E. Galliera

G. Goi

L. Massaccesi

M.M. Corsi Romanelli

N. Papini

V. Ragone

English

M. M. Corsi Romanelli

AIR Universita degli studi di Milano

species in RAW are hydrogen peroxide, nitrous acid and complexdecaying into peroxynitrite and peroxynitrous acid during notless than 4 days. The reaction with active species in RAW lastsup to 4 days. Active species have both oxidizing and reducingproperties, exhibit a strong antimicrobial effect. There is a delayin the action of radiation, since a significant concentration ofactive species is achieved in water not less than 1 min afterswitching on the radiation source. RAW characteristics are: pH~ 3, ORP = 790 mV (SHE), conductivity ~ 1 mS/cm. The specificenergy cost of producing RAW is about 10 times less than PAW.*The authors marked with an asterisk equally contributed to thework.P-41-019O-GlcNAcylation as marker of antioxidanteffects of vitamin E-stabilized ultra highmolecular weight polyethylene in humanosteoblastL. Massaccesi1, V. Ragone2, N. Papini3, M. M. CorsiRomanelli1,4, G. Goi5, E. Galliera1,61Department of Biomedical Sciences for Health, Universita DegliStudi di Milano, Milano, Italy, 2Permedica S.p.A, Research andDevelopment Department, Merate (Lecco), Italy, 3Department ofMedical Biotechnology and Translational Medicine, Universitadegli Studi di Milano, Milano, Italy, 4IRCCS Policlinico SanDonato, U.O.C SMEL-1 Patologia Clinica, San Donato(Milano), Italy, 5Department of Biomedical, Surgical and DentalSciences, Universita degli Studi di Milano, Milano, Italy, 6IRCCSGaleazzi Orthopaedic Institute, Milano, ItalyHigh Crosslink process was introduced in the development of jointprosthetic devices, in order to decrease the wear rate of ultrahighmolecular weight polyethylene (UHMWPE) but it also triggers theformation of free radicals and oxidative stress (OS), which affectsthe physiological bone remodeling, leading to osteolysis. VitaminE stabilization of UHMWPE was proposed to provide oxidationresistance without affecting mechanical properties and fatiguestrength. The aim of this study is to evaluate the antioxidant effectof vitamin E added to UHMWPE on OS induced osteolysis,focusing in particular on the evaluation (by western blot analysis)of protein O-GlcNAcylation, OGA and OGT levels consideredmarkers of cellular response to OS. O-GlcNAcylation levelsincreased in presence of vitamin E blended UHMWPE, in particu-lar with not crosslinked vitamin E stabilized UHMWPE(P < 0.005) while, conversely, they fall in absence of vitamin E.Significant increase (P < 0.01) of OGT protein was found in pres-ence of not crosslinked Vitamin E blended UHMWPE, whereas asignificant increase (P < 0.05) of OGA enzyme was observed inVitamin E absence. The OGT/OGA expression ratio show abehavior consistent with the observed O-GlcNAcylation levels.Our results suggest that the Vitamin E stabilization of UHMWPE:(i) seems to improve the ability of osteoblast to respond to oxida-tive stress, inducing cellular mechanism of defense, such asdynamic O-GlcNAcylation in order to promote cell survival; (ii)could contribute to reduce oxidation- induced osteolysis and theconsequent loosening of the prosthetic device, therefore improvingthe longevity of total joint replacements.P-41-020Inhibition of adenosine deaminase activity bynovel synthesized piperazine class compoundsL. Karapetyan1, A. Bakaryan1, N. Hakobyan2, A. Antonyan1,S. Mardanyan1, G. Gevorgyan21H. Buniatyan Institute of Biochemistry of NAS RA, Yerevan,Armenia, 2A.L. Mnjoyan Institute of Fine Organic Chemistry ofNAS RA, Yerevan, ArmeniaAdenosine deaminase (ADA) is an important enzyme in purinemetabolism. The increase of the ADA activity is documented insuch pathologies as diabetes, tuberculosis, cancer, rheumatoidarthritis, etc. The substrate of ADA, the adenosine, suppressesthe inflammation and plays the role in the protection againstinjuries. The increasing of ADA activity in the extracellular med-ium or pathological effusions results in decrease of adenosineconcentration and aggravation of inflammation. Therefore, theinhibition of ADA is considered as beneficial tool for regulationand remission of inflammation. This work describes the in vitroinhibition of purified from bovine lung ADA by novel synthe-sized tertiary amino alcohols substituted by piperazine ring. Thescreening of 15 compounds was carried out. Among them, thecompounds, containing in the piperazine ring the phenyl andheliotropine substitutes were more effective. The IC50 values for6 potent compounds in inhibition of ADA were between 3.5–15.5 lg/mL. The average value of IC50, for the compounds, intheir turn, containing in the heliotropine ring different substituentgroups was of 20 lg/mL. The inhibition of ADA by effective one– GGN 322*HCl the IC50 value was equal to 15.2 lg/mL. Theinhibition was of competitive nature with Ki = 2.5 lM. For thecompounds containing as a substitute phenyl group in the piper-azine ring, the average IC50 value was of 10 lg/mL. The mostsignificant results among them was registered for thePO191*2HCl, with benzhydryl (diphenylmethyl) as a substitutegroup. The IC50 value for it in inhibition of ADA was a ratherlow 3.5 lg/mL (6.5 lM). The Ki for this compound was 1.5 lMand the nature of inhibition was competitive. The constant ofbimolecular interaction of PO191*2HCl with the tryptophan resi-dues in ADA (KSV) was evaluated from the fluorescence quench-ing, using Stern–Volmer equation as of 0.145  0.027.P-41-021Interaction of PCID2 and NudC proteins ofDrosophila melanogaster in vitroA. Glukhova, D. KopytovaInstitute of Gene Biology, Russian Academy of Sciences, Moscow119334, RussiaThere are many proteins and protein complexes involved in themRNA transport from the nucleus to the cytoplasm in Droso-phila melanogaster cells. PCID2 protein as one of the partici-pants in this process binds mRNA in the nucleus and enters thecytoplasm, changing partners in the transport complex. In ourlaboratory we purified the PCID2 complex from cytoplasm anddetected its interaction with the NudC (nuclear distribution pro-tein). We investigated interaction between PCID2 and NudC pro-teins. First of all we generated antibodies to NudC andconfirmed PCID2 and NudC proteins interaction by immunopre-cipitation. We continued by testing whether PCID2 protein has aseparate domain for interaction with NudC protein. We dividedPCID2 into the following domains: N-terminal, C-terminal (WD-domain) and the “middle” domain (MID) located between them.Moreover, MID- and WD-domains together constitute a func-tional PCI-domain. We also divided NudC protein into N- andC-terminal domains in the same way. We expressed the 6xHis-tagged proteins related to said domains and full-size PCID2 andGeneral topics – various Posters414 FEBS Open Bio 9 (Suppl. 1) (2019) 65–431 DOI: 10.1002/2211-5463.12675ª 2019 The Authors. FEBS Open Bio ª 2019 FEBS

O-GlcNAcylation as marker of antioxidant effects of vitamin E-stabilized ultra high molecular weight polyethylene in human osteoblast

https://air.unimi.it/bitstream/2434/658666/2/FEBS_OpenBio_O_GicNAcylation_2019.pdf

O-GlcNAcylation as marker of antioxidant effects of vitamin E-stabilized ultra high molecular weight polyethylene in human osteoblast

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