The pa raoxo na se 1, 2 and 3 in hu ma ns Ma ri ja Gr dic Raj ko vic * , La da Ru mo ra, Kar me la Ba ri sic De par tme nt of Me di cal Bioc he mis try and He ma to lo gy, Fa cul ty of Phar ma cy and Bioc he mis try, Uni ver si ty of Zag reb, Zag reb, Croa tia *Cor res pon di ng aut hor: m_gr dic @ya hoo.com Ab stra ctThe pa raoxo na se ge ne fa mi ly in hu ma ns in clu des three mem be rs: PON1, PO N2 and PO N3. The pro duc ts of tho se three ge nes are the fol lowi ng enzymes: pa raoxo na se 1 (PO N1), pa raoxo na se 2 (PO N2) and paraoxo na se 3 (PO N3). PO N1 is main ly as so cia ted wi th a hi gh den si ty li pop ro tein (HDL). A sma ll amou nt of this en zyme is al so bou nd to ve ry low-den si ty li pop ro tein (VLDL) and pos tpran dial chylo mic ro ns. PO N1 pos se ss or ga nop hos pha tase, aryles te ra se and lac to na se ac ti vi ty and it hydro lyzes ma ny diff e re nt sub stra tes. It is al so known that PO N1 may ha ve an tiat he ro ge nic fun ction. Com pa red to the PO N1, PO N2 and PO N3 are mu ch le ss stu died and des cri bed. PO N2 is ubiqui tous ly expres sed in tra cel lu lar pro tein, whi le PO N3 is bou nd to HDL, li ke PO N1. The bo th en zymes pos se ss an tioxi da nt pro per ties.

Extracellular Hsp70 (eHsp70) can act as damage-associated molecular pattern (DAMP) via Toll-like receptors TLR2 and TLR4, and stimulate immune and inflammatory responses leading to sterile inflammation and propagation of already existing inflammation. It was found elevated in the blood of patients with chronic obstructive pulmonary disease (COPD), who might suffer occasional bacterial colonizations and infections. We used a monocytic THP-1 cell line as a cellular model of systemic compartment of COPD to assess inflammatory effects of eHsp70 when present alone or together with bacterial products lypopolysaccharide (LPS) and lypoteichoic acid (LTA). THP-1 cells were differentiated into macrophage-like cells and treated with various concentrations of recombinant human Hsp70 protein (rhHsp70), LPS (TLR4 agonist), LTA (TLR2 agonist), and their combinations for 4, 12, 24, and 48 h. Concentrations of IL-1α, IL-6, IL-8, and TNF-α were determined by ELISA. Cell viability was assessed by MTS assay, and mode of cell death by luminometric measurements of caspases-3/7, -8, and -9 activities. rhHsp70 showed cell protecting effect by suppressing caspases-3/7 activation, while LPS provoked cytotoxicity through caspases-8 and -3/7 pathway. Regarding inflammatory processes, rhHsp70 alone induced secretion of IL-1α and IL-8, but had modulatory effects on release of all four cytokines when applied together with LPS or LTA. Combined effect with LPS was mainly synergistic, and with LTA mainly antagonistic, although it was cytokine- and time-dependent. Our results confirmed pro-inflammatory function of extracellular Hsp70, and suggest its possible implication in COPD exacerbations caused by bacterial infection through desensitization or inappropriate activation of TLR2 and TLR4 receptors.

Extracellular heat shock protein 70 (eHsp70) might modulate immune responses in chronic obstructive pulmonary disease (COPD). The aim of the study was to explore eHsp70 concentration in stable COPD, its association with disease severity and smoking status as well as its diagnostic performance in COPD assessment. Plasma samples were collected from 137 COPD patients and 95 healthy individuals, and concentration of eHsp70 was assessed by commercially available enzyme-linked immunosorbent assay (ELISA) kit (Enzo Life Science, Farmingdale, NY, USA). COPD patients were subdivided regarding airflow obstruction severity and symptoms severity according to the Global Initiative for COPD (GOLD) guidelines. eHsp70 concentration increased in COPD patients when compared to controls and increased with the severity of airflow limitation as well as symptoms burden and exacerbation history. eHsp70 concentration did not differ among COPD patients based on smoking status, yet it increased in healthy smokers compared to healthy nonsmokers. In addition, eHsp70 negatively correlated with lung function parameters forced expiratory volume in one second (FEV1) and FEV1/ forced vital capacity (FVC), and positively with COPD multicomponent indices BODCAT (BMI, airflow obstruction, dyspnea, CAT score), BODEx (BMI, airflow obstruction, dyspnea, previous exacerbations), CODEx (Charlson’s comorbidity index, airflow obstruction, dyspnea, previous exacerbations) and DOSE (dyspnea, airflow obstruction, smoking status, previous exacerbations) With great predictive value (OR = 7.63) obtained from univariate logistic regression, eHsp70 correctly classified 76% of cases. eHsp70 is associated with COPD prediction and disease severity and might have the potential for becoming an additional biomarker in COPD assessment.

Potentiometric analysis of fluoride content (as F- ion) in solutions by using fluoride ion-selective electrode is simple, reliable and cheap. Very small concentrations of fluoride-ions (to 10-6 mol/dm3) can be determined by fluoride selective electrode, with regulation of ion strength of a solution and control of concentration of hydroxide ions and interfering ions of metals. The influence of pH and complexing ions of metals can be successfully regulated by the TISAB solution and by preserving pH value in the range from 5.00 to 7.00. The content of fluorides in the samples can be determined by the method of direct potentiometer, and in the case of very low concentration by standard addition method. In this paper it was analyzed the determination of fluoride ions concentration in bottled mineral waters and water from Belgrade plumbing in two Belgrade districts (Palilula and Novi Beograd) and in tea, by using the fluoride selective electrode. It was determined that the content of fluoride ions in bottled mineral water significantly differs from values given on declaration, and that content of fluoride ions varies over a period of time. The content of fluoride ions in water from plumbing in two Belgrade districts at the time of analysis was significantly increased and exceeded values given in Regulation for drinking water quality. The received results from the analysis of fluorides in teas show that fluorides exist in teas in different concentrations. There are also differences between the same kinds of tea, which is noted with mint (Mentha piperitae folium), as a consequence of differences between soils where it was planted. As taking of fluorides, according to World Health Organisation recommendation (WHO), is limited in the range from 2 to 4 mg per day, it is necessary to give the content of fluorides on all products that are used in human consumption

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