RESUMO
OBJECTIVE: To quantify the burden of maternal and neonatal conditions in low- and middle-income countries (LMICs) that could be averted by full access to quality first-level obstetric surgical procedures. DESIGN: Burden of disease and epidemiological modelling. SETTING: LMICs from all global regions. POPULATION: The entire population in 2010. METHODS: We included five conditions in our analysis: maternal haemorrhage; obstructed labour; obstetric fistula; abortion(1) ; and neonatal encephalopathy. Demographic and epidemiological data were obtained from the Global Burden of Disease 2010 study. We split the disability-adjusted life years (DALYs) of these conditions into surgically 'avertable' and 'non-avertable' burdens. We applied the lowest age-specific fatality rates from all global regions to each LMIC region to estimate the avertable deaths, assuming that the differences of death rates between each region and the lowest rates reflect the gap in surgical care. MAIN OUTCOME MEASURES: Deaths and DALYs avertable. RESULTS: Of the estimated 56.6 million DALYs (i.e. 56.6 million years of healthy life lost) of the selected five conditions, 21.1 million DALYs (37%) are avertable by full coverage of quality obstetric surgery in LMICs. The avertable burden in absolute term is substantial given the size of burden of these conditions in LMICs. Neonatal encephalopathy constitutes the largest portion of avertable burden (16.2 million DALYs) among the five conditions, followed by abortion (2.1 million DALYs). CONCLUSIONS: Improving access to quality surgical care at first-level hospitals could reduce a tremendous burden of maternal and neonatal conditions in LMICs.
Assuntos
Traumatismos do Nascimento/prevenção & controle , Efeitos Psicossociais da Doença , Países em Desenvolvimento , Expectativa de Vida , Modelos Estatísticos , Complicações na Gravidez/cirurgia , Fístula Vesicovaginal/cirurgia , Traumatismos do Nascimento/complicações , Traumatismos do Nascimento/epidemiologia , Parto Obstétrico , Feminino , Procedimentos Cirúrgicos em Ginecologia , Acessibilidade aos Serviços de Saúde , Humanos , Hipóxia Encefálica/epidemiologia , Hipóxia Encefálica/etiologia , Hipóxia Encefálica/prevenção & controle , Recém-Nascido , Gravidez , Complicações na Gravidez/epidemiologia , Anos de Vida Ajustados por Qualidade de Vida , Fístula Vesicovaginal/epidemiologiaRESUMO
Infection with Helicobacter pylori cagA-positive strains is associated with gastric adenocarcinoma. Intestinal metaplasia is a precancerous lesion of the stomach characterized by transdifferentiation of the gastric mucosa to an intestinal phenotype. The H. pylori cagA gene product, CagA, is delivered into gastric epithelial cells, where it undergoes tyrosine phosphorylation by Src family kinases. Tyrosine-phosphorylated CagA specifically binds to and activates SHP-2 phosphatase, thereby inducing cell-morphological transformation. We report here that CagA physically interacts with E-cadherin independently of CagA tyrosine phosphorylation. The CagA/E-cadherin interaction impairs the complex formation between E-cadherin and beta-catenin, causing cytoplasmic and nuclear accumulation of beta-catenin. CagA-deregulated beta-catenin then transactivates beta-catenin-dependent genes such as cdx1, which encodes intestinal specific CDX1 transcription factor. In addition to beta-catenin signal, CagA also transactivates p21(WAF1/Cip1), again, in a phosphorylation-independent manner. Consequently, CagA induces aberrant expression of an intestinal-differentiation marker, goblet-cell mucin MUC2, in gastric epithelial cells that have been arrested in G1 by p21(WAF1/Cip1). These results indicate that perturbation of the E-cadherin/beta-catenin complex by H. pylori CagA plays an important role in the development of intestinal metaplasia, a premalignant transdifferentiation of gastric epithelial cells from which intestinal-type gastric adenocarcinoma arises.
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Caderinas/metabolismo , Transformação Celular Neoplásica/metabolismo , Mucosa Gástrica/metabolismo , Lesões Pré-Cancerosas/metabolismo , Neoplasias Gástricas/etiologia , beta Catenina/metabolismo , Adenocarcinoma/etiologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Antígenos de Bactérias/análise , Proteínas de Bactérias/análise , Caderinas/análise , Linhagem Celular , Núcleo Celular/química , Núcleo Celular/metabolismo , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Citoplasma/química , Citoplasma/metabolismo , Mucosa Gástrica/patologia , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Mucina-2 , Mucinas/metabolismo , Fosforilação , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Ativação Transcricional , Tirosina/metabolismo , beta Catenina/análiseRESUMO
Thymus and activation-regulated chemokine (TARC) is a recently identified lymphocyte-directed CC chemokine which specifically chemoattracts T helper type 2 CD4(+) T cells in human. To establish the pathophysiological roles of TARC in vivo, we investigated whether a monoclonal antibody (mAb) against TARC could inhibit the induction of hepatic lesions in murine model using Propionibacterium acnes and lipopolysaccharide (LPS). P. acnes-induced intrahepatic granuloma formation in the priming phase is essential to the subsequent liver injury elicited by a low dose of LPS. The priming phase appears to be dominated by Th1 type immune responses determined by the profile of chemokine and chemokine receptor expression. TARC was selectively produced by granuloma-forming cells, and CC chemokine receptor 4 (CCR4)-expressing CD4(+) T cells migrated into the liver after LPS administration. In vivo injection of anti-TARC mAb just before LPS administration protected the mice from acute lethal liver damage, which was accompanied by a significant reduction of both CCR4 mRNA expression and IL-4 production by liver-infiltrating CD4(+) T cells. Moreover, both TNF-alpha and Fas ligand expressions in the liver were decreased by anti-TARC treatment. These results suggest that recruitment of IL-4-producing CCR4(+) CD4(+) T cells by granuloma-derived TARC into the liver parenchyma may be a key cause of massive liver injury after systemic LPS administration.
Assuntos
Quimiocinas CC/fisiologia , Infecções por Bactérias Gram-Positivas/complicações , Encefalopatia Hepática/fisiopatologia , Lipopolissacarídeos/toxicidade , Propionibacterium acnes , Células Th1/imunologia , Células Th2/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Biomarcadores , Quimiocina CCL17 , Quimiocinas CC/antagonistas & inibidores , Quimiocinas CC/imunologia , Feminino , Granuloma/etiologia , Granuloma/fisiopatologia , Encefalopatia Hepática/etiologia , Encefalopatia Hepática/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Receptores CCR4 , Receptores de Quimiocinas/metabolismo , Organismos Livres de Patógenos Específicos , Células Th2/metabolismo , Fatores de Transcrição/metabolismoRESUMO
A method for the screening of tetanus and diphtheria antibodies in serum using anatoxin (inactivated toxin) instead of toxin was developed as an alternative to the in vivo toxin neutralization assay based on the toxin-binding inhibition test (TOBI test). In this study, the serum titers (values between 1.0 and 19.5 IU) measured by a modified TOBI test (Modi-TOBI test) and toxin neutralization assays were correlated (P < 0.0001). Titers of tetanus or diphtheria antibodies were evaluated in serum samples from guinea pigs immunized with tetanus toxoid, diphtheria-tetanus or triple vaccine. For the Modi-TOBI test, after blocking the microtiter plates, standard tetanus or diphtheria antitoxin and different concentrations of guinea pig sera were incubated with the respective anatoxin. Twelve hours later, these samples were transferred to a plate previously coated with tetanus or diphtheria antitoxin to bind the remaining anatoxin. The anatoxin was then detected using a peroxidase-labeled tetanus or diphtheria antitoxin. Serum titers were calculated using a linear regression plot of the results for the corresponding standard antitoxin. For the toxin neutralization assay, L+/10/50 doses of either toxin combined with different concentrations of serum samples were inoculated into mice for anti-tetanus detection, or in guinea pigs for anti-diphtheria detection. Both assays were suitable for determining wide ranges of antitoxin levels. The linear regression plots showed high correlation coefficients for tetanus (r(2) = 0.95, P < 0.0001) and for diphtheria (r(2) = 0.93, P < 0.0001) between the in vitro and the in vivo assays. The standardized method is appropriate for evaluating titers of neutralizing antibodies, thus permitting the in vitro control of serum antitoxin levels.
Assuntos
Antitoxina Diftérica/sangue , Vacina contra Difteria e Tétano/imunologia , Vacina contra Difteria, Tétano e Coqueluche/imunologia , Antitoxina Tetânica/sangue , Animais , Antitoxina Diftérica/imunologia , Feminino , Cobaias , Masculino , Camundongos , Testes de Neutralização/métodos , Padrões de Referência , Reprodutibilidade dos Testes , Antitoxina Tetânica/imunologiaRESUMO
Adenosine is known to modulate the function of neostriatal neurons. Adenosine acting on A(2A) receptors increases the phosphorylation of dopamine- and cAMP-regulated phosphoprotein of M(r) 32 kDa (DARPP-32) at Thr34 (the cAMP-dependent protein kinase [PKA] site) in striatopallidal neurons, and opposes dopamine D2 receptor signaling. In contrast, the role of adenosine A(1) receptors in the regulation of dopamine/DARPP-32 signaling is not clearly understood. Here, we investigated the effect of adenosine A(1) receptors on D(1), D(2) and A(2A) receptor signaling using mouse neostriatal slices. An A(1) receptor agonist, 2-chloro-N(6)-cyclopentyladenosine (100 nM), caused a transient increase, followed by a transient decrease, in DARPP-32 Thr34 phosphorylation. Our data support the following model for the actions of the A(1) receptor agonist. The A(1) receptor-induced early increase in Thr34 phosphorylation was mediated by presynaptic inhibition of dopamine release, and the subsequent removal of tonic inhibition by D(2) receptors of A(2A) receptor/G(olf)/cAMP/PKA signaling. The A(1) receptor-induced late decrease in Thr34 phosphorylation was mediated by a postsynaptic G(i) mechanism, resulting in inhibition of D(1) and A(2A) receptor-coupled G(olf)/cAMP/PKA signaling in direct and indirect pathway neurons, respectively. In conclusion, A(1) receptors play a major modulatory role in dopamine and adenosine receptor signaling.
Assuntos
Neostriado/fisiologia , Receptor A1 de Adenosina/fisiologia , Receptor A2A de Adenosina/metabolismo , Receptores de Dopamina D1/metabolismo , Transdução de Sinais/fisiologia , Adenosina/análogos & derivados , Adenosina/farmacologia , Agonistas do Receptor A1 de Adenosina , Antagonistas do Receptor A1 de Adenosina , Antagonistas do Receptor A2 de Adenosina , Animais , Benzazepinas/farmacologia , Antagonistas de Dopamina/farmacologia , Fosfoproteína 32 Regulada por cAMP e Dopamina/metabolismo , Interações Medicamentosas , Expressão Gênica/efeitos dos fármacos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Neostriado/citologia , Fenetilaminas/farmacologia , Racloprida/farmacologia , Transdução de Sinais/efeitos dos fármacos , Treonina/metabolismo , Triazinas/farmacologia , Triazóis/farmacologia , Xantinas/farmacologiaRESUMO
The differential effects (BLM) on cycling and noncycling cells were investigated with a mouse ascites tumor in vivo. An i.p. injection of 37.0 or 111.1 mug BLM per g caused a decrease in tumor cell number but an increase in percentage of tumor cells in mitosis. There are no significant differences between the percentage labeled mitoses at various times after pulse labeling by tritiated thymidine of BLM-treated tumor cells and by that of an untreated control, except that the height of the second peak was significantly lower in the treated cells. Hence BLM may be cell cycle nonspecific, and the BLM-induced decrease in cell number, i.p., may stimulate some nondividing cells to reenter the division cycle. However, the fact that percentage of cells in mitosis versus time after the administration of BLM showed two peaks indicates the possibility that another cause of the increase in mitotic figures might be a relative increase of cycling cells due to higher sensitivity of noncycling cells to the agent. Autoradiographic studies on the intracellular distribution of [14C]BLM revealed the following. (a) There were few necrotic cells in mitosis that incorporated much [14C]BLM into the cytoplasm at each time point and the mitotic figures gradually increased with time after i.p. injection of the isotope, while necrotic cells other than in mitosis, most of which were heavily labeled, increased in number with time. These findings seem to be related to the possibility that cycling cells may be less sensitive to BLM. The mode of intracellular distribution of [14C]BLM in mitotic cells changed with time and appeared to reflect the drug susceptibility depending on the cell cycle phase when labeled.
Assuntos
Bleomicina/metabolismo , Mitose/efeitos dos fármacos , Neoplasias Experimentais/metabolismo , Animais , Bleomicina/farmacologia , Camundongos , Neoplasias Experimentais/patologiaRESUMO
N-Glycolylneuraminic acid (NeuGc) is distributed in most animals except humans and chickens. However, human and chicken cancerous tissues often synthesize this heterophilic sialic acid as a tumor-associated Hanganutziu-Deicher antigen [M. Naiki and H. Higashi, Adv. Exp. Med. Biol., 152: 445-456, 1982; H. Higashi et al., Cancer Res., 45: 3796-3802, 1985]. In this paper, NeuGc in human cancerous tissues and chicken Marek's disease lymphoma cell lines was determined quantitatively with gas chromatography-mass spectrometry analysis using mass fragmentography. The detectable limit of NeuGc was 40 pg (0.12 pmol) in each injection using 5 ng of trideuteriomethyl ester trideuteriomethyl glycoside of the sialic acid as an internal standard sample when a pair of ions at m/e 386 and 389 was chosen for ion monitoring. NeuGc was detected in ganglioside-rich fractions of various human cancerous tissues from 5 of 8 patients examined but was not detected in glycosphingolipids of normal human tissues. The contents of NeuGc in these cancerous tissues ranged from 0.02 to 0.5% of the total sialic acid content. NeuGc was also detected in freeze-dried samples of 5 different cell lines from chicken Marek's disease lymphomas but was not detected in a cell line from chicken lymphoid leukosis lymphoma and normal chicken skeletal muscle tissue. The contents of NeuGc in the positive cell lines ranged from 0.03 to 0.11% of the total sialic acid content. These results indicate that NeuGc can be synthesized in both humans and chickens in some cancers.
Assuntos
Biomarcadores Tumorais , Doença de Marek/metabolismo , Neoplasias/metabolismo , Ácidos Neuramínicos/metabolismo , Animais , Linhagem Celular , Galinhas , Cromatografia , Neoplasias do Colo/metabolismo , Humanos , Técnicas In Vitro , Neoplasias Hepáticas/metabolismo , Neoplasias Pulmonares/metabolismo , Linfoma/metabolismo , Espectrometria de Massas , Ácido N-Acetilneuramínico , Ácidos Siálicos/análise , Ácidos Siálicos/biossíntese , Neoplasias Gástricas/metabolismoRESUMO
Peripheral lymphocytes from cancer patients receiving mitomycin C treatment were examined for cytogenetic effects. The treatment consisted of i.v. injections of mitomycin C at a dose of 4 mg given twice a week for 2 weeks. The lymphocytes were cultured in vitro for 72 hr with phytohemagglutinin and 5-bromodeoxyuridine, and then sister chromatid exchanges were scored. Before treatment with mitomycin C, the frequencies of sister chromatid exchanges in lymphocytes of cancer patients were similar to those of healthy controls. After the first and second treatments in vivo with mitomycin C, the frequencies of sister chromatid exchnages increased with time, reached a peak in about 24 hr, and then returned to the pretreatment level in about 48 hr, in contrast to the case of in vitro exposure to mitomycin C. After the third and fourth injections, however, the frequency increased further and did not return to the original level. The significance of the specific kinetics of change in the sister chromatid exchnage frequency after in vivo treatments is discussed in relation to cancer chemotherapy.
Assuntos
Troca Genética/efeitos dos fármacos , Mitomicinas/efeitos adversos , Neoplasias/tratamento farmacológico , Troca de Cromátide Irmã/efeitos dos fármacos , Adulto , Idoso , Aberrações Cromossômicas , Feminino , Humanos , Linfócitos/efeitos dos fármacos , Linfócitos/ultraestrutura , Masculino , Pessoa de Meia-Idade , Neoplasias/genética , Fatores de TempoRESUMO
Hanganutziu-Deicher (HD) antigen-active N-glycolylneuraminic acid (NeuGc)-containing gangliosides were isolated and characterized from human colon cancer tissues. The antigenic gangliosides were detected by thin-layer chromatography by our newly developed method (H. Higashi, Y. Fukui, S. Ueda, S. Kato, Y. Hirabayashi, M. Matsumoto, and M. Naiki. J. Biochem., 95: 1517-1520, 1984) of enzyme immunostaining using affinity-purified chicken antibody against hematoside containing NeuGc (II3NeuGc-LacCer) and horseradish peroxidase-conjugated rabbit anti-chicken lgG. One to six species of the antigenic gangliosides were isolated from seven of 16 cases of colon cancer, whereas no antigenic compound was detected in all apparently normal colorectal tissues from 17 individuals without colorectal cancer. Tissues from different patients showed different patterns of molecular species of the antigenic gangliosides. Densitometric determination indicated that HD antigenic sialic acid, NeuGc, accounted for about 1% or less of the total lipid-bound sialic acids. Four species of antigenic gangliosides were identified as hematoside and hematoside-containing O-acyl ester (II3NeuGc-LacCer and II3 4- or 7-O-acyl-NeuGc-LacCer), GM2-containing NeuGc (II3NeuGc-GgOse3Cer), and sialylparagloboside (IV3NeuGc-nLcOse4Cer) by their behaviors on 2-dimensional thin-layer chromatography, and the effects of mild alkaline treatment, sialidase treatment, periodate oxidation, and endo-beta-galactosidase treatment.
Assuntos
Antígenos Heterófilos/análise , Neoplasias do Colo/imunologia , Gangliosídeos/análise , Antígenos Heterófilos/isolamento & purificação , Densitometria , Gangliosídeo G(M2)/análise , Gangliosídeos/imunologia , Glicolipídeos/análise , HumanosRESUMO
We present a case of successful management for severe respiratory failure during thoracic aortic aneurysm repair by applying extracorporeal membrane oxygenation (ECMO). The patient was a 71-year-old man who was diagnosed as thoracic aortic aneurysm and coronary artery stenosis. Severe respiratory failure occurred during operation because of pulmonary hemorrhage, and it was difficult to wean from cardiopulmonary bypass. ECMO was provided for improvement of oxygenation and CO2 removal. Pulmonary hemorrhage was controlled by strict management of coagulation system, and ECMO was discontinued after improvement of oxygenation on the 4th postoperative day. It is considered that early application of ECMO was effective in this case.
Assuntos
Aneurisma da Aorta Torácica/cirurgia , Implante de Prótese Vascular , Estenose Coronária/complicações , Oxigenação por Membrana Extracorpórea , Complicações Intraoperatórias/terapia , Insuficiência Respiratória/terapia , Idoso , Aneurisma da Aorta Torácica/complicações , Procedimentos Cirúrgicos Cardíacos/métodos , Ponte Cardiopulmonar , Oxigenação por Membrana Extracorpórea/normas , Hemorragia/terapia , Humanos , Pneumopatias/terapia , Masculino , Insuficiência Respiratória/etiologiaRESUMO
The retinoblastoma protein (pRB) and the related pocket proteins, p107 and p130, play crucial roles in mammalian cell cycle control. Recent studies indicate that these pocket proteins are also involved in cellular differentiation processes. We demonstrate in this work that the pRB-related p130 selectively accumulates during the in vitro differentiation of the myeloid progenitor cell, 32Dcl3, into granulocyte in response to granulocyte-colony stimulating factor (G-CSF). This G-CSF-dependent granulocytic differentiation is blocked by the adenovirus E1A oncoprotein, which binds to and inactivates the pRB family of pocket proteins including p130. Furthermore, enforced overexpression of p130 but not pRB inhibits the myeloid cell proliferation that is concomitantly associated with granulocytic differentiation morphologically characterized by nuclear segmentation. However, simple G1-cell cycle arrest induced by cytokine deprivation or ectopic overexpression of the p27 cyclin-dependent kinase inhibitor, or inhibition of E2F activities by dominant negative DP-1 is not sufficient to trigger granulocytic differentiation. The differentiation-promoting activity of p130 in myeloid cells requires both the pocket domain and the spacer domain. Our results indicate that the pRB-related p130 plays a critical role in myeloid cell differentiation and suggest that coupling of cell cycle exit with the cellular differentiation program may be specifically achieved by p130.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Transporte , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Fase G1/fisiologia , Fator Estimulador de Colônias de Granulócitos/farmacologia , Granulócitos/citologia , Células-Tronco Hematopoéticas/citologia , Fosfoproteínas/fisiologia , Proteínas , Proteínas E1A de Adenovirus/fisiologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Fatores de Transcrição E2F , Fase G1/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Camundongos , Fosfoproteínas/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/fisiologia , Proteína do Retinoblastoma/fisiologia , Proteína 1 de Ligação ao Retinoblastoma , Proteína p130 Retinoblastoma-Like , Fator de Transcrição DP1 , Fatores de Transcrição/fisiologiaRESUMO
Cyclin-dependent kinase 2 (Cdk2) controls the transition from the G1 to the S phase in the mammalian cell cycle. We found by immunoblotting that anti-Cdk2 antibodies recognize three Cdk2 proteins (of 33, 34 and 39 kDa) in FRTL-5 and FRTL-Tc cells (malignantly transformed FRTL cells). Although 33 kDa protein is a phosphorylated form of 34 kDa protein previously reported, the nature of 39 kDa protein is unknown. In order to determine the nature of this protein, we screened a FRTL-5 cDNA library. Two cDNA clones of the rat homologue (rat Cdk2-alpha and -beta) of human Cdk2 were isolated. The open reading frame of rat Cdk2-alpha cDNA encoded a protein with 428 amino acids and has a high degree of conservation with human Cdk2. The calculated molecular weight of Cdk2-alpha protein is 33892 Da. The rat Cdk2-beta cDNA was identical to Cdk2-alpha cDNA except that it had extra 144 bp; this coincided with insertion of 48 amino acids into Cdk2-alpha protein between Met 196 and Val 197. The calculated molecular weight of Cdk2-beta protein is 39087 Da. Northern blot analysis indicated that the sizes of rat Cdk2-alpha and -beta mRNAs are approximately 2.5 kb and 3.0 kb, respectively. Partial proteolytic mapping showed that Cdk2-beta gene product is 39 kDa Cdk2 in the immunoblotting. We also found that Cdk2-beta protein binds to cyclin A and suc1 proteins. During G1-S phase in FRTL-Tc cells, Cdk2-alpha protein level is constant, but is gradually phosphorylated. In contrast, the level of Cdk2-beta protein increases through the S phase and decreases at the early G2 phase. These results suggest that a variant form of Cdk2 protein might be required for entry into the S phase of the cell cycle in FRTL-Tc cells.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Quinases Ciclina-Dependentes/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Ciclo Celular , Transformação Celular Neoplásica , Clonagem Molecular , Quinase 2 Dependente de Ciclina , Quinases Ciclina-Dependentes/imunologia , Humanos , Dados de Sequência Molecular , Mapeamento de Peptídeos , Proteínas Serina-Treonina Quinases/imunologia , Ratos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Glândula Tireoide/enzimologiaRESUMO
Butyrolactone I is a selective inhibitor of the cyclin-dependent kinase (cdk) family. It inhibits both cdk2 and cdc2 kinase, but scarcely affects C-kinase, A-kinase, casein kinases, MAP kinase or EGF receptor-tyrosine kinase (Kitagawa et al., 1993, Oncogene, 8, 2425-2432). We studied the effects of butyrolactone I on the cell cycle as well as on phosphorylation of retinoblastoma protein (pRB). Butyrolactone I inhibited phosphorylation of pRB catalyzed by cyclin A-cdk2 produced by baculovirus in vitro. Furthermore, it inhibited phosphorylation of pRB and cell cycle progression from G1 to S phase in WI38 cell cultures. WI38 cells arrested at the G0 phase by serum starvation progressed in the cell cycle after serum stimulation. pRB was phosphorylated after 10 h serum stimulation. Incorporation of [3H]thymidine into the cells began to increase after 16 h serum stimulation. These processes were inhibited by butyrolactone I. Flow cytometric analysis showed that exposure to butyrolactone I inhibited progression of the cell cycle from G1 to S phase. These data suggested that initiation of DNA synthesis was inhibited by butyrolactone I and that the cell cycle was arrested in the G1 phase. Butyrolactone I also inhibited H1 histone phosphorylation in human WI38 cells and their G2/M progression. tsFT210 cells, a temperature-sensitive cdc2 mutant cell line, were synchronized at G2/M at a nonpermissive temperature, butyrolactone I inhibited the cell cycle progression of these cells at G2/M at the permissive temperature. Thus butyrolactone I, a cyclin-dependent kinase family inhibitor, which prevented the phosphorylations of the cell cycle-regulating proteins pRB and H1 histone, inhibited the cell cycle at G1/S and G2/M, respectively. These results suggest that the phosphorylations of pRB and H1 histone may play crucial roles in G1/S and G2/M progression, respectively, although it is possible that phosphorylations of other proteins by cdks are involved in G1/S and G2/M progression.
Assuntos
4-Butirolactona/análogos & derivados , Inibidores de Proteínas Quinases , Proteína do Retinoblastoma/metabolismo , 4-Butirolactona/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Histonas/metabolismo , Humanos , Fosforilação , Timidina/metabolismoRESUMO
We screened cdc2 kinase inhibitors from cultured mediums of micro organisms using purified mouse cyclin B-cdc2 kinase and a specific substrate peptide for cdc2 kinase. A selective inhibitor of cdc2 kinase was isolated from the cultured medium of Aspergillus species F-25799, and identified as butyrolactone I. Butyrolactone I inhibited cdc2 and cdk2 kinases but it had little effect on mitogen-activated protein kinase, protein kinase C, cyclic-AMP dependent kinase, casein kinase II, casein kinase I or epidermal growth factor-receptor tyrosine kinase. Its inhibitory effect was found to be due to competition with ATP. Butyrolactone I selectively inhibited the H1 histone phosphorylation in nuclear extracts. It also inhibited the phosphorylation of the product of retinoblastoma susceptibility gene in nuclear extracts and intact cells. Thus butyrolactone I should be very useful for elucidating the function of cdc2 and cdk2 kinases in cell cycle regulation.
Assuntos
4-Butirolactona/análogos & derivados , Proteína Quinase CDC2/antagonistas & inibidores , Quinases relacionadas a CDC2 e CDC28 , Quinases Ciclina-Dependentes , Inibidores de Proteínas Quinases , Proteínas Serina-Treonina Quinases , 4-Butirolactona/farmacologia , Sequência de Aminoácidos , Aspergillus/química , Proteínas Quinases Dependentes de Cálcio-Calmodulina , Quinase 2 Dependente de Ciclina , Histonas/metabolismo , Técnicas In Vitro , Cinética , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Protamina Quinase/antagonistas & inibidores , Proteína Quinase C/antagonistas & inibidores , Proteína do Retinoblastoma/metabolismo , Especificidade por SubstratoRESUMO
Transcription factor E2F-1 has a putative consensus sequence for phosphorylation by cyclin dependent kinase (Ser-Pro-X-Lys/Arg). Therefore, we studied the phosphorylation of E2F-1 in vivo and in vitro and its biological functions. E2F-1 was prepared by immunoprecipitation with anti-E2F-1 antibody from IMR32 lysates and was effectively phosphorylated by human cyclin A-cdk2 which was expressed in insect cells using baculovirus system. GST-E2F-1 was phosphorylated by cyclin A-cdk2 more efficiently than by cyclin E-cdk2. Cyclin D1-cdk4 phosphorylated pRB but scarcely phosphorylated GST-E2F-1 or H1 histone. The 60 kd protein precipitated with anti-E2F-1 antibody was phosphorylated in vivo. Phospho-peptide mapping indicated that its cleavage profile was identical with that of E2F-1 phosphorylated by cyclin A-cdk2 in vitro. This 60 kd protein, which is likely to be E2F-1, was not phosphorylated during the G0 and early G1 phase. Phosphorylation of E2F-1 began from the S phase while phosphorylation of pRB started nearly at G1/S. The in vivo phosphorylation of E2F-1 was inhibited by butyrolactone I, a cyclin-dependent kinase inhibitor (Kitagawa et al., 1993, Oncogene, 8, 2425-2432). The binding of E2F-1 to E2 promoter was found to be reduced by phosphorylation of E2F-1 by cyclin A-cdk2, suggesting that phosphorylation of E2F-1 may induce shut off of gene expression at the transcriptional level. These results suggest that E2F-1 is phosphorylated by cyclin A-cdk2 in the S phase in vivo as well as in vitro and that its phosphorylation by cyclin A-cdk2 may modulate its activity.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Transporte , Proteínas de Ciclo Celular , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Baculoviridae/genética , Sequência de Bases , Ciclo Celular , Quinase 2 Dependente de Ciclina , DNA Recombinante , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Regulação da Expressão Gênica , Humanos , Técnicas In Vitro , Substâncias Macromoleculares , Dados de Sequência Molecular , Mapeamento de Peptídeos , Fosforilação , Testes de Precipitina , Proteína 1 de Ligação ao Retinoblastoma , Fator de Transcrição DP1 , Células Tumorais CultivadasRESUMO
It has been postulated that the product (pRB) of the retinoblastoma gene dissociates from the E2F-pRB complex upon phosphorylation by cyclin-dependent kinase(s) (cdk). However, there is no direct evident for the regulation of formation of the E2F-pRB complex via phosphorylation by purified cdk. Therefore, we investigated the regulation of formation of this complex by phosphorylation using pRB and purified cyclin A-cdk2, cyclin E-cdk2 or cyclin D1-cdk4. Purified pRB was incubated with nuclear extracts prepared from pRB-defective cells and then subjected to gel mobility shift assays. We confirmed that unphosphorylated pRB associated with various types of E2F but pRB has been phosphorylated by cyclin A-cdk2 did not. We found that E2F-pRB complexes were disrupted as a consequence of phosphorylation by cyclin A-cdk2, and the levels of the free forms of E2Fs increased. We also found that not only the E2F-pRB complexes but also the E2F-p107 complexes were disrupted upon phosphorylation by cyclin A-cdk2. Furthermore, E2F-pRB complexes were disrupted through phosphorylation by cyclin D1-cdk4 and cyclin E-cdk2, as well as by cyclin A-cdk2. These results clearly demonstrate that the phosphorylation of pRB and p107 by cdks regulates the formation of complexes between E2F and pRB or p107.
Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular , Quinases Ciclina-Dependentes/metabolismo , Proteínas Nucleares/metabolismo , Proteína do Retinoblastoma/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Bases , Linhagem Celular , Primers do DNA/química , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição E2F , Humanos , Técnicas In Vitro , Substâncias Macromoleculares , Dados de Sequência Molecular , Fosforilação , Proteína 1 de Ligação ao Retinoblastoma , Proteína p107 Retinoblastoma-Like , Fator de Transcrição DP1RESUMO
Sensitive staining methods with wheat germ agglutinin were developed for the detection of glycosphingolipids of neolacto series (A) and gangliosides with a terminal N-acetylneuraminyl residue (B) on thin-layer chromatograms. (A) Neolacto series glycosphingolipids were treated by beta-galactosidase on the chromatograms in the presence of taurodeoxycholate. Then the chromatograms were incubated with biotinated wheat germ agglutinin followed by incubation with a complex of avidin and biotinated horseradish peroxidase, and the reaction was detected by 4-chloro-1-naphthol. In the case of gangliosides, sialidase treatment on the chromatograms was performed before the beta-galactosidase treatment. The sensitivity of the method for Lc3Cer, nLc4Cer, sialyl-nLc4Cer, and sialyl-nLc6Cer was 4 pmol, 7.6 pmol, 2.9 pmol and 1.4 pmol, respectively. (B) The gangliosides on the chromatograms were oxidized by periodic acid and reduced by NaBH4. Then the chromatograms were stained with wheat germ agglutinin as mentioned above. As little as 0.5 pmol of GM3, NeuAc-nLc4Cer, and NeuAc-nLc6Cer was detected by this method, whereas the detected limits for these gangliosides were 10 pmol, 10 pmol and 2 pmol, respectively, when periodate oxidation was omitted. GM4, GD3 and GD1a were an order less reactive than GM3, GM2, GM1 or GD1b were not stained under the same condition. In contrast to NeuAc-containing gangliosides, any gangliosides with N-glycolylneuraminic acid were not stained by the method in (B).
Assuntos
Gangliosídeos/análise , Glicoesfingolipídeos/análise , Ácidos Siálicos/análise , Cromatografia em Camada Fina/métodos , Indicadores e Reagentes , Microquímica , Ácido N-Acetilneuramínico , Coloração e Rotulagem , Relação Estrutura-Atividade , Aglutininas do Germe de TrigoRESUMO
An immunochemical method has been developed for sensitive detection and determination of ganglio-series gangliosides using thin-layer chromatography/enzyme-immunostaining. After chromatography of gangliosides, the plate was treated with Arthrobacter ureafaciens sialidase to remove all sialic acids from ganglio-series gangliosides. For the complete hydrolysis of gangliosides, sodium taurodeoxycholate was found to be required. The resulting asialo-glycolipids, GA2 and GA1 were reacted first with affinity-purified anti-GA2 and anti-GA1, respectively, and second with horseradish peroxidase-conjugated anti-rabbit IgG. Being highly sensitive and reproducible, it allows the characterization of gangliosides in cerebrospinal fluid which cannot be detected by classical methods.
Assuntos
Gangliosídeos/líquido cefalorraquidiano , Animais , Fenômenos Químicos , Química , Cromatografia em Camada Fina , Humanos , Técnicas Imunoenzimáticas , Métodos , Neuraminidase/metabolismo , Coelhos , Ácido Taurodesoxicólico/farmacologiaRESUMO
The mesencephalic trigeminal nucleus (MesV) contains the somata of primary afferent neurons innervating masticatory muscle spindles and the periodontal membrane. MesV afferent somata are unique in receiving synaptic inputs. Intracellular recordings in coronal pontine slices from adult rats were made from MesV neurons identified by having Cs-sensitive inward rectification and pseudounipolar morphology. Stimuli near the MesV evoked either a cluster of action potentials superimposed on a postsynaptic potential (PSP) or an antidromic spike at resting membrane potential (RMP). Membrane hyperpolarization revealed that each cluster of action potentials consisted of an antidromic spike and a subsequent PSP. Evoked PSPs in slices and miniature postsynaptic currents (mPSCs) recorded using whole-cell patch in dissociated MesV neurons were resistant to glutamate antagonists and strychnine but were reversibly abolished by 40 microM bicuculline. Superfusion of 1-10 mM GABA decreased input resistance and depolarized the membrane. Reversal potentials for evoked PSPs and GABA-induced depolarizations were similar and close to that for mPSCs which matched the Cl- equilibrium potential. Thus activation of synapses on MesV somata evokes GABAergic PSPs that generate action potentials at RMP in the adult. These data also indicate that primary afferent MesV neurons can act as interneurons in the central control of mastication.
Assuntos
Mastigação/fisiologia , Neurônios Aferentes/metabolismo , Transmissão Sináptica/fisiologia , Núcleos do Trigêmeo/metabolismo , Ácido gama-Aminobutírico/metabolismo , Potenciais de Ação/fisiologia , Animais , Estimulação Elétrica , Potenciais Pós-Sinápticos Excitadores/fisiologia , Técnicas In Vitro , Masculino , Músculos da Mastigação/inervação , Mesencéfalo/fisiologia , Microeletrodos , Técnicas de Patch-Clamp , RatosRESUMO
To compare neuroprotective effects of lidocaine and procaine against ischemic insult, intracellular recordings were made from rat hippocampal CA1 pyramidal neurons in slice preparations. Superfusion of the slices with oxygen- and glucose-deprived medium (in vitro ischemia) produced a rapid depolarization 6 min from the onset. When oxygen and glucose were reintroduced, the membrane depolarized further until it reached 0 mV, and thereafter the membrane showed no functional recovery. Pretreatment with lidocaine (10 microM), but not procaine (50 microM), restored the membrane potential after the reintroduction of oxygen and glucose. Lidocaine, compared to procaine, significantly inhibited the reduction in both tissue ATP content and flavoprotein fluorescence during and after in vitro ischemia. Under electron microscopy, only lidocaine well preserved the structure of mitochondria in the CA1 pyramidal cell body. Extracellular recordings revealed that procaine reduced the field postsynaptic potential whereas lidocaine augmented it. Both drugs reduced the presynaptic volley dose-dependently. Neither lidocaine nor procaine significantly affected a rapid rise of the intracellular Ca2+ level produced by in vitro ischemia in the CA1 region. All the results suggest that the neuroprotective lidocaine action is due to the protection of the mitochondria to maintain the tissue ATP content during and after in vitro ischemia.