Proteomic analysis of human mesenchymal stromal cells derived from adipose tissue undergoing osteoblast differentiation.

BACKGROUND AIMS: Stem cells derived from human adipose tissue (ASC) have the capacity for renewal, are easily obtained and have plasticity properties that allow them to differentiate into several cell types, including osteoblast cells. With the aim of understanding the issue of the osteogenic process and finding reliable biomarkers in cells undergoing the osteogeneic differentiation process, this work took advantage of a proteomic approach to identify proteins involved in osteogenesis. METHODS: For this purpose, ASC were analyzed under three conditions: S0, in the absence of stimulation; S1, with 2 weeks of osteogenic medium stimulation; and S2, with 4 weeks of osteogenic medium stimulation. The identification of ASC was carried out by flow cytometry using antibodies specific to known undifferentiated stem cell-surface markers. Cell viability, enzymatic activity, mineral deposition, collagen structure and production and gene analyzes were evaluated for each condition. RESULTS: Phenotypic modifications were observed during the in vitro osteogenic differentiation process by two-dimensional (2-D) differential image gel electrophoresis (DIGE). The proteins were identified by mass espectrometry in tandem (MS/MS) analyzes using Matrix-assisted laser desorption/ionization with TOF/TOF is a tandem mass spectrometry method where two time-of-flight mass spectrometers are used consecutively (MALDI-TOF/TOF). A total of 51 differentially expressed proteins was identified when comparing the three observed conditions. Sixteen different spots were identified in the S0 stage compared with S2, while 28 different spots were found in S2 compared with S0. S1 expressed seven different spots compared with S0 and S2. CONCLUSIONS: These findings suggest the involvement of several proteins directly related to the osteogenic pathway, which can be used to improve understanding of the osteogenic process.

Dose-dependent effects of triiodothyronine on the osteogenic differentiation of rat bone marrow mesenchymal stem cells.

BACKGROUND/AIMS: The aim of this study was to investigate the dose-dependent effects of triiodothyronine (T3) on the osteogenic differentiation of mesenchymal stem cells(MSCs). METHODS: MSCs that express CD73, CD54 (intercellular adhesion molecule-1) and CD90 were cultured in triplicate (1 x 10(5)/well) in osteogenic medium with T3 (1, 10, 10(3) or 10(5) pM) or without T3 (control) for 7, 14 and 21 days. Alkaline phosphatase activity, conversion of MTT into formazan crystals, collagen synthesis, collagen maturation, the number of mineralized nodules and their diameters were all determined, and the means were compared by the Student-Newman-Keuls test. RESULTS: A dose of 10(5) pM T3 resulted in a negative effect on MSC osteogenic differentiation, with less collagen synthesis. The 1 pM T3 dose resulted in greater collagen synthesis and alkaline phosphatase activity and more mineralized nodules than in the control group, similar to the 10 pM dose. Nevertheless, the 10 pM dose demonstrated better results than the 1 pM dose with regard to MSC osteogenic differentiation, with greater MTT reduction, better collagen maturation and a larger mean diameter of mineralized nodules. CONCLUSIONS: The effect of T3 on MSC differentiation is dose-dependent, with the 10 pM dose promoting better bone marrow MSC osteogenic differentiation.

Addition of ficoll and disaccharides to vitrification solutions improve in vitro viability of vitrified equine embryo.

The aim of the present study was to evaluate the in vitro viability of equine embryos vitrified in three different solutions. Day 6 and 6.5 embryos were measured and morphologically evaluated. Only grade 1 or 2 morulae and early blastocysts were vitrified. Eighteen embryos were distributed in Group 1: 40 percent ethylene glycol in PBS, Group: 2 and 3: 40 percent ethylene glycol, 18 percent Ficoll, 0.3M sucrose or 0.3M trehalose in PBS, respectively. The vitrified embryos were loaded individually into 0.25 ml straws, which were cooled and immersed in liquid nitrogen. After warming at 20 degree C for 20s, the embryos were expelled out into 0.5M sucrose in PBS and transferred to PBS solution. The embryonic diameter was measured again and morphology and viability were evaluated with Propidium iodide and Hoechst 33258 dyes. Embryos vitrified with sucrose (19.2 percent) and trehalose (26.7 percent ) showed the highest percentage of viable cells and morphological quality.

Behavior of mesenchymal stem cells stained with 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) in osteogenic and non osteogenic cultures.

4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) is a DNA dye widely used to mark and trace stem cells in therapy. We here studied the effect of DAPI staining on the behavior of mesenchymal stem cells cultured in either a control, non-osteogenic medium or in an osteogenic differentiation medium. In the control medium, the number of stem cells/field, as well as the number of fluorescent cells/field increased up to the sixth day in both control and DAPI-treated cultures. Afterwards, both the number of fluorescent cells and their fluorescence intensity decreased. Control cells were fusiform and with some long extensions that apparently linked them to neighboring cells, while DAPI-treated cells were mostly round cells with fine and short extensions. The trypan-blue exclusion method showed 99% cell viability in both groups, however, both alkaline phosphatase activity and the thiazolyl blue formazan assay (indicative of mitochondrial metabolism) gave significantly lower values in DAPI-marked cells. The mitochondrial mass, as indicated by specific staining and flow cytometry, showed no differences between groups. Mesenchymal stem cells gave origin to mineralized nodules in the osteogenic differentiation medium and there were not DAPI-marked cells on the ninth day of culture. Alkaline phosphatase activity, viability assay and number of cells/field and of mineralized nodules/field were similar in both groups. So, DAPI treatment did not change cell viability and proliferation during osteogenic differentiation of mesenchymal stem cells. However, since these cells loose DAPI marking after 9 days in osteogenic cultures suggests that DAPI may not be an effective marker for mesenchymal stem cells implanted in bone tissue for long periods.

Mimotopes of mutalysin-II from Lachesis muta snake venom induce hemorrhage inhibitory antibodies upon vaccination of rabbits.

Mutalysin-II (mut-II) from Lachesis muta snake venom is an endopeptidase with hemorrhagic activity. A mAb against mutalysin-II that neutralized the hemorrhagic effect was produced previously. To identify the mAb epitopes, sets of 15-mer overlapping peptides covering the mut-II amino acid sequence were synthesized using the SPOT method and tested but failed to react with the mAb. Using a phage-display approach seventeen clones reactive with mAb were identified. Additional immunoassays with the peptides and mAb identified the QCTMDQGRLRCR, TCATDQGRLRCT, HCFHDQGRVRCA, HCTMDQGRLRCR and SCMLDQGRSRCR sequences as possible epitopes. Immunization of rabbits with these peptides induced antibodies that recognize mut-II and protected against the hemorrhagic effects of Lachesis venom.

Molecular cloning and characterization of cDNA encoding cardosin B, an aspartic proteinase accumulating extracellularly in the transmitting tissue of Cynara cardunculus L.

Cardosins A and B are related aspartic proteinases from the pistils of Cynara cardunculus L., whose milk-clotting activity has been exploited for the manufacture of cheese. Here we report the cloning of cardosin B cDNA and its organ, tissue and cytological localization. The cDNA-derived amino acid sequence has 73% similarity with that of cardosin A and displays several distinguishing features. Cardosin B mRNA was detected in young inflorescences but not in pistils of fully opened inflorescences, indicating that its expression is developmentally regulated. The proteinase, however, accumulates in the pistil until the later stages of floral development. Immunocytochemistry with a monospecific antibody localized cardosin B to the cell wall and extracellular matrix of the floral transmitting tissue. The location of cardosin B in the pistil is therefore clearly different from that of cardosin A, which was found at protein storage vacuoles of the stigmatic papillae and has been suggested to be involved in RGD-mediated proteolytic mechanisms. In view of these results the possible functions of cardosin B in the transmitting tissue are discussed.

Subsite specificity of memapsin 2 (beta-secretase): implications for inhibitor design.

Memapsin 2 is the protease known as beta-secretase whose action on beta-amyloid precursor protein leads to the production of the beta-amyloid (Abeta) peptide. Since the accumulation of Abeta in the brain is a key event in the pathogenesis of Alzheimer's disease, memapsin 2 is an important target for the design of inhibitory drugs. Here we describe the residue preference for the subsites of memapsin 2. The relative k(cat)/K(M) values of residues in each of the eight subsites were determined by the relative initial cleavage rates of substrate mixtures as quantified by MALDI-TOF mass spectrometry. We found that each subsite can accommodate multiple residues. The S(1) subsite is the most stringent, preferring residues in the order of Leu > Phe > Met > Tyr. The preferences of other subsites are the following: S(2), Asp > Asn > Met; S(3), Ile > Val > Leu; S(4), Glu > Gln > Asp; S(1)', Met > Glu > Gln > Ala; S(2)', Val > Ile > Ala; S(3)', Leu > Trp > Ala; S(4)', Asp > Glu > Trp. In general, S subsites are more specific than the S' subsites. A peptide comprising the eight most favored residues (Glu-Ile-Asp-Leu-Met-Val-Leu-Asp) was found to be hydrolyzed with the highest k(cat)/K(M) value so far observed for memapsin 2. Residue preferences at four subsites were also studied by binding of memapsin 2 to a combinatorial inhibitor library. From 10 tight binding inhibitors, the consensus preferences were as follows: S(2), Asp and Glu; S(3), Leu and Ile; S(2)', Val; and S(3)', Glu and Gln. An inhibitor, OM00-3, Glu-Leu-Asp-LeuAla-Val-Glu-Phe (where the asterisk represents the hydroxyethylene tansition-state isostere), designed from the consensus residues, was found to be the most potent inhibitor of memapsin 2 so far reported (K(i) of 3.1 x 10(-10) M). A molecular model of OM00-3 binding to memapsin 2 revealed critical improvement of the interactions between inhibitor side chains with enzyme over a previous inhibitor, OM99-2 [Ghosh, A. K., et al. (2000) J. Am. Chem. Soc. 14, 3522-3523].

Behavior of mesenchymal stem cells stained with 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) in osteogenic and non osteogenic cultures

4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) is a DNA dye widely used to mark and trace stem cells in therapy. We here studied the effect of DAPI staining on the behavior of mesenchymal stem cells cultured in either a control, non-osteogenic medium or in an osteogenic differentiation medium. In the control medium, the number of stem cells/field, as well as the number of fluorescent cells/field increased up to the sixth day in both control and DAPI-treated cultures. Afterwards, both the number of fluorescent cells and their fluorescence intensity decreased. Control cells were fusiform and with some long extensions that apparently linked them to neighboring cells, while DAPI-treated cells were mostly round cells with fine and short extensions. The trypan-blue exclusion method showed 99% cell viability in both groups, however, both alkaline phosphatase activity and the thiazolyl blue formazan assay (indicative of mitochondrial metabolism) gave significantly lower values in DAPI-marked cells The mitochondrial mass, as indicated by specific staining and flow cytometry, showed no differences between groups. Mesenchymal stem cells gave origin to mineralized nodules in the osteogenic differentiation medium and there were not DAPI-marked cells on the ninth day of culture. Alkaline phosphatase activity, viability assay and number of cells/field and of mineralized nodules/field were similar in both groups. So, DAPI treatment did not change cell viability and proliferation during osteogenic differentiation of mesenchymal stem cells. However, since these cells loose DAPI marking after 9 days in osteogenic cultures suggests that DAPI may not be an effective marker for mesenchymal stem cells implanted in bone tissue for long periods.

Perfusao antiblástica hipertérmica regional no melanoma de membros em adultos / Regional hyperthermic antiblastic perfusion in extremities melanoma in adults

The first antiblastic regional perfusion was performed in 1957, in New Orleans, to treat a patient with locally advanced melanoma. This technique allows to use high doses of antiblastic drugs and was improved about 10 years later by additional hyperthermia, an idea suggested by Cavaliere et al and by Stehlin. Three types of isolated perfusion are possible: normothermic antiblastic perfusion. The use of adjuvant hyperthermic antiblastic perfusion offers to patients with extremity melanomas an excellent chance of cure. To patients with ® in transit ¼ metastases, hyperthermic antiblastic perfusion offers the most effective treatment with limb salvage. The main complication in isolated perfusion is the escape of the perfusate to the systemic circulation, but methods for escape evaluation are improving with the utilization of radiolabelled serum albumin and gamma-detecting probes. The hyperthermia is one of the most promising means of cancer therapy and it can be a local, regional or systemic treatment. Several retrospective and a few prospective and randomized studies have demonstrated the superiority of using hyperthermic antiblastic perfusion in extremity melanoma as compared to other forms of treatment. The World Health Organization and the European Organization for Research and Treatment of Cancer are performing randomized and controlled studies to evaluate the role of hyperthermic antiblastic perfusion in melanoma