Crystallization and preliminary X-ray studies of ornithine decarboxylase from Trypanosoma brucei

Trypanosoma brucei ornithine decarboxylase, expressed and purified from E. coli, has been crystallized by the vapor diffusion method using PEG 3350 as a precipitant. The crystals belong to the monoclinic space group P2₁ and have cell constants of a = 66.3 Å, b = 151.8 Å, c = 83.7 Å, and β = 101.2°. While larger crystals are twinned, smaller crystals (0.4 × 0.3 × 0.05 mm³) are single.     

RdgB acts to avoid chromosome fragmentation in Escherichia coli

Bacterial RecA protein is required for repair of two-strand DNA lesions that disable whole chromosomes. recA mutants are viable, suggesting a considerable cellular capacity to avoid these chromosome-disabling lesions. recA-dependent mutants reveal chromosomal lesion avoidance pathways. Here we characterize one such mutant, rdgB/yggV, deficient in a putative inosine/xanthosine triphosphatase, conserved throughout kingdoms of life. The rdgB recA lethality is suppressed by inactivation of endonuclease V (gpnfi) specific for DNA-hypoxanthines/xanthines, suggesting that RdgB either intercepts improper DNA precursors dITP/dXTP or works downstream of EndoV in excision repair of incorporated hypoxathines/xanthines. We find that DNA isolated from rdgB mutants contains EndoV-recognizable modifications, whereas DNA from nfi mutants does not, substantiating the dITP/dXTP interception by RdgB. rdgB recBC cells are inviable, whereas rdgB recF cells are healthy, suggesting that chromosomes in rdgB mutants suffer double-strand breaks. Chromosomal fragmentation is indeed observed in rdgB recBC mutants and is suppressed in rdgB recBC nfi mutants. Thus, one way to avoid chromosomal lesions is to prevent hypoxanthine/xanthine incorporation into DNA via interception of dITP/dXTP.     

Lateral heterogeneity of photosystems in thylakoid membranes studied by Brownian dynamics simulations

The aggregation and segregation of photosystems in higher plant thylakoid membranes as stromal cation-induced phenomena are studied by the Brownian dynamics method. A theoretical model of photosystems lateral movement within the membrane plane is developed, assuming their pairwise effective potential interaction in aqueous and lipid media and their diffusion. Along with the screened electrostatic repulsive interaction the model accounts for the van der Waals-type, elastic, and lipid-induced attractive forces between photosystems of different sizes and charges. Simulations with a priori estimated parameters demonstrate that all three studied repulsion-attraction alternatives might favor the local segregation of photosystems under physiologically reasonable conditions. However, only the lipid-induced potential combined with the size-corrected screened Coulomb interaction provides the segregated configurations with photosystems II localized in the central part of the grana-size simulation cell and photosystems I occupying its margins, as observed experimentally. Mapping of thermodynamic states reveals that the coexistence curves between isotropic and aggregated phases are the sigmoidlike functions regardless of the effective potential type. It correlates with measurements of the chlorophyll content of thylakoid fragments. Also the universality of the phase curves characterizes the aggregation and segregation of photosystems as order-disorder phase transitions with the Debye radius as a governing parameter.     

Spatiotemporal dynamics of contact activation factors of blood coagulation

A new in vitro model is proposed for studying the spatiotemporal distributions of activated clotting factors, in which clotting is activated in a thin layer of non-stirred plasma supplemented with a fluorogenic substrate and is monitored by fluorescence from its cleavage product. Analysis of the spatiotemporal dynamics of factor XIa and kallikrein in glass-activated human plasma provides evidence that both contact factors remain restricted to the glass surface and possibly a narrow boundary zone (<0.1 mm). The kinetics of factor XIa and kallikrein studied by a new method (in non-stirred plasma) coincided with those studied fluorimetrically with full stirring: their concentrations rapidly rose for the first few minutes after activation and then slowly declined. Factor XI and prekallikrein activation is likely to be restricted by the limited number of sites available for binding to the surface. The maximum concentration of the active factors was estimated at 2 x 10(8) molecules per mm(2) at the glass surface (irrespective of stirring). At the plastic surface, this value was 15--30 times lower.     

Isolation and characterization of high affinity aptamers against DNA polymerase iota

Human DNA-polymerase iota (Pol ι) is an extremely error-prone enzyme and the fidelity depends on the sequence context of the template. Using the in vitro systematic evolution of ligands by exponential enrichment (SELEX) procedure, we obtained an oligoribonucleotide with a high affinity to human Pol ι, named aptamer IKL5. We determined its dissociation constant with homogenous preparation of Pol ι and predicted its putative secondary structure. The aptamer IKL5 specifically inhibits DNA-polymerase activity of the purified enzyme Pol ι, but did not inhibit the DNA-polymerase activities of human DNA polymerases beta and kappa. IKL5 suppressed the error-prone DNA-polymerase activity of Pol ι also in cellular extracts of the tumor cell line SKOV-3. The aptamer IKL5 is useful for studies of the biological role of Pol ι and as a potential drug to suppress the increase of the activity of this enzyme in malignant cells.     

Does Parental Fin Digging Improve Feeding Opportunities for Offspring in the Convict Cichlid?

The function of the fin digging behaviour in increasing food availability for the offspring was analysed in the convict cichlid, Cichlasoma (Archocentrus) nigrofasciatum. Consistent individual differences in the frequency of fin digging were found in the parental fish. Examination of the gastrointestinal tract of young revealed that higher frequency of parental fin digging was associated with higher consumption of large and more profitable prey (Diptera larvae), which inhabited deep horizons of the bottom substrate and possibly were difficult to access without parental assistance. Thus, parental fin digging was initially associated with a significant increase of the offspring growth rate. However, at later brood intervals, when parental care ceased, the young of the high-digging parents were characterised by a poorer consumption of small larvae that were most accessible for them without parental aid and represented an increasingly more important component of their ration than large larvae. Offspring of the low-digging parents, on the other hand, presumably as a result of their individual experience, showed a considerably better consumption of small larvae, increasing their growth rate. As a consequence, prior parental fin digging did not affect the offspring body size after independence. Thus, there exist pronounced individual differences and alternative parental styles in the convict cichlid.     

Linking ecology,morphology, and metabolism: Niche differentiation in sympatric populations of closely related species of the genus Littorina (Neritrema)

Divergence of ecological niches in phylogenetically closely related species indicates the importance of ecology in speciation, especially for sympatric species are considered. Such ecological diversification provides an advantage of alleviating interspecies competition and promotes more efficient exploitation of environmental resources, thus being a basis for ecological speciation. We analyzed a group of closely related species from the subgenus Neritrema (genus Littorina, Caenogastropoda) from the gravel‐bouldery shores. In two distant sites at the Barents and Norwegian Sea, we examined the patterns of snail distribution during low tide (quantitative sampling stratified by intertidal level, presence of macrophytes, macrophyte species, and position on them), shell shape and its variability (geometric morphometrics), and metabolic characteristics (metabolomic profiling). The studied species diversified microbiotopes, which imply an important role of ecological specification in the recent evolution of this group. The only exception to this trend was the species pair L. arcana / L. saxatilis, which is specifically discussed. The ecological divergence was accompanied by differences in shell shape and metabolomic characteristics. Significant differences were found between L. obtusata versus L. fabalis and L. saxatilis / L. arcana versus L. compressa both in shell morphology and in metabolomes. L. saxatilis demonstrated a clear variability depending on intertidal level which corresponds to a shift in conditions within the occupied microhabitat. Interestingly, the differences between L. arcana (inhabiting the upper intertidal level) and L. compressa (inhabiting the lower one) were analogous to those between the upper and lower fractions of L. saxatilis. No significant level‐dependent changes were found between the upper and lower fractions of L. obtusata, most probably due to habitat amelioration by fucoid macroalgae. All these results are discussed in the contexts of the role of ecology in speciation, ecological niche dynamics and conservatism, and evolutionary history of the Neritrema species.     

Simultaneous assay of metformin and glibenclamide in human plasma based on extraction-less sample preparation procedure and LC/(APCI)MS

Separation of metformin and glibenclamide was achieved within a single chromatographic run on a Zorbax CN column, under isocratic conditions, using acetonitrile and aqueous component (0.01 moles/L ammonium acetate adjusted at pH 3.5 with acetic acid) in volumetric ratio 1/1. Plasma sample preparation is based on protein precipitation by means of organic solvent addition. 1,3,5-Triazine-2,4,6-triamine (IS1) was used as internal standard for metformin, while gliquidone (IS2) played the same role for glibenclamide. Detection was performed with an ion trap mass analyzer, using atmospheric pressure chemical ionization (APCI). A single MS stage was used for detection of metformin and IS1, by extracting ion chromatograms corresponding to molecular ions. MS/MS detection in the SRM mode was used for glibenclamide (m/z transition from 494 to 369 Da) and IS2 (m/z transition from 528 to 403 Da). The method produces linear responses up to 2000 ng/mL for metformin and 400 ng/mL for glibenclamide, respectively. Low limits of quantification were found in the 40 ng/mL range for metformin and at the 4 ng/mL level for glibenclamide. Precision was characterized by relative standard deviations (RSD%) below 9%. The analytical method was successfully applied to a single dose, open-label, randomized, two-period, two-sequence, crossover bioequivalence study of two commercially available anti-diabetic combinations containing 400 mg metformin and 2.5 mg of glibenclamide per coated tablet.