Hyaluronan degradation by copper(II) chloride and ascorbate: rotational viscometric, EPR spin-trapping, and MALDI-TOF mass spectrometric investigations

The degradation of high-molar-mass hyaluronan (HA) by copper(II) chloride and ascorbate was studied by means of rotational viscometry. It was found that even small amounts of CuCl(2) present in the oxidative system led to the pronounced degradation of HA, reflected in a rapid decrease of the dynamic viscosity of the biopolymer solution. Such degradation was induced by free radicals generated in elevated amounts in the presence of copper ions. Electron paramagnetic resonance investigations performed on a model oxidative system containing Cu(II) and ascorbic acid proved the formation of relatively stable ascorbate anion radicals resulting from the reaction of ascorbic acid with hydroxyl radicals. In this way, by scavenging the hydroxyl radicals, ascorbic acid protected HA from their degradative action. Matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry was applied to analyze the degraded HA. The results showed that only regular fragmentation of hyaluronan occurred using the mentioned oxidative system that led to the formation of HA oligomers with unaffected primary chemical structure.     

Late Glacial and Holocene history of Plešné Lake and its surrounding landscape based on pollen and palaeoalgological analyses

Pollen analysis has been carried out on a 549 cm thick sediment profile from lake Ple?né jezero (Ple?né Lake) in the Bohemian Forest (?umava, Czech Republic; 1090 m a.s.l.; 48°47′ N; 13°52′ E). Analyses of 67 samples characterise the development of the lake biotope and the surrounding landscape during the last ca. 14,000 years. The pollen diagram shows a very distinct transition between the Late Glacial and the Holocene biostratigraphic units at a depth of ca. 312 cm. In the surroundings of Ple?né Lake the vegetation was treeless during the entire Late Glacial. The alpine tree limit, formed by Betula and Pinus with undergrowth of shrubs, might have been at ca. 500 m a.s.l. Pollen transported from long distances was significant due to the openness of the landscape, coming from southern Europe and even Africa, and including high numbers of Artemisia, Poaceae, Chenopodiaceae, and some other herbs and shrubs from steppe and forest-steppe areas in southern Europe or Africa (likely Ephedra, certainly Lygeum spartum). The expansion of shrubs, particularly Juniperus, preceded the expansion of trees near the end of the Late Glacial. Afforestation of the region by thin stands of Betula and Pinus occurred during the Preboreal. Significant warming in the Boreal resulted in the expansion of Corylus, Quercetum mixtum (QM) trees, and probably also Picea and Alnus. Picea as well as QM trees were further expanding during the Early Atlantic. Picea was the dominant tree during the Late Atlantic and Fagus started to spread towards its end. Abrupt expansion of Abies marks the Subboreal. A high degree of afforestation (Abies, Fagus, Picea) was characteristic for the Early Subatlantic. During Late Subatlantic, pollen of synanthropic plants appears. Phases of the lake biotope development were defined on the basis of coccal green algae and Isoëtes.     

Proton magnetic resonance spectroscopy in patients with solitary and sporadic temporal lobe epileptic seizures

Proton magnetic resonance spectroscopy (¹H MRS) is an optional diagnostic method for potential epilepsy surgery candidates. The aim of this study was to determine the credibility of ¹H MRS examination in a group of patients suffering from solitary and sporadic epileptic seizures generated in temporal lobe. We recorded a 100% sensitivity of ¹H MRS in a group of ten patients in terms of detection of a pathological process in the temporal lobe. ¹H MRS also enabled determination of lateralization of the pathological process in three patients with bilateral epileptiform abnormalities on electroencephalography. Based on these results we suggest new perspectives on ¹H MRS as a part of standard diagnostic algorithm for solitary and sporadic temporal lobe epileptic seizures, particularly in cases with normal electroencephalography and magnetic resonance imaging findings.     

Preparation and in vitro screening of symmetrical bispyridinium cholinesterase inhibitors bearing different connecting linkage—initial study for Myasthenia gravis implications

Reversible inhibitors (e.g., pyridostigmine bromide, neostigmine bromide) of carbamate origin are used in the early treatment of Myasthenia gravis (MG) to block acetylcholinesterase (AChE) native function and conserve efficient amount of acetylcholine for decreasing number of nicotinic receptors. Carbamate inhibitors are known for many undesirable side effects related to the reversible inhibition of AChE. In contrast, this paper describes 20 newly prepared bispyridinium inhibitors of potential concern for MG. Although some compounds from this series have been known before, they were not assayed for cholinesterase inhibition yet.The newly prepared compounds were evaluated in vitro on human erythrocyte AChE and human plasmatic butyrylcholinesterase (BChE). Their inhibitory ability was expressed as IC₅₀ and compared to standard carbamate drugs. Three compounds presented promising inhibition (in μM range) of both enzymes in vitro similar to the used standards. The novel inhibitors did not present selectivity between AChE and BChE. Two newly prepared compounds were chosen for docking studies and confirmed apparent π–π or π–cationic interactions aside enzyme’s catalytic sites. The kinetics assay confirmed non-competitive inhibition of AChE by two best newly prepared compounds.     

Ion chromatography of azide in pharmaceutical protein samples with high chloride concentration using suppressed conductivity detection

Methods based on reversed-phase liquid chromatography with UV detection of 4-nitrobenzoyl- or 3,5-dinitrobenzoyl azide derivatives lack in accuracy and stability of derivatives to be applied for azide determination in pharmaceutical protein samples with high sodium chloride concentrations. This paper describes a sensitive and selective ion chromatographic method, with simple sample preparation and suppressed conductivity detection, developed for trace determination of azide in protein samples containing sodium chloride in concentrations as high as 11.6 g L(-1). Anion exchange stationary phase with quaternary alkyl amine functional groups and gradient elution with sodium hydroxide enabled good resolution of anions with similar retention times: azide, bromide and nitrate, as well as chloride whose retention time was shorter than azide's. Anions with high affinity to stationary phase (phosphate and citrate) were also eluted within acceptable analysis time of 32 min. The stability of sample solutions and the method selectivity, accuracy, precision and sensitivity satisfied the validation criteria of international organizations competent for pharmaceutical industry. The detection and quantitation limit ranges of sodium azide in protein samples were 0.007-0.02 mg L(-1) and 0.02-0.06 mg L(-1), respectively. Both limits increased with the concentration of sodium chloride.     

Frequency of ventricular premature beats and ventricular tachycardia in STEMI treated with fibrinolytics

To determine in acute myocardial infarction with an ST elevation (STEMI) treated with fibrinolytics frequency of ventricular premature beats (VPBs) and ventricular tachycardia (VT) according to the damaged area and residual cardiac function. With anterolateral infarction with ejection fraction (EF) < 45%, incidence of VPBs < 10/h was statistically significantly reduced (p < 0.001) while incidence of VPBs > or = 10/h as well as VPBs in a pair and VT was increased (p < 0.001). With anteroseptal infarction with EF < 45%, incidence of VPBs < 10/h was statistically reduced (p = 0.06) and incidence of VPBs > 10/h, VPBs in a pair and VT was increased (p = 0.06). With inferior and inferoposterior infarction with EF < 45%, incidence of VPBs < 10/h was reduced and incidence of VPBs > or = 10/h, VPBs in a pair and VT was increased. However, such difference was not statistically significant. Along with reduced residual cardiac function, one can also expect increase in frequency of VPBs and VT in all forms of STEMI regardless the area of damage. Such frequency is significant with all forms of anterior infarction, that is to say, slightly more with anterolateral infarction in relation to anteroseptal one. However, with inferior and inferoposterior infarction this frequency of VPBs i VT is not significant.     

Seasonal variability of oxidative stress markers in city bus drivers. Part I. Oxidative damage to DNA

We investigated the seasonal variability of 8-oxodeoxyguanosine (8-oxodG), a marker of oxidative damage to DNA, in urine of 50 bus drivers and 50 controls in Prague, Czech Republic, in three seasons with different levels of air pollution: winter 2005, summer 2006 and winter 2006. The exposure to environmental pollutants (carcinogenic polycyclic aromatic hydrocarbons, c-PAHs, particulate matter (PM), and volatile organic compounds (VOC)) was monitored by personal and/or stationary monitors. For the analysis of 8-oxodG levels, the ELISA technique was used. Bus drivers were exposed to significantly higher levels of c-PAHs in winter 2006, while in the other two seasons the exposure of controls was unexpectedly higher than that of bus drivers. We did not see any difference in VOC exposure between both groups in summer 2006 and in winter 2006; VOC were not monitored in winter 2005. 8-OxodG levels were higher in bus drivers than in controls in all seasons. The median levels of 8-oxodG (nmol/mmol creatinine) in bus drivers vs. controls were as follows: winter 2005: 7.79 vs. 6.12 (p=0.01); summer 2006: 6.91 vs. 5.11 (p<0.01); winter 2006: 5.73 vs. 3.94 (p<0.001). Multivariate logistic regression analysis identified PM2.5 and PM10 levels, measured by stationary monitors during a 3-day period before urine collection, as the only factors significantly affecting 8-oxodG levels, while the levels of c-PAHs had no significant influence.     

Initiation factor eIF2B not p70 S6 kinase is involved in the activation of the PI-3K signalling pathway induced by the v-src oncogene

Our data show that in hamster fibroblasts transformed by Rous sarcoma virus (RSV), the phosphoinositide 3'-kinase (PI-3K)/Akt/glycogen synthase kinase 3 antiapoptotic pathway is upregulated and involved in increased protein synthesis through activation of initiation factor eIF2B. Upon inhibition of PI-3K by wortmannin, phosphorylation of 70-kDa ribosomal protein S6 kinase (p70 S6k) and its physiological substrate, ribosomal protein S6, decreased in the non-transformed cells but not in RSV-transformed cells. Thus PI-3K, which is thought to be involved in regulation of p70 S6k, signals to p70 S6k in normal fibroblasts, but it does not appear to be an upstream effector of p70 S6k in fibroblasts transformed by v-src oncogene, suggesting that changes in the PI-3K signalling pathway upstream of p70 S6k are induced by RSV transformation.     

Effect of salt stress on the production and properties of extracellular polysaccharides produced by Cryptococcus laurentii

The composition, main structural features and molecular properties of exopolysaccharides (EP) produced by Cryptococcus laurentii var. laurentii CCY 17-3-16 under optimal (EPo) and NaCI-stress conditions (EPs) as well as their subfractions isolated by gel chromatography were studied using chemical, FT-IR and NMR spectroscopy methods. The results showed that under stress conditions the yeast produced EP with a lower content of protein and phosphorus. In comparison to EPo, the EPs exhibited a substantially larger proportion of high molecular mass populations. NMR analysis of EPs revealed a higher degree of branching with single xylose side chains of the heteromannan components. The increase of the molecular mass and degree of branching of the macromolecular chains of the heteromannan components might in part be related to the function of EPs to protect the yeast cells from water loss and maintain growth conditions under the salt stress.