Oligomerization state and supramolecular structure of the HIV-1 Vpu protein transmembrane segment in phospholipid bilayers

HIV‐1 Vpu is an 81‐residue protein with a single N‐terminal transmembrane (TM) helical segment that is involved in the release of new virions from host cell membranes. Vpu and its TM segment form ion channels in phospholipid bilayers, presumably by oligomerization of TM helices into a pore‐like structure. We describe measurements that provide new constraints on the oligomerization state and supramolecular structure of residues 1–40 of Vpu (Vpu_1–40), including analytical ultracentrifugation measurements to investigate oligomerization in detergent micelles, photo‐induced crosslinking experiments to investigate oligomerization in bilayers, and solid‐state nuclear magnetic resonance measurements to obtain constraints on intermolecular contacts between and orientations of TM helices in bilayers. From these data, we develop molecular models for Vpu TM oligomers. The data indicate that a variety of oligomers coexist in phospholipid bilayers, so that a unique supramolecular structure can not be defined. Nonetheless, since oligomers of various sizes have similar intermolecular contacts and orientations, molecular models developed from our data are most likely representative of Vpu TM oligomers that exist in host cell membranes.     

Influence of inbreeding depression on a lake population of Nymphoides peltata after restoration from the soil seed bank

The negative effects of inbreeding depression on fragmented small populations are likely to be expressed more strongly after restoration efforts if regeneration processes have been highly restricted in degraded habitats. We examined the potential influences of inbreeding depression on a population of Nymphoides peltata (Menyanthaceae) restored from the remnant soil seed bank. A hand-pollination experiment demonstrated self-compatibility of a single remaining homostyle genet and significant inbreeding depression in selfed progeny, especially in parameters related to seedling growth (–0.6 for biomass, and –0.4 for relative growth rate). Our genetic analysis indicated that the presumed number of parents contributing to the current soil seed bank was only 2–8 genets and that a single sib-family dominated at each of three sampling sites. The results also showed that the selfed progeny of the homostyle genet were overwhelmingly dominant at two sites (86.8 and 94.7%). As a result, the growth performance of the seed bank seedlings was significantly reduced, to a level as low as that of the selfed progeny. Active restoration efforts to minimize the negative effects of the genetic bottleneck and continuous monitoring based on genetic and demographic study are recommended.     

Molecular Evidence for an Extreme Genetic Bottleneck During Introduction of an Invading Grass to California

Although exotic species cause tremendous economic and ecological loss, we know relatively little about the post-introduction evolutionary dynamics of the invasive species themselves. Barbed goatgrass, Aegilops triuncialis L., is a cleistogamous annual grass with a native range throughout the Mediterranean Basin and Asia and introduced to California during the last century. It is considered a serious noxious range weed and is one of the few exotic plant species that is invading serpentine soil habitats. We examined whether patterns of molecular variation are consistent with a single or multiple introduction events into California and further, if individual populations show evidence for a genetic bottleneck during introduction. Fingerprinting patterns, using microsatellite loci derived from Triticum aestivum, were investigated for 57 Eurasian accessions, broadly spanning the native range and for 108 individuals from 11 localities in California. There is strong evidence for an extreme bottleneck in this species as it colonizes its new range because we detected only three multilocus genotypes occurring in California and 36 genotypes in Eurasia. In California one of the genotypes differs from one other by only one fragment and only occurs in one individual. This suggests two separate introductions. Each population is composed of highly uniform individuals and the two main genotypes are geographically separated. A. triuncialis is still expanding its range in California despite genomic uniformity after a strong bottleneck and its recently increased rate of spread is not correlated to a high within-population variability created by multiple introductions.     

Evaluation of manometric temperature measurement, a process analytical technology tool for freeze-drying: Part I, product temperature measurement

This study examines the factors that may cause systematic errors in the manometric temperature measurement (MTM) procedure used to evaluate product temperature during primary drying. MTM was conducted during primary drying using different vial loads, and the MTM product temperatures were compared with temperatures directly measured by thermocouples. To clarify the impact of freeze-drying load on MTM product temperatures, simulation of the MTM vapor pressure rise was performed, and the results were compared with the experimental results. The effect of product temperature heterogeneity in MTM product temperature determination was investigated by comparing the MTM product temperatures with directly measured thermocouple product temperatures in systems differing in temperature heterogeneity. Both the simulated and experimental results showed that at least 50 vials (5 mL) were needed to give sufficiently rapid pressure rise during the MTM data collection period (25 seconds) in the freeze dryer, to allow accurate determination of the product temperature. The product temperature is location dependent, with higher temperature for vials on the edge of the array and lower temperature for the vials in the center of the array. The product temperature heterogeneity is also dependent upon the freeze-drying conditions. In product temperature heterogeneous systems, MTM measures a temperature close to the coldest product temperature, even, if only a small fraction of the samples have the coldest product temperature. The MTM method is valid even at very low product temperature (−45°C). Published: February 10, 2006     

Statistical assessment of DNA extraction reagent lot variability in real‐time quantitative PCR

Aims: The aim of this study was to evaluate the variability in lots of a DNA extraction kit using real‐time PCR assays for Bacillus anthracis, Francisella tularensis and Vibrio cholerae. Methods and Results: Replicate aliquots of three bacteria were processed in duplicate with three different lots of a commercial DNA extraction kit. This experiment was repeated in triplicate. Results showed that cycle threshold values were statistically different among the different lots. Conclusions: Differences in DNA extraction reagent lots were found to be a significant source of variability for qPCR results. Steps should be taken to ensure the quality and consistency of reagents. Minimally, we propose that standard curves should be constructed for each new lot of extraction reagents, so that lot‐to‐lot variation is accounted for in data interpretation. Significance and Impact of the Study: This study highlights the importance of evaluating variability in DNA extraction procedures, especially when different reagent lots are used. Consideration of this variability in data interpretation should be an integral part of studies investigating environmental samples with unknown concentrations of organisms.     

Fluorescence quenching of serum albumin by rifamycin antibiotics and their analytical application.

In neutral medium, rifamycin antibiotics such as rifapentin (RFPT), rifampicin (RFP), rifandin (RFD) and rifamycin SV (RFSV) can bind with human serum albumin (HSA) and bovine serum albumin (BSA) to form complexes, resulting in the quenching of the intrinsic fluorescence (lambda(ex)/lambda(em) = 285/355 nm) of the BSA and HSA. The quenching intensity (DeltaF) is directly proportional to the concentration of the rifamycin antibiotics. Therefore, a new analytical method was established to determine trace rifamycin antibiotics. The method had fairly high sensitivity and the detecting limits (3sigma) for RFPT, RFP, RFD and RFSV were 0.85, 0.98, 1.83, 1.89 ng/mL, respectively, for the HSA system and 0.76, 0.89, 1.55, 1.77 ng/mL, respectively, for the BSA system. All relative standard deviations (RSDs) were <3.8%. In this work, the characteristics of the fluorescence spectra were studied and the optimum reaction conditions and influencing factors were investigated. The influence of coexisting substances was tested and the results showed that the method had good selectivity and could be applied to determine trace rifamycin antibiotics in medicine capsules and urine samples. Taking the RFSV-serum albumin system as an example, the reaction mechanisms, such as binding constants, binding sites, binding distance and the type of fluorescence quenching, were investigated.     

Genetic signatures of pre-expansion bottleneck in the Choctaw population of Oklahoma

Previous research showed that the Choctaw Indians of Oklahoma exhibit considerable linkage disequilibria (LD) in a number of regions of the genome that has allowed genetic fine mapping for potential susceptibility genes for the autoimmune connective tissue disease scleroderma, or systemic sclerosis (SSc). In principle, such enhanced background LD in the Choctaws could be caused by population bottleneck event(s) followed by recent population expansion. This investigation utilizes genome-scan data on 175 dinucleotide loci from 76 Choctaw individuals to seek genetic evidence of the demographic history of the Choctaw Nation. Of the 175 loci examined, 105 are in Hardy-Weinberg equilibrium. The average unbiased homozygosity over the 105 loci for the Choctaws (29.3%) is significantly higher than that in the European descent group (20.9%); and when adjusted for sample-size differences, the Choctaw also exhibit a significantly smaller number of segregating alleles (6.65 vs. 8.14) at these loci. Both of these observations are consistent with the trend expected in an isolated population. Comparison of the allele size variance and gene diversity yields an imbalance index (lnbeta) of 0.811 in the Choctaw. Of the 105 loci examined, 93 exhibit excess expected homozygosity in comparison to the expectations of a stepwise mutation model in a population of constant size. Taken together, these observations are consistent with a signature of the recent population size expansion of the Choctaws, preceded by bottleneck event(s).     

The extended multidomain solution structures of the complement protein Crry and its chimeric conjugate Crry-Ig by scattering,analytical ultracentrifugation and constrained modelling: implications for function and therapy

Complement receptor-related gene/protein y (Crry) is a cell membrane-bound regulator of complement activation found in mouse and rat. Crry contains only short complement/consensus repeat (SCR) domains. X-ray and neutron scattering was performed on recombinant rat Crry containing the first five SCR domains (rCrry) and mouse Crry with five SCR domains conjugated to the Fc fragment of mouse IgG1 (mCrry-Ig) in order to determine their solution structures at medium resolution. The radius of gyration R(G) of rCrry was determined to be 4.9-5.0 nm, and the R(G) of the cross-section was 1.2-1.5 nm as determined by X-ray and neutron scattering. The R(G) of mCrry-Ig was 6.6-6.7 nm, and the R(G) of the cross-section were 2.3-2.4 nm and 1.3 nm. The maximum dimension of rCrry was 18 nm and that for mCrry-Ig was 26 nm. The neutron data indicated that rCrry and mCrry-Ig have molecular mass values of 45,000 Da and 140,000 Da, respectively, in agreement with their sequences, and sedimentation equilibrium data supported these determinations. Time-derivative velocity experiments gave sedimentation coefficients of 2.4S for rCrry and 5.4S for mCrry-Ig. A medium-resolution model of rCrry was determined using homology models that were constructed for the first five SCR domains of Crry from known crystal and NMR structures, and linked by randomly generated linker peptide conformations. These trial-and-error calculations revealed a small family of extended rCrry structures that best accounted for the scattering and ultracentrifugation data. These were shorter than the most extended rCrry models as the result of minor bends in the inter-SCR orientations. The mCrry-Ig solution data were modelled starting from a fixed structure for rCrry and the crystal structure of mouse IgG1, and was based on conformational searches of the hinge peptide joining the mCrry and Fc fragments. The best-fit models showed that the two mCrry antennae in mCrry-Ig were extended from the Fc fragment. No preferred orientation of the antennae was identified, and this indicated that the accessibility of the antennae for the molecular targets C4b and C3b was not affected by the covalent link to Fc. A structural comparison between Crry and complement receptor type 1 indicated that the domain arrangement of Crry SCR 1-3 is as extended as that of the CR1 SCR 15-17 NMR structure.     

How do helix–helix interactions help determine the folds of membrane proteins? Perspectives from the study of homo‐oligomeric helical bundles

The final, structure-determining step in the folding of membrane proteins involves the coalescence of preformed transmembrane helices to form the native tertiary structure. Here, we review recent studies on small peptide and protein systems that are providing quantitative data on the interactions that drive this process. Gel electrophoresis, analytical ultracentrifugation, and fluorescence resonance energy transfer (FRET) are useful methods for examining the assembly of homo-oligomeric transmembrane helical proteins. These methods have been used to study the assembly of the M2 proton channel from influenza A virus, glycophorin, phospholamban, and several designed membrane proteins-all of which have a single transmembrane helix that is sufficient for association into a transmembrane helical bundle. These systems are being studied to determine the relative thermodynamic contributions of van der Waals interactions, conformational entropy, and polar interactions in the stabilization of membrane proteins. Although the database of thermodynamic information is not yet large, a few generalities are beginning to emerge concerning the energetic differences between membrane and water-soluble proteins: the packing of apolar side chains in the interior of helical membrane proteins plays a smaller, but nevertheless significant, role in stabilizing their structure. Polar, hydrogen-bonded interactions occur less frequently, but, nevertheless, they often provide a strong driving force for folding helix-helix pairs in membrane proteins. These studies are laying the groundwork for the design of sequence motifs that dictate the association of membrane helices.     

Enzyme I: the gateway to the bacterial phosphoenolpyruvate:sugar phosphotransferase system.

Regulatory aspects of the bacterial phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS) are reviewed. The structure and conformational stability of the first protein (enzyme I) of the PTS, as well as the requirement for enzyme I to dimerize for autophosphorylation by PEP in the presence of MgCl2 are discussed.