , Animais , Cálcio/farmacocinética , Gerbillinae , Hipocampo/irrigação sanguínea , Masculino , Permeabilidade , Receptores de N-Metil-D-Aspartato/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Neuronal plasticity can be defined as adaptive changes in structure and function of the nervous system, an obvious example of which is the capacity to remember and learn. Long-term potentiation and long-term depression are the experimental models of memory in the central nervous system (CNS), and have been frequently utilized for the analysis of the molecular mechanisms of memory formation. Extensive studies have demonstrated that various kinases and phosphatases regulate neuronal plasticity by phosphorylating and dephosphorylating proteins essential to the basic processes of adaptive changes in the CNS. These proteins include receptors, ion channels, synaptic vesicle proteins, and nuclear proteins. Multifunctional kinases (cAMP-dependent protein kinase, Ca2+/phospholipid-dependent protein kinase, and Ca2+/calmodulin-dependent protein kinases) and phosphatases (calcineurin, protein phosphatases 1, and 2A) that specifically modulate the phosphorylation status of neuronal-signaling proteins have been shown to be required for neuronal plasticity. In general, kinases are involved in upregulation of the activity of target substrates, and phosphatases downregulate them. Although this rule is applicable in most of the cases studied, there are also a number of exceptions. A variety of regulation mechanisms via phosphorylation and dephosphorylation mediated by multiple kinases and phosphatases are discussed.
Assuntos
Encéfalo/fisiologia , Plasticidade Neuronal/fisiologia , Fosfoproteínas Fosfatases/metabolismo , Proteínas Quinases/metabolismo , Medula Espinal/fisiologia , Animais , Proteínas de Ligação ao GTP/fisiologia , Homeostase , Humanos , Potenciação de Longa Duração/fisiologia , Fosforilação , Receptores de Superfície Celular/fisiologiaRESUMO
EDG-3 is a receptor for sphingosine-1-phosphate mapped on human chromosome 9q22.1-q22.2. We used the compact Fugu genome for its linkage analysis. The Fugu EDG-3 was composed of one intron and two exons, encoding a 384 amino acid protein that has 56.9% homology with the human EDG-3. Approximately 3 kb apart, a neuronal Shc (N-Shc) gene was identified. It spans 7 kb containing 12 coding exons, and has an overall 53.4% similarity with the human protein. We mapped the human N-Shc gene to chromosome 9q21.3-q22.2. This is the first report of the genomic structure and the linkage of these two genes conserved between Fugu and human.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas I-kappa B , Neuropeptídeos/genética , Sequência de Aminoácidos , Animais , Mapeamento Cromossômico , Cromossomos Humanos Par 9 , Clonagem Molecular , Peixes Venenosos/genética , Ligação Genética , Genoma Humano , Humanos , Dados de Sequência Molecular , Inibidor de NF-kappaB alfa , Receptores de Lisofosfolipídeos , Homologia de Sequência de Aminoácidos , Proteínas Adaptadoras da Sinalização Shc , Proteína 3 de Transformação que Contém Domínio 2 de Homologia de SrcRESUMO
Nine src family members are known including c-Src, c-Yes, c-Lck, c-Fyn, c-Hck, c-Lyn, c-Blk, c-Fgr and c-Yrk. They encode proteins with molecular weights of 55-62 kilodaltons (kDa), which are either cytoplasmic or membrane-associated protein tyrosine kinases. A close correlation exists between an elevated pp60c-src tyrosine kinase activity and cell transformation. However, the level of activation of pp60c-src in non-small cell lung cancers (NSCLC) remains obscure. The aim of this study was to examine the level of activity of pp60c-src in NSCLC. pp60c-src expression and in vitro protein tyrosine kinase activity in lung cancer tissue samples were measured by western blotting and in vitro kinase assays and compared with those in the surrounding non-tumour lung tissue from the same patient. pp60c-src phosphorylation was assessed by two-dimensional tryptic phosphopeptide mapping. The kinase activity of pp60c-src was significantly activated in NSCLC, especially in adenocarcinomas. In addition, the pp60c-src kinase activity increased with the size of the adenocarcinoma. Two-dimensional tryptic phosphopeptide mapping showed dephosphorylation of pp60c-src at Tyr 530 in adenocarcinomas. The proto-oncogene product, pp60c-src, was activated in NSCLC, especially in adenocarcinomas, in part through the dephosphorylation of Tyr 530. Our results suggest that activation of pp60c-src might play an important role in the progression of lung adenocarcinomas.
Assuntos
Adenocarcinoma/enzimologia , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma de Células Escamosas/enzimologia , Neoplasias Pulmonares/enzimologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proto-Oncogene Mas , Células Tumorais CultivadasRESUMO
Mammalian brains contain a cde2-like protein kinase which is a heterodimer of cyclin-dependent kinase 5 (Cdk5) and a brain-specific regulatory subunit with a molecular weight of 35,000. In this study, we examined the temporal and spatial expression patterns of p35nck5a in the developing rat brain. Northern blot analysis showed that p35nck5a messenger RNA expression was low in the brain of 12-day postcoitum rats, and increased to a much higher level from 18 days postcoitum to two weeks after birth, and then declined at three weeks after birth. These developmental changes in p35nck5a expression correlated with the changes in Cdk5-associated kinase activity during brain development. These data suggest that p35nck5a is the specific activator for Cdk5 in the brain. Immunohistochemical and in situ hybridization studies demonstrated the presence of p35nck5a protein in postmitotic neurons but not in glial cells at all stages of brain development, indicating that p35nck5a is a neuron-specific protein. In the adult brain, the protein was rich in cell bodies and dendrites, and only very low amounts were detected in axons. In fetal and neonatal brains, however, axonal pathways such as the corpus callosum and external capsule were also stained with anti-p35nck5a antibody. Our findings suggest that p35nck5a is neuron specific, and a specific activator for Cdk5, and the subcellular localization of the two is strictly regulated depending on brain development. Neuronal Cdc2-like kinase may play key roles in neuronal maturation, synaptic formation, and neuronal plasticity.
Assuntos
Encéfalo/metabolismo , Fosfotransferases/metabolismo , Animais , Animais Recém-Nascidos/metabolismo , Feminino , Imuno-Histoquímica , Hibridização In Situ , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
A cDNA encoding a putative ion channel protein was isolated from a rat hippocampus library. This gene, termed phosphohippolin (Php), contains 318 bp open reading frame encoding a single transmembrane protein with a 5' signal peptide region. The deduced amino acid sequence shares 48.1% homology with phospholemman (Plm). Expression sequence tag database (dEST) search identified a mouse (AA521976) and human (AA209241) Php gene homologues. The tissue distribution studies of Php mRNA showed its abundant expression in rat brain and kidney, and in the brain, high expression was observed in hippocampus and cerebellum.
Assuntos
Hipocampo/química , Hipocampo/fisiologia , Canais Iônicos/genética , Proteínas de Membrana/genética , Fosfoproteínas/genética , Animais , Sequência de Bases , Cerebelo/química , Cerebelo/fisiologia , Clonagem Molecular , Hibridização In Situ , Masculino , Dados de Sequência Molecular , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Homologia de Sequência de AminoácidosRESUMO
Bone morphogenetic proteins (BMPs) are a group of proteins which induce bone formation from mesenchymal cells. The existence of BMPs in the nervous system as well as in bone tissue has recently been reported. In this study, we show that BMP-6 is neuron-specific, and describe the temporal and spatial expression patterns of BMP-6 mRNA in the developing rat and gerbil brain. Northern blot analysis showed that the BMP-6 transcript level was specifically high from newborn to 3 weeks after birth compared with those in fetal and adult rats. In situ hybridization showed that most of the neurons possessed high levels of BMP-6 mRNA in the neonatal brain, while in the adult brain, BMP-6 mRNA level was significantly decreased in most of the neurons except those in hippocampus which retained high levels. Furthermore, to show that the BMP-6 expression was specific to neurons, we induced delayed neuronal cell death and compensative glial cell proliferation in the gerbil hippocampus by transient ischemia. Our findings collectively suggest that BMP-6 is neuron-specific and may play important roles in neuronal maturation and synapse formation.
Assuntos
Prosencéfalo/crescimento & desenvolvimento , Biossíntese de Proteínas , Animais , Proteínas Morfogenéticas Ósseas , Isquemia Encefálica/metabolismo , Gerbillinae , Hipocampo/metabolismo , Hibridização In Situ , Fatores de Crescimento Neural/biossíntese , Neurônios/metabolismo , Prosencéfalo/metabolismo , RNA Mensageiro/análise , RatosRESUMO
Treatment of 2-(2'-bromo-beta-phenethyl)isocarbostyrils 7 with AIBN-Bu(3)SnH in boiling benzene gave 8-oxoberbines 3 in good yields. A similar treatment of 2-(2'-bromo-beta-phenethyl)isoquinolinium bromides 6 and their nor- and homoanalogues (10,11) induced 6-, 5-, and 7-exo radical closures in a one-pot manner to give protoberberines 2, dibenzo[b,g]indolizidine 14a and, dibenzo[a, h]-1-azabicyclo[5.4.0]undecane 15a, respectively. A one-pot radical cyclization of 1-(2'-bromobenzyl)isoquinoline methiodide 18a gave a pavine alkaloid, (+/-)-algemonine (19a).