Changes in the reactivity of human serum albumin Cys34 thiol group during fatty acids binding in vitro and in carbonyl stress

Humani serum albumin (HSA) je najzastupljeniji protein plazme sa udelom od oko 50 do 60 % svih proteina plazme. Vezuje i transportuje mnoge endogene i egzogene molekule, i doprinosi antioksidativnom kapacitetu seruma jer na površini molekula poseduje jednu slobodnu tiolnu grupu ostatka Cys34. Slobodne masne kiseline (MK), koje transportuje HSA, i karbonilni stres mogli bi, kroz promenu reaktivnosti tiolne grupe, uticati na njen potencijal kao hvatača reaktivnih karbonilnih vrsta. Stoga su ciljevi ove teze bili: in vitro ispitivanje uticaja 1) vezivanja MK, različite dužine lanca i zasićenosti na (i) reaktivnost Cys34-SH grupe, (ii) njen potencijal kao hvatača reaktivnih α-oksoaldehida, tj. na stepen karbonilacije i (iii) reaktivnost tiolne grupe HSA karbonilovanog metilglioksalom (model-sistem za molekule HSA modifikovane u karbonilnom stresu); 2) vezivanja MK ribljeg ulja na reaktivnost Cys34-SH grupe (sagledavanje mogućnosti modulacije svojstava HSA pomoću suplemenata); 3) određivanje sadržaja Cys34-SH grupe, korelacija sa sadržajem HbA1c i glukoze u karbonilnim stresom; 4) određivanje reaktivnosti Cys34-SH grupe HSA izolovanog iz seruma dijabetičara i zdravih osoba; 5) razvijanje qTLC metode za određivanje sadržaja MK, vezanih za HSA, koji je izolovan iz realnih uzoraka. Vezivanje MK (različite dužine lanca i zasićenosti: miristinske (MYR), palmitinske (PLM), stearinske (STE), oleinske (OLA), eikozapentenske (EPA) i dokozaheksaenske kiseline (DHA)) za HSA in vitro, dovodi do povećanja vrednosti konstanti brzine reakcije (k) Cys34 tiolne grupe (i DTNB) (k vrednosti kompleksa HSAMK: od 14,58±0,19 x 10-3 do 26,02±1,06 x 10-3 s-1 u odnosu na k HSA: 7,52±0,04 x 10- 3 s-1), odnosno njene reaktivnosti za 2 do 3,5 puta. STE i OLA ispoljavaju slične efekte na reaktivnost HSA-SH, a najjači efekat ispoljava polinezasićena EPA. Od zasićenih MK, najveća vrednost k dobijena je pri vezivanju MYR za HSA, što može biti posledica izuzetne komplementarnosti njenog molekula sa vezivnim mestima na molekulu HSA, koja najviše utiču na izloženost tiolne grupe rastvaraču. Između vrednosti konstanti brzine reakcije Cys34-SH, dobijenih pri vezivanju različitih MK, i izračunatih vrednosti dostupnosti Cys34 tiolne grupe rastvaraču nađen je visok stepen korelacije (r=0,927)...Human serum albumin (HSA) is the most abundant protein of human plasma, accounting for 50-60% of total plasma proteins. HSA binds and transport many endogenous and exogenous substances, and contributes to the antioxidative pool in serum because of Cys34 free thiol group on the surface of its molecule. Free fatty acids (FAs), transported by HSA, and carbonyl stress could influence, through changed reactivity of the thiol group, on its potential as the scavenger of the reactive carbonyl species. Therefore, the aims of this thesis were: in vitro examination of the influence 1) of different (in terms of chain length and saturation) FAs binding on the: (i) reactivity of the Cys34-SH group, (ii) its potential as the scavenger of the reactive α-oxoladehydes, and (iii) on the reactivity of the thiol group of HSA carbonylated with methylglyoxal (the model-system for HSA molecules modified during carbonyl stress); 2) of FAs from fish oil on the Cys34-SH group reactivity (overviewing the possibility of the modulation of the HSA scavenger properties with supplements); 3) determination of the Cys34-SH group content, correlation with the HbA1c and glucose level of persons with elevated carbonyl stress; 4) determination of the Cys34-SH group reactivity of the HSA isolated from the sera of diabetic patients and healthy persons; 5) development of qTLC method for determination of the FAs content, bound to the HSA, which was isolated from sera samples. Binding of FAs of different chain lengths and saturation (myristic (MYR), palmitic (PLM), stearic (STE), oleic (OLA), eicosapentaenoic (EPE) and docosahexaenoic acid (DHA)) to the HSA in vitro resulting in increasing of the kinetics constant (k) of the Cys34 thiol group reaction (with DTNB)(k values for HSA-FAs complexes: from 14.58±0.19 ×10-3 to 26.02±1.06 × 10-3 s-1 comparing to k of the HSA solely 7.52 ± 0.04 × 10-3 s-1), i.e. its reactivity for 2 to 3.5 times. STE and OLA show similar effects to the HSA-SH reactivity, and the strongest effect shows polyunsaturated EPA. From saturated FAs binding to the HSA, the strongest effect shows MYR, and that could be explained with extremely good fitting of the MYR molecule to the HSA FAs binding pockets which has the most influences to the exposure/accessibility of the thiol group to solvent..

Variability of the chloroplast dna of sessile oak (Quercus petraea agg. Ehrendorfer, 1967) in Serbia

Genetic variability of sessile oak (Quercus petraea agg. Ehrendorfer, 1967) in Serbia is estimated applying cpDNA universal primer pairs that were characterized by a high informative level for chloroplast genome variability assessment in previous investigations. Five different haplotypes were detected in the analyzed sample material from populations in Serbia

HSA carbonylation with methylglyoxal and the binding/release of copper(II) ions

The potential of carbonylation with methylglyoxal to alter HSA's binding affinity for copper(II) ions and its influence on the release of copper(II) ions from copper-HSA complexes were studied. The affinity of HSA to coordinate copper(II) decreased upon carbonylation of the Cys34-SH group. Carbonylation of copper-HSA complexes caused a decrease in Cys34-SH content, conformational changes and the release of copper(II) ions. The ratio between the percentage of reduction in the Cys34-SH group content and the percentage of release of copper(II) from complexes is 2.12 +/- 0.28. Because the same ratio (1.96 +/- 0.36) was obtained upon oxidation of the Cys34-SH group (with no changes in HSA conformation), the binding/release of copper (II) by HSA depended mainly on the redox state of the Cys34-SH group. The contents of Cys34-SH and HSA-bound copper(II) ions in the diabetic group (0.457 +/- 0.081 mol SH per mol HSA, 10.7 +/- 0.01 mmol per mol HSA, resp.) were significantly lower (p lt 0.01) compared to the control group (0.609 +/- 0.027 mol SH per mol HSA; 13.4 +/- 0.01 mmol per mol HSA, resp.). Very strong correlations between the values for HSA-SH and glycated haemoglobin, HbA1c, (R = -0.803, p lt 0.01), and between the values for the HSA-bound copper(II) content and HSA-SH content (R = 0.841, p lt 0.002) were found in the diabetic group. Thus, HSA carbonylation leads to decrease in HSA-SH content and to the impairment of its copper(II) binding capacity that could contribute to further enhancement of oxidative and carbonyl stress in diabetes (as well as in other diseases with carbonyl stress)

NOS3 gene variants and male infertility: Association of 4a/4b with oligoasthenozoospermia

Results of recent studies confirmed that oxidative stress negatively affects sperm motility and causes sperm DNA damage. Produced by nitric oxide synthase 3 (NOS3), nitric oxide is considered to be one of the important mediators of oxidative stress in testis tissue. The aim of this study was to assess the possible association of three genetic variants (rs2070744, rs1799983 and intron variant 4a/4b) in NOS3 gene and infertility occurrence in two groups of infertile men (idiopathic azoospermia and oligoasthenozoospermia) and fertile controls. Genotypes for the single-nucleotide genetic variants rs1799983 and rs2070744 were determined by PCR-RFLP, while genotyping of intron 4 variant 4a/4b was performed by gel electrophoresis of PCR products. Statistical analysis was performed by SNPStats software. No significant association between the three genetic variants of the NOS3 gene and infertility risk was determined comparing allele and genotype frequencies among group of patients diagnosed with azoospermia and the control group. Nevertheless, there was a significant positive association between 4a/4b and infertility in the group of males diagnosed with oligoasthenozoospermia, under overdominant genetic model. Our findings suggest that tandem repeat variant within intron 4 of the NOS3 gene is associated with an increased risk of infertility in men diagnosed with idiopathic oligoasthenozoospermia.Andrologia (2017): e1281

Clinical pattern of ocular toxoplasmosis treated in a referral centre in Serbia

Purpose To analyze the clinical pattern of ocular toxoplasmosis (OT) in a referral centre in Serbia. Patients and methods The medical records of consecutive patients admitted for OT to the single referral centre for uveitis in Serbia between 2006 and 2010 were retrospectively analyzed. OT was diagnosed on the basis of typical fundus lesions and positive serology for Toxoplasma. Results In a total of 457 uveitis patients, OT was the third leading cause, with 59 patients (12.9%). Most OT cases (73%) were monocular. An active primary retinal lesion was observed in 36% and recurrent OT in 64% patients. Localization of lesions was central/paracentral (44%), juxtapapillar (27%), peripheral (19%), and multifocal (10%). Other ocular manifestations of inflammation included vitritis (44%), anterior uveitis (19%), and retinal vasculitis (10%). Complications included choroidal neovascularization in two and exudative retinal detachment with cataract, glaucoma, and cystoid macular oedema in one patient each. The detection of Toxoplasma-specific IgM antibodies in a single patient indicates a low rate of OT concomitant with acute infection. After treatment, the mean best-corrected visual acuity (BCVA) increased significantly. However, 14 (24%) patients ended up legally blind in the affected eye, of which 2 (3%) with bilateral blindness, all with a very poor BCVA (0.047 +/- 0.055) at presentation. Visual impairment and treatment outcome were both associated with central localization of lesions (P lt 0.0001 and P = 0.006, respectively). Conclusion OT is a significant cause of posterior uveitis in Serbia. Patients should be aware of the recurring nature of OT and react immediately if symptoms occur. Eye (2012) 26, 723-728; doi: 10.1038/eye.2012.20; published online 24 February 201

Corrigendum to "European contribution to the study of ROS:A summary of the findings and prospects for the future from the COST action BM1203 (EU-ROS)" [Redox Biol. 13 (2017) 94-162]

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed

New test of modulated electron capture decay of hydrogen-like 142Pm ions: Precision measurement of purely exponential decay

An experiment addressing electron capture (EC) decay of hydrogen-like 142Pm60+ions has been conducted at the experimental storage ring (ESR) at GSI. The decay appears to be purely exponential and no modulations were observed. Decay times for about 9000 individual EC decays have been measured by applying the single-ion decay spectroscopy method. Both visually and automatically analysed data can be described by a single exponential decay with decay constants of 0.0126(7)s−1for automatic analysis and 0.0141(7)s−1for manual analysis. If a modulation superimposed on the exponential decay curve is assumed, the best fit gives a modulation amplitude of merely 0.019(15), which is compatible with zero and by 4.9 standard deviations smaller than in the original observation which had an amplitude of 0.23(4)

Determination of Fluorine Ions Penetration Degree Into Enamel by Empa.

Exogen of different preparations was applied to completely healthy teeth of children up to 11 years. A month after application of fluorine, teeth were extracted and prepared by standard methods for investigation by electron microprobe ARL, type SEMQ. The results obtained showed that the highest degree of fluorine ions migration was stimulated by organic fluorite' (amin fluoride). Very poor results were obtained by NaF, while the values obtained by 'fluor-protector' ranges between these two preparates. All up to date investigations showed that protective characteristics of fluorine against caries formation were reached only in case when enamel surface contained fluorine concentration of 10**3, mu g. Thus, our results proved that the first condition for good prophylaxy is obtained by application of aminofluoride and then by 'fluor-protector'

The interlaminar strength of the glass fiber polyester composite

The paper outlines the method and the results of the experimental investigation of interlaminar strength of glass fibre reinforced polyester composites, with the aim of determining the influence that the structure, a reinforcement type and a sort of resin exert upon the interlaminar strength. The tested specimens were fabricated under different formation conditions, namely in eight composition patterns and with three sorts of resin used for polymerization