Transgenetic studies implicate interactions between homologous PrP isoforms in scrapie prion replication

Transgenic (Tg) mice expressing both Syrian hamster (Ha) and mouse (Mo) prion protein (PrP) genes were used to probe the mechanism of scrapie prion replication. Four Tg lines expressing HaPrP exhibited distinct incubation times ranging from 48 to 277 days, which correlated inversely with HaPrP mRNA and HaPrPC. Bioassays of Tg brain extracts showed that the prion inoculum dictates which prions are synthesized de novo. Tg mice inoculated with Ha prions had approximately 10(9) ID50 units of Ha prions per gram of brain and less than 10 units of Mo prions. Conversely, Tg mice inoculated with Mo prions synthesized Mo prions but not Ha prions. Similarly, Tg mice inoculated with Ha prions exhibited neuropathologic changes characteristic of hamsters with scrapie, while Mo prions produced changes similar to those in non-Tg mice. Our results argue that species specificity of scrapie prions resides in the PrP sequence and prion synthesis is initiated by a species-specific interaction between PrPSc in the inoculum and homologous PrPC.     

Effect of grafted polyethylene glycol (PEG) on the size, encapsulation efficiency and permeability of vesicles

Liposomes have been prepared by the vesicle extrusion method (VETs) from mixtures of dipalmitoylphosphatidylcholine (DPPC), phosphatidylinositol (PI) and dipalmitoylphosphatidylethanolamine with covalently linked poly(ethylene glycol) molecular mass 5000 and 2000 (DPPE-PEG 5000 and DPPE-PEG 2000) covering a range of 0-7.5 mole%. The encapsulation of D-glucose has been studied and found to be markedly dependent on the mole% DPPE-PEG. The permeability of the liposomes to D-glucose has been measured both as a function of temperature and liposome composition. The permeability coefficients for D-glucose increase with mole% DPPE-PEG 5000 and with temperature over the range 25-50 degrees C. The activation energies for glucose permeability range from 90 to 23 kJ mol(-1). The decrease in activation energy with increasing temperature is attributed to an increasing number of bilayer defects as the liposome content of PEG-grafted lipid is increased. The dependence of D-glucose encapsulation as a function of PEG-grafted lipid content is discussed in terms of the conformation of the PEG molecules on the inner surface of the bilayer. For liposomes containing DPPE-PEG 5000 the relative percentage encapsulation of glucose, assuming that the PEG surface layer excludes glucose, is comparable to that predicted from the mushroom and brush conformational models.     

Human pendrin expressed in Xenopus laevis oocytes mediates chloride/formate exchange

Pendred syndrome,characterized by congenital sensorineural hearing loss and goiter, isone of the most common forms of syndromic deafness. The gene causingPendred syndrome (PDS) encodes a protein designated pendrin,which is expressed in the thyroid, kidney, and fetal cochlea. Pendrinfunctions as an iodide and chloride transporter, but its role in thedevelopment of hearing loss and goiter is unknown. In this study, weexamined the mechanism of pendrin-mediated anion transport inXenopus laevis oocytes. Unlabeled formate added to the uptakemedium inhibited pendrin-mediated ³⁶Cl uptake in X. laevis oocytes. In addition, the uptake of[¹⁴C]formate was stimulated in oocytes injected with PDScRNA compared with water-injected controls. These results indicate thatformate is a substrate for pendrin. Furthermore, chloride stimulatedthe efflux of [¹⁴C]formate and formatestimulated the efflux of ³⁶Cl in oocytes expressingpendrin, results consistent with pendrin-mediated chloride/formateexchange. These data demonstrate that pendrin is functionally similarto the renal chloride/formate exchanger, which serves as an importantmechanism of chloride transport in the proximal tubule. A similarprocess could participate in the development of ion gradients withinthe inner ear.

     

Inactivation of Isocitrate Lyase Leads to Increased Production of Medium-Chain-Length Poly(3-Hydroxyalkanoates) in Pseudomonas putida

Medium-chain-length (mcl) poly(3-hydroxyalkanoates) (PHAs) are storage polymers that are produced from various substrates and accumulate in Pseudomonas strains belonging to rRNA homology group I. In experiments aimed at increasing PHA production in Pseudomonas strains, we generated an mcl PHA-overproducing mutant of Pseudomonas putida KT2442 by transposon mutagenesis, in which the aceA gene was knocked out. This mutation inactivated the glyoxylate shunt and reduced the in vitro activity of isocitrate dehydrogenase, a rate-limiting enzyme of the citric acid cycle. The genotype of the mutant was confirmed by DNA sequencing, and the phenotype was confirmed by biochemical experiments. The aceA mutant was not able to grow on acetate as a sole carbon source due to disruption of the glyoxylate bypass and exhibited two- to fivefold lower isocitrate dehydrogenase activity than the wild type. During growth on gluconate, the difference between the mean PHA accumulation in the mutant and the mean PHA accumulation in the wild-type strain was 52%, which resulted in a significant increase in the amount of mcl PHA at the end of the exponential phase in the mutant P. putida KT217. On the basis of a stoichiometric flux analysis we predicted that knockout of the glyoxylate pathway in addition to reduced flux through isocitrate dehydrogenase should lead to increased flux into the fatty acid synthesis pathway. Therefore, enhanced carbon flow towards the fatty acid synthesis pathway increased the amount of mcl PHA that could be accumulated by the mutant.     

Influence of Salts on Virus Adsorption to Microporous Filters

We investigated the direct and indirect effects of mono-, di-, and trivalent salts (NaCl, MgCl₂, and AlCl₃) on the adsorption of several viruses (MS2, PRD-1, X174, and poliovirus 1) to microporous filters at different pH values. The filters studied included Millipore HA (nitrocellulose), Filterite (fiberglass), Whatman (cellulose), and 1MDS (charged-modified fiber) filters. Each of these filters except the Whatman cellulose filters has been used in virus removal and recovery procedures. The direct effects of added salts were considered to be the effects associated with the presence of the soluble salts. The indirect effects of the added salts were considered to be (i) changes in the pH values of solutions and (ii) the formation of insoluble precipitates that could adsorb viruses and be removed by filtration. When direct effects alone were considered, the salts used in this study promoted virus adsorption, interfered with virus adsorption, or had little or no effect on virus adsorption, depending on the filter, the virus, and the salt. Although we were able to confirm previous reports that the addition of aluminum chloride to water enhances virus adsorption to microporous filters, we found that the enhanced adsorption was associated with indirect effects rather than direct effects. The increase in viral adsorption observed when aluminum chloride was added to water was related to the decrease in the pH of the water. Similar results could be obtained by adding HCl. The increased adsorption of viruses in water at pH 7 following addition of aluminum chloride was probably due to flocculation of aluminum, since removal of flocs by filtration greatly reduced the enhancement observed. The only direct effect of aluminum chloride on virus adsorption that we observed was interference with adsorption to microporous filters. Under conditions under which hydrophobic interactions were minimal, aluminum chloride interfered with virus adsorption to Millipore, Filterite, and 1MDS filters. In most cases, less than 10% of the viruses adsorbed to filters in the presence of a multivalent salt and a compound that interfered with hydrophobic interactions (0.1% Tween 80 or 4 M urea).     

The inference of phased haplotypes for the immunoglobulin H chain V region gene loci by analysis of VDJ gene rearrangements

The existence of many highly similar genes in the lymphocyte receptor gene loci makes them difficult to investigate, and the determination of phased "haplotypes" has been particularly problematic. However, V(D)J gene rearrangements provide an opportunity to infer the association of Ig genes along the chromosomes. The chromosomal distribution of H chain genes in an Ig genotype can be inferred through analysis of VDJ rearrangements in individuals who are heterozygous at points within the IGH locus. We analyzed VDJ rearrangements from 44 individuals for whom sufficient unique rearrangements were available to allow comprehensive genotyping. Nine individuals were identified who were heterozygous at the IGHJ6 locus and for whom sufficient suitable VDJ rearrangements were available to allow comprehensive haplotyping. Each of the 18 resulting IGHV│IGHD│IGHJ haplotypes was unique. Apparent deletion polymorphisms were seen that involved as many as four contiguous, functional IGHV genes. Two deletion polymorphisms involving multiple contiguous IGHD genes were also inferred. Three previously unidentified gene duplications were detected, where two sequences recognized as allelic variants of a single gene were both inferred to be on a single chromosome. Phased genomic data brings clarity to the study of the contribution of each gene to the available repertoire of rearranged VDJ genes. Analysis of rearrangement frequencies suggests that particular genes may have substantially different yet predictable propensities for rearrangement within different haplotypes. Together with data highlighting the extent of haplotypic variation within the population, this suggests that there may be substantial variability in the available Ab repertoires of different individuals.     

Common variants of xeroderma pigmentosum genes and prostate cancer risk

The genetic basis of prostate cancer (PC) is complex and appears to involve multiple susceptibility genes. A number of studies have evaluated a possible correlation between several NER gene polymorphisms and PC risk, but most of them evaluated only single SNPs among XP genes and the results remain inconsistent. Out of 94 SNPs located in seven XP genes (XPA–XPG) a total of 15 SNPs were assayed in 720 unselected patients with PC and compared to 1121 healthy adults. An increased risk of disease was associated with the XPD SNP, rs1799793 (Asp312Asn) AG genotype (OR = 2.60; p < 0.001) and with the AA genotype (OR = 531; p < 0.0001) compared to the control population. Haplotype analysis of XPD revealed one protective haplotype and four associated with an increased disease risk, which showed that the A allele (XPD rs1799793) appeared to drive the main effect on promoting prostate cancer risk. Polymorphism in XPD gene appears to be associated with the risk of prostate cancer.     

Divergent post‐breeding distribution and habitat associations of fledgling and adult Black‐footed Albatrosses Phoebastria nigripes in the North Pacific

Past tracking studies of marine animals have primarily targeted adults, biasing our understanding of at‐sea habitat use toward older life stages. Anthropogenic threats persist throughout the at‐sea ranges of all life stages and it is therefore of interest to population ecologists and managers alike to understand spatiotemporal distributions and possible niche differentiation between age‐classes. In albatrosses, particularly little is known about the juvenile life stage when fledglings depart the colonies and venture to sea with no prior experience or parental guidance. We compared the dispersal of 22 fledgling Black‐footed Albatross Phoebastria nigripes between 2006 and 2008 using satellite telemetry and 16 adults between 2008 and 2009 using geolocaters from Midway Atoll National Wildlife Refuge, Northwest Hawaiian Islands. Following tag deployment, all fledglings spent several days within the calm atoll waters, then travelled northward until reaching 750–900 km from the colony. At this point, fledgling distributions approached the productive North Pacific Transition Zone (NPTZ). Rather than reaching the high chlorophyll a densities on the leading edge of this zone, however, fledglings remained in areas of low productivity in the subtropical gyre. In contrast, adult albatrosses from the same breeding colony did not utilize the NPTZ at this time of year but rather ranged throughout the highly productive northern periphery of the Pacific Ocean Basin among the shelf regions off Japan and the Aleutian Islands. The dichotomy in habitat use between fledglings and adults from Midway Atoll results in complete spatial segregation between age‐classes and suggests ontogenetic niche separation in this species. This research fills a large knowledge gap in at‐sea habitat use during a little known yet critical life stage of albatrosses, and contributes to a more comprehensive understanding of differential mortality pressure between age‐classes and overall conservation status for the vulnerable Black‐footed Albatross.     

Fin-tail coordination during escape and predatory behavior in larval zebrafish

Larval zebrafish innately perform a suite of behaviors that are tightly linked to their evolutionary past, notably escape from threatening stimuli and pursuit and capture of prey. These behaviors have been carefully examined in the past, but mostly with regard to the movements of the trunk and tail of the larvae. Here, we employ kinematics analyses to describe the movements of the pectoral fins during escape and predatory behavior. In accord with previous studies, we find roles for the pectoral fins in slow swimming and immediately after striking prey. We find novel roles for the pectoral fins in long-latency, but not in short-latency C-bends. We also observe fin movements that occur during orienting J-turns and S-starts that drive high-velocity predatory strikes. Finally, we find that the use of pectoral fins following a predatory strike is scaled to the velocity of the strike, supporting a role for the fins in braking. The implications of these results for central control of coordinated movements are discussed, and we hope that these results will provide baselines for future analyses of cross-body coordination using mutants, morphants, and transgenic approaches.