Structure Elucidation and Functional Studies of a Novel β-hairpin Antimicrobial Peptide from the Marine Polychaeta Capitella teleta

Endogenous antimicrobial peptides (AMPs) are evolutionary ancient molecular factors of innate immunity that play a key role in host defense. Among the most active and stable under physiological conditions AMPs are the peptides of animal origin that adopt a β-hairpin conformation stabilized by disulfide bridges. In this study, a novel BRICHOS-domain related AMP from the marine polychaeta Capitella teleta, named capitellacin, was produced as the recombinant analogue and investigated. The mature capitellacin exhibits high homology with the known β-hairpin AMP family—tachyplesins and polyphemusins from the horseshoe crabs. The β-hairpin structure of the recombinant capitellacin was proved by CD and NMR spectroscopy. In aqueous solution the peptide exists as monomeric right-handed twisted β-hairpin and its structure does not reveal significant amphipathicity. Moreover, the peptide retains this conformation in membrane environment and incorporates into lipid bilayer. Capitellacin exhibits a strong antimicrobial activity in vitro against a wide panel of bacteria including extensively drug-resistant strains. In contrast to other known β-hairpin AMPs, this peptide acts apparently via non-lytic mechanism at concentrations inhibiting bacterial growth. The molecular mechanism of the peptide antimicrobial action does not seem to be related to the inhibition of bacterial translation therefore other molecular targets may be assumed. The reduced cytotoxicity against human cells and high antibacterial cell selectivity as compared to tachyplesin-1 make it an attractive candidate compound for an anti-infective drug design.     

Study of Structure–Activity Relationships of the Marine Alkaloid Fascaplysin and Its Derivatives as Potent Anticancer Agents

Marine alkaloid fascaplysin and its derivatives are known to exhibit promising anticancer properties in vitro and in vivo. However, toxicity of these molecules to non-cancer cells was identified as a main limitation for their clinical use. Here, for the very first time, we synthesized a library of fascaplysin derivatives covering all possible substituent introduction sites, i.e., cycles A, C and E of the 12H-pyrido[1-2-a:3,4-b’]diindole system. Their selectivity towards human prostate cancer versus non-cancer cells, as well as the effects on cellular metabolism, membrane integrity, cell cycle progression, apoptosis induction and their ability to intercalate into DNA were investigated. A pronounced selectivity for cancer cells was observed for the family of di- and trisubstituted halogen derivatives (modification of cycles A and E), while a modification of cycle C resulted in a stronger activity in therapy-resistant PC-3 cells. Among others, 3,10-dibromofascaplysin exhibited the highest selectivity, presumably due to the cytostatic effects executed via the targeting of cellular metabolism. Moreover, an introduction of radical substituents at C-9, C-10 or C-10 plus C-3 resulted in a notable reduction in DNA intercalating activity and improved selectivity. Taken together, our research contributes to understanding the structure–activity relationships of fascaplysin alkaloids and defines further directions of the structural optimization.     

Mechanism of Action and Therapeutic Potential of the β-Hairpin Antimicrobial Peptide Capitellacin from the Marine Polychaeta Capitella teleta

Among the most potent and proteolytically resistant antimicrobial peptides (AMPs) of animal origin are molecules forming a β-hairpin structure stabilized by disulfide bonds. In this study, we investigated the mechanism of action and therapeutic potential of the β-hairpin AMP from the marine polychaeta Capitella teleta, named capitellacin. The peptide exhibits a low cytotoxicity toward mammalian cells and a pronounced activity against a wide range of bacterial pathogens including multi-resistant bacteria, but the mechanism of its antibacterial action is still obscure. In view of this, we obtained analogs of capitellacin and tachyplesin-inspired chimeric variants to identify amino acid residues important for biological activities. A low hydrophobicity of the β-turn region in capitellacin determines its modest membranotropic activity and slow membrane permeabilization. Electrochemical measurements in planar lipid bilayers mimicking the E. coli membrane were consistent with the detergent-like mechanism of action rather than with binding to a specific molecular target in the cell. The peptide did not induce bacterial resistance after a 21-day selection experiment, which also pointed at a membranotropic mechanism of action. We also found that capitellacin can both prevent E. coli biofilm formation and destroy preformed mature biofilms. The marked antibacterial and antibiofilm activity of capitellacin along with its moderate adverse effects on mammalian cells make this peptide a promising scaffold for the development of drugs for the treatment of chronic E. coli infections, in particular those caused by the formation of biofilms.     

Modification of 0+ perch foraging behaviour by indirect cues of predation risk

Abstract –  Intake rate and prey size selection of 0+ perch, Perca fluviatilis , from Lake Wallersee (Austria) was studied at different illumination (day light: 400 lx, twilight: 2 lx) during 5 days of habituation to novel surroundings in aquaria. The hypothesis was tested that high illumination and novelty of surroundings (transfer from holding to test aquaria) as indirect cues of predation risk influences 0+ perch foraging behaviour. Significantly lower total intake and lower proportion of large Daphnia magna (two prey size groups, 2.9 and 1.2 mm, were used) were observed at higher illumination and under novel surroundings. Habituation to novelty caused an increase in consumption of large prey and decrease in consumption of small prey. During the whole period of habituation, more large prey was ingested at twilight than at daylight; no light-induced difference in ingestion of small prey was found. Foraging 0+ perch responded to indirect nonspecific cues of predation risk by reduction of intake of large prey, which are costly in terms of handling time. This allows fish to be more vigilant without ceasing their foraging activity even in potentially dangerous situations. In the lake, young perch are most vulnerable to abundant piscivorous fish and birds during the day; in twilight perch can afford a more time-consuming foraging activity.     

Induced hydrogen ion transport in lipid membranes as origin of toxic effect of pentachlorophenol in an alga

Pentachlorophenol (PCP) decreases the rate of carbon assimilation in the alga Selenastrum capricornutum. In parallel with the reduction of carbon assimilation in this alga there is a decrease of electrical resistance of lipid membranes and development of negative membrane surface charge. The experimental results suggest that PCP toxicity to algae is due to adsorption of negatively charged PCP ions at the membrane surface that act as carriers of hydrogen ion across the membrane. This protonophoretic action of PCP causes the decrease of membrane electrical resistance and the dissipation of hydrogen ion electrochemical potential gradients across cellular and subcellular membranes, which reduces the ability of algae to assimilate carbon.     

Late Mousterian persistence near the Arctic Circle

Palaeolithic sites in Russian high latitudes have been considered as Upper Palaeolithic and thus representing an Arctic expansion of modern humans. Here we show that at Byzovaya, in the western foothills of the Polar Urals, the technological structure of the lithic assemblage makes it directly comparable with Mousterian Middle Palaeolithic industries that so far have been exclusively attributed to the Neandertal populations in Europe. Radiocarbon and optical-stimulated luminescence dates on bones and sand grains indicate that the site was occupied during a short period around 28,500 carbon-14 years before the present (about 31,000 to 34,000 calendar years ago), at the time when only Upper Palaeolithic cultures occupied lower latitudes of Eurasia. Byzovaya may thus represent a late northern refuge for Neandertals, about 1000 km north of earlier known Mousterian sites.     

Seasonal patterns of cellulase concentration in desert soil

The concentration of cellulose in plant material greatly affects the decomposition rate of plant-derived litter and hence carbon availability. The disappearance of pure cellulose in soil was studied as a measure of plant decomposition and carbon turnover. Our objective was to understand the effect of various cellulose concentrations and plant material added to soil and collected during different seasons, on cellulase concentrations under laboratory conditions (e.g. constant soil moisture and incubation temperature). The percentage of recovery of the enzyme in the control soil and in samples amended with known amounts of cellulose powder was estimated. Several methods for estimating soil cellulase concentrations/activity are available, most based on the determination of released reducing sugars. The method used in this study is based on the cleavage of a cellulose-azure substrate by cellulase to spectrophotometrically detectable fragments. Our results showed a significant correlation (p<0.05) between cellulose concentration and cellulase levels in soil, which varied along the study period. When pure cellulose was added to the soil, cellulase was detected after 7 days of incubation, whereas when plant material was added to the soil, cellulase was detected after 14 days. The recovery of cellulase from soil was also found to be seasonally dependent. The method of cellulase determination used in this study was simple, safe and rapid. From the results presented in this study, it can be assumed that there are seasonally dependent factors that affect the existence and concentration of cellulase in soils of the arid Negev Desert, in addition to organic matter, water and temperature.     

Identification of species-specific DNA in feedstuffs

Due to the menace of transmission of spongiform encephalopathies, feed components intended for ruminant nutrition must be checked for the presence of ruminant-derived materials. A sensitive method for the identification of bovine- and ovine- and also swine- and chicken-specific mitochondrial DNA sequences based on Polymerase Chain Reaction (PCR) has been developed. The specificity of the primers for PCR has been tested using samples of DNA of other vertebrate species, which may also be present in rendering plant products intended for feed manufacture. The method allows the detection in concentrate mixtures of 0.01% of the target species derived material. The identity of a sample containing 0.1% of bovine, ovine, swine, and chicken meat-and-bone meal has further been confirmed by sequencing.     

Manganese Biogeochemistry in a Central Czech Republic Catchment

Mn biogeochemistry was studied from 1994 to 2003 in a small forested catchment in the central Czech Republic using the watershed mass balance approach together with measurements of internal stores and fluxes. Mn inputs in bulk deposition were relatively constant during a period of sharply decreasing acidic deposition, suggesting that the Mn source was terrestrial, and not from fossil fuel combustion. Mn inputs in bulk deposition and Mn supplied by weathering each averaged 13 mg m^?2 year^?1 (26 mg m^?2 year^?1 total input), whereas Mn export in streamwater and groundwater averaged 43 mg m^?2 year^?1. Thus an additional Mn source is needed to account for 17 mg m^?2 year^?1. Internal fluxes and pools of Mn were significantly greater than annual inputs and outputs. Throughfall Mn flux was 70 mg m^?2 year^?1, litterfall Mn flux was 103 mg m^?2 year^?1, and Mn net uptake by vegetation was 62 mg m^?2 year^?1. Large pools of labile or potentially labile Mn were present in biomass and surficial soil horizons. Small leakages from these large pools likely supply the additional Mn needed to close the watershed mass balance. This leakage may reflect an adjustment of the ecosystem to recent changes in atmospheric acidity.