Biomedical subjects

Paola Narducci

Publications and source records attributed to Paola Narducci.

10 recordsLinked to original sources

PubMed2006

Constitutive traffic of melanocortin-4 receptor in Neuro2A cells and immortalized hypothalamic neurons.

Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor (GPCR) that binds alpha-melanocyte-stimulating hormone (alpha-MSH) and has a central role in the regulation of appetite and energy expenditure. Most GPCRs are endocytosed following binding to the agonist and receptor desensitization. Other GPCRs are internalized and recycled back to the plasma membrane constitutively, in the absence of the agonist. In unstimulated neuroblastoma cells and immortalized hypothalamic neurons, epitopetagged MC4R was localized both at the plasma membrane and in an intracellular compartment. These two pools of receptors were in dynamic equilibrium, with MC4R being rapidly internalized and exocytosed. In the absence of alpha-MSH, a fraction of cell surface MC4R localized together with transferrin receptor and to clathrin-coated pits. Constitutive MC4R internalization was impaired by expression of a dominant negative dynamin mutant. Thus, MC4R is internalized together with transferrin receptor by clathrin-dependent endocytosis. Cell exposure toalpha-MSH reduced the amount of MC4R at the plasma membrane by blocking recycling of a fraction of internalized receptor, rather than by increasing its rate of endocytosis. The data indicate that, in neuronal cells, MC4R recycles constitutively and that alpha-MSH modulates MC4R residency at the plasma membrane by acting at an intracellular sorting step.

Animals↗

PubMed2006

Morphological features of osteoclasts derived from a co-culture system.

The interaction between the receptor activator of NfKB (RANK) and its ligand receptor activator of NfKB ligand (RANKL) has recently been proven to be pivotal for osteoclast differentiation and activation. The influence of RANK-RANKL signaling on osteoclast formation was established by co-culturing murine osteoblasts (type CRL-12257) and murine mononuclear monocytes (RAW 264.7). The aim of the present study was to examine, by means of morphological techniques, the interaction between these two cell lines grown in the absolute absence of exogenous cytokines and other stimulating factors. Moreover, we wanted to show that our model could provide a system to analyze the bone resorption process. Mineralized matrix induced morphological changes of osteoclasts (OC) by the formation of organized ruffled-border and a large number of secondary lysosomal vesicles. On the contrary, OC grown on glass coverslips without dentin showed no organized ruffled border or secondary lysosomes. The study of the relationship between these two cell types could establish new approaches for a potential pharmacological control of these cell types and tissues in health and disease.

Animals↗

PubMed2005

Rabphilin localizes with the cell actin cytoskeleton and stimulates association of granules with F-actin cross-linked by {alpha}-actinin.

In endocrine cell, granules accumulate within an F-actin-rich region below the plasma membrane. The mechanisms involved in this process are largely unknown. Rabphilin is a cytosolic protein that is expressed in neurons and neuroendocrine cells and binds with high affinity to members of the Rab3 family of GTPases localized to synaptic vesicles and dense core granules. Rabphilin also interacts with alpha-actinin, a protein that cross-links F-actin into bundles and networks and associates with the granule membrane. Here we asked whether rabphilin, in addition to its granule localization, also interacts with the cell actin cytoskeleton. Immunofluorescence and immunoelectron microscopy show that rabphilin localizes to the sub-plasmalemmal actin cytoskeleton both in neuroendocrine and unspecialized cells. By using purified components, it is found that association of rabphilin with F-actin is dependent on added alpha-actinin. In an in vitro assay, granules, unlike endosomes or mitochondria, associate with F-actin cross-linked by alpha-actinin. Rabphilin is shown to stimulate this process. Rabphilin enhances by approximately 8-fold the granule ability to localize within regions of elevated concentration of cross-linked F-actin. These results suggest that rabphilin, by interacting with alpha-actinin, organizes the cell cytoskeleton to facilitate granule localization within F-actin-rich regions.

Actinin↗

PubMed2005

Different levels of the neuronal nitric oxide synthase isoform modulate the rate of osteoclastic differentiation of TIB-71 and CRL-2278 RAW 264.7 murine cell clones.

It has been clearly established that osteoclasts, which play a crucial role in bone resorption, differentiate from hematopoietic cells belonging to the monocyte/macrophage lineage in the presence of macrophage-colony stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL). We have here investigated the M-CSF- and RANKL-induced osteoclastic differentiation of two distinct clones of the murine monocytic/macrophagic RAW 264.7 cell line, known as TIB-71 and CRL-2278, the latter cell clone being defective for the expression of the inducible nitric oxide synthase isoform in response to interferon-gamma or lipopolysaccharide. CRL-2278 cells demonstrated a more rapid osteoclastic differentiation than TIB-71 cells, as documented by morphology, tartrate-resistant acid phosphatase positivity, and bone resorption activity. The enhanced osteoclastic differentiation of CRL-2278 was accompanied by a higher rate of cells in the S/G2-M phases of cell cycle as compared to TIB-71. The analysis of nitric oxide synthase (NOS) isoforms clearly demonstrated that only neuronal NOS was detectable at high levels in CRL-2278 but not in TIB cells under all tested conditions. Moreover, the broad inhibitor of NOS activity L-NAME significantly inhibited osteoclastic differentiation of CRL-2278 cells. Altogether, these results demonstrate that a basal constitutive neuronal NOS activity positively affects the RANKL/M-CSF-related osteoclastic differentiation.

Acid Phosphatase↗

PubMed2005

Immunolocalisation of bilitranslocase in mucosecretory and parietal cells of the rat gastric mucosa.

Bilitranslocase is a plasma membrane carrier localised at the vascular pole of the rat liver cell, where it mediates uptake of organic anions from the blood into the liver. This carrier is also present in the epithelium of the rat gastric mucosa, with similar molecular mass and functional properties. An immunohistochemical study reveals that both the mucus-secreting cells of the gastric pit and the H+-secreting parietal cells express bilitranslocase. These data point to a possible role of bilitranslocase and of its food-borne substrates (anthocyanins and nicotinic acid) in regulating the function and the permeability of the gastric mucosa.

Animals↗

PubMed2003

Tumour necrosis factor-related apoptosis-inducing ligand sequentially activates pro-survival and pro-apoptotic pathways in SK-N-MC neuronal cells.

The SK-N-MC neuroblastoma cell line, which expresses surface tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors TRAIL-R2 and TRAIL-R4, was used as a model system to examine the effect of TRAIL on key intracellular pathways involved in the control of neuronal cell survival and apoptosis. TRAIL induced distinct short-term (1-60 min) and long-term (3-24 h) effects on the protein kinase B (PKB)/Akt (Akt), extracellular signal-regulated kinase (ERK), cAMP response element-binding protein (CREB), nuclear factor kappa B (NF-kappaB) and caspase pathways. TRAIL rapidly (from 20 min) induced the phosphorylation of Akt and ERK, but not of c-Jun NH2-terminal kinase (JNK). Moreover, TRAIL increased CREB phosphorylation and phospho-CREB DNA binding activity in a phosphatidylinositol 3-kinase (PI 3K)/Akt-dependent manner. At later time points (from 3 to 6 h onwards) TRAIL induced a progressive degradation of inhibitor of kappaB (IkappaB)beta and IkappaBepsilon, but not IkappaBalpha, coupled to the nuclear translocation of NF-kappaB and an increase in its DNA binding activity. In the same time frame, TRAIL started to activate caspase-8 and caspase-3, and to induce apoptosis. Remarkably, caspase-dependent cleavage of NF-kappaB family members as well as of Akt and CREB proteins, but not of ERK, became prominent at 24 h, a time point coincident with the peak of caspase-dependent apoptosis.

Apoptosis↗

PubMed2002

Binding of elements of protein kinase C-alpha regulatory domain to lamin B1.

Previous results from our laboratory have demonstrated that lamin B1 is a protein kinase C (PKC)-binding protein. Here, we have identified the regions of PKC-alpha that are important for this binding. By means of overlay assays and fusion proteins made of glutathione-S-transferase (GST) fused to elements of the regulatory domain of rat PKC-alpha, we have established that binding occurs through both the V1 region and a portion of the C2 region (i.e., the calcium-dependent lipid binding [CaLB] domain) of the kinase. In particular, we have found that amino acids 200-217 of the CaLB domain are essential for binding lamin B1, as a synthetic peptide corresponding to this stretch of amino acids prevented the interaction between the CaLB domain of PKC-alpha and lamin B1. In agreement with the results of other investigators, we have determined that binding of regulatory elements of PKC-alpha to lamin B1 does not require the presence of cofactors such as PS and Ca(2+). We have also found that the binding site of lamin B1 for PKC-alpha is localized in the carboxyl-terminus of the lamin. Our findings may prove to be important in shedding more light on the mechanisms that regulate PKC functions within the nuclear compartment and may also lead to the synthesis of isozyme-specific pharmacological tools to attenuate or reverse PKC-dependent nuclear signalling pathways important for the pathogenesis of cancer.

3T3 Cells↗

PubMedDate not supplied

Morphological observations and morphometric analysis in three human fetuses with bilateral cervical cystic hygroma.

Three human fetuses (crown-rump length, CRL, ranging from 71 to 77 mm), presenting bilateral cervical cystic hygroma were examined. The specimens were cleared and double-stained with alcian blue and alizarin red S for detecting the ossification growth patterns in the vertebral column, ribs, ischium, limbs, and face. Longitudinal measurements of some long bones in the upper (humerus, ulna, radius) and lower (femur, tibia, fibula) limb were taken. The values of both the total length (TL) and the ossified part (OL) of each long bone, as well as the OL/TL per cent ratio were considered. Reference points were located on the mandible, i.e. condylar process (Pcl), coronoid process (Pco), gnathion (GN), gonion (GO), inferior interdental point (IDI) for measuring linear dimensions. All values obtained were related with those relative to a group of fetuses, without any detectable malformation and chromosomal abnormalities, with CRL mean value 75 mm, in order to assess the presence of further anomalies, besides the cystic hygroma, in the three fetuses considered.

Anthropometry↗

PubMedDate not supplied

Immunochemical and immunocytochemical expression of protein kinase c isoenzymes alpha, delta, epsilon and zeta in primary adherent cultures of chick chondrocytes.

The family of protein kinase C (PKC) comprises serine/threonine isoenzymes involved in various biological processes, including cell proliferation and differentiation. On the bases of previous investigations performed by us on the expression of various PKC isoforms in the endochondral ossification process of the vertebral column, the aim of the present work was to investigate the expression of various PKC-isoenzymes in chick primary chondrocyte cultures i.e. the most used chondrocyte culture model in vitro. Immunochemical and immunocytochemical experiments were performed to detect the expression of PKC-alpha, -delta, -epsilon and -zeta. Chondrocyte cultures were examined two weeks after cell collection from tibiae of 6-day old chick embryos. By means of morphological observations associated with the immunocytochemical expression of type II collagen, two different cell phenotypes were identified, i.e. fibroblast-like and polygonal-roundish-shaped cells. As far as PKC-isoenzyme expression was concerned, PKC-zeta revealed a stronger immunochemical and immunocytochemical expression; PKC-alpha exhibited a positivity less marked than PKC-zeta, whereas PKC-delta and -epsilon were less expressed in this culture stage. It is reasonable that a major role could be played by PKC-alpha and -zeta in this phase of the chondrogenic process, whereas PKC-delta and -epsilon could be involved in different stages of chondrocyte differentiation in vitro.

Animals↗

PubMedDate not supplied

Expression of protein kinase C (PKC) alpha, delta, epsilon, zeta in primary chick chondrocyte cultures: immunocytochemical study.

PKC is a family of 12 serine/threonine isoenzymes that plays a pivotal role in signal transduction in a large number of biological processes. In the present work we have investigated the expression of PKC (alpha, delta, epsilon, zeta) in chick chondrocyte primary cultures at different differentiation times, i.e. at 48, 55, 62 and 69 days after cell collection from tibiae of 6-day old chick embryos. We would also detect cell differentiation stages towards the osteoblast-like cell phenotype by observing the immunocytochemical expression of the specific osteoblast marker, type I collagen. At the considered culture times, cells exhibited immunocytochemical positivity for type I collagen, thus showing their differentiation towards the osteoblast-like phenotype. PKC-zeta was the isoenzyme that exhibited the most relevant immunocytochemical expression in all considered culture times, whereas PKC-epsilon always less expressed in comparison to the other PKC-isoforms. No relevant differences were observed for the immunocytochemical expressions of PKC-alpha and PKC-delta. On the basis of the immunocytochemical data obtained from the present investigation, we could affirm that PKC-alpha, -delta, -epsilon, and -zeta may play peculiar roles in the differentiation process of chick chondrocytes towards the osteoblast-like cell phenotype.

Animals↗