Aggregation of cateslytin beta-sheets on negatively charged lipids promotes rigid membrane domains. A new mode of action for antimicrobial peptides?

Cateslytin, a positively charged (5+) arginine-rich antimicrobial peptide (bCgA, RSMRLSFRARGYGFR), was chemically synthesized and studied against membranes that mimic bacterial or mammalian systems. Circular dichroism, polarized attenuated total reflection infrared spectroscopy, (1)H high-resolution MAS NMR, and (2)H and (31)P solid state NMR were used to follow the interaction from peptide and membrane points of view. Cateslytin, which is unstructured in solution, is converted into antiparallel beta-sheets that aggregate mainly flat at the surface of negatively charged bacterial mimetic membranes. Arginine residues are involved in the binding to negatively charged lipids. Following the interaction of the cateslytin peptide, rigid and thicker membrane domains enriched in negatively charged lipids are found. Much less interaction is detected with neutral mammalian model membranes, as reflected by only minor percentages of beta-sheets or helices in the peptide secondary structure. No membrane destruction was detected for both bacterial and mammalian model membranes. A molecular model is proposed in which zones of different rigidity and thickness bring about phase boundary defects that ultimately lead to permeability induction and peptide crossing through bacterial membranes.     

Lithium fluxes in Paramecium and their relationship to chemoresponse.

Paramecia respond to environmental stimuli by altering swimming behavior to disperse from or accumulate in the vicinity of the stimulus. We have found, using the T-maze assay, that treatment of paramecia with LiCl in a time- and concentration-dependent manner modifies the normal response to folate, acetate, and lactate from attraction to no response or even repulsion. Responses to NH4Cl were unaffected and to cAMP were variably affected by LiCl. Cells incubated in the presence of K+, or both Na+ and K+, but not Na+ alone reliably recovered attraction to folate. Treatment of cells with 4 mM LiCl for 1 h dramatically slowed swimming speed from about 1 mm/s in NaCl or KCl (control) to 0.18 mm/s in LiCl. Li-treated cells subsequently incubated in 4 mM NaCl, KCl or sequentially in KCl and NaCl for a total of 20 min increased their swimming speed to 0.35, 0.45 and 0.67 mm/s, respectively. Paramecia readily took up Li+ in Na(+)- and K(+)-free media reaching intracellular concentrations of 5-10 mM in 10 mM extracellular Li+. Efflux of intracellular Li+ was stimulated 35% by extracellular 10 mM NaCl and 185% by 10 mM KCl over 10 mM choline chloride. Incubation of cells in 10 mM LiCl for 1 h inhibited the rate of Ca2+ efflux by 44% compared to cells in 10 mM NaCl. This may relate to the mechanism by which Li+ perturbs chemoresponse. A mutant with defects in Ca homeostasis responds normally to NH4Cl, but not to any of the stimuli that are affected by LiCl.     

Cholesterol synthesis in rat intestine: effect of fasting and of porphyrogenic chemical allylisopropylacetamide

(a) Administration of allylisopropylacetamide to fasting rats stimulates intestinal sterol synthesis as measured by incorporation of ¹⁴C from [1-¹⁴C]sodium acetate. Stimulatory effect of AIA is confined to the acetate to mevalonate segment of cholesterol biosynthetic pathway.(b) It is also shown that the suppression of sterol synthesis in the ileum of intact rats produced by fasting is of the same order of magnitude as that observed for liver sterol synthesis due to fasting.     

Using Classical Population Genetics Tools with Heterochroneous Data: Time Matters!

Background

New polymorphism datasets from heterochroneous data have arisen thanks to recent advances in experimental and microbial molecular evolution, and the sequencing of ancient DNA (aDNA). However, classical tools for population genetics analyses do not take into account heterochrony between subsets, despite potential bias on neutrality and population structure tests. Here, we characterize the extent of such possible biases using serial coalescent simulations.

Methodology/Principal Findings

We first use a coalescent framework to generate datasets assuming no or different levels of heterochrony and contrast most classical population genetic statistics. We show that even weak levels of heterochrony (∼10% of the average depth of a standard population tree) affect the distribution of polymorphism substantially, leading to overestimate the level of polymorphism θ, to star like trees, with an excess of rare mutations and a deficit of linkage disequilibrium, which are the hallmark of e.g. population expansion (possibly after a drastic bottleneck). Substantial departures of the tests are detected in the opposite direction for more heterochroneous and equilibrated datasets, with balanced trees mimicking in particular population contraction, balancing selection, and population differentiation. We therefore introduce simple corrections to classical estimators of polymorphism and of the genetic distance between populations, in order to remove heterochrony-driven bias. Finally, we show that these effects do occur on real aDNA datasets, taking advantage of the currently available sequence data for Cave Bears (Ursus spelaeus), for which large mtDNA haplotypes have been reported over a substantial time period (22–130 thousand years ago (KYA)).

Conclusions/Significance

Considering serial sampling changed the conclusion of several tests, indicating that neglecting heterochrony could provide significant support for false past history of populations and inappropriate conservation decisions. We therefore argue for systematically considering heterochroneous models when analyzing heterochroneous samples covering a large time scale.     

Complexes of [Re(CO)3Cl] with different oxidation states of the polyfunctional bmtz/H2bmtz ligand system (bmtz=3,6-bis(2-pyrimidyl)-1,2,4,5-tetrazine)

Combining fac-[Re(CO)₃Cl] with components of the ligand redox system bmtz/bmtz⁻/H₂bmtz/H₂bmtz⁻ (bmtz=3,6-bis(2-pyrimidyl)-1,2,4,5-tetrazine) has led to the isolation of the complexes (H₂bmtz)Re(CO)₃Cl, (μ-H₂bmtz)[Re(CO)₃Cl]₂ and (μ-bmtz)[Re(CO)₃Cl]₂. Other species characterized were (bmtz)Re(CO)₃Cl (UV/Vis, IR), [(H₂bmtz)Re(CO)₃Cl]⁻ (UV/Vis, IR, EPR), {(μ-H₂bmtz)[Re(CO)₃Cl]₂}⁻ (UV/Vis, IR, EPR) and {(μ-bmtz)[Re(CO)₃Cl]₂}⁻ (UV/Vis, IR, X band and high-field EPR). The results confirm bmtz as very strong and H₂bmtz as moderate π acceptor ligand versus one or two chelate-bonded low-valent metal centers. Reactivity is observed in terms of oxidative proton and reductive chloride dissociation.     

Increased myocardial oxygen consumption by TNF-alpha is mediated by a sphingosine signaling pathway

The present study investigated the effect of tumor necrosis factor (TNF)-alpha on myocardial energy metabolism as estimated by myocardial oxygen consumption (MVo(2)). MVo(2) of electrically stimulated isolated trabeculae of right ventricular Wistar rat myocardium was analyzed using a Clark-type oxygen probe. After the initial data collection in the absence of TNF-alpha, measurements were repeated after TNF-alpha stimulation. In separate experiments, pretreatment with the nitric oxide (NO) synthase inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME) or the ceramidase inhibitor n-oleoylethanolamine (NOE) was done to investigate NO/sphingosine-related effects. TNF-alpha impaired myocardial economy at increasing stimulation frequencies without altering baseline MVo(2). Incubation with TNF-alpha in the presence of l-NAME further impaired myocardial economy. NOE preincubation abrogated the TNF-alpha effect on myocardial economy. Moreover, the negative inotropic effect of TNF-alpha was observed in NOE-pretreated but not l-NAME-pretreated muscle fibers. Exogenous sphingosine mimicked the TNF-alpha effect on mechanics and energetics. We conclude that TNF-alpha impairs the economy of chemomechanical energy transduction primarily through a sphingosine-mediated pathway.     

Power of neutrality tests to detect bottlenecks and hitchhiking

The power of several neutrality tests to reject a simple bottleneck model is examined in a coalescent framework. Several tests are considered including some relying on the frequency spectrum of mutations and some reflecting the linkage disequilibrium structure of the data. We evaluate the effect of the age and of the strength of the bottleneck, and their interaction. We contrast two qualitatively different bottleneck effects depending on their strength. In genealogical terms, during severe bottlenecks, all lineages coalesce leading to a star-like gene genealogy of the sample. Some time after the bottleneck, once new mutations have arisen, they tend to show an excess of rare variants and a slight excess of haplotypes. On the contrary, more moderate bottlenecks allow several lineages to survive the demographic crash, leading to a balanced genealogy with long internal branches. Soon after the event, data tend to show an excess of intermediate frequency variants and a deficit of haplotypes. We show that for moderate sequencing efforts, severe bottlenecks can be detected only after an intermediate time period has allowed for mutations to occur, preferably by frequency spectrum statistics. Moderate bottlenecks can be more easily detected for more recent events, especially using haplotype statistics. Finally, for a single locus, the bottleneck results closely approximate those of a simple hitchhiking model. The main difference concerns the frequency distribution of mutations and haplotypes after moderate perturbations. Hitchhiking increases the number of rare ancestral mutations and leads to a more predominant major haplotype class. Thus, despite a number of common features between the two processes, hitchhiking cannot be strictly modeled by bottlenecks.     

Reliable microsatellite genotyping of the Eurasian badger (Meles meles) using faecal DNA

The potential link between badgers and bovine tuberculosis has made it vital to develop accurate techniques to census badgers. Here we investigate the potential of using genetic profiles obtained from faecal DNA as a basis for population size estimation. After trialling several methods we obtained a high amplification success rate (89%) by storing faeces in 70% ethanol and using the guanidine thiocyanate/silica method for extraction. Using 70% ethanol as a storage agent had the advantage of it being an antiseptic. In order to obtain reliable genotypes with fewer amplification reactions than the standard multiple-tubes approach, we devised a comparative approach in which genetic profiles were compared and replication directed at similar, but not identical, genotypes. This modified method achieved a reduction in polymerase chain reactions comparable with the maximum-likelihood model when just using reliability criteria, and was slightly better when using reliability criteria with the additional proviso that alleles must be observed twice to be considered reliable. Our comparative approach would be best suited for studies that include multiple faeces from each individual. We utilized our approach in a well-studied population of badgers from which individuals had been sampled and reliable genotypes obtained. In a study of 53 faeces sampled from three social groups over 10 days, we found that direct enumeration could not be used to estimate population size, but that the application of mark-recapture models has the potential to provide more accurate results.