Rheological studies on mixed phospholipid sols and their interaction with penicillins I. Continuous shear viscometry

The effects of sonication and ageing of phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) sols have been examined. Ageing was found to significantly alter the nature of the flow curves obtained for sonicated mixtures; only after 48 hr was reproducibility obtained. Rheological studies on aged mixed sols indicated behaviour similar to that previously reported. Addition of urea and guanidine hydrochloride in concentrations up to 8 M indicated hydrophobic interaction between PC and LPC.

Addition of Penicillin G and Ampicillin to mixed PC/LPC sols indicated that Penicillin G interacted to a greater degree than Ampicillin; pronounced Penicillin G interaction was observed in a mixed sol containing 60% PC, 40% LPC, a system where the asymmetry of the aggregates is at a minimum. Ampicillin appeared to interact to a greater degree with mixtures containing more LPC than PC. Differences in hydrophobic interaction were indicated by use of urea and guanidine hydrochloride, which is of interest in the light of present knowledge regarding the in vivo activity of these antibiotics.     

Modeling extent and distribution of zygotic disequilibrium: implications for a multigenerational canine pedigree

Unlike gametic linkage disequilibrium defined for a random-mating population, zygotic disequilibrium describes the nonrandom association between different loci in a nonequilibrium population that deviates from Hardy-Weinberg equilibrium. Zygotic disequilibrium specifies five different types of disequilibria simultaneously that are (1) Hardy-Weinberg disequilibria at each locus, (2) gametic disequilibrium (including two alleles in the same gamete, each from a different locus), (3) nongametic disequilibrium (including two alleles in different gametes, each from a different locus), (4) trigenic disequilibrium (including a zygote at one locus and an allele at the other), and (5) quadrigenic disequilibrium (including two zygotes each from a different locus). However, because of the uncertainty on the phase of the double heterozygote, gametic and nongametic disequilibria need to be combined into a composite digenic disequilibrium and further define a composite quadrigenic disequilibrium together with the quadrigenic disequilibrium. To investigate the extent and distribution of zygotic disequilibrium across the canine genome, a total of 148 dogs were genotyped at 247 microsatellite markers located on 39 pairs of chromosomes for an outbred multigenerational pedigree, initiated with a limited number of unrelated founders. A major portion of zygotic disequilibrium was contributed by the composite digenic and quadrigenic disequilibrium whose values and numbers of significant marker pairs are both greater than those of trigenic disequilibrium. All types of disequilibrium are extensive in the canine genome, although their values tend to decrease with extended map distances, but with a greater slope for trigenic disequilibrium than for the other types of disequilibrium. Considerable variation in the pattern of disequilibrium reduction was observed among different chromosomes. The results from this study provide scientific guidance about the determination of the number of markers used for whole-genome association studies.     

Fatty acid amide hydrolase inhibitors. Surprising selectivity of chiral azetidine ureas

We report the discovery of a novel, chiral azetidine urea inhibitor of Fatty Acid Amide Hydrolase (FAAH,) and describe the surprising species selectivity of VER-156084 versus rat and human FAAH and also hCB1.     

Structure-Function Analysis of Inositol Hexakisphosphate-induced Autoprocessing in Clostridium difficile Toxin A

The action of Clostridium difficile toxins A and B depends on inactivation of host small G-proteins by glucosylation. Cellular inositol hexakisphosphate (InsP6) induces an autocatalytic cleavage of the toxins, releasing an N-terminal glucosyltransferase domain into the host cell cytosol. We have defined the cysteine protease domain (CPD) responsible for autoprocessing within toxin A (TcdA) and report the 1.6 Å x-ray crystal structure of the domain bound to InsP6. InsP6 is bound in a highly basic pocket that is separated from an unusual active site by a β-flap structure. Functional studies confirm an intramolecular mechanism of cleavage and highlight specific residues required for InsP6-induced TcdA processing. Analysis of the structural and functional data in the context of sequences from similar and diverse origins highlights a C-terminal extension and a π-cation interaction within the β-flap that appear to be unique among the large clostridial cytotoxins.Clostridium difficile is a Gram-positive, spore-forming anaerobe that infects the colon and causes a range of disorders, including diarrhea, pseudomembranous colitis, and toxic megacolon (1, 2). Two large toxins, TcdA² and TcdB (308 and 270 kDa, respectively) are recognized as the main virulence factors of C. difficile, although their relative importance is the subject of on-going study (3, 4). These proteins belong to a class of homologous toxins called large clostridial toxins (LCTs) and have been classified more broadly as AB toxins, wherein a B moiety is involved in the delivery of an enzymatic A moiety into the cytosol of a target cell. In LCTs, the A subunit is an N-terminal glucosyltransferase that inactivates small G-proteins, such as Rho, leading to cell rounding and apoptosis of the intoxicated cell (5, 6). The B subunit corresponds to the remainder of the toxin and is responsible for binding the target cell through a C-terminal receptor-binding domain (7–9) and forming the membrane pore needed for translocation of the A subunit (10, 11). Unlike other known AB toxins, the glucosyltransferase A domains of LCTs are released from the B subunits by an autoproteolytic cleavage event (12). Cleavage is triggered by host inositol phosphates and the reducing environment of the cytosol (12).In LCTs, autoproteolysis has been attributed to a cysteine protease activity located within the N-terminal region of the B subunit (13). This region was identified based on homology with the cysteine protease domain (CPD) found in the multifunctional autoprocessing repeats in toxins (MARTX) toxins from Gram-negative bacteria (14). Autoprocessing in the MARTX toxin from Vibrio cholera (VcRTx) is also stimulated by InsP6 (15). A recent crystal structure of VcRTx CPD bound to InsP6 suggests a novel mechanism of InsP6-induced allosteric activation (16). The CPDs of TcdA and VcRTx share only 19% sequence identity. To gain insight into the mechanistic commonalities between these entirely different toxins and to delineate the LCT-specific modes of InsP6-induced processing, we performed structural and functional analyses on the cysteine protease from TcdA.     

Tracking macaroni penguins during long foraging trips using ‘behavioural geolocation’

The movement of marine vertebrates has been tracked using a variety of techniques, all of which depend on the external attachment of a transmitting or recording device. However, these devices can have negative effects on the subject animals, limiting both the quantity and quality of data collected. We present a new method for monitoring large-scale movement of marine vertebrates that uses behavioural data stored on a surgically implanted data logger. The technique (‘behavioural geolocation’) relies on the principles of light-based geolocation but rather than measuring ambient light levels, changes in diving behaviour associated with sunrise and sunset are used to infer daylength and time of local sunrise, and hence location. We present data from a trial, post-hoc, analysis of diving data collected from macaroni penguins Eudyptes chrysolophus during long foraging trips associated with incubation and preparation for moult. Our results showed that the penguins usually travelled to the polar frontal zone to the north of their breeding colony at South Georgia, an area broadly consistent with previously measured behaviour and the availability of preferred prey at this period of the annual cycle.     

Application of the self-organizing map as a prediction tool for an integrated constructed wetland agroecosystem treating agricultural runoff

A self-organizing map (SOM) model was applied as a prediction tool for the performance of an integrated constructed wetland (ICW) agroecosystem treating agricultural runoff to protect receiving watercourses. By utilizing the SOM model, the time-consuming to measure expensive biochemical oxygen demand outflow concentrations were predicted well by other inexpensive variables, which were quicker and easier to measure. Correct predictions for the outflow biochemical oxygen demand concentrations were between 89% and 100%. This novel approach allows for the real time control of the outflow water quality of the ICW and potentially also of other treatment system applications. Moreover, the missing values and outliers from the large but incomplete ICW data set were replaced accurately by most likely values determined by the SOM model. This was important because the proportions of unusable entries for chemical oxygen demand, suspended solids and biochemical oxygen demand were very high: 41%, 54%, and 61%, respectively.     

Trans-species polymorphism of the Mhc class II DRB-like gene in banded penguins (genus Spheniscus)

The Major Histocompatibility Complex (Mhc) class II DRB locus of vertebrates is highly polymorphic and some alleles may be shared between closely related species as a result of balancing selection in association with resistance to parasites. In this study, we developed a new set of PCR primers to amplify, clone, and sequence overlapping portions of the Mhc class II DRB-like gene from the 5′UTR end to intron 3, including exons 1, 2, and 3 and introns 1 and 2 in four species (20 Humboldt, six African, five Magellanic, and three Galapagos penguins) of penguin from the genus Spheniscus (Sphe). Analysis of gene sequence variation by the neighbor-joining method of 21 Sphe sequences and 20 previously published sequences from four other penguin species revealed overlapping clades within the Sphe species, but species-specific clades for the other penguin species. The overlap of the DRB-like gene sequence variants between the four Sphe species suggests that, despite their allopatric distribution, the Sphe species are closely related and that some shared DRB1 alleles may have undergone a trans-species inheritance because of balancing selection and/or recent rapid speciation. The new primers and PCR assays that we have developed for the identification of the DRB1 DNA and protein sequence variations appear to be useful for the characterization of the molecular evolution of the gene in closely related Penguin species and might be helpful for the assessment of the genetic health and the management of the conservation and captivity of these endangered species. The nucleotide sequence and amino acid sequence data reported in this paper have been submitted to the DDBJ database and have been assigned the accession numbers AB301478, AB301944–AB301950, AB302087–AB302090, AB302190–AB302192, AB302843, AB302844, and AB303942–AB303945.