RESUMO
The present study has evaluated the use of distortion product otoacoustic emission (DPOAE) responses in the detection of cisplatin-induced ototoxicity in a Sprague Dawley rat animal model. The cisplatin was administered as a 16 mg/kg, dose introduced by a slow 30-min intraperitoneal infusion. Data from three DP-gram protocols, DPOAE input-output responses at 8 kHz, and auditory brainstem responses (ABRs) at 8, 12 and 16 kHz were collected before and 72 h after treatment. The post-treatment ABRs at 16 kHz showed the greatest mean threshold shift of 33.6 dB. The post-treatment DP-gram data showed significant reduction of the signal to noise ratios in the majority of the frequencies tested, across all tested protocols. The data suggest that the most sensitive DPOAE procedure for the early detection of the cisplatin-induced ototoxic damage is the DPOAE I/O protocol. Morphological analyses indicated that the inner hair cells remained intact, while several types of alterations were observed in the arrangement of the stereocilia in the outer hair cells.
Assuntos
Antineoplásicos/efeitos adversos , Cisplatino/efeitos adversos , Cóclea/efeitos dos fármacos , Emissões Otoacústicas Espontâneas/efeitos dos fármacos , Animais , Antineoplásicos/administração & dosagem , Percepção Auditiva/efeitos dos fármacos , Limiar Auditivo/efeitos dos fármacos , Cisplatino/administração & dosagem , Cóclea/ultraestrutura , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Células Ciliadas Auditivas Internas/ultraestrutura , Células Ciliadas Auditivas Externas/efeitos dos fármacos , Células Ciliadas Auditivas Externas/ultraestrutura , Injeções Intraperitoneais , Masculino , Distribuição Aleatória , Ratos , Ratos Sprague-DawleyRESUMO
The aim of this study is to outline the mechanisms leading cochlear cells to die. We utilized an immortalized cell line (OC-k3 cells) derived from the organ of Corti of transgenic mice in order to perform in-depth biochemical studies with no limitations on sample size and number. We probed these cells with cisplatin and gentamicin, two drugs which display in vivo undesired ototoxic side-effects. We investigated cell viability, reactive oxygen species (ROS) production and glutathione (GSH) levels and tested the effects of different concentrations of cisplatin and gentamicin from 0 to 48 h. Results show that cells undergo a dose- and treatment-time-dependent apoptosis characterized by nuclear fragmentation, integrity of the cell membrane and mitochondria, and absence of DNA endonuclease activity. During the early part of treatment, ROS production increases and intracellular GSH decreases, probably due to the activation of protein kinase C alpha. Use of antioxidants such as N-acetylcysteine, GSH and vitamin C rescues cells from apoptosis almost completely. Overall, these data indicate that ROS generation might play a central role in inducing inner ear cell apoptosis and may have an additive role in the ageing process.
Assuntos
Apoptose/fisiologia , Órgão Espiral/patologia , Animais , Morte Celular , Linhagem Celular , Camundongos , Camundongos TransgênicosRESUMO
Several studies have indicated that mild to moderate hyperhomocystinemia is a common cause of arterial occlusive disease. Whether hyperhomocystinemia per se is an independent risk factor for vein thromboembolism (VTE) is still somewhat controversial. Both genetic and nutritional factors influence plasma homocysteine levels. Therefore, we evaluated plasma total homocysteine (tHcy), folate, and vitamin B12 levels and established, by polymerase chain reaction, the presence of the C677T mutation (A223V) in the methylenetetrahydrofolate reductase (MTHFR) gene in 220 cases with VTE without well-established prothrombotic defects. As a control group, 220 healthy subjects from the same geographic area as the cases were investigated. Hyperhomocystinemia was defined as a plasma tHcy level above the 95th percentile in the controls (18.05 micromol/L). Hyperhomocystinemia was found in 16% of cases (odds ratio=3.59; P<0.001); deficiencies of folate (<2.47 ng/mL) or vitamin B12 (<165 pg/mL), defined as values below the 5th percentile in controls, were found in 17.7% (P<0.001) and 12.3% (P=0.015) of cases, respectively. The homozygous condition for the MTHFR mutation (VV) was present in 28.2% of cases and 17.7% of controls (odds ratio=1.82; P=0.013). Comparing only the idiopathic forms of VTE (n=80/220; 36.3%) with normal controls, individuals with hyperhomocystinemia, or individuals homozygous for MTHFR mutation increased the odds ratios to 4.03 (P=0.005) and 2.11 (P=0.018), respectively. No statistically significant difference was observed in the MTHFR genotype distribution of cases and controls with hyperhomocystinemia (P=0.386); however, the normal MTHFR genotype (AA) appeared in control subjects only when tHcy levels were below the 80th percentile (10.57 micromol/L) of the distribution, whereas in case patients, it was present at the highest tHcy levels. A strong association between mutated homozygosity (VV), low folate levels, and hyperhomocystinemia was found in both groups. We conclude that in patients with VTE who do not have coexisting prothrombotic defects, hyperhomocystinemia increases the risk of developing idiopathic and venous thrombosis; the homozygous condition for the MTHFR mutation confers a moderate risk but, together with low folate levels, it is the main determinant of mild hyperhomocystinemia in normal and thromboembolic populations.
Assuntos
Ácido Fólico/sangue , Homocistina/sangue , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Tromboembolia/sangue , Trombose Venosa/sangue , Adulto , Idoso , Feminino , Genótipo , Humanos , Masculino , Metilenotetra-Hidrofolato Redutase (NADPH2) , Pessoa de Meia-Idade , MutaçãoRESUMO
The effect of differentiating doses of all-trans retinoic acid (ATRA, 10(-6) M) and vitamin D3 (10(-7) M) was investigated on the nuclear levels of endogenous ceramide and protein kinase C-zeta (PKC-zeta) catalytic activity in HL-60 myeloid cells. ATRA induced a parallel increase of ceramide and catalytically active PKC-zeta into the nuclear compartment of HL-60 cells (peak at 72 h). On the other hand, vitamin D3 increased the levels of nuclear ceramide and PKC-zeta activity to a lesser extent and with a delayed kinetics compared to ATRA (peak at 96 h). Pretreatment of HL-60 cells with high pharmacological concentrations of exogenously-added C2-ceramide (10(-6) M) completely blocked the ATRA-mediated activation of nuclear PKC-zeta. Exogenous C2-ceramide (10(-6) M) also inhibited the granulocytic differentiation induced by ATRA, whereas it did not affect monocytic differentiation mediated by vitamin D3. Transient transfection experiments performed with a plasmid construct containing a constitutively active mutated form of the PKC-zeta cDNA fused in 3' to a fluorescent tag protein (pEGFP-PKC-zeta) demonstrated that the overexpression of catalytically active PKC-zeta was not accompanied by the appearance of a differentiated morphology. These findings suggest that nuclear PKC-zeta is necessary but not sufficient to induce granulocytic differentiation of HL-60 myeloid malignant cells.
Assuntos
Colecalciferol/farmacologia , Granulócitos/citologia , Proteína Quinase C/metabolismo , Tretinoína/farmacologia , Western Blotting , Diferenciação Celular , Núcleo Celular/metabolismo , Ceramidas/metabolismo , Células HL-60 , HumanosRESUMO
The biological basis for the pleiotropic activity of extracellular human immunodeficiency virus (HIV)-1 Tat protein on lymphoid T cell survival is not well understood. We have here demonstrated that the addition in culture of 0.1-10 nM Tat protein to 36-h serum-starved lymphoblastoid Jurkat T cells rapidly stimulates the catalytic activity of phosphatidylinositol 3-kinase (PI 3-K). The peak of activation was observed 30 min after Tat addition. Extracellular Tat also stimulated the catalytic activity of the Akt/PKB kinase, a major target of PI 3-K lipid products. Pretreatment of serum-starved Jurkat cells with 100 nM wortmannin (WT) or 10 microM LY294002, two unrelated pharmacological inhibitors of PI 3-K, markedly suppressed the catalytic activity of both PI 3-K and Akt/PKB in Jurkat cells. Moreover, at low concentrations (0.1-1 nM), extracellular Tat showed a small but reproducible protection of Jurkat cells from apoptosis induced by serum deprivation (p < 0.05), while the combination of Tat plus 100 nM WT significantly (p < 0.05) increased the percentage of apoptosis with respect to cells left untreated or treated with Tat alone. Taken together, these data suggest that the anti-apoptotic activity of low concentrations of Tat protein on Jurkat cells is mediated by a PI 3-kinase/Akt pathway.
Assuntos
Linfócitos T CD4-Positivos/enzimologia , Espaço Extracelular/virologia , Produtos do Gene tat/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Apoptose/efeitos dos fármacos , Western Blotting , Linfócitos T CD4-Positivos/virologia , Ativação Enzimática/efeitos dos fármacos , Espaço Extracelular/enzimologia , Humanos , Células Jurkat , Ativação Linfocitária/efeitos dos fármacos , Microscopia Confocal , Fosfatidilinositol 3-Quinases/isolamento & purificação , Inibidores de Fosfoinositídeo-3 Quinase , Testes de Precipitina , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-aktRESUMO
To establish whether protein kinase C was involved in the nuclear events underlying cell differentiation and proliferation, rat pheochromocytoma PC12 cells, serum-starved for 24 h, were treated with either differentiating doses of nerve growth factor or high serum concentrations, which represented a powerful mitogenic stimulus. Western blot analysis with isoform-specific antibodies, performed on whole cell homogenates, cytoplasms, and purified nuclei, showed that PKC isotypes alpha, beta I, beta II, delta, epsilon, eta, and zeta were expressed in PC12 cells and that all of them, except for beta I, were found at the nuclear level, variably modulated depending on the cell treatment. Compared to serum-stimulated cells, in which an early (1 day) and marked rise of protein kinase C activity was followed by a plateau, nerve growth factor-treated cells showed a progressive increase of protein kinase C activity coincident with the onset and maintenance of the differentiated phenotype. Western blot analysis of nuclei isolated from fully differentiated cells demonstrated an increase of protein kinase C alpha, paralleled by enhanced phosphotransferase activity along with the nerve growth factor treatment, and complete loss of the delta isotype. In contrast, in nuclei of proliferating PC12 cells, after an early but modest increase at 1 day of mitogenic stimulation, protein kinase C activity reached a plateau. Isotype-specific analysis indicated a concomitant increase of protein kinase C beta II, delta, and zeta and the appearance of protein kinase C epsilon and eta at the nuclear level. Considering the relative intensity of the cytoplasmic and nuclear immunoreactive bands under the three conditions examined, clear-cut translocation to the nucleus occurred for PKC epsilon and eta in serum-stimulated cells. Additional nuclear accumulation of PKC by translocation from the cytoplasm was prominently induced for the zeta isoform after mitogenic stimulation and for PKC alpha during prolonged NGF treatment. Our data suggest that nuclear translocation and selective activation of distinct protein kinase C isoforms play a relevant role in the control of proliferation and differentiation of the same cell type and that nuclear protein kinase C is crucial to the induction and persistence of the differentiated neuronal phenotype of PC12 cells.
Assuntos
Núcleo Celular/enzimologia , Isoenzimas/análise , Neurônios/citologia , Proteína Quinase C/análise , Sequência de Aminoácidos , Animais , Diferenciação Celular , Divisão Celular , Núcleo Celular/ultraestrutura , Meios de Cultura , Dados de Sequência Molecular , Fatores de Crescimento Neural , Células PC12 , RatosRESUMO
A method for ceramide (CER) and diradylglycerol (DG) determination after normal-phase HPLC separation was developed. The free oxydril group of ceramide and diradylglycerol is coupled to the carboxylic group of the fluorescent label (+)-6-methoxy-alpha-methyl-2 naphthaleneacetic acid (NAP), using as catalytic agents 4-dimethylaminopyridine and N,N'-dicyclohexylcarbodiimide. The use of NAP-free acid instead of the halide-activated form ensures higher stability of the reagent, lower reaction temperatures, and improved yield and reproducibility. The yield of the reaction is greater than 90% after a period of 3 h at the temperature of -20 degrees C. Over 85% of the starting material is recovered at the end of HPLC separation. The lower detection limit is below 5 ng for CER and 150 ng for DG. Under the conditions employed in the assay, no significant hydrolysis of triglycerides, sphingolipids, or phospholipids occurs and the esterification reaction is not affected by components of crude lipid extracts. Since separation and/or purification steps are not required, cellular levels of CER and DG can be easily and rapidly measured.
Assuntos
Ceramidas/análise , Cromatografia Líquida de Alta Pressão/métodos , Diglicerídeos/análise , Linhagem Celular , Ceramidas/química , Ceramidas/isolamento & purificação , Cromatografia em Camada Fina , Diglicerídeos/química , Diglicerídeos/isolamento & purificação , Estudos de Avaliação como Assunto , Humanos , Indicadores e Reagentes , Microquímica , NaproxenoRESUMO
The subcellular distribution of PI-PLC beta 1, gamma 1, and delta 1 has been investigated in rat liver by western blot and immunohistochemical analysis with a panel of isoform-specific antibodies. The data obtained in situ on cryo-sectioned tissue indicate that PI-PLC beta 1 is predominantly nuclear, while gamma 1 is largely cytoplasmic and delta 1 is sharply restricted to the cytoplasm. In fractionation experiments, the Western blot analysis indicated that the recovery of the nuclear isoforms beta 1 and gamma 1 was not affected by the removal of the nuclear membrane, and that the two enzymes persisted in nuclear matrix and lamina, obtained after nuclease digestion and extraction with high salt and detergent. The assay of the phosphodiesterase activity in different cell fractions correlates with the observed relative abundance of the enzymes, and specific inhibition with neutralizing anti-beta 1 and -gamma 1 isoforms confirms that these are the enzymes active at the nuclear level. These results demonstrate that in rat liver cells, as in other cell types, different members of the PI-PLC family show a discrete intracellular distribution, and suggest that PI-PLC beta 1 and gamma 1 play a central role in modulating the nuclear phosphoinositide cycle.
Assuntos
Isoenzimas/metabolismo , Fígado/enzimologia , Fosfatidilinositóis/metabolismo , Transdução de Sinais , Fosfolipases Tipo C/metabolismo , Animais , Western Blotting , Núcleo Celular/enzimologia , Imunofluorescência , Fígado/citologia , Fígado/ultraestrutura , Microscopia Eletrônica , Diester Fosfórico Hidrolases/metabolismo , Testes de Precipitina , Ratos , Frações Subcelulares/enzimologiaRESUMO
We investigated the effect of extracellular Tat protein of human immunodeficiency virus-type 1 (HIV-1) on the phosphatidylinositol (PI) cycle, which represents a major signal transduction pathway in lymphoid cells. Recombinant Tat, recombinant HIV-1 p24 and cross-linked anti-CD3 monoclonal antibody (mAb) were added in culture for 1-60 min to Jurkat lymphoblastoid CD4+ T cells. The stimulation of T cell receptor by cross-linked anti-CD3 mAb resulted in a rapid increase of the phosphatidylinositol-specific phospholipase C (PI-PLC) activity in whole cell lysates. On the other hand, Tat protein, either alone or in combination with anti-CD3 mAb, showed little effect on the PI turnover of whole cell extracts. Tat, however, selectively stimulated a nuclear-specific PI-PLC with a peak of activity after 30 min from the addition in culture to Jurkat cells. Interestingly, this time corresponded to that required for the uptake and nuclear localization of recombinant Tat protein, as demonstrated by electron microscope immunocytochemistry experiments with anti-Tat mAb. Moreover, exogenous Tat reached the nucleus of Jurkat cells in a bioactive form, as shown in a HIV-1 long terminal repeat-chloramphenicol acetyl transferase transactivation assay. The specific increase of a nuclear PI-PLC activity was further demonstrated by the ability of Tat to stimulate PI turnover also when added directly to isolated nuclei. As a whole, these data demonstrate that Tat selectively stimulates a nuclear polyphosphoinositide hydrolysis, which appears to be independent of the cellular PI turnover. The relevance of these findings for a better understanding of the biological functions of extracellular Tat is discussed.
Assuntos
Produtos do Gene tat/metabolismo , Fosfatidilinositóis/metabolismo , Linfócitos T/metabolismo , Fosfolipases Tipo C/metabolismo , Transporte Biológico , Linhagem Celular , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Ativação Enzimática/efeitos dos fármacos , Produtos do Gene tat/farmacologia , Produtos do Gene tat/ultraestrutura , Humanos , Imuno-Histoquímica , Microscopia Eletrônica , Linfócitos T/ultraestruturaRESUMO
Membrane-depleted rat liver nuclei contain diacylglycerol (DAG) kinase showing a specific activity which doubles that of the whole homogenate. In contrast, cytoplasmic and plasma membrane marker enzymes attain a specific activity of 0.4% at the most, when nuclear DAG kinase approaches 4.5% of the total tissue activity. The enzyme shows a Km of 161 and 200 microM for ATP in both nuclei and microsomes whereas the Km for DAG is 75 microM in nuclei and 658 microM in microsomes. Octylglucoside, CHAPS and Triton X-100 behave mainly as inhibitors, while deoxycholate stimulates the enzyme activity in both cellular fractions, increasing specific activity (3.2-fold in nuclei and 29.1-fold in microsomes) and decreasing Km for DAG (39 microM in nuclei and 237 microM in microsomes). Phospholipids and ceramide stimulate the enzyme activity in isolated nuclei, while no effect occurs in the microsomal fraction. At variance, sphingosine behaves as an inhibitor in both cellular fractions. DAG kinase also utilizes endogenous substrates mobilized by Bacillus cereus phospholipase C, which hydrolyses nuclear phosphatidylcholine and phosphatidylethanolamine and by phosphatidylinositol-specific phospholipase C, which hydrolyses nuclear PI and PIP. These data indicate that nuclear DAG can be controlled by converting it into phosphatidic acid by the action of a nuclear enzyme and support the contention that protein kinase C activity can be modulated at the nuclear level by a discrete system involving phospholipase C and DAG kinase that could operate independently from the cytoplasm.
Assuntos
Núcleo Celular/enzimologia , Fígado/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Animais , Detergentes/farmacologia , Diacilglicerol Quinase , Ativação Enzimática , Inibidores Enzimáticos , Estabilidade Enzimática , Masculino , Microssomos Hepáticos/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fator de Ativação de Plaquetas/antagonistas & inibidores , Pirimidinonas/farmacologia , Ratos , Ratos Sprague-Dawley , Especificidade por Substrato , Tiazóis/farmacologiaRESUMO
Rat pheochromocytoma PC12 cells were permanently transfected with a plasmid vector, containing the tat gene of human immunodeficiency virus type 1 (HIV-1). Various clones were obtained showing the production of different levels of bioactive Tat protein (Tat) after transient cotransfection with an HIV-1 long terminal repeat-chloramphenicol acetyltransferase reporter plasmid. Under conditions of serum starvation, tat-positive PC12 clones expressing high levels of Tat showed a significantly (P < 0.05) higher proliferation rate with respect to both mock-transfected PC12 cells and tat-positive PC12 cells expressing lower levels of Tat. Moreover, all tat-positive PC12 cell clones showed a partial morphological differentiation into sympathetic-like neurons, when seeded in low density (5 x 10(3) cells/cm2) cultures. On the other hand, mock-transfected PC12 cells showed the round shaped morphology typical of untreated PC12 cells and displayed signs of neuronal differentiation only after treatment with 100 ng/ml of nerve growth factor. The addition of 5 micrograms/ml of anti-Tat monoclonal antibody to the culture medium of tat-positive PC12 cell clones almost completely blocked their increased proliferation rate (P < 0.05), but did not affect neuronal differentiation. A significant (P < 0.05) increase in cell proliferation was consistently observed in PC12 cells supplemented with low concentrations of Tat (5 to 25 ng/ml), whereas neuronal differentiation was hardly affected by exogenous Tat. Our data strongly suggest that Tat exerts a complex influence on the proliferation and differentiation of PC12 cells, and this might help in increasing understanding of the pathogenesis of the frequent neurological disorders observed in AIDS patients.
Assuntos
Produtos do Gene tat/farmacologia , HIV-1 , Interleucina-6/farmacologia , Fatores de Crescimento Neural/farmacologia , Células PC12/efeitos dos fármacos , Sistema Nervoso Simpático/crescimento & desenvolvimento , Animais , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Clonais , Relação Dose-Resposta a Droga , Produtos do Gene tat/genética , HIV-1/genética , Neurônios/citologia , Ratos , Proteínas Recombinantes/farmacologia , Sistema Nervoso Simpático/citologia , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
The confocal microscope is becoming increasingly important as an apparatus to analyze the 3-D topography of the cell. Main reasons are the high resolution optical sectioning capacity, the non-invasiveness which leaves the object intact, and the imaging capabilities. This chapter introduces a description of the confocal principle, the basic concepts of confocal fluorescence microscopy and some criteria for cell preservation. Optimization of in situ immunofluorescence, hybridization and detection procedures in combination with new digital microscope techniques can fully express their capacities only if the preparation of biological specimens is accurate for 3-D analysis. Some applications of confocal microscopy to the study of intranucleolar antigens, enzyme translocations and fluorescence in situ hybridization, are described in association with 3-D software image processing, as a useful framework for the study of the 3-D visualization of proteins and chromatin domains.
Assuntos
Hibridização In Situ , Microscopia/instrumentação , Técnicas de Preparação Histocitológica , Imuno-Histoquímica , Microscopia de FluorescênciaRESUMO
Protein phosphorylation catalysed by rat brain protein kinase C (PKC) has been studied in nuclei isolated from normal and regenerating rat liver. Histone H1 and a 40,000 molecular weight protein were hyperphosphorylated at all the explored regeneration times, ranging from 3 to 22 h after partial hepatectomy. Phosphorylation of the two substrates was totally dependent on calcium and lipids and was abolished by low concentration of staurosporine. The observed early change of phosphate content of histone H1 and of the 40,000 molecular weight protein on the time scale of liver regeneration suggests that PKC might be involved in the initial nuclear events leading to cell proliferation.
Assuntos
Encéfalo/enzimologia , Núcleo Celular/metabolismo , Regeneração Hepática , Fígado/enzimologia , Proteína Quinase C/metabolismo , Animais , Fígado/metabolismo , Masculino , Peso Molecular , Fosforilação , Proteínas/análise , Proteínas/metabolismo , Ratos , Ratos EndogâmicosRESUMO
Nuclear matrix isolated from murine erythroleukemia cells (Friend cells) has been phosphorylated with gamma 32P-ATP and purified protein kinase C in order to identify specific nuclear substrates for the enzyme. HMBA has been employed to induce the cell to differentiate and to compare the changes of phosphorylation profile after erythroid differentiation. Lamin B has been found to be hyperphosphorylated by rat brain PK-C in nuclear matrix purified from uninduced cells. This difference characterizes the cells from 14 to 72 hrs of HMBA treatment and indicates that the ability of lamin B to be phosphorylated by PK-C is linked to the differentiated state. The involvement of PK-C in lamin phosphorylation might represent an early step of the signalling pathway utilized by erythroid differentiating agents to target the cell nucleus.
Assuntos
Acetamidas/farmacologia , Leucemia Eritroblástica Aguda/metabolismo , Proteínas Nucleares/metabolismo , Proteína Quinase C/metabolismo , Animais , Cálcio/metabolismo , Diferenciação Celular , Núcleo Celular/metabolismo , Ácido Egtázico/farmacologia , Imunoeletroforese Bidimensional , Lamina Tipo B , Laminas , Leucemia Eritroblástica Aguda/patologia , Camundongos , Membrana Nuclear/metabolismo , Matriz Nuclear/metabolismo , Fosforilação , Proteínas Quinases , Células Tumorais CultivadasRESUMO
The incorporation of 32Pi into phospholipids was studied in Friend erythroleukemia cells either induced or not to erythroid differentiation with 4 mM hexamethylenebisacetamide (HMBA). The effect of the differentiating agent on the recovery of radiolabelled phospholipids was compared in whole cells, isolated nuclei and nuclear matrix after in vivo labelling for 1 hr. The procedure employed for the isolation of nuclei was demonstrated to allow only negligible lipid redistribution caused by cell manipulations. Among the lipids extractable from nuclei, acidic phospholipids, and particularly polyphosphoinositides, were more represented than in whole cells, while small differences were found in the other phospholipid classes examined. The comparison between the uninduced and induced condition showed that the relative amounts of nuclear inositol lipids were modified by HMBA treatment of the cells, with a decreased recovery of phosphatidylinositol 4,5 bisphosphate. These results indicate that phosphatidylinositol and its phosphorylation products synthesized in vivo show a different metabolism in nuclei and whole cells. They appear to be tightly bound nuclear components, also present in membrane-deprived nuclei and nuclear matrix, and are probably related to the nuclear events involved in erythroid differentiation.
Assuntos
Núcleo Celular/metabolismo , Fosfatos de Inositol/metabolismo , Leucemia Eritroblástica Aguda/metabolismo , Fosfatidilinositóis/metabolismo , Acetamidas/farmacologia , Diferenciação Celular , Linhagem Celular , Vírus da Leucemia Murina de Friend , Matriz Nuclear/metabolismoRESUMO
Subcellular distribution of inositol lipids has been studied in Friend Erythroleukemia Cells following induction to erythroid differentiation with hexamethylenebisacetamide, after labelling with [3H]myo-inositol. In situ autoradiography indicated that inositol-derived molecules were present also in the nuclear compartment of uninduced and induced cells. Fractionation studies showed that the nuclear polyphosphoinositides were deeply changed after short induction times, while the whole cell inositol lipids resulted only slightly modified by the inducer. The nuclear recovery of phosphatidylinositol 4,5-bisphosphate was largely increased after 2 hrs of induction, suggesting that inositol lipid metabolism is involved in the early differentiation events occurring at the nuclear level.
Assuntos
Núcleo Celular/metabolismo , Inositol/metabolismo , Leucemia Eritroblástica Aguda/patologia , Fosfatidilinositóis/metabolismo , Acetamidas/farmacologia , Diferenciação Celular , Linhagem Celular , Vírus da Leucemia Murina de Friend , Lipídeos de Membrana/metabolismo , Membrana Nuclear/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologiaRESUMO
The incorporation of phosphatidyl[2-3H]inositol ([3H]PI) from vesicles or microsomal membranes into rat liver nuclei is greatly stimulated by phosphatidylinositol transfer protein (PI-TP). The nuclei are able to phosphorylate [3H]PI, with the production of phosphatidylinositol 4-phosphate (PIP). Recovery of tritiated inositol trisphosphate, inositol phosphate, glycerophosphoinositol and inositol, suggests that in isolated nuclei a large set of enzymes of the PI cycle is present, similar to the enzymes involved in the plasma membrane PI cycle. Incubation with [gamma-32P]ATP shows that isolated nuclei are able to phosphorylate endogenous PI to PIP and phosphatidylinositol 4,5-bisphosphate (PIP2). In the presence of exogenous PI and detergent the synthesis of PIP is increased, indicating that in nuclei the PI pool is suboptimal for the PI-kinase activity. The present study suggests that PI-TP may be involved in providing substrates for PI metabolism at the nuclear level.
Assuntos
Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Fígado/metabolismo , Proteínas de Membrana , Fosfatidilinositóis/metabolismo , Animais , Fracionamento Celular , Cromatografia em Camada Fina , Cinética , Lipossomos/metabolismo , Fígado/ultraestrutura , Masculino , Proteínas de Transferência de Fosfolipídeos , Fosforilação , Ratos , Ratos EndogâmicosRESUMO
Isolated rat liver nuclei and subnuclear fractions synthesize polyphosphoinositides in vitro in a mode dependent on the presence of nuclear membrane, detergent and exogenous substrates. The nuclear membrane is not essential as a source of lipid kinases, since the addition of exogenous phosphatidylinositol or phosphatidylinositol monophosphate to reaction mixtures lacking membranes restores the synthesis of phosphatidylinositol mono- and bisphosphate, respectively. Inositide phosphorylation is best accomplished by high-salt extracted nuclei and pre-detergent lamina. These data suggest that the nucleus, and especially the nuclear periphery, is a cell compartment in which polyphosphoinositide synthesis occurs; this might be related to the progression of phosphatidylinositol metabolism-dependent signals to the genetic apparatus.