Identification of the major autophosphorylation site of the Met/hepatocyte growth factor receptor tyrosine kinase.

The MET proto-oncogene encodes a transmembrane tyrosine kinase receptor for HGF (p190MET). In this work, p190MET was immunoprecipitated, allowed to phosphorylate in the presence of [gamma-32P]ATP, and digested with trypsin. A major phosphopeptide was purified by reverse phase chromatography. The phosphorylated tyrosine was identified as residue 1235 (Tyr1235) by Edman covalent radiosequencing. A synthetic peptide derived from the corresponding MET sequence was phosphorylated by p190MET in an in vitro assay and coeluted in reverse phase chromatography. Tyr1235 lies within the tyrosine kinase domain of p190MET, within a canonical tyrosine autophosphorylation site that shares homology with the corresponding region of the insulin, CSF-1 and platelet-derived growth factor receptors, and of p60src and p130gag-fps. The p190MET kinase is constitutively phosphorylated on tryosine in a gastric carcinoma cell line (GTL16), due to the amplification and overexpression of the MET gene. Metabolic labeling of GTL-16 cells with [32P]orthophosphate followed by immunoprecipitation and tryptic phosphopeptide mapping of p190MET showed that Tyr1235 is a major site of tyrosine phosphorylation in vivo as well. Since phosphorylation activates p190MET kinase, we propose a regulatory role for Tyr1235

Simulated performance of a xenohybrid bone graft (Smartbone®) in the treatment of acetabular prosthetic reconstruction

open6Total hip arthroplasty (THA) is a surgical procedure for the replacement of hip joints with artificial prostheses. Several approaches are currently employed in the treatment of this kind of defect. Overall, the most common method involves using a quite invasive metallic support (a Burch–Schneider ring). Moreover, valid alternatives and less invasive techniques still need to be supported by novel material development. In this work, we evaluated the performance of SmartBone®, a xenohybrid bone graft composed of a bovine bone matrix reinforced with biodegradable polymers and collagen, as an effective support in acetabular prosthesis reconstruction. Specifically, the material’s mechanical properties were experimentally determined (E = ~1.25 GPa, Ef = ~0.34 GPa, and Et = ~0.49 GPa) and used for simulation of the hip joint system with a SmartBone® insert. Moreover, a comparison with a similar case treated with a Burch–Schneider ring was also conducted. It was found that it is possible to perform THA revision surgeries without the insertion of an invasive metal support and it can be nicely combined with SmartBone®’s osteointegration characteristics. The material can withstand the loads independently (σmax = ~12 MPa) or be supported by a thinner titanium plate in contact with the bone in the worst cases. This way, improved bone regeneration can be achieved.openGrottoli C.F.; Cingolani A.; Zambon F.; Ferracini R.; Villa T.; Perale G.Grottoli, C. F.; Cingolani, A.; Zambon, F.; Ferracini, R.; Villa, T.; Perale, G

Intracellular calcium regulates the tyrosine kinase receptor encoded by the MET oncogene.

Previous work (Gandino, L., Di Renzo, M. F., Giordano, S., Bussolino, F., and Comoglio, P.M. (1990) Oncogene 5, 721-725) has shown that the tyrosine kinase activity of the receptor encoded by the MET protooncogene is negatively modulated by protein kinase C (PKC). We now show that an increase of intracellular Ca2+ has a similar inhibitory effect in vivo, via a PKC-independent mechanism. In GTL-16 cells the p145MET kinase is overexpressed and constitutively phosphorylated on tyrosine. A rapid and reversible decrease of p145MET tyrosine phosphorylation was induced by treatment with the calcium ionophores A23187 or ionomycin. Experiments performed with the ionophores in absence of extracellular calcium showed that a rise in cytoplasmic Ca2+ concentration to 450 nM (due to release from intracellular stores) resulted in a similar effect. These Ca2+ concentrations had no effect on p145MET autophosphorylation in an in vitro kinase assay. This suggests that the effect of Ca2+ on p145MET tyrosine phosphorylation is not direct but may be mediated by Ca(2+)-activated proteins(s). Involvement of Ca(2+)-dependent tyrosine phosphatases was ruled out by experiments carried out in presence of Na2VO4. In vivo labeling with [32P]orthophosphate showed that the rise of intracellular Ca2+ induces serine phosphorylation of p145MET on a specific phosphopeptide. This suggests that Ca2+ negatively modulates p145MET kinase through the phosphorylation of a critical serine residue by a Ca(2+)-activated serine kinase distinct from PKC

The Geometric Osteotomy: Joint Preservation in Juxta-Articular Surface Bone Neoplasms

Purpose. To present the oncologic and functional results of a consecutive series of patients treated by geometric osteotomy and allograft reconstruction for juxta-articular surface bone neoplasms

Regeneration of articular cartilage: Scaffold used in orthopedic surgery. A short handbook of available products for regenerative joints surgery

Introduction: Restoring defects of load-bearing connective tissues such as articular cartilage resulting from traumas, degenerative or age-related diseases remains a significant clinical challenge for clinicians due to the limited inherent repair capacity of articular cartilage. Tissue engineering has emerged as a potential alternative to the traditional surgical techniques, as it can be effectively used to regenerate bone, cartilage and the bone-cartilage interface. Several scaffold strategies have been developed and evaluated for osteochondral defect repair. Materials and methods: The classes of polymers (scaffold-based proteins, scaffold-base polysaccharides and synthetic scaffolds) and Hydrogels have been reviewed trough literature and market search. The study focused on their respective properties and analyzed advantages and disadvantages of each of them. Discussion: Clinical studies demonstrated improved cartilage regeneration thanks to the implantation of biomaterials after bone marrow stimulation. New cartilage can be engineered in vivo by transplanting chondrocytes seeded into a three-dimensional scaffold and this novel scaffold has mechanical properties that can be comparable to native cartilage and could be used to repair large osteochondral joints defects. Anyway, there is still space for improvement regarding clinical outcome and tissue quality

CD38 and bone marrow microenvironment.

This review summarizes the events ruled by CD38 shaping the bone marrow environment, recapitulating old and new aspects derived from the body of knowledge on the molecule. The disease models considered were myeloma and chronic lymphocytic leukemia (CLL). CD38 has been analyzed considering its twin function as receptor and enzyme, roles usually not considered in clinics, where it is used as a routine marker. Another aspect pertaining basic science concerns the role of the molecule as a member of an ectoenzyme network, potentially metabolizing soluble factors not yet analyzed (e.g., NAD+, ATP, NAM) or influencing hormone secretion (e.g., oxytocin). The last point is focused on the use of CD38 as a target of an antibody-mediated therapeutic approach in myeloma and CLL. A recent observation is that CD38 may run an escape circuit leading to the production of adenosine. The generation of local anergy may be blocked by using anti-CD38 antibodies. Consequently, not only might CD38 be a prime target for mAb-mediated therapy, but its functional block may contribute to general improvement in cancer immunotherapy and outcomes

LIGHT as regulator of bone homeostasis during osteolytic bone metastasis formation in non-small cell lung cancer patients

Tumor necrosis factor superfamily member 14 (TNFSF14), LIGHT is one of the cytokines produced by tumor and immune cells, which promotes homeostasis of lymphoid organs, liver and bone. Nonsmall cell lung cancer (NSCLC) commonly metastasizes bone, altering bone homeostasis and causing osteolysis. Here we investigated the role of LIGHT in NSCLC-induced osteolytic bone disease. The LIGHT expression in monocytes was higher in patients with metastatic bone lesions than in non-bone metastatic ones (66.5 ± 24.5 vs 43.3 ± 25.2 mean ± SD, p = 0.001), in healthy donors (66.5 ± 24.5 vs 8.5 ± 4.6 p = 0.0002), and in non-bone metastatic patients than in healthy donors (43.3 ± 25.2 vs 8.5 ± 4.6, p = 0.0001). Serum LIGHT levels were also significantly higher in bone metastatic patients than in non-bone metastatic ones (186.8 ± 191.2 pg/ml vs 115.8 ± 73 pg/ml, p = 0.04) and in healthy donors (186.8 ± 191.2 pg/ml vs 85.7 ± 38.4 pg/ml, p = 0.04). A neutralizing mAb anti-LIGHT added to osteoclast (OC) cultures of both bone and non-bone metastases inhibited osteoclastogenesis, but the decrease was statistically significant only for bone metastatic patients (272 ± 98 vs 132 ± 74, p = 0.01). To investigate the role of LIGHT in NSCLC- induced bone lesion in vivo, we performed an intratibial injection of a mouse lung cancer cell line LLC-1, in wild-type (WT) and LIGHT KO mice. The WT-injected mice displayed a significant reduction of about 20% for BV/TV, Tb.N, Tb.Th, and Tb.Sp compared to the WT-vehicle mice (pb 0.01). These parameters did not show significant variation for KO-injected mice vs vehicle or for WT-injected mice vs KO-injected mice. These data indicate LIGHT as a regulator of bone homeostasis during NSCLC metastatic invasion, thus it may be a novel therapeutic target in osteolytic bone metastases