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1.
Hypertension ; 16(3): 245-51, 1990 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-2394484

RESUMO

Cytosolic free calcium concentration ([Ca2+]i) and muscle tension were simultaneously measured in aortic tissue isolated from spontaneously hypertensive rats (SHR), normotensive Wistar-Kyoto (WKY) rats, and SHR chronically treated with a novel angiotensin converting enzyme inhibitor, CS-622. In the presence of 2.5 mM Ca2+ in the bathing solution, aortic [Ca2+]i measured with fura-2 was higher in SHR than in WKY rats, and it was almost the same in CS-622-treated SHR and untreated WKY rats. Increase of external Ca2+ concentration from zero to 2.5 mM elicited a contraction in SHR aortas but not in aortas from both CS-622-treated SHR and untreated WKY rats. When the aortas were contracted by 60 mM K+, however, [Ca2+]i as well as developed tension was similar in the three groups. CGP-28392 (10(-6) M), a Ca2+ channel activator, induced a rhythmic activity superimposed on a gradual increase of [Ca2+]i and tension in SHR aortas but not in the aortas of CS-622-treated SHR or untreated WKY rats. Nicardipine (10(-7) M) decreased the resting [Ca2+]i and the resting tone in SHR aortas, but not in WKY rat aortas. These results suggest that SHR aortas have a higher myogenic tone due to increased [Ca2+]i than WKY rat aortas and that the increased [Ca2+]i is attributed to alterations of dihydropyridine-sensitive Ca2+ channels in SHR aortas. Further, the decrease of the vascular tone induced by long-term administration of the angiotensin converting enzyme inhibitor may be due to a reduction of increased [Ca2+]i in SHR.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Aorta/análise , Cálcio/análise , Hipertensão/metabolismo , Tiazepinas/farmacologia , Animais , Aorta/efeitos dos fármacos , Citosol/análise , Masculino , Contração Muscular/efeitos dos fármacos , Nicardipino/farmacologia , Piridinas/farmacologia , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY
2.
Hepatology ; 12(3 Pt 1): 547-52, 1990 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-2401459

RESUMO

Animals with end-to-side portacaval shunts and sham-operated animals, wherein the body weight and liver weight of the animals varied spontaneously over a considerable range, were studied. The relationships between hepatic androgen- and estrogen-receptor content, serum testosterone and estradiol levels and hepatic mass were characterized. Animals with portacaval shunts were smaller than those without shunts. Moreover, they had reduced serum levels of testosterone and estradiol. The reduction in serum testosterone was greater than that of estradiol. As a result, the calculated estrogen/testosterone ratio of the two groups of animals was greater for the portacaval shunt animals than for the controls. The dissociation constant values for the androgen receptor and estrogen receptor in the liver did not differ between groups. The activity of the androgen receptor (p less than 0.01) and estrogen receptor (p less than 0.05) was reduced markedly in the animals with portacaval shunts compared with controls. Moreover, the hepatic cytosolic estrogen receptor activity--but not that of the androgen receptor--correlated with the measured hepatic mass in both groups of animals. These data suggest that hepatic feminization is either associated with or is a hepatic regenerative signal in the rat.


Assuntos
Estradiol/sangue , Feminização/metabolismo , Fígado/metabolismo , Animais , Peso Corporal/fisiologia , Citosol/análise , Citosol/metabolismo , Feminização/etiologia , Fígado/análise , Masculino , Tamanho do Órgão/fisiologia , Derivação Portocava Cirúrgica , Ratos , Ratos Endogâmicos , Receptores Androgênicos/análise , Receptores Androgênicos/metabolismo , Receptores de Estrogênio/análise , Receptores de Estrogênio/metabolismo , Testículo/fisiologia , Testosterona/sangue , Fatores de Tempo
3.
Endocrinology ; 127(3): 1167-75, 1990 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-2201532

RESUMO

We have synthesized three peptides with amino acid sequences corresponding to amino acids 533-547, 597-611, and 765-779 of the human progesterone receptor (hPR). These peptides were conjugated to keyhole limpet hemocyanin and injected into mice and rabbits to develop antibodies to hPR. Antibodies to the undenatured form of PR were elicited only by the peptide with amino acid sequence 533-547. Fusion of SP2/0 myeloma cells with spleen cells from mice immunized with this peptide produced several active clones. Rabbit sera from immunized animals produced one antiserum that reacted with the undenatured form of PR. One monoclonal antibody (PR-AT 4.14) and one antiserum (PR-AT533) raised against peptide-(533-547) were characterized. Binding of these antibodies to the undenatured form of PR was demonstrated by analysis of the antibody-receptor complexes on sucrose density gradients and by immunoprecipitation techniques. Binding of PR to the antibodies was inhibited by excess peptide. The antibodies did not react with estrogen, glucocorticoid, or androgen receptors, but recognized PR from human breast cancer as well as calf, rabbit, mouse, and rat uteri, indicating that this epitope was conserved among these species. Based on sucrose density gradient analysis of PR prepared and labeled in the presence of proteolysis inhibitors and sodium molybdate, the antibodies bound to a site on the intact undenatured PR, but failed to bind to partially degraded steroid-binding form of the receptor, suggesting that the antibody-binding domain is at or near a site sensitive to proteolysis.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos/imunologia , Fragmentos de Peptídeos/imunologia , Peptídeo Hidrolases/metabolismo , Receptores de Progesterona/imunologia , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Antígenos/imunologia , Sítios de Ligação de Anticorpos , Neoplasias da Mama/análise , Bovinos , Centrifugação com Gradiente de Concentração , Citosol/análise , Feminino , Hemocianinas/imunologia , Humanos , Técnicas de Imunoadsorção , Masculino , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Ratos , Ratos Endogâmicos , Receptores de Progesterona/análise , Receptores de Progesterona/metabolismo , Especificidade da Espécie
4.
Biochem Pharmacol ; 40(4): 811-5, 1990 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-2386549

RESUMO

An endogenous inhibitor of monoamine oxidase (MAO) was separated by gel-filtration from 105,000 g supernate of T4-treated rat liver cytosol. The inhibition by this inhibitor was concentration-dependent and more potent for A-form MAO than for B-form MAO. The mode of inhibition was competitive either with 5-hydroxytryptamine or beta-phenylethylamine. The molecular weight of this inhibitor was estimated to be 600-700 by gel filtration. The pI value was determined to be 3.0 by isoelectric focusing. This inhibitor was proved to be heat-stable and resistant to protease treatment. MAO inhibition activity was much lower in the cytosol of thyroidectomized, non-T4-treated rats than T4-treated rats, suggesting that this inhibitor is induced by thyroid hormone T4. MAO activity in rat liver might be regulated by the level of this inhibitor.


Assuntos
Citosol/análise , Fígado/análise , Inibidores da Monoaminoxidase/isolamento & purificação , Tiroxina/farmacologia , Animais , Ponto Isoelétrico , Masculino , Inibidores da Monoaminoxidase/farmacologia , Ratos , Ratos Endogâmicos
5.
Biol Reprod ; 43(2): 219-28, 1990 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-2378933

RESUMO

The microheterogeneity seen when rat androgen-binding protein (rABP) is analyzed by two-dimensional polyacrylamide gel electrophoresis is attributable, at least in part, to the differential glycosylation of a single promoter. Further insight into the chemical nature of the oligosaccharide units on rABP was obtained by serial lectin chromatography. When rABP was chromatographed on immobilized Concanavalin A (Con-A), it was fractionated into three classes: (1) one that did not bind to the lectin (about 44% of the rABP), (2) one that was bound and could be eluted with 10 mM 1-O-methyl alpha-D-glucopyranoside (glucoside), about 34%, and (3) one that could be eluted with 0.5 M methyl alpha-D-mannopyranoside (mannoside), about 23%. Binding to Con-A indicates the presence of asparagine-linked oligosaccharides. Chromatography of the glucoside-eluted peak on lentil lectin (LcH) indicated that the rABP in that fraction contained a fucose residue on the chitobiose core. Chromatography of the mannoside-eluted peak on wheat germ agglutinin (WGA) indicated the presence of rABP with high mannose- (44%) and hybrid-type (56%) glycans attached. Chromatography on Ricinus communis I (RCA-I) lectin indicated a species containing galactosylated complex-type oligosaccharide chains. Treatment of rABP forms with exoglycosidases confirmed the presence of externally disposed fucose, sialic acid, mannose, and galactose residues. LcH chromatography indicated that about 30% of the rABP that did not bind to Con-A possessed triantennary oligosaccharides with fucose on the chitobiose core. About 28% of the rABP was retarded when it was chromatographed on Phaseolus vulgaris E lectin, suggesting the presence of bisected biantennary chains with terminal galactose residues. We were unable to detect rABP species with serine- or threonine-linked oligosaccharide chains in this fraction. Other forms of rABP in the nonretained fraction of Con-A were not resolved. Western blotting did not reveal major differences in relative molecular weight (Mr) among the rABP species; some differences in the ratio of the heavy to the light subunit of the molecule were detectable.


Assuntos
Proteína de Ligação a Androgênios/análise , Oligossacarídeos/análise , Lectinas de Plantas , Animais , Configuração de Carboidratos , Sequência de Carboidratos , Cromatografia de Afinidade , Concanavalina A , Citosol/análise , Eletroforese em Gel de Poliacrilamida , Epididimo/análise , Glicosilação , Lectinas , Masculino , Dados de Sequência Molecular , Ratos , Aglutininas do Germe de Trigo
6.
Toxicology ; 63(2): 199-213, 1990 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-2399535

RESUMO

In order to clarify whether peroxisomal hydrogen peroxide (H2O2) plays an important role in peroxisome proliferator-induced hepatocarcinogenesis, we examined the change in metabolism of peroxisomal H2O2 in vivo and in vitro using male Fischer-344 rats fed clofibrate, bezafibrate and di(2-ethylhexyl)phthalate (DEHP) for up to 78 weeks. Hepatic peroxisomal fatty acyl-CoA oxidase activity increased 12-20-fold after 2 or 4 weeks treatment; later this level gradually decreased toward controls, and at 78 weeks activity was 3-10-times of control. Although hepatic H2O2 levels were increased slightly by clofibrate, bezafibrate and DEHP, the changes did not correlate with the changes in peroxisomal fatty acyl-CoA oxidase activity. In isolated hepatocytes, the rate of leakage of peroxisomal H2O2 from peroxisomes into the cytosol and the hepatocellular H2O2 content was measured. The rate of leakage of peroxisomal H2O2 into cytosol increased 2.5-4-fold when peroxisomal beta-oxidation activity was induced by peroxisome proliferators, and the increases in this rate corresponded with changes in the peroxisomal beta-oxidation activity. In contrast, the hepatocellular H2O2 contents were not affected by induced peroxisomal beta-oxidation. These data show that H2O2 leaking from peroxisome into cytosol would be quickly decomposed, and thus peroxisomal H2O2 does not appear to play an important role in hepatocarcinogenesis by such an oxidative stress mechanism after the long-term treatment with peroxisome proliferators.


Assuntos
Bezafibrato/toxicidade , Clofibrato/toxicidade , Dietilexilftalato/toxicidade , Peróxido de Hidrogênio/metabolismo , Fígado/efeitos dos fármacos , Microcorpos/efeitos dos fármacos , Ácidos Ftálicos/toxicidade , Acil-CoA Oxidase , Animais , Células Cultivadas , Citosol/análise , Ativação Enzimática/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Fígado/ultraestrutura , Neoplasias Hepáticas Experimentais/análise , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/patologia , Masculino , Microcorpos/metabolismo , Microcorpos/patologia , Microscopia Eletrônica , Oxirredutases/metabolismo , Ratos , Ratos Endogâmicos F344 , Fatores de Tempo
7.
Tumori ; 76(4): 360-4, 1990 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-2399565

RESUMO

Serum levels of tissue polypeptide antigen (TPA) are related to the proliferative activity and to the mass of the malignancy, differently from any other available tumor marker. We therefore evaluated TPA in comparison with CA15.3 and MCA (mucinous-like carcinoma-associated antigen) in patients with primary breast cancer. TPA was measured in tumor cytosol and in serum. Cytosol and serum TPA levels were not significantly correlated. Serum TPA was higher in patients with locally more advanced disease and in receptor-negative cases. The relation between TPA and disease spread was not directly dependent on tumor bulk, whereas CA15.3 and MCA were highly correlated to the number of positive lymph nodes and tumor size. No correlations were found between TPA and CA15.3 or MCA, and the positivity concordance rate between TPA and CA15.3 or MCA was very low. Patients with higher TPA serum levels showed a worse prognosis in cases with and in those without axillary metastases. From our data we conclude that TPA provides information different from that obtained with breast-specific tumor markers and could therefore be useful in association with CA15.3 and/or MCA in the management of patients with breast cancer.


Assuntos
Antígenos de Neoplasias/sangue , Antígenos Glicosídicos Associados a Tumores/sangue , Biomarcadores Tumorais/sangue , Neoplasias da Mama/sangue , Peptídeos/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Neoplasias/análise , Antígenos Glicosídicos Associados a Tumores/análise , Biomarcadores Tumorais/análise , Neoplasias da Mama/análise , Citosol/análise , Estudos de Avaliação como Assunto , Feminino , Humanos , Pessoa de Meia-Idade , Peptídeos/análise , Antígeno Polipeptídico Tecidual
8.
Biochem J ; 269(3): 723-8, 1990 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-2390064

RESUMO

Cytosol and membrane fractions from human neutrophils, monocytes, lymphocytes and platelets were separated by SDS/PAGE, blotted on to nitrocellulose and assayed for selective binding of phosphatidylserine (PS). Two PS-binding proteins with apparent molecular masses of 115 kDa and 100 kDa were identified in the cytosol of neutrophils, monocytes and lymphocytes. Corresponding bands along with other PS-binding proteins were detected in platelets in both cytosol and membrane fractions. These proteins were also found to bind protein kinase C (PKC) provided that PS was present. The 115 kDa and 100 kDa proteins (PS-p115/110) were partially purified from neutrophils and were used for the study of PS and PKC binding. The binding of PS did not require Ca2+ or Mg2+ and was inhibited by phosphatidic acid, by 1-alkyl-2-acetylphosphocholine and, to a lesser extent, by other lipids. The binding of PKC, however, was strictly PS- and Ca2(+)-dependent and seems to occur secondarily to PS binding.


Assuntos
Proteínas Sanguíneas/isolamento & purificação , Proteínas de Transporte/sangue , Leucócitos/metabolismo , Fosfatidilserinas/sangue , Proteínas Sanguíneas/metabolismo , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Colódio/metabolismo , Citosol/análise , Citosol/metabolismo , Eritrócitos/metabolismo , Eritrócitos/ultraestrutura , Humanos , Leucócitos/enzimologia , Leucócitos/ultraestrutura , Neutrófilos/análise , Neutrófilos/metabolismo , Neutrófilos/ultraestrutura , Fosfatidilserinas/isolamento & purificação , Fosfatidilserinas/metabolismo , Ligação Proteica , Proteína Quinase C/sangue
9.
Biochem J ; 269(3): 729-34, 1990 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-2390065

RESUMO

We describe the isolation, lipid-binding properties and partial amino acid sequence of PS-p68, a novel 68 kDa phosphatidylserine-binding protein from human platelets. PS-p68 is an abundant constituent of platelets, accounting for 0.5-0.75% of total cell protein. It was purified from platelet cytosol by affinity chromatography. Amino acid sequence analysis yielded no similarity to identified proteins. In contrast with most known phospholipid-binding proteins, PS-p68 does not bind Ca2+ and does not require Ca2+ for its binding of phosphatidylserine. Phosphatidylserine binding to PS-p68 was inhibited by phosphatidic acid and by alkylphospholipids. PS-p68 was isolated as a major phosphoprotein from 32P-labelled platelets and was found to function as a protein kinase C substrate in vitro. However, treatment of intact platelets with phorbol 12-myristate 13-acetate, thrombin or carbacyclin did not increase PS-p68 phosphorylation. Platelets appear to be the only blood cells containing PS-p68, which was not detected in neutrophils, monocytes and lymphocytes.


Assuntos
Plaquetas/análise , Proteínas Sanguíneas/isolamento & purificação , Proteínas de Transporte/sangue , Sequência de Aminoácidos , Plaquetas/ultraestrutura , Proteínas Sanguíneas/metabolismo , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Colódio/metabolismo , Citosol/análise , Humanos , Dados de Sequência Molecular , Fosfoproteínas/sangue , Fosfoproteínas/isolamento & purificação , Fosfoproteínas/metabolismo , Fosforilação , Ligação Proteica , Distribuição Tecidual
10.
J Biol Chem ; 265(22): 12763-6, 1990 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-2198277

RESUMO

Antibodies are found to transfer rapidly between antigen samples attached to separate solid supports. The half-times for this antibody exchange at 20 and 37 degrees C were 7 and 1 h, respectively. Antibody exchange was exhibited by all 17 polyclonal and monoclonal antibodies tested and was readily detected by enzyme-linked immunosorbent assay, immunoblotting, or immunocytochemical assays. Taking advantage of this phenomenon, antibodies can be affinity-purified using less than a picomole of antigen and small amounts of antibody.


Assuntos
Anticorpos Monoclonais/imunologia , Reações Antígeno-Anticorpo , Proteínas Associadas aos Microtúbulos/imunologia , Animais , Química Encefálica , Bovinos , Linhagem Celular , Citosol/análise , Imuno-Histoquímica , Técnicas Imunológicas , Cinética , Proteínas Associadas aos Microtúbulos/análise , Microtúbulos/ultraestrutura , Tubulina (Proteína)/análise , Tubulina (Proteína)/imunologia
11.
J Immunol ; 145(3): 945-51, 1990 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-2115550

RESUMO

The dormant O2(-)-generating oxidase in plasma membranes from unstimulated neutrophils becomes activated in the presence of arachidonate and a multicomponent cytosolic fraction. This process is stimulated by nonhydrolyzable GTP analogues and may involve a pertussis toxin insensitive GTP-binding protein. Our studies were designed to characterize the putative GTP-binding protein, localizing it to either membrane or cytosolic fraction in this system. Exposure of the isolated membrane fraction to guanosine-5'-(3-O-thio)triphosphate (GTP gamma S), with or without arachidonate, had no effect on subsequent NADPH oxidase activation by the cytosolic fraction. Preexposure of the cytosolic fraction to GTP gamma S alone did not enhance activation of the membrane oxidase. However, preexposure of the cytosol to GTP gamma S then arachidonate caused a four-fold enhancement of its ability to activate the membrane oxidase. This enhancement was evident after removal of unbound GTP gamma S and arachidonate, and was not augmented by additional GTP gamma S during membrane activation. A reconstitution assay was developed for cytosolic component(s) responsible for the GTP gamma S effect. Cytosol preincubated with GTP gamma 35S then arachidonate was fractionated by anion exchange chromatography. A single peak of protein-bound GTP gamma 35S was recovered that had reconstitutive activity. Cytosol preincubated with GTP gamma 35S alone was similarly fractionated and the same peak of protein-bound GTP gamma 35S was observed. However, this peak had no reconstitutive activity. We conclude that the GTP-binding protein regulating this cellfree system is located in the cytosolic fraction. The GTP gamma S-liganded form of this protein may be activated or stabilized by arachidonate.


Assuntos
Citosol/análise , Proteínas de Ligação ao GTP/análise , NADH NADPH Oxirredutases/metabolismo , Neutrófilos/análise , Ácido Araquidônico , Ácidos Araquidônicos/farmacologia , Membrana Celular/enzimologia , Ativação Enzimática , Ácidos Graxos/farmacologia , Guanosina 5'-O-(3-Tiotrifosfato) , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/farmacologia , Humanos , NADPH Oxidases , Tionucleotídeos/farmacologia
12.
Surgery ; 108(2): 191-7, 1990 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-2200154

RESUMO

The purpose of this study was to demonstrate that the pancreas of the hamster contains a growth factor(s) that can induce cells associated with the ductular epithelium to differentiate along an endocrine pathway and thereby provide a means of regenerating a functioning islet cell mass. We have shown previously that partial obstruction of the pancreatic duct leads to the induction of nesidioblastosis. A cytosol extract prepared from the partially obstructed hamster pancreas was injected at a dose of 4000 microliters intraperitoneally twice a day for 2 days and produced significant increases in pancreatic weight, protein, and deoxyribonucleic acid of 18%, 18% and 42% respectively, over saline-treated control animals. To assess the effects of this extract on morphology, 150 microliters intraperitoneally twice a day was administered for 21 days. Tissue was processed for histologic, morphometric, and autoradiographic analysis. Budding of endocrine cells from cells of the terminal ductules was observed in cytosol-injected animals and the number of islets per square millimeter was determined to be increased by 100% compared with saline-treated controls (p less than 0.01). Tritiated thymidine uptake by ductal and islet cells was increased tenfold and sixfold, respectively, over that of control animals (p less than 0.01). Cytosol extract was also administered to hamsters rendered diabetic by streptozocin. Survival in these animals was 100% compared with only 60% for saline-treated control animals (p less than 0.05). Furthermore, the blood levels of glucose in cytosol-treated animals was significantly less than the levels in saline-treated controls (p less than 0.05). We conclude that the pancreas does indeed contain a growth factor(s) responsible for the induction of nesidioblastosis and the new islet tissue is functionally capable of stabilizing a diabetic state.


Assuntos
Diabetes Mellitus Experimental/terapia , Substâncias de Crescimento/fisiologia , Ilhotas Pancreáticas/fisiopatologia , Pâncreas/metabolismo , Ductos Pancreáticos/fisiopatologia , Animais , Autorradiografia , Cricetinae , Citosol/análise , Feminino , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Cinética , Mesocricetus , Pâncreas/análise , Regeneração , Extratos de Tecidos/farmacologia
13.
Cancer ; 66(2): 341-6, 1990 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-2369715

RESUMO

Breast cancer tissue from 95 women was simultaneously assayed for three receptors: cytosolic estrogen (CER), cytosolic progesterone (CPR), and nuclear estrogen (NER). The main objective was to determine whether the addition of NER assay to the currently accepted practice with only CER and CPR could improve the predictive capacity of receptors. Forty-two patients were studied for response to hormone therapy and 95 patients were studied for survival; the median follow-up period was 73 months (range, 8 to 300 months). The incidence of CER+, CPR+, and NER+ was 74%, 70%, and 52%, respectively. Each receptor appeared more frequently, although not significantly so, in higher age groups. Forty percent of tumors had all three receptors positive and 14% had all negative; the remaining tumors showed all possible combinations of receptors. Both the rate of response and survival curves among 70 patients with CER+ did not show any significant difference whether NER was positive or negative. Also, among 38 patients with CER+, CPR+, and NER+, there was no significant difference in the clinical outcome as compared to 17 patients with CER+, CPR+, and NER-. Among 25 patients with CER- the rare occurrence of NER+ in only three patients did not suggest any clinical implication. It is concluded, therefore, that on overall clinical grounds the current series does not support the addition of NER assay whenever data is available on both CER and CPR.


Assuntos
Neoplasias da Mama/metabolismo , Receptores de Estrogênio/análise , Receptores de Progesterona/análise , Fatores Etários , Idoso , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Núcleo Celular/análise , Citosol/análise , Feminino , Seguimentos , Humanos , Menopausa , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Análise de Sobrevida
14.
Toxicol Appl Pharmacol ; 104(3): 521-32, 1990 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-2385841

RESUMO

The hepatotoxicity of acetaminophen is believed to be mediated by the metabolic activation of acetaminophen to N-acetyl-p-benzoquinone imine which covalently binds to cysteinyl residues on proteins as 3-(cystein-S-yl)acetaminophen adducts. The formation of these adducts in hepatic protein correlates with the hepatotoxicity. In this study, the formation of 3-(cystein-S-yl)acetaminophen adducts in specific cellular proteins was investigated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detected using affinity-purified antisera specific for 3-(cystein-S-yl)acetaminophen adducts on immunoblots. These techniques were used to investigate the liver 10,000g supernatant and serum from B6C3F1 mice that received hepatotoxic doses of acetaminophen. More than 15 proteins containing 3-(cystein-S-yl)acetaminophen adducts were detected in the liver 10,000g supernatant. The most prominent protein containing 3-(cystein-S-yl)acetaminophen adducts in the hepatic 10,000g supernatant had a relative molecular mass of 55 kDa. Serum proteins containing 3-(cystein-S-yl)acetaminophen adducts had molecular masses similar to those found in the liver 10,000g supernatant (55, 87, and approximately 102 kDa). These data, combined with our previous findings describing the temporal relationship between the appearance of 3-(cystein-S-yl)acetaminophen adducts in protein in the serum and the decrease in the levels of 3-(cystein-S-yl)acetaminophen adducts in protein in the liver, suggested that liver adducts were released into the serum following lysis of hepatocytes. The temporal relationship between the formation of specific adducts and hepatotoxicity in mice following a hepatotoxic dose of acetaminophen was examined using immunoblots of mitochondria, microsomes, cytosol, and plasma membranes. Hepatotoxicity indicated by serum alanine aminotransferase levels was increased at 2 and 4 hr after dosing. The cytosolic fraction contained numerous proteins with 3-(cystein-S-yl)acetaminophen adducts, the most intensely stained of which was a 55-kDa protein. 3-(Cystein-S-yl)acetaminophen adducts were detected in the 55-kDa liver protein 30 min after dosing and prior to the development of significant toxicity. Examination of gels suggested that maximal levels of immunochemically detectable adducts in the 55-kDa protein occurred at 1-2 hr, with a decrease in intensity 4 hr after dosing. The presence of 3-(cystein-S-yl)acetaminophen adducts in proteins prior to hepatotoxicity suggests a threshold for adduct formation in the development of toxicity. Protein in microsomes which contained 3-(cystein-S-yl)acetaminophen adducts ranged in molecular weight from 38 to approximately 106 kDa. The major proteins containing 3-(cystein-S-yl)acetaminophen adducts in the mitochondria had molecular masses of 39, 50, 68, and 79 kDa.(ABSTRACT TRUNCATED AT 400 WORDS)


Assuntos
Acetaminofen/metabolismo , Fígado/análise , Alanina Transaminase/análise , Animais , Ligação Competitiva , Western Blotting , Membrana Celular/análise , Citosol/análise , Eletroforese em Gel de Poliacrilamida , Masculino , Camundongos , Camundongos Endogâmicos , Microssomos/análise , Mitocôndrias/análise , Proteínas/análise
15.
Eur J Immunol ; 20(7): 1563-7, 1990 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-2167226

RESUMO

Interleukin 1 (IL 1) is a potent protein mediator of inflammation. Among other things it increases the number of lymphocytes adhering to endothelial cell monolayers. We analyzed the signal transduction during IL 1-induced lymphocyte binding. Dibutyryl cyclic AMP, which is a cAMP analog able to penetrate into the cytosol, increased lymphocyte binding to the same extent as IL 1. Direct activation of adenylate cyclase by forskolin enhanced also lymphocyte binding. IL 1 increased the level of cytosolic cAMP in a time- and dose-dependent manner measured with radioimmunoassay. 2',5'-Dideoxyadenosine, which is an inhibitor of adenylate cyclase, decreased both the IL 1-induced lymphocyte binding to endothelial cells and elevation in cytosolic cAMP levels. Lymphocyte binding increased with cytosolic cAMP levels in accordance with elevation of IL 1 concentration. These results suggest that cAMP is essential in signal transduction during IL 1-induced lymphocyte binding to cultured endothelial cell monolayers.


Assuntos
AMP Cíclico/fisiologia , Endotélio Vascular/imunologia , Interleucina-1/fisiologia , Linfócitos/fisiologia , Animais , Adesão Celular/imunologia , AMP Cíclico/metabolismo , Citosol/análise , Técnicas In Vitro , Ratos , Ratos Endogâmicos , Sistemas do Segundo Mensageiro/imunologia
16.
J Steroid Biochem ; 36(4): 333-43, 1990 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-2168008

RESUMO

We have observed that ATP induces a second type of oestradiol binding site with slightly lower affinity (Ka 3.3 x 10(8) M-1) and lower sedimentation coefficient (4 S) in cytosol from immature lamb uterus and MCF-7 cells. A factor isolated from immature lamb uterine nuclear extract was found to decrease the steroid binding activity of oestradiol receptor that had been purified by heparin Sepharose and oestradiol-Sepharose chromatography. Inhibition of this factor by known phosphatase inhibitors, indicated that this factor may be a phosphatase. Another factor isolated from immature lamb uterine cytosol was found to enhance the effect of ATP on receptor binding in cytosol from immature lamb uterus and MCF-7 cells. The ability of this factor to phosphorylate a partially purified cytosol receptor from immature lamb uterus when incubated with [gamma 32P]ATP, indicates that this factor is a phosphokinase. The phosphorylated products after labeling with [3H]tamoxifen aziridine were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Three phosphorylated proteins with molecular weights 150, 97, and 67 kDa bound [3H]tamoxifen aziridine. Ammonium sulphate precipitated cytosol oestradiol receptor from immature lamb uterus was inactivated with receptor inactivating factor and then reactivated with receptor activating factor in the presence of [gamma 32P]ATP and substantially affinity labelled with [3H]tamoxifen aziridine. The affinity labelled oestradiol receptor was immunopurified with the monoclonal antibody JS 34/32. Three proteins with molecular weights 67, 50 and 43 kDa specifically bound [3H]tamoxifen aziridine and only 43 kDa receptor fragment was phosphorylated. The relevance of inactivation/reactivation of oestradiol receptor to the dephosphorylation/phosphorylation of receptor is discussed.


Assuntos
Trifosfato de Adenosina/farmacologia , Estradiol/metabolismo , Receptores de Estradiol/metabolismo , Marcadores de Afinidade , Animais , Neoplasias da Mama , Núcleo Celular/análise , Citosol/análise , Feminino , Humanos , Técnicas de Imunoadsorção , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/farmacologia , Fosforilação , Proteínas Quinases/farmacologia , Receptores de Estradiol/efeitos dos fármacos , Ovinos , Tamoxifeno/análogos & derivados , Tamoxifeno/metabolismo , Células Tumorais Cultivadas , Útero/ultraestrutura
17.
Biochemistry ; 29(28): 6662-70, 1990 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-2168738

RESUMO

Sucrose density gradient shift assays were adapted to permit determination of the affinity of interaction between human glucocorticoid receptors (GR) and DNA under conditions of DNA excess. Saturation analyses were performed to ascertain dissociation constants for the interaction of activated human GR with each of five DNA fragments. Centrifugation of GR-DNA complexes on sucrose gradients under nearly isotonic salt conditions revealed similar affinities with dissociation constants in the range of 2-16 nM for GR interaction with DNA fragments containing glucocorticoid response elements (GREs) exhibiting partial dyad symmetry. By contrast, GR exhibited virtually no affinity for non-GRE-containing DNA or for DNA containing only GRE half-sites. Additionally, GR showed evidence of multiple-site interaction with a DNA fragment containing two partially symmetric GREs, but interacted at only one site of an MMTV LTR DNA fragment containing a single partially symmetric GRE along with a cluster of three half-GREs. Together these data indicate that under physiologically relevant conditions, glucocorticoid receptors have high selectivity and affinity only for DNA containing specific partially symmetric GREs and further suggest that this high affinity for such DNA sites may be sufficient to account for the selective regulation of gene expression observed in glucocorticoid-responsive cells.


Assuntos
Proteínas de Transporte/metabolismo , DNA/metabolismo , Receptores de Glucocorticoides/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Proteínas de Transporte/isolamento & purificação , Citosol/análise , DNA Viral/genética , DNA Viral/metabolismo , Regulação da Expressão Gênica , Células HeLa , Vírus do Tumor Mamário do Camundongo/genética , Dados de Sequência Molecular , Proteínas de Neoplasias/metabolismo , Concentração Osmolar , Ligação Proteica , Ratos , Receptores de Glucocorticoides/isolamento & purificação , Sequências Reguladoras de Ácido Nucleico , Sequências Repetitivas de Ácido Nucleico
18.
Brain Res Mol Brain Res ; 8(2): 105-11, 1990 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-2169565

RESUMO

A protein stimulating the GTPase activity of rhoB p20, a ras p21-like GTP-binding protein (G protein), was partially purified from the cytosol fraction of bovine brain. This protein, designated as rhoB p20 GTPase-activating protein (GAP), did not stimulate the GTPase activity of other ras p21/ras p21-like G proteins including c-Ha-ras p21, smg p21 and smg p25A. The activities of c-Ha-ras p21 GAP and smg p21 GAP were also detected in the cytosol fraction of bovine brain and rhoB p20 GAP was separated from these GAPs. The activity of rhoB p20 GAP was eliminated by tryptic digestion or boiling. The Mr value of rhoB p20 GAP was estimated to be 150-200 x 10(3) and 37 x 10(3) by gel filtration and sucrose density gradient ultracentrifugation, respectively. These results indicate that there is rhoB p20 GAP in addition to c-Ha-ras p21 GAP and smg p21 GAP in bovine brain. In rat brain, about 50% of rhoB p20 GAP was found with the highest specific activity in the P2 fraction containing myelin, synaptosomes and mitochondria. In the P2 fraction, about 30% of rhoB p20 GAP was found in the P2C fraction containing mainly synaptosomes. rhoB p20 GAP was detected in the cytosol and particulate fractions of not only rat brain but also other rat tissues.


Assuntos
Química Encefálica , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Membrana/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas/isolamento & purificação , Animais , Bovinos , Citosol/análise , Feminino , Proteínas Ativadoras de GTPase , Família Multigênica , Proteínas/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas p21(ras) , Ratos , Especificidade da Espécie , Frações Subcelulares/análise , Proteínas Ativadoras de ras GTPase , Proteína rhoB de Ligação ao GTP
19.
Biochem Biophys Res Commun ; 170(2): 673-83, 1990 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-2116795

RESUMO

In bovine aortic smooth muscle, about 50% of total GTP-binding activity was present in the cytosol fraction. A major GTP-binding protein (G protein) with a Mr value of about 21,000 (21K G) in this fraction was purified to near homogeneity and characterized. 21K G bound maximally about 0.8 mol of [35S]guanosine 5'-(3-O-thio)triphosphate/mol of protein with a Kd value of about 20 nM. 21K G showed GTPase activity with a turnover number of about 0.007 min-1. 21K G was ADP-ribosylated by botulinum ADP-ribosyltransferase and about 0.4 mol of ADP-ribose was maximally incorporated into 1 mol of 21K G. 21K G and the bovine brain rhoA gene product (rhoA p21) were eluted at the same retention time on C4 reversed-phase high performance liquid chromatography and migrated at the same positions on two-dimensional gel electrophoresis. These results indicate that the major G protein in bovine aortic smooth muscle cytosol is rhoA p21.


Assuntos
Proteínas de Ligação ao GTP/análise , Proteínas de Membrana/análise , Músculo Liso Vascular/análise , Adenosina Difosfato Ribose/metabolismo , Animais , Aorta/análise , Bovinos , Cromatografia , Cromatografia Líquida de Alta Pressão , Citosol/análise , Eletroforese em Gel Bidimensional , Proteína rhoA de Ligação ao GTP
20.
Biochem J ; 269(2): 513-9, 1990 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-2117443

RESUMO

A description is given of the methodology, and problems encountered, for the use of a new fluorescent Ca2(+)-indicator dye, fluo-3, in neutrophils and platelets. The higher Kd and longer excitation wavelength of fluo-3 can have significant advantages over fura-2. Although neutrophils and platelets are used as examples, these observations will be applicable to other cell types. The Kd of fluo-3 for binding Ca2+ at 37 degrees C was measured and found to be 864 nM; the previously published value was 400 nM at 22 degrees C. The Kd of fluo-3, like that of fura-2, is therefore very temperature-dependent. Protocols for loading cells, and preventing leakage of fluo-3, are described; probenecid, known to inhibit fura-2 leakage from cells, was found to be essential to get good fluo-3 signals from platelets. Calibration of fluo-3 fluorescence signals to [Ca2+] and methods for obtaining maximum and minimum fluorescence signals are described; these methods differ from those used with fura-2. Agonist-stimulated responses of fluo-3-loaded neutrophils and platelets are shown, and the calculated cytosolic [Ca2+] is comparable with that previously obtained with fura-2. Responses of cells in the presence of plasma are also shown; such measurements, unobtainable with quin2, fura-2 or indo-1, are possible with fluo-3, owing to its longer excitation wavelengths. Co-loading of cells with bis-(o-aminophenoxy)ethane-NNN'N'-tetra-acetic acid and fluo-3 is included as an example of how cytosolic [Ca2+] can be buffered and manipulated. Many of these observations will be of value when using fluo-3 (or other Ca2(+)-indicator dyes) in most cell types.


Assuntos
Compostos de Anilina , Plaquetas/análise , Cálcio/sangue , Citosol/análise , Corantes Fluorescentes , Neutrófilos/análise , Xantenos , Benzofuranos , Sangue , Plaquetas/ultraestrutura , Soluções Tampão , Digitonina/farmacologia , Ácido Egtázico/farmacologia , Fura-2 , Humanos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/ultraestrutura , Probenecid/farmacologia , Espectrometria de Fluorescência , Trombina/farmacologia
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