Clathrin-dependent endocytosis of membrane-bound RANKL in differentiated osteoclasts.

Bone is continuously repaired and remodelled through well-coordinated activity of osteoblasts that form new bone and osteoclasts, which resorb it. Osteoblasts synthesize and secrete two key molecules that are important for osteoclast differentiation, namely the ligand for the receptor of activator of nuclear factor kappaB (RANKL) and its decoy receptor osteoprotegerin (OPG). Active membrane transport is a typical feature of the resorbing osteoclast during bone resorption.

Receptor Activator for Nuclear Factor kappa B Ligand (RANKL) as an osteoimmune key regulator in bone physiology and pathology.

The strength and integrity of the human skeleton depends on a delicate equilibrium between bone resorption and bone formation. Bone resorption is an elementary cellular activity in the modelling of the skeleton during growth and development. Later in life a most important physiological process in the skeleton is bone remodelling, which is locally initiated by resorption.

Breast adenocarcinoma MCF-7 cell line induces spontaneous osteoclastogenesis via a RANK-ligand-dependent pathway.

The metastasis of breast cancer to the skeleton is a serious clinical problem resulting in hypercalcemia, bone fragility and insurmountable pain. The invasion of bony tissue by neoplastic cells usually very rapidly affects the balance between bone apposition and bone resorption. In order to elucidate a mechanism for cancer-induced osteoclastogenesis, cells from a human breast cancer line, MCF-7, were directly co-cultured with murine monocytes RAW 264.

Effects of neridronic acid on osteoclasts derived by physiological dual-cell cultures.

Increased osteoclastic activity is observed in many osteopathic disorders - including postmenopausal osteoporosis, Paget's disease, primary bone tumours, lytic bone metastases, multiple myeloma and rheumatoid arthritis - that involve increased bone resorption and a loss of bone mass. Bisphosphonates are highly effective inhibitors of bone resorption that selectively affect the osteoclasts. The aim of this study was to obtain more information about the mechanism of action of bisphosphonates such as neridronic acid using a dual-cell culture model.

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).

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.

Constitutively active Akt1 protects HL60 leukemia cells from TRAIL-induced apoptosis through a mechanism involving NF-kappaB activation and cFLIP(L) up-regulation.

TRAIL is a member of the tumor necrosis factor superfamily which induces apoptosis in cancer but not in normal cells. Akt1 promotes cell survival and blocks apoptosis. The scope of this paper was to investigate whether a HL60 human leukemia cell clone (named AR) with constitutively active Akt1 was resistant to TRAIL.

Diacylglycerol kinases in nuclear lipid-dependent signal transduction pathways.

Several independent groups have shown that lipid-dependent signal transduction systems operate in the nucleus and that they are regulated independently from their membrane and cytosolic counterparts. A sizable body of evidence suggests that nuclear lipid signaling controls critical biological functions such as cell proliferation and differentiation. Diacylglycerol is a fundamental lipid second messenger which is produced in the nucleus.

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.

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.

Molecular characterization of protein kinase C-alpha binding to lamin A.

Previous results from our laboratory have identified lamin A as a protein kinase C (PKC)-binding protein. Here, we have identified the regions of PKC-alpha that are crucial for this binding. By means of overlay assays and fusion proteins made of glutathione-S-transferase (GST) fused to elements of rat PKC-alpha, we have established that binding occurs through both the V5 region and a portion of the C2 region (i.

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.

Localization of the small monomeric GTPases Rab3D and Rab3A in the AtT-20 rat pituitary cell line.

We investigated the cellular localization of the small GTPases Rab3D and Rab3A in AtT-20 cells treated with the drug Brefeldin A. Brefeldin A induces the redistribution of the Golgi complex into the endoplasmic reticulum and tubulation of endosomes. However, in Brefeldin A-treated wild-type AtT-20 cells, both Rab3D and Rab3A retained their distribution, indicating that they belong to a nonendosomal, post-Golgi compartment.

Morphological features and measurements in a human fetus with karyotype 69, XXX: comparison with fetuses of the same CRL, without chromosomal anomalies.

A triploid fetus (karyotype 69, XXX) with crown-rump length (CRL) 94 mm, presenting micro- and retrognathia, low-set ears and crooked feet, was cleared and double-stained with alizarin red S and alcian blue for detecting the ossification 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.

Further considerations on the intranuclear distribution of HMGI/Y proteins.

We have investigated the intranuclear distribution of High-mobility group proteins I/Y by means of immunofluorescent staining employing a variety of cell lines. Confocal scanning laser microscopy analysis revealed that High-mobility group proteins I/Y are present in an intranuclear fibrogranular network and mitotic chromosomes. In Caski, C4I, Flow 2002, and K562 cell lines, High-mobility group proteins I/Y were absent from nucleoli, whereas in HeLa cells they were present in this nuclear domain.

Phosphoinositide-specific phospholipase Cbeta1 expression is not linked to nerve growth factor-induced differentiation, cell survival or cell cycle control in PC12 rat pheocromocytoma cells.

Recent reports have highlighted that phosphoinositide-specific phospholipase Cbeta1 expression is linked to neuronal differentiation in different experimental models. We sought to determine whether or not this is also true for nerve growth factor (NGF)-induced neuronal differentiation of rat PC12 cells. However, we did not find differences in the expression of both the forms of phosphoinositide-specific phospholipase Cbeta1 (a and b) during sympathetic differentiation of these cells.

Re-examination of the mechanisms regulating nuclear inositol lipid metabolism.

Although inositol lipids constitute only a very minor proportion of total cellular lipids, they have received immense attention by scientists since it was discovered that they play key roles in a wide range of important cellular processes. In the late 1980s, it was suggested that these lipids are also present within the cell nucleus. Albeit the early reports about the intranuclear localization of phosphoinositides were met by skepticism and disbelief, compelling evidence has subsequently been accumulated convincingly showing that a phosphoinositide cycle is present at the nuclear level and may be activated in response to stimuli that do not activate the inositol lipid metabolism localized at the plasma membrane.

Nuclear changes in necrotic HL-60 cells.

Cell death in eukaryotes can occur by either apoptosis or necrosis. Apoptosis is characterized by well-defined nuclear changes which are thought to be the consequence of both proteolysis and DNA fragmentation. On the other hand, the nuclear modifications that occur during necrosis are largely less known.

Rab3A and Rab3D control the total granule number and the fraction of granules docked at the plasma membrane in PC12 cells.

Rab proteins are Ras-like GTPases that regulate traffic along the secretory or endocytic pathways. Within the Rab family, Rab3 proteins are expressed at high levels in neurons and endocrine cells where they regulate release of dense core granules and synaptic vesicles. Immunoelectron microscopy shows that Rab3A and Rab3D can coexist on the same granule before and after docking.

In vitro evaluation of the biocompatibility of dental alloys: fibronectin expression patterns and relationships to cellular proliferation rates.

Objective: This short-term (72- to 96-hour) in vitro study on fibroblasts evaluated the biocompatibility of 3 single-phase dental alloys by determining cellular proliferation rates and the expression of a glycoprotein, fibronectin, which is involved in cellular adhesion processes.

Method And Materials: Flow 2002 fibroblasts were cultured together with 3 single-phase dental alloys of different composition. Proliferation rates were determined by 5-bromodeoxyuridine incorporation.

Immunocytochemical expression of protein kinase C isoenzymes alpha, delta, epsilon and zeta in differentiating chick chondrocytes in vitro.

In our previous work we have investigated the expression of the serine-threonine kinase protein kinase C (PKC) in the vertebral column of mouse foetuses. In the present work we would verify the expression of four PKC-isoenzymes (alpha, delta, epsilon, zeta) in two distinct phases of the chondrogenesis and the endochondral osteogenesis in vitro. We performed primary cultures of chondrocytes collected from tibiae of 6-day old chick embryos.

Biocompatibility evaluation of dental metal alloys in vitro: expression of extracellular matrix molecules and its relationship to cell proliferation rates.

The biocompatibility in vitro of dental biomaterials has been widely studied, with consideration of cell viability and cell proliferation rates. In the present study we evaluated the biocompatibility in vitro of three single-phase dental metal alloys, all provided by the same manufacturer. To this aim, we considered the percentage of proliferating cells revealed by 5-bromodeoxyuridine incorporation in human fibroblast cultures in the presence of these biomaterials, performing a short time test (72 h).

Behavior of nucleolar proteins during the course of apoptosis in camptothecin-treated HL60 cells.

By means of immunofluorescence and immunoelectron microscopy we have studied the fate of different nucleolar components during the apoptotic process in camptothecin-treated HL60 cells. We have found that RNA polymerase I disappeared while UBF was associated with previously described fibrogranular threaded bodies. In contrast, fibrillarin, C23/nucleolin, and B23/nucleophosmin remained detectable in granular material present amid micronuclei of late apoptotic cells.