The role of calcium and nicotinic acid adenine dinucleotide phosphate (NAADP) in human osteoclast formation and resorption.

Osteoclasts are specialised bone resorbing cells which form by fusion of circulating mononuclear phagocyte precursors. Bone resorption results in the release of large amounts of calcium into the extracellular fluid (ECF), but it is not certain whether changes in extracellular calcium concentration [Ca(2+)]e influence osteoclast formation and resorption. In this study, we sought to determine the effect of [Ca(2+)]e and NAADP, a potent calcium mobilising messenger that induces calcium uptake, on human osteoclast formation and resorption. CD14+ human monocytes were cultured with M-CSF and RANKL in the presence of different concentrations of calcium and NAADP and the effect on osteoclast formation and resorption evaluated. We found that the number of TRAP+ multinucleated cells and the extent of lacunar resorption were reduced when there was an increase in extracellular calcium and NAADP. This was associated with a decrease in RANK mRNA expression by CD14+ cells. At high concentrations (20 mM) of [Ca(2+)]e mature osteoclast resorption activity remained unaltered relative to control cultures. Our findings indicate that osteoclast formation is inhibited by a rise in [Ca(2+)]e and that RANK expression by mononuclear phagocyte osteoclast precursors is also [Ca(2+)]e dependent. Changes in NAADP also influence osteoclast formation, suggesting a role for this molecule in calcium handling. Osteoclasts remained capable of lacunar resorption, even at high ECF [Ca(2+)]e, in keeping with their role in physiological and pathological bone resorption.

Thermolability of mutant MMACHC protein in the vitamin B12-responsive cblC disorder.

Methylmalonic aciduria and homocystinuria, cblC type, is the most common inborn error of cellular vitamin B12 metabolism. We previously showed that the protein carrying the mutation responsible for late-onset cblC (MMACHC-R161Q), treatable with high dose OHCbl, is able to bind OHCbl with wild-type affinity, leaving undetermined the disease mechanism involved [Froese et al., Mechanism of responsiveness, Mol. Genet. Metab. (2009).]. To assess whether the mutation renders the protein unstable, we investigated the thermostability of the wild-type and mutant MMACHC proteins, either unbound or bound to different cobalamins (Cbl), using differential scanning fluorimetry. We found that MMACHC-wt and MMACHC-R161Q are both very thermolabile proteins in their apo forms, with melting temperatures (T(m)) of 39.3+/-1.0 and 37.1+/-0.7 degrees C, respectively; a difference confirmed by unfolding of MMACHC-R161Q but not MMACHC-wt by isothermal denaturation at 35 degrees C over 120 min. However, with the addition of OHCbl, MMACHC-wt becomes significantly stabilized (Delta T(m max)=8 degrees C, half-maximal effective ligand concentration, AC(50)=3 microM). We surveyed the effect of different cobalamins on the stabilization of the wild-type protein and found that AdoCbl was the most stabilizing, exerting a maximum increase in T(m) of approximately 16 degrees C, followed by MeCbl at approximately 13 degrees C, each evaluated at 50 microM cofactor. The other cobalamins stabilized in the order (CN)(2)Cbi>OHCbl>CNCbl. Interestingly, the AC(50)'s for AdoCbl, MeCbl, (CN)(2)Cbi and OHCbl were similar and ranged from 1-3 microM, which compares well with the K(d) of 6 microM for OHCbl [Froese et al., Mechanism of responsiveness, Mol. Genet. Metab. (2009).]. Unlike MMACHC-wt, the mutant protein MMACHC-R161Q is only moderately stabilized by OHCbl (Delta T(m max)=4 degrees C). The dose-response curve also shows a lower effectivity of OHCbl with respect to stabilization, with an AC(50) of 7 microM. MMACHC-R161Q showed the same order of stabilization as MMACHC-wt, but each cobalamin stabilized this mutant protein less than its wild-type counterpart. Additionally, MMACHC-R161Q had a higher AC(50) for each cobalamin form compared to MMACHC-wt. Finally, we show that MMACHC-R161Q is able to support the base-off transition for AdoCbl and CNCbl, indicating this mutant is not blocked in that respect. Taken together, our results suggest that protein stability, as well as propensity for ligand-induced stabilization, contributes to the disease mechanism in late-onset cblC disorder. Our results underscore the importance of cofactor stabilization of MMACHC and suggest that even small increases in the concentration of cobalamin complexed with MMACHC may have therapeutic benefit in children with the late-onset, vitamin responsive cblC disease.

Dynamic protein methylation in chromatin biology.

Post-translational modification of chromatin is emerging as an increasingly important regulator of chromosomal processes. In particular, histone lysine and arginine methylation play important roles in regulating transcription, maintaining genomic integrity, and contributing to epigenetic memory. Recently, the use of new approaches to analyse histone methylation, the generation of genetic model systems, and the ability to interrogate genome wide histone modification profiles has aided in defining how histone methylation contributes to these processes. Here we focus on the recent advances in our understanding of the histone methylation system and examine how dynamic histone methylation contributes to normal cellular function in mammals.

Medium- and short-chain dehydrogenase/reductase gene and protein families : the SDR superfamily: functional and structural diversity within a family of metabolic and regulatory enzymes.

Short-chain dehydrogenases/reductases (SDRs) constitute a large family of NAD(P)(H)-dependent oxidoreductases, sharing sequence motifs and displaying similar mechanisms. SDR enzymes have critical roles in lipid, amino acid, carbohydrate, cofactor, hormone and xenobiotic metabolism as well as in redox sensor mechanisms. Sequence identities are low, and the most conserved feature is an alpha/beta folding pattern with a central beta sheet flanked by 2 - 3 alpha-helices from each side, thus a classical Rossmannfold motif for nucleotide binding. The conservation of this element and an active site, often with an Asn-Ser-Tyr-Lys tetrad, provides a platform for enzymatic activities encompassing several EC classes, including oxidoreductases, epimerases and lyases. The common mechanism is an underlying hydride and proton transfer involving the nicotinamide and typically an active site tyrosine residue, whereas substrate specificity is determined by a variable C-terminal segment. Relationships exist with bacterial haloalcohol dehalogenases, which lack cofactor binding but have the active site architecture, emphasizing the versatility of the basic fold in also generating hydride transfer-independent lyases. The conserved fold and nucleotide binding emphasize the role of SDRs as scaffolds for an NAD(P)(H) redox sensor system, of importance to control metabolic routes, transcription and signalling.

Pleomorphic liposarcoma of bone: a rare primary malignant bone tumour.

BACKGROUND: Liposarcoma is an extremely rare primary bone sarcoma. CASE PRESENTATION: We report a case of primary pleomorphic liposarcoma that arose in an 18 year old male in the metaphysis of the left tibia. Plain radiographs showed a partly sclerotic lesion and MR imaging a heterogeneous tumour predominantly isointense on T1- and high-signal on T2-weighted sequences with focal areas of increased T1 signal that suppressed with fat saturation. PET/CT showed marked FDG uptake (SUV = 17.1) in the primary tumour as well as a metastasis in the right distal femur and multiple small pulmonary metastases. Histologically, the tumour was a pleomorphic liposarcoma containing large tumour cells with vacuolated cytoplasm and hyperchromatic pleomorphic nuclei as well as numerous lipoblasts and scattered brown fat-like cells. Tumour cells strongly expressed FABP4/aP2, a marker of adipocyte differentiation, and UCP1, a marker of brown fat, but not S100. The case was treated with neoadjuvant MAP chemotherapy, resulting in extensive (> 95%) necrosis in the primary tumour and almost complete resolution of the femoral and pulmonary metastases. CONCLUSIONS: Pleomorphic liposarcoma can present as a sclerotic primary malignant bone tumour; markers of adipose differentiation are useful in histological diagnosis and neoadjuvant MAP chemotherapy results in significant tumor necrosis.

Non-competitive cyclic peptides for targeting enzyme-substrate complexes.

Affinity reagents are of central importance for selectively identifying proteins and investigating their interactions. We report on the development and use of cyclic peptides, identified by mRNA display-based RaPID methodology, that are selective for, and tight binders of, the human hypoxia inducible factor prolyl hydroxylases (PHDs) - enzymes crucial in hypoxia sensing. Biophysical analyses reveal the cyclic peptides to bind in a distinct site, away from the enzyme active site pocket, enabling conservation of substrate binding and catalysis. A biotinylated cyclic peptide captures not only the PHDs, but also their primary substrate hypoxia inducible factor HIF1-α. Our work highlights the potential for tight, non-active site binding cyclic peptides to act as promising affinity reagents for studying protein-protein interactions.

Utility of VS38c in the diagnostic and prognostic assessment of osteosarcoma and other bone tumours/tumour-like lesions.

BACKGROUND: VS38c is a monoclonal antibody that recognises a rough endoplasmic reticulum (rER) intracellular antigen termed cytoskeleton-linking membrane protein 63. rER is typically found in viable tumour cells and is abundant in osteosarcoma cells. The aim of this study was to determine the diagnostic and prognostic utility of VS38c in the histological assessment of osteosarcoma and other bone tumours/tumour-like leisons. METHODS: Immunohistochemical staining with VS38c was carried out on formalin-fixed specimens of osteosarcoma (pre/post-chemotherapy) and a wide range of benign and malignant bone lesions. In addition, VS38c staining of cultures of MG63 and Sa0S2 osteosarcoma cell cultures. (±cisplatin and actinomycin D-treatment) was analysed. RESULTS: VS38c strongly stained tumour cells in all low-grade and high-grade osteosarcomas and in undifferentiated sarcomas and high-grade chondrosarcomas. There was little or no VS38c staining of low-grade chondrosarcomas or chordomas and variable staining of Ewing sarcomas. Osteoblasts in benign bone-forming tumours and mononuclear stromal cells in chondroblastomas, giant cell tumours and non-ossifying fibromas strongly stained for VS38c. VS38c staining was absent in cisplatin and actinomycin D treated Sa0S2 and MG63 cells. In specimens of osteosarcoma post-neoadjuvant therapy, VS38c staining was absent in most morphologically necrotic areas of tumor although some cells with pyknotic nuclei stained for VS38c in these areas. Most tumour cells exhibiting atypical nuclear forms were not stained by VS38c. CONCLUSIONS: Our findings show that VS38c is a sensitive but not specific diagnostic marker of osteosarcoma. Staining with VS38c identifies viable osteosarcoma cells, a feature which may be useful in the assessment of percentage tumour necrosis post-neoadjuvant chemotherapy.

Dendritic and mast cell involvement in the inflammatory response to primary malignant bone tumours.

BACKGROUND: A chronic inflammatory cell infiltrate is commonly seen in response to primary malignant tumours of bone. This is known to contain tumour-associated macrophages (TAMs) and lymphocytes; dendritic cells (DCs) and mast cells (MCs) have also been identified but whether these and other inflammatory cells are seen commonly in specific types of bone sarcoma is uncertain. METHODS: In this study we determined the nature of the inflammatory cell infiltrate in 56 primary bone sarcomas. Immunohistochemistry using monoclonal antibodies was employed to assess semiquantitatively CD45+ leukocyte infiltration and the extent of the DC, MC, TAM and T and B lymphocyte infiltrate. RESULTS: The extent of the inflammatory infiltrate in individual sarcomas was very variable. A moderate or heavy leukocyte infiltrate was more commonly seen in conventional high-grade osteosarcoma, undifferentiated pleomorphic sarcoma and giant cell tumour of bone (GCTB) than in Ewing sarcoma, chordoma and chondrosarcoma. CD14+/CD68+ TAMs and CD3+ T lymphocytes were the major components of the inflammatory cell response but (DC-SIGN/CD11c+) DCs were also commonly noted when there was a significant TAM and T lymphocyte infiltrate. MCs were identified mainly at the periphery of sarcomas, including the osteolytic tumour-bone interface. DISCUSSION: Our findings indicate that, although variable, some malignant bone tumours (e.g. osteosarcoma, GCTB) are more commonly associated with a pronounced inflammatory cell infiltrate than others (e.g. chondrosarcoma. Ewing sarcoma); the infiltrate is composed mainly of TAMs but includes a significant DC, T lymphocyte and MC infiltrate. CONCLUSION: Tumours that contain a heavy inflammatory cell response, which includes DCs, TAMs and T lymphocytes, may be more amenable to immunomodulatory therapy. MCs are present mainly at the tumour edge and are likely to contribute to osteolysis and tumour invasion.

Dentine matrix protein 1 (DMP-1) is a marker of bone formation and mineralisation in soft tissue tumours.

Dentine matrix protein 1 (DMP-1) is a non-collagenous matrix protein found in dentine and bone. It is highly expressed by osteocytes and has been identified in primary benign and malignant osteogenic bone tumours. Bone formation and matrix mineralisation are seen in a variety of benign and malignant soft tissue tumours and tumour-like lesions, and in this study, we analysed immunohistochemically the DMP-1 expression in a wide range of soft tissue lesions (n = 254) in order to assess whether DMP-1 expression is useful in the histological diagnosis of soft tissue tumours. Matrix staining of DMP-1 was seen in all cases of myositis ossificans, fibro-osseous tumour of the digits, extraskeletal soft tissue osteosarcoma and in most cases of ossifying fibromyxoid tumour. DMP-1 was also noted in dense collagenous connective tissue of mineralising soft tissue lesions such as tumoural calcinosis, dermatomyositis and calcific tendinitis. DMP-1 was expressed in areas of focal ossification and calcification in synovial sarcoma and other soft tissue tumours. With few exceptions, DMP-1 was not expressed in other benign and malignant soft tissue tumours. Our findings indicate that DMP-1 is a matrix marker of bone formation and mineralisation in soft tissue tumours. DMP-1 expression should be particularly useful in distinguishing extraskeletal osteosarcoma and ossifying fibromyxoid tumour from other sarcomas and in identifying areas of osteoid/bone formation and mineralisation in soft tissue tumours.

Selective inhibition of human type 1 11beta-hydroxysteroid dehydrogenase by synthetic steroids and xenobiotics.

Functional analyses were performed with microsomal human 11beta-hydroxysteroid dehydrogenase type 1 overexpressed in the yeast Pichia pastoris. Cell extracts or microsomes from transformed strains displayed dehydrogenase and reductase activities, which were up to 10 times higher than in human liver microsomes, while for whole cells cortisone reduction but no dehydrogenase activity was observed. The synthetic glucocorticoids prednisolone and prednisone were efficiently metabolized by subcellular fractions, whereas no activity was observed with dexamethasone, budesonide and deflazacort. Inhibitors found to be effective towards the recombinant 11beta-hydroxysteroid dehydrogenase include synthetic steroids and xenobiotic compounds, revealing selective inhibition of the reaction direction, useful for development of specific inhibitors.