Structural analysis of the alpha-D-glucan (EPS35-5) produced by the Lactobacillus reuteri strain 35-5 glucansucrase GTFA enzyme

The neutral exopolysaccharide EPS35-5 (reuteran) produced from sucrose by the glucansucrase GTFA enzyme from Lactobacillus reuteri 35-5 was found to be a (1-->4,1-->6)-alpha-D-glucan, with no repeating units present. Based on linkage analysis and 1D/2D 1H and 13C NMR spectroscopy of intact EPS35-5, as well as MS and NMR analysis of oligosaccharides obtained by partial acid hydrolysis and enzymatic hydrolysis, using pullulanase M1 (Klebsiella planticola), of EPS35-5, a composite model, that includes all identified structural elements, was formulated as follows: [Formula: see text].     

Influence of sodium nitroprusside on human erythrocyte membrane water permeability: an NMR study

Sodium nitroprusside (SNP) is a nitric oxide (?NO) donor in vitro and in vivo. In this paper the time variation of the intracellular water proton nuclear magnetic resonance (NMR) effective relaxation time T'(2a) in SNP-treated human erythrocyte suspensions, containing 10 mM membrane impermeable paramagnetic MnCl2, has been measured. The observed T'(2a) time-course was analyzed in terms of the two mechanisms by which released ?NO affects T'(2a). These are, respectively, enhancement of the intracellular water proton intrinsic NMR relaxation rate 1/T(2a) by paramagnetism of ?NO subsequently bonded to iron atoms of intracellular deoxyhemoglobin, and suppression of diffusional water permeability P(d) as a consequence of nitrosylation of aquaporin-1 (AQP1) channel Cys189, either by direct reaction with ?NO or with one of the ?NO oxidation products, such as N2O3. The bound ?NO on the Cys189 thiol residue appears to impose a less efficient barrier to water permeation through AQP1 than the larger carboxyphenylmercuryl residue from p-chloromercuribenzoate. The effect of ?NO on P(d) is discussed in terms of NO-induced vasodilation.     

Structural analysis of bioengineered alpha-D-glucan produced by a triple mutant of the Glucansucrase GTF180 enzyme from Lactobacillus reuteri strain 180: generation of (alpha1-->4) linkages in a native (1-->3)(1-->6)-alpha-D-glucan

Site-directed mutagenesis of the glucansucrase gtf180 gene from Lactobacillus reuteri strain 180 was used to transform the active site region. The alpha-D-glucan ( mEPS-PNNS) produced by the triple mutant V1027P:S1137N:A1139S differed in structure from that of the wild-type alpha-D-glucan ( EPS180). Besides (alpha1-->3) and (alpha1-->6) linkages, as present in EPS180, mEPS-PNNS also contained (alpha1-->4) linkages. Linkage analysis, periodate oxidation, and 1D/2D (1)H NMR spectroscopy of the intact mEPS-PNNS, as well as MS and NMR analysis of oligosaccharides obtained by partial acid hydrolysis of mEPS-PNNS afforded a composite model, which includes all identified structural features.     

Fitness profile of elite Croatian female taekwondo athletes

The aim of the study was to assess fitness profile of elite Croatian female taekwondo athletes and to determine which physical, physiological and motor characteristics differentiate mostly the successful from the less successful fighters. Thirteen national taekwondo champions were divided into two groups according to their senior international competitive achievements. Physiological characteristics, including maximal oxygen uptake (VO2max), were assessed during a continuous progressive treadmill test. The measured motor abilities included explosive and elastic leg strength, maximal strength, muscular endurance, anaerobic alactic power, agility and flexibility. Differences between the successful and less successful athletes were determined using independent t-test. Even though the differences were not statistically significant, the successful athletes had somewhat less fat (2.3%) and were taller by 5.8 cm. The successful athletes achieved significantly higher maximum running speed (15.8 +/- 0.5 versus 14.9 +/- 0. 7 km h(-1); p < 0.05), their ventilatory anaerobic threshold was significantly higher (41.4 +/- 4.1 versus 37.6 +/- 2.0 ml kg(-1) min(-1); p < 0.05) at a significantly lower heart rate (166.8 +/- 6.8 versus 171.0 +/- 8.2 beats min(-1); p < 0.05) than in the less successful athletes. Significant differences were also found in three tests of explosive power (p < 0.05), anaerobic alactic power (p < 0.01), and lateral agility (p < 0.05). The performance of taekwondo female athletes primarily depends on the anaerobic alactic power, explosive power expressed in the stretch-shortening cycle movements, agility and aerobic power.     

Quantification of binding of some thiol-reactive clenbuterol analogues to bovine serum albumin by electron paramagnetic resonance spectroscopy

The only free thiol group of bovine serum albumin (BSA) was coupled in a high yield with some novel thiol-reactive clenbuterol analogues. The unreacted cysteines were probed with maleimide spin label to determine the yield of the coupling reaction. A novel approach to determining free thiol groups of BSA quantitatively by electron paramagnetic resonance spectroscopy and spectral decomposition without the usual gel-filtration step or extensive dialysis is presented.     

4,6-α-Glucanotransferase activity occurs more widespread in Lactobacillus strains and constitutes a separate GH70 subfamily

Family 70 glycoside hydrolase glucansucrase enzymes exclusively occur in lactic acid bacteria and synthesize a wide range of α-d-glucan (abbreviated as α-glucan) oligo- and polysaccharides. Of the 47 characterized GH70 enzymes, 46 use sucrose as glucose donor. A single GH70 enzyme was recently found to be inactive with sucrose and to utilize maltooligosaccharides [(1→4)-α-d-glucooligosaccharides] as glucose donor substrates for α-glucan synthesis, acting as a 4,6-α-glucanotransferase (4,6-αGT) enzyme. Here, we report the characterization of two further GH70 4,6-αGT enzymes, i.e., from Lactobacillus reuteri strains DSM 20016 and ML1, which use maltooligosaccharides as glucose donor. Both enzymes cleave α1→4 glycosidic linkages and add the released glucose moieties one by one to the non-reducing end of growing linear α-glucan chains via α1→6 glycosidic linkages (α1→4 to α1→6 transfer activity). In this way, they convert pure maltooligosaccharide substrates into linear α-glucan product mixtures with about 50% α1→6 glycosidic bonds (isomalto/maltooligosaccharides). These new α-glucan products may provide an exciting type of carbohydrate for the food industry. The results show that 4,6-αGTs occur more widespread in family GH70 and can be considered as a GH70 subfamily. Sequence analysis allowed identification of amino acid residues in acceptor substrate binding subsites +1 and +2, differing between GH70 GTF and 4,6-αGT enzymes.     

Biochemical and crystallographic characterization of a glucansucrase from Lactobacillus reuteri 180

Glucansucrases are large extracellular transglycosidases secreted by lactic acid bacteria. Using sucrose as a substrate they synthesize high molecular mass α-glucans or, in the presence of suitable acceptor molecules, low molecular mass oligosaccharides. Although about 60 glucansucrases have been classified in glycoside hydrolase family GH70, no three-dimensional structure has been reported for any. With the aim of solving the first structure of a GH70 glucansucrase, purification and crystallization experiments were performed with a fully active, 117 kDa N-terminally truncated fragment of glucansucrase GTF180 from Lactobacillus reuteri 180 (residues 742–1772). Crystallization experiments yielded crystals that belong to two different triclinic crystal forms (space group P1) and one orthorhombic crystal form (space group P2₁2₁2₁). Native data sets for both triclinic and the orthorhombic crystals were collected at 1.7 and 2.0 Å resolution, respectively. Enzyme activity assays, pH and temperature optima show comparable values for both the full-length and the N-terminally truncated GTF180.     

Plitvice Lakes National park harbors ancient,yet endangered diversity of trout (genus Salmo)

National park Plitvice Lakes is the oldest, the largest and the most visited national park in Croatia, well known for its stunning karstic watersheds that supply Plitvice Lakes and exceptionally valuable biodiversity. Lack of knowledge on the taxonomic status, population structure and viability of trout in the Plitvice Lakes National park, as well as the possibility of existence of cryptic diversity, motivated this study which aimed to determine: taxonomic status and phylogenetic relationship of Plitvice Lakes trout with other Salmo species, genetic structure and diversity of their populations, as well as future viability of populations. 150 samples of trout were obtained from 17 localities. Two mitochondrial gene markers, gene for cytochrome b and mitochondrial control region were used in phylogenetic reconstruction, as well as in population genetic investigation. Intraspecific and intrapopulation genetic diversity was described by calculating several measures of genetic polymorphism, while gene flow among populations was estimated as the migration rates and as the number of immigrants per generation. Phylogenetic trees revealed that Plitvice Lakes watershed is inhabited by two evolutionary independent units of the genus Salmo, representing two species, Salmo labrax Pallas, 1814 and Salmo trutta Linnaeus, 1758. Possibly ‘pure’ populations of both species were found alongside several admixed ones in which both species are present. Our data has shown low intraspecific and intrapopulational diversities for Plitvice Lakes trout and restricted gene flow. Small effective sizes of populations and weak interconnections between them, which are most likely a consequence of fragmentation and habitat degradation, accentuating the need for urgent conservation measures.     

The effect of temperature on the duration of the effective pollination period in ‘Oblica’ olive (Olea europaea) cultivar

The effective pollination period (EPP) was determined for ‘Oblica’ olive trees in response to sequential pollinations at anthesis and at increasing days after. The role played by each of three components of the EPP, namely the stigmatic receptivity, pollen tube growth and ovule longevity was examined and the duration of the EPP under different environmental conditions was calculated. An increase in temperature in the field was caused by enclosing bearing trees within polyethylene cages. The duration of the EPP varied between years and environmental conditions. Initial fruit set (IFS) declined gradually in response to sequential cross‐pollination. The significant decrease with respect to the maximum levels of IFS occurred 5 days after anthesis (DAA) in 2007 and between 4 and 5 DAA in 2010 at both environmental conditions. However, the fruit set decrease with sequential pollinations was sharper for the trees inside the cages than for trees growing at the open air, suggesting that high temperatures induce earlier and more pronounced decrease in flower fertility. The stigma receptivity was quite extended decreasing slowly with time. In 2010, the stigma receptivity was prolonged beyond sampling date (7 DAA) in both environmental conditions. Stigmas first lost the capacity to support pollen germination and later their capacity to adhere pollen grains. Pollen tube growth occurred rapidly and fertilization took place 1 or 2 days after pollination. Cross‐pollination resulted with higher fertilization levels, whereas fertilization was reduced and delayed under self‐pollination. Enclosing the trees within the cages had small effect on cross‐pollination but impaired the self‐pollination which resulted with reduced levels of fertilization. The ovule longevity was prolonged beyond the end of sampling in both years and conditions (beyond 9 DAA in 2009 or 7 DAA in 2010). Discrepancy between the estimates of the duration of the EPP based on analyses of its components and those indicated by the declines in fruit set from delayed pollination suggests that a different component of the pistil, the style, could have the critical role in determining the duration of the EPP.     

Maternal immune system adaptation to pregnancy - a potential influence on the course of diabetic retinopathy

Background  

Progression of diabetic retinopathy occurs at least temporarily during pregnancy. Although the cause of this progression is not entirely understood, the immune phenomenon and chronic inflammation may play a significant role. During pregnancy in order to avoid fetus rejection, certain components of the immune system that are knowingly implicated in the pathogenesis of diabetic retinopathy are activated including generalized leukocyte activation and an increase in certain cytokine plasma levels. Activated leukocytes with up regulated adhesion molecules have an increased potential to bind to the endothelium cells of blood vessels. Leukocyte-endothelial interaction and the consequent leukostasis with capillary occlusion, ischemia and vascular leakage have a substantial role in the development of diabetic retinopathy. Furthermore, certain increased cytokines are known to cause blood-retinal-barrier breakdown whilst others promote angiogenic and fibrovascular proliferation and thereby can also be implicated in the pathogenesis of this diabetic complication.