Binding of LL-37 to model biomembranes: insight into target vs host cell recognition

Pursuing the molecular mechanisms of the concentration dependent cytotoxic and hemolytic effects of the human antimicrobial peptide LL-37 on cells, we investigated the interactions of this peptide with lipids using different model membranes, together with fluorescence spectroscopy for the Trp-containing mutant LL-37(F27W). Minimum concentrations inhibiting bacterial growth and lipid interactions assessed by dynamic light scattering and monolayer penetration revealed the mutant to retain the characteristics of native LL-37. Although both LL-37 and the mutant intercalated effectively into zwitterionic phosphatidylcholine membranes the presence of acidic phospholipids caused augmented membrane binding. Interestingly, strongly attenuated intercalation of LL-37 into membranes containing both cholesterol and sphingomyelin (both at X=0.3) was observed. Accordingly, the distinction between target and host cells by LL-37 is likely to derive from i) acidic phospholipids causing enhanced association with the former cells as well as ii) from attenuated interactions with the outer surface of the plasma membrane of the peptide secreting host, imposed by its high content of cholesterol and sphingomyelin. Our results further suggest that LL-37 may exert its antimicrobial effects by compromising the membrane barrier properties of the target microbes by a mechanism involving cytotoxic oligomers, similarly to other peptides forming amyloid-like fibers in the presence of acidic phospholipids.     

Microplate screening assay for binding of ligands to bovine or reindeer beta-lactoglobulins

Several analytical methods have been used to determine whether ligands bind to bovine beta-lactoglobulin (betaLG). The most common methods are based on fluorescence quenching. We have miniaturised this method from a quartz cell to a 96-well plate. The miniaturisation was evaluated using retinol. The binding constants between the two methods demonstrated a good correlation. The 96-well plate method is much faster and allows many references to be used in the same analysis. The miniaturised method was used to study the binding of three different ligands (4-HPR, arotinoid, warfarinyl palmitate) modelled to bind to betaLG. The binding data showed that all of these ligands bound to betaLG. The method was further used to demonstrate that reindeer betaLG could also bind the four ligands in the same way as bovine betaLG. Because one aim is to use bovine and reindeer betaLG as a binder molecule for aliments in e.g. functional food or for drugs, the influence of pH was also studied and demonstrated that short-term acidic conditions had only a slight effect on the binding properties.     

Symbiotic nitrogen fixation in eight Acacia senegal provenances in dryland clays of the Blue Nile Sudan estimated by the 15N natural abundance method

The symbiotic biological N₂fixation by Acacia senegal was estimated using the ¹⁵N natural abundance (δ ¹⁵N) procedure on eight provenances collected from different environments and soil types grown in a clay soil in the Blue Nile region, Sudan. Balanites aegyptiaca (a non-legume) was used as a non-N₂-fixing reference plant to allow ¹⁵N-based estimates of the proportion of the Acacia N derived from atmospheric N₂ (N_dfa) to be calculated. Results show variation in leaf δ ¹⁵N between A. senegal and the reference plant and among years. The relative δ ¹⁵N values (‰) were higher in B. aegyptiaca than in the N₂-fixing acacia provenances. Provenances originally collected from clay soils fixed little N in the first year, but the amount fixed increased as the trees aged. All provenances showed a decrease in δ ¹⁵N with age. The N_dfa varied between 24% (Mazmoom provenance) and 61% (Rahad provenance) 4 years after planting. There was no significant difference in δ ¹⁵N between provenance groups based on soil type or rainfall at original growing site. The amount of N_dfa increased significantly with age in all provenances. The above-ground contribution of fixed N to foliage growth in a 4-year-old A. senegal was highest in the Rahad sand–soil provenance (46.7 kg N ha⁻¹) and lowest in the Mazmoom clay-soil provenance (28.7 kg N ha⁻¹). Our study represents the first use of the δ ¹⁵N method for estimating the N input by A. senegal to the clay plain soils of the gum belt in the Sudan.     

Young flying squirrels (Pteromys volans) dispersing in fragmented forests

Dispersal is a key determinant of the population dynamics ofspecies. Thus, a better understanding of how dispersal is affectedby the landscape structure and how animals make decisions aboutmoving across different landscapes is needed. We studied thedispersal of 60 radio-collared juvenile Siberian flying squirrels(Pteromys volans) in southern Finland. The effect of landscapestructure on selected dispersal direction, dispersal distance,and straightness of dispersal path was studied. Flying squirrelswere capable of dispersing over long distances in fragmentedforest landscapes. The patches used as temporary roosting sitesduring dispersal were of a lower quality than were those usedas finally occupied patches. The patches used were larger thanwere patches on average in the study areas. There was a veryclear directional bias in the dispersal path (i.e., it was nearlya straight line), which remained over a large scale, but wide-openareas obstructed the straightness of the path. As the distancesbetween crossed patches increased, short-distance disperserswere found further away from their natal home range. However,there were no differences in the landscape that could explainthe differences between individuals in decisions to remain philopatricor to become short- or long-distance dispersers. In addition,whereas short-distance dispersers dispersed in random directions,long-distance dispersers started to disperse in directions dominatedby preferred habitat. Thus, there were behavioral differencesbetween dispersers. Our results supported the hypotheses statingthat individuals decide to disperse long or short distancesbefore the onset of dispersal.     

Experimental increase of predation risk induces breeding dispersal of Tengmalm's owl

Nest predation and its avoidance are critical components of an individual's fitness and play an important role in life history evolution. Almost all studies on this topic have been observational, and thus have not been able to separate the effects of individual quality, habitat selection and predation risk of given nest sites from each other. More experimental studies on nest predation and breeding dispersal, therefore, are needed to avoid confusing interpretations of the results. In western Finland, pine marten (Martes martes) predation risk was experimentally simulated at the nests of Tengmalm's owls (Aegolius funereus) by using a caged American mink (Mustela vison) as a predator. Nests without exposure to a mink served as controls. In accordance with our predictions and earlier observational studies, males exposed to simulated predation risk increased nest-hole shift and breeding dispersal distances compared to control males. Nest-hole shift and long breeding dispersal distances probably decrease the risk of nest predation, because pine martens are known to revisit nest-holes they have found.     

Generation of mice with a conditional allele for the p75(NTR) neurotrophin receptor gene

The p75(NTR) neurotrophin receptor has been implicated in multiple biological and pathological processes. While significant advances have recently been made in understanding the physiologic role of p75(NTR) , many details and aspects remain to be determined. This is in part because the two existing knockout mouse models (Exons 3 or 4 deleted, respectively), both display features that defy definitive conclusions. Here we describe the generation of mice that carry a conditional p75(NTR) (p75(NTR-FX) ) allele made by flanking Exons 4-6, which encode the transmembrane and all cytoplasmic domains, by loxP sites. To validate this novel conditional allele, both neural crest-specific p75(NTR) /Wnt1-Cre mutants and conventional p75(NTR) null mutants were generated. Both mutants displayed abnormal hind limb reflexes, implying that loss of p75(NTR) in neural crest-derived cells causes a peripheral neuropathy similar to that seen in conventional p75(NTR) mutants. This novel conditional p75(NTR) allele will offer new opportunities to investigate the role of p75(NTR) in specific tissues and cells.     

A vertebrate model for the study of lipid binding/transfer protein function: Conservation of OSBP-related proteins between zebrafish and human

Oxysterol-binding protein (OSBP) and OSBP-related (ORP) or OSBP-like (OSBPL) proteins constitute a family of lipid-binding/transfer proteins (LTPs) present in eukaryotes from yeast to man. The mechanisms of ORP function have remained incompletely understood. However, several ORPs are present at membrane contact sites and act as either lipid transporters or sensors that control lipid metabolism, cell signaling, and vesicle transport. Zebrafish, Danio rerio, has gained increasing popularity as a model organism in developmental biology, human disease, toxicology, and drug discovery. However, LTPs in the fish are thus far unexplored. In this article we report a series of bioinformatic analyses showing that the OSBPL gene family is highly conserved between the fish and human. The OSBPL subfamily structure is markedly similar between the two organisms, and all 12 human genes have orthologs, designated osbpl and located on 11 chromosomes in D. rerio. Interestingly, osbpl2 and osbpl3 are present as two closely related homologs (a and b), due to gene duplication events in the teleost lineage. Moreover, the domain structures of the distinct ORP proteins are almost identical between zebrafish and man, and molecular modeling in the present study suggests that ORD liganding by phosphatidylinositol-4-phosphate (PI4P) is a feature conserved between yeast Osh3p, human ORP3, and zebrafish Osbpl3. The present analysis identifies D. rerio as an attractive model to study the functions of ORPs in vertebrate development and metabolism.     

The responsiveness of TrkB to BDNF and antidepressant drugs is differentially regulated during mouse development

Background

Previous studies suggest that the responsiveness of TrkB receptor to BDNF is developmentally regulated in rats. Antidepressant drugs (AD) have been shown to increase TrkB signalling in the adult rodent brain, and recent findings implicate a BDNF-independent mechanism behind this phenomenon. When administered during early postnatal life, ADs produce long-lasting biochemical and behavioural alterations that are observed in adult animals.

Methodology

We have here examined the responsiveness of brain TrkB receptors to BDNF and ADs during early postnatal life of mouse, measured as autophosphorylation of TrkB (pTrkB).

Principal Findings

We found that ADs fail to induce TrkB signalling before postnatal day 12 (P12) after which an adult response of TrkB to ADs was observed. Interestingly, there was a temporally inverse correlation between the appearance of the responsiveness of TrkB to systemic ADs and the marked developmental reduction of BDNF-induced TrkB in brain microslices ex vivo. Basal p-TrkB status in the brain of BDNF deficient mice was significantly reduced only during early postnatal period. Enhancing cAMP (cyclic adenosine monophosphate) signalling failed to facilitate TrkB responsiveness to BDNF. Reduced responsiveness of TrkB to BDNF was not produced by the developmental increase in the expression of dominant-negative truncated TrkB.T1 because this reduction was similarly observed in the brain microslices of trkB.T1 ^−/− mice. Moreover, postnatal AD administration produced long-lasting behavioural alterations observable in adult mice, but the responses were different when mice were treated during the time when ADs did not (P4-9) or did (P16-21) activate TrkB.

Conclusions

We have found that ADs induce the activation of TrkB only in mice older than 2 weeks and that responsiveness of brain microslices to BDNF is reduced during the same time period. Exposure to ADs before and after the age when ADs activate TrkB produces differential long-term behavioural responses in adult mice.     

Activation of the β-carbonic anhydrase from the protozoan pathogen Trichomonas vaginalis with amines and amino acids

We report the first activation study of the β-class carbonic anhydrase (CA, EC 4.2.1.1) encoded in the genome of the protozoan pathogen Trichomonas vaginalis, TvaCA1. Among 24 amino acid and amine activators investigated, derivatives incorporating a second carboxylic moiety, such as L-Asp, L- and D-Glu, were devoid of activating effects up to concentrations of 50 µM within the assay system, whereas the corresponding compounds with a CONH₂ moiety, i.e. L-Gln and L-Asn showed modest activating effects, with activation constants in the range of 26.9 − 32.5 µM. Moderate activation was observed with L- and D-DOPA, histamine, dopamine, serotonin, (2-Aminoethyl)pyridine/piperazine and morpholine (K_A‘s ranging between 8.3 and 14.5 µM), while the best activators were L-and D-Trp, L-and D-Tyr and 4-amino-Phe, which showed K_A‘s ranging between 3.0 and 5.1 µM. Understanding in detail the activation mechanism of β-CAs may be relevant for the design of enzyme activity modulators with potential clinical significance.