Biomedical subjects

Fraser McDonald

Publications and source records attributed to Fraser McDonald.

8 recordsLinked to original sources

PubMed2006

Interaction between store-operated and arachidonate-activated calcium entry.

A ubiquitous pathway for cellular Ca(2+) influx involves 'store-operated channels' that respond to depletion of intracellular Ca(2+) pools via an as yet unknown mechanism. Due to its wide-spread expression, store-operated Ca(2+) entry (SOCE) has been considered a principal route for Ca(2+) influx. However, recent evidence has suggested that alternative pathways, activated for example by lipid metabolites, are responsible for physiological Ca(2+) influx. It is not clear if these messenger-activated Ca(2+) entry routes exist in all cells and what interaction they have with SOCE. In the present study we demonstrate that HEK-293 cells and Saos-2 cells express an arachidonic acid (AA)-activated Ca(2+) influx pathway that is distinct from SOCE on the basis of sensitivity to pharmacological blockers and depletion of cellular cholesterol. We examined the functional interaction between SOCE and the arachidonate-triggered Ca(2+) influx (denoted non-SOCE). Both Ca(2+) entry routes could underlie substantial long-lasting Ca(2+) elevations. However, the two pathways could not operate simultaneously. With cells that had an on-going SOCE response, addition of arachidonate gave two profound effects. Firstly, it rapidly inhibited SOCE. Secondly, the mode of Ca(2+) influx switched to the non-SOCE mechanism. Addition of arachidonate to naïve cells resulted in rapid activation of the non-SOCE pathway. However, this Ca(2+) entry route was very slowly engaged if the SOCE pathway was already operative. These data indicate that the SOCE and arachidonate-activated non-SOCE pathways interact in an inhibitory manner. We probed the plausible mechanisms by which these two pathways may communicate.

Acetamides↗

PubMed2006

Runx2 and dental development.

The Runx2 gene is a master transcription factor of bone and plays a role in all stages of bone formation. It is essential for the initial commitment of mesenchymal cells to the osteoblastic lineage and also controls the proliferation, differentiation, and maintenance of these cells. Control is complex, with involvement of a multitude of factors, thereby regulating the expression and activity of this gene both temporally and spatially. The use of multiple promoters and alternative splicing of exons further extends its diversity of actions. RUNX2 is also essential for the later stages of tooth formation, is intimately involved in the development of calcified tooth tissue, and exerts an influence on proliferation of the dental lamina. Furthermore, RUNX2 regulates the alveolar remodelling process essential for tooth eruption and may play a role in the maintenance of the periodontal ligament. In this article, the structure of Runx2 is described. The control and function of the gene and its product are discussed, with special reference to developing tooth tissues, in an attempt to elucidate the role of this gene in the development of the teeth and supporting structures.

Ameloblasts↗

PubMed2004

Calmidazolium and arachidonate activate a calcium entry pathway that is distinct from store-operated calcium influx in HeLa cells.

Agonists that deplete intracellular Ca2+ stores also activate Ca2+ entry, although the mechanism by which store release and Ca2+ influx are linked is unclear. A potential mechanism involves 'store-operated channels' that respond to depletion of the intracellular Ca2+ pool. Although SOCE (store-operated Ca2+ entry) has been considered to be the principal route for Ca2+ entry during hormonal stimulation of non-electrically excitable cells, recent evidence has suggested that alternative pathways activated by metabolites such as arachidonic acid are responsible for physiological Ca2+ influx. It is not clear whether such messenger-activated pathways exist in all cells, whether they are truly distinct from SOCE and which metabolites are involved. In the present study, we demonstrate that HeLa cells express two pharmacologically and mechanistically distinct Ca2+ entry pathways. One is the ubiquitous SOCE route and the other is an arachidonate-sensitive non-SOCE. We show that both these Ca2+ entry pathways can provide long-lasting Ca2+ elevations, but that the channels are not the same, based on their differential sensitivity to 2-aminoethoxydiphenyl borate, LOE-908 [(R,S)-(3,4-dihydro-6,7-dimethoxy-isochinolin-1-yl)-2-phenyl-N,N-di[2-(2,3,4-trimethoxyphenyl)ethyl]acetamid mesylate] and gadolinium. In addition, non-SOCE and not SOCE was permeable to strontium. Furthermore, unlike SOCE, the non-SOCE pathway did not require store depletion and was not sensitive to displacement of the endoplasmic reticulum from the plasma membrane using jasplakinolide or ionomycin pretreatment. These pathways did not conduct Ca2+ simultaneously due to the dominant effect of arachidonate, which rapidly curtails SOCE and promotes Ca2+ influx via non-SOCE. Although non-SOCE could be activated by exogenous application of arachidonate, the most robust method for stimulation of this pathway was application of the widely used calmodulin antagonist calmidazolium, due to its ability to activate phospholipase A2.

Arachidonic Acid↗

PubMed2004

A preliminary optical and electron microscopic study of the beta(1) integrin distribution pattern of human osteosarcoma-derived cells.

Immunogold labelling was used to study the organisation of the beta(1) integrins on osteosarcoma-derived osteoblasts (Saos-2 and MG-63). Monolayers of cells were prepared in multiwell culture plates on both uncovered and collagen-covered coverslips, and beta(1) integrins were primarily labelled using mouse monoclonal antibodies to beta(1) integrins. Indirect immunofluorescence labels using an anti-mouse fluorescein-conjugated goat antibody showed an even distribution of the beta(1) integrins on the cell membranes of all cell types used. A concentration of 2 microg/ml of the primary antibodies and a 1:100 dilution of the secondary antibodies were determined as the optimal concentration for labelling to use with indirect localisation of the primary antibodies gold conjugated to goat anti-mouse antibodies and viewed under an electron microscope. Ten nanometre gold particles were used for transmission electron microscopy (TEM) and 40 nm gold particles for scanning electron microscopy. TEM showed that beta(1) integrins were mainly clustered on the cell membrane processes with less labelling on the cell membranes themselves. The distribution of beta(1) integrins on osteosarcoma cells supports the concept that integrins may function by forming focal adhesions at the site of the cytoplasmic membrane processes.

Bone Neoplasms↗

PubMed2004

The localization of estrogen receptors on human osteoblasts: a preliminary optical and electron microscopic technique.

Attempts to identify estrogen target cells in bone by immunocytochemistry using antibodies to the receptor have proved to be controversial. The aim of this study was therefore to determine whether immunogold labeling can be used as a technique for the localization of estrogen receptors (ER) on a human osteoblast-like cell line. The aim was also to determine the distribution of ER on the cell surface by using a scanning electron microscope (SEM) and intracellularly by using a transmission electron microscope (TEM). Labeling of the cytoplasmic material was seen around areas that appeared to be a disrupted plasma membrane. No nuclear or perinuclear labeling could be detected. The conclusion can be made that SEM immunogold labeling combined with TEM can be regarded as a practical technique for localizing ER on human osteoblasts. This article clearly demonstrated that osteoblast-like cells do express ER at low levels and that, although cytoplasmic immunoreactivity could be detected, no nuclear or perinuclear labeling was found.

Cell Line↗

PubMed2003

Regulation of InsP3 receptor activity by neuronal Ca2+-binding proteins.

Inositol 1,4,5-trisphosphate receptors (InsP(3)Rs) were recently demonstrated to be activated independently of InsP(3) by a family of calmodulin (CaM)-like neuronal Ca(2+)-binding proteins (CaBPs). We investigated the interaction of both naturally occurring long and short CaBP1 isoforms with InsP(3)Rs, and their functional effects on InsP(3)R-evoked Ca(2+) signals. Using several experimental paradigms, including transient expression in COS cells, acute injection of recombinant protein into Xenopus oocytes and (45)Ca(2+) flux from permeabilised COS cells, we demonstrated that CaBPs decrease the sensitivity of InsP(3)-induced Ca(2+) release (IICR). In addition, we found a Ca(2+)-independent interaction between CaBP1 and the NH(2)-terminal 159 amino acids of the type 1 InsP(3)R. This interaction resulted in decreased InsP(3) binding to the receptor reminiscent of that observed for CaM. Unlike CaM, however, CaBPs do not inhibit ryanodine receptors, have a higher affinity for InsP(3)Rs and more potently inhibited IICR. We also show that phosphorylation of CaBP1 at a casein kinase 2 consensus site regulates its inhibition of IICR. Our data suggest that CaBPs are endogenous regulators of InsP(3)Rs tuning the sensitivity of cells to InsP(3).

Amino Acid Sequence↗

PubMed2002

Cytokine release by osteoblast-like cells cultured on implant discs of varying alloy compositions.

OBJECTIVE: The aims of this study were (i). to assess the morphological features of osteo-blast-like, osteosarcoma cells (cell line SaOS-2) cultured on implant surfaces of varying alloys and (ii). to evaluate the biological activity of these cells, following their adhesion onto these surfaces. MATERIALS AND METHODS: SaOS-2 cells (6 x 104) were grown on titanium discs (diameter 30 mm), each with a surface of differing composition and roughness (commercially pure titanium, titanium-aluminium-vanadium alloy, oxide-blasted titanium and Astra-Tech special treatment titanium; the alloys are directly comparable with those used to construct implants). The cells were grown for time periods of 1, 3, 5 and 7 days, the media were collected and the cells were fixed with 2.5% glutaraldehyde. The media were then assayed (using enzyme-linked immunosorbant assay) for the levels of interleukin (IL)-1, interleukin-6, interleukin-18 and osteoprotegerin (OPG) produced by the cells. The discs, with the cells fixed on them, were viewed under scanning electron microscopy (SEM, x 2.0 k) to evaluate cell morphology. RESULTS: Following attachment, the cells changed their morphology and released local factors known to activate osteoclasts. Commercially pure titanium stimulated the cells the most and titanium-aluminium-vanadium alloy the least. All implant materials stimulated production of IL-1, IL-6, IL-18 above that produced by cells grown on Petri dishes (polystyrene). The titanium-aluminium-vanadium alloy allowed cell attachment but levels of IL-1 in this medium were significantly lower (31.5 +/- 5.2 pg/ml on same day) than cultures with pure titanium (201.8 +/- 11.5 pg/ml at day 5). The same pattern was observed with the IL-6, IL-18, and OPG with polystyrene appearing to stimulate most production of OPG. Titanium-aluminium-vanadium produced the least biological response.

Alloys↗

PubMed2002

A cephalometric analysis of patients with recessive dystrophic epidermolysis bullosa.

Patients diagnosed with Recessive Dystrophic Epidermolysis Bullosa (RDEB) suffer from severe growth inhibition due to reduced food intake as a result of severe oropharyngeal and esophageal blistering. This investigation examined the patterns of facial growth in a group of 42 children with RDEB for whom lateral skull radiographs were available. The differences between RDEB patients and patients with normal cephalometric values were also assessed. Lateral skull radiographs were digitized and a number of orthodontic indices were compared to the published normal values. The RDEB patients examined demonstrated smaller maxillae than normal (length 41.3 +/- 2.9 mm compared to 47.4 +/- 2.5 mm) and smaller mandibles than normal (length 82.3 +/- 6.1 mm compared to 93.1 +/- 4.2 mm). This impaired growth may result from reduced food intake or severe orofacial scarring associated with RDEB. This contributes significantly to dento-alveolar disproportion and dental crowding and puts patients at increased risk of dental caries.

Adolescent↗