RESUMO
In this study we aimed to examine a role for interleukin 6 (IL-6) and its receptor (IL-6R) in peripheral nerve regeneration in vivo. We first observed that cultured mouse embryonic dorsal root ganglia exhibited dramatic neurite extension by simultaneous addition of IL-6 and soluble IL-6R (sIL-6R), a complex that is known to interact with and activate a signal transducing receptor component, gp130. After injury in the hypoglossal nerve in adult mice by ligation, immunoreactivity to IL-6 was upregulated in Schwann cells at the lesional site as well as in the cell bodies of hypoglossal neurons in the brain stem. In the latter, upregulation of the immunoreactivity to IL-6R was also observed. Regeneration of axotomized hypoglossal nerve in vivo was significantly retarded by the administration of anti-IL-6R antibody. Surprisingly, accelerated regeneration of the axotomized nerve was achieved in transgenic mice constitutively expressing both IL-6 and IL-6R, as compared with nontransgenic controls. These results suggest that the IL-6 signal may play an important role in nerve regeneration after trauma in vivo.
Assuntos
Antígenos CD/biossíntese , Gânglios Espinais/fisiologia , Regulação da Expressão Gênica , Nervo Hipoglosso/fisiologia , Interleucina-6/biossíntese , Regeneração Nervosa/imunologia , Neurônios/fisiologia , Receptores de Interleucina/biossíntese , Animais , Anticorpos Monoclonais/farmacologia , Antígenos CD/imunologia , Antígenos CD/fisiologia , Células Cultivadas , Embrião de Mamíferos , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/imunologia , Nervo Hipoglosso/imunologia , Imunoglobulina G/farmacologia , Interleucina-6/farmacologia , Camundongos , Camundongos Endogâmicos ICR , Neuritos/efeitos dos fármacos , Neuritos/imunologia , Neuritos/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/imunologia , Receptores de Interleucina/imunologia , Receptores de Interleucina/fisiologia , Receptores de Interleucina-6RESUMO
We reported that interleukin (IL) 6 alone cannot induce osteoclast formation in cocultures of mouse bone marrow and osteoblastic cells, but soluble IL-6 receptor (IL-6R) strikingly triggered osteoclast formation induced by IL-6. In this study, we examined the mechanism of osteoclast formation by IL-6 and related cytokines through the interaction between osteoblastic cells and osteoclast progenitors. When dexamethasone was added to the cocultures, IL-6 could stimulate osteoclast formation without the help of soluble IL-6R. Osteoblastic cells expressed a very low level of IL-6R mRNA, whereas fresh mouse spleen and bone marrow cells, both of which are considered to be osteoclast progenitors, constitutively expressed relatively high levels of IL-6R mRNA. Treatment of osteoblastic cells with dexamethasone induced a marked increase in the expression of IL-6R mRNA. By immunoblotting with antiphosphotyrosine antibody, IL-6 did not tyrosine-phosphorylate a protein with a molecular mass of 130 kD in osteoblastic cells but did so in dexamethasone-pretreated osteoblastic cells. Osteoblastic cells from transgenic mice constitutively expressing human IL-6R could support osteoclast development in the presence of human IL-6 alone in cocultures with normal spleen cells. In contrast, osteoclast progenitors in spleen cells from transgenic mice overexpressing human IL-6R were not able to differentiate into osteoclasts in response to IL-6 in cocultures with normal osteoblastic cells. These results clearly indicate that the ability of IL-6 to induce osteoclast differentiation depends on signal transduction mediated by IL-6R expressed on osteoblastic cells but not on osteoclast progenitors.
Assuntos
Antígenos CD/fisiologia , Interleucina-6/farmacologia , Osteoblastos/metabolismo , Receptores de Interleucina/fisiologia , Regulação para Cima , Animais , Antígenos CD/biossíntese , Antígenos CD/genética , Células da Medula Óssea , Diferenciação Celular/efeitos dos fármacos , Técnicas de Cocultura , Dexametasona/farmacologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoclastos/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Quinases/metabolismo , RNA Mensageiro/biossíntese , Receptores de Interleucina/biossíntese , Receptores de Interleucina/genética , Receptores de Interleucina-6 , Proteínas Recombinantes/biossíntese , Transdução de Sinais , Crânio/citologia , Baço/citologia , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismoRESUMO
We described recently the activation of the Janus kinasesignal transducer and activator of transcription (JakSTAT) and mitogen-activated protein (MAP) kinase pathways by leukemia inhibitory factor (LIF) through gp130, a signal transducer of IL-6-related cytokines, that transduces hypertrophic signals in cardiac myocytes. In addition, stimulation of gp130 by IL-6-related cytokines is known to exert a cytoprotective effect. In the present study, we investigated the possibility that activation of gp130 initiates activation of the cytoprotective genes in cardiac myocytes. Incubation of cardiac myocytes with LIF induced the expression of bcl-x, and the isoform that was induced by LIF was identified as bcl-xL. Induction of bcl-xL protein was also identified by Western blotting. Antisense oligonucleotide against bcl-x mRNA inhibited protective effect of LIF accompanied with the reduction in bclxL protein. We constructed bcl-x promoter-luciferase reporter gene plasmids (-639/+10- or -161/+10-luciferase), and transfected them to cardiac myocytes. LIF stimulation increased the luciferase activity of -639/+10-luciferase plasmids. Although -161/+10-luciferase plasmids presented comparable responsiveness to LIF, the basal transcription level was impaired. The LIF-responsive cis-element was localized to a DNA fragment (positions -161 to +10) that contains an interferon-gamma activation site (GAS) motif (GGA) at position -41 of the bcl-x gene promoter. This motif bound to STAT1, not to STAT3, and site-directed mutagenesis revealed that this motif was essential for LIF-responsive promoter activity. These data suggest that LIF induces bcl-x mRNA via STAT1 binding cis-element in cardiac myocytes, presenting cytoprotective effect.
Assuntos
Antígenos CD/fisiologia , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Interleucina-6 , Glicoproteínas de Membrana/fisiologia , Miocárdio/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais , Transativadores/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Receptor gp130 de Citocina , DNA/metabolismo , Inibidores do Crescimento/farmacologia , Fator Inibidor de Leucemia , Linfocinas/farmacologia , Camundongos , Norepinefrina/farmacologia , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes , Fator de Transcrição STAT1 , Proteína bcl-XRESUMO
Pituitary apoplexy occurs as a very rare complication of the pituitary function test. We have experienced two cases of pituitary apoplexy following anterior pituitary function tests for preoperative assessment: a triple bolus test and a TRH test. To elucidate such a rare complication, we outline our two cases and review 28 cases from the literature. The clinical characteristics, etiology, pathophysiology, and diagnostic and therapeutic implications are also discussed. The combined data suggest that pituitary function tests have the potential to precipitate pituitary apoplexy, and its manifestations range from a clinically benign event to a catastrophic presentation with permanent neurological deficits or even death, although most patients may fortunately have a good outcome. We suggest that the pituitary function test should not be done as a routine test, and when such a test is planned, the patient should be observed with caution for any symptomatic changes for at least 2 hours following the test for appropriate treatment. Further, MRI, especially enhanced studies, may provide an earlier diagnosis of the pituitary apoplexy since CT scan images often fail to demonstrate either density changes or obvious enlargement of the pituitary adenoma at the acute stage.
Assuntos
Adenoma Acidófilo/cirurgia , Hormônio Liberador de Gonadotropina/efeitos adversos , Apoplexia Hipofisária/induzido quimicamente , Testes de Função Hipofisária/efeitos adversos , Neoplasias Hipofisárias/cirurgia , Hormônio Liberador de Tireotropina/efeitos adversos , Adenoma Acidófilo/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Diagnóstico Diferencial , Feminino , Humanos , Hipofisectomia , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Apoplexia Hipofisária/diagnóstico , Apoplexia Hipofisária/cirurgia , Hipófise/patologia , Neoplasias Hipofisárias/diagnóstico , Cuidados Pré-Operatórios , Reoperação , Tomografia Computadorizada por Raios XRESUMO
We have shown previously that NAD/poly(ADP-ribose) polymerase-deficient cells that overexpress Mr 78,000 glucose-regulated stress protein (GRP78) are resistant to topoisomerase II inhibitors, such as etoposide, m-amsacrine, and doxorubicin. However, these cells have been found to be hypersensitive to DNA cross-linking agents, including melphalan, cisplatin, and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). These observations prompted us to examine whether overexpression of GRP78 is associated with modulation of cytotoxicity of clinically useful DNA-cross-linking agents such as melphalan, BCNU, and cisplatin. We up-regulated GRP78 in V79 Chinese hamster cells by 2-5-fold using two independent approaches that include exposure to 6-aminonicotinamide, or 2-deoxyglucose. Subsequently, these GRP78-overexpressing cells were trypsinized, plated in regular medium without GRP78-inducing agents, and allowed a 5-h attachment time before being treated with melphalan, BCNU, or cisplatin for 1 h to determine clonogenic survivals. In addition, repair of DNA cross-links induced by those agents were determined by alkaline elution assay. Our results show that the GRP78-overexpressing V79 cells are hypersensitive to DNA cross-linking agents compared to the control V79 cells. Furthermore, repair of drug-induced DNA cross-links appears to be considerably slower in these cells relative to that found in control V79 cells. Thus, our results suggest that (a) up-regulation of GRP78 is associated with an impairment of DNA cross-link repair, (b) up-regulation of GRP78 is associated with potentiation of cytotoxicity induced by alkylating and platinating agents, and (c) up-regulation of GRP78 can be considered as a potentially useful tool to modulate the cytotoxicity of clinically useful alkylating and platinating agents.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Proteínas de Transporte/metabolismo , Reparo do DNA , DNA de Neoplasias/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Regulação para Cima , 6-Aminonicotinamida/farmacologia , Animais , Antimetabólitos/farmacologia , Carmustina/farmacologia , Linhagem Celular/efeitos dos fármacos , Cisplatino/farmacologia , Cricetinae , Desoxiglucose/farmacologia , Chaperona BiP do Retículo Endoplasmático , Melfalan/farmacologia , Teratogênicos/farmacologiaRESUMO
We detected a novel kind of bile acid in the content of chronic subdural hematoma. This substance was specifically found in chronic subdural hematoma, and not in subdural hygroma, which is pathologically similar except for the lack of capsular membrane. The compound was identified as 7 alpha-hydroxy-3-oxo-4-cholestenoic acid by high performance liquid chromatography, gas chromatography-mass spectrometry, and nuclear magnetic resonance spectrometry. The structure was confirmed by the comparison with the chemically synthesized compound. The average contents in chronic subdural hematoma were 658.09 +/- 137.53 ng/ml, while those in normal human plasma were 126.27 +/- 17.73 ng/ml. It was not detected in normal cerebrospinal fluid. The higher level in chronic subdural hematoma than human plasma strongly suggests the local, extrahepatic production of this type of C27 bile acids.
Assuntos
Ácidos e Sais Biliares/metabolismo , Colestenonas/metabolismo , Hematoma Subdural/metabolismo , Ácidos e Sais Biliares/sangue , Ácidos e Sais Biliares/líquido cefalorraquidiano , Ácidos e Sais Biliares/química , Colestenonas/sangue , Colestenonas/líquido cefalorraquidiano , Colestenonas/química , Cromatografia Líquida de Alta Pressão , Doença Crônica , Cromatografia Gasosa-Espectrometria de Massas , Hematoma Subdural/etiologia , Humanos , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Espectrofotometria UltravioletaRESUMO
BACKGROUND: Mice with cardiac-specific overexpression of signal transducer and activator of transcription 3 (STAT3) are resistant to doxorubicin-induced damage. The STAT3 signal may be involved in the detoxification of reactive oxygen species (ROS). METHODS AND RESULTS: The effects of leukemia inhibitory factor (LIF) or adenovirus-mediated transfection of constitutively activated STAT3 (caSTAT3) on the intracellular ROS formation induced by hypoxia/reoxygenation (H/R) were examined using rat neonatal cardiomyocytes. Either LIF treatment or caSTAT3 significantly suppressed the increase of H/R-induced ROS evaluated by 2',7'-dichlorofluorescin diacetate fluorescence. To assess whether ROS are really involved in H/R-induced cardiomyocyte injury, the amount of creatine phosphokinase in cultured medium was examined. Both LIF treatment and caSTAT3 significantly decreased H/R-induced creatine phosphokinase release. These results indicate that the gp130/STAT3 signal protects H/R-induced cardiomyocyte injury by scavenging ROS generation. To investigate the mechanism of scavenging ROS, the effects of LIF on the induction of antioxidant enzymes were examined. LIF treatment significantly increased the expression of manganese superoxide dismutase (MnSOD) mRNA, whereas the expression of the catalase and glutathione peroxidase genes were unaffected. This induction of MnSOD mRNA expression was completely blocked by adenovirus-mediated transfection of dominant-negative STAT3. Moreover, caSTAT3 augmented MnSOD mRNA and its enzyme activity. In addition, the antisense oligodeoxyribonucleotide to MnSOD significantly inhibited both LIF and caSTAT3-mediated protective effects. CONCLUSIONS: The activation of STAT3 induces a protective effect on H/R-induced cardiomyocyte damage, mainly by inducting MnSOD. The STAT3-mediated signal is proposed as a therapeutical target of ROS-induced cardiomyocyte injury.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Interleucina-6 , Miocárdio/metabolismo , Estresse Oxidativo , Superóxido Dismutase/metabolismo , Transativadores/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Proteínas de Ligação a DNA/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Inibidores do Crescimento/farmacologia , Fator Inibidor de Leucemia , Linfocinas/farmacologia , Miocárdio/citologia , Estresse Oxidativo/efeitos dos fármacos , Oxigênio/farmacologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fator de Transcrição STAT3 , Superóxido Dismutase/genética , Transativadores/genética , Regulação para Cima/efeitos dos fármacosRESUMO
PURPOSE: We conducted a phase I and pharmacologic study of a weekly 96-hour infusion of irinotecan to determine the maximum-tolerated dose, define the toxicity profile, and characterize the clinical pharmacology of irinotecan and its metabolites. PATIENTS AND METHODS: In 26 adult patients with solid tumors, the duration and dose rate of infusion were escalated in new patients until toxicity was observed. RESULTS: In 11 patients who were treated with irinotecan at 12.5 mg/m(2)/d for 4 days weekly for 2 of 3 weeks, dose-limiting grade 3 diarrhea occurred in three patients and grade 3 thrombocytopenia occurred in two patients. The recommended phase II dose is 10 mg/m(2)/d for 4 days given weekly for 2 of 3 weeks. At this dose, the steady-state plasma concentration (Css) of total SN-38 (the active metabolite of irinotecan) was 6.42 +/- 1.10 nmol/L, and the Css of total irinotecan was 28.60 +/- 17.78 nmol/L. No patient experienced grade 3 or 4 neutropenia during any cycle. All other toxicities were mild to moderate. The systemic exposure to SN-38 relative to irinotecan was greater than anticipated, with a molar ratio of the area under the concentration curve (AUC) of SN-38 to irinotecan of 0.24 +/- 0.08. One objective response lasting 12 months in duration was observed in a patient with metastatic colon cancer. CONCLUSION: The recommended phase II dose of irinotecan of 10 mg/m(2)/d for 4 days weekly for 2 of 3 weeks was extremely well tolerated. Further efficacy testing of this pharmacologic strategy of administering intermittent low doses of irinotecan is warranted.
Assuntos
Camptotecina/análogos & derivados , Neoplasias/tratamento farmacológico , Adulto , Idoso , Antineoplásicos Fitogênicos/administração & dosagem , Antineoplásicos Fitogênicos/efeitos adversos , Antineoplásicos Fitogênicos/farmacocinética , Camptotecina/administração & dosagem , Camptotecina/efeitos adversos , Camptotecina/sangue , Camptotecina/farmacocinética , Camptotecina/farmacologia , Esquema de Medicação , Feminino , Seguimentos , Doenças Hematológicas/induzido quimicamente , Humanos , Infusões Intravenosas , Irinotecano , Masculino , Pessoa de Meia-Idade , Náusea/induzido quimicamente , Neoplasias/sangue , Vômito/induzido quimicamenteRESUMO
gp130 was initially identified as a signal-transducing receptor component that associates with the interleukin 6 receptor (IL-6R) when the receptor is occupied by interleukin 6 (IL-6). It has been revealed that the receptor complexes for IL-6, IL-11, leukemia inhibitory factor (LIF), oncostatin M (OM), and ciliary neurotrophic factor (CNTF) utilize this gp130 protein as a common signal-transducing component, explaining how these cytokines mediate overlapping biological function. Recent observations with mice lacking gp130 or having continuously activated gp130 protein have disclosed an important biological function of gp130 in cardiovascular system: the former mice show extremely hypoplastic development of the ventricular myocardium at 16.5 days postcoitum (dpc), and the latter exhibit hypertrophy of myocardium. These cardiovascular abnormalities are considered to be the results of the perturbation of gp130, which also transduces the signal of cardiotrophin-1 (CT-1), a recently isolated factor causing hypertrophy in cultured cardiomyocytes and having sequence similarity with IL-6, IL-11, LIF, CNTF, and OM. In fact, CT-1 shares gp130 with these cytokines as a critical signaling component. Besides various well-established mechanisms by which cardiac growth and development are regulated, a gp130 signaling may be a newly discovered mechanism that regulates these events.
RESUMO
Recombinant human granulocyte colony-stimulating factor (rhG-CSF) (10 ng/mL) prolonged human neutrophil survival in culture by at least 36 hours. The addition of H-series compounds at concentrations that are considered to inhibit both protein kinase C (PKC) and cyclic adenylate monophosphate (cAMP)-dependent protein kinase (PKA) counteracted the effect of rhG-CSF. Concomitantly, the inhibition of nucleosomal DNA fragmentation by rhG-CSF was canceled. At lower concentrations, presumably capable of inhibiting only PKA, however, the compounds exhibited marginal effects on rhG-CSF-mediated increase of cell survival. These PKC inhibitors did not influence the priming effect of rhG-CSF significantly, as determined by O2- production stimulated by N-formyl-L-methionyl-L-leucyl phenylalanine (fMLP). Our results suggest that PKC plays an important role in the mechanism by which rhG-CSF promotes neutrophil survival, in striking contrast with the priming effect elicited by rhG-CSF.
Assuntos
Sobrevivência Celular , Fator Estimulador de Colônias de Granulócitos/farmacologia , Neutrófilos/fisiologia , Proteína Quinase C/sangue , Adulto , Fosfatase Alcalina/sangue , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , AMP Cíclico/farmacologia , DNA/sangue , Humanos , Cinética , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases , Proteínas Quinases/sangue , Proteínas Recombinantes/farmacologia , Superóxidos/sangueRESUMO
An in vitro study was performed on the effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) and adenine on the survival of purified human neutrophils. The addition of rhG-CSF (1 to 100 ng/mL) or adenine (100 microM) enhanced the survival of neutrophils. The maintenance of O2- production in response to N-formylmethionyl-leucyl-phenyl-alanine (FMLP) suggested that these neutrophils were functionally alive. Neutrophils in cultures had shown two distinct biochemical changes during cell death: DNA fragmentation and depletion of cellular adenosine triphosphate (ATP) pools. Treatment with rhG-CSF (10 ng/mL) significantly delayed the appearance of DNA fragmentation as measured quantitatively by diphenylamine or by agarose gel electrophoresis. On the other hand, adenine had no effect on the generation of DNA fragmentation. The decrease of ATP during incubation for 12 hours was similar in control and rhG-CSF-treated neutrophils, while rhG-CSF prevented the further decline of ATP seen in control cultures. In contrast, adenine (100 microM) preserved ATP at levels significantly higher than in controls at both 12 hours and 24 hours of incubation. Our results suggest that rhG-CSF and adenine promote the survival of neutrophils in vitro by different mechanisms.
Assuntos
Adenina/farmacologia , Fator Estimulador de Colônias de Granulócitos/farmacologia , Neutrófilos/citologia , Trifosfato de Adenosina/metabolismo , Adulto , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Citidina Trifosfato/metabolismo , Dano ao DNA/efeitos dos fármacos , Guanosina Trifosfato/metabolismo , Humanos , Neutrófilos/metabolismo , Proteínas Recombinantes/farmacologia , Superóxidos/sangue , Superóxidos/metabolismo , Uridina Trifosfato/metabolismoRESUMO
We investigated the effects of stem cell factor (SCF) on the growth of blast clonogenic cells from 27 patients with acute myeloblastic leukemia (AML) and 3 patients with chronic myelocytic leukemia in myeloid crisis. SCF alone showed a significant stimulatory activity in 15 of 30 patients (50%). A marked reduction in the number of blast cell colonies supported by SCF alone was noted by the addition of neutralizing antibody (Ab) against granulocyte-macrophage colony-stimulating factor (GM-CSF). Ab against interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) also moderately reduced the number of colonies, whereas Ab against granulocyte CSF (G-CSF) failed to do so. All four Ab together completely abolished the growth in 5 of 6 patients tested. c-kit antisense oligonucleotides reduced the colony formation supported by IL-3 or G-CSF or, in the absence of growth factor, in only 2 of 10 patients tested. SCF caused stimulation by acting synergistically with G-CSF, GM-CSF, IL-3, IL-6, IL-9, IL-11, and IL-12 in 20 of 27 (74%), 17 of 27 (63%), 14 of 28 (50%), 9 of 28 (32%), 1 of 15 (7%), 3 of 28 (11%), and 2 of 15 (13%) patients, respectively. Thus, SCF alone or in combination with some other factor stimulated the growth in 27 of 30 (90%) patients. Of 3 nonresponders, 2 were AML, M3 at presentation. G-CSF at the optimal concentration increased the sensitivity of blasts to SCF. Taken together, SCF acting in combination with other factors, but not alone, stimulates the growth of blast clonogenic cells. GM-CSF, IL-6, and TNF-alpha may be produced endogenously, whereas G-CSF and SCF may be supplied exogenously. Autocrine regulation of the growth of blasts seems to increase the responsiveness of the cells to any of these factors, allowing them to achieve a highly active growth state.
Assuntos
Crise Blástica/tratamento farmacológico , Ensaio de Unidades Formadoras de Colônias , Fatores Estimuladores de Colônias/farmacologia , Interleucinas/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Fator de Células-Tronco/farmacologia , Sequência de Bases , Crise Blástica/patologia , Humanos , Leucemia Mieloide Aguda/patologia , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/farmacologia , Células Tumorais CultivadasRESUMO
We investigated the effect of aphidicolin, an inhibitor of DNA polymerase alpha and delta, on the induction of apoptosis by arabinosyl nucleosides in a human promyelocytic leukemia cell line, HL-60. Pretreatment of HL-60 cells with aphidicolin (2 microM) significantly increased the number of morphologically apoptotic cells induced by 1-beta-D arabinofuranosylcytosine (ara-C) during 4 hr of incubation. This is consistent with the appearance of DNA fragmentation as determined quantitatively by diphenylamine or by agarose gel electrophoresis. The inhibition of cell growth on day 3 after drug exposure was correlated with the degree of apoptosis: Such synergistic interaction between aphidicolin and ara-C has also been observed in other human myeloid leukemia cell lines, U937 and KG-1. In addition, the induction of apoptosis by 9-beta-D arabinofuranosyladenine or 9-beta-D arabinofuranosylguanine is augmented by aphidicolin.
Assuntos
Afidicolina/farmacologia , Apoptose/efeitos dos fármacos , Arabinonucleosídeos/farmacologia , Arabinonucleosídeos/biossíntese , DNA/metabolismo , Sinergismo Farmacológico , Eletroforese em Gel de Ágar , Humanos , Leucemia Mieloide , Células Tumorais Cultivadas/efeitos dos fármacos , Vidarabina/farmacologiaRESUMO
We examined the effect of pyrrolidine dithiocarbamate (PDTC), which potently blocks the activation of nuclear factor kappa B (NF-kappa B), on the induction of apoptosis by a variety of agents. Treatment of a human promyelocytic leukemia cell line, HL-60, with 10 micrograms/mL etoposide or 2 microM 1-beta-D-arabinofuranosylcytosine induced NF-kappa B activation within 1 hr and subsequently caused apoptosis within 3-4 hr. The simultaneous addition of 50-500 microM PDTC with these agents blocked NF-kappa B activation and completely abrogated both morphologically apoptotic changes and internucleosomal DNA fragmentation for up to 6 hr. However, PDTC failed to inhibit the endonuclease activity contained in the whole cell lysates. The inhibitory effect of PDTC was also observed in etoposide- and dexamethasone-induced apoptosis in human thymocytes at a concentration of 1-10 microM. Since PDTC has both antioxidant and metal-ion chelating activities, we tested the effects of N-acetyl-L-cysteine (NAC) (antioxidant) or o-phenanthroline (OP) (metal-ion chelator) on the induction of apoptosis. Pretreatment of HL-60 cells or thymocytes with 100-500 microM OP for 2 hr, but not 10-60 mM NAC, suppressed subsequent occurrence of apoptosis induced by etoposide. These results suggest that the activation of NF-kappa B plays an important role in the apoptotic process of human hematopoietic cells.
Assuntos
Apoptose/efeitos dos fármacos , Leucemia Promielocítica Aguda/patologia , NF-kappa B/antagonistas & inibidores , Pirrolidinas/farmacologia , Linfócitos T/efeitos dos fármacos , Tiocarbamatos/farmacologia , Acetilcisteína/farmacologia , Sequência de Bases , Citarabina/farmacologia , Endonucleases/antagonistas & inibidores , Etoposídeo/farmacologia , Humanos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Fenantrolinas/farmacologia , Linfócitos T/citologia , Timo/citologia , Células Tumorais CultivadasRESUMO
Synergistic antiproliferative effect has been proven in vitro when mouse leukemic cells were sequentially treated with MTX and ara-C. The mechanism of this combination effect not well elucidated but the intracellular uptake of ara-C was higher when cells were pre-exposed to MTX. In this experiment, the intracellular ara-CTP was measured by HPLC after MTX and ara-C were sequentially administered to BDF1 mice bearing L1210 leukemic cell, either being sensitive or resistant to MTX. When MTX at the dose of 12 mg/kg was preceded 6 h and 3 h to ara-C at the dose of 25 mg/kg, the intracellular levels of ara-CTP were found to be significantly higher as compared with those of ara-C alone as control group. At 1 h after ara-C, ara-CTP was measured about 165 and 130% of the control levels, respectively, and at 12 h, ara-CTP was over 4 times higher of control level with group of mice to which MTX was preceded 6 h prior to MTX. On the contrary, the enhancement of ara-CTP production was definitely diminished with MTX-resistant cells in the same administrative model. From our present experiment, the time sequential modulation of intracellular ara-CTP production by MTX was reconfirmed in vivo, and this modulation might depend upon the sensitivity of MTX of leukemic cells.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Arabinofuranosilcitosina Trifosfato/biossíntese , Arabinonucleotídeos/biossíntese , Citarabina/administração & dosagem , Metotrexato/administração & dosagem , Animais , Nucleotídeos de Desoxicitosina/análise , Esquema de Medicação , Leucemia L1210/metabolismo , Masculino , Camundongos , Camundongos EndogâmicosRESUMO
Williams syndrome (WS) is characterized by distinct facial changes, growth deficiency, mental retardation, and congenital heart defect (particularly supravalvular aortic stenosis), associated at times with infantile hypercalcemia. Molecular genetic studies have indicated that hemizygosity at the elastin locus (7q11.23) causes WS. The purpose of this study was to confirm that this regional deletion, involving the elastin locus, is the cause of WS in Japan, and to clarify the correlation between the phenotype and the elastin locus. Thirty-two patients with WS and thirty of their relatives were examined by fluorescent in situ hybridization (FISH), using the WS chromosome region (WSCR) probe. All patients had cardiovascular disease (100%), 30 had typical WS facial changes (94%), 31 had mental retardation or developmental delay (97%), 16 were small-for-date at birth (50%), 14 had short stature (44%), and 13 had dental anomalies (41%). No relatives showed any manifestation of WS. Hemizygosity for a region of 7q11.23, involving the elastin locus, was found in all WS patients, but was not found in the 30 relatives.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 7/genética , Hibridização In Situ/métodos , Síndrome de Williams/diagnóstico , Síndrome de Williams/genética , Adolescente , Adulto , Angiocardiografia , Criança , Pré-Escolar , Sondas de DNA , Elastina/genética , Feminino , Humanos , Lactente , Masculino , Síndrome de Williams/diagnóstico por imagemRESUMO
Various fatty acyl-CoAs are involved as intermediates or precursors of sex pheromone components in the biosynthetic pathway of the pheromones in many lepidopteran insects. We have purified a 10-kDa protein from the cytosolic fraction of Bombyx mori pheromone glands by using affinity chromatography with a palmitoyl-CoA-agarose column and reversed-phase HPLC. Amino acid sequence analysis of the fragment peptides obtained from the purified protein, and a homology search, revealed that this protein was a member of acyl-CoA-binding proteins (ACBPs). MALDI-TOF mass spectral analysis of the purified protein and cloning of the gene from a pheromone gland cDNA library confirmed B. mori ACBP to be a 90 amino acid protein with 78.9% identity to that of Manduca sexta ACBP. The secondary structure of the recombinant B. mori ACBP was determined by NMR spectroscopy. Northern blot analysis demonstrated that B. mori ACBP was predominantly expressed in the pheromone gland and the corresponding transcript was expressed from the day before adult eclosion. Present results suggest that ACBP plays a significant role in the production of sex pheromones regulated by the neurohormone, pheromone biosynthesis activating neuropeptide (PBAN).
Assuntos
Acil Coenzima A , Bombyx/química , Proteínas de Transporte/análise , Atrativos Sexuais , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting/métodos , Proteínas de Transporte/genética , Bovinos , DNA Complementar , Inibidor da Ligação a Diazepam , Humanos , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Análise de Sequência de DNA , Análise de Sequência de Proteína , Homologia de Sequência de AminoácidosRESUMO
Systemic administration of propentofylline (PPF), an adenosine uptake inhibitor, has been demonstrated to protect CA1 pyramidal cells from death following transient cerebral ischemia in gerbils. In order to examine the direct effects of this inhibitor, we tested whether or not PPF administered into the hippocampus in situ through a microdialysis probe could attenuate ischemia-induced excitatory amino acid (EAA) release and prevent subsequent death of CA1 pyramidal cells in the gerbil. The EAA release and death of CA1 pyramidal cells observed in the hippocampus were compared with those in the contralateral hippocampus of the same animal into which vehicle alone was administered. The results indicated that pre- as well as post-treatments with PPF inhibited the death of CA1 pyramidal cells after 5-min ischemia in a dose-dependent manner, but did not significantly alter the EAA release during ischemia and reperfusion in the same animals. While the neuroprotective effect of PPF against ischemic damage has commonly been ascribed to attenuation of EAA release during ischemia, other actions of adenosine such as those influencing the synaptic responses, neuronal excitation, and local cerebral circulation, or as yet unidentified actions may be involved in the observed neuroprotective effects of PPF.
Assuntos
Aminoácidos/metabolismo , Hipocampo/efeitos dos fármacos , Ataque Isquêmico Transitório/patologia , Xantinas/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Gerbillinae , Hipocampo/metabolismo , Hipocampo/patologia , Ataque Isquêmico Transitório/metabolismo , Microdiálise , Células Piramidais/efeitos dos fármacosRESUMO
Purple-coloured dandelion (Taraxacum officinale) callus cultures producing anthocyanin pigments were established on a cytokinin-rich medium under the light. When the cells were placed in the dark, only grey cells proliferated. Anthocyanin productivity of these cells was partially restored in the light. The major pigment was identified as cyanidin 3-(6"-malonylglucoside). The lower stem of the original plant contained the same pigment. Chalcone synthase (CHS) activity was detected in the extracts of these purple cells, whereas no activity was observed in grey cells propagated in the dark. When the CHS-active cell-free extract was combined with the extract of Escherichia coli over expressing polyketide reductase (PKR) cDNA of licorice (Glycyrrhiza echinata), isoliquiritigenin (a 6'-deoxychalcone), in addition to naringenin (a 5-hydroxyflavanone), was detected as the reaction product from 4-coumaroyl-CoA, malonyl-CoA and NADPH. This result confirms the catalytic function of the PKR gene product.
Assuntos
Aciltransferases/metabolismo , Antocianinas/biossíntese , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Proteínas de Bactérias/metabolismo , Células Cultivadas , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Glycyrrhiza/enzimologia , Extratos Vegetais/metabolismo , Extratos Vegetais/farmacologia , Plantas/enzimologia , Plantas MedicinaisRESUMO
Xanthogranulomas of the dura presenting with clinical symptoms are rare. We report here a case of a huge dural xanthogranuloma in the middle cranial fossa, which caused hemiparesis through marked displacement and stenosis of the middle cerebral artery. Although such tumors usually arise in association with histiocytosis X or familial hyperlipoproteinemia, the present case was not associated with these diseases.