Biomedical subjects

N M Maraldi

Publications and source records attributed to N M Maraldi.

At least 19 recordsLinked to original sources

PubMed1997

Increase of nuclear phosphatidylinositol 4,5-bisphosphate and phospholipase C beta 1 is not associated to variations of protein kinase C in multidrug-resistant Saos-2 cells.

The multidrug resistance (MDR) phenotype that is mediated by an overexpression of P-glycoprotein, has been suggested to be related also to an increased activity of protein kinase C (PKC) and to changes in phospholipid pattern. By electron microscope quantitative immunocytochemistry, we investigated whether PKC and other elements of the polyphosphoinositide signal transduction system are affected in an MDR variant of the human osteosarcoma cell line Saos-2. These cells, which are characterized by an increased expression of P-glycoprotein not only at the plasma membrane but also at the nuclear level, showed increased intranuclear amounts of phosphatidylinositol 4,5-bisphosphate and of phospholipase C beta 1, while both the amount and activity of both nuclear and cellular PKC were not modified with respect to sensitive cells. These results suggest that, in this model, the changes observed in the elements of nuclear signal transduction could be related to previously reported modifications of the MDR phenotype, but that P-glycoprotein phosphorylation is not dependent from increased PKC activity.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1997

Redistribution of DNA topoisomerase II beta after in vitro stabilization of human erythroleukemic nuclei by heat or Cu++ revealed by confocal microscopy.

Using confocal laser scanning microscope and a monoclonal antibody we have examined by means of indirect immunofluorescence techniques the distribution of DNA topoisomerase II beta (the 180-kDa nucleolar isoform of topoisomerase II) following stabilization of isolated nuclei by exposure to moderate heat (37 degrees or 42 degrees C) or Cu++. In intact cells the antibody specifically decorated the nucleoli. The same pattern was maintained if nuclei were incubated at 0 degree C in a buffer containing spermine/spermidine/KCl or stabilized by means of 0.5 mM Cu++ for 10 minutes at 0 degree C in the same buffer. On the contrary, if stabilization was performed by incubating the nuclei either at 37 degrees or 42 degrees C, the immunoreactivity dispersed all over the nucleus, forming numerous speckles. This phenomenon was not detected if, in addition to spermine/spermidine/KCl, the incubation buffer also contained 5 mM Mg++ and the temperature was 37 degrees C. If the stabilization was performed at 42 degrees C, Mg++ failed to maintain the original distribution of DNA topoisomerase II beta, as seen in intact cells. The analysis on 2-D optical section showed the alteration of the nucleolar profile, particularly at 37 degrees C, even when the samples were treated with Mg++. The 3-D reconstruction figured out the irregularity of the surface at 37 degrees C and the variations of the volume occupied by the fluorescent figures. These were in close proximity to each other both in intact cells and in 0 degree C incubated nuclei; they showed a certain degree of shrinkage in 0 degree C plus Cu++ exposed samples (-20% of the volume), and, on the contrary, the labeled structures were scattered in a volume increased two- or threefold when exposed to 37 degrees or 42 degrees C, respectively. The addition of Mg++ restored the original spatial relationship and volume at 37 degrees C, but not at 42 degrees C, where the volumetric analysis showed an increase of about 50%. Our results demonstrate that heat stabilization of isolated nuclei in a buffer without Mg++ (i.e., a technique often employed to prepare the nuclear matrix or scaffold) cannot be considered an optimal procedure to maintain the original distribution of protein within the nucleus.

Antibodies, Monoclonal↗

PubMed1997

Localization of the laminin alpha 2 chain in normal human skeletal muscle and peripheral nerve: an ultrastructural immunolabeling study.

A particular form of congenital muscular dystrophy is associated with a deficiency of the tissue-specific basement membrane protein laminin alpha 2. A more precise knowledge of the normal distribution and localization of laminin alpha 2 would be useful in further elucidating the development of this disorder. In this study we used specific electron microscopic techniques, i.e., thin-section fracture labeling and cryoultramicrotomy in combination with immunogold labeling for laminin alpha 2, to determine its ultrastructural localization in normal human muscle and peripheral nerve. Both in muscle and in peripheral nerve, laminin alpha 2 is found to be associated solely with the basal lamina of myofibers and Schwann cells, respectively. Of special interest is the finding that in peripheral nerve, laminin alpha 2 is associated only with myelinated and not with unmyelinated nerve fibers.

Humans↗

PubMed1997

Enhanced resolution of specific chromosome and nuclear regions by reflectance laser scanning confocal microscopy.

DNA sequences digested by HaeIII and reconstructed by in situ nick translation employing digoxigenin-labelled nucleotides are usually revealed either by horseradish peroxidase or FITC fluorescence. To obtain a significant improvement in terms of resolution, sensitivity and specificity, colloidal gold has been used instead of FITC (as the reporter molecule) to reveal the labelled DNA. Colloidal gold and propidium iodide were visualised by employing the reflectance mode and the 488-nm laser line of a confocal laser scanning microscope. In chromosomes, the fluorescent reaction pattern showed diffuse areas of labelling in which it was impossible to identify any specific kind of banding along the arms. In some chromosomes and, in particular, 1 and 9, a C-negative banding due to the negativity of the centromeric areas was seen. A more accurate localisation on chromosomes, including telomeric regions, often organised in spot pairs that resembled an R-like banding, was detected using 1-nm colloidal gold. A fine labelling was also demonstrated in nuclei, especially at their peripheral heterochromatin. The non-fading properties of colloidal gold combined with visualisation by reflectance confocal laser scanning microscopy demonstrated the possibility of obtaining a higher spatial resolution than when using conventional fluorophores or higher laser wavelength. This improved way to study the localization of HaeIII digestion sites in single chromosomes and in interphase nuclei made the reaction a valuable tool for the detection of antigens or of specific DNA sequences in biological preparations.

Cell Nucleus↗

PubMed1997

Intracellular detection of laminin alpha 2 chain in skin by electron microscopy immunocytochemistry: comparison between normal and laminin alpha 2 chain deficient subjects.

The aim of this study is to localize the alpha 2 laminin chain in normal human skin. The methods used were immuno-gold cytochemistry on cryo-ultramicrotomy sections and thin-section-fracture-label, together with electron microscopy observation. Results were compared with light microscopy peroxidase immuno-staining. Both normal skin samples and skin biopsies from laminin alpha 2 chain deficient congenital muscular dystrophy affected patients were studied. The results show that, in normal skin, the laminin alpha 2 chain is spread throughout the cytoplasm of basal keratinocytes, while it appears associated with desmosomal tonofilaments in the spinous and granular epidermal layers; in skin samples from dystrophic patients the laminin alpha 2 chain was not detectable. These data suggest that the function of the laminin alpha 2 chain is different in the epidermis as compared to that in muscle and peripheral nerve, where it is localized in the basement membrane.

Humans↗

PubMed1997

Superficial and deep defects in dyschondroplastic and degenerated pig articular cartilage.

Normal and pathological (osteochondrotic and osteoarthrotic) pig articular cartilages from medial humeral and femoral condyles were studied by scanning electron microscopy. The pathological cartilages showed primitive osteochondrotic lesions with progressive aspects according to the severity of the pathology (flaking, fibrillation and cracks) and other superficial changes (micro-undulation, micro-fissurations, clefts) that appeared to be a consequence of the action of intense mechanical stresses such as shearing forces, compressive deformation, friction of the articular surfaces. The observation of deep lesions such as cracks and fractures at or near the interface between cartilage and calcified zone, frequently observed both at medial condyle and at intercondylar crista of the humerus, often evolving in cartilagineous flaps, were related to excessive tangential and shear forces induced by an abnormal articular topography resulting in joint instability. This pathological joint dynamic could be also worsened by an anomalous leg conformation (cross-legs) and/or by an increased occurrence of environmental micro- and macrotrauma (impact loading). Also in this case, the frequency and severity of the lesions can be increased if the deep layer is affected by osteo-chondrotic lesions. The results stress the pathogenetic importance of mechanical load in initiating and worsening the articular lesions in pigs; they also suggest that the resulting alterations can be influenced by a pre-existing different maturity or pathological condition of the cartilage.

Animals↗

PubMed1997

Protein kinase C isoforms undergo quantitative variations during rat spermatogenesis and are selectively retained at specific spermatozoon sites.

Signal transduction elements, including protein kinase C, have been identified in mammalian spermatozoa. In order to evaluate the pattern of expression and the subcellular localization of nine different protein kinase C isoforms in the course of spermatogenesis, we utilized quantitative electron microscopy immunocytochemistry on thin sections of rat seminiferous tubules. The results indicate a progressive reduction of the protein kinase C isoforms present in the early stages of spermatogenesis, so that in late spermatids none of them is present in the nucleus, while the isoforms alpha, gamma and beta II are specifically retained in the acrosome, the isoforms beta I and zeta in the neck, and the isoform epsilon in the tail. These isoforms, except for beta II, are maintained at the same sites in spermatozoa. Western blotting analysis indicates the presence of alpha and gamma isoforms in the head subfraction, and of beta I, zeta and epsilon isoforms in the tail subfraction of spermatozoa. These findings suggest that specific protein kinase C isoforms may be functionally involved in some events of spermatozoa differentiation and, eventually, in the fertilization process.

Animals↗

PubMed1997

Immunolocalization of several laminin chains in the normal human central and peripheral nervous system.

Using specific monoclonal antibodies against different subunits of laminin, we studied the differential distribution pattern of several laminin chains in the central (CNS) and peripheral (PNS) nervous system. Laminin chains alpha 1, beta 1 and gamma 1, were found in the basement membrane (BM) of blood vessels in both CNS and PNS. In contrast, laminin alpha 2 though present in the BM of capillaries in the CNS, was completely absent from PNS capillaries. Laminins alpha 2, beta 1, gamma 1 could be detected in peripheral nerve, in the BM of Schwann cells, which did not contain Laminin alpha 1. The possible importance of laminin alpha 2 for myelination in the PNS as well as in the function of the blood-brain barrier in the CNS, and its potential relevance to the pathology of congenital muscular dystrophy associated with deficiency of this laminin chain, is discussed.

Antibodies, Monoclonal↗

PubMed1996

Influence of specimen preparation on the identification of phospholipids by the phospholipase A2-gold method in mineralizing cartilage and bone.

The role of phospholipids in biological mineralization has been hypothesized but not fully elucidated. In order to identify phospholipids at the ultrastructural level in the mineralizing extracellular matrix, rat epiphyseal cartilage and metaphyseal bone have been labeled with the phospholipase A(2) (PLA(2))-gold method. The specificity and the efficiency of phospholipid detection have been evaluated by postembedding labeling of sections from epoxy- or hydrophilic resin-embedded samples, and by preembedding labeling of cryosectioned samples. The efficiency of the labeling was higher in cryosections than in hydrophilic resin-embedded specimens, while lower efficiency was found in epoxy resin-embedded samples. A 2- to 6-fold increase of the labeling density in calcified with respect to uncalcified areas of cartilage and bone has been found, depending on the specimen preparation used. The labeling intensity was significantly higher, at the periphery of the calcifying nodules in the epiphyseal cartilage matrix and in the calcifying osteoid, while the fully calcified bone matrix presented a weak labeling. Matrix vesicles, which are considered a possible source of extracellular phospholipids, appeared labeled in cryosections and in epoxy resin-embedded samples after a preincubation with PLA(2), which also increased the labeling of the intracellular membranes. The localization of phospholipids in the areas of initial mineralization suggests some hypotheses on the possible involvement of these molecules in the mineral phase deposition process.

Animals↗

PubMed1996

Interleukin-1 alpha induces variations of the intranuclear amount of phosphatidylinositol 4,5-bisphosphate and phospholipase C beta 1 in human osteosarcoma Saos-2 cells.

Some key elements of signal transduction have been identified within the nucleus and demonstrated to be responsive to specific agonists in numerous cell types. In particular, mitogenic stimuli have been reported to induce a transient increase of the nuclear phospholipase C beta 1 activity, causing the release of inositide-derived second messengers, whereas differentiating stimuli induced a decrease of the enzyme activity and an increase of nuclear phosphatidylinositol 4,5-bisphosphate (PIP2). Recently, we reported evidence, in human osteosarcoma Saos-2 cell lines, on the presence of specific nuclear phospholipase C isoforms and on the activation of phospholipase C beta 1 in the nucleus following the exposure to interleukin-1 alpha. In this study we report immunocytochemical ultrastructural evidence on quantitative variations of PIP2 and phospholipase C beta 1 amounts in the nucleus of Saos-2 cells at different times of exposure to interleukin-1 alpha. After short periods of culture in the presence of the agonist, the intranuclear amount of PIP2 is decreased, while a translocation of phospholipase C beta 1 occurs from the cytoplasm to the nucleus, in correspondence with the increased hydrolyzing activity of the enzyme. After longer periods of incubation with interleukin-1 alpha, on the other hand, the intranuclear amount of PIP2 is restored to initial level, while the amount of phospholipase C beta 1 is increased both at the nuclear and cytoplasmic level, when its activation is no longer effective. The results, compared with those obtained in other cell types responsive to given agonists, account for a cell-specific modulation of signal transduction based on polyphosphoinositide breakdown at the nuclear level.

Bone Neoplasms↗

PubMed1996

Cytoplasmic and nuclear localization sites of phosphatidylinositol 3-kinase in human osteosarcoma sensitive and multidrug-resistant Saos-2 cells.

The intracellular localization of phosphatidyl-inositol 3-kinase (PI 3-kinase) has been analyzed by western blotting, confocal, and electron microscopy immunocytochemistry in human osteosarcoma Saos-2 cells. By western blotting, the enzyme appears to be present in both the cytoplasmic and nuclear subfractions. By confocal microscope immunocytochemistry, the cytoplasmic fluorescence is localized in the perinuclear region and on a network of filaments, while a diffused signal is present in the nucleus, except for the nucleolar areas. Ultrastructural analyses on whole cells and on in situ matrix preparations reveal that nuclear PI 3-kinase is localized in interchromatin domains, in stable association with inner nuclear matrix components, while the enzyme diffused in the cytosol is partly associated with the cytoskeletal filaments. Quantitative evaluations indicate that, in a multidrug-resistant variant obtained by continuous exposure of Saos-2 cells to doxorubicin, the amount of nuclear and cytoplasmic PI 3-kinase is significantly lower than in the sensitive parental cell line. The nuclear localization of PI 3-kinase and its variation in multidrug-resistant cells, characterized by a reduced mitotic index, are consistent with the data on the existence of a nuclear inositol lipid cycle, which could also utilize 3-phosphorylated inositides to modulate signal transduction for the control of some key functional activities.

Blotting, Western↗

PubMed1996

Inositol lipid cycle and autonomous nuclear signalling.

The involvement of phospholipids and in particular polyphosphoinositides in cellular signalling has been documented in detail in the last 20 years. In addition to the plasma membrane localization also the nucleus is shown to be a site for both synthesis and hydrolysis of the phosphorylated forms of phosphatidylinositol. Previous observation have established that the nucleus possesses a specific PLC for inositol lipids, i.e., the PLC beta 1 isoform, which undergoes rapid and transient activation after IGF-I stimulation of quiescent Swiss 3T3 cells and is down-regulated after treatment of Friend erythroleukemia cells with DMSO. Here we have reviewed: (i) the potential of nuclear PLC beta 1 to be a target for anti-cancer drug, (ii) the capability of this PLC isoform, when activated by IGF-I, to be a key signalling molecule in the onset of DNA synthesis, via DAG generation and PKC alpha translocation to the nucleus, (iii) the chromosome mapping of PLC beta 1 gene. The differentiation program of Friend cells can be activated by other agents besides DMSO including tiazofurin, an anti-tumor drug, also capable of affecting the nuclear inositol lipid cycle. Tiazofurin induces a lowering of the activity of PLC beta 1 due to down regulation of this isoform as revealed by both Western blotting and Northern blotting analyses. Using Swiss 3T3 cells stably transformed with an antisense PLC beta 1 construct, the knock-out of the PLC beta 1 gene induces both a loss of PLC beta 1 expression, as determined by Western blots, and a loss of the mitogenic responsiveness to IGF-I. These events show a direct relationship between nuclear PLC beta 1 evoked signals and IGF-I induced cell proliferation. Finally, the assignment of the PLC beta 1 gene to the band q35-36 of rat chromosome 3 paves the way for further genetic studies given the fact that the region where PLC beta 1 gene maps is a hot spot for genetic alterations in a number of experimentally induced rat tumors. Taken as a whole, these results assign a key role to the regulation of nuclear PLC activity and expression both in growth-factor activated mitogenesis and in in vitro erythroid differentiation.

Antineoplastic Agents↗

PubMed1996

Transfected Saos-2 cells overexpressing phosphoinositidase C beta 1 isoform accumulate it within the nucleus.

The subcellular partitioning of the phosphoinositidase C (PIC) isoforms involved in signal transduction, with the selective localization of the PIC beta 1 isoform in the nucleus, represents a crucial aspect of the complex mechanism of cell response to agonists. In order to further elucidate this phenomenon, we utilized human osteosarcoma Saos-2 cells, transfected with the cDNA for rat PIC beta 1. In the cells overexpressing this isoform, immunocytochemical analyses at the electron microscope level reveal an increased synthesis at the cytoplasm and a significant accumulation within the nucleus of the protein. Interestingly, the sites of intranuclear localization are, as in wild type cells, the interchromatin domains. These results indicate that the transfected cells maintain the capability of accumulating the enzyme within the nucleus and can be considered a model for functional studies on the nuclear signal transduction also in response to specific agonists.

Cell Nucleus↗

PubMed1996

Combined use of malachite green fixation and PLA2-gold complex technique to localize phospholipids in areas of early calcification of rat epiphyseal cartilage and bone.

Two cytochemical methods (malachite green fixation and PLA2-gold complex technique) were combined in order to detect the presence and ultrastructural distribution of phospholipids in epiphyseal cartilage and metaphyseal bone of 25-day-old rats. Chondrocytes and osteoblasts showed a more intense PLA2-gold complex labeling of rough endoplasmic reticulum than mitochondria and plasma membranes. Roundish osmiophilic, electron-dense, lightly labeled structures were visible at the periphery of the cells. In areas of early mineralization of cartilage, not all the matrix vesicles were labeled by PLA2-gold complex. Calcification nodules showed intense labeling in comparison with the surrounding, lightly labeled uncalcified matrix; gold particles were chiefly found at their periphery. Calcification nodules of metaphyseal bone were deeply electron-dense after glutaraldehyde-malachite green fixation; PLA2-gold labeling was mainly found in connection with crystals, whereas the uncalcified matrix was weakly labeled. Calcified bone matrix showed a heavy labeling with randomly scattered gold particles. When pre-embedding decalcification was carried out in the presence of malachite green, a good preservation of organic component was obtained. The PLA2-gold positivity of decalcified areas of cartilage and bone confirmed the presence of phospholipids in mineralized matrix.

Animals↗

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

Nuclear phosphoinositide-specific phospholipase C, phosphatidylinositol 4,5-bisphosphate and protein kinase C during rat spermatogenesis.

Presence and intracellular distribution of phosphoinositide-specific phospholipase C, phosphatidylinositol 4,5-bisphosphate and protein kinase C have been investigated in rat maturing germ cells and spermatozoa. The isoforms beta 1 and gamma 1 of phosphoinositide-specific phospholipase C were immunologically identified and found to be predominantly nuclear or cytoplasmic and nuclear, respectively. The two enzymes were present in the maturing cell lineage of the seminiferous tubule, except for the nucleus of late spermatids, and absent in spermatozoa, in which, however, a phosphoinositide-specific phospholipase C activity persisted, due to yet uncharacterized enzyme(s). Protein kinase C paralleled these developmental changes, and was completely down-regulated in both total cell homogenates and isolated nuclei obtained from spermatozoa. On the contrary, phosphatidylinositol 4,5-bisphosphate, present at the nuclear level in all cell types, accumulated in the nuclei of late spermatids and spermatozoa. These data support the contention that the spermatozoon nucleus stores a lipid-dependent signaling apparatus which could be reactivated either during sperm maturation or at fertilization.

Animals↗

PubMed1996

Ultrastructural features and P-glycoprotein immunolocalization in Saos-2/DX580 multidrug-resistant human osteosarcoma cells.

The multiple drug type of resistance to anticancer agents (MDR) is mediated by an over-expression of the MDR1 gene product, the P-glycoprotein. This is largely present at the cell surface of MDR cells, mediating the active efflux of cytotoxic molecules, but may be found also intracellularly. In this paper, using Saos-2 human osteosarcoma cells as a model, we provide further evidence of increased presence of P-glycoprotein at the plasma membrane and in the nucleus of MDR cells, where it is closely bound to the nuclear matrix. The structural changes observed in Saos-2 MDR cells, including an increase of the cell surface by the formation of blebs, and a peculiar clustering of chromatin, which are similar to those observed in other MDR cell lines, are likely to be associated with the observed overexpression of the P-glycoprotein at the cell membrane and nuclear level. These findings suggest the existence of more complex, still undetermined, mechanisms underlying the MDR phenomenon.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

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↗