RESUMO
To examine the potentially chemopreventive effects of alpha-tocopherol on hepatocarcinogenesis, we fed the transgenic mice line MT42, which overexpresses transforming growth factor-alpha (TGF-alpha) and which has been established as having a high incidence of liver tumor, with different concentrations of alpha-tocopherol and examined the hepatic tumorigenesis of these mice. At 3 weeks of age, MT42 male mice received a single intraperitoneal injection of diethylnitrosamine (DEN), 5 mg/kg body weight, to initiate the formation of liver tumors. The mice were divided into three groups: group A, control diet (20 mg/kg of alpha-tocopherylacetate); group B, deficient diet (less than 1 mg/kg); group C, supplemented diet (500 mg/kg). Neoplastic change was determined at 40 weeks of age. The incidence of adenomas (p < 0.05), the maximum tumor size (p < 0.01), the mean relative liver weight (p < 0.01), and the proliferating cell nuclear antigen (PCNA) labeling indices of the non-tumor sites (p < 0.01) of group B were significantly higher than those of group C. No toxic effects of alpha-tocopherol were found. Alpha-tocopherol-deficient diet accelerated the hepatocarcinogenesis of TGF-alpha transgenic mice treated with DEN. At best, these data demonstrate that alpha-tocopherol-deficiency is not beneficial for prevention of hepatocarcinogenesis in this model. Alpha-tocopherol may be useful for the chemoprevention for liver cancer.
Assuntos
Alquilantes/uso terapêutico , Antioxidantes/uso terapêutico , Dietilnitrosamina/toxicidade , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/prevenção & controle , Fator de Crescimento Transformador alfa/análise , Fator de Crescimento Transformador alfa/efeitos dos fármacos , alfa-Tocoferol/uso terapêutico , Animais , Quimioprevenção , Masculino , Camundongos , Camundongos TransgênicosRESUMO
In an earlier report, we developed a rapid, sensitive and clean method consisting of non-chloroform extraction, clean-up on a commercial multifunctional cartridge column and HPLC with fluorescence detection for the analyses of aflatoxins. In this report, we applied this method to analyze aflatoxins in nuts, giant corn, cereals, spice and black teas. The method was effective for macadamia nuts, walnuts, hazelnuts, brazil nuts, giant corn, rice, wheat and buckwheat, and the recoveries of aflatoxins B1, B2, G1 and G2 spiked in them at the level of 10 ng/g were 85-106%. However, in the chromatograms of spices and black tea, many background peaks were observed. Therefore, we added a purification step with an affinity column to the clean-up of these samples with the multifunctional cartridge column. After the additional purification, most of the background peaks were gone. The recoveries of aflatoxins B1, B2 and G1 spiked at the level of 10 ng/g were 71-112% except for the case of B2 in white pepper (48%). The recoveries of G2 were 49-95%.
Assuntos
Aflatoxinas/análise , Análise de Alimentos/métodos , Cromatografia Líquida de Alta Pressão , Grão Comestível/química , Nozes/química , Especiarias/análise , Chá/química , Zea mays/químicaRESUMO
Male reproductive performance is composed of two principal elements, copulation and spermatogenesis. A wealth of literature has described the intricate web of endocrine events underlying these biological processes. In the present study we show that puromycin-sensitive aminopeptidase (Psa)-deficient mice are infertile, lack copulatory behavior, and have impaired spermatogenesis. The reproductive deficits of the mutants are not restored by androgen administration, although no aberrant localization of the sex steroid receptors was detectable in their brains and testes. Considering the strong expression of the Psa gene in the brain and Sertoli cells and the degenerative morphology of Sertoli cells in Psa-deficient mice, Psa may participate in testosterone-mediated reproductive signal pathways in the brain and testis.
Assuntos
Aminopeptidases/metabolismo , Infertilidade Masculina/enzimologia , Comportamento Sexual Animal , Espermatogênese , Testículo/fisiologia , Aminopeptidases/deficiência , Aminopeptidases/genética , Animais , Feminino , Citometria de Fluxo , Hormônio Foliculoestimulante/farmacologia , Genótipo , Hipotálamo/citologia , Hipotálamo/metabolismo , Immunoblotting , Imuno-Histoquímica , Infertilidade Masculina/genética , Inibinas/metabolismo , Hormônio Luteinizante/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Prolactina/farmacologia , Receptores Androgênicos/metabolismo , Receptores de Estrogênio/metabolismo , Glândulas Seminais/citologia , Glândulas Seminais/efeitos dos fármacos , Túbulos Seminíferos/citologia , Túbulos Seminíferos/efeitos dos fármacos , Espermatogênese/efeitos dos fármacos , Testículo/metabolismo , Testículo/ultraestrutura , Testosterona/farmacologiaRESUMO
Method-performance studies were conducted for the notified revised analytical method of clofentezine. Clofentezine spiked in azuki beans, apple, orange, banana, grape, tea powder and tea extract at the level of 0.2 microgram/g (2 micrograms/g for tea) was analyzed in replicate in 6 laboratories. Mean values of recovery from 7 crops ranged from 78.4 to 85.2%. Repeatability relative standard deviation values ranged from 2.2 to 4.6% and reproducibility standard deviation values ranged from 4.8 to 10.3%. The detection limits were 0.005-0.01 microgram/g. These results show the notified analytical method has good performance.
Assuntos
Clorobenzenos/análise , Inseticidas/análise , Fabaceae/química , Frutas/química , Malus/química , Musa/química , Reprodutibilidade dos Testes , Chá/química , Vitis/químicaRESUMO
BACKGROUND: The potential of higher plants as sources for new immunosuppressive medications is well recognized. In our experiments we investigated the immunosuppressive effect of a highly refined and potent extract of a Chinese herbal preparation, CMX-13, on inhibiting acute allograft rejection (AR) in a highly histoincompatible rat lung transplant model, BN-->LEW, and on lymphocyte activation and cytokine gene expression in vitro. METHODS: Left lung transplants: the control group (group 1) received only dimethylsulfoxide (DMSO) which is the solvent for CMX-13. Group 2 received intramuscular cyclosporin A (CsA, 25 mg/kg) on day 2 posttransplant. Group 3 and 4 received i.p. CMX-13 (0.5 mg/day, low dose and 5 mg/day, high dose, respectively) on day 1, 2, and 3 posttransplant. All animals were killed on day 6 posttransplant. Several pathological categories of inflammation were examined. In vitro experiments: rat spleen cells were incubated with Con A or irradiated stimulator cells with/without serial dilutions of CMX-13 or CsA. Cell proliferation was measured by 3H-thymidine incorporation. mRNA expression of interleukin-2 and interferon-gamma was examined by reverse transcriptase-polymerase chain reaction. RESULTS: The severity of AR in animals receiving high dose CMX-13 was significantly reduced (stage II, P<0.05) compared with controls (stage IV). Significant differences were also seen when more specific parameters of inflammation were examined (necrosis, 0 vs. 1.7+/-1.0, P<0.05; interalveolar hemorrhage, 0 vs. 3.0+/-0.9, P<0.05). The responses seen in the animals treated with high dose CMX-13 were similar to those in the CsA group. CMX-13 inhibited T cell proliferative responses induced by Con A and alloantigen stimulation in a dose-dependent manner that were similar to CsA. Interleukin-2, and interferon-gamma mRNA expression in Con A-stimulated spleen cells was not inhibited by CMX-13 although CsA showed significant inhibition. CONCLUSIONS: 1) CMX-13 significantly reduces the stage of AR and parameters of inflammation in a highly histoincompatible rat lung transplant model. 2) CMX-13 has equal potency to CsA in the inhibition of Con A and alloantigen stimulated rat spleen cell proliferation. 3) CMX-13 showed no inhibitory effects on IL-2 and gamma-IFN mRNA expression, suggesting that its mechanism of action is different from CsA. 4) CMX-13 or derivatives may have potential utility as an immunosuppressive agent(s) in modulation of AR and management of other inflammatory and immunological disorders.
Assuntos
Medicamentos de Ervas Chinesas/uso terapêutico , Rejeição de Enxerto/prevenção & controle , Imunossupressores/uso terapêutico , Transplante de Pulmão/imunologia , Doença Aguda , Animais , Divisão Celular , Ciclosporina/farmacologia , Expressão Gênica/efeitos dos fármacos , Histocompatibilidade , Interferon gama/genética , Interleucina-2/genética , Transplante de Pulmão/patologia , Ativação Linfocitária/efeitos dos fármacos , Masculino , RNA/isolamento & purificação , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/citologia , Estatística como Assunto , Transplante Homólogo/patologiaRESUMO
BACKGROUND: Iron overload in the presence of increasing concentrations of iron is one of the indicators of poor response to interferon therapy in chronic hepatitis C. In order to analyze the effect of iron on hepatitis C virus (HCV) replication, we measured replication in an HCV-infected cell line. METHODS AND RESULTS: Cells from a non-neoplastic HCV-infected human hepatocyte line (PH5CH8) susceptible to HCV infection and supportive of HCV replication were used in this study. The replication of HCV RNA was measured by reverse transcription-nested polymerase chain reaction (RT-nested PCR). PH5CH8 cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. PH5CH8 cells were incubated with 0, 1, 10, 50, and 100 microM of FeSO4 at 37 degrees C with 5% CO2. Forty-eight hours after iron supplementation, the quantity of HCV RNA in the cells incubated in 50 and 100 microM of FeSO4 was approximately ten times that of the cells with no iron supplementation. Similar changes were observed beginning at 12 h from supplementation with FeSO4 and continued for at least 72 h after supplementation. MTT assay indicated that iron did not have cytotoxic effects on the PH5CH8 cells. CONCLUSION: Iron enhances HCV replication in a hepatocyte cell line. The results suggest that iron deposition in hepatocytes could facilitate HCV infection in the liver.
Assuntos
Hepacivirus/crescimento & desenvolvimento , Ferro/farmacologia , Fígado/virologia , Replicação Viral , Linhagem Celular Transformada , Sobrevivência Celular/efeitos dos fármacos , Corantes/metabolismo , Primers do DNA/química , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Humanos , Fígado/citologia , Fígado/efeitos dos fármacos , RNA Viral/análise , RNA Viral/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sais de Tetrazólio/metabolismo , Tiazóis/metabolismo , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
Histamine release inhibitors in watercress (Nasturtium officinale) were isolated using a monitoring system with antigen-stimulated RBL-2H3 cells. Of the 15 compounds isolated, flavonols and megastigmanes significantly inhibited histamine release. Two flavonols, 3-O-sophorosides of rhamnetin and rhamnazin, were new compounds. To investigate the inhibitory mechanism, the effects of rhamnetin, rhamnetin 3-O-sophoroside and an isolated megastigmane glucoside on the increase in the intracellular free calcium concentration were examined at a concentration providing 60% inhibition of histamine release. The results suggest that these compounds did not affect the calcium influx at that concentration. The structure-activity relationships of the megastigmanes on histamine release were also investigated.
Assuntos
Antagonistas dos Receptores Histamínicos/farmacologia , Liberação de Histamina/efeitos dos fármacos , Rosales/química , Animais , Cálcio/metabolismo , Flavonoides/isolamento & purificação , Flavonoides/farmacologia , Flavonóis , Extratos Vegetais/farmacologia , Ratos , Células Tumorais CultivadasAssuntos
Ciclosporina/uso terapêutico , Medicamentos de Ervas Chinesas/uso terapêutico , Rejeição de Enxerto/prevenção & controle , Imunossupressores/uso terapêutico , Transplante de Pulmão/imunologia , Animais , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Hemorragia , Transplante de Pulmão/patologia , Masculino , Necrose , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Transplante HomólogoRESUMO
Three new cardenolide glycosides were isolated from the seeds of Corchorus olitorius L. On the basis of chemical and spectroscopic evidence, their structures were established as cannogenol 3-O-beta-D-glucopyranosyl-(1-->4)-O-beta-D-boivinopyranoside, periplogenin 3-O-beta-D-glucopyranosyl-(1-->4)-O-beta-D-digitoxopyranoside and digitoxigenin 3-O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl-(1-->4)-O-beta - D-digitoxopyranoside.
Assuntos
Cardenolídeos/química , Cardenolídeos/isolamento & purificação , Glicosídeos/química , Glicosídeos/isolamento & purificação , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Plantas Medicinais/química , Sequência de Carboidratos , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Sementes/química , Espectrometria de Massas de Bombardeamento Rápido de ÁtomosRESUMO
Epidermal growth factor (EGF) induces tyrosine phosphorylation of the Shc adapter protein, which plays an important role in EGF-stimulated mitogenesis. Shc stimulates Ras/mitogen-activated protein kinase (MAPK) through forming a complex with Grb2 at the phosphorylated tyrosine (Y) residue 317. In this study, we identified novel phosphorylation sites of Shc, at Y239 and Y240. To define the Shc pathway further, we used NIH 3T3 cells expressing the previously characterized mutant EGF receptor (EGF-R) which lacks all known autophosphorylation sites but retains EGF-stimulated mitogenesis with selective phosphorylation of Shc. We constructed wild-type (WT) or mutant Shc cDNAs in which Y317 or/and Y239 and Y240 are replaced with phenylalanine (F) and introduced them into NIH 3T3 cells expressing WT or mutant EGF-R. In the WT EGF-R-expressing cells, the Y239/240/317F Shc, but not Y317F or Y239/240F Shc, decreased EGF-stimulated cell growth. In the mutant EGF-R-expressing cells, Y317F Shc or Y239/240F Shc decreased EGF-stimulated cell growth significantly, though Y317F was a little more potent than Y239/240F. Although cells expressing the Y317F Shc hardly activated MAPK in response to EGF, cells expressing the Y239/240F Shc fully activated MAPK. In contrast, Y239/240F Shc, but not Y317F Shc, reduced the EGF-induced c-myc message. These results suggest that Shc activates two distinct signaling pathways, Y317 to Ras/MAPK and Y239 and Y240 to another pathway including Myc, and that both are involved in EGF-induced mitogenic signaling.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Proteínas/química , Proteínas/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , DNA Complementar/genética , Ativação Enzimática , Receptores ErbB/genética , Receptores ErbB/metabolismo , Genes myc , Humanos , Camundongos , Mitógenos/farmacologia , Mutagênese Sítio-Dirigida , Fosforilação , Proteínas/genética , Transdução de Sinais , Tirosina/química , Proteínas ras/metabolismoRESUMO
We examined the effect of alpha-linolenic acid (18:3 (n-3)) pretreatment on the metabolism of omega-3 and omega-6 polyunsaturated fatty acids and histamine content and release of RBL-2H3 cells. RBL-2H3 cells grew without reduction in number when incubated with subculture media for 3 d and then placed again in serum-free medium with bovine serum albumin (BSA) and epidermal growth factor (EGF). Cholesterol pullulan (10 micrograms/ml) emulsified alpha-linolenic acid (20 micrograms/ml) was recommended as an additional form serum free medium. We determined the fatty acid composition in all neutral lipids, free fatty acids and all phospholipids in alpha-linolenic acid-treated cells. In all cases the concentration of alpha-linolenic acid and docosahexenoic acid (DHA, 22:6 (n-3)) was increased, while linolenic acid (18:2 (n-6)) was slightly and arachidonic acid (20:4 (n-6)) was markedly decreased. Content of histamine in alpha-linolenic acid-treated cells was remarkably lower than that of untreated cells. Accordingly, net histamine release stimulated by antigen or A23187 was also markedly decreased in the alpha-linolenic acid-treated cells, as was the percent histamine release stimulated by antigen. Results from our in vitro experiment suggest that the anti-allergic effect of alpha-linolenic acid may be caused either by the decrease in histamine content or by inhibition of the release of chemical mediator resulting from changes in the fatty acid composition.
Assuntos
Gorduras Insaturadas na Dieta/metabolismo , Ácidos Graxos Ômega-3/metabolismo , Ácidos Graxos Insaturados/metabolismo , Liberação de Histamina/efeitos dos fármacos , Ácido alfa-Linolênico/farmacologia , Animais , Basófilos/citologia , Basófilos/efeitos dos fármacos , Basófilos/metabolismo , Calcimicina/farmacologia , Divisão Celular/efeitos dos fármacos , Colesterol/química , Cromatografia Líquida de Alta Pressão , Dinitrofenóis/farmacologia , Relação Dose-Resposta a Droga , Emulsões , Fator de Crescimento Epidérmico/química , Ácidos Graxos Ômega-6 , Glucanos/química , Haptenos/farmacologia , Antagonistas dos Receptores Histamínicos H1/farmacologia , Leucemia Basofílica Aguda/patologia , Metabolismo dos Lipídeos , Fosfolipídeos/metabolismo , Ratos , Soroalbumina Bovina/química , Soroalbumina Bovina/farmacologia , Células Tumorais CultivadasRESUMO
Borate was directly chelate-extracted from foods with 5% 2-ethyl-1,3-hexanediol (EHD) in n-hexane-n-butyl acetate mixture (8 + 2), from which borate was selectively transferred into 1% NaOH, since EHD-chelated boron did not react with curcumin to develop color. Finally, an aliquot of the alkaline solution was acidified with HCl and reacted with curcumin in a rotary evaporator. Color development was increased by heating for 8 min at 80 degrees C under reduced pressure of 16 mm Hg. Frozen shrimp and prawns (peeled and with shells) and salted jelly fish were analyzed by the proposed method. Results were compared with the contemporary official method of Japan based on curcumin reaction on an incinerated sample. Over 90% of the boric acid was recovered by the proposed method when samples were fortified with 20 ppm boric acid. Recoveries were superior to those of the official method especially for shrimp and prawns with shells and salted jelly fish. Detection limit of boric acid is 1 ppm. Moreover, the method requires only about 1 hr for analysis of one sample, making it suitable for routine analysis.