Morphology of sodium deoxycholate-solubilized apolipoprotein B-100 using negative stain and vitreous ice electron microscopy

The primary and secondary structures of apolipoprotein B-100 (apoB-100) are well established. Previous morphological studies have suggested that apoB is a long, flexible, threadlike molecule that encircles the low density lipoprotein (LDL) particle. Several large domain regions of the protein have been observed in frozen hydrated LDL and may be involved in anchoring of the protein to the lipid surface of LDL. Calorimetric studies of sodium deoxycholate (NaDC)-solubilized apoB indicated a similar number of independently melting domains. We therefore undertook a morphological study of NaDC-solubilized apoB-100 using negative stain and vitreous ice cryoelectron microscopy, a nonperturbing preservation technique. Negative staining experiments were performed in two ways: 1) grids were pulled through NaDC-containing buffer surfaces on which monolayers of apoB had been promoted, or 2) apoB molecules were allowed to diffuse onto carbon surfaces of grids that were floated on sample droplets. Vitrified molecules of apoB were obtained by plunging a thin fluid layer of protein adhered to a holey carbon-coated grid into supercooled ethane and by preserving the molecules in liquid nitrogen. The majority of molecules prepared in negative stain and vitreous ice were curved or arced and had alternating thin and thick regions. In negative stain, the apoB molecules lay on the grid perpendicular to the electron beam and had a mean length of 650 A. In vitreous ice the molecules were randomly oriented and their images ranged from 160 to 650 A in length. Vitrified molecules provided visualization of one or two beaded regions. Similar regions were observed in negative stain but the overall thickness was two to three times greater. Some vitrified molecules contained ribbon-like portions.Our study supports previously obtained data on molecule length but suggests that negative staining overestimates molecule width. These first images of vitrified NaDC-solubilized apoB-100 confirm the long, flexible, beaded thread morphology of the molecule and support the unique potential of this technique when coupled with proper molecule orientation and antibody labeling to correlate the tertiary structure of apoB seen in the intact particle with that of the isolated molecule.     

Hyaluronic acid (HA) binding to CD44 activates Rac1 and induces lamellipodia outgrowth

Both cell adhesion protein CD44 and its main ligand hyaluronic acid (HA) are thought to be involved in several processes ultimately requiring cytoskeleton rearrangements. Here, we show that the small guanine nucleotide (GTP)-binding protein, Rac1, can be activated upon HA binding to CD44. When applied locally to a passive cell edge, HA promoted the formation of lamellipodial protrusions in the direction of the stimulus. This process was inhibited by the prior injection of cells with dominant-negative N17Rac recombinant protein or by pretreatment of cells with monoclonal anti-CD44 antibodies, interfering with HA binding, implying the direct involvement of CD44 in signaling to Rac1.     

High-pressure and stark hole-burning studies of chlorosome antennas from Chlorobium tepidum

Results from high-pressure and Stark hole-burning experiments on isolated chlorosomes from the green sulfur bacterium Chlorobium tepidum are presented, as well as Stark hole-burning data for bacteriochlorophyll c (BChl c) monomers in a poly(vinyl butyral) copolymer film. Large linear pressure shift rates of -0.44 and -0.54 cm(-1)/MPa were observed for the chlorosome BChl c Q(y)-band at 100 K and the lowest Q(y)-exciton level at 12 K, respectively. It is argued that approximately half of the latter shift rate is due to electron exchange coupling between BChl c molecules. The similarity between the above shift rates and those observed for the B875 and B850 BChl a rings of the light-harvesting complexes of purple bacteria is emphasized. For BChl c monomer, fDeltamu++ = 0.35 D, where Deltamu+ is the dipole moment change for the Q(y) transition and f is the local field correction factor. The data establish that Deltamu+ is dominated by the matrix-induced contribution. The change in polarizability (Deltaalpha) for the Q(y) transition of the BChl c monomer is estimated at 19 A(3), which is essentially identical to that of the Chl a monomer. Interestingly, no Stark effects were observed for the lowest exciton level of the chlorosomes (maximum Stark field of 10(5) V/cm). Possible explanations for this are given, and these include consideration of structural models for the chlorosome BChl c aggregates.     

Kinetics of local helix formation in poly-L-glutamic acid studied by time-resolved photoacoustics: neutralization reactions of carboxylates in aqueous solutions and their relevance to the problem of protein folding

Photoactivatable caged protons have been used to trigger proton transfer reactions in aqueous solutions of acetate, glutamate, and poly-L-glutamic acid, and the volumetric and enthalpic changes have been detected and characterized by means of time-resolved photoacoustics. Neutralization of carboxylates in aqueous solutions invariably results in an expansion of the solution due to the disappearance of two charges and is accompanied by little enthalpic change. The reactions occur with thermally activated, apparent bimolecular rates on the order of 10(10) M(-1)s(-1). In the case of aqueous solutions of poly-L-glutamic acid at pH around the pK(a) of the coil-to-helix transition, diffusional binding of a proton by carboxylates is followed by a sequential reaction with rate 1.06 (+/- 0.05) x 10(7)s(-1). This step is not thermally activated in the temperature range we have investigated and is likely related to local formation of hydrogen bonds near the protonation site. This structural event may constitute a rate-limiting step in helix propagation.     

Fine mapping of the chromosome 12 late-onset Alzheimer disease locus: potential genetic and phenotypic heterogeneity

Apolipoprotein E (APOE) is the only confirmed susceptibility gene for late-onset Alzheimer disease (AD). In a recent genomic screen of 54 families with late-onset AD, we detected significant evidence for a second late-onset AD locus located on chromosome 12 between D12S373 and D12S390. Linkage to this region was strongest in 27 large families with at least one affected individual without an APOE-4 allele, suggesting that APOE and the chromosome 12 locus might have independent effects. We have since genotyped several additional markers across the region, to refine the linkage results. In analyzing these additional data, we have addressed the issue of heterogeneity in the data set by weighting results by clinical and neuropathologic features, sibship size, and APOE genotype. When considering all possible affected sib pairs (ASPs) per nuclear family, we obtained a peak maximum LOD score between D12S1057 and D12S1042. The magnitude and location of the maximum LOD score changed when different weighting schemes were used to control for the number of ASPs contributed by each nuclear family. Using the affected-relative-pair method implemented in GENEHUNTER-PLUS, we obtained a maximum LOD score between D12S398 and D12S1632, 25 cM from the original maximum LOD score. These results indicate that family size influences the location estimate for the chromosome 12 AD gene. The results of conditional linkage analysis by use of GENEHUNTER-PLUS indicated that evidence for linkage to chromosome 12 was stronger in families with affected individuals lacking an APOE-4 allele; much of this evidence came from families with affected individuals with neuropathologic diagnosis of dementia with Lewy bodies (DLB). Taken together, these results indicate that the chromosome 12 locus acts independently of APOE to increase the risk of late-onset familial AD and that it may be associated with the DLB variant of AD.     

Absence of sodA Increases the Levels of Oxidation of Key Metabolic Determinants of Borrelia burgdorferi

Borrelia burgdorferi, the causative agent of Lyme disease, alters its gene expression in response to environmental signals unique to its tick vector or vertebrate hosts. B. burgdorferi carries one superoxide dismutase gene (sodA) capable of controlling intracellular superoxide levels. Previously, sodA was shown to be essential for infection of B. burgdorferi in the C3H/HeN model of Lyme disease. We employed two-dimensional electrophoresis (2-DE) and immunoblot analysis with antibodies specific to carbonylated proteins to identify targets that were differentially oxidized in the soluble fractions of the sodA mutant compared to its isogenic parental control strain following treatment with an endogenous superoxide generator, methyl viologen (MV, paraquat). HPLC-ESI-MS/MS analysis of oxidized proteins revealed that several proteins of the glycolytic pathway (BB0057, BB0020, BB0348) exhibited increased carbonylation in the sodA mutant treated with MV. Levels of ATP and NAD/NADH were reduced in the sodA mutant compared with the parental strain following treatment with MV and could be attributed to increased levels of oxidation of proteins of the glycolytic pathway. In addition, a chaperone, HtpG (BB0560), and outer surface protein A (OspA, BBA15) were also observed to be oxidized in the sodA mutant. Immunoblot analysis revealed reduced levels of Outer surface protein C (OspC), Decorin binding protein A (DbpA), fibronectin binding protein (BBK32), RpoS and BosR in the sodA mutant compared to the control strains. Viable sodA mutant spirochetes could not be recovered from both gp91/phox ^−⁄− and iNOS deficient mice while borrelial DNA was detected in multiple tissues samples from infected mice at significantly lower levels compared to the parental strain. Taken together, these observations indicate that the increased oxidation of select borrelial determinants and reduced levels of critical pathogenesis-associated lipoproteins contribute to the in vivo deficit of the sodA mutant in the mouse model of Lyme disease. This study, utilizing the sodA mutant, has provided insights into adaptive capabilities critical for survival of B. burgdorferi in its hosts.     

Emergency Department Presentations following Tropical Cyclone Yasi

Introduction

Emergency departments see an increase in cases during cyclones. The aim of this study is to describe patient presentations to the Emergency Department (ED) of a tertiary level hospital (Townsville) following a tropical cyclone (Yasi). Specific areas of focus include changes in: patient demographics (age and gender), triage categories, and classification of diseases.

Methods

Data were extracted from the Townsville Hospitals ED information system (EDIS) for three periods in 2009, 2010 and 2011 to coincide with formation of Cyclone Yasi (31 January 2011) to six days after Yasi crossed the coast line (8 February 2012). The analysis explored the changes in ICD10-AM 4-character classification and presented at the Chapter level.

Results

There was a marked increase in the number of patients attending the ED during Yasi, particularly those aged over 65 years with a maximum daily attendance of 372 patients on 4 Feb 2011. The most marked increases were in: Triage categories - 4 and 5; and ICD categories - diseases of the skin and subcutaneous tissue (L00-L99), and factors influencing health care status (Z00-Z99). The most common diagnostic presentation across all years was injury (S00-T98).

Discussion

There was an increase in presentations to the ED of TTH, which peaked in the first 24 – 48 hours following the cyclone and returned to normal over a five-day period. The changes in presentations were mostly an amplification of normal attendance patterns with some altered areas of activity. Injury patterns are similar to overseas experience.     

High Prevalence of the Liver Fluke Amphimerus sp. in Domestic Cats and Dogs in an Area for Human Amphimeriasis in Ecuador

Background

Amphimerus sp. is a liver fluke which recently has been shown to have a high prevalence of infection among an indigenous group, Chachi, who reside in a tropical rainforest in the northwestern region of Ecuador. Since it is unknown which animals can act as a reservoir and/or definitive hosts for Amphimerus sp. in this endemic area, a study was done to determine the prevalence of infection in domestic cats and dogs. This information is important to understand the epidemiology, life cycle and control of this parasite.

Methodology/Findings

In July 2012, three Chachi communities located on Rio Cayapas, province of Esmeraldas, were surveyed. A total of 89 of the 109 registered households participated in the study. Of the 27 cats and 43 dogs found residing in the communities, stool samples were collected from 14 cats and 31 dogs (total of 45 animals) and examined microscopically for the presence of Amphimerus eggs. The prevalence of infection was 71.4% in cats and 38.7% in dogs, with similar rates of infection in all three communities. Significantly more cats were infected than dogs (p = 0.042).

Conclusions/Significance

The data show a high rate of Amphimerus sp. infection in domestic cats and dogs residing in Chachi communities. It can be concluded that these animals act as definitive and reservoir hosts for this liver fluke and that amphimeriasis is a zoonotic disease. These findings provide important epidemiological data which will aid in the development and implementation of control strategies against the transmission of Amphimerus.     

Comparing land surface phenology derived from satellite and GPS network microwave remote sensing

The land surface phenology (LSP) start of season (SOS) metric signals the seasonal onset of vegetation activity, including canopy growth and associated increases in land-atmosphere water, energy and carbon (CO₂) exchanges influencing weather and climate variability. The vegetation optical depth (VOD) parameter determined from satellite passive microwave remote sensing provides for global LSP monitoring that is sensitive to changes in vegetation canopy water content and biomass, and insensitive to atmosphere and solar illumination constraints. Direct field measures of canopy water content and biomass changes desired for LSP validation are generally lacking due to the prohibitive costs of maintaining regional monitoring networks. Alternatively, a normalized microwave reflectance index (NMRI) derived from GPS base station measurements is sensitive to daily vegetation water content changes and may provide for effective microwave LSP validation. We compared multiyear (2007–2011) NMRI and satellite VOD records at over 300 GPS sites in North America, and their derived SOS metrics for a subset of 24 homogenous land cover sites to investigate VOD and NMRI correspondence, and potential NMRI utility for LSP validation. Significant correlations (P?<?0.05) were found at 276 of 305 sites (90.5 %), with generally favorable correspondence in the resulting SOS metrics (r ²?=?0.73, P?<?0.001, RMSE = 36.8 days). This study is the first attempt to compare satellite microwave LSP metrics to a GPS network derived reflectance index and highlights both the utility and limitations of the NMRI data for LSP validation, including spatial scale discrepancies between local NMRI measurements and relatively coarse satellite VOD retrievals.     

Phylogeography and marine retention

A number of studies now point to the association of patterns of phylogeography with discontinuities in coastal current patterns. If such phylogeographic patterns are indicative of populations that retain local diversity, as has been predicted by recent modelling, such results may be of use in marine reserve planning. Here we show that there is a distributional correlation on the Pacific coast of North America between marine reserve placement and phylogeographic patterns. A number of factors could contribute to this correlation, but its existence suggests the utility of genetic studies in marine conservation planning.