Identification, phylogenetic relationships and a new maximum size of two rudd populations (Scardinius, Cyprinidae) from the Adriatic Sea drainage, Croatia

In order to determine an unknown fish population from the Vrana Lake, mitochondrial cytochrome b gene and non-coding nuclear region Cyfun P were investigated. Stabile population of Bulldog rudd, Scardinius dergle Heckel & Kner, the endemic Croatian freshwater fish in the Krka River, was genetically characterized with the same markers in order to compare it with the material from the Vrana Lake. Genetic markers were sequenced and aligned with the similar ones obtained from the GenBank in order to determine taxonomic and phylogenetic position of these two species. A significant discrepancy between nuclear genetic markers of our specimens and the sequence from the GenBank was found. Phylogenetic analysis suggested that the specimens from the Vrana Lake belong to the species S. hesperidicus. Morphometric characteristics, the maximum length and body mass showed new maximum values for both S. dergle and S. hesperidicus.     

A sensitive fluorigenic substrate for selective in vitro and in vivo assay of leukotriene A4 hydrolase activity

Leukotriene A4 hydrolase (LTA4H) is a bifunctional zinc-dependent metalloprotease bearing both an epoxide hydrolase, producing the pro-inflammatory LTB4 leukotriene, and an aminopeptidase activity, whose physiological relevance has long been ignored. Distinct substrates are commonly used for each activity, although none is completely satisfactory; LTA4, substrate for the hydrolase activity, is unstable and inactivates the enzyme, whereas aminoacids β-naphthylamide and para-nitroanilide, used as aminopeptidase substrates, are poor and nonselective. Based on the three-dimensional structure of LTA4H, we describe a new, specific, and high-affinity fluorigenic substrate, PL553 [l-(4-benzoyl)phenylalanyl-β-naphthylamide], with both in vitro and in vivo applications. PL553 possesses a catalytic efficiency (k_cat/K_m) of 3.8 ± 0.5 × 10⁴ M⁻¹ s⁻¹ using human recombinant LTA4H and a limit of detection and quantification of less than 1 to 2 ng. The PL553 assay was validated by measuring the inhibitory potency of known LTA4H inhibitors and used to characterize new specific amino-phosphinic inhibitors. The LTA4H inhibition measured with PL553 in mouse tissues, after intravenous administration of inhibitors, was also correlated with a reduction in LTB4 levels. This authenticates the assay as the first allowing the easy measurement of endogenous LTA4H activity and in vitro specific screening of new LTA4H inhibitors.     

Field and laboratory studies on the life-cycle, growth and feeding preference in the hairy snail Trochulus hispidus (L., 1758) (Gastropoda: Pulmonata: Hygromiidae)

For the first time the life cycle of the common land snail Trochulus hispidus was completely described in Central Europe (Poland). This is a semelparous species predominantly with an annual life cycle and the reproductive period lasting from April till October. The first young snails hatch in spring, grow rapidly in summer and reach ca. 4 whorls until winter. In spring of the next year they mature and reproduce. After that they die. There is hardly any growth from late autumn till early spring. The average proportional growth rate is ca. 0.3 whorl/month in the wild. The fastest growth is present in the youngest snails and then gradually decreases over the course of their age. Laboratory and field observations allowed for establishing the following life cycle parameters: eggs calcified, almost spherical, ca. 1.5 mm, laid in spring and summer in batches of between 1 and 47. Time to hatching is 6–24 days, hatching is asynchronous; newly-hatched snails have approximately 1.5 whorls. Analysis of food preferences revealed, that T. hispidus tends to restrict its diet during the life. Generally the youngest snails equally consumed leaves of all four tree species offered (Fraxinus excelsior, Acer pseudoplatanus, Tilia cordata and A. platanoides) whereas adults preferred F. excelsior over A. pseudoplatanus and A. platanoides.     

Detection and quantification of probiotic strain Lactobacillus gasseri K7 in faecal samples by targeting bacteriocin genes

Lactobacillus gasseri K7 is a probiotic strain that produces bacteriocins gassericin K7 A and K7 B. In order to develop a real-time quantitative PCR assay for the detection of L. gasseri K7, 18 reference strains of the Lactobacillus acidophilus group and 45 faecal samples of adults who have never consumed strain K7 were tested with PCR using 14 pairs of primers specific for gassericin K7 A and K7 B gene determinants. Incomplete gassericin K7 A or K7 B gene clusters were found to be dispersed in different lactobacilli strains as well as in faecal microbiota. One pair of primers was found to be specific for the total gene cluster of gassericin K7A and one for gassericin K7B. The real-time PCR analysis of faecal samples spiked with K7 strain revealed that primers specific for the gene cluster of the gassericin K7 A were more suitable for quantitative determination than those for gassericin K7 B, due to the lower detection level. Targeting of the gassericin K7 A or K7 B gene cluster with specific primers could be used for detection and quantification of L. gasseri K7 in human faecal samples without prior cultivation. The results of this study also present new insights into the prevalence of bacteriocin-encoding genes in gastrointestinal tract.     

Search for new tools to combat Gram-negative resistant bacteria among amine derivatives of 5-arylidenehydantoin

A series of amine-alkyl derivatives of 5-arylidenehydantoin 3–21 was evaluated for their ability to improve antibiotic effectiveness in two strains of Gram-negative Enterobacter aerogenes: the reference strain (ATCC-13048) and the chloramphenicol-resistant derivative over-producing the AcrAB-TolC efflux pump (CM-64). Three antibiotics, chloramphenicol, nalidixic acid and sparfloxacin were used as markers of efflux pump activity. New compounds (5–16) were obtained within 3–4 step synthesis using Knoevenagel condensation, Mitsunobu reaction and microwave aided N-alkylation. Molecular modeling based structure–activity relationship (SAR) studies were performed. The most active compounds: (Z)-5-(4-(diethylamino)benzylidene)-3-(2-hydroxy-3-(4-(2-hydroxyethyl)piperazin-1-yl)propyl)imidazolidine-2,4-dione (14) and (Z)-5-(2,4-dimethoxybenzylidene)-3-(2-hydroxy-3-(isopropylamino)propyl)imidazolidine-2,4-dione (15) induced fourfold decrease of minimal inhibition concentration (MIC) of all tested antibiotics in the strain CM-64 overexpressing the AcrAB-TolC pump.     

Current advances in research of cytochrome c oxidase

The function of cytochrome c oxidase as a biomolecular nanomachine that transforms energy of redox reaction into protonmotive force across a biological membrane has been subject of intense research, debate, and controversy. The structure of the enzyme has been solved for several organisms; however details of its molecular mechanism of proton pumping still remain elusive. Particularly, the identity of the proton pumping site, the key element of the mechanism, is still open to dispute. The pumping mechanism has been for a long time one of the key unsolved issues of bioenergetics and biochemistry, but with the accelerating progress in this field many important details and principles have emerged. Current advances in cytochrome oxidase research are reviewed here, along with a brief discussion of the most complete proton pumping mechanism proposed to date, and a molecular basis for control of its efficiency.     

The hazards of DAPI photoconversion: effects of dye, mounting media and fixative, and how to minimize the problem

Immunocytochemistry is a powerful tool for detection and visualization of specific molecules in living or fixed cells, their localization and their relative abundance. One of the most commonly used fluorescent DNA dyes in immunocytochemistry applications is 4′,6-diamidino-2-phenylindole dihydrochloride, known as DAPI. DAPI binds strongly to DNA and is used extensively for visualizing cell nuclei. It is excited by UV light and emits characteristic blue fluorescence. Here, we report a phenomenon based on an apparent photoconversion of DAPI that results in detection of a DAPI signal using a standard filter set for detection of green emission due to blue excitation. When a sample stained with DAPI only was first imaged with the green filter set (FITC/GFP), only a weak cytoplasmic autofluorescence was observed. Next, we imaged the sample with a DAPI filter set, obtaining a strong nuclear DAPI signal as expected. Upon reimaging the same samples with a FITC/GFP filter set, robust nuclear fluorescence was observed. We conclude that excitation with UV results in a photoconversion of DAPI that leads to detection of DAPI due to excitation and emission in the FITC/GFP channel. This phenomenon can affect data interpretation and lead to false-positive results when used together with fluorochrome-labeled nuclear proteins detected with blue excitation and green emission. In order to avoid misinterpretations, extra precaution should be taken to prepare staining solutions with low DAPI concentration and DAPI (UV excitation) images should be acquired after all other higher wavelength images. Of various DNA dyes tested, Hoechst 33342 exhibited the lowest photoconversion while that for DAPI and Hoechst 33258 was much stronger. Different fixation methods did not substantially affect the strength of photoconversion. We also suggest avoiding the use of mounting medium with high glycerol concentrations since glycerol showed the strongest impact on photoconversion. This photoconversion effect cannot be avoided even when using narrow bandpass filter sets.     

Population specific fitness response of Drosophila subobscura to lead pollution

Abstract Differences in heavy metal tolerance among separate populations of the same species have often been interpreted as local adaptation. Persistence of differences after removing the stressor indicates that mechanisms responsible for the increased tolerance were genetically determined. Drosophila subobscura Collin (Diptera: Drosophilidae) populations were sampled from two localities with different history of heavy metal pollution, and reared for eight generations in the laboratory on a standard medium and on media with different concentrations of lead (Pb). To determine whether flies from different natural populations exposed to the Pb‐contaminated media in the laboratory show population specific variability in fitness components over generations, experimental groups with different concentrations of lead were assayed in three generations (F₂, F₅, and F₈) for fecundity, developmental time, and egg‐to‐adult viability. On the contaminated medium, fecundity was reduced in later generations and viability was increased, irrespective of the environmental origin of populations. For both populations, developmental time showed a tendency of slowing down on media with lead. Faster development was observed in later generations. Preadaptation to contamination, meaning higher fecundity, higher viability, and faster egg to adult development in all studied generations, was found in D. subobscura originating from the locality with a higher level of heavy metal pollution.