Genomic analysis of Pseudomonas aeruginosa phages LKD16 and LKA1: establishment of the phiKMV subgroup within the T7 supergroup

Lytic Pseudomonas aeruginosa phages LKD16 and LKA1 were locally isolated and morphologically classified as Podoviridae. While LKD16 adsorbs weakly to its host, LKA1 shows efficient adsorption (ka = 3.9 x 10(-9) ml min(-1)). LKA1, however, displays a narrow host range on clinical P. aeruginosa strains compared to LKD16. Genome analysis of LKD16 (43,200 bp) and LKA1 (41,593 bp) revealed that both phages have linear double-stranded DNA genomes with direct terminal repeats of 428 and 298 bp and encode 54 and 56 genes, respectively. The majority of the predicted structural proteins were experimentally confirmed as part of the phage particle using mass spectrometry. Phage LKD16 is closely related to bacteriophage phiKMV (83% overall DNA homology), allowing a more thoughtful gene annotation of both genomes. In contrast, LKA1 is more distantly related, lacking significant DNA homology and showing protein similarity to phiKMV in 48% of its gene products. The early region of the LKA1 genome has diverged strongly from phiKMV and LKD16, and intriguing differences in tail fiber genes of LKD16 and LKA1 likely reflect the observed discrepancy in infection-related properties. Nonetheless, general genome organization is clearly conserved among phiKMV, LKD16, and LKA1. The three phages carry a single-subunit RNA polymerase gene adjacent to the structural genome region, a feature which distinguishes them from other members of the T7 supergroup. Therefore, we propose that phiKMV represents an independent and widespread group of lytic P. aeruginosa phages within the T7 supergroup.     

Giant unilamellar vesicles electroformed from native membranes and organic lipid mixtures under physiological conditions

In recent years, giant unilamellar vesicles (GUVs) have become objects of intense scrutiny by chemists, biologists, and physicists who are interested in the many aspects of biological membranes. In particular, this "cell size" model system allows direct visualization of particular membrane-related phenomena at the level of single vesicles using fluorescence microscopy-related techniques. However, this model system lacks two relevant features with respect to biological membranes: 1), the conventional preparation of GUVs currently requires very low salt concentration, thus precluding experimentation under physiological conditions, and 2), the model system lacks membrane compositional asymmetry. Here we show for first time that GUVs can be prepared using a new protocol based on the electroformation method either from native membranes or organic lipid mixtures at physiological ionic strength. Additionally, for the GUVs composed of native membranes, we show that membrane proteins and glycosphingolipids preserve their natural orientation after electroformation. We anticipate our result to be important to revisit a vast variety of findings performed with GUVs under low- or no-salt conditions. These studies, which include results on artificial cell assembly, membrane mechanical properties, lipid domain formation, partition of membrane proteins into lipid domains, DNA-lipid interactions, and activity of interfacial enzymes, are likely to be affected by the amount of salt present in the solution.     

Characterization of chicken cystatin binding to rat renal brush-border membranes

Chicken cystatin, a homologue of human cystatin C, like other low-molecular-weight proteins is metabolized by renal proximal tubule cells. However, the precise mechanism(s) of this process has not been elucidated yet. To characterize chicken cystatin binding to renal brush-border membranes, the incubation of fluorescein labelled protein with rat cortical homogenate was performed. Saturation-dependent and reversible binding with low affinity (K_d = 3.67–4.07 μM) and high capacity (B_max = 2.32–2.79 nmol/mg) was observed. Bovine albumin was the most potent competitor (K_i = 0.7 μM) among other megalin/cubilin ligands tested. The presence of Ca^+ 2 ions was necessary to effective cystatin binding by brush-border membranes. Obtained data strongly support the hypothesis that chicken cystatin is a novel ligand for megalin/cubilin receptors tandem on proximal tubular cells.     

Structural basis of hierarchical multiple substates of a protein. II: Monte Carlo simulation of native thermal fluctuations and energy minimization

Conformational fluctuations in a globular protein, bovine pancreatic trypsin inhibitor, in the time range between picoseconds and nanoseconds are studied by a Monte Carlo simulation method. Multiple energy minima are derived from sampled conformations by minimizing their energy. They are distributed in clusters in the conformational space. A hierarchical structure is observed in the simulated dynamics. In the time range between 10(-14) and 10(-10) seconds dynamics is well represented by a superposition of vibrational motions within an energy well with transitions among minima within each cluster. Transitions among clusters take place in the time range of nanoseconds or longer.     

Classification method for disease risk mapping based on discrete hidden Markov random fields

Risk mapping in epidemiology enables areas with a low or high risk of disease contamination to be localized and provides a measure of risk differences between these regions. Risk mapping models for pooled data currently used by epidemiologists focus on the estimated risk for each geographical unit. They are based on a Poisson log-linear mixed model with a latent intrinsic continuous hidden Markov random field (HMRF) generally corresponding to a Gaussian autoregressive spatial smoothing. Risk classification, which is necessary to draw clearly delimited risk zones (in which protection measures may be applied), generally must be performed separately. We propose a method for direct classified risk mapping based on a Poisson log-linear mixed model with a latent discrete HMRF. The discrete hidden field (HF) corresponds to the assignment of each spatial unit to a risk class. The risk values attached to the classes are parameters and are estimated. When mapping risk using HMRFs, the conditional distribution of the observed field is modeled with a Poisson rather than a Gaussian distribution as in image segmentation. Moreover, abrupt changes in risk levels are rare in disease maps. The spatial hidden model should favor smoothed out risks, but conventional discrete Markov random fields (e.g. the Potts model) do not impose this. We therefore propose new potential functions for the HF that take into account class ordering. We use a Monte Carlo version of the expectation-maximization algorithm to estimate parameters and determine risk classes. We illustrate the method's behavior on simulated and real data sets. Our method appears particularly well adapted to localize high-risk regions and estimate the corresponding risk levels.     

Testing parasite ‘intimacy’: the whipworm Trichuris muris in the European house mouse hybrid zone

Host‐parasite interaction studies across hybrid zones often focus on host genetic variation, treating parasites as homogeneous. ‘Intimately’ associated hosts and parasites might be expected to show similar patterns of genetic structure. In the literature, factors such as no intermediate host and no free‐living stage have been proposed as ‘intimacy’ factors likely constraining parasites to closely follow the evolutionary history of their hosts. To test whether the whipworm, Trichuris muris, is intimately associated with its house mouse host, we studied its population genetics across the European house mouse hybrid zone (HMHZ) which has a strong central barrier to gene flow between mouse taxa. T. muris has a direct life cycle and nonmobile free stage: if these traits constrain the parasite to an intimate association with its host we expect a geographic break in the parasite genetic structure across the HMHZ. We genotyped 205 worms from 56 localities across the HMHZ and additionally T. muris collected from sympatric woodmice (Apodemus spp.) and allopatric murine species, using mt‐COX1, ITS1‐5.8S‐ITS2 rDNA and 10 microsatellites. We show four haplogroups of mt‐COX1 and three clear ITS1‐5.8S‐ITS2 clades in the HMHZ suggesting a complex demographic/phylogeographic history. Microsatellites show strong structure between groups of localities. However, no marker type shows a break across the HMHZ. Whipworms from Apodemus in the HMHZ cluster, and share mitochondrial haplotypes, with those from house mice. We conclude Trichuris should not be regarded as an ‘intimate’ parasite of the house mouse: while its life history might suggest intimacy, passage through alternate hosts is sufficiently common to erase signal of genetic structure associated with any particular host taxon.     

Linear, Single-Stranded Deoxyribonucleic Acid Isolated from Kilham Rat Virus

Kilham rat virus (KRV) was grown in a rat nephroma cell line and was purified by two isopycnic centrifugations in cesium chloride. The virus contains single-stranded deoxyribonucleic acid (DNA) with a molecular weight of approximately 1.6 x 10(6). The DNA was extracted from the virion by both phenol extraction and by 2% sodium dodecyl sulfate at 50 C. KRV DNA, extracted by both procedures, was observed in an electron microscope by using a cytochrome c or diethylaminoethyldextran monolayer. The DNA was also exposed to exonuclease I, an enzyme which hydrolyzes specifically linear, single-stranded DNA. Hydrolysis of 70 to 80% of the DNA was observed. Both the enzymatic and the electron microscope studies support the conclusion that extracted KRV DNA is a single-stranded, linear molecule. The length of the DNA was measured in the electron microscope and determined to be 1.505 +/- 0.206 mum.     

A locus for familial hypertrophic cardiomyopathy is closely linked to the cardiac myosin heavy chain genes, CRI-L436, and CRI-L329 on chromosome 14 at q11-q12

We report that a gene responsible for familial hypertrophic cardiomyopathy (HC) is closely linked to the cardiac alpha and beta myosin heavy chain (MHC) genes on chromosome 14q11. We have recently shown that probe CRI-L436, derived from the anonymous DNA locus D14S26, detects a polymorphic restriction fragment that segregates with familial HC in affected members of a large Canadian family. Using chromosomal in situ hybridization, we have mapped CRI-L436 to chromosome 14 at q11-q12. Because the cardiac MHC genes also map to this chromosomal band, we have determined the genetic distances between the cardiac beta MHC gene, D14S26, and the familial HC locus. Data presented here show that these three loci are linked within 5 centimorgans on chromosome 14 at q11-q12. The possibility that defects in either the cardiac alpha or beta MHC genes are responsible for familial HC is discussed.