RESUMO
The aim of the current study was to examine whether analysis of the appearance of specific lectin-positive substances in the quail embryonic cloacal gland would be useful for evaluating the androgenic and antiandrogenic effects of chemicals. Fertilized Japanese quail eggs were injected with 0 to 75 µg of cyproterone acetate (CA), an antiandrogenic compound, on d 12 of incubation (d 12), followed by injection of 0 to 300 µg of testosterone propionate (TP) on d 13. Experimental groups consisted of a control group (corn oil injections on d 12 and 13), a TP-L group [corn oil and a low dose (L; 30 µg) of TP], a TP-H group [corn oil and a high dose (H; 300 µg) of TP], a CA-L + TP-H group [a low dose (L;7.5 µg) of CA + TP-H], and a CA-H + TP-H group [a high dose (H; 75 µg) of CA + TP-H]. The cloacal tissues were collected on d 16, processed into paraffin sections, and stained using 14 different biotinylated lectins. The Vicia villosa (VVA) lectin most strongly stained the developing cloacal glandular cells in TP-H. Western blotting analysis showed 1 VVA-positive band of approximately 75 kDa. The ratio of VVA-positive areas per unit square examined microscopically by image analysis was significantly greater in the TP-H group than in the control group in both males and females. The ratio was significantly decreased in the CA-L + TP-H and CA-H + TP-H groups compared with the TP-H group in both males and females. Furthermore, the ratio was smaller in females than in males within a TP-L or TP-H treatment group. These results suggest that lectin histochemistry on quail embryonic cloaca using VVA is useful for evaluating the androgenic and antiandrogenic effects of chemical compounds.
Assuntos
Coturnix , Acetato de Ciproterona/toxicidade , Imuno-Histoquímica/veterinária , Lectinas/metabolismo , Propionato de Testosterona/toxicidade , Antagonistas de Androgênios/toxicidade , Androgênios/toxicidade , Animais , Coturnix/embriologia , Feminino , MasculinoRESUMO
The lipid modifications which occur on Bombyx mori Ras proteins BmRas1, BmRas2 and BmRas3 were studied by metabolic labelling in an insect cell-free protein synthesis system and in a baculovirus expression system, using specific inhibitors of protein prenylation and protein palmitoylation. In addition, the subcellular localization of BmRas proteins was examined using EGFP fusion proteins of constitutively active forms of BmRas proteins transiently expressed in Sf9 cells. As a result, it was revealed that the three B. mori Ras proteins BmRas1, BmRas2 and BmRas3 are neither farnesylated nor palmitoylated but are geranylgeranylated for localization to the plasma membrane of insect cells. Thus, the mechanism of membrane binding of insect Ras proteins is quite different from that reported for mammalian Ras proteins.
Assuntos
Bombyx/metabolismo , Proteínas de Insetos/metabolismo , Lipoilação , Prenilação , Proteínas ras/metabolismo , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Bombyx/citologia , Linhagem Celular , Membrana Celular/metabolismo , Sistema Livre de Células , Evolução Molecular , Vetores Genéticos/genética , Humanos , Proteínas de Insetos/química , Proteínas de Insetos/genética , Dados de Sequência Molecular , Biossíntese de Proteínas , Transporte Proteico , Coloração e Rotulagem , Proteínas ras/química , Proteínas ras/genéticaRESUMO
The aim of the current study was to establish the sensitive embryonic endpoints and a test system for detecting androgenic and anti-androgenic potential of chemicals using an in ovo treatment assay in Japanese quail. In ovo injection with 0 to 75 microg of cyproterone acetate (CA) was performed on d 12 of incubation, followed by 0 to 300 microg of testosterone propionate (TP) injection on d 13 and histological examination on d 16. Experimental groups were composed of control (twice injected corn oil injections; on d 12 and d 13, respectively), TP-L (corn oil and 30 microg of TP), TP-H (corn oil and 300 microg of TP), CA-L + TP-H (7.5 microg of CA and 300 microg of TP), and CA-H+ TP-H (75 microg of CA and 300 microg of TP). Histological examinations were performed in the cloacal gland, liver, kidneys, testes, ovaries, uropygial gland, and bursa of Fabricius. The cloacal gland consists of many glandular units (tubular gland structures) lined by developed or undeveloped glandular cells. The developed glandular cells were tall in height and contained mucous substance in the cytoplasm. The glandular units containing developed glandular cells were termed as the developing glandular units. The developing glandular units were observed in the TP-H, CA-L + TP-H, and CA-H + TP-H groups, but not in the control and TP-L groups, in both males and females. The ratio of developing glandular units to the total number of glandular units was significantly greater in TP-H than control and TP-L and was significantly decreased in CA-L + TP-H and CA-H + TP-H compared with TP-H in both males and females. The ratio was significantly greater in males than in females of CA-L + TP-H. No significant structural differences were observed in the other organs. These results suggest that the most sensitive endpoint of androgenic effects in quail embryo appeared in the cloacal glands. The ratio of the developing glandular units could be used for evaluation of androgenic and anti-androgenic effects of compounds.
Assuntos
Antagonistas de Androgênios/toxicidade , Androgênios/toxicidade , Coturnix/embriologia , Acetato de Ciproterona/toxicidade , Óvulo/efeitos dos fármacos , Propionato de Testosterona/toxicidade , Animais , Feminino , Masculino , Caracteres SexuaisRESUMO
A sleeve lobectomy is an established general thoracic surgical procedure. To improve clinical outcomes following the procedure, we reviewed the records of 60 patients who underwent a bronchoplasty procedure in our department from 1992 to 2007. Induction chemotherapy was performed for 20, of whom 10 underwent radiotherapy as well. For all subjects, the postoperative mortality and morbidity rates were 1.7% and 33.3%, respectively. Induction therapy did not significantly affect those rates, though complications related to bronchial anastomoses occurred exclusively in subjects who received that therapy. The overall 5-year survival rate was 51.0%, while subjects with pN0 (67.9%) and pN1 (60.0%) disease, and those in stage I (79.1%) and stage II (59.9%) had better survival as compared with patients with pN2 (16.9%) disease, and those in stage III (21.8%) and stage IV (0%). Furthermore, the survival rate of yp-stage I and II patients was significantly greater than that of those in yp-stage III and IV (59.9% vs. 14.3%, p = 0.0158). We concluded that patients in stages I, II or with pN0-1 disease are good candidates for a bronchoplasty procedure, though induction therapy should be considered thereafter. In addition, due diligence for postoperative complications is necessary.
Assuntos
Brônquios/cirurgia , Neoplasias Pulmonares/cirurgia , Procedimentos de Cirurgia Plástica/métodos , Procedimentos Cirúrgicos Torácicos/métodos , Adolescente , Adulto , Idoso , Terapia Combinada , Feminino , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Pneumonectomia , Complicações Pós-Operatórias , Prognóstico , Procedimentos de Cirurgia Plástica/mortalidade , Estudos Retrospectivos , Taxa de Sobrevida , Procedimentos Cirúrgicos Torácicos/mortalidadeRESUMO
Human pro-tumor necrosis factor (pro-TNF) is a type II transmembrane protein with a highly conserved 76-residue leader sequence. We have analyzed the behavior, both in a microsomal translocational system and by transfection, of a series of mutants with deletions from the cytoplasmic, transmembrane, and linking domains. Cytoplasmic deletions included the Arg doublet at -49 and -48 and/or the Lys doublet at -58 and -57; additional mutants included deletion of residues -73 to -55 and -73 to -55, -49, and -48. The transmembrane and linking domain mutants included deletions in the -42 to -35 region, combined with the deletion of residues -32 to -1. Two hybrid mutants combined the cytoplasmic deletions with the deletion of residues -32 to -1. All of the cytoplasmic deletion mutants were properly translocated, as were the transmembrane deletion mutants with deletions up to residues -36, -35, -32 to -1, although the last one exhibited reduced efficiency; further incremental deletions, including deletions of residues -38 to -35 and -32 to -1, completely blocked translocation. Both hybrid mutants were effectively translocated; furthermore, transfection analysis revealed competent expression and maturation of both the cytoplasmic and hybrid mutants. Thus, proper expression and maturation of human pro-TNF can be accomplished with as few as approximately 12 of the 26 residues of the native transmembrane domain and with a net negative charge in the cytoplasmic domain flanking the transmembrane region.
Assuntos
Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico , Membrana Celular/metabolismo , Sistema Livre de Células , Células Cultivadas , Chlorocebus aethiops , Cães , Retículo Endoplasmático Rugoso/metabolismo , Humanos , Íons , Dados de Sequência Molecular , RNA Mensageiro/genética , Deleção de Sequência , Relação Estrutura-AtividadeRESUMO
PURPOSE: Stage shift is considered a major reason for more favorable outcomes in patients with screen-detected breast cancer. However, even after adjusting for clinical stage, unresolved issues concerning the reasons for a survival benefit associated with screening programs remain. This study aims to evaluate differences in subtype distribution and outcomes among patients with screen-detected and symptomatic invasive breast cancer and assess whether variations in subtype distribution could explain differences in prognosis. METHODS: Survival analysis was performed to estimate the likelihood of distant recurrence and death in 1132 patients. Subtypes were defined as luminal A [estrogen receptor (ER)+ and/or progesterone receptor (PR)+, human epidermal growth factor receptor 2 (HER2)-, and Ki67 low], luminal B (HER2-) (ER+ and/or PR+, HER2-, and Ki67 high), luminal B (HER2+) (ER+ and/or PR+ and HER2+), HER2 overexpressing (ER-, PR-, and HER2+), and triple negative (ER-, PR-, and HER2-). RESULTS: Screen-detected cancers had favorable clinicopathological characteristics, such as smaller tumor size and a lower frequency of lymph node involvement. Women with screen-detected cancers had a survival advantage. Subtype distribution differed significantly among women with screen-detected and symptomatic cancer. Screen-detected cancers were more likely to be luminal A and less likely to be HER2 overexpressing or triple negative cancer compared with symptomatic cancers (luminal A 61.3 vs. 44.2%, HER2 overexpressing 4.0 vs. 8.0%, triple negative 8.0 vs. 15.9%). Node status, mode of detection, and subtype were independent prognostic factors in the multivariate analysis. CONCLUSIONS: Differences in subtype distribution between screen-detected and symptomatic cancer could partially explain differences in outcomes.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/mortalidade , Detecção Precoce de Câncer/mortalidade , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Feminino , Seguimentos , Humanos , Pessoa de Meia-Idade , Invasividade Neoplásica , Prognóstico , Estudos Retrospectivos , Taxa de SobrevidaRESUMO
The liposomal delivery of recombinant human tumor necrosis factor (rHuTNF) has been shown to be effective in reducing its toxic effects and in its targeting of organs rich in cells of the reticuloendothelial system. However, native recombinant human TNF shows only poor affinity for liposomes, presumably due to its low hydrophobicity. In an attempt to increase the efficiency of association with liposomes, we have modified TNF to increase its hydrophobicity by selective substitution of its amino groups with fatty acid side chains. N-hydroxysuccinimide esters of saturated fatty acids ranging from C8 to C18 were reacted with recombinant human TNF. Modification with esters of C8 to C14 acids occurred as determined by consumption of positively charged amino groups monitored by native polyacrylamide gel electrophoresis; however, esters of longer chain lengths (C16, C18) were much less capable of introducing these chains via amide linkages, and thus these adducts were not further characterized. Biological assays revealed that retention of activity was inversely dependent both on the number of chains introduced and on their length; activity was most conserved (greater than 50%) in a TNF preparation modified with only approximately 1-2.5 caprylic acid (C8) residues/trimer. This preparation was found to bind with high efficiency (approximately 50%) to preformed dipalmitoylphosphatidylcholine-small unilamellar vesicles. The extent of binding closely paralleled both the number of chains introduced and their length; binding was even more efficient (80-90%) for TNF modified either with approximately 3.5 caprylic acid residues/trimer or with approximately 1.5 residues of myristic acid (C14). However, the biological activity of these acylated TNFs was further reduced by this more extensive chemical modification (less than 50% activity for C8 and less than 10% for C14). The biological activity of dipalmitoylphosphatidylcholine-small unilamellar vesicle-C8-TNF was found to be comparable to that of the nonliposomal C8-TNF. Thus, biologically active preparations of liposomal-lipophilic TNF can be prepared with high efficiency.
Assuntos
Fator de Necrose Tumoral alfa/química , 1,2-Dipalmitoilfosfatidilcolina/química , Acilação , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Técnicas In Vitro , Células L , Lipossomos , Camundongos , Proteínas Recombinantes , Solubilidade , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/administração & dosagem , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Steroid sulfatase (STS) hydrolyzes several sulfated steroids such as estrone sulfate, dehydroepiandrosterone sulfate, and cholesterol sulfate. In the present study, we have measured STS mRNA levels in 97 breast cancers by reverse transcription-PCR using a fluorescent primer in the presence of an internal standard RNA and evaluated its association with disease-free and overall survival. The median value was 728.0 amol/ng RNA (range, 0-11,778 amol/ng RNA). Levels were significantly higher in tumors demonstrating lymph node metastasis than in those without nodal involvement (P = 0.033) and in patients who experienced a recurrence during the follow-up period (mean, 40.8 months; median, 39 months) compared with those with no evidence of further disease (mean, 49.2 months; median, 48 months; P = 0.029). No significant associations were found between STS mRNA expression and age, menopausal status, tumor size, histological grade, estrogen receptor status, or postoperative adjuvant therapy. High levels of STS mRNA proved to be a significant predictor of reduced relapse-free survival as a continuous variable (log STS mRNA; P = 0.028). As a dichotomous variable with an optimized cutoff point of 1,240 amol/ng RNA, expression was also associated with a significantly shorter relapse-free survival rate (P = 0.002), but no significant correlation was found between the STS mRNA level and overall survival. Expression was found to be an independent factor for predicting relapse-free survival on multivariate analysis. The results thus support a putative role of STS in breast cancer growth and metastasis.
Assuntos
Arilsulfatases/genética , Neoplasias da Mama/enzimologia , Recidiva Local de Neoplasia/enzimologia , Adulto , Idoso , Arilsulfatases/fisiologia , Neoplasias da Mama/mortalidade , Feminino , Humanos , Pessoa de Meia-Idade , RNA Mensageiro/análise , Receptores de Estrogênio/análise , Esteril-Sulfatase , Taxa de SobrevidaRESUMO
The interaction of ricin and of its constituent polypeptides, the A- and B-chain, with dipalmitoylphosphatidylcholine (DPPC) vesicles was investigated. The A- and B-chain were individually associated with DPPC vesicles, although the intact ricin was not associated. The maximum binding and association constants were evaluated to be 154 micrograms per mg of DPPC and Ka = 2.30 X 10(5) M-1 for the A-chain, and 87 micrograms per mg of DPPC and Ka = 14.5 X 10(5) M-1 for the B-chain, respectively. The A-chain could induce the phase transition release of carboxyfluorescein from DPPC vesicles to a greater extent than the B-chain, whereas the release induced by the intact ricin was negligible. The evidence indicated that the hydrophobic regions on the A-chain and on the B-chain were buried inside when the two chains constituted the intact ricin molecule through one interchain disulfide bond, and that the A-chain caused perturbation of the DPPC bilayer at the phase transition temperature with consequent leakage of carboxyfluorescein.
Assuntos
Bicamadas Lipídicas , Surfactantes Pulmonares , Ricina , Radioisótopos do Iodo , Cinética , Substâncias Macromoleculares , Microscopia de Fluorescência , Modelos Biológicos , Ligação ProteicaRESUMO
2-Methoxyoestrogen sulphamates are a new class of compounds, which inhibit breast cancer cell proliferation and are also potent inhibitors of steroid sulphatase (STS) activity. In the present study, we have used two cell proliferation assays (MTS and AB) to identify potent new compounds in this class. Similar IC(50) values were obtained using these assays with two of the most potent compounds identified being 2-methoxyoestradiol-bis-sulphamate (2-MeOE2bisMATE) and 2-methoxyoestradiol-17beta-cyanomethyl-3-O-sulphamate (2-MeOE2CyMATE). Both compounds inhibited the proliferation of MCF-7 (ER+) and MDA-MB-231 (ER-) breast cancer cells. Using the AB assay, which allows repeat measurements of cell proliferation without killing cells, both compounds were shown to inhibit cell proliferation in an irreversible manner. As STS may be involved in the removal of the sulphamoyl moiety of these compounds, which could reduce their potency, their ability to inhibit the proliferation of MCF-7 cells transfected with the cDNA for STS was also examined. Although the STS activity was 20-fold higher in these cells than in non-transfected MCF-7 cells, no decrease in the ability of these compounds to inhibit cell proliferation was detected. To test the efficacy of these compounds in vivo, nude mice were inoculated with MCF-7 cells in Matrigel and stimulated to grow with oestradiol. Three weeks after the oral administration of 2-MeOE2bisMATE or 2-MeOE2CyMATE (20mg/kg/day, 5 days/week) tumour volumes had regressed by 52% and 22%, respectively. Both compounds also inhibited liver and tumour STS activity by >90%. The potent anti-proliferative effects of these compounds, and their ability to inhibit tumour growth and STS activity in vivo, indicates that they are suitable for development as novel therapeutic agents, which should be active against a wide range of cancers.
Assuntos
Neoplasias da Mama/patologia , Divisão Celular/efeitos dos fármacos , Estradiol/análogos & derivados , Estradiol/farmacologia , Estrona/análogos & derivados , Estrona/farmacologia , Linhagem Celular Tumoral , Estradiol/síntese química , Feminino , Humanos , Esteril-Sulfatase/antagonistas & inibidoresRESUMO
The role of amino functions in the expression of the biological activity of recombinant human TNF (rHuTNF) was studied by chemical modification. rHuTNF is a homotrimer of 17 kD subunits, each of which contains an N-terminal valine and six lysyl residues: two of these lysyl residues are known to be involved in intra- or intersubunit interactions. Chemically reactive amino functions were modified with the N-hydroxysuccinimide ester of acetic acid; modification of amino groups to amide, and the concomitant loss of charge, was monitored by native PAGE. When rHuTNF was reacted with the active ester at increasing mole ratios, up to 12 amino groups per trimer could be modified. When the biological activity of acetylated rHuTNF was determined, a strong correlation between the extent of modification and loss of biological activity was observed. One to three amino groups per trimer could be modified with nearly complete retention (approximately 80-95%) of biological activity; activity was essentially completely destroyed at the highest levels of modification. These results reveal important functions for the amino groups of rHuTNF and significant constraints on strategies involving their modification in development of second-generation-TNF variants.
Assuntos
Fator de Necrose Tumoral alfa/química , Acetilação , Aminas/química , Morte Celular , Humanos , Técnicas In Vitro , Proteínas Recombinantes/química , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/toxicidadeRESUMO
The expression of aromatase (estrogen synthetase) is tissue specifically regulated through the alternative use of multiple exons 1 and promotors. We have determined the amounts of aromatase messenger ribonucleic acid (mRNA) and which type of multiple exons 1 of the human aromatase gene is used in breast tissues of 49 patients with breast cancer by reverse transcription-PCR analysis. The aromatase mRNA levels in these breast cancer tissues (4.53 +/- 0.66 x 10(-3) attomoles/micrograms RNA) were significantly (P < 0.01) higher than those in 16 nonmalignant breast tissues (1.73 +/- 0.40 x 10(-3) amol/micrograms RNA). Aromatase mRNA in all nonmalignant breast tissue were transcribed from skin fibroblast/fetal liver-specific exon 1 (exon 1b) of the gene. In 23 breast cancer tissues, the utilization of multiple exons 1 in the aromatase mRNA was the same as that in nonmalignant breast tissues, whereas in the other 26 cases, it changed from exon 1b to ovary-specific exon 1 (exon 1c). Such switching of tissue-specific exons 1 may affect strict regulation of the tissue-specific expression of aromatase, leading to abnormal expression of the aromatase. The consequent overproduction of local estrogen might promote carcinogenesis or the proliferation of breast cancers.
Assuntos
Processamento Alternativo , Aromatase/genética , Neoplasias da Mama/genética , Genes , Transcrição Gênica , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Neoplasias da Mama/química , Neoplasias da Mama/patologia , Éxons , Feminino , Humanos , Imunoquímica/métodos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Sondas de Oligonucleotídeos/genética , RNA Mensageiro/análise , RNA Mensageiro/genéticaRESUMO
One-third of human breast cancers exhibit estrogen-dependent proliferation. It appears that estrogen functions as a mitogenic factor in these carcinomas. As aromatase is the rate-limiting enzyme in estrogen biosynthesis. It could play an important role in the pathogenesis of estrogen-dependent breast cancer. The aromatase gene consists of at least six exons 1, each containing a promoter, and the tissue-specific expression is regulated by alternative use of these multiple promoters. The expression of aromatase in the breast and abdominal adipose tissues is regulated by a promoter flanking exon 1b. Molecular and epidemiological analyses of tissue-specific utilization of multiple exons 1 and promoters revealed a switching from use of the adipose-specific exon 1b to exon 1c in adipose tissues adjacent to the carcinomas in most breast cancer patients. Exon 1c has been shown to be specific for the ovary. Aromatase mRNA in adipose tissues distal to the tumour of the same patients was normally transcribed from exon 1b as was breast tissue in healthy controls. It is noteworthy that a switching from exon 1b to exon 1c was often observed in breast cancer patients having metastatic lymph nodes. These data suggest that switching from an adipose-specific exon 1b to exon 1c could cause a deviation from strict regulation of tissue-specific expression of the adipose aromatase leading to over-expression of the adipose aromatase. Consequently overproduction of local estrogen may promote carcinogenesis or proliferation of breast cancer cells.
Assuntos
Aromatase/biossíntese , Aromatase/genética , Neoplasias da Mama/epidemiologia , Neoplasias da Mama/genética , Éxons , Tecido Adiposo/enzimologia , Adolescente , Adulto , Encéfalo/enzimologia , Neoplasias da Mama/enzimologia , DNA Complementar , Feminino , Feto , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Placenta/enzimologia , Gravidez , Próstata/enzimologia , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Pele/enzimologia , Testículo/enzimologiaRESUMO
The interaction of ricin with ganglioside GM1 or glycoprotein containing liposomes was investigated. At neutral pH, ricin bound to galactose moieties on the surface of the liposomes to form ricin-liposomes complexes, but did not associate with their lipid bilayers. When these ricin-liposomes complexes were exposed to a pH below 5, ricin bound to GM1-liposomes became associated with the lipid bilayer, whereas ricin bound to glycoprotein-liposomes (containing human erythrocyte Band 3) was only rarely associated. Association of ricin with the lipid bilayer of GM1-liposomes did not occur in the presence of lactose, which inhibits the binding of ricin to ganglioside GM1. Using a hydrophobic probe, 8-amino-1-naphthalene sulfonic acid (ANS), it was revealed that an acidity below pH 5 resulted in exposure of hydrophobic regions on the ricin molecule. These results strongly suggest that association of ricin with the lipid bilayer of GM1-liposomes at acidic pH is mediated by the binding of ricin to ganglioside GM1 at neutral pH and occurs through interaction between the exposed hydrophobic region on the ricin molecule and the lipid bilayer of GM1-liposomes at low pH.
Assuntos
Gangliosídeo G(M1)/metabolismo , Bicamadas Lipídicas/metabolismo , Ricina/metabolismo , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Galactose/metabolismo , Galactose/farmacologia , Concentração de Íons de Hidrogênio , Lactose/farmacologia , Lipossomos/metabolismoRESUMO
The interaction of ricin and its constituent polypeptides, the A- and B-chain, with small unilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC) or dimyristoylphosphatidylcholine (DMPC) was investigated by means of differential scanning calorimetry measurements. The A-chain, at neutral pH, entirely shifted the endothermic peak of small unilamellar vesicles of DPPC from 37 degrees C to 41 degrees C at low protein/lipid ratios. The potency of either ricin or the B-chain to induce the shift of endothermic peak was much less than that of the A-chain. The A-chain was also found to cause mixing of endothermic peaks of DMPC vesicles and DPPC vesicles. These data strongly suggest that the A-chain has the ability to induce fusion of phospholipid vesicles.
Assuntos
Fusão de Membrana/efeitos dos fármacos , Fosfatidilcolinas , Ricina/farmacologia , Varredura Diferencial de Calorimetria , Concentração de Íons de Hidrogênio , Bicamadas LipídicasRESUMO
Involvement of oxygen radicals in the pathogenesis of various inflammatory diseases has been the focus of recent attention. Since lipid peroxidation of cell membranes is postulated to be one of the major reasons for radical-induced tissue injury, inhibition of oxygen toxicity at or near plasma membranes is important. To metabolize extracellular superoxide radicals effectively at or near cell membranes, we synthesized amphipathic superoxide dismutase (SOD) derivatives (AC-SOD) by covalently linking hydrophobic fatty acids with different chain lengths, such as caprylic acid, capric acid, lauric acid and myristic acid, to the lysyl amino groups of the enzyme. When incubated with erythrocytes or polymorphonuclear leukocytes (PMNs), AC-SOD, but not SOD, bound to plasma membranes of these cells. When topically instilled to the eye, AC-SOD also bound to corneal epithelial cell surface. Upon activation by phorbolmyristyl acetate, extracellular cytochrome c was rapidly reduced by PMNs which were pretreated with SOD. In contrast, PMNs preincubated with AC-SOD failed to catalyze the reduction of cytochrome c under the same experimental conditions. These results suggested that AC-SOD bound to cell membranes and effectively dismutated superoxide radicals at or on the outer surface of plasma membranes.
Assuntos
Superóxido Dismutase/metabolismo , Superóxidos/metabolismo , Acilação , Túnica Conjuntiva/metabolismo , Córnea/metabolismo , Membrana Eritrocítica/enzimologia , Humanos , Técnicas In Vitro , Neutrófilos/metabolismo , Peptídeo Hidrolases/metabolismo , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Apoptosis, a naturally occurring programmed cell death or cell 'suicide', has been paid much attention as one of the critical mechanisms for morphogenesis and tissue remodeling. Activation of cysteine aspartases (caspases) is one of the critical steps leading to apoptosis. Although a mitochondria-mediated pathway has been postulated to be one of the activation mechanism of caspase-3, another subcellular compartment might be involved in the activation of the enzyme. The present study shows that the supernatant fraction of digitonin-treated lysosomes strongly activates Ac-DEVD-CHO inhibitable caspase-3-like protease. Activation of caspase-3-like protease by digitonin-treated lysosomal fractions was specifically suppressed by leupeptin and E-64, inhibitors of cysteine protease. These results indicate that leakage of lysosomal cysteine protease(s) into the cytosolic compartment might be involved in the activation of caspase-3-like protease.
Assuntos
Apoptose , Caspases , Cisteína Endopeptidases/metabolismo , Digitonina/farmacologia , Lisossomos/enzimologia , Animais , Caspase 3 , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Precursores Enzimáticos/metabolismo , Indicadores e Reagentes/farmacologia , Lisossomos/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
A hydrophilic enzyme, lysozyme, was myristoylated in vitro by the N-hydroxysuccinimide ester of myristic acid, and the monomyristoylated lysozyme was isolated by CM-cellulose cation-exchange column chromatography. The monomyristoylated lysozyme associated with phospholipid vesicles, whereas the association of native lysozyme was negligible. The membrane-associated monomyristoylated lysozyme was phosphorylated with partially purified rat brain Ca2+- and phospholipid-dependent protein kinase (protein kinase C) in the presence of Ca2+, phosphatidylserine and phorbolmyristate acetate. Thus, the myristoylated lysozyme became a substrate of protein kinase C through its hydrophobic association with the membrane. The present results suggest that the myristoylation of cytoplasmic proteins may have an important role in signal transduction.
Assuntos
Encéfalo/enzimologia , Lipoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Muramidase/metabolismo , Ácidos Mirísticos/metabolismo , Proteína Quinase C/metabolismo , Animais , Cinética , Ácido Mirístico , Fosforilação , Proteína Quinase C/isolamento & purificação , RatosRESUMO
Human transmembrane tumor necrosis factor (pro-TNF) was examined for protein acylation. The cDNA encoding pro-TNF was expressed in both COS-1 cells and Sf9 cells and metabolic labeling with [(3)H]myristic or [(3)H]palmitic acid was attempted. The 17 kDa mature TNF secreted from the transfected cells was not labeled, whereas the 26 kDa pro-TNF was specifically labeled with [(3)H]palmitic acid. The [(3)H]palmitic acid labeling of pro-TNF was eliminated by treatment with hydroxylamine, indicating that the labeling was due to palmitoylation of a cysteine residue via a thioester bond. Site-directed mutagenesis of the two cysteine residues residing in the leader sequence of pro-TNF demonstrated that palmitoylation of pro-TNF occurs solely at Cys-47, located at the boundary between the transmembrane and cytoplasmic domains of pro-TNF. Thus, pro-TNF interacts with the plasma membrane via both its proteinaceous transmembrane domain and a lipid anchor.
Assuntos
Ácidos Graxos Monoinsaturados/metabolismo , Processamento de Proteína Pós-Traducional , Fator de Necrose Tumoral alfa/metabolismo , Acilação , Animais , Células COS , Cisteína/metabolismo , Citoplasma/metabolismo , Insetos , Estrutura Terciária de Proteína , Transdução de Sinais , Transfecção , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/genéticaRESUMO
BACKGROUND: We reported the increased nitric oxide (NO) level in exhaled air of rat lung transplant recipients during acute rejection (AR). The aim of this study was to determine the site and level of NO production in the rejected graft. METHODS: Rat lung transplantation was performed in isografts and allografts. RESULTS: In isografts, no AR and no significant increase in NO production was identified. In allografts, grades I-II of AR was seen on postoperative day (POD) 3 and grade III on POD 5. NO produced by BAL cells increased on both POD 3 (11.8+/-2.0 parts per billion [ppb]) and POD 5 (115.3+/-66.9 ppb). There was a highly significant correlation between the level of NO and the severity of AR (p=0.862, P<0.005). BAL cells from allografts expressed iNOS mRNA. Among them, macrophages, lymphocytes and neutrophils were immunostained for iNOS. CONCLUSION: NO produced by BAL cells was detected in the early stages of rejection. Therefore, it may serve as a sensitive indicator of AR in lung transplantation.