L-tüüpi püruvaadi kinaasi N-terminaalse domeeni regulatoorne roll

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Püruvaat on oluline metabolismi substraat ja seetõttu on püruvaadi kinaaside aktiivsuse regulatsioon oluline raku energia- ja süsinikuvoogude suunamise seisukohast. Maksas, kus samaaegselt toimivad need mõlemad metabolismi rajad, esineb koe-spetsiifiline püruvaadi kinaaas (L-PK), mis on aktiveeritud homotroopselt selle ensüümi substraadi fosfoenoolpüruvaadi (PEP) poolt ning heterotroopselt fruktoos-1,6-bisfosfaadi (FBP) poolt. Samas on need mõlemad efektid omakorda reguleeritud selle ensüümi N-terminaalse domeeni fosforüleerimise kaudu. Käesolevas töös uuriti selle regulatoorse fosforüleerimise nähtuse tagamaid, kasutades selleks klassikalisi valgukeemia meetodeid ning keemilise kineetika rakendusi koos ligandide seostumise modelleerimisega arvutil. Töö tulemused näitasid, et mitte-fosforüülitud L-PK poolt katalüüsitud reaktsioon on PEP korral iseloomustatud tavalise hüperboolse Michelis-Menteni reaktsioonikiiruse võrrandiga (KPEP = 0.11 mM). Samas lülitas cAMP-sõltuva proteiinkinaasi poolt katalüüsitud valgu fosforüülimine sisse L-PK kooperatiivsuse PEP suhtes ning ka selle ensüümi aktiivsuse allosteerilise regulatsiooni. Nii iseloomustas stöhhiomeetriliselt fosforüleeritud ensüümi, kus ühe valgu tetrameerse molekuli kohta esines neli fosfaatrühma, KPEP väärtus 2.2 mM ja Hilli koefitsient n = 2.5. Samuti ilmnes, et kooperatiivsed omadused lülitusid sisse tetrameerse valgu esimese subühiku fosforüleerimisel, kusjuures edasine fosfaatrühmade lisamine ainult moduleeris seda efekti. Samuti leiti, et mitte-fosforüülitud L-PK aktiivsust ei mõjutanud ka allosteeriline aktivaator FBP. Kuna fosforüleeritud L-PK aktiivsus suureneb oluliselt allosteerilise aktivaatori FBP juuresolekul, siis järeldati, et fosforüleerimine toimib ka selle ensüümi allosteerilise regulatsiooni molekulaarse lülitina. Regulatoorne fosforüülimine toimub seriini nr 12 juures, mis asub N-terminaalses valgujärjestuses MEGPAGYLRR10AS12VAQLTQEL20GTAFF. Selle valgujärjestuse rolli selgitamiseks uuriti fosforüülitava seriini ümbruses asuvate aminohapete punktmutatsioonide mõju valgu katalüütilistele omadustele. Muutused olid positsioonides 9, 10 ja 13 ning asenduseks kasutati aminohappeid A, L, Q ja E, mis võimaldas muuta selle domeeni summaarset laengut ja uurida, kas ioonsete rühmade ümberjaotumine ja uute ioonsete rühmade sisseviimine võib imiteerida seriini jäägi fosforüülimise efekti. Ilmnes, et mõned asendused mõjutasid valgu aktiivsust, vähendades selle afiinsust PEP suhtes ning sellega imiteerisid fosforüülimist. Saadud tulemus on kooskülas A. Fentoni ja kaastöötajate poolt pakutud oletusega (Fenton & Tang, 2009; Holyoak et al., 2013), et N-domeen võib seostuda valgumolekuli mingis muus piirkonnas ja selliselt mõjutada substraadi sidumist. Selle sisemolekulaarse kompleksi lõhub aga domeeni fosforüülimine. Samas aga ilmnes tõsiasi, et N-domeeni struktuuri ja kogulaengu muutmine ei indutseeri ensüümi kooperatiivsust PEP suhtes. Lähtudes oletusest, et N-domeen võib tänu oma paindlikkusele toimida valgumolekuli teiste osadega, teostati arvutil modelleerimise teel võimalike sidumiskohtade otsing. Selleks uuriti regulatoorse domeeni fragmendi RRASVA ja selle fosforüülitud analoogi RRAS(Pi)VA seostumist L-PK alaühikuga. Leiti, et RRASVA seostumine võib toimuda ensüümi aktiivtsentri piirkonnas ning see võib tõepoolest olla oluline PEP sidumise seisukohast. Samal ajal toimus fosfopeptiidi sidumine subühiku C-domeenil, kus seostub allosteeriline ligand FBP. Seega võib sisemolekulaarsete komplekside teke tõepoolest nihutada tasakaalu ensüümi aktiivse R-vormi ja vähemaktiivse T-vormi vahel. Samas võib kooperatiivsuse sisselülitamine toimuda fosforüleeritud N-domeeni seostumisel L-PK allosteerilises tsentris. Molekulaarne lülitusmehhanism allosteerilise ja mitte-allosteerilise L-PK vormide vahel, mis põhineb regulatoorse domeeni fosforüülimisel, võib omada olulist tähtsust maksas toimuva glükoosi metabolism mõistmisel.Pyruvate is an essential substrate in metabolic processes, and regulation of the activity of pyruvate kinases is essential for control over the energy and carbon fluxes in living cells. In liver tissue, where both of these glycolysis and glyconeogenesis pathways are in function, the tissue-specific pyruvate kinase (L-PK) is activated homotropically by phosphoenolpyruvate (PEP) and heterotropically by fructose-1,6-bisphosphate (FBP), and both of these effects depend on phosphorylation of the N-terminal domain of the enzyme. In this dissertation the regulatory role of the N-terminal domain has been studied by using conventional methods of protein chemistry and chemical kinetics along with computational ligand docking analysis. The results of this study revealed that the activity of the non-phosphorylated L-PK was characterised by a common hyperbolic Michaelis-Menten plot in the case of PEP (KPEP = 0.11 mM) and the activity of the enzyme was not regulated by FBP. The cooperativity of the enzyme for PEP was switched on by phosphorylation of the protein by cAMP-dependent protein kinase. The stoichiometrically phosphorylated enzyme, containing four moles of phosphate per tetrameric L-PK molecule, was characterised by KPEP = 2.2 mM and the Hill coefficient n = 2.5. It was also observed that enzyme cooperativity was mostly engaged by phosphorylation of the first subunit in the tetrameric protein, while further phosphorylation only modulated this effect. Further, it was found that the activity of the non-phosphorylated L-PK was not elevated by FBP, and this ligand acted as a relatively weak reversible inhibitor. As the phosphorylated L-PK is a subject of significant allosteric activation by FBP, it was concluded that phosphorylation should also function as a molecular switch in the case of the allosteric properties of L-PK. The regulatory phosphorylation occurs in the serine residue in position 12 of the N-terminal sequence MEGPAGYLRR10AS12VAQLTQEL20GTAFF, and in this study the influence of point mutations around the phosphorylation site in positions 9, 10 and 13 on the catalytic properties of the mutants was studied. Amino acids A, L, Q and E were introduced into these positions to alter the net charge of the regulatory domain and to check whether redistribution and/or introduction of new ionic groups can mimic the effect of serine phosphorylation. It was found that some of these mutations affected the catalytic activity of the enzyme by reducing the effectiveness of phosphoenolpyruvate binding, simulating thus the effect of regulatory phosphorylation. This result was in agreement with the ideas suggested by A. Fenton & Tang (Fenton & Tang, 2009) that the flexible N-domain may interact with the main body of the enzyme affecting substrate binding, and this interaction is interfered with by phosphorylation. However, differently from phosphorylation, the cooperativity of the enzyme was not switched on by these mutations in the N-domain. Finally, following the suggestion that the N-domain interacts with the main body of the L-PK molecule, computational analysis was made to identify putative binding sites. The docking site of the N-domain peptide RRASVA was found in the vicinity of the enzyme active centre, while docking of the phosphorylated analogue RRAS(Pi)VA occurred on the C domain of the L-PK molecule, in a site overlapping with the allosteric site for FBP binding. This means that formation of these intramolecular complexes may indeed change equilibrium between the active R-state and the less active T-state of the enzyme, while induction of cooperativity by the N-domain phosphorylation could be connected with phosphopeptide intramolecular binding with the enzyme allosteric site. The phenomenon of switching between non-allosteric and allosteric forms of L-PK and the possibility of modulating of the enzyme allostery by phosphorylation may have some implication for further understanding of the regulation of glycolysis in the liver

Solid Phase Sorption of Phenols on Metals Acetylacetonates

The solid phase extraction properties of surface layers of Eu(III), Al(III), Fe(III), Cr(III) acetylacetonates are compared for sorption some phenols and chlorophenols. The effects of the energies of adsorption and complexation on the retention of various sorbates were calculated. GC methods with preconcentration are proposed to evaluate phenols by means of solid-phase extraction on a sorbent with a surface layer of Eu acetylacetonate with extraction effectiveness of 85%

Synthesis and study of the composition and properties of chelate complexes of benzoyl acetonates of rare-earth elements and use of these in gas chromatography

Chelate complex cerium [Ce(BA)3)] and europium [Eu(BA)3] benzoyl acetonates were synthesized. According to IR spectroscopic data and elemental analyses, the composition of the complexes is described by the empirical formulas M(BA)3·2H2O. Sorbents based on a mesoporous silica gel modified with cerium and europium benzoyl acetonates were obtained. The nitrogen porosimetry demonstrated that, as the complexes are deposited onto the surface of the starting support, the specific surface area and the pore volume become smaller. The polarity of the sorbents Chromaton N-AW + SiO2 + M(BA)3 with respect to all classes of test compounds decreases in the order CeIII > EuIII. According to the data obtained for the capacity factors, the sorbent modified with cerium benzoyl acetonate selectively separates alcohols, and Chromaton N-AW + SiO2 with deposited chelates Eu(BA)3 shows the best separating capacity for alkanes and alcohols. It was shown that the sorbents can be used in practice for separating gaseous mixtures of light hydrocarbons

Effect of solution pH on the surface morphology of sol-gel derived silica gel

We have examined the effect of solution acidity on the textural characteristics of silica gels prepared by sol–gel synthesis using tetraethyl orthosilicate (TEOS) as a silica precursor and cetyltrimethylammonium bromide (CTAB) as a template. Using IR spectroscopy, we have studied micellar TEOS solutions and the synthesized silica gel samples. The results demonstrate that, in an alkaline medium in a water–ethanol solution, SiO2 experiences short-range ordering on the surface of micelles formed by CTAB molecules, whereas in an acid medium the process is not influenced by the presence of CTAB. Nitrogen porosimetry and electron microscopy data indicate that the silica gel obtained at pH 2 is microporous, with an average pore size of 2 nm. In an alkaline medium at pH 10, we obtained mesoporous SiO2 (18 nm) with a narrow pore size distribution and a specific surface area of 110 m2/g

Predictors of Negative Self-Attitude in Patients Who Tolerated a Surgical Repair

One of the main reasons for seeking a medical aid of a plastic surgeon is the negative attitude toward physical appearance and personality overall in patients with acquired defects of appearance. Correction of physical defects of a face and a body involuntarily associates with subsequent “automatic” growth in self-confidence and self-acceptance in those affected. The attempt undertaken to identify and evaluate factors that determine a level of the negative self-attitude on different stages of reconstructive-restorative treatment. The sample consisted of 58 respondents (mean age – 38,8 + 10,7 years), 27 persons (20 female, 7 male) were with visible differences on a face and a head, 31 persons (28 female, 3 male) were with defects of appearance located on a body. Respondents were interviewed one week before the treatment began, 2 weeks and 6-10 weeks after correcting physical defects. The mathematical analysis carried out using non-parametric tests as well as multiple regression analysis techniques. There was a statistically significant decrease in the levels of self-accusation and internal conflict (χ2emp. from 9,270 to 15,876; p<0,01). The regression equations are constructed and predictors of the negative self-attitude selected, which provides with valid information for creating a program of psychological support for such patients

SUSCEPTIBILITY OF PLANKTONIC AND FILM FORMS OF CANDIDA GLABRATA AND CANDIDA ALBICANS TO CATIONIC SURFACTANT ANTISEPTICS

The aim of the study was to investigate the sensitivity of planktonic and film forms of C. albicans and C. glabrata to cationic surfactant antiseptics. Materials and methods. The study was based on investigating 20 clinical strains of C. albicans and 15 C. glabrata isolated from surgical inpatientsю. The sensitivity of planktonic forms of investigated strains to antiseptic agents was quantitatively evaluated by two-fold serial dilutions (macrodilution) in Sabouraud liquid nutrient medium. Biofilm-forming properties of clinical strains C. albicans and C. glabrata were assessed by using the Christensen’s spectrophotometric method (MtP-test “microtiter plate test”). The influence of the antiseptics on C. albicans and C. glabrata film forms was assessed by the reproduction of the biofilms according to the above-described procedure with adding antiseptics in sub-bacteriostatic concentrations and the subsequent spectrophotometric ODU assessment. In the study we used antiseptics based on cationic surfactants, chlorhexidine digluconate 0.05 (Chlorhexidine-KR, manufactured by PJSC “Khimfarmzavod “Chervona zirka"”, Kharkiv, Ukraine (CHH)) and decamethoxin 0.2 (Decasan, produced by Yuria-Farm LLC ", Kyiv, Ukraine (DCM)). Results. According to the research results, lower sensitivity of C. glabrata strains to CHH was found, compared to the sensitivity of C. albicans strains. In addition, the activity of DCM in the investigated representatives of Candida spp. did not differ significantly. Clinical strains of C. glabrata were more susceptible to DCM compared to their susceptibility to CHH. C. albicans strains showed medium film-forming properties, while C. glabrata - high. The investigated cationic surfactant antiseptics possessed the same degree of activity on the film-forming properties of clinical strains of Candida spp. Conclusions. Cationic surfactant antiseptics (CHH and DCM) possess antifungal activity against planktonic and film forms of C. albicans and C. glabrata

Correlation of Susceptibility to Antiseptics With Biofilm-forming Properties in Acinetobacter baumannii as a Pathogen of Surgical Infection

Introduction: The purpose of this research was to study the biofilm-forming properties of clinical strains of A.baumannii, isolated from burn wounds in patients of ICU, and their sensitivity to antiseptics. Methods: 220 clinicalstrains of A. baumannii isolated from intensive care unit infected burn patients were the object of the study. Antiseptic sensitivity of Acinetobacter spp. (decamethoxine, chlorhexidine, miramistin, povidone iodine) was investigatedusing double serial dilutions according to the standard procedure. The study of biofilm-forming properties of clinicalAcinetobacter isolates was performed using the spectrophotometric technique by G.D. Christensen. In order to determine the relationship between the antiseptic sensitivity and biofilm-forming properties of A. baumannii strains,we determined the correlation coefficient (r-Pearson), the absolute value of which characterised the binding force.Results: Among 435 burn persons, who were involved in the investigated group, representatives of Acinetobacterspp. were found in 220 (50.6%), that has revealed the etiological significance of the opportunistic pathogens of Acinetobacter isolates in the development of infectious complications of burns in intensive care units. Clinical strains of A. baumannii have shown variable susceptible to decamethoxine, chlorhexidine, miramistin, povidone iodine and have been found to possess high biofilm-forming properties. The r-Pearson coefficient between sensitivity of A. baumannii to investigated antiseptics and biofilm-formation pointed out positive moderate and strong correlations. Conclusion: Biofilm-formation of Acinetobacter spp. is correlated with their susceptibility to chlorhexidine and povidone iodine strongly. However, as a more powerful antimicrobial activity of antiseptic against A. baumannii was as weaker correlation had been establishe

Endocrine Disorders Associated with Medicinal Products: Approaches to Preclinical Safety Assessment

The endocrine system coordinates almost all organs and other systems in vertebrates. In particular, it regulates such important biological functions as metabolism, development, reproduction, and behaviour. To date, a significant amount of information has accumulated on endocrine disorders associated with chemical compounds (endocrine disruptors) used in various fields of human activity. The aim of this study was to evaluate the possibility of preclinical risk assessment for the endocrine function disorders attributable to new medicinal products. Endocrine disruptors are associated with a wide range of adverse events, including developmental problems arising from functional abnormalities of the endocrine system. Endocrine disorders caused by endocrine-disrupting chemicals are characterised by a long latency period between exposure and manifestation of a dysfunction; a nonlinear dose–response relationship; and a linear correlation of damage severity to exposure timing and duration. The chemicals influence the endocrine system through multiple mechanisms, the main of which being the interaction with cellular receptors sensitive to certain hormones and the influence on gene expression, intracellular signalling, and hormone transport, etc. This paper discusses the possibility of using hormone levels as indicators of endocrine disruption and presents the literature and authors’ own data on normal levels of relevant hormones in the blood of animals. An analysis of animal blood hormone levels in preclinical programmes will provide an opportunity to evaluate potential iatrogenic risks