Thermodynamics of the interactions of tryptophan-rich cathelicidin antimicrobial peptides with model and natural membranes

Tritrpticin and indolicidin are short 13-residue tryptophan-rich antimicrobial peptides that hold potential as future alternatives for antibiotics. Isothermal titration calorimetry (ITC) has been applied as the main tool in this study to investigate the thermodynamics of the interaction of these two cathelicidin peptides as well as five tritrpticin analogs with large unilamellar vesicles (LUVs), representing model and natural anionic membranes. The anionic LUVs were composed of (a) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPE/POPG) (7:3) and (b) natural E. coli polar lipid extract. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was used to make model zwitterionic membranes. Binding isotherms were obtained to characterize the antimicrobial peptide binding to the LUVs, which then allowed for calculation of the thermodynamic parameters of the interaction. All peptides exhibited substantially stronger binding to anionic POPE/POPG and E. coli membrane systems than to the zwitterionic POPC system due to strong electrostatic attractions between the highly positively charged peptides and the negatively charged membrane surface, and results with tritrpticin derivatives further revealed the effects of various amino acid substitutions on membrane binding. No significant improvement was observed upon increasing the Tritrp peptide charge from +4 to +5. Replacement of Arg residues with Lys did not substantially change peptide binding to anionic vesicles but moderately decreased the binding to zwitterionic LUVs. Pro to Ala substitutions in tritrpticin, allowing the peptide to adopt an alpha-helical structure, resulted in a significant increase of the binding to both anionic and zwitterionic vesicles and therefore reduced the selectivity for bacterial and mammalian membranes. In contrast, substitution of Trp with other aromatic amino acids significantly decreased the peptide's ability to bind to anionic LUVs and essentially eliminated binding to zwitterionic LUVs. The ITC results were consistent with the outcome of fluorescence spectroscopy membrane binding and perturbation studies. Overall, our work showed that a natural E. coli polar lipid extract as a bacterial membrane model was advantageous compared to the simpler and more widely used POPE/POPG lipid system.     

Arginine vasopressin in fever: a still unsolved puzzle

(1) Administration of arginine vasopressin (AVP) in the ventral septal area (VSA) or intracerebroventricularly (i.c.v.) is thought to attenuate lipopolysaccharide (LPS) or prostaglandin (PG) E₂ fevers in rabbits and rats by acting on the V₁ receptor. (2) We found that the fever response of rabbits to intravenous LPS (200 ng/kg) or intra-VSA PGE₂ (500 ng) was not attenuated but enhanced by intra-VSA AVP (5 μg); a pharmacological analysis showed that this fever-enhancing effect was mediated by the V₂ receptor. (3) The febrile response of rats to intraperitoneal (50 μg/kg) or i.c.v. (100 ng) LPS was unaffected by i.c.v. AVP (2.5–100 ng). (4) The role of AVP in fever should be re-examined.     

A new UV-B absorbing mycosporine with photo protective activity from the lichenized ascomycete Collema cristatum.

A novel photo protective mycosporine was isolated from the lichenized ascomycete Collema cristatum. Biological activity was measured in terms of protection against UV-B induced membrane destruction and pyrimidine dimer formation in cultured human keratinocytes, and prevention of UV-B induced erythema. It was found that the pure isolated compound prevented UV-B induced cell destruction in a dose-dependent manner, that the compound partially prevented pyrimidine dimer formation and completely prevented UV-B induced erythema when applied to the skin prior to irradiation.     

Distinct Stages of Protein Evolution as Suggested by Protein Sequence Analysis

Evolution of proteins encoded in nucleotide sequences began with the advent of the triplet code. The chronological order of the appearance of amino acids on the evolution scene and the steps in the evolution of the triplet code have been recently reconstructed (Trifonov, 2000b) on the basis of 40 different ranking criteria and hypotheses. According to the consensus chronology, the pair of complementary GGC and GCC codons for the amino acids alanine and glycine appeared first. Other codons appeared as complementary pairs as well, which divided their respective amino acids into two alphabets, encoded by triplets with either central purines or central pyrimidines: G, D, S, E, N, R, K, Q, C, H, Y, and W (Glycine alphabet G) and A, V, P, S, L, T, I, F, and M (Alanine alphabet A). It is speculated that the earliest polypeptide chains were very short, presumably of uniform length, belonging to two alphabet types encoded in the two complementary strands of the earliest mRNA duplexes. After the fusion of the minigenes, a mosaic of the alphabets would form. Traces of the predicted mosaic structure have been, indeed, detected in the protein sequences of complete prokaryotic genomes in the form of weak oscillations with the period 12 residues in the form of alteration of two types of 6 residue long units. The next stage of protein evolution corresponded to the closure of the chains in the loops of the size 25–30 residues (Berezovsky et al., 2000). Autocorrelation analysis of proteins of 23 complete archaebacterial and eubacterial genomes revealed that the preferred distances between valine, alanine, glycine, leucine, and isoleucine along the sequences are in the same range of 25–30 residues, indicating that the loops are primarily closed by hydrophobic interactions between the ends of the loops. The loop closure stage is followed by the formation of typical folds of 100–200 amino acids, via end-to-end fusion of the genes encoding the loop-size chains. This size was apparently dictated by the optimal ring closure for DNA. In both cases the closure into the ring (loop) rendered evolutionarily advantageous stability to the respective structures. Further gene fusions lead to the formation of modern multidomain proteins. Recombinational gene splicing is likely to have appeared after the DNA circularization stage. Received: 21 December 2000 / Accepted: 28 February 2001     

Self-Regulation Phenomena Applied to Bacterial Reaction Centers: 2. Nonequilibrium Adiabatic Potential: Dark and Light Conformations Revisited

Experimental and theoretical results in support of nonlinear dynamic behavior of photosynthetic reaction centers under light-activated conditions are presented. Different conditions of light adaptation allow for preparation of reaction centers in either of two different conformational states. These states were detected both by short actinic flashes and by the switching of the actinic illumination level between different stationary state values. In the second method, the equilibration kinetics of reaction centers isolated from Rhodobacter sphaeroides were shown to be inherently biphasic. The fast and slow equilibration kinetics are shown to correspond to electron transfer (charge separation) at a fixed structure and to combined electron-conformational transitions governed by the bounded diffusion along the potential surface, respectively. The primary donor recovery kinetics after an actinic flash revealed a pronounced dependence on the time interval (Δt) between cessation of a lengthy preillumination of a sample and the actinic flash. A pronounced slow relaxation component with a decay half time of more than 50 s was measured for Δt > 10 s. This component corresponds to charge recombination in reaction centers for which light-induced structural changes have not relaxed completely before the flash. The amplitude of this component depended on the conditions of the sample preparation, specifically on the type of detergent used in the preparation. The redox potential parameters as well as the structural diffusion constants were estimated for samples prepared in different ways.     

Phosphatases and phosphodiesterases isolated from the red king crab (Paralithodes camtschatica) hepatopancreas

Five enzymes have been isolated from the hepatopancreas of the red king crab Paralithodes camtschatica by means of ion exchange and gel chromatography: two acid (AcP) and one alkaline (AlkP) phosphomonoesterases, one alkaline phosphodiesterase (AlkPI), and one acid phosphodiesterase (AcPD). The pH optimum values of these enzymes are: AlkPs and AlkPD, 7.5; AcP, 5.5; and AcPD, 5.0. The activity of AlkP and AlkPD demands Mg²⁺ ions. The molecular weights of the enzymes (kDa) are the following: AlkP, 80; AcPs, 80 and 82; AlkPD, 51; and AcPD, 57. The enzymes are relatively thermostable (ID 50 from 47 to 62°C). AlkP is inhibited by NaCl (IC 50 at 0.4 M). The AcP, AcPD, and AlkPD activities are tolerant of high ionic strength.     

Effects of food type, feeding frequency, and temperature on juvenile survival and growth of Marisa cornuarietis (Mollusca: Gastropoda)

Abstract. The present experiments are part of a larger study designed to investigate the influence of husbandry parameters on the life history of the ramshorn snail, Marisa cornuarietis, in order to identify suitable husbandry conditions for maintaining multi‐generation populations in the laboratory for use in ecotoxicological testing. In this paper we focus on the effects of a combination of food types and feeding frequencies (i.e., the frequency with which the snails were offered food) on juvenile growth and survival at different temperatures. Offspring produced in the laboratory by wild specimens of M. cornuarietis, from Puerto Rico, were used to test the effects of three types of food (lettuce, alginate with fish food, alginate with snail mix) fed at three frequencies (given ad libitum on 4/4, 2/4, or 1/4 d) on juvenile survival and growth. The 4‐d feeding regimens were repeated four times, giving a total of 16 d for the experiments. The experiments were conducted at two temperatures (22° and 25°C) under a 12 h light:12 h dark photoperiod. Juvenile growth rates increased with increasing feeding frequency for all food types. The most rapid growth rates occurred in the high‐frequency lettuce treatments and the slowest growth rates in the low‐frequency lettuce and alginate with snail mix treatments. Juvenile snails grew faster at 25° than at 22°C, and mortality was about twice as high at the lower temperature. Growth rates were used to provide a rough estimate of time to maturity, which was determined to take about twice as long at 22° than at 25°C. The results showed that lettuce is the best food if supplied in abundance, but effects on growth are very dependent on feeding frequency and temperature. We conclude that 25°C is a more appropriate temperature for maintaining populations than 22°C, that lettuce provides a suitable food source, and that food should be supplied continuously for husbandry and toxicity testing of populations of M. cornuarietis.