Real-time monitoring of melittin-induced pore and tubule formation from supported lipid bilayers and its physiological relevance

The temporal evolution of effects of antimicrobial peptide melittin on supported phospholipid bilayers (SPBs) containing negatively charged phospholipids was monitored by ellipsometry and laser scanning microscopy together with measurements of lipid mobility by Z-scan fluorescence correlation spectroscopy. Under all conditions used in our study, we observed reproducibly two effects. The first one is formation of pores in the SPB, which occupy approximately 40% of the bilayer. The formation of pores was accompanied by a decrease in lateral diffusion coefficient of the lipids to approximately 60% of its initial value. The second, simultaneous, effect is the formation of tubules of approximately 30 nm radius and length of the order of 10 μm. Flushing of the sample with excess of buffer removes most of the tubules, but it does not affect the pores. Further experiments performed under various conditions demonstrated reproducibility of both phenomena.     

Variability of foraging and roosting activities in adult females of Daubenton’s bat (<Emphasis Type="Italic">Myotis daubentonii</Emphasis>) in different seasons

We radio-tracked fifteen reproductive females (5 pregnant, 5 lactating, 5 in post-lactation) of the Daubenton’s bat in summer 2005 in order to reveal the effect of reproductive state on their foraging and roosting activity. Spatial activity of females decreased from pregnancy to lactation and increased again in the post-lactation period. Overall time spent foraging did not differ among the three study periods. However, while pregnant and lactating females spent similar proportion of the night length foraging, females in the post-lactation period were foraging for shorter part of night. The frequency of nightly visits to roosts was highest during lactation but there was a trend towards shortening of particular visits during that period. All but one roost were in tree hollows excavated by woodpeckers in spatially restricted area of ca 0.7 km². Tree cavities used during pregnancy were located higher on a tree trunk and had larger entrance area than the cavities used in the two later periods. Bats switched roosts every 2–3 days (range 1–8) and moved to a new roost up to 800 m apart. Pregnant females tended to switch roosts more frequently than females in the two later periods. We did not observe a significant effect of minimum nightly temperature on the activity of radio-tracked Daubenton’s bats. Therefore, we suggest that observed seasonal changes in the pattern of behaviour of Daubenton’s bat females were driven by their changing energetic demands rather than by some extrinsic factors (e.g. weather conditions).     

Mapping the peptide and protein immune response in the larvae of the fleshfly Sarcophaga bullata.

We chose the larvae of fleshfly Sarcophaga bullata to map the peptide and protein immune response. The hemolymph of the third-instar larvae of S. bullata was used for isolation. The larvae were injected with bacterial suspension to induce an antimicrobial response. The hemolymph was separated into crude fractions, which were subdivided by RP-HPLC, gel electrophoresis, and free-flow electrophoresis. In several fractions, we determined significant antimicrobial activities against the pathogenic bacteria Escherichia coli, Staphylococcus aureus, or Pseudomonas aeruginosa. Among antimicrobially active compounds we identified dipeptide beta-alanyl-L-tyrosine, protein transferrin, and two variants of peptide sapecin. We also partially characterized two novel antimicrobially active polypeptides; odorant-binding protein 99b, and a peptide which remains unidentified.     

Helicosporidium infection of the great European spruce bark beetle, Dendroctonus micans (Coleoptera: Scolytidae)

An infection of the great European spruce bark beetle Dendroctonus micans (from Turkey) by the parasitic green alga Helicosporidium is described. This is the first time that Helicosporidium has been found to infect a bark beetle (Scolytidae) and the first time that a naturally infected beetle has been reported for the Eurasian continent. The typical cysts of Helicosporidium contain three ovoid cells and one helical, filamentous cell. The morphological characteristics are revealed by light and electron microscopy. Distinct electron-dense inclusions with a peculiar ultrastructure may represent pyrenoids. Since D. micans is an important pest, the discovery of a natural pathogen may offer a chance for biological control.     

Synthesis of tumor necrosis factor alpha-induced protein 6 in porcine preovulatory follicles: a study with A38 antibody

We have previously shown that the heavy chains (HCs) of inter-alpha-trypsin inhibitor (IalphaI) become covalently linked to hyaluronan (HA) during in vivo and in vitro expansion of porcine oocyte-cumulus cell complexes (OCCs). We have now studied by immunoblotting the synthesis of tumor necrosis factor alpha-induced protein 6 (TNFAIP6), which is essential for catalyzing this reaction in expanding mouse OCCs. Expanding OCCs were collected from preovulatory follicles of naturally cycling pigs and also after in vitro culture (24 or 42 h) in medium supplemented with FSH and pig serum. After isolation, OCCs were treated with Streptomyces hyaluronidase or Chondroitinase ABC. Matrix, cell pellet, and total extracts were analyzed by Western blotting. A band of about 35 kDa and a doublet of about 120 kDa, corresponding to the molecular weight of the native and HC-linked forms of TNFAIP6, respectively, were detected by a rabbit anti-human TNFAIP6 polyclonal antibody in matrix extracts of expanded cumuli. Moreover, we found by using a cell-free assay that porcine follicular fluid collected from follicles at 24 h after hCG stimulation contains HC-HA coupling activity. This activity was abolished by the rat anti-human monoclonal antibody A38, which has an epitope within the Link module domain of TNFAIP6. These experiments suggest that free TNFAIP6 protein was present in follicular fluid aspirated from porcine follicles 24 h after hCG stimulation. In contrast to mouse, we show that the A38 monoclonal antibody does not affect in vitro cumulus expansion of porcine OCCs.     

Synthesis, cytostatic, and antiviral activity of novel 6-[2-(dialkylamino)ethyl]-, 6-(2-alkoxyethyl)-, 6-[2-(alkylsulfanyl)ethyl]-, and 6-[2-(dialkylamino)vinyl]purine nucleosides

An efficient and facile synthesis of a large series of diverse 6-[2-(dialkylamino)vinyl]-, 6-[2-(dialkylamino)ethyl]-, 6-(2-alkoxyethyl)-, and 6-[2-(alkylsulfanyl)ethyl]purine nucleosides (35 examples of both ribo- and 2'-deoxyribonucleosides) was developed. The key transformations involved conjugate nucleophilic additions of amines, alcoholates, or thiolates to Tol-protected 6-alkylylpurine or 6-vinylpurine nucleosides. 6-[(2-Dialkylamino)vinyl]- and some 6-[(2-dialkylamino)ethyl]purine ribonucleosides exerted significant cytostatic effects and some anti-HCV activity with low selectivity.