892 research outputs found
Improving water utilization from a catchment perspective
Water management / Water scarcity / Water use efficiency / Catchment areas / Calibrations / Hydrology / Models / River basins / Participatory management / Water balance / Case studies / Asia / Africa / South Africa / Zimbabwe
Expression patterns of chondrocyte genes cloned by differential display in tibial dyschondroplasia
AbstractTibial dyschondroplasia (TD) appears to involve a failure of the growth plate chondrocytes within growing long bones to differentiate fully to the hypertrophic stage, resulting in a mass of prehypertrophic chondrocytes which form the avascular TD lesion. Many biochemical and molecular markers of chondrocyte hypertrophy are absent from the lesion, or show reduced expression, but the cause of the disorder remains to be identified. As differentiation to the hypertrophic state is impaired in TD, we hypothesised that chondrocyte genes that are differentially expressed in the growth plate should show altered expression in TD. Using differential display, four genes, B-cadherin, EF2, HT7 and Ex-FABP were cloned from chondrocytes stimulated to differentiate to the hypertrophic stage in vitro, and their differential expression confirmed in vivo. Using semi-quantitative RT-PCR, the expression patterns of these genes were compared in chondrocytes from normal and TD growth plates. Surprisingly, none of these genes showed the pattern of expression that might be expected in TD lesion chondrocytes, and two of them, B-cadherin and Ex-FABP, were upregulated in the lesion. This indicates that the TD phenotype does not merely reflect the absence of hypertrophic marker genes, but may be influenced by more complex developmental mechanisms/defects than previously thought
Identification of a novel splice variant of the haloacid dehalogenase: PHOSPHO1
PHOSPHO1, a new member of the haloacid dehalogenase superfamily, has recently been implicated in the mineralization process in both osteoblasts and chondrocytes. In this study we describe the identification of a novel, alternatively spliced PHOSPHO1 transcript (PHOSPHO1-3a). This transcript contains the three exons of the previously published variant, however exon 3 contains a retained, 127bp section of intron 2. This forms an in-frame start site, producing an open reading frame of 879bp and predicting a protein of 292 amino acids. The novel 40 amino acid N-terminal region of PHOSPHO1-3a contains a relatively strong secretory signal, however all three domains of the HAD superfamily are retained in exon 3. The expression of this splice variant was confirmed in both human and mouse osteoblast-like cells and also in the chondrogenic ATDC5 cell line. The data within this study indicate a possible function relating to chondrocyte differentiation/mineralization as with the previously published variant
Extracellular Matrix Mineralization Promotes E11/gp38 Glycoprotein Expression and Drives Osteocytic Differentiation
Osteocytes are terminally differentiated osteoblasts which reside in a mineralized extracellular matrix (ECM). The factors that regulate this differentiation process are unknown. We have investigated whether ECM mineralization could promote osteocyte formation. To do this we have utilised MLO-A5 pre-osteocyte-like cells and western blotting and comparative RT-PCR to examine whether the expression of osteocyte-selective markers is elevated concurrently with the onset of ECM mineralization. Secondly, if mineralization of the ECM is indeed a driver of osteocyte formation, we reasoned that impairment of ECM mineralization would result in a reversible inhibition of osteocyte formation. Supplementation of MLO-A5 cell cultures with ascorbic acid and phosphate promoted progressive ECM mineralization as well as temporally associated increases in expression of the osteocyte-selective markers, E11/gp38 glycoprotein and sclerostin. Consistent with a primary role for ECM mineralization in osteocyte formation, we also found that inhibition of ECM mineralization, by omitting phosphate or adding sodium pyrophosphate, a recognized inhibitor of hydroxyapatite formation, resulted in a 15-fold decrease in mineral deposition that was closely accompanied by lower expression of E11 and other osteocyte markers such as Dmp1, Cd44 and Sost whilst expression of osteoblast markers Ocn and Col1a increased. To rule out the possibility that such restriction of ECM mineralization may produce an irreversible modification in osteoblast behaviour to limit E11 expression and osteocytogenesis, we also measured the capacity of MLO-A5 cells to re-enter the osteocyte differentiation programme. We found that the mineralisation process was re-initiated and closely allied to increased expression of E11 protein after re-administration of phosphate or omission of sodium pyrophosphate, indicating an ECM mineralization-induced restoration in osteocyte formation. These results emphasise the importance of cell-ECM interactions in regulating osteoblast behaviour and, more importantly, suggest that ECM mineralization exerts pivotal control during terminal osteoblast differentiation and acquisition of the osteocyte phenotype
Correction to ::Optimised isolation method for RNA extraction suitable for RNA sequencing from feline teeth collected in a clinical setting and at post mortem
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