Receptors for advanced glycosylation endproducts

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Exogenous nitric oxide inhibits shedding of ADAM17 substrates

Both ADAM17, the secretase responsible for the shedding of ectodomains of numerous membrane proteins including TNF and its receptors, as well as nitric oxide synthesized by inducible nitric oxide synthase play regulatory roles in inflammation and tumor progression. We analyzed the effect of endogenous and exogenous nitric oxide on the expression and activity of ADAM17 in murine endothelial cells and a monocyte/macrophage cell line. We found that endogenous nitric oxide influenced neither ADAM17 mRNA level nor the shedding of two ADAM17 substrates, TNF and TNFR1. Exogenous NO significantly diminished the release of TNF and TNFR1 without affecting the ADAM17 transcript level. Our data seem contrary to a previous report that showed the activation of ADAM17 by nitric oxide (Zhang et al., 2000, J Biol Chem 275: 15839-15844). We discuss potential mechanisms of NO-mediated inhibition of ectodomain shedding and possible reasons of discrepancy between our results and the previous report

Studies of new purine derivatives with acetic acid moiety in human keratinocytes

Recently we described a group of purine derivatives based on theophylline structure with acetic acid moiety. Studies in a group of these compounds demonstrated their analgesic and anti-inflammatory properties. Taking into account wide spectrum of theophylline derivatives activity and searching for their new properties. the aim of the study was to evaluate safety of newly synthesized derivatives in human keratinocytes model. The effect of new purine derivatives with acetic acid moiety: 2-(8-methoxy-1,3-dimethyl-2,6-dioxo-purin-7-yl) acetic acid and 2-(1,3-dimethyl-2,6,8-trioxo-9H-purin-7-yl) acetic acid on proliferation rate and the ability of keratinocytes to migration was carried out. The results clearly demonstrate that purine derivatives with acetic acid moiety did not affect basic keratinocytes functions. Our compounds do not inhibit cells proliferation rate as well as their ability to migration. It can be therefore concluded that new purine derivatives with acetic acid moiety are safe versus normal cells. This observation opens up additional prospects in searching for their new applications

Minichromosome maintenance (MCM) and AgNOR proteins expression in desmoid tumours: a tissue microarray analysis.

In the present study, nuclear proliferative proteins: MCM2, MCM5, MCM7, Ki-67 and AgNORs expression was assessed in paraffin sections from sporadic desmoid tumours using a tissue microarray (TMA)-based immuno- and histochemistry, respectively. Nuclear expression of MCM7, where the percentage of positive cells was 0.87% (Âą 1.64) (range 0-5%), was found in 4/20 (20.0%) cases. In 32/32 (100%) of the examined desmoid cases no expression of nuclear proteins MCM2 and MCM5 was detected. Nuclear expression of Ki-67 was observed in 4/21 (19%) cases. Paraffin sections from 30 cases of desmoid tumours were silver-stained to visualize AgNORs. The following AgNOR parameters were calculated: mean AgNOR number per nucleus (N), mean AgNOR area per nucleus, mean AgNOR dot area per nucleus (A), and mean AgNOR content (C = N/A). In the investigated group the mean values of AgNOR parameters were the following number: 4.34 (Âą 0.11); area: 0.74 Îźm2 (Âą 0.19); dot area: 0.18 m2 (Âą 0.01), and AgNOR content: 23.73 (Âą 1.85). The mean AgNOR number per nucleus and mean AgNOR content in desmoid tumours were statistically significantly higher as compared to the controls (tonsil tissue) (

ADAM17 promotes motility, invasion, and sprouting of lymphatic endothelial cells

Tumor-associated lymphatic vessels actively participate in tumor progression and dissemination. ADAM17, a sheddase for numerous growth factors, cytokines, receptors, and cell adhesion molecules, is believed to promote tumor development, facilitating both tumor cell proliferation and migration, as well as tumor angiogenesis. In this work we addressed the issue of whether ADAM17 may also promote tumor lymphangiogenesis. First, we found that ADAM17 is important for the migratory potential of immortalized human dermal lymphatic endothelial cells (LEC). When ADAM17 was stably silenced in LEC, their proliferation was not affected, but: (i) single-cell motility, (ii) cell migration through a 3D Matrigel/collagen type I matrix, and (iii) their ability to form sprouts in a 3D matrix were significantly diminished. The differences in the cell motility between ADAM17-proficient and ADAM17-silenced cells were eliminated by inhibitors of EGFR and HER2, indicating that ADAM17-mediated shedding of growth factors accounts for LEC migratory potential. Interestingly, ADAM17 depletion affected the integrin surface expression/functionality in LEC. ADAM17-silenced cells adhered to plastic, type I collagen, and fibronectin faster than their ADAM17-proficient counterparts. The difference in adhesion to fibronectin was abolished by a cyclic RGD peptide, emphasizing the involvement of integrins in the process. Using a soluble receptor array, we identified BIG-H3 among several candidate proteins involved in the phenotypic and behavioral changes of LEC upon ADAM17 silencing. In additional assays, we confirmed the increased expression of BIG-H3, as well as TGFβ2 in ADAM17-silenced LEC. The antilymphangiogenic effects of ADAM17 silencing in lymphatic endothelial cells suggest further relevance of ADAM17 as a potential target in cancer therapy

Selected physico-chemical properties of composite scaffolds of sintered submicrocrystalline corundum and bioglass

Presented paper contains description and interpretation of the results of selected physicochemical and structural properties of two types of composite sinters. They were constituted of a mixture of sintered microcrystalline corundum and bioglass CaO-P2O5-SiO2-Na2O system intended for scaffolds to cell culture of human chondrocytes. The composites contained a mixture of both above-mentioned components in the volumetric proportion of 50:50 (W5) and 30:70 (W7). They were obtained using powder metallurgy by free sintering in air atmosphere. Phase analysis of composites and verification of theoretical identification using X-ray diffraction were performed. The same phases were found in both cases (Al2O3 SiO2 CaAl2Si2O8, Ca3 (PO4)2, Ca2Al4O7 and NaAlSiO4). Microscopic tests of composite surfaces were performed and some differences were found. W5 sample was not completely covered with bioglass, whilst W7 sample was completely covered with bioglass with few fine pores. Tests of surface topography confirmed the presence of large and small pores. Composite surfaces immersed for 30 days in artificial blood plasma were tested and then electron microscopy analysis was performed. It was found that no significant changes occurred on the surface of the W5 composite, probably partial corrosion of the glass happened. Spherical forms characteristic of HA-hydroxyapatites were observed on the surface of sample W7. Human articular chondrocyte cells were seeded on both types of sinters and proliferation assay was performed. Results indicate that tested scaffolds support cellular attachment and proliferation of chondrocytes

shRNAs targeting mouse Adam10 diminish cell response to proinflammatory stimuli independently of Adam10 silencing

RNA interference is one of the common methods of studying protein functions. In recent years critical reports have emerged indicating that off-target effects may have a much greater impact on RNAi-based analysis than previously assumed. We studied the influence of Adam10 and Adam17 silencing on MC38CEA cell response to proinflammatory stimuli. Eight lentiviral vector-encoded shRNAs that reduced ADAM10 expression, including two that are specific towards ADAM17, caused inhibition of cytokine-induced Nos2 expression presumably via off-target effects. ADAM10 silencing was not responsible for this effect because: (i) CRISPR/Cas9 knockdown of ADAM10 did not affect Nos2 levels; (ii) ADAM10 inhibitor increased rather than decreased Nos2 expression; (iii) overexpression of ADAM10 in the cells with shRNA-silenced Adam10 did not reverse the effect induced by shRNA; (iv) shRNA targeting ADAM10 resulted in decrease of Nos2 expression even in ADAM10-deficient cells. The studied shRNAs influenced transcription of Nos2 rather than stability of Nos2 mRNA. They also affected stimulation of Ccl2 and Ccl7 expression. Additionally, we used vectors with doxycycline-inducible expression of chosen shRNAs and observed reduced activation of NF-κB and, to a lesser extent, AP-1 transcription factors. We discuss the requirements of strict controls and verification of results with complementary methods for reliable conclusions of shRNA-based experiments

The effect of 1-methylnicotinamide (1-MNA) on wound healing

1-MNA uważany był przez długi czas za nieaktywny biologicznie metabolit nikotynamidu. Badania wykazały jednak, ze 1-MNA może działać jako czynnik przeciwzakrzepowy i przeciwzapalny in vivo. Celem niniejszej pracy było zbadanie wpływu 1-metylonikotynamidu (1-MNA) na gojenie ran. Oceniono efekt działania 1-MNA in vitro na ludzkie keratynocyty i fibroblasty oraz in vivo w modelu gojenia ran u myszy.Wyniki przeprowadzonych badań in vitro dowodzą, że 1-MNA nie jest toksyczny wobec komórek skóry nawet w wysokich stężeniach. 1-MNA nie wpływa w istotny sposób na proliferację komórek skóry i aktywność metaboliczną keratynocytów. Nieznaczne efekty wywierane na aktywność metaboliczną fibroblastów nie tłumaczą efektów terapeutycznych obserwowanych in vivo.Ogólnoustrojowa terapia 1-MNA znacząco przyspiesza gojenie ran u myszy i jest skuteczniejsza niż miejscowe podawanie 1-MNA. Miejscowa terapia 1-MNA skutkowała jedynie tendencją do szybszego gojenie ran. Działanie 1-MNA podczas gojenia ran nie wydaje się być wynikiem zdolności 1-MNA do wiązania się do glikozaminoglikanów.For many years 1-methylnicotinamide (1-MNA) has been considered to be a biologically inactive metabolite of nicotinamide. It was demonstrated, however, that 1-MNA can act as an antithrombotic and anti-inflammatory agent in vivo. The aim of this study was to investigate the effect of 1-MNA on cutaneous wound healing. For this purpose in vitro analysis of the effect of 1-MNA on human keratinocytes and fibroblasts, as well as experiments in vivo in a murine model of wound healing were employed. Results of the in vitro studies show that 1-MNA is not toxic to both types of cells from human skin even at high concentrations. Moreover, 1-MNA does not affect the proliferation of both types of cells and the metabolic activity of keratinocytes in a significant way. Slight effects exerted on the metabolic activity of fibroblasts do not explain the therapeutic effects observed in vivo.Systemic treatment of 1-MNA significantly accelerated wound healing in mice and was more effective than topical administration. Topical 1-MNA therapy resulted only in a tendency to improve wound healing. The action of 1-MNA during wound healing does not appear to be a result of the ability of 1-MNA to bind to glycosaminoglycans