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 TempoRESUMO
The early stages of the carcinogenic process induced by aflatoxin B1 (AFB1) in rat liver during 24 weeks of feeding and the resulting tumours have been studied with respect to cytokeratin (CK) expression. A previously uncharacterized monoclonal antibody, MRCTU/J1, has been shown to recognize rat CK18 and together with antibodies against human CK8, 18 and 19, has been used to examine the possible lineage of tumour cells and also to identify the altered foci that might be most relevant to tumorigenesis. Results suggested that AFB1-induced transformation in liver may occur in more than one cell type, since tumours with the normal hepatocyte CK pattern and those with bile duct or oval cell CK phenotype were identified. Additionally, hepatocytes with a bile duct CK phenotype appeared during the early stages of carcinogenesis. The in vivo pattern of CK expression also appeared to be maintained in one normal and one hepatoma-derived cell line. Overexpression of CKs (particularly of CK19) was a much more selective marker for altered foci, compared to gamma-glutamyltranspeptidase, and was more consistently expressed at high levels in tumours, suggesting that it might be a more reliable way of identifying those cells involved in the transformation process.
Assuntos
Citoesqueleto/análise , Filamentos Intermediários/análise , Queratinas/análise , Neoplasias Hepáticas Experimentais/análise , Fígado/análise , Aflatoxina B1 , Aflatoxinas , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antineoplásicos/imunologia , Transformação Celular Neoplásica/análise , Transformação Celular Neoplásica/induzido quimicamente , Reações Cruzadas/imunologia , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/imunologia , Masculino , Ratos , Ratos Endogâmicos F344 , Células Tumorais Cultivadas/análise , Células Tumorais Cultivadas/imunologiaRESUMO
An immunohistochemical study of c-myc and c-erbA products (p-myc and p-erbA) in preneoplastic and neoplastic rat liver lesions showed that the longer the time of hepatocarcinogenic treatment, the higher the proportion of lesions, whatever their type, showing p-erbA positive cells (p-erbA+). The proportion of p-myc positive foci (the majority of which are also p-erbA+), which was low at the focus stage, increased at the nodule and tumor stages. The incidence of p-myc positivity (alone or combined with erbA positivity) in the nodules decreased from the nodule to the tumor stage. For tumors, three types of altered phenotypes were found, namely: p-myc+, p-erbA+ or p-myc+/p-erbA+. Nevertheless, there were regions in these tumors that were negative. At a given stage, all types of lesions exhibited about the same incidence of immunopositivity for the two oncogene products (expressed either alone or together), the presence of which did not correlate with proliferative activity. Since the fraction of lesions that undergo full malignant progression is much smaller than the proportion of lesions that express c-myc and/or c-erbA proteins, our data exclude the possibility that increased incidence of p-myc/ and/or p-erbA+ cells might be sufficient for inducing full malignancy. A significant proportion of foci and nodules, and of regions of these lesions and of tumors, were unlabeled, whatever the stage at which they are found. This indicates that, if implicated, the positive phenotype(s) would not be required for the maintenance of those liver alterations.
Assuntos
Neoplasias Hepáticas Experimentais/análise , Lesões Pré-Cancerosas/análise , Proteínas Proto-Oncogênicas/análise , Animais , Dietilnitrosamina , Feminino , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/patologia , Lesões Pré-Cancerosas/induzido quimicamente , Lesões Pré-Cancerosas/patologia , Proteínas Proto-Oncogênicas c-myc , Ratos , Ratos Endogâmicos , Receptores dos Hormônios TireóideosRESUMO
The early cellular and molecular changes in the Solt-Farber model of hepatocarcinogenesis with and without initiation was studied by using histochemical, immunohistochemical, and in situ hybridization techniques. Increased cellularity was observed in the periductal space in both models 32 to 56 h after partial hepatectomy. These periductal cells and Ito cells were the only cells that became labeled with tritiated thymidine in the uninitiated liver model. Forty-five to 60% of the labeled periductal cells were positive for gamma-glutamyltranspeptidase. From the periductal area the cells that were positive for antibody raised against oval cells (OV-6) infiltrated into liver parenchyma and were followed by desmin-positive Ito cells. The number of Ito cells in the uninitiated model 6 days after partial hepatectomy was 3.5 times higher in the area occupied by oval cells than elsewhere in the liver. The first alpha-fetoprotein (AFP)-positive cells appeared either as individual cells or as pseudoductal formations 32 or 56 h after partial hepatectomy at the periphery of the periductal space in both initiated and uninitiated animals. A combination of in situ and immunohistochemistry revealed that the OV-6-positive cells were AFP positive, whereas desmin-positive cells were AFP negative. Glutathione S-transferase P (GST-P) transcripts could be found mainly in OV-6-positive oval cells. Bile duct cells were positive for GST-P and negative for transforming growth factor beta 1, whereas cells in the periductal space were positive for both of these transcripts. The GST-P-positive early preneoplastic lesions showed a similar distribution pattern as that of oval cells; the preexisting hepatocytes became trapped between small basophilic hepatocytes that showed either irregular or pseudoalveolar arrangement. This raises the question as to whether cells which are stem cell-like are among the target cells in the Solt-Farber model of hepatocarcinogenesis. Proliferation of transforming growth factor beta 1-producing, desmin-positive cells (Ito cells) and multipotent oval cells in a close proximity to each other indicates an intricate relationship between Ito cells and oval cells in liver that warrants further investigation.
Assuntos
Glutationa Transferase/análise , Neoplasias Hepáticas Experimentais/análise , Fatores de Crescimento Transformadores/análise , gama-Glutamiltransferase/análise , 2-Acetilaminofluoreno , Animais , Hepatectomia , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/patologia , Masculino , Ratos , Ratos Endogâmicos F344 , alfa-Fetoproteínas/análiseRESUMO
Murine hybridomas were generated by immunizing mice with purified alpha-fetoprotein (AFP) to determine the cross-reactivity of AFP antibodies with human serum albumin (HSA). Eighteen out of 23 hybridoma products were cross-reactive with HSA. All 23 monoclonal antibodies (MAbs) could be subgrouped into 3 groups by their binding affinity to AFP and HSA, and one MAb from each group, IIB3, IIIB1 and IIIC6, were chosen for this study. The affinity to AFP was highest for IIB3 followed by IIIC6 and IIIB1, and that to HSA was in the order IIIB1 greater than IIB3. Immunohistochemical staining revealed no cross-reactions of these antibodies with HSA at the tissue level. IIB3 was the most efficient in the quantitative assays, indicating high affinity and specificity (non-cross-reactivity) of the antibodies, both of which are prerequisites for in vitro assays. Scans with good tumor localization were obtained from mice which received 131I-labeled IIB3 or IIIC6 after xenografting of the human hepatocellular carcinoma. The images obtained with IIB3 were inhibited in the presence of either AFP or HSA in the circulation. These findings indicated that the use of cross-reactive and/or high-affinity antibodies against AFP provides an inhibitory factor for tumor localization. MAb IIIC6, because of its high specificity and moderate affinity to AFP, seems to be the best candidate for immunoscintigraphy of the tumors in future studies.
Assuntos
Anticorpos Monoclonais/imunologia , Neoplasias Hepáticas Experimentais/diagnóstico por imagem , Albumina Sérica/imunologia , alfa-Fetoproteínas/imunologia , Animais , Afinidade de Anticorpos , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Hibridomas , Imuno-Histoquímica , Neoplasias Hepáticas Experimentais/análise , Camundongos , Camundongos Endogâmicos BALB C , Peso Molecular , Testes de Precipitina , Radioimunoensaio , Cintilografia , Células Tumorais Cultivadas , alfa-Fetoproteínas/análiseRESUMO
A partial, progressive loss of I-compounds (age-dependent, putative indigenous DNA modifications) has been observed recently during hepatocarcinogenesis induced in rats by 2,3,7,8-tetrachlorodibenzo-p-dioxin, choline-devoid diet or peroxisome proliferators. It was of interest, therefore, to investigate the status of I-compounds in hepatic neoplasms. I-compounds were measured by 32P-postlabeling in eight transplantable rat (Morris) hepatomas of different growth rates and in host liver. Most I-compounds seen in liver were not detected in any of the hepatomas, and those present exhibited low levels. Hepatomas displayed an overall level of one I-compound in 2 x 10(8) DNA nucleotides, which was 7-16 times lower than liver values. The extent of I-compound deficiency did not correlate with tumor growth rate. These results, taken together with previously documented pronounced tissue-, sex-, strain- and species-specificity of I-compound profiles, suggest that I-compounds are normal DNA modifications and that their deficiency may contribute to development and maintenance of neoplasia.
Assuntos
DNA de Neoplasias/isolamento & purificação , DNA/isolamento & purificação , Neoplasias Hepáticas Experimentais/análise , Fígado/crescimento & desenvolvimento , Envelhecimento , Animais , Deficiência de Colina/patologia , Feminino , Fígado/análise , Neoplasias Hepáticas Experimentais/patologia , Dibenzodioxinas Policloradas , Ratos , Ratos Endogâmicos ACI , Ratos Endogâmicos BUF , Ratos EndogâmicosRESUMO
The effects of selenium (Na2SeO3) on aflatoxin B1 (AFB1)-induced hepatic neoplasia were studied in the rat. Putative preneoplastic foci and nodules composed of basophilic, eosinophilic, and clear cells developed early. Basophilic foci were seen first; in the later stages basophilic and eosinophilic nodules predominated. At each stage the AFB1 + Se groups showed fewer and smaller foci and nodules than the AFB1 - Se group. The number of foci in the AFB1 + 3 ppm Se group and their mean area were smaller than those in the 6 ppm Se + AFB1 group. At the end of the experiment hepatocellular carcinoma (HCC) was found in 11/18 rats (61%) of the AFB1 - Se group. HCC was not found in either of the groups given AFB1 + Se. We conclude that Se had an inhibitory effect on the initiation and promotion stages of AFB1-induced preneoplastic foci and nodules. Se also prevented progression of these nodules to HCC even after cessation of AFB1 administration. The inhibitory effect of Se at 3 ppm was greater than at 6 ppm. The 6 ppm Se group also showed evidence of toxicity.
Assuntos
Aflatoxinas/toxicidade , Carcinógenos/toxicidade , Neoplasias Hepáticas Experimentais/prevenção & controle , Selênio/uso terapêutico , Aflatoxina B1 , Aflatoxinas/antagonistas & inibidores , Animais , Peso Corporal/efeitos dos fármacos , Neoplasias Hepáticas Experimentais/análise , Neoplasias Hepáticas Experimentais/patologia , Masculino , Ratos , Ratos Endogâmicos , Selênio/análise , Selênio/sangueRESUMO
The immunochemical localization of hamster liver nucleolar antigens in subcellular fractions (nuclei, 10,000 x g pellet, 100,000 x g pellet and supernatant), nuclear substructures (chromatin, nuclear matrix, nuclear envelope, nucleoli, RNP particles and nucleosomes), and three classes of nonhistone chromosomal proteins with different affinities to DNA (NHCP1, NHCP2 and NHCP3) from nuclease-sensitive and nuclease-resistant chromatin fractions of hamster liver were studied. Six main nucleolar antigens with mol. wts 27,000; 29,000; 30,000; 36,000; 45,000; and 46,000 were found in subcellular fractions, nuclear substructures and classes of non-histone proteins of hamster liver. The antigens with mol.wts of approx. 27,000; 29,000; and 36,000 which were absent in hamster pancreas, spleen and Kirkman--Robbins hepatoma nuclei, seem specific for liver tissue.
Assuntos
Antígenos/análise , Nucléolo Celular/ultraestrutura , Fígado/ultraestrutura , Proteínas Nucleares/análise , Animais , Fracionamento Celular , Nucléolo Celular/análise , Cricetinae , Eletroforese em Gel de Poliacrilamida , Técnicas Imunoenzimáticas , Fígado/análise , Neoplasias Hepáticas Experimentais/análise , Neoplasias Hepáticas Experimentais/ultraestrutura , Mesocricetus , Peso Molecular , Matriz Nuclear/análise , Matriz Nuclear/ultraestruturaAssuntos
Compostos de Aminobifenil/farmacologia , Carcinógenos/farmacologia , Dano ao DNA , DNA/genética , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias da Bexiga Urinária/induzido quimicamente , 2-Acetilaminofluoreno/farmacologia , 2-Acetilaminofluoreno/toxicidade , Compostos de Aminobifenil/análise , Compostos de Aminobifenil/toxicidade , Animais , Carcinógenos/toxicidade , Análise Mutacional de DNA , DNA de Neoplasias/análise , DNA Recombinante/análise , Desoxiguanosina/análogos & derivados , Desoxiguanosina/análise , Escherichia coli/genética , Feminino , Fluorenos/análise , Neoplasias Hepáticas Experimentais/análise , Neoplasias Hepáticas Experimentais/epidemiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Especificidade de Órgãos , Fatores Sexuais , Neoplasias da Bexiga Urinária/análise , Neoplasias da Bexiga Urinária/epidemiologiaRESUMO
The properties of glucose-6-phosphate dehydrogenase (G6PDH; glucose-6-phosphate-NADP-oxidoreductase, EC 1.1.1.49) in the liver tissue of intact rats and those with ascites Zaidel hepatoma were compared. The specific activity of the enzyme from hepatoma was 7 times as high as that of the enzyme from the normal liver. The G6PDH preparations with the specific activity of 1.5 U/mg of protein were obtained by the three-stage purification. No differences in kinetic properties, coenzyme specificity and electrophoretic mobility of the enzymes from hepatoma and normal liver were observed.
Assuntos
Glucosefosfato Desidrogenase/análise , Neoplasias Hepáticas Experimentais/enzimologia , Fígado/análise , Animais , Eletroforese , Neoplasias Hepáticas Experimentais/análise , Masculino , RatosRESUMO
Polyclonal antibodies were raised against membrane-associated calcium-binding proteins (apparent molecular masses 65000 and 67000 (CBP 65/67) and 33000 and 35000 (CBP 33 and CBP 35)), which were isolated from rat liver and Morris hepatoma. Using immunoblotting, various amounts of CBP 33 and CBP 35 as well as CBP 65/67 were detected in most rat organs. Using alkaline phosphatase and monoclonal-anti-alkaline phosphatase antibodies (APAAP), all the calcium-binding proteins were detected by immunohistochemical techniques in the plasma membranes of many cells, such as vascular endothelial cells, lymphocytes, epididymal principal cells, secretory and excretory duct cells of certain exocrine glands, straight distal tubular cells of the kidney, and in the cytoplasm of muscle cells and fibres as well as nerve cells and chondrocytes, and in connective tissue elements. Immunohistochemical analysis also showed that in polarized epithelial cells, e.g., renal tubular cells, epididymal principal cells or excretory duct cells, these calcium-binding proteins are present exclusively or mostly in the luminal plasma membrane.
Assuntos
Proteínas de Ligação ao Cálcio/isolamento & purificação , Neoplasias Hepáticas Experimentais/análise , Fígado/análise , Animais , Anticorpos/imunologia , Membrana Celular/análise , Feminino , Immunoblotting , Imuno-Histoquímica , Fígado/ultraestrutura , Neoplasias Hepáticas Experimentais/ultraestrutura , Masculino , Proteínas de Membrana/análise , Peso Molecular , Ratos , Ratos Endogâmicos BUF , Ratos Endogâmicos F344 , Ratos Endogâmicos , Distribuição TecidualAssuntos
Proteínas de Transporte/isolamento & purificação , Neoplasias Hepáticas Experimentais/análise , Proteínas de Neoplasias/isolamento & purificação , Proteínas de Transporte Nucleocitoplasmático , 2-Acetilaminofluoreno/toxicidade , Animais , Anticorpos Antineoplásicos/imunologia , Biomarcadores Tumorais/análise , Proteínas de Transporte/imunologia , Cocarcinogênese , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fígado/análise , Fígado/efeitos dos fármacos , Neoplasias Hepáticas Experimentais/induzido quimicamente , Camundongos , Proteínas de Neoplasias/imunologia , Fenobarbital/toxicidade , Ratos , Ratos Endogâmicos ACI , Ratos Endogâmicos BUF , Ratos Endogâmicos , Especificidade da Espécie , Fator de Transcrição RelA , Células Tumorais CultivadasRESUMO
A 65K protein, known for promoting nucleocytoplasmic mRNA transport in a cell-free system, was previously found in fetal and tumor cells of the rat. The primary objective of this study was to show specificity of immunohistochemical staining for the 65K protein in the livers of rats subjected to a hepatocarcinogenesis protocol. Altered hepatic foci were induced by feeding male weanling Sprague-Dawley rats 2-acetylaminofluorene (AAF) followed by a phenobarbital (PB) diet. It was shown, using polyclonal antibodies produced in rabbits, that the 65K protein was present in the cells of rat liver putative preneoplastic foci, with little or none being detected in the surrounding cells.
Assuntos
Antígenos de Neoplasias/análise , Proteínas de Transporte/análise , Neoplasias Hepáticas Experimentais/análise , Fígado/análise , Lesões Pré-Cancerosas/análise , RNA Mensageiro/metabolismo , Animais , Imuno-Histoquímica , Ratos , Ratos EndogâmicosRESUMO
Previous studies have documented nuclear insulin accumulation in a variety of cell types. The present investigation extends these observations by demonstrating that insulin associates with the matrix fraction of H35 rat hepatoma cell nuclei. Nuclei were isolated from [125I]insulin-loaded cells and extracted with DNase I, RNase A and high salt. The resulting matrix fraction was found to contain greater than 75% of the radiolabel initially present. Ultrastructural studies to confirm these findings were carried out using an agarose-encapsulated nuclear matrix preparation. Electron microscopic immunocytochemistry specifically detected insulin in matrices prepared from insulin-treated cells. No reaction was observed in matrices obtained from non-insulin-treated (control) cells. Further biochemical analysis revealed that matrix-associated insulin could be solubilized with 1% sodium dodecyl sulfate (SDS) or in the presence of high urea concentrations. Gel filtration analysis of urea-solubilized matrix material revealed the presence of apparently intact [125I]insulin and a higher molecular weight peak. It is hypothesized that the latter may represent a tightly associated complex of insulin with some matrix protein(s).
Assuntos
Insulina/análise , Neoplasias Hepáticas Experimentais/análise , Matriz Nuclear/análise , Animais , Cromatografia em Gel , Imuno-Histoquímica , Insulina/metabolismo , Microscopia Eletrônica , Matriz Nuclear/metabolismo , Matriz Nuclear/ultraestrutura , Ratos , Células Tumorais CultivadasRESUMO
The rat insulin-like growth factor II (rIGFII) gene produces, in addition to three major mRNA species 3.6 kilobases (kb), 4.6 kb and 3.8 kb in length which represent transcripts from three independent leader-exons, multiple smaller-sized products that distribute broadly in the 1-3 kb region on Northern blots. Structural constituents of these RNAs were analyzed by hybridization with region-specific probes prepared from the entire rIGFII genome. Most of these shorter RNAs contained both 5'-untranslated and coding regions, but only parts of the 3'-untranslated region. At least nine protected sites were mapped within a single 3'-most exon E6 by S1 nuclease analysis. Some but not all of these sites were associated with the upstream polyadenylation signal, AATAAA, or its variants. Since none of the shorter subspecies contained intronic sequences, aberration in splicing is not involved in their generation. Thus, the main parts of submature materials are a collection of discrete species of RNAs, most, if not all, of which are produced by alternative polyadenylation site selection.
Assuntos
Éxons , Fator de Crescimento Insulin-Like II/genética , Poli A/metabolismo , RNA/metabolismo , Somatomedinas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sondas de DNA , Íntrons , Neoplasias Hepáticas Experimentais/análise , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Poli A/genética , RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RatosRESUMO
The transport of messenger RNA (mRNA) in response to normal adult (35 kDa) and oncofetal (60 kDa) transport factors has been studied in a reconstituted cell-free system. Poly(A)+ mRNA sequences transported by the 35 kDa and 60 kDa transport factors were compared by cDNA:RNA hybridization kinetics. Heterologous hybridization reactions indicated that a proportion of messengers transported in response to the 35 kDa factor were absent or at a markedly reduced abundance in the mRNA released by the 60 kDa factor. Recombinant DNA probes containing cDNA inserts were used to quantitate transport of rat-liver-specific alpha 2 mu-globulin and albumin mRNA from isolated nuclei in presence of the normal and tumor-specific transport factors. More alpha 2 mu-globulin and albumin messenger sequences were transported in response to the 35 kDa transport factor as compared to the 60 kDa factor. These results indicate that the 35 kDa transport protein isolated from rat liver cytosol and the 60 kDa transport protein isolated from hepatoma cytosol, differ significantly in specificity for the classes of RNA sequences released from nuclei. Monoclonal antibodies against the 60 kDa factor do not cross-react with the 35 kDa factor or other proteins as determined by the immunobioassay and by the Western blot technique.
Assuntos
Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Fígado/ultraestrutura , Hibridização de Ácido Nucleico , RNA Mensageiro/metabolismo , Albuminas/genética , alfa-Globulinas/genética , Animais , Anticorpos Monoclonais , Especificidade de Anticorpos , Transporte Biológico/efeitos dos fármacos , Western Blotting , Proteínas de Transporte/análise , Proteínas de Transporte/imunologia , Citosol/análise , Sondas de DNA , Feminino , Neoplasias Hepáticas Experimentais/análise , Peso Molecular , Ratos , Ratos EndogâmicosRESUMO
With the aim of identifying proteins involved in linking microtubules to other cytoplasmic structures, microtubule-binding proteins were isolated from rat liver extracts by a taxol-dependent procedure. The major non-tubulin component, a 58-kDa protein (designated 58K), was purified to homogeneity by gel filtration chromatography. To aid further characterization of 58K, purified preparations of the protein were used as immunogen for the production of monoclonal antibodies. Five different monoclonals were obtained, and each of these reacted on immunoblots of liver homogenates with a single band that comigrated with 58K. Based on the results of immunochemical, peptide mapping, and microsequencing experiments, 58K was found to be unrelated structurally to similarly sized cytoskeleton-associated proteins, such as tubulin, tau, vimentin, or keratin, and to represent a new protein species. Several in vitro properties of 58K were found to be characteristic of microtubule-associated proteins. For instance, 58K cosedimented quantitatively with microtubules out of liver extracts, stimulated polymerization of tubulin, and bound to microtubules in a saturable manner. In contrast to traditional microtubule-associated proteins, however, 58K was not found to be distributed uniformly along microtubules in cells. Immunofluorescence microscopy of cultured hepatoma cells revealed, instead, that 58K is associated principally with the Golgi apparatus. Moreover, Golgi membranes isolated from rat liver were observed by immunoblotting to contain significant levels of 58K, which, upon subfractionation of the membranes, partitioned as if it were a peripheral membrane protein exposed to the cytoplasmic side of the Golgi. These collective results have been evaluated in terms of earlier evidence that the intracellular position and structural integrity of the Golgi relies on the presence and organization of microtubules. In that context, the observations reported here suggest that the in vivo function of 58K is to provide an anchorage site for microtubules on the outer surface of the Golgi.
Assuntos
Complexo de Golgi/análise , Fígado/análise , Proteínas Associadas aos Microtúbulos/isolamento & purificação , Microtúbulos/análise , Proteínas/isolamento & purificação , Animais , Química Encefálica , Bovinos , Fracionamento Celular , Complexo de Golgi/ultraestrutura , Neoplasias Hepáticas Experimentais/análise , Microscopia Eletrônica , Microtúbulos/ultraestrutura , Peso Molecular , Mapeamento de Peptídeos , RatosRESUMO
Using specific probes we show that sequences homologous to NADH dehydrogenase Subunit 6, and Cytochrome oxidase Subunits I, II, and III mitochondrial genes are present in nuclear DNA from various tissues. These mitochondrial-like sequences are also present in rat hepatoma nuclear DNA but with an abnormal organization and a higher copy number than in normal hepatocytes.
Assuntos
Sequência de Bases , Núcleo Celular/análise , DNA Mitocondrial/genética , Neoplasias Hepáticas Experimentais/análise , Neoplasias Hepáticas/análise , Mitocôndrias Hepáticas/análise , Homologia de Sequência do Ácido Nucleico , Animais , Clonagem Molecular , Sondas de DNA , DNA Mitocondrial/isolamento & purificação , Feminino , Hibridização de Ácido Nucleico , Ratos , Ratos Endogâmicos , Células Tumorais Cultivadas/análiseRESUMO
Analysis of mouse Swiss/3T3 fibroblasts and rat hepatoma H35 cells using the affinity cross-linking method revealed multiple forms of 125I-insulin binding components (Mr greater than 300,000) in the absence of reducing agents. The same analysis, in the presence of reducing agents, revealed two major components (Mr = 125,000 and Mr = 30,000). The Mr = 125,000 component appeared to be the alpha-subunit of the high-affinity insulin receptor, whereas the small insulin-binding component of Mr = 30,000 was not a degradation product of the alpha-subunit but was apparently associated with the insulin receptor. We suggest that it is likely a novel component for regulating the function of insulin receptor.