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Cys34 thiol group of human serum albumin (HSA) represents major plasma antioxidant. Its reactivity is influenced by multiple factors. The influence of fatty acids (FA; saturated, mono, and poly unsaturated acids from fish oil) binding to HSA, on copper(II) binding affinity and Cys34 thiol group accessibility/reactivity, in the presence of carbonylation agent (methylglyoxal, MG) was examined. HSA-copper(II) content, thiol group reactivity, and HSA carbonylation level were monitored spectrophotometrically. Changes in HSA were followed by fluorescence spectroscopy and native PAG electrophoresis. FA/HSA molar ratio was screened by GC. Together, binding of copper(II) ions and FA to HSA increase the reactivity of Cys34 thiol group (depending on the type of FA), with constant contribution of copper(II) ions of one-third. Carbonylation of FA-HSA-Cu(II) complexes caused a decrease in the Cys34 thiol group content, accompanied by a decrease in the content of HSA-bound copper. The carbonylation level of guanidine groups was not affected by FAs and copper(II) binding. Fluorescent emission spectra of FA-HSA-Cu(II)-MG complexes showed conformational changes in HSA molecule. Although binding of fatty acids and copper ions caused a significant increase in the thiol group reactivity, Cys34 thiol from FA-HSA-Cu(II) complexes reacted with MG in smaller extent than expected, probably as a consequence of conformational changes introduced by carbonylation. Increase in the percentage of reacted-free thiol groups with MG (due to FA and copper binding) may not seem to be very significant, but it is very important in complex biological systems, where catalytic metal is present. [GRAPHICS] .

Antipsychotic drugs interfere with the antioxidant defense system provoking complex and often toxicological effects. Here we examined differences in plasma albumin reduced free thiol (SH) group content and its reactivity as a consequence of clozapine (CLZ) and ziprasidone (ZIP) binding. Chronic administration of CLZ reduced, whereas treatment with ZIP increased albumin-SH content in rats. Regardless of the ratio of stearic acid (SA) bound to protein, in vitro binding of ZIP to human serum albumin (HSA) increased both the SH group level and reactivity. In contrast, the effect of CLZ on HSA-SH reactivity was dependent on HSA to SA molar ratio. CLZ binding was accompanied by an increase in HSA-SH reactivity in samples with normal, but a reduction of itsreactivity level with higher SA/HSA ratio, compared to drug-free samples. We demonstrate by steady-state fluorescence quenching studies that an increase in SA binding to HSA is associated with a significant reduction of binding constant for both antipsychotics. In addition, this is the first report of quantitative characterization of ZIP binding to HSA. Our findings suggest that albumin-SH content and reactivity is modulated by ZIP towards an increased antioxidant defense capacity in circulation, as opposed to CLZ, which can contribute to the safer, more effective treatment of schizophrenia. This is the peer-reviewed version of the following article: Uzelac, T. N.; Nikolić-Kokić, A. L.; Spasić, S. D.; Mačvanin, M. T.; Nikolić, M. R.; Mandić, L. M.; Jovanović, V. B. Opposite Clozapine and Ziprasidone Effects on the Reactivity of Plasma Albumin SH-Group Are the Consequence of Their Different Binding Properties Dependent on Protein Fatty Acids Content. Chemico-Biological Interactions 2019, 311, 108787, [https://doi.org/10.1016/j.cbi.2019.108787] The published version: [http://cer.ihtm.bg.ac.rs/handle/123456789/3053]

This paper describes the results achieved by high school seniors on an item which involves translation of the equationP=kTinto a corresponding pictorial external representation. The majority of students (the classes of 2011, 2012 and 2013) did not give the correct answer to the multiple choice part of the translation item. They chose pictorial representations of the other gas laws (P=k/V, orV=kT) instead. Failure to choose the correct answer was surprising considering that the symbol for volume was absent which should have been the key clue. Through the analysis of students' explanations (the classes of 2011 and 2012) and interviews (the class of 2013) we considered the reasoning applied by students who chose the correct answer or distractors for the multiple choice part of the item. Among the students who answered correctly there were explanations which contained misconceptions. Several factors that lead to the unsuccessful translation between external representations have been discovered. Students interpreted the change in one quantity based on the notation for the change in another one because of deep rooted cognitive schemas about changing two quantities (volume and pressure, pressure and temperature, temperature and volume), without consideration that for such changes to be valid for gases all three quantities need to be considered for a certain amount of substance. Those cognitive schemas interfered with mathematical reasoning,i.e.students possessed limited understanding of the equations.

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).

Congeneric set of thirty-eight 4-aryl-4-oxo-2-(N-aryl/cycloalkyl)butanamides has been designed, synthesized and evaluated for acetyl- and butyrylcholinesterase inhibitory activity. Structural variations included cycloalkylamino group attached to C2 position of butanoyl moiety, and variation of amido moiety of molecules. Twelve compounds, mostly piperidino and imidazolo derivatives, inhibited AChE in low micromolar range, and were inactive toward BChE. Several N-methylpiperazino derivatives showed inhibition of BChE in low micromolar or submicromolar concentrations, and were inactive toward AChE. Therefore, the nature of the cycloalkylamino moiety governs the AChE/BChE selectivity profile of compounds. The most active AChE inhibitor showed mixed-type inhibition modality, indicating its binding to free enzyme and to enzyme-substrate complex. Thorough docking calculations of the seven most potent AChE inhibitors from the set, showed that the hydrogen bond can be formed between amide -NH- moiety of compounds and -OH group of Tyr 124. The 10 ns unconstrained molecular dynamic simulation of the AChE- compound 18 complex shows that this interaction is the most persistent. This is, probably, the major anchoring point for the binding. (C) 2014 Elsevier Masson SAS. All rights reserved. Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/2906] This is the peer-reviewed version of the article: Vitorović-Todorović, M. D.; Koukoulitsa, C.; Juranić, I. O.; Mandić, L. M.; Drakulić, B. J. Structural Modifications of 4-Aryl-4-Oxo-2-Aminylbutanamides and Their Acetyl- and Butyrylcholinesterase Inhibitory Activity. Investigation of AChE-Ligand Interactions by Docking Calculations and Molecular Dynamics Simulations. European Journal of Medicinal Chemistry 2014, 81, 158–175. [https://doi.org/10.1016/j.ejmech.2014.05.008]

ObjectiveThe objective of the present study was to determine if vitamin D intake and status are associated with pre-eclampsia in a country without a vitamin D fortification policy.DesignA case–control study of pregnancies with (case) and without (control) pre-eclampsia was conducted from January to April when UVB is minimal. Maternal and cord blood obtained at delivery were measured for plasma 25-hydroxycholecalciferol (25-OH-D3), 3-epimer of 25-OH-D3(3-epi-25-OH-D3) and 24,25-dihydroxycholecalciferol (24,25-(OH)2D3) by LC–MS/MS and maternal 1,25-dihydroxyvitamin D (1,25-(OH)2D). Differences between groups were tested with ANOVA and Bonferronipost hoctests (PSettingClinical Center of Serbia.SubjectsPregnant women with and without pre-eclampsia (n60) and their infants.ResultsExogenous vitamin D intake (0·95–16·25 µg/d (38–650 IU/d)) was not significantly different between groups. Women with pre-eclampsia delivered infants at an earlier gestational age and had significantly lower mean total plasma 25-hydroxyvitamin D (25-OH-D; case: 11·2 (sd5·1); control: 16·1 (sd5·7) ng/ml;P=0·0006), 25-OH-D3(case: 10·0 (sd4·9); control: 14·2 (sd5·8) ng/ml;P=0·002), 3-epi-25-OH-D3(case: 0·5 (sd0·2); control: 0·7 (sd0·2) ng/ml;P=0·0007) and 1,25-(OH)2D (case: 56·5 (sd26·6); control: 81·0 (sd25·7) pg/ml;P=0·018), while 24,25-(OH)2D3was not different between groups. Infants did not differ in total plasma 25-OH-D, 25-OH-D3, 3-epi-25-OH-D3and 24,25-(OH)2D3, but the mean proportion of 3-epi-25-OH-D3was higher in the infant case group (case: 7·9 (sd1·1); control: 7·0 (sd1·4) % of total 25-OH-D3;P=0·005).ConclusionsA high prevalence of vitamin D deficiency, as defined by plasma 25-OH-D

Non-esterified fatty acids bound to the human serum albumin (HSA) contribute to several HSAs properties of special concern in pathologies, for instance to the reactivity of the free HSA-Cys34 thiol group (important antioxidative thiol pool in plasma), and to the affinity for binding of molecules and ions (for example cobalt as a prominent biomarker in heart ischemia). Therefore, the method for determination of FAs bound to HSA was developed. FAs were released from HSA (previously isolated from serum by ammonium sulfate precipitation) using acidic copper(II) sulfate in phosphoric acid, extracted by n-heptane-chloroform (4:1, v/v) mixture, spotted on TL silica-gel and then developed with n-heptane-chloroform-acetic acid (5:3:03, v/v/v). Common office flatbed scanner and software solution for densitometric image analysis, developed in R, were used. The linearity of calibration curve in concentration range from 0.1 to 5.0 mmol/L stearic acid was achieved. The method was proved to be precise (with RSD of 1.4-4.7%) and accurate. Accuracy was examined by standard addition method (recoveries 97.2-102.5%) and by comparison to results of GC. The method is sample saving, technically less demanding, and cheap, and therefore suitable for determination of FAs/HSA ratio when elevated concentrations of free FAs are reliable diagnostic/risk parameter of pathological states. (C) 2016 Elsevier B.V. All rights reserved.

The interaction of polyphenolic molecules with human serum albumin (HSA) could lead to changes in the reactivity of the HSA Cys34 thiol group (HSA-SH). The influences of enterolactone (EL) and enterodiol (ED) binding on HSA-SH reactivity in fatty acid (FA)-free HSA, and in HSA with bound stearic acid (S) in S/HSA molar ratios of 1 : 1 and 4 : 1, were investigated by the determination of the pseudo first order rate constants (k') for the thiol reaction with 5,5'-dithiobis-(2-nitrobenzoic acid). The binding affinities and binding sites of EL and ED were also determined, using fluorescence measurements of the intrinsic fluorescence of Trp214 and diazepam (binding site marker). EL and ED binding to HSA increased the reactivity of HSA-SH in all assayed HSA-enterolignan complexes by 9.1-33.1%. The strongest effects were obtained for FA-free HSA-enterolignan complexes. S modulated/reduced the effect of EL on HSA-SH reactivity, while its influence on the effect of ED was negligible. The binding of enterolignans to HSA was investigated: the binding constants were the highest for FA-free HSA (EL: 11.64 x 10(4) M-1 and ED: 5.59 x 10(4) M-1 at 37 degrees C) and the lowest for S/HSA 4 : 1-enterolignan complexes (EL: 2.43 x 10(4) M-1 and ED: 1.92 x 10(4) M-1). When the S/HSA ratio was increased, the binding affinities and number of binding sites for EL and ED were decreased. At the same time, a high correlation between binding constants and increased Cys34 reactivity was found (r = 0.974). Competitive experiments using diazepam indicated that the binding of ED and of EL was located in the hydrophobic pocket of site II in HSA. Overall, it is evident that stearic acid could modulate the enterolignan effects on HSA-SH reactivity as well as their binding to HSA. This finding could be important for pharmacokinetics and the expression of enterolignan antioxidant effects in vivo after an intake of lignan rich food.

Beer was chosen as a teaching tool to maximize students' class participation and systemize and enhance their knowledge of chemistry. Viewing beer as a complex mixture allowed the students to learn how to directly apply their chemistry knowledge. Before the "Beer Unit" students were instructed to research beer and acquire data on beer composition and properties. They were also asked to propose a hypothesis about possible chemical links between the components of beer and suggest qualitative analytical experiments. During the Laboratory and classroom periods, the students performed experiments, analyzed the results, tested their hypotheses, and solved problems. The multilevel approach generated more discussion topics and acquisition of new chemistry knowledge. The students were also encouraged to point out negative consequences caused by uncontrolled consumption of beer. As a result of this unit, the students obtained the correct answers during chemistry classes, and they gained powerful arguments for discussions and were able to make proper health and life choices.

Propranolol, a widely used beta-blocker, inhibits the reaction of enzyme cholinesterase hydrolysis. Measurements of the hydrolysis rate differences between and inhibited reactions, allows the development of a kinetic method for its determination. Both systems, enzyme-substrate-chromogen and enzyme-substrate-chromogen-inhibitor, were characterized through biochemical kinetic parameters (KM = 0.326-0.330 mmol/L; Vmax = 40-42.99 ?mol/Lmin), inhibition type was recognized as competitive, and inhibition constant, Ki, was determined to be 22.60 ?mol/L. The detection and quantification limits were calculated as 0.004 and 0.0136 ?mol/L, respectively. Accuracy and precision of proposed methods were tested. Proposed method is characterized with good sensitivity, selectivity, simplicity and rapidity, thus it is convenient for clinical applications.

Summary In the present study, we examined the effects of fish oil supplementation in 3 months old male Wistar rats on changes in plasma and liver lipid metabolism and oxidative stress parameters. Twenty Wistar rats were randomly divided into two groups of ten animals: control group and intervention group, treated for 6 weeks with fish oil capsules containing 45 mg eicosapentanoic acid and 30 mg docosahexanoic acid. After intervention, biochemical parameters in plasma [triglycerides (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL) and total cholesterol, urea, creatinine and uric acid], fatty acid (FAs) profile of liver phospholipids and parameters of oxidative stress in liver [activity of catalase, superoxide dismutase and paraoxonase (PON1), concentration of nitrites, lipid peroxidation (LPO), free thiol (SH) groups and lactate dehydrogenase (LDH) izoenzymes were determined. Treatment with fish oil improved FAs profile of liver phospholipids, increasing n-3 FAs and decreasing n-6/n-3 ratio. Significant decrease in plasma TG and LDL concentration, and increase in the level of HDL and uric acid were found in intervention group at the end of the study. Catalase activity, LPO, and nitrites concentration in liver were significantly decreased, after the supplementation, together with elevated PON1 activity. Applied treatment significantly improved plasma lipid profile, liver FAs composition and parameters of oxidative stress in male Wistar rats.

This study examines the relationship between erythrocyte phospholipid FA composition and insulin sensitiv­ity in obese patients on a typical Serbian diet. In 30 patients, two insulin sensitivity groups were formed and their FAs analyzed. The shares of 22:5n-3, 22:6n-3, and total n-3 PUFAs, as well as a measure of Δ5 desaturase activity (the 20:4n-6/20:3n-6 ratio) were lower in the insulin resistance group compared to the normal glucose tolerance group. The fasting insulin level and HOMA value were inversely related to the percentage of 22:5n-3, 22:6n-3, and total n-3 PUFAs. Our data indicate that the percentage of n-3 PUFAs in erythrocyte phospholipids is linked with insulin sensitivity parameters in obese patients. Masnokiselinski sastav membrana, koji se bar delimično može modifikovati dijetarnim unosom masnih kiselina, igra važnu ulogu u senzitivnosti na insulin. Cilj ove studije bio je ispitivanje veze između senzitivnosti na insulin i masnokiselinskog sastava fosfolipida eritrocita kod gojaznih osoba. Trideset umereno gojaznih/gojaznih nedijabetičara sa hiperlipoproteinemijama (ukupni holesterol gt 5.2 mmol/l, LDL-holesterol gt 3.4 mmol/l) podeljeni su u dve grupe, koristeći HOMA vrednost kao parametar senzitivnosti na insulin: grupu sa normalnom tolerancom na glukozu (NTG) i grupu sa rezistencijom na insulin (IR). Ukupni lipidi eritrocita su estrahovani i fosfolipidi izolovani tankoslojnom hromatografijom. Posle metilovanja masne kiseline analizirane su gasno-tečnom hromatografijom. Zastupljenost 22:5n-3 (p lt 0.01), 22:6n-3 (p lt 0.01), ukupnih n-3 polinezasićenih masnih kiselina (p lt 0.001) i odnos zastupljenosti 20:4n-6/20:3n-6 (pokazatelja aktivnosti delta-5 desaturaze) (p lt 0.05) su sniženi u IR grupi u poređenju sa NGT grupom. Nivo insulina natašte i HOMA vrednost inverntno su povezani sa zastupljenošću 22:5n-3 (p lt 0.05), 22:6n-3 (p lt 0.01) i ukupnim n-3 polinezasićenim kiselinama (p lt 0.01). Naši rezultati pokazuju da je nivo n-3 masnih kiselina u fosfolipidima eritrocita povezan sa senzitivnošću na insulin kod gojaznih osoba na ishrani tipičnoj za podneblje Srbije.

ObjectiveThe objective of the present study was to examine the external validity of an FFQ designed to estimate dietary vitamin D intake compared with a plasma biomarker and three repeated 24 h dietary recalls in women of reproductive age in Serbia, where there is no exposure to food fortified with vitamin D. The method of triads was applied.DesignIn a cross-sectional study, 422 women completed the Women and Reproductive Health FFQ (WRH-FFQ) during the winter months. From a representative subgroup (n44), three 24 h dietary recalls and anthropometric parameters were collected as well as a fasting blood sample for vitamin D biomarker analyses. Correlation coefficients were calculated between each of the dietary methods. Validity coefficients, as a correlation between the measured and estimated ‘true’ exposure, were calculated using the method of triads. Bland–Altman plots were also constructed.SettingThree major universities in Serbia.SubjectsHealthy young women (n422) aged 18–35 years.ResultsThe WRH-FFQ estimate of vitamin D intake for all participants was 4·0 (sd3·3) µg/d and 3·1 (sd2·3) µg/d for the subgroup. Bland–Altman plots for these intakes showed high agreement. Validity coefficients for the FFQ, 24 h recall and biomarker wereρQI=0·847 (95 % CI 0·564, 0·928),ρRI=0·810 (95 % CI 0·537, 0·997) andρBI=0·499 (95 % CI 0·190, 0·840), while the correlation coefficients were 0·686, 0·422 and 0·404.ConclusionsThe FFQ applied in the present study is a valid tool for assessing dietary vitamin D intake in women living in Serbia, a region without mandatory vitamin D food fortification.

Pancuronium bromide (PCBr) inhibition effect on enzyme cholinesterase from pooled human serum (Che, EC 3.1.1.8 acylcholine acylhydrolase) was used for development of a spectrophotometric kinetic method for PCBr determination in human serum and urine. Optimal conditions for the basic and inhibitor reactions were established: pH=7.7 and substrate concentration c(benzoylcholine chloride)=1.33 mmol/L. Kinetic parameters were also determined: Michaelis-Menten's constant K(M)=0.40 mmol/L, maximal reaction rate V(max)=52.2 micromol/L min, inhibition constant K(i)=0,56 micromol/L and IC(50)=1.31 micromol/L. Linear dependence between the reaction rate and inhibitor concentration exists in PCBr concentration range 8.20-68.25 nmol/L, which corresponds to the real sample concentrations from 0.328 to 2.730 micromol/L. The method detection and quantification limits were 2.01 nmol/L and 6.67 nmol/L, respectively. Precision of the method was tested for three pancuronium concentrations (10.70, 29.35 and 51.25 nmol/L). Relative standard deviation (RSD) was in the range 0.15-7.45%. Accuracy was examined by standard addition method. Influence of the substances usually present in serum and urine on the reaction rate was tested. The developed method was applied for PCBr content determination in serum model samples, urine model samples and in urine taken during surgery. The method has good sensitivity, accuracy, precision and it is suitable for clinical practice.

Objectives: To have a reliable diagnostic test, the influence of urine pH on the determination of the total activity of N-acetyl-beta-D-glucosarninidase (NAG) and NAG isoenzyme activities was studied. Design and methods: After ultrafiltration and dialysis of the acidic and alkaline urines, the B, A, and A(2) forms of NAG were separated by ion-exchange chromatography on DEAE cellulose. Results: A significant decrease in the total activity of NAG in alkaline urines (pH around 8 or higher) was found, which makes this determination unreliable. Analysis of the isoenzymic profiles obtained for weakly acidic and alkaline urines (in the pH range from 5.5. to 10.0) showed that the percent fractions of the individual isoenzyme activities in the total NAG activity and their ratios changed only at pH values above 9.5. Conclusions: The determination of the denoted isoenzymes of urinary NAG after ultrafiltration, dialysis, and chromatographic separation on DEAE cellulose is reliable in a wide range of alkaline pH values Of urine. (c) 2005 The Canadian Society of Clinical Chemists. All rights reserved.

The influence of urinary pigments and urine pH on the spectrophotometric determination of N‐acetyl‐β‐d‐glucosaminidase (NAG; EC 3.2.1.30) activity with 2‐methoxy‐4‐(2′‐nitrovinyl)‐phenyl‐N‐acetyl‐β‐d‐glucosaminide as a substrate was studied. The investigation was performed with human and rabbit urine samples. It was found that alkaline urine pH values influenced NAG activity in two ways: 1) NAG activity decreased due to enzyme instability with pH increase, and 2) NAG activity increased because of the contribution of urinary pigments to absorbance of 2‐methoxy‐4‐(2′‐nitrovinyl)‐phenol (MNP) at 505 nm. It was shown that besides the maximum (I) in the range of 350–360 nm of the absorption spectra of alkaline urine, there was a maximum (II) in the range of 380–460 nm. With the increase of pH, maximum II was shifted toward higher wavelengths and contributed to MNP absorption (5–90%). On the other hand, the maximum of MNP absorption was shifted toward lower wavelengths (495–400 nm) with increasing pH. Two procedures to eliminate the influence of urinary pigments are presented. The justification of applying a correction to the values of NAG activity in human and rabbit urine (a model system for studying the toxic effects of cadmium) was discussed. J. Clin. Lab. Anal. 19:260–266, 2005. © 2005 Wiley‐Liss, Inc.

In the present study was assessed the influence of occupational exposure to mercury vapours on the basic haematological parameters (erythrocyte, leukocyte and platelet count, haemoglobin concentration, haematocrit, MCV, MCH and MCHC). Studies were carried out on 138 workers involved in the production of chlorine using the mercuric electrolysis method (divided into three groups: permanently, periodically and earlier exposed to mercury vapours), as well as on 38 healthy workers. The shift time - weighted averages for mercury was determined in the workplace air before research; mean value was significantly over maximum tolerated dose. The mercury content in the blood and urine of exposed workers was determined by atomic absorption spectrophotometry. In all three groups 95th percentile values of mercury in blood and urine are significantly over MTD. Peripheral blood cell parameters were determined using an automatic cell counter. In the group exposed to mercury vapours, was found a statistically significant increase of erythrocyte count with a concomitant decrease in MCV. The mean values of haemoglobin concentration, MCHC and platelet count were higher in the group of workers exposed to mercury vapours, but the difference was not statistically significant. There were no significant differences in haematocrit, MCH and leukocytes between the studied groups. Our results indicate that long-term and permanent exposure to mercury vapours induces changes in the important haematological parameters of the peripheral blood.

In order to improve the ability for application of knowledge of chemistry (acquired in the existing educational system) in real life the Model for Consideration of Ecological Issues was developed and applied in high school. The Model consists of the continuous text (“It Happened, What’s the Problem?”) and the test with non-continuous text (“A Guide Through the Problem”), which are prepared for consideration of the problem of eutrophication. All results obtained (average achievement of 70.9±14.3 %) showed that the application of the Model enabled: understanding of an ecological problem based on scientific representations of the term eutrophication given in the continuous text, realization that the pollution of our environment may be directly related to modern life, application of acquired knowledge of chemistry to observe and understand the cause and effect of eutrophication in our environment, to draw a scientific conclusion, and understanding the importance of science and technology discoveries for solving ecological problems. In addition, Model contributed to the development of student’s environmental literacy (ecological knowledge and cognitive skills), ability to think critically, and provided possibilities for classroom knowledge to become applicable in real life. [Projekat Ministarstva nauke Republike Srbije, br. 179048]

An acid phosphatase from an extract of mugwort (Artemisia vulgaris) pollen was purified by a factor of 48 by a combination of ion exchange and gel-chromatography. The molecular weights of the enzyme were 76 kDa and 73 kDa, determined by gel filtration on a Sephadex G-100 sf column and by SDS PAGE(under reducing and non-reducing conditions), respectively. In analytical isoelectrofocusing, the enzyme appears as two very close bands pI at about 4.2. The optimum pH for the enzyme is 5.4. The apparent Km for p-nitrophenyl phosphate was estimated to be 0.16mM. The purified enzyme has broad specificity, and hydrolyses p-nitrophenyl phosphate and α-naphthyl phosphate. Pyrophosphate and O-phospho-L-tyrosine were estimated to be the best substrates for this enzyme as potential in vivo substrates. The enzyme is inhibited competitively by phosphate (Ki = 1.25 mM), molybdate (Ki = 0.055 mM) and pyrophosphate (Ki = 6.7 mM) and non-competitively by fluoride (Ki = 9.8 mM). Metal ions such as Hg2+, Cu2+ and Zn2+ express an inhibitory effect on the enzyme, while the enzyme is slightly activated by non-ionic detergents, Tween 20 and Triton X-100. There is no change in the enzyme activity in the presence of tartrate, citrate, EDTA, 1,10-phenanthroline and sulfhydryl-group modifiers such as p-chloromercuribenzoate and N-ethylmaleimide. Kisela fosfataza ekstrakta polena visokog korova (Artemisia vulgaris) je prečišćena 48 puta kombinacijom jonoizmenjivačke i gel-hromatografije. Molekulska težina enzima je 76 kDa i 73 kDa, određena gel-filtracijom na matriksu Sephadex G-100 sf i SDS PAG elektroforezom (pri redukujućim i neredukujućim uslovima), respektivno. Pri izoelektrofokusiranju, enzim se sastoji iz dve vrlo bliske trake pI vrednosti oko 4,2. Optimalno pH za aktivnost enzima je 5,4. PrividnoKmza hidrolizu p-nitrofenil-fosfata je procenjeno da je 0,16 mM. Prečišćeni enzim ima široku specifičnost hidrolizuje p-nitrofenil-fosfat i α-naftil-fosfat. Pirofosfat i O-fosfo-L-tirozin su procenjeni kao najbolji od potencijalnih in vivo supstrata ovog enzima. Enzim je inhibiran kompetitivno fosfatom (Ki=1,25 mM), molibdatom (Ki=0,055 mM) i pirofosfatom (Ki=6,7 mM) a nekompetitivno fluoridom (Ki= 9,8mM). Joni metala, kao što su Hg2+, Cu2+ i Zn2+ iskazuju inhibitorni efekat na enzim, dok je efekat ne-jonskih detergenata, kao što su Tween 20 i Triton X-100 blago aktivirajuć i. Nema promene u aktivnosti enzima u prisustvu tartarata, citrata, EDTA 1,10-fenantrolina i modifikatora sulfhidrilnih grupa kao što su p-hloromerkuribenzoat i N-etilmaleimid.

Fatty acids (FAs) binding to human serum albumin (HSA) could lead to the changes of Cys-34 thiol group accessibility and reactivity, i.e. its scavenger capacity and antioxidant property. The influence of saturated, mono and poly unsaturated, and fish oil FAs binding to HSA on the carbonylation level and the reactivity of HSA-SH and HSA modified with methylglyoxal (MG-HSA-SH) was investigated. Changes of thiol group reactivity were followed by determination of pseudo first order rate constant (k') for thiols reaction with 5,5'-dithiobis(2-nitrobenzoic acid). HSA changes were monitored using native PAG electrophoresis and fluorescence spectroscopy. For FA/HSA molar ratios screening, qTLC and GC were used. FAs increase thiol group carbonylation levels from 8% to 20%. The k' values obtained for FAs-free HSA-SH and FAs-free MG-HSA-SH are almost equal (7.5 x 10(-3) and 7.7 x 10(-3) resp.). Binding of all FAs amplify the reactivity (k' values from 14.6 x 10(-3) to 26.0 x 10(-3) s(-1)) of HSA-SH group for 2-3.5 times in the order: palmitic, docosahexaenoic, fish oil extract, stearic, oleic, myristic and eicosapentaenoic acid, due to HSA conformational changes. FAs-bound MG-HSA-SH samples follow that pattern, but their k' values (from 9.8 x 10(-3) to 14.3 x 10(-3) s(-1)) were lower compared to unmodified HSA due to additional conformation changes of HSA molecules during carbonylation. Carbonylation level and reactivity of Cys34 thiol group of unmodified and carbonylated HSA depend on type of FAs bound to HSA, which implies the possibility for modulation of -SH reactivity (scavenger capacity and antioxidant property) by FAs as a supplement. (C) 2014 Elsevier Ireland Ltd. All rights reserved.

alpha-Oxoaldehydes, which are produced in higher quantities in diabetes, uremia, oxidative stress, inflammation and aging, react with the amino, guanidine and thiol groups of proteins and cause the formation of advanced glycated end-products and protein cross-linking. To prevent these reactions, the efficiency of tow molecular mass thiols with an alpha-amino-beta-mercapto-ethane group (Cys, penicillamine and N-acetylcysteine (NAcCys, with a blocked amino group)) as scavengers of methylglyoxal, compared with glutathione (GSH) and the biguanidine derivative metformin, was investigated. The time courses of the reactions of the aforementioned compounds with methylglyoxal were assayed. The reactivity of their thiol and amino groups decreased in the order of Cys gt penicillamine gt GSH gt NAcCys and penicillamine gt Cys gt GSH, respectively. Human serum albumin (HSA) carbonylation in the absence or presence of methylglyoxal scavengers were monitored by the determination of the amino, guanidine and thiol groups' contents, as well as by spectrofluorimetry, CD and native and SDS PAGE. Cys and penicillamine were highly efficient in the prevention of the carbonylation of the HSA-amino (for 80%) and guanidine (for 84% and 55%, respectively) groups and the formation of fluorescent AGEs. GSH and metformin exhibited medium efficiency (reduction of amino group's carbonylation for 60% and guanidine for about 30%); the least efficient was NAcCys. The presence of Cys, penicillamine and NAcCys led to an almost complete protection of the HSA-thiol group's carbonylation, whereas metformin was inefficient. The efficiency in the prevention of protein cross-linking increased in the order of metformin, NAcCys lt GSH lt penicillamine lt Cys. Thus, the substances with an alpha-amino-beta-mercapto-ethane group as a pharmacophore exhibit great potential as an efficient methylglyoxal scavengers, and are thus promising compounds for medicinal chemistry. In addition, they protect the HSA-SH group and preserve its antioxidative potential, which is very important for the HSA's function in vivo.

During investigation of the changes of the Cys34 thiol group of human serum albumin (HSA) (isolated by affinity chromatography with Cibacron Blue (CB)) in diabetes, we found that the HSA-SH content was higher (11-33%) than the total serum thiol content. The influence of fatty acids (FA) binding to HSA on this discrepancy was investigated in vitro (using fluorescence and CD spectroscopy and GC) and with HSA samples from diabetic (n=20) and control groups (n=17). HSA-bound FA determine the selection of HSA molecules by CB and enhance reactivity and/or accessibility of the SH group. A high content of polyunsaturated FA (35.6%) leads to weaker binding of HSA molecules to CB. Rate constants of DTNB reaction with the SH group of HSA applied to a CB column, bound-HSA and unbound-HSA fractions, were 4.8 x 10(-3), 21.6 x 10(-3), and 11.2 x 10(-3) s(-1), respectively. The HSA-SH group of diabetics is more reactive compared with control individuals (rate constants 20.9 x 10(-3)+/- 4.4 x 10(-3) vs 12.9 x 10(-3)+/- 2.6 x 10(-3) s(-1), P lt 0.05). Recovery values of the SH group obtained after chromatography of HSA with bound stearic acid ranged from 110 to 140%, while those for defatted HSA were from 98.5 to 101.7%. Thus, HSA-bound FA leads to an increase of HSA-SH content and a contribution to total serum thiols, which make the determination of the thiol group unreliable. (C) 2013 Elsevier Inc. All rights reserved.

The thiol (Cys34) content of human serum albumin (HSA-SH) decreases during oxidative and carbonyl stress and, therefore, could represent a useful parameter in clinical practice. Nevertheless, the reliability of HSA-thiol determination with Ellman's method depends on the purity of isolated HSA. Determination of total serum thiols (mmol/L) and HSA-SH content (mmol -SH/mmol HSA) after HSA isolation from diabetic patient and control sera by a two-step precipitation with ammonium sulfate (AS), as well as HSA-SH contribution (%) to total serum thiols, was assessed. Purity and yield of isolated HSA were monitored spectrophotometrically and by native polyacrylamide gel electrophoresis. Precipitation of HSA from serum via a two-step method with AS produced HSA with 91.9 +/- 3.6% purity and 69.7 +/- 4.4% yield, allowing for precise (relative standard deviation of 3.2%) and reliable (comparing with total serum thiols) measurement of HSA-SH content with DTNB [5,5'-dithiobis-(2-nitrobenzoic acid)]. The content of the HSA-SH group in patients with type 2 diabetes was significantly (P lt 0.05) lower compared with that of the healthy cohort (0.483 +/- 0.067 vs. 0.561 +/- 0.054 mmol -SH/mmol HSA). Because the proposed method of HSA isolation is simple, time-efficient, and technically less demanding, and it also enables reliable determination of HSA-SH content, it is suitable for clinical practice.

The c-Jun NH2-terminal kinases (JNKs) are important stress-responsive kinases. They regulate cellular activities by sequential phosphorylation and activation through a mitogen-activated protein kinase cascade, whereas JNKs activation is altered in response to various stressors. In the present study, we used immunoblotting to assess the effect of 21 day of social isolation as the chronic stressor, either sole and in combination with 2 h of acute immobilization or cold (4A degrees C) stress on circulating corticosterone level and phosphorylation status of p46 (phospho-p46/total p46) and p54 (phospho-p54/total p54) JNK isoforms in the cytosolic and nuclear fraction of the prefrontal cortex and hippocampus of male Wistar rats. Also, the phosphorylation status of JNK nuclear down-stream target c-Jun (p-c-Jun/c-Jun) on Ser63 was examined. Both acute stressors with elevated CORT levels led to increased phosphorylation status of cytosolic p54 JNK isoforms but not p46 JNK isoforms only in the hippocampus and no change in phosphorylation status of c-jun in both brain regions. Chronic isolation with unaltered CORT level and reduced responsiveness to novel acute stressors, led to unchanged or reduced phosphorylation status of p46 and p54 JNK isoforms in both fractions and both brain regions, whereas the decrease of c-Jun phosphorylation status was found only in the prefrontal cortex. Our results suggest that compromised JNKs activation following chronic isolation may lead to interruption of JNK signaling, which could be related with neuropsychiatric disorders such as depression or long-lasting neuronal remodeling.

Objectives: Carbonylation of the protein amino, guanidino and thiol groups inane of the important causes of vascular complications in diabetes. We developed a simple spectrophotometric method for monitoring of the changes in the protein amino group contents during carbonylation. Design and methods: The method is based on the reaction of amino group with p-benzoquinone in the slightly alkaline media. It was applied during carbonylation in vitro with methylglyoxal and in vivo in 13 patients with type 2 diabetes and 20 healthy persons. Results: The method is simple, fast, precise (RSD in the range of 1.2-1.8%) and accurate (recovery about 100%). The content of amino groups in human serum albumin isolated from diabetics was significantly lower (p lt 0.01) in comparison with a control group. Conclusion: The method developed is suitable for quantification of protein amino groups during in vitro carbonylation as well as for clinical practice. (C) 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Methylglyoxal (MG), a reactive alpha-oxoaldehyde that is produced in higher quantities in diabetes, uremia, oxidative stress, aging and inflammation, reacts with the thiol groups (in addition to the amino and guanidino groups) of proteins. This causes protein modification, formation of advanced glycated end products (AGEs) and cross-linking. Low molecular mass thiols can be used as competitive targets for MG, preventing the reactions mentioned above. Therefore, this paper investigated how the microenvironment of the thiol group in low molecular mass thiols (cysteine, N-acetylcysteine (NAcCys), carboxymethylcysteine (CMC) and glutathione (GSH)) and human serum albumin (HSA) affected the thiol reaction with MG. The SH group reaction course was monitored by (1)H-NMR spectroscopy and spectrophotometric quantification. Changes in the HSA molecules were monitored by SDS-PAGE. The microenvironment of the SH group had a major effect on its reactivity and on the product yield. The reactivity of SH groups decreased in the order Cys gt GSH gt NAcCys. CMC did not react. The percentages of the reacted SH groups in the equilibrium state were almost equal, regardless of the ratio of thiol compound/MG (1:1, 1:2, 1:5): 38.1 +/- 0.9%; 38.2 +/- 0.7% and 39.0 +/- 0.8% for Cys; 26.5 +/- 0.6%; 26.6 +/- 2.6% and 27.4 +/- 2.5% for GSH; 10.8 +/- 0.9%; and 11.2 +/- 0.7% and 12.2 +/- 0.9% for NAcCys, respectively. Our results explain why substances containing alpha-amino-beta-mercapto-ethane as a pharmacophore are successful scavengers of MG. In equilibrium, HSA SH reacted in high percentages both with an insufficient amount and with an excess of MG (55% and 65%, respectively). An analysis of the hydrophobicity of the microenvironment of the SH group on the HSA surface showed that it could contribute to high levels of SH modification, leading to an increase in the scavenging activity of the albumin thiol. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Synthesis and anticholinesterase activity of 4-aryl-4-oxo-N-phenyl-2-aminylbutyramides, novel class of reversible, moderately potent cholinesterase inhibitors, are reported. Simple substituent variation on aroyl moiety changes anti-AChE activity for two orders of magnitude; also substitution and type of hetero(ali)cycle in position 2 of butanoic moiety govern AChE/BChE selectivity. The most potent compounds showed mixed-type inhibition, indicating their binding to free enzyme and enzyme-substrate complex. Alignment-independent 3D QSAR study on reported compounds, and compounds having similar potencies obtained from the literature, confirmed that alkyl substitution on aroyl moiety of molecules is requisite for inhibition activity. The presence of hydrophobic moiety at close distance from hydrogen bond acceptor has favorable influence on inhibition potency. Docking studies show that compounds probably bind in the middle of the AChE active site gorge, but are buried deeper inside BChE active site gorge, as a consequence of larger BChE gorge void. (C) 2009 Elsevier Ltd. All rights reserved.

The connection between changes in the activity of serum N-acetyl-beta-D-glucosaminidase (NAG, E.C.3.2.1.30) and iso-enzymes and degree of secondary complications was analyzed in four groups of type 1 diabetic patients (n=69): without complications (n=22); with retinopathy (n=16); with retinopathy and polyneuropathy (n= 13), and with retinopathy, neuropathy, and nephropathy (n=18). In all groups statistically significant higher (P lt 0.001) percent fraction of A form (83.84 +/- 6.09, 84.37 +/- 5.74, 81.76 +/- 6.02, 76.37 +/- 7.38%, resp.) and lower (P lt 0.001, P lt 0.01) fraction of B form (15.87 +/- 5.65, 15.66 +/- 5.74, 18.33 +/- 5.98, 23.63 +/- 7.38, resp.) in total NAG compared with the control (A = 69.38 +/- 4.79%, B = 30.61 +/- 4.78%) were found. The differences in A as well as B forms between diabetic groups were not statistically significant. Significant strong positive correlations between total NAG and glycemia (0.494-0.623), total NAG and A form (0.934-0.966), and A form and glycemia (0.512-0.638) were found in all groups. No correlation was found between the fractions of B and A forms, except in the fourth group. The A form of diabetic patients in the fourth group was more acidic compared with the control and other diabetic groups. It was concluded that the changes in serum NAG and isoenzymic profiles in diabetes are the consequence of its increased exocytose, especially of the A form, in hyperglycemia and posttranslational modifications of iso-enzymes. The total activity of serum NAG and iso-enzymic profiles cannot be used for monitoring the development and distinction of type 1 diabetes secondary complications.

The kinetic spectrophotometric method for Au(III) determination was developed and validated. It was based on the catalytic effect of gold on the oxidation of methylene blue B (3,7di-(dimethyl amino)-10-dehydro-phenotiazin chloride) by ammonium peroxo-disulfate in citric buffer solution. There was the linearity of the calibration curve in the concentration range from 0.09 to 2.90 mu g ml(-1) Au(III). The relative standard deviation was 2.50% and correlation coefficient of 0.9999. The limit of detection was determined as signal to noise ratio (3: 1) and it was 5.5 ng ml(-1). The limit of quantification, based on signal to noise ratio 10: 1 was 19.25 ng ml(-1). The selectivity was tested on the basis of influence of known amounts of different ions in the reaction mixture, upon the reaction rate. Kinetic and thermodynamic parameters were reported for both catalytic and non-catalytic reactions. The method was verified by Au(III) determination in anti-rheumatic drug "Thuredon" and in human urine samples, using ICP-AES as the comparative method. As the method is accurate, reliable, quick and simple it could be useful for clinical and toxicological practice. (c) 2004 Elsevier B.V. All rights reserved.

The iso-enzyme profiles of urinary N-acetyl-b-D-glucosoaminidase (NAG) were studied in the workers from a chloro-alkaline electrolysis plant that had been continuously exposed to elemental mercury. The same workers then had a work break of four months and were later re-exposed to mercury for another five months. The activities of urinary NAG in workers exposed to mercury before and after the work break and in the control group were 2.099 / 1.03 IU/L; 0.909 / 0.52 IU/L and 1.139 / 0.35 IU/L, respectively. The fraction of the dominant A form activity in the total activity, in the workers exposed to mercury after a work break (85.319 / 5.32%), was almost equal to the fraction of A in the control group (84.649 / 2.75%). The percent fraction of A forms (A and A2) during continuous exposure was almost equal to the sum of the fractions A and A2 in the control group and for exposure after the work break. The fraction of the B form activity in the total activity during exposure to elemental mercury for five months after the work break (7.419 / 3.45%) was lower (P B / 0.1) than the fraction of the B form in the control group (8.629 / 2.19%). The decrease in the fraction of the B form compared with the control group was more significant (P B / 0.001) in the case of continuously exposed workers (5.259 / 2.55%). Beside the major A and B iso-enzymes of NAG, the A2form was also isolated, and its fraction in the control group (6.739 / 2.15%) was not negligible. For workers exposed to mercury for five months, it was 7.289 / 3.52%. It is concluded that mercury affected the increased exocytosis of iso-enzyme A and the inhibition of B iso-enzyme of NAG. The increase in activity of urinary NAG in subjects exposed to mercury was a consequence of the increased excretion of A form. It was established also that the four months work break was sufficient for the repair of renal damages that occurred during continuous exposure, and which were monitored by the activity of NAG. Thus, the effects of mercury seem to have been reversible.

A rapid and sensitive HPLC-RP method for simultaneous determination of tiazofurin, its 5'-O acetyl and benzoyl esters and their active metabolite thiazole-4-carboxamide adenine dinucleotide was developed and validated. The method allowed determination and quantification of nanomolar quantities of these substances in cell extracts of treated cells, and was also used in kinetic studies of cellular uptake of tiazofurin and its esters from the cultivation medium. Separation of the analyzed substances from unidentified peaks from both biological materials was achieved by gradient elution, thus reducing the possibility of interference. The mobile phase consisted of a 0.1 M sodium-hydrogen phosphate, pH 5.1 and methanol. Run time was 22 min, with 5 min equilibration time. (C) 2002 Elsevier Science B.V. All rights reserved.

The activity of the isoenzymes of N-acetyl-beta-D-glucosaminidase (NAG, EC 3.2.1.30) is determined in the serum of insulin-dependent (IDDM) and non-insulin-dependent diabetics (NIDDM) with or without diabetic complications. The increase in total activity of serum NAG in diabetics is proportional to the A form activity (r = 0.976, p < 0.0001). The contribution of the A form activity (65.87 +/- 5.99%) to total NAG activity of IDDM and NIDDM diabetics with and without complications does not change considerably compared to the control group. The contribution of the B form activity depends on the state of metabolic monitoring and diabetic complications. A significantly lower activity of the serum B form was found in IDDM (p < 0.001) and NIDDM diabetics (p < 0.05) compared to the healthy individuals, as well as the higher activity ratios of the A/B forms. A decrease in serum B form is correlated with the occurrence and abundance of the intermediary I forms (r = 0.665). These changes are particularly significant in the individuals with the pronounced microangiopathy.