Diverse chemotactic responses of Dictyostelium discoideum amoebae in the developing (temporal) and stationary (spatial) concentration gradients of folic acid,cAMP, Ca(2+) and Mg(2+)

The responses of Dictyostelium discoideum amoebae to developing (temporal) and stationary (spatial) gradients of folic acid, cAMP, Ca(2+), and Mg(2+) were studied using the methods of computer-aided image analysis. The results presented demonstrate that the new type of experimental chambers used for the observation of single cells moving within the investigated gradients of chemoattractants permit time lapse recording of single amoebae and determination of the trajectories of moving cells. It was found that, besides folic acid and cAMP (natural chemoattractants for Dictyostelium discoideum amoebae), also extracellular Ca(2+) and Mg(2+) are potent inducers of these cells' chemotaxis, and the amoebae of D. discoideum can respond to various chemoattractants differently. In the positively developing gradients of folic acid, cAMP, Ca(2+), and Mg(2+) oriented locomotion of amoebae directed towards the higher concentration of the tested chemoattractants was observed. However, in the negatively developing (temporal) and stationary linear (spatial) gradients, the univocal chemotaxis of amoebae was recorded only in the case of the Mg(2+) concentration gradient. This demonstrates that amoebae can respond to both developing and stationary gradients, depending upon the nature of the chemoattractant. We also investigated the effects of chosen inhibitors of signalling pathways upon chemotaxis of D. discoideum amoebae in the positively developing (temporal) gradients of tested chemoattractants. Verapamil was found to abolish the chemotaxis of amoebae only in the Ca(2+) gradients. Pertussis toxin suppressed the chemotactic response of cells in the gradients of folic acid and cAMP but did not prevent chemotaxis in those of Ca(2+) and Mg(2+), while quinacrine inhibited chemotaxis in the gradients of folic acid, cAMP, and Ca(2+) but only slightly affected chemotaxis in the Mg(2+) gradient. None of the tested inhibitors causes inhibition of cell random movement, when applied in isotropic solution. Also EDTA and EGTA up to 50 mM concentration did not inhibit locomotion of amoebae in control isotropic solutions.     

Capacitance and resistance of the bilayer lipid membrane formed of phosphatidylcholine and cholesterol

Capacity and electric resistance of lipid membranes composed of lecithin and cholesterol were determined. The components were chosen for the study because they were present in biological membranes. Capacitance of the lecithin and cholesterol membranes amounts to 0.38 and 0.61 microF/cm(2), and resistance to 1.44(10(4)and 2.12(10(6)Omega cm(2), respectively. A 1:1 complex appears as a result of lecithin-cholesterol membrane formation. Parameters of the membrane formed of the lecithin-cholesterol complex were determined: surface concentration (Gamma(3)), capacitance (C(3)), and conductance (R;(3)(-1), as well as the stability constant (K) of the complex. The mean values of those magnitudes are as follows: 4.265(10(-6)mol/m(2), 0.54 microF/cm(2), 1.381(10(-6)Omega(-1)cm(-2)and 3.748(10(7), respectively.     

The DCX-domain tandems of doublecortin and doublecortin-like kinase

The doublecortin-like domains (DCX), which typically occur in tandem, are novel microtubule-binding modules. DCX tandems are found in doublecortin, a 360-residue protein expressed in migrating neurons; the doublecortin-like kinase (DCLK); the product of the RP1 gene that is responsible for a form of inherited blindness; and several other proteins. Mutations in the gene encoding doublecortin cause lissencephaly in males and the 'double-cortex syndrome' in females. We here report a solution structure of the N-terminal DCX domain of human doublecortin and a 1.5 A resolution crystal structure of the equivalent domain from human DCLK. Both show a stable, ubiquitin-like tertiary fold with distinct structural similarities to GTPase-binding domains. We also show that the C-terminal DCX domains of both proteins are only partially folded. In functional assays, the N-terminal DCX domain of doublecortin binds only to assembled microtubules, whereas the C-terminal domain binds to both microtubules and unpolymerized tubulin.     

Acid-base equilibria at interface separating electrolyte solution and lipid bilayer formed from phosphatidylcholine

The dependence of the interfacial tension of a lipid membrane formed from phosphatidylcholine on the pH of the aqueous solution has been studied. The model describing the H(+) and OH(-) ions adsorption in the bilayer lipid surface has been presented in this work. We take suitable equations to describe the dependence of interfacial tension of a lipid bilayer membrane on H(+) and OH(-) ion concentrations. A theoretical equation is derived to describe this dependence in the whole pH range.     

Chiral aspects of metabolism of antiinflammatory drug flobufen in human hepatocytes

The metabolism of the nonsteroidal antiinflammatory drug flobufen, 4-(2',4'-difluorobiphenyl-4-yl)-2-methyl-4-oxobutanoic acid, was studied in primary cultures of human hepatocytes prepared by two-step collagenase perfusion of livers from four donors. Racemic flobufen or its individual enantiomers, R-(+)- and S-(-)-flobufen were used as substrates. Aliquots of culture medium were collected during 24-h incubation. The time-dependent disappearance of flobufen enantiomers and the formation of metabolites (stereoisomers of dihydroflobufen (DHF)) in hepatocytes were measured by chiral HPLC. The reduction of flobufen in human hepatocytes was stereoselective ((+)-R-flobufen was preferentially metabolized) and stereospecific ((2R;4S)-DHF and (2S;4S)-DHF stereoisomers were mostly formed). Although the structure of flobufen is different from the profens (2-arylpropionates), flobufen undergoes chiral inversion in human hepatocytes. The inversion of R-(+)-flobufen to S-(-)-flobufen predominates. The individual DHF stereoisomers were incubated in hepatocyte cultures and their biotransformation studied. The unidirectional chiral inversion of (2S;4S)-DHF to (2R;4S)-DHF and (2R;4R)-DHF to (2S;4R)-DHF was observed. Stereoselective oxidation of the DHFs to flobufen was also detected. Thus, flobufen metabolism in primary cultures of human hepatocytes is much more complicated (via chiral inversion and DHF re-oxidation) than was presumed from a preliminary achiral point of view.     

Conformational Studies of PACAP(1–27) and Its Fragments

Summary Conformational features of the neuropeptide pituitary adenylate cyclase activating polypeptide (1–27) (PACAP(1–27)) and its shorter fragments (1–5), (7–11) and (14–27) were studied by circular dichroism (CD) and fluorescence spectroscopy. The obtained CD spectra revealed that only PACAP(1–27) and the fragment (14–27) possess some content of an organized structure — the α-helix. This C-terminal, helical part of the peptides is important for receptor binding as it provides a stable structure that can reside in the ordered lipid region of the receptor site in the membrane, while the primary biological function of the hormone resides in the N-terminal, disordered part. Fluorescence studies have revealed that the tyrosine residue located in the helical region of PACAP has a higher quantum yield and a longer average lifetime than the tyrosine in the N-terminus, probably due to a ‘shielding’ effect of the hydrophobic cluster around Tyr²².     

Compensatory changes in Photosystem II electron turnover rates protect photosynthesis from photoinhibition

Exposure of algae or higher plants to bright light can result in a photoinhibitory reduction in the number of functional PS II reaction centers (n) and a consequential decrease in the maximum quantum yield of photosynthesis. However, we found that light-saturated photosynthetic rates (P_max) in natural phytoplankton assemblages sampled from the south Pacific ocean were not reduced despite photoinhibitory decreases in n of up to 52%. This striking insensitivity of P_max to photoinhibition resulted from reciprocal increases in electron turnover ( )through the remaining functional PS II centers. Similar insensitivity of P_max was also observed in low light adapted cultures of Thalassiosira weissflogii (a marine diatom), but not in high light adapted cells where P_max decreased in proportion to n. This differential sensitivity to decreases in n occurred because was close to the maximum achievable rate in the high light adapted cells, whereas was initially low in the low light adapted cells and could thus increase in response to decreases in n. Our results indicate that decreases in plant productivity are not necessarily commensurate with photoinhibition, but rather will only occur if decreases in n are sufficient to maximize or incident irradiance becomes subsaturating.     

Differences in Energy Balance-Related Behaviours in European Preschool Children: The ToyBox-Study

Background

The aim of the current study was to compare levels of energy balance-related behaviours (physical activity, sedentary behaviour, and dietary behaviours (more specifically water consumption, sugar-sweetened beverage consumption and unhealthy snacking)) in four- to six-year-old preschoolers from six European countries (Belgium, Bulgaria, Germany, Greece, Poland, and Spain) within the ToyBox cross-sectional study.

Methods

A sample of 4,045 preschoolers (4.77 ± 0.43 years; 52.2% boys) had valid physical activity data (steps per day), parents of 8,117 preschoolers (4.78 ± 0.46 years; 53.0% boys) completed a parental questionnaire with questions on sedentary behaviours (television viewing, computer use, and quiet play), and parents of 7,244 preschoolers (4.77 ± 0.44 years; 52.0% boys) completed a food frequency questionnaire with questions on water consumption, sugar-sweetened beverage consumption and unhealthy snacking.

Results

The highest levels of physical activity were found in Spain (12,669 steps/day on weekdays), while the lowest levels were found in Bulgaria and Greece (9,777 and 9,656 steps/day on weekdays, respectively). German preschoolers spent the least amount of time in television viewing (43.3 min/day on weekdays), while Greek preschoolers spent the most time in television viewing (88.5 min/day on weekdays). A considerable amount of time was spent in quiet play in all countries, with the highest levels in Poland (104.9 min/day on weekdays), and the lowest levels in Spain (60.4 min/day on weekdays). Belgian, German, and Polish preschoolers had the lowest intakes of water and the highest intakes of sugar-sweetened beverages. The intake of snacks was the highest in Belgian preschoolers (73.1 g/day) and the lowest in Greek preschoolers (53.3 g/day).

Conclusions

Across six European countries, differences in preschoolers’ energy balance-related behaviours were found. Future interventions should target European preschoolers’ energy balance-related behaviours simultaneously, but should apply country-specific adaptations.     

TLR9 2848 GA Heterozygotic Status Possibly Predisposes Fetuses and Newborns to Congenital Infection with Human Cytomegalovirus

Background

Some single nucleotide polymorphisms (SNP), located in Toll-like receptor (TLR) genes, were reported to be associated with human cytomegalovirus (HCMV) infections. The study was aimed to assess the correlation of SNPs at TLR4 and TLR9 genes with the occurrence of congenital cytomegaly, based on available samples.

Methods

Reported case-control study included both HCMV infected and non-infected fetuses and newborns. The specimens were classified to the molecular analyses, based on serological features of the recent infection and HCMV DNAemia in body fluids. TLR SNPs were studied, using multiplex nested PCR-RFLP assay, and determined genotypes were confirmed by sequencing. Hardy-Weinberg equilibrium was assessed for the identified genotypes. The linkage disequilibrium was also estimated for TLR4 SNPs. A relationship between the status of TLR genotypes and congenital cytomegaly development was estimated, using a logistic regression model.

Results

Hardy Weinberg equilibrium was observed for almost all SNPs, both infected and non-infected patients, with exception of TLR4 896 A>G polymorphism in the control group (P≤0.050). TLR4 896 A>G and 1196 C>T SNPs were found in linkage disequilibrium in both study groups (P≤0.050). The CC genotype at TLR4 1196 SNP and the GA variant at TLR9 2848 G>A SNP were significantly associated with HCMV infection (P≤0.050). The risk of congenital cytomegaly was higher in heterozygotes at TLR9 SNP than in the carriers of other genotypic variants at the reported locus (OR 4.81; P≤0.050). The GC haplotype at TLR4 SNPs and GCA variants at TLR4 and TLR9 SNPs were significantly associated with HCMV infection (P≤0.0001). The ACA variants were more frequent among fetuses and neonates with symptomatic, rather than asymptomatic cytomegaly (P≤0.0001).

Conclusions

TLR4 and TLR9 polymorphisms may contribute to the development of congenital infection with HCMV in fetuses and neonates. The TLR9 2848 GA heterozygotic status possibly predisposes to HCMV infection, increasing the risk of congenital cytomegaly development.     

Changes in Non-Enzymatic Antioxidants in the Blood Following Anaerobic Exercise in Men and Women

Purpose

The aim of this study was to compare changes in total oxidative status (TOS), total antioxidative capacity (TAC) and the concentration of VitA, VitE, VitC, uric acid (UA), reduced (GSH) and oxidized glutathione (GSSG) in blood within 24 hours following anaerobic exercise (AnEx) among men and women.

Methods

10 women and 10 men performed a 20-second bicycle sprint (AnEx). Concentrations of oxidative stress indicators were measured before AnEx and 3, 15 and 30 minutes and 1 hour afterwards. UA, GSH and GSSH were also measured 24 hours after AnEx. Lactate and H⁺ concentrations were measured before and 3 minutes after AnEx.

Results

The increase in lactate and H⁺ concentrations following AnEx was similar in both sexes. Changes in the concentrations of all oxidative stress indicators were significant and did not differ between men and women. In both sexes, TOS, TAC, TOS/TAC and VitA and VitE concentrations were the highest 3 minutes, VitC concentration was the highest 30 minutes, and UA concentration was the highest 1 hour after AnEx. GSH concentration was significantly lower than the initial concentration from 15 minutes to 24 hour after AnEx. GSSG concentration was significantly higher, while the GSH/GSSG ratio was significantly lower than the initial values 1 hour and 24 hour after AnEx.

Conclusions

With similar changes in lactate and H⁺ concentrations, AnEx induces the same changes in TAC, TOS, TOS/TAC and non-enzymatic antioxidants of low molecular weight in men and women. Oxidative stress lasted at least 24 hours after AnEx.