Eight new microsatellite markers in Atlantic cod (Gadus morhua L.) derived from an enriched genomic library

One hundred and fifty random clones from an enriched genomic library of Atlantic cod were sequenced. Primer pairs were designed for 15 microsatellites containing perfect di‐, tri‐, tetra‐ and hexanucleotide repeats and characterized in 96 unrelated fish. Eight markers were successfully amplified, with the number of alleles ranging from two to nine per locus and observed heterozygosity ranging from 0.341 to 0.977. Loci Gmo‐G13 and Gmo‐G14 had a significant excess of homozygotes. All loci conformed to the Hardy–Weinberg equilibrium. Genetic linkage disequilibrium analysis between all pairs of the loci showed no significant departure from the null hypothesis between any of the loci.     

Combination of abundant protein depletion and multi-lectin affinity chromatography (M-LAC) for plasma protein biomarker discovery

We report on the development of a robust and relatively high-throughput method for in-depth proteomic analysis of human plasma suitable for biomarker discovery. The method consists of depletion of albumin and IgG and multi-lectin affinity chromatography (M-LAC), followed by nanoLC-MS/MS analysis of digested proteins and label-free comparative quantitation of proteins. The performance of the method is monitored by multiple quality control points to ensure reproducibility of the analysis. The method identifies proteins that are reported to be present in normal plasma at concentrations of 10-100 ng/mL and that may be of particular interest when studying a variety of disease conditions. Numerous tissue leakage proteins of potentially even lower concentrations are also identified. When the method was used in a study to identify potential biomarkers of psoriasis, the differential abundance of proteins present at low mug/mL level was quantitated and later verified by ELISA measurements.     

Dynamics of Membrane-Bound G12V-KRAS from Simulations and Single-Molecule FRET in Native Nanodiscs

Recent studies have shown that the small GTPase KRAS adopts multiple orientations with respect to the plane of anionic model membranes, whereby either the three C-terminal helices or the three N-terminal β-strands of the catalytic domain face the membrane. This has functional implications because, in the latter, the membrane occludes the effector-interacting surface. However, it remained unclear how membrane reorientation occurs and, critically, whether it occurs in the cell in which KRAS operates as a molecular switch in signaling pathways. Herein, using data from a 20 μs-long atomistic molecular dynamics simulation of the oncogenic G12V-KRAS mutant in a phosphatidylcholine/phosphatidylserine bilayer, we first show that internal conformational fluctuations of flexible regions in KRAS result in three distinct membrane orientations. We then show, using single-molecule fluorescence resonance energy transfer measurements in native lipid nanodiscs derived from baby hamster kidney cells, that G12V-KRAS samples three conformational states that correspond to the predicted orientations. The combined results suggest that relatively small energy barriers separate orientation states and that signaling-competent conformations dominate the overall population.     

Effects of herbivorous snails and macroalgal canopy on recruitment and early survivorship of the barnacle Semibalanus balanoides (L.)

This study investigated the effects of grazing by the herbivorous snail Littorina littorea (L.) and the presence of a macroalgal canopy on recruitment and early survivorship of the barnacle Semibalanus balanoides (L.) at four sites of various distances from the mouth of Toothacher Cove on Swans Island in the Gulf of Maine in 1998. Recruitment plates were attached to the substrate at each site in all possible treatment combinations, including either presence of absence of macroalgal canopy, presence or absence of the herbivore, and presence or absence of a cage. Significant differences in recruitment were observed among sites in April and May, but not in June. The average number of recruits also varied significantly among herbivore treatments in May and June, and in the May macroalgaexherbivore interaction. Survivorship did not differ among sites, but did vary significantly among herbivore treatments. These differences were due almost entirely to consistently low recruitment and survival in the uncaged or open controls. Comparisons of the caged and open treatments explained between 88 and 99% of the variation in the respective main effects tests. The presence or absence of L. littorea or of macroalgal canopy had little effect on S. balanoides recruitment or survivorship. Results support previous conclusions that recruitment is greater on more exposed shores and suggest that exclusion cages may protect barnacle recruits from factors such as desiccation and the whiplash effect of macroalgal fronds.     

Immunoassay for viable Cryptosporidium parvum oocysts in turbid environmental water samples

Cryptosporidium parvum oocysts in drinking water have been implicated in outbreaks of diarrheal disease. Current methods for monitoring environmental exposures to C. parvum only account for total number of oocysts without regard for the viability of the parasite. Measurement of oocyst viability, as indicated by an oocyst's ability to excyst, is useful because over time oocysts lose the ability to excyst and become noninfective. Thus, correlating the number of viable oocysts in drinking water with incidence and risk for disease should be more reliable than using the total number of oocysts. We have developed a quantitative assay capable of detecting low numbers of excystable, sporozoite-releasing C. parvum oocysts in turbid water samples. Monoclonal (CP7) and polyclonal antibodies have been developed against a sporozoite antigen released only during excystation or when the oocyst is mechanically disrupted. CP7 is specific for C. parvum and does not react with C. baileyi, C. muris, C. serpentis, Giardia spp., Eimeria spp., or E. nieschulzi. In this assay, oocysts in the test sample are first excysted and then centrifuged. The soluble sporozoite antigen is captured by CP7 attached to a magnetic bead. The captured antigen is then detected by ruthenium-labeled polyclonal antibodies via electrochemiluminescence. The CP7 viability assay can detect as few as 50 viable oocysts in a 1-ml assay sample with a turbidity as high as 200 Nephelometric turbidity units. This sensitive, turbidity-tolerant assay for oocyst viability may permit a better assessment of the disease risk associated with the presence of environmental oocysts.     

A bigger mouse? The rat genome unveiled

Rattus norvegicus is an important experimental organism and interesting to evolutionary biologists. The recently published draft rat genome sequence provides us with insights into both the rat's evolution and its physiology. We learn more about genome evolution and, in particular, the adaptive significance of gene family expansions and the evolution of rodent genomes, which appears to have decelerated since the divergence of mouse and rat. An important observation is that some regions of genomes, many in noncoding regions, show very high sequence conservation, while others show unexpectedly fast evolution. Both of these may be pointers to functional significance.     

Floral biology, pollination and fertilisation in temperate zone fruit species and grape. * Kozma P, Nyeki J, Soltesz M, Szabo Z. 2003. * Budapest: Akademiai Kiado. $98 (hardback).621 pp.

The primary focus of this book is to discuss the floral biologyof the fruit crops including apple, grape, Ribes, Rubus, strawberry,the stone fruits and several     

Longitudinal study of fecal shedding of Escherichia coli O157:H7 in feedlot cattle: predominance and persistence of specific clonal types despite massive cattle population turnover

Identification of the sources and methods of transmission of Escherichia coli O157:H7 in feedlot cattle may facilitate the development of on-farm control measures for this important food-borne pathogen. The prevalence of E. coli O157:H7 in fecal samples of commercial feedlot cattle in 20 feedlot pens between April and September 2000 was determined throughout the finishing feeding period prior to slaughter. Using immunomagnetic separation, E. coli O157:H7 was isolated from 636 of 4,790 (13%) fecal samples in this study, with highest prevalence earliest in the feeding period. No differences were observed in the fecal or water trough sediment prevalence values of E. coli O157:H7 in 10 pens supplied with chlorinated drinking water supplies compared with nonchlorinated water pens. Pulsed-field gel electrophoresis of XbaI-digested bacterial DNA of the 230 isolates obtained from eight of the pens revealed 56 unique restriction endonuclease digestion patterns (REDPs), although nearly 60% of the isolates belonged to a group of four closely related genetic subtypes that were present in each of the pens and throughout the sampling period. The other REDPs were typically transiently detected, often in single pens and on single sample dates, and in many cases were also closely related to the four predominant REDPs. The persistence and predominance of a few REDPs observed over the entire feeding period on this livestock operation highlight the importance of the farm environment, and not necessarily the incoming cattle, as a potential source or reservoir of E. coli O157:H7 on farms.     

A new class of potent non-imidazole H(3) antagonists: 2-aminoethylbenzofurans

2-aminoethylbenzofurans constitute a new class of H(3) antagonists that are more rotationally constrained than most previously reported H(3) antagonists. They retain high potency at human and rat receptors, with efficient CNS penetration observed in 35. The SAR of the basic amine moiety was compared in three different series of analogues. The greatest potency was found in analogues bearing a 2-methylpyrrolidine, a 2,5-dimethylpyrrolidine, or a 2,6-dimethylpiperidine.