Entrapment of Ovalbumin into Liposomes—Factors Affecting Entrapment Efficiency,Liposome Size,and Zeta Potential

Various amounts of Ovalbumin (OVA) were encapsulated into positively and negatively charged multilamellar liposomes, with the aim to investigate the entrapment efficiency in different buffers and to study their effects on the liposome size and zeta potential. Results showed that the entrapment efficiency of OVA in anionic liposomes was the same in 10 mM Phosphate Buffer (PB) as in Phosphate-Buffered Saline (PBS; PB?+?0.15 M NaCl). Also, liposome size was approximately 1200 nm for all anionic liposomes incorporating OVA. The entrapment efficiency of OVA in cationic liposomes was highly dependent on ionic strength. The size of cationic liposomes was approximately 1200 nm in PBS, regardless of protein content, but increased with the amount of the incorporated protein in PB. Aggregation of cationic liposomes in PB was observed when the mass of the protein was 2.5 mg or greater. The zeta potential of anionic liposomes was negative and of cationic liposomes positive in the whole range of protein mass tested. These results show how different compositions of lipid and aqueous phases can be used to vary the entrapment efficiency, liposome size, and zeta potential—the factors that are of great importance for the use of liposomes as drug carriers.     

Putative silicon transport vesicles in the cytoplasm of the diatom Synedra acus during surge uptake of silicon

We studied the growth of the araphid pennate diatom Synedra acus subsp. radians (Kützing) Skabichevskii using a fluorescent dye N ¹,N ³-dimethyl-N ¹-(7-nitro-2,1,3-benzoxadiazol-4-yl)propane-1,3-diamine (NBD-N2), which stains growing siliceous frustules but does not stain other subcellular organelles. We used a clonal culture of S. acus that was synchronized by silicon starvation. Epifluorescence microscopy was performed in two different ways with cells stained by the addition of silicic acid and the dye. Individual cells immobilized on glass were observed during the first 15–20 min following the replenishment of silicic acid after silicon starvation. Alternatively, we examined cells of a batch culture at time intervals during 36 h after the replenishment of silicic acid using fluorescence and confocal microscopy. The addition of silicic acid and NBD-N2 resulted in the rapid (1–2 min) formation of several dozen green fluorescent submicrometer particles (GFSPs) in the cytoplasm, which was accompanied by the accumulation of fluorescent silica inside silica deposition vesicles (SDVs) along their full length. In 5–15 min, GFSPs disappeared from the cytoplasm. Mature siliceous valves were formed within the SDVs during the subsequent 14–16 h. In the next 8–10 h, GFSPs appeared again in the cytoplasm of daughter cells. The data obtained confirm observations about the two-stage mechanism of silicon assimilation, which includes rapid silicon uptake (surge uptake) followed by slow silica deposition. It is likely that the observed GFSPs are silicon transport vesicles, which were first proposed by Schmid and Schulz in (Protoplasma 100:267–288, 1979).     

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.     

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.     

Improving bioorthogonal protein ubiquitylation by click reaction

Posttranslational modification of proteins with ubiquitin (ubiquitylation) regulates numerous cellular processes. Besides functioning as a signal for proteasomal degradation, ubiquitylation has also non-proteolytic functions by altering the biochemical properties of the modified protein. To investigate the effect(s) of ubiquitylation on the properties of a protein, sufficient amounts of homogenously and well-defined ubiquitylated proteins are required. Here, we report on the elaboration of a method for the generation of high amounts of site-specifically mono-ubiquitylated proteins. Firstly, a one-step affinity purification scheme was developed for ubiquitin containing the unnatural amino acid azidohomoalanine at the C-terminal position. This ubiquitin was conjugated in a click reaction to recombinant DNA polymerase β, equipped with an alkyne function at a distinct position. Secondly, addition of defined amounts of SDS to the reaction significantly improved product formation. With these two technical improvements, we have developed a straight forward procedure for the efficient generation of site-specifically ubiquitylated proteins that can be used to study the effect(s) of ubiquitylation on the activities/properties of a protein.     

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.