No abstract

The nucleotide pyrophosphatases/phosphodiesterases NPP1 and NPP2/autotaxin are structurally related eukaryotic ecto-enzymes, but display a very different substrate specificity. NPP1 releases nucleoside 5'-monophosphates from various nucleotides, whereas NPP2 mainly functions as a lysophospholipase D. We have used a domain-swapping approach to map substrate-specifying determinants of NPP1 and NPP2. The catalytic domain of NPP1 fused to the N- and C-terminal domains of NPP2 was hyperactive as a nucleotide phosphodiesterase, but did not show any lysophospholipase D activity. In contrast, chimaeras of the catalytic domain of NPP2 and the N- and/or C-terminal domains of NPP1 were completely inactive. These data indicate that the catalytic domain as well as both extremities of NPP2 contain lysophospholipid-specifying sequences. Within the catalytic domain of NPP1 and NPP2, we have mapped residues close to the catalytic site that determine the activities towards nucleotides and lysophospholipids. We also show that the conserved Gly/Phe-Xaa-Gly-Xaa-Xaa-Gly (G/FXGXXG) motif near the catalytic site is required for metal binding, but is not involved in substrate-specification. Our data suggest that the distinct activities of NPP1 and NPP2 stem from multiple differences throughout the polypeptide chain.

Recent studies suggested that boron has a chemo-preventive role in prostate cancer. In the present report, we investigated the effects of calcium fructoborate (CF) and boric acid (BA) on activation of the apoptotic pathway in MDA-MB-231 human breast cancer cells. Exposure to BA and CF inhibited the proliferation of breast cancer cells in a dose-dependent manner. Treatment with CF but not BA resulted in a decrease in p53 and bcl-2 protein levels. Furthermore, after the treatment with CF, augmentation of pro-caspase-3 protein expression, cytosolic cytochrome c level, and caspase-3 activity were observed, indicating apoptotic cell death induction. This was also demonstrated by terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick-end-labeling assay. In conclusion, our data provide arguments to the fact that both BA and CF inhibited the growth of breast cancer cells, while only CF induced apoptosis. Additional studies will be needed to identify the underlying mechanism responsible for the observed cellular responses to these compounds and to determine if BA and CF may be further evaluated as chemotherapeutic agents for human cancer.

Green bio-based polymeric membranes are rapidly emerging as materials of choice for a number of biomedical applications such as in the osseointegration processes. In this work, we report our preliminary studies on the covalent immobilization of sericin on to green cellulose membranes for potential applications in the osseointegration field. Initially, the surface of the cellulose acetate membrane was immobilized with the amino-propyl-triethoxysilane (APTS) functional group, while the protein was immobilized through glutaraldehyde that was used as a linker between amino-propyl-triethoxysilane and sericin. The functionalized membranes were thoroughly characterized by different characterization techniques such as infrared spectroscopy (FT-IR); Raman spectroscopy, x-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and thermogravimetric analysis (TGA/DTG). All these techniques confirmed the successful functionalization and uniform immobilization of the protein onto the membrane surface. Subsequently, the performance of the membranes was characterized both in terms of flows and retention of bovine serum albumin and haemoglobin in the case of immobilized protein membranes. The retention was found to be more than 90% after 90 minutes of process. Since in these membranes cases, their speed of degradation is essential in the physiological pH conditions, so the degradation was also studied over a period of three months and the degradation mechanism is also explained. Furthermore, the membrane functionalized with sericin has proven to hold great promise for application in bone regeneration.

Modern strategies in adipose tissue engineering (ATE) take advantage of the easy harvest, abundance and differentiation potential towards mesenchymal lineages of hADSCs. The controlled conversion of hADSCs to committed adipogenic precursors and further mature adipocytes formation is important for good long-term results in soft tissue regeneration. Thus, in this study, we report: (i) the isolation of the processed lipoaspirate (PLA) cells from adipose tissue and sanguine fractions; (ii) the phenotypic characterization of the PLA descendants; (iii) the design of a novel protocol for the modulation of adipogenic conditions in the perspectives of ATE applications. To modulate the differentiation rate through our protocol, we propose to selectively modify the formulation of the adipogenic media in accordance with the evolution of the process. Therefore, we aimed to ensure the long-term proliferation of the precursor cells and to delay the late adipogenic events. The status of differentiation was characterized in terms of intracellular lipid accumulation and reorganization of the cytoskeleton simultaneously with perilipin protein expression. Moreover, we studied the sequential activation of PPARγ2, FAS, aP2 and perilipin genes which influence the kinetics of the adipogenic process. The strategies developed in this work are the prerequisites for prospective 3D regenerative systems.