RESUMO
SARS-CoV-2 infection is considered as a multi-organ disease, and several studies highlighted the relevance of the virus infection in the induction of vascular injury and tissue morphological alterations, including placenta. In this study, immunohistochemical analyses were carried out on placenta samples derived from women with COVID-19 infection at delivery (SARS-CoV-2 PCR+) or women healed from a COVID-19 infection (SARS-CoV-2 negative at delivery, SARS-CoV-2 PCR-) or women who gave birth before 2019 (Control). Angiotensin Converting Enzyme 2 (ACE2) receptor, Cluster of differentiation 147 (CD147), endothelial CD34 marker, Vascular Endothelial Growth Factor (VEGF) and total Microtubule-associated protein 1 Light Chain 3B marker (LC3B) were investigated in parallel with SPIKE protein by standard IHC. Multiplexed Immunohistochemical Consecutive Staining on Single Slide (MICSSS) was used to examine antigen co-expression in the same specimen. SPIKE protein was detected in villi and decidua from women with ongoing infection, with no significant differences in SPIKE staining between both biopsy sites. VEGF was significantly increased in SARS-CoV-2 PCR + biopsies compared to control and SARS-CoV-2 PCR- samples, and MICSSS method showed the co-localization of SPIKE with VEGF and CD34. The induction of autophagy, as suggested by the LC3B increase in SARS-CoV-2 PCR + biopsies and the co-expression of LC3B with SPIKE protein, may explain one of the different mechanisms by which placenta may react to infection. These data could provide important information on the impact that SARS-CoV-2 may have on the placenta and mother-to-fetus transmission.
RESUMO
Human islet allografts are well known to induce full and sustained remission of hyperglycemia, with complete normalization of key metabolic parameters. Nevertheless, acquiring human islets, even from cadaveric human donor pancreases, remains a significant impediment to successful transplantation therapy for diabetes. To overcome this difficulty, neonatal porcine cell clusters (NPCCs) have been considered for human islet substitutes because they are easily obtained by collagenase digestion of the neonatal piglet pancreas. Currently, the major hurdle in using NPCCs for xenograft is the delay (time lag) in achieving the posttransplant normalization of blood glucose levels in animal diabetic recipients. The present work is the first attempt to evaluate whether incubation of NPCCs in simulated microgravity, in the presence or absence of Sertoli cells (SC), may reduce the maturation time lag of beta-cells by differentiation acceleration in vitro, thereby expediting production, viability, and acquisition of functional competence of pretransplantation beta-cell-enriched islets. Following a 3-day incubation period, NPCCs maintained in conventional culture, NPCCs incubated in simulated microgravity in the HARV biochamber, and NPCCs plus co-incubated SC in simulated microgravity were examined for viability, morphology, and insulin secretion. Results show that NPCCs grown alone in the HARV biochamber are superior in quality, both in terms of viability and functional competence, when compared to other culture pretreatment protocols. This finding strongly suggests that NPCC pretreatment in simulated microgravity may enhance the transplantation success of NPCCs in the diabetic recipient.
Assuntos
Ilhotas Pancreáticas , Células de Sertoli/citologia , Simulação de Ausência de Peso , Animais , Animais Recém-Nascidos , Sobrevivência Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura , Glucose/química , Imuno-Histoquímica , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/fisiologia , Ilhotas Pancreáticas/ultraestrutura , Masculino , Microscopia Eletrônica , Células de Sertoli/ultraestrutura , SuínosRESUMO
Phosphoinositide 3-kinase (PI 3-K) activity increases in HL-60 cells that are induced to granulocytic differentiation by all-trans-retinoic acid. Immunochemical and immunocytochemical analyses by confocal microscopy also reveal an increase in the amount of the enzyme, which is particularly evident at the nuclear level. Inhibition of PI 3-K activity by nanomolar concentrations of wortmannin and of its expression by transfection with an antisense fragment of p85alpha prevented the differentiative process. The data obtained indicate that PI 3-K activity plays an essential role in promoting granulocytic differentiation.
Assuntos
Antineoplásicos/farmacologia , Granulócitos/efeitos dos fármacos , Granulócitos/enzimologia , Células HL-60/efeitos dos fármacos , Células HL-60/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Tretinoína/farmacologia , Diferenciação Celular/efeitos dos fármacos , Regulação para Baixo , Granulócitos/citologia , Células HL-60/citologia , Humanos , Microscopia Confocal , Fosfatos de Fosfatidilinositol/metabolismo , Frações Subcelulares/enzimologiaRESUMO
Previous investigations have demonstrated the existence of an autonomous intranuclear inositide cycle endowed with conventional lipid kinases and phospholipase C (PLC) which is the isoform beta in Swiss 3T3 cells, PC12 pheochromocytoma cells, human osteosarcoma SaOS-2 cells, and rat liver. The presence of PLC has been investigated in nuclei of Friend erythroleukemia cells. Both beta and gamma isoforms are present in these nuclei. When Friend cells undergo terminal erythroid differentiation in the presence of dimethyl sulfoxide the PLC beta isoform is down-regulated as shown by immunochemical and immunocytochemical analysis, by determination of enzymatic activity directly and in the presence of neutralizing monoclonal antibodies and also by Northern blot for PLC beta message. By contrast, the amount of PLC gamma and its activity are unaffected by erythroid differentiation. Thus, the presence of a nuclear PLC beta, the activity and expression of which are modulated during differentiation of erythroleukemia cells, implicates a role for nuclear phosphoinositide signaling in the processes of cell determination and indicates the nuclear PLC beta as a key enzyme of the cycle in relation to the erythroid differentiative commitment of murine erythroleukemia cells.
Assuntos
Núcleo Celular/enzimologia , Vírus da Leucemia Murina de Friend , Isoenzimas/metabolismo , Leucemia Eritroblástica Aguda/enzimologia , Leucemia Eritroblástica Aguda/patologia , Fosfolipases Tipo C/metabolismo , Animais , Anticorpos Monoclonais , Western Blotting , Diferenciação Celular , Regulação para Baixo , Camundongos , RNA Mensageiro/metabolismo , Células Tumorais CultivadasRESUMO
The prototypic tumor suppressor gene, the retinoblastoma gene (RB/ p105), is mutated in a variety of human tumors. However, to date, mutational data on retinoblastoma family members p107 and RB2/p130 in tumors is lacking. We studied the expression of pRb2/p130 by immunocytochemistry and Western blot analysis in a panel of human osteosarcoma and lymphoid cell lines. Only the lymphoid cell lines showed an abnormal cytoplasmic localization of pRb2/p130, suggesting possible alterations within the region of nuclear localization signaling. We screened these cell lines for genetic alterations of the RB2/p130 gene in the region of the putative bipartite nuclear localization signal (NLS). This region is highly homologous with that of the RB/p105 gene. In addition, we screened four primary Burkitt's lymphomas for genetic alterations in the RB2/p130 gene. Naturally occurring mutations, which disrupt the putative bipartite NLS, were found in lymphoma cell lines and primary tumors, but not in the osteosarcoma cell lines, where normal nuclear localization of the protein was detectable. Site-directed mutagenesis and transfection assay using NLS mutants displayed markedly reduced biological activity as measured by flow cytometric analysis. This study clearly describes RB2/ p130 as an important target for mutations and subsequent inactivation in lymphoma pathogenesis, thus validating that RB2/p130 is a classical tumor suppressor gene.
Assuntos
Linfoma de Burkitt/genética , Núcleo Celular/metabolismo , Mutação , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas , Substituição de Aminoácidos , Núcleo Celular/patologia , Éxons , Feminino , Mutação da Fase de Leitura , Humanos , Imuno-Histoquímica , Células Jurkat , Leucemia , Linfoma , Masculino , Mutagênese Sítio-Dirigida , Fosfoproteínas/análise , Mutação Puntual , Polimorfismo Conformacional de Fita Simples , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/biossíntese , Proteína p130 Retinoblastoma-Like , Transfecção , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-TroncoRESUMO
We have previously demonstrated that rat liver nuclei contain PI-PLC beta1 and gamma1 in the inner nuclear matrix and lamina associated with specific phosphodiesterase activity (Bertagnolo et al., 1995, Cell Signall. 7, 669-678). Since compensatory hepatic growth is an informative and well characterized model for natural cell proliferation, the presence of specific PI-PLC isoforms and their activity as well as PIP2 recovery were studied at various regenerating times, ranging from 3 to 22 h after partial hepatectomy. Three PI-PLC isoforms (beta1, gamma1, delta1) were examined in control and regenerating liver cells by using specific antibodies. By means of in situ immunocytochemistry and confocal microscopy, PI-PLC beta1 was found mainly in the nucleoplasm and this pattern was not modified after hepatectomy. On the contrary, the nuclear gamma1 isoform showed a marked decrease at 3 and 16 h after hepatectomy, but a clear increase at 22 h covering with bright intensity the whole nucleus. The PI-PLC delta1 isoform, which is exclusively cytoplasmic, was not altered during rat liver regeneration. By western blotting analysis on whole cell homogenates, none of the PI-PLC isozymes under study showed proliferation-linked modification. However, analyses of isolated nuclei identified changes in the nucleus associated PI-PLC gamma1 that paralleled the in situ observation whereas the beta1 isoform was unmodified at all the times examined. Nuclear phosphodiesterase activity on PIP2 was lower at 3 and 16 h, in comparison with sham operated rats, increased at 6 h and reached the highest value after 22 h. Consistently, the recovery of PIP2, obtained in conditions that optimise PIP-kinase activity, showed a marked decrease at 3 h and an increase up to 16 h of liver regeneration, followed by a further decrease at 22 h. These data are consistent with a close relationship between cell proliferation and the nuclear inositide cycle, depending, in rat liver, predominantly on the modulation of the gamma1 isoform of PI-PLC.
Assuntos
Regeneração Hepática/fisiologia , Fígado/enzimologia , Proteínas Nucleares/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Western Blotting , Divisão Celular , Imunofluorescência , Imuno-Histoquímica , Isoenzimas , Fígado/citologia , Microscopia Confocal , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositol Diacilglicerol-Liase , Fosfoinositídeo Fosfolipase C , Fosforilação , RatosRESUMO
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
Changes in the metabolism of nuclear inositides phosphorylated in the D3 position of the inositol ring, which may act as second messengers, mainly have been linked to cell differentiation. To clarify a possible role of this peculiar class of inositides also during cell proliferation and/or apoptosis, we have examined the issue of whether or not in the osteoblast-like clonal cell line MC3T3-E1 it may be observed an insulin-like growth factor-I (IGF-I)- and platelet-derived growth factor (PDGF)-dependent nuclear translocation of an active phosphatidylinositol 3-kinase (PI 3-K). We found that both the growth factors increased rapidly and transiently both the amount and the activity of immunoprecipitable nuclear PI 3-K. Intranuclear PI 3-K exhibited a massive tyrosine phosphorylation on the p85 regulatory subunit. Moreover, by means of coimmunoprecipitation experiments, we showed the presence, in isolated nuclei, of the p110beta catalytic subunit of PI 3-K. Enzyme translocation was blocked by the specific PI 3-K inhibitor LY294002. In contrast, intranuclear translocation of PI 3-K did not occur in response to the proapoptotic cytokine tumor necrosis factor alpha (TNF-alpha). IGF-I was able to counteract the apoptotic stimulus of TNF-alpha and this was accompanied by the intranuclear translocation of PI 3-K. LY294002 inhibited both intranuclear translocation of PI 3-K and the rescuing effect of IGF-I. These findings strongly suggest that an important step in the signaling pathways that mediate both cell proliferation and survival is represented by the intranuclear translocation of PI 3-K.
Assuntos
Núcleo Celular/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Osteoblastos/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/enzimologia , Cromonas/farmacologia , Citoplasma/efeitos dos fármacos , Citoplasma/enzimologia , Citoplasma/metabolismo , Imuno-Histoquímica , Cinética , Camundongos , Morfolinas/farmacologia , Osteoblastos/citologia , Osteoblastos/enzimologia , Osteoblastos/metabolismo , Fosfatidilinositol 3-Quinases/química , Inibidores de Fosfoinositídeo-3 Quinase , Fosfotirosina/metabolismo , Testes de Precipitina , Subunidades Proteicas , Transdução de Sinais/efeitos dos fármacosRESUMO
The increasing evidence of discrete roles of phosphoinositidase C (PIC) isoforms and the assessment of their localization in the cytoskeleton and in the nucleus support the involvement of particular isotypes of this enzyme in signal transduction at multiple levels. PC12 rat pheochromocytoma is one of the few cell lines expressing three immunologically distinct isoforms of PIC. We have analyzed the subcellular distribution of the PIC beta 1, gamma 1 and delta 1 isoforms using confocal and electron microscope immunocytochemistry. PIC beta 1 is mainly found in the nucleus and is associated with interchromatin domains. On the other hand, the PIC gamma 1 isoform is found in the nucleus and in the cytosol, while PIC delta 1 is exclusively cytoplasmic. Immunoblot and immunocytochemical experiments indicate that the various PIC isoforms are differently bound to structural cell compartments, such as cytoskeletal and nuclear matrix elements. In fact, PIC beta 1 and PIC gamma 1 isoforms are tightly associated with the nuclear matrix, while only about 50% of PIC gamma 1 is associated with the cytoskeleton after DNase I and high salt extractions. PIC gamma 1 is almost completely soluble under these conditions. These results further confirm the complexity of the inositide signal transduction mechanism, which involves several PIC isoforms, specifically localized in different cell compartments and support the existence of a membrane-unrelated inositol lipid-dependent signalling in the nuclear interior.
Assuntos
Compartimento Celular/fisiologia , Citoesqueleto/química , Isoenzimas/análise , Matriz Nuclear/enzimologia , Diester Fosfórico Hidrolases/análise , Animais , Immunoblotting , Imuno-Histoquímica , Células PC12 , Ratos , Especificidade por SubstratoRESUMO
Human metaphase chromosomes were isolated and digested in situ with HaeIII restriction enzyme to detect cytosine and guanine-rich sequences (CpG islands), which are known to be associated with most of the mammalian genes. Digested DNA was reconstructed by in situ nick translation employing digoxigenin-labeled nucleotides. The DNA sequences were revealed by antibodies conjugated either with fluorescein isothiocyanate or 1-nm colloidal gold. DNA was counterstained with propidium iodide. A sensitive, high resolution method for visualizing three signals, simultaneously excited by a single argon laser line of 488 nm has been developed. The green fluorescence of fluorescein isothiocyanate was detected in combination with the red fluorescence of propidium iodide, and the third signal was imaged by employing the reflectance mode of the confocal microscope after silver enhancement of the gold beads. The high reflectance intensity, the accurate localization and the non-fading properties of colloidal gold made the reaction a valuable tool for the detection of antigens and, as a consequence, of specific DNA sequences in chromosome preparations. Overlaying of three signals allowed the simultaneous observation of distinct structures: total DNA, as well as fluorescein- and gold-labeled sequences after in situ nick translation, or total DNA and centromeric sequences of two different chromosome pairs (17 and X) after in situ hybridization. The use of HaeIII restriction enzyme that cut CpG islands combined with in situ nick translation identified the chromosome sites where active, inactive or housekeeping genes can be located. In chromosomes, the fluorescent reaction pattern showed large areas of labeling, while a more defined staining, often organized in spot pairs that resembled an R-like banding, was detected when the reflected mode was used. These results are confirmed by the observation that R-like bands actually are multiple symmetrical spots localized on sister chromatids. In addition, some chromosomes, and in particular 1 and 9, displayed a C-negative banding due to the negativity of the centromeric areas. Reflectance confocal scanning microscopy and in situ nick translation represent a powerful tool to study the in situ genome organization.
Assuntos
Cromossomos Humanos/ultraestrutura , Ilhas de CpG , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Microscopia/métodos , Análise de Sequência de DNA/métodos , Cromossomos Humanos Par 17 , DNA Satélite/isolamento & purificação , Técnicas Genéticas , Humanos , Hibridização in Situ Fluorescente , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Cromossomo XRESUMO
Two different clones of Swiss 3T3 cells belonging to the same original cell line have been obtained, one of which was unresponsive to mitogenic stimulation (e.g. insulin-like growth factor-I, bombesin, insulin-like growth factor-I + bombesin), while the other clone showed a very high rate of DNA synthesis under identical conditions as demonstrated by 5-bromodeoxyuridine incorporation. Both types of cells expressed the IGF-I receptor and showed high contact inhibition. When highly purified nuclei from responsive cells, treated for a short time with bombesin and insulin-like growth factor-I or insulin-like growth factor-I alone, were incubated with [gamma-32P]adenosine triphosphate, the labelling of phosphatidylinositol-mono- and diphosphate decreased when compared to controls, while this transient effect did not appear in the nuclei from unresponsive cells. Similarly nuclear protein kinase C is activated only in responsive cells. Therefore, it seems that a direct link exists between polyphosphoinositide metabolism, protein kinase C activation and the early events leading to cell division, since the rapid changes in the labelling of both phosphatidylinositol mono- and di-phosphate occur only in nuclei from Swiss 3T3 cells, which respond to the mitogenic stimulus determined by insulin-like growth factor-I on its own, or in combination with bombesin.
Assuntos
Bombesina/farmacologia , Núcleo Celular/fisiologia , Replicação do DNA/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/farmacologia , Mitógenos , Ácidos Fosfatídicos/metabolismo , Fosfatos de Fosfatidilinositol , Fosfatidilinositóis/metabolismo , Proteínas Quinases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Cinética , Camundongos , Fosfatidilinositol 4,5-DifosfatoRESUMO
An active phosphatidylinositol 3-kinase (PI3K) has been shown in nuclei of different cell types. The products of this enzyme, i.e. inositides phosphorylated in the D3 position of the inositol ring, may act as second messengers themselves. Nuclear PI3K translocation has been demonstrated to be related to an analogous translocation of a PtdIns(3,4,5)P(3) activated PKC, the zeta isozyme. We have examined the issue of whether or not in the osteoblast-like clonal cell line MC3T3-E1 there may be observed an insulin-like growth factor-I- (IGF-I) and platelet-derived growth factor- (PDGF) dependent nuclear translocation of an active Akt/PKB. Western blot analysis showed a maximal nuclear translocation after 20 min of IGF-I stimulation or after 30 min of PDGF treatment. Both growth factors increased rapidly and transiently the enzyme activity of immunoprecipitable nuclear Akt/PKB on a similar time scale and after 60 min the values were slightly higher than the basal levels. Enzyme translocation was blocked by the specific PI3K inhibitor, LY294002, as well as cell entry into S-phase. Confocal microscopy showed an evident increase in immunostaining intensity in the nuclear interior after growth factor treatment but no changes in the subcellular distribution of Akt/PKB when a LY294002 pre-treatment was administered to the cells. These findings strongly suggest that the intranuclear translocation of Akt/PKB is an important step in signalling pathways that mediate cell proliferation.
Assuntos
Núcleo Celular/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/enzimologia , Cromonas/farmacologia , Ativação Enzimática/efeitos dos fármacos , Fibroblastos , Imunofluorescência , Cinética , Camundongos , Morfolinas/farmacologia , Osteoblastos/citologia , Osteoblastos/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt , Fase S/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , CrânioRESUMO
To determine the subcellular distribution of PKC after GFs treatment we have employed a combined immunochemical and in situ confocal microscopy analysis. In quiescent Swiss 3T3 cells only a faint PKC positivity was observable in the nucleus while a strong reaction was seen in the cytoplasm. IGF-I and to a lesser extent PDGF and EGF induced, after 45 min of treatment, a nuclear translocation of PKC detected by a pan-anti-PKC antibody and nuclear fluorescence was distributed in the nuclear interior except for the nucleolar regions. Bombesin and FGF did not affect the sub-cellular distribution of the enzyme. To further the understanding of which PKC isoform was involved in the translocation process, we have tested nine isozyme-specific anti-PKC antibodies. Immunoblotting analysis revealed the presence in Swiss 3T3 fibroblasts of alpha, beta I, epsilon and zeta isoforms. In isolated nuclei from GF-exposed cells only the alpha isozyme was detected: immunostaining was very intense after IGF-I treatment and clearly observable after PDGF and EGF stimulation. This result was strongly supported by the in situ confocal microscopy which parallels the Western blot analysis. These data demonstrate that several, but not all, GFs acting through tyrosine kinase receptor induce the intranuclear translocation of PKC alpha and, because of the dramatic effect of IGF-I, strengthen the case for a link between the activation of nuclear inositol lipid cycle and PKC translocation induced by this GF.
Assuntos
Núcleo Celular/metabolismo , Substâncias de Crescimento/farmacologia , Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Transporte Biológico/efeitos dos fármacos , Compartimento Celular , Fator de Crescimento Epidérmico/farmacologia , Imunofluorescência , Fator de Crescimento Insulin-Like I/farmacologia , Camundongos , Microscopia/métodos , Dados de Sequência Molecular , Proteínas Nucleares/isolamento & purificação , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteína Quinase C/isolamento & purificaçãoRESUMO
Morphological changes that occur in K562 cells after natural killing produce profound changes in cellular light scattering properties. The possibility of gating out all the effector cells by thresholding on perpendicular light scatter and the subsequent identification of two distinct clusters of cells, which correspond to dead and viable targets, have permitted the measurement of natural killer activity in vitro. The changes in scattering properties after cell death are mainly determined by the variation of internal refractive index of the dying cell. A comparison of the scattering and propidium iodide staining procedures showed good correlation. The morphological detection and measurement of cellular death is therefore used to estimate NK lytic activity. This methodology permits the measurement of NK activity without staining the target and the measurement of perpendicular light scatter provides an alternative approach to the study of lytic processes in vitro.
Assuntos
Citotoxicidade Imunológica , Citometria de Fluxo , Células Matadoras Naturais/fisiologia , Humanos , Luz , Espalhamento de Radiação , Células Tumorais Cultivadas/patologiaRESUMO
The distribution of three nuclear scaffold proteins (of which one is a component of a particular class of nuclear bodies) has been studied in intact K562 human erythroleukemia cells, isolated nuclei, and nuclear scaffolds. Nuclear scaffolds were obtained by extraction with the ionic detergent lithium diidosalicylate (LIS), using nuclei prepared in the absence of divalent cations (metal-depleted nuclei) and stabilized either by a brief heat exposure (20 min at 37C or 42C) or by Cu++ ions at 0C. Proteins were visualized by in situ immunocytochemistry and confocal microscopy. Only a 160-kD nuclear scaffold protein was unaffected by all the stabilization procedures performed on isolated nuclei. However, LIS extraction and scaffold preparation procedures markedly modified the distribution of the polypeptide seen in intact cells, unless stabilization had been performed by Cu++. In isolated nuclei, only Cu++ treatment preserved the original distribution of the two other antigens (M(r), 125 and 126 kD), whereas in heat-stabilized nuclei we detected dramatic changes. In nuclear scaffolds reacted with antibodies to 125 and 126-kD proteins, the fluorescent pattern was always disarranged regardless of the stabilization procedure. These results, obtained with nuclei prepared in the absence of Mg+2 ions, indicate that heat treatment per se can induce changes in the distribution of nuclear proteins, at variance with previous suggestions. Nevertheless, each of the proteins we have studied behaves in a different way, possibly because of its specific association with the nuclear scaffold.
Assuntos
Autoantígenos/metabolismo , Cobre/farmacologia , Proteínas Nucleares/metabolismo , Antígenos Nucleares , Autoantígenos/química , Biomarcadores/química , Western Blotting , Temperatura Alta , Humanos , Leucemia Eritroblástica Aguda/metabolismo , Microscopia Confocal , Peso Molecular , Proteínas Nucleares/química , Células Tumorais CultivadasRESUMO
The nuclear matrix, a proteinaceous network believed to be a scaffolding structure determining higher-order organization of chromatin, is usually prepared from intact nuclei by a series of extraction steps. In most cell types investigated the nuclear matrix does not spontaneously resist these treatments but must be stabilized before the application of extracting agents. Incubation of isolated nuclei at 37C or 42C in buffers containing Mg++ has been widely employed as stabilizing agent. We have previously demonstrated that heat treatment induces changes in the distribution of three nuclear scaffold proteins in nuclei prepared in the absence of Mg++ ions. We studied whether different concentrations of Mg++ (2.0-5 mM) affect the spatial distribution of nuclear matrix proteins in nuclei isolated from K562 erythroleukemia cells and stabilized by heat at either 37C or 42C. Five proteins were studied, two of which were RNA metabolism-related proteins (a 105-kD component of splicing complexes and an RNP component), one a 126-kD constituent of a class of nuclear bodies, and two were components of the inner matrix network. The localization of proteins was determined by immunofluorescent staining and confocal scanning laser microscope. Mg++ induced significant changes of antigen distribution even at the lowest concentration employed, and these modifications were enhanced in parallel with increase in the concentration of the divalent cation. The different sensitivity to heat stabilization and Mg++ of these nuclear proteins might reflect a different degree of association with the nuclear scaffold and can be closely related to their functional or structural role.
Assuntos
Magnésio/farmacocinética , Proteínas Nucleares/metabolismo , Antígenos Nucleares , Nucléolo Celular/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Temperatura Alta , Humanos , Processamento de Imagem Assistida por Computador , Leucemia Eritroblástica Aguda/metabolismo , Microscopia Confocal , Proteínas Nucleares/efeitos dos fármacos , Ribonucleoproteínas/metabolismo , Células Tumorais CultivadasRESUMO
We analyzed the incorporation of bromodeoxyuridine (BrdUrd) into DNA in exponentially growing murine erythroleukemia cells (FLC-745), using fluorescent anti-BrdUrd antibodies with light microscopy and flow cytometry. The fine localization of the DNA replicating sites was investigated at the ultrastructural level by using a second antibody conjugated with colloidal gold. The latter approach, which does not require acidic denaturation of the DNA, enables preservation of good morphology and obtains a better resolution power than that of electron microscopic autoradiography, the percentage of labeled cells obtained with the two techniques being comparable. After short BrdUrd pulses, characteristic distribution of the labeling can be identified in the heterochromatin, in interchromatin domains, or at the boundary between the dispersed and the condensed chromatin. Similar patterns are also observable in the nuclear structures which condense after acid denaturation, suggesting that DNA replication takes place at fixed sites associated with the nuclear matrix.
Assuntos
Anticorpos/análise , Especificidade de Anticorpos , Bromodesoxiuridina , Cromatina/análise , DNA de Neoplasias/biossíntese , Animais , Bromodesoxiuridina/imunologia , Ciclo Celular , Replicação do DNA , Citometria de Fluxo , Imunofluorescência , Leucemia Eritroblástica Aguda , Camundongos , Microscopia Eletrônica , Células Tumorais CultivadasRESUMO
In recent there has been increasing interest in the definition of hormone influence on the immune system. Physical stress provides a suitable model for studying the interactions between the immune system and the neuroendocrine factors which have been shown to modulate the lymphoid cellular compartment. Our approach has been devoted to defining the stable modifications induced in the immune system in athletes during agonistic training. The results show that the circulating compartment of the immune system tends to modulate its different subsets under the continuous influence of stress hormones, together with a specific functional impairment of the helper subset in the proliferative response after stimulation with PHA, and particularly with PWM.
Assuntos
Ativação Linfocitária , Aptidão Física , Anticorpos Monoclonais , Antígenos de Superfície , Separação Celular , Células Cultivadas , Citometria de Fluxo , Imunofluorescência , Antígenos HLA-DR/análise , Humanos , Fenótipo , Fito-Hemaglutininas/metabolismo , Mitógenos de Phytolacca americana/metabolismo , Receptores de Interleucina-2/análiseRESUMO
A growing body of evidence, accumulated over the past 15 years, has highlighted that the protein kinase C family of isozymes is capable of translocating to the nucleus or is resident within the nucleus. The comprehension of protein kinase C isoform regulation within this organelle is under development. At present, it is emerging that lipid second messengers may play at least two roles in the control of nuclear protein kinase C: on one side they serve as chemical attractants, on the other they directly modulate the activity of specific isoforms. One of the best characterized lipid second messenger that could be involved in the regulation of nuclear PKC activity is DAG. The existence of two separate pools of nuclear DAG suggests that this lipid second messenger might be involved in distinct pathways that lead to different cell responses. Nuclear phosphatidylglycerol, D-3 phosphorylated inositol lipids and nuclear fatty acids are involved in a striking variety of critical biological functions which may act by specific PKC activation. The fine tuning of PKC regulation in cells subjected to proliferating or differentiating stimuli, might prove to be of great interest also for cancer therapy, given the fact that PKC-dependent signaling pathways are increasingly being seen as possible pharmacological target in some forms of neoplastic diseases. In this article, we review the current knowledge about lipid second messengers that are involved in regulating the translocation and/or the activity of different protein kinase C isoforms identified at the nuclear level.
Assuntos
Núcleo Celular/fisiologia , Proteína Quinase C/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Animais , Núcleo Celular/metabolismo , Diglicerídeos/metabolismo , Ácidos Graxos/metabolismo , Humanos , Inositol/metabolismo , Isoenzimas/metabolismo , Fosfatidilgliceróis/metabolismoRESUMO
It is becoming increasingly clear that the cell nucleus is a highly structurized organelle. Because of its tight compartmentalization, it is generally believed that a framework must exist, responsible for maintaining such a spatial organization. Over the last twenty years many investigations have been devoted to identifying the nuclear framework. Structures isolated by different techniques have been obtained in vitro and are variously referred to as nuclear matrix, nucleoskeleton or nuclear scaffold. Many different functions, such as DNA replication and repair, mRNA transcription, processing and transport have been described to occur in close association with these structures. However, there is still much debate as to whether or not any of these preparations corresponds to a nuclear framework that exists in vivo. In this article we summarize the most commonly-used methods for obtaining preparations of nuclear frameworks and we also stress the possible artifacts that can be created in vitro during the isolation procedures. Emphasis is placed also on the protein composition of the frameworks as well as on some possible signalling functions that have been recently described to occur in tight association with the nuclear matrix.