Biomedical subjects

L M Neri

Publications and source records attributed to L M Neri.

At least 55 records · Page 3Linked to original sources

PubMed1996

Multiple fluorescence and reflectance simultaneous detection by confocal microscopy of HaeIII digested DNA sequences.

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.

Chromosomes, Human↗

PubMed1996

The nuclear matrix: a critical appraisal.

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.

Animals↗

PubMed1995

Analysis by confocal microscopy of the behavior of heat shock protein 70 within the nucleus and of a nuclear matrix polypeptide during prolonged heat shock response in HeLa cells.

By means of confocal laser scanning microscopy and indirect fluorescence experiments we have examined the behavior of heat-shock protein 70 (HSP70) within the nucleus as well as of a nuclear matrix protein (M(r) = 125 kDa) during a prolonged heat-shock response (up to 24 h at 42 degrees C) in HeLa cells. In control cells HSP70 was mainly located in the cytoplasm. The protein translocated within the nucleus upon cell exposure to hyperthermia. The fluorescent pattern revealed by monoclonal antibody to HSP70 exhibited several changes during the 24-h-long incubation. The nuclear matrix protein showed changes in its location that were evident as early as 1 h after initiation of heat shock. After 7 h of treatment, the protein regained its original distribution. However, in the late stages of the hyperthermic treatment (17-24 h) the fluorescent pattern due to 125-kDa protein changed again and its original distribution was never observed again. These results show that HSP70 changes its localization within the nucleus conceivably because it is involved in solubilizing aggregated polypeptides present in different nuclear regions. Our data also strengthen the contention that proteins of the insoluble nucleoskeleton are involved in nuclear structure changes that occur during heat-shock response.

Antibodies, Monoclonal↗

PubMed1995

Immunocytochemical evaluation of protein kinase C translocation to the inner nuclear matrix in 3T3 mouse fibroblasts after IGF-I treatment.

The complex pathway which links the agonist-cell membrane receptor binding to the response at the genome level involves, among other elements, protein kinase C (PKC). Agonists acting at the cell membrane can affect an autonomous nuclear polyphosphoinositide signaling system inducing an activation of nuclear phosphoinositidase activity and a subsequent translocation of PKC to the nuclear region. The fine localization of PKC has been investigated by means of electron microscopy quantitative immunogold labeling in 3T3 mouse fibroblasts, mitogenically stimulated by IGF-I. The enzyme, which in untreated cells is present in the cytoplasm, except for the organelles, and in the nucleoplasm, after IGF-I treatment is reduced in the cytoplasm and almost doubled in the nucleus. The PKC isoform translocated to the nucleus is the alpha isozyme, which is found not only associated with the nuclear envelope but mainly with the interchromatin domains. By using in situ matrix preparations, PKC appears to be retained at the nuclear matrix level, both at the nuclear lamina and at the inner nuclear matrix, suggesting a direct involvement in the phosphorylation of nuclear proteins which are responsible for the regulation of DNA replication.

3T3 Cells↗

PubMed1995

The effect of sodium tetrathionate stabilization on the distribution of three nuclear matrix proteins in human K562 erythroleukemia cells.

Using both conventional fluorescence and confocal laser scanning microscopy we have investigated whether or not stabilization of isolated human erythroleukemic nuclei with sodium tetrathionate can maintain in the nuclear matrix the same spatial distribution of three polypeptides (M(r) 160 kDa and 125 kDa, previously shown to be components of the internal nuclear matrix plus the 180-kDa nucleolar isoform of DNA topoisomerase II) as seen in permeabilized cells. The incubation of isolated nuclei in the presence of 2 mM sodium tetrathionate was performed at 0 degrees C or 37 degrees C. The matrix fraction retained 20-40% of nuclear protein, depending on the temperature at which the chemical stabilization was executed. Western blot analysis revealed that the proteins studied were completely retained in the high-salt resistant matrix. Indirect immunofluorescence experiments showed that the distribution of the three antigens in the final matrix closely resembled that detected in permeabilized cells, particularly when the stabilization was performed at 37 degrees C. This conclusion was also strengthened by analysis of cells, isolated nuclei and the nuclear matrix by means of confocal laser scanning microscopy. We conclude that sodium tetrathionate stabilization of isolated nuclei does not alter the spatial distribution of some nuclear matrix proteins.

Blotting, Western↗

PubMed1995

Identification of PI-PLC beta 1, gamma 1, and delta 1 in rat liver: subcellular distribution and relationship to inositol lipid nuclear signalling.

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.

Animals↗

PubMed1994

Selective nuclear translocation of protein kinase C alpha in Swiss 3T3 cells treated with IGF-I, PDGF and EGF.

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.

3T3 Cells↗

PubMed1994

Phosphoinositide signaling in nuclei of Friend cells: phospholipase C beta down-regulation is related to cell differentiation.

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.

Animals↗

PubMed1994

Discrete localization of different DNA topoisomerases in HeLa and K562 cell nuclei and subnuclear fractions.

Monoclonal antibodies raised against DNA topoisomerase I and against topoisomerase II alpha and beta isoforms, which have been previously demonstrated to be highly specific and capable of detecting cell cycle-related variations of the topoisomerase II isoforms (Negri et al., 1992, Exp. Cell Res. 200, 452-459), have been utilized for a fine subcellular localization. Immunocytochemistry by confocal and electron microscopy have been used for a topological and quantitative evaluation of the fine distribution of the different topoisomerases in HeLa and K562 cells. Topoisomerase I and topoisomerase II alpha are present both in the nucleoplasm and in the nucleolus, though at different relative ratios, while topoisomerase II beta is exclusively present at the nucleolar level. This is further confirmed by immunoblotting and immunocytochemical quantitative evaluations performed on purified nuclear matrix fractions obtained from K562 cells. In fact, the amount of topoisomerase I and topoisomerase II alpha present in the whole cell nuclei is partly lost in isolated nuclei but, while topoisomerase I is further significantly reduced in nuclear matrix preparations, the topoisomerase II alpha content is only slightly decreased. On the other hand, the great majority of topoisomerase II beta is retained in the nuclear matrix and can be detected exclusively in association with the nucleolar remnant. These results are consistent with specific functional roles hypothesized for the different topoisomerase types.

Antibodies, Monoclonal↗

PubMed1994

In vitro heat exposure induces a redistribution of nuclear matrix proteins in human K562 erythroleukemia cells.

By using both conventional and confocal laser scanning microscopy with three monoclonal antibodies recognizing nuclear matrix proteins we have investigated by means of indirect fluorescence whether an incubation of isolated nuclei at the physiological temperature of 37 degrees C induces a redistribution of nuclear components in human K562 erythroleukemia cells. Upon incubation of isolated nuclei for 45 min at 37 degrees C, we have found that two of the antibodies, directed against proteins of the inner matrix network (M(r) 125 and 160 kDa), gave a fluorescent pattern different from that observed in permeabilized cells. By contrast, the fluorescent pattern did not change if nuclei were kept at 0 degrees C. The difference was more marked in case of the 160-kDa polypeptide. The fluorescent pattern detected by the third antibody, which recognizes the 180-kDa nucleolar isoform of DNA topoisomerase II, was unaffected by heat exposure of isolated nuclei. When isolated nuclear matrices prepared from heat-stabilized nuclei were stained by means of the same three antibodies, it was possible to see that the distribution of the 160-kDa matrix protein no longer corresponded to that observable in permeabilized cells, whereas the fluorescent pattern given by the antibody to the 125-kDa polypeptide resembled that detectable in permeabilized cells. The 180-kDa isoform of topoisomerase II was still present in the matrix nucleolar remnants. We conclude that a 37 degrees C incubation of isolated nuclei induces a redistribution of some nuclear matrix antigens and cannot prevent the rearrangement in the spatial organization of one of these antigens that takes place during matrix isolation in human erythroleukemia cells. The practical relevance of these findings is discussed.

Antibodies↗

PubMed1994

6-Iodoacetamidofluorescein labelling to assess the state of sulphhydril groups after thermal stabilization of isolated nuclei.

Isolated nuclei and nuclear matrices, prepared from mouse erythroleukaemia cells, were reacted with the sulphhydryl-specific dye 6-iodoacetamidofluorescein. To determine whether in vitro formation of disulphide bonds might play a role in the nuclear matrix stabilization triggered by exposure of isolated nuclei to the physiological temperature of 37 degrees C, a variety of techniques were employed to assess the state of cysteinyl residues after such an incubation. Both flow cytometry and confocal microscopy quantitative analysis did not reveal major differences in the fluorescence intensity of nuclei incubated at 37 degrees C in comparison with those maintained at 0 degrees C. Confocal scanning laser microscopy revealed that 6-iodoacetamidofluorescein labelled a fibrogranular network in isolated nuclei. The fluorescent pattern of the network was not affected by a 37 degrees C exposure of nuclei. However, such a network was not detectable in isolated nuclear matrices, thus suggesting a possible protein re-arrangement during matrix preparation. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of fluorescent-labelled nuclear proteins showed no difference between heat-exposed and control samples. We conclude that oxidation of cysteinyl residues is not a major factor leading to the stabilization of nuclei incubated at 37 degrees C.

Animals↗

PubMed1994

An immunohistochemical study of protein kinase C distribution in fetal mouse vertebral column.

Using polyclonal antibodies we have studied the distribution of protein kinase C in fetal mouse low thoracic vertebrae. By means of a pan protein kinase C antiserum recognizing the catalytic domain of the enzyme, we show that protein kinase C is markedly expressed in chondrocytes before birth. The enzyme seems to be very abundant in the more mature cells that are close to ossification centres as well as the periphery of the intervertebral disc, although it can also be detected in chondrocytes. In order to establish which protein kinase C isoenzyme(s) the chondrocytes produce, we employed polyclonal isoenzyme-specific antisera developed against three calcium-dependent isoforms (alpha, beta, gamma) and three calcium-independent isoforms (delta, epsilon, zeta). Secondary antibody conjugated to alkaline phosphatase revealed that chondrocytes markedly express the beta-isoform. Cells were also weakly stained by the anti-epsilon serum. The immunostaining was completely abolished by pre-incubating primary antibodies with the peptide antigens to which they were raised. These results suggest that protein kinase C (and particularly the beta isoform) could play an important role in mouse fetal chondrogenesis of the vertebral column.

Alkaline Phosphatase↗

PubMed1994

Immunocytochemical detection of the specific association of different PIC isoforms with cytoskeletal and nuclear matrix compartments in PC12 cells.

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.

Animals↗

PubMed1994

Nuclear translocation of phosphatidylinositol 3-kinase in rat pheochromocytoma PC 12 cells after treatment with nerve growth factor.

Immunocytochemical analysis of PI 3-kinase localization in PC 12 cells demonstrates that the enzyme translocates to the nucleus after cell treatment with differentiating doses of NGF. The association of PI 3-kinase to the nucleus occurs rapidly (within minutes) and increases with the time of exposure of NGF. We suggest that PI-3 kinase specific localization may determine the production of novel phosphoinositides in cell compartments targeted to effect diverse cell responses. The nuclear translocation is consistent with accumulating data on the existence of a nuclear inositol lipid cycle which could also include 3-phosphorylated inositides, participating to the modulation of the cell response to extracellular stimuli.

Animals↗

PubMed1993

Absence of high levels of DNA polymerase alpha activity in the nuclear matrix prepared from mouse erythroleukemia cells.

We have examined the association of DNA polymerase alpha activity with the nuclear matrix prepared by different techniques from mouse erythroleukemia cells. At variance with the data obtained using other cell types we have found that only a small amount (less than 2%) of nuclear DNA polymerase alpha activity resisted extraction with high-ionic strength buffers, even if nuclei were heat-stabilized by incubation at 37 degrees C for 45 min prior to subfractionation. The recovery of DNA polymerase alpha activity bound to the matrix was unaffected by the type of extracting agent used (NaCl or (NH4)2 SO4), by the extraction sequence or by the method employed for obtaining nuclei. These results could indicate that in some types of cells the nuclear matrix is not involved in DNA replication.

Animals↗

PubMed1993

Immunocytochemical analysis of phosphatidylinositol-specific phospholipase C in PC12 cells: predominance of the delta isoform during neural differentiation.

The rat pheochromocytoma PC12 cell line, which differentiates into sympathetic neurons under nerve growth factor (NGF) treatment, contains at least three phosphoinositidase C (PIC) isozymes, PIC beta, PIC gamma, PIC delta. These isozymes have been previously shown to display a different subcellular localization. To determine whether or not NGF induces changes in the presence and/or distribution of PIC isozymes during PC12 neural differentiation, studies were carried out by means of in situ immunocytochemistry. After NGF administration the proliferative activity was progressively reduced to very low levels, as measured by bromodeoxy Uridine incorporation, and a neuron-like morphology was displayed by almost all cells. In unstimulated PC12 cells, PIC beta was detected in the nucleus whereas PIC delta was only cytoplasmic; PIC gamma was found in both cell compartments. In cells treated with NGF for 3 days, neural processes extended to twice the diameter of the cell body; the gamma isoform was concentrated near the nucleus, while the immunoreactivity of the beta form remained constant and the delta form was increased. After 10 days of treatment with NGF, PIC beta was hardly detectable and PIC gamma immunostaining was considerably decreased. On the contrary, PIC delta progressively increased and, after 14 days of NGF exposure, fully differentiated cells displayed an intense labelling of cell body and neurites. In the same cells, PIC beta and PIC gamma were almost negative. These results suggest that NGF dependent neural differentiation is related to the selective down regulation of PIC beta and gamma and the increase of PIC delta isozyme associated with the decrease of cell proliferation.

Animals↗

PubMed1993

Influence of the human immunodeficiency virus type 1 Tat protein on the proliferation and differentiation of PC12 rat pheochromocytoma cells.

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.

Animals↗

PubMed1992

Discrete subcellular localization of phosphoinositidase C beta, gamma and delta in PC12 rat pheochromocytoma cells.

Phosphoinositidase C activity was revealed in nuclei isolated from PC12 rat pheochromocytoma cells incubated with tritiated phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. Phosphoinositide breakdown was found to be optimal at neutral pH and Ca++ concentrations ranging from endogenous levels to millimolar values. To characterize the enzymes involved, three monoclonal antibodies directed against the beta, gamma and delta phosphoinositidase C isoforms were employed. A combination of Western blot immunochemical analysis on cytoplasmic and nuclear fractions and of in situ immunocytochemistry on intact cells and isolated nuclei indicated that phosphoinositidase C gamma, though predominantly cytoplasmic, was present in both cell compartments. On the contrary, phosphoinositidase C beta was exclusively localized in the nucleus, whereas phosphoinositidase C delta was restricted to the cytoplasm. These data suggest that inositol lipid breakdown is controlled by different phosphoinositidase C isozymes in the various cell compartments, and support the notion that a separate phosphoinositide signalling system is located in the nucleus.

Animals↗