PREPARATION OF PLASMA MEMBRANE FROM ISOLATED NEURONS

A bulk fraction enriched with respect to neuronal cell bodies was used as starting material for the isolation of neuronal plasma membrane The cells were gently homogenized in isotonic sucrose and a crude membrane containing fraction sedimented at 3000 g. Subsequently, the membrane fraction was purified on a discontinuous sucrose density gradient between 35% and 25 5% sucrose (w/w). Enzymatic analyses showed a 4–5-fold enrichment in plasma membrane markers, and a 10–15% contamination of mitochondrial and microsomal material. Electron micrographs of the membrane fraction confirmed the enzymatic data Fragmented membranes were found, mainly in vesicular form No ribosomes, but a few mitochondria and some multilamellar membranes were seen     

Seasonal carbon allocation to arbuscular mycorrhizal fungi assessed by microscopic examination, stable isotope probing and fatty acid analysis

Background and Aim

Climate change models are limited by lack of baseline data, in particular carbon (C) allocation to – and dynamics within – soil microbial communities. We quantified seasonal C-assimilation and allocation by plants, and assessed how well this corresponds with intraradical arbuscular mycorrhizal fungal (AMF) storage and structural lipids (16:1ω5 NLFA and PLFA, respectively), as well as microscopic assessments of AMF root colonization.

Methods

Coastal Hypochoeris radicata plants were labeled with ¹³CO₂ in February, July and October, and ¹³C-allocation to fine roots and NLFA 16:1ω5, as well as overall lipid contents and AM colonization were quantified.

Results

C-allocation to fine roots and AMF storage lipids differed seasonally and mirrored plant C-assimilation, whereas AMF structural lipids and AM colonization showed no seasonal variation, and root colonization exceeded 80 % throughout the year. Molecular analyzes of the large subunit rDNA gene indicated no seasonal AMF community shifts.

Conclusions

Plants allocated C to AMF even at temperatures close to freezing, and fungal structures persisted in roots during times of low C-allocation. The lack of seasonal differences in PLFA and AM colonization indicates that NLFA analyses should be used to estimate fungal C-status. The implication of our findings for AM function is discussed.     

Anatomy,relationships and distribution of Taiwanassiminea affinis (Böttger) (Caenogastropoda: Assimineidae), with a reassessment of Cyclotropis Tapparone-Canefri

Assiminea affinis (Mousson ms) Böttger, 1887 Böttger, O. (1887) Aufzählung der zur Gattung Assiminea Fleming gehörigen Arten. Jahrbücher der Deutschen Malakozoologischen Gesellschaft 14, 147–234, pl. 6. [Google Scholar](=A. queenslandica [Pilsbry ms] Thiele, 1927 Thiele, J. (1927) Über die Schneckenfamilie Assimineidae. Zoologische Jahrbücher, Abteilung für Systematik, Ökologie und Geographie der Tiere 53, 114–146, pl. 1. [Google Scholar]), a previously unrecognised Australian assimineid species, is described anatomically and allocated to the genus Taiwanassiminea Kuroda and Habe, 1950 Kuroda, T. & Habe, T. (1950) Nomenclatural notes. Illustrated Catalogue of Japanese Shells 1, 16. [Google Scholar], first described from Taiwan. This is the first record of the genus from Australia. Taiwanassiminea affinis is found in slightly brackish waters in the upper tidal reaches of the larger rivers from northern Queensland to the Shoalhaven River in the southern half of New South Wales. The terrestrial Cyclotropis Tapparone-Canefri, 1883, which has somewhat similar shell and radular characters, is redefined and several species (Assiminea bedaliensis Rensch, 1934; Paludinella javana Thiele, 1927 Thiele, J. (1927) Über die Schneckenfamilie Assimineidae. Zoologische Jahrbücher, Abteilung für Systematik, Ökologie und Geographie der Tiere 53, 114–146, pl. 1. [Google Scholar]; Assiminea lentula, A. riparia and A. sororcula, all Benthem Jutting, 1963 Benthem Jutting, W.S.S. van. (1963) Non-marine Mollusca of west New Guinea Part 1, Mollusca from fresh and brackish waters. Nova Guinea, Zoology 20, 409–521. [Google Scholar]) previously included in Cyclotropis are transferred to Taiwanassiminea.     

An asparagine-rich protein from blood stages of Plasmodium falciparum shares determinants with sporozoites.

We describe a cDNA clone derived from mRNA of asexual blood-stages of the malaria parasite Plasmodium falciparum. This clone, designated Ag319, expresses a P.falciparum antigen fused to beta-galactosidase in Escherichia coli. Human antibodies from Papua New Guinea were affinity-purified by adsorption to extracts of Ag319 immobilized on CNBr-Sepharose. The antibodies reacted predominantly with P. falciparum polypeptides of Mr 220,000 and 160,000, and a number of ill-defined lower molecular weight species. Antibodies reacted in indirect immunofluorescence with all asexual blood-stages although the antigen appeared to be most abundance in the schizont. Surprizingly the antibodies also reacted with sporozoites. The amino acid sequence predicted from the complete nucleotide sequence of this clone is remarkable because 40% of the residues are Asn, and so the antigen has been termed the Asparagine-Rich Protein (ARP). Like other P. falciparum antigens, ARP contains tandemly repetitive sequences, based on the tetrapeptide Asn-Asn-Asn-Met and we have confirmed that these represent natural epitopes by reaction of the corresponding synthetic peptides with human antibodies. Surprisingly, ARP is also rich in Asn outside the tandem repeats.     

Structure of the L1 protuberance in the ribosome

The L1 protuberance of the 50S ribosomal subunit is implicated in the release/disposal of deacylated tRNA from the E site. The apparent mobility of this ribosomal region has thus far prevented an accurate determination of its three-dimensional structure within either the 50S subunit or the 70S ribosome. Here we report the crystal structure at 2.65 A resolution of ribosomal protein L1 from Sulfolobus acidocaldarius in complex with a specific 55-nucleotide fragment of 23S rRNA from Thermus thermophilus. This structure fills a major gap in current models of the 50S ribosomal subunit. The conformations of L1 and of the rRNA fragment differ dramatically from those within the crystallographic model of the T. thermophilus 70S ribosome. Incorporation of the L1-rRNA complex into the structural models of the T. thermophilus 70S ribosome and the Deinococcus radiodurans 50S subunit gives a reliable representation of most of the L1 protuberance within the ribosome.     

Macromolecular Drug Delivery: Basic Principles and Therapeutic Applications

Macromolecular drugs hold great promise as novel therapeutics of several major disorders, such as cancer and cardiovascular disease. However, their use is limited by lack of efficient, safe, and specific delivery strategies. Successful development of such strategies requires interdisciplinary collaborations involving researchers with expertise on e.g., polymer chemistry, cell biology, nano technology, systems biology, advanced imaging methods, and clinical medicine. This poses obvious challenges to the scientific community, but also provides opportunities for the unexpected at the interface between different disciplines. This review summarizes recent studies of macromolecular delivery that should be of interest to researchers involved in macromolecular drug synthesis as well as in vitro and in vivo drug delivery studies.     

Differential expression analysis for sequence count data

High-throughput sequencing assays such as RNA-Seq, ChIP-Seq or barcode counting provide quantitative readouts in the form of count data. To infer differential signal in such data correctly and with good statistical power, estimation of data variability throughout the dynamic range and a suitable error model are required. We propose a method based on the negative binomial distribution, with variance and mean linked by local regression and present an implementation, DESeq, as an R/Bioconductor package.     

Walking speed and area utilization of red king crab (<Emphasis Type="Italic">Paralithodes camtschaticus</Emphasis>) introduced to the Barents Sea coastal ecosystem

The red king crab (Paralithodes camtschaticus) was introduced in the Barents Sea in the 1960s and soon established a viable population. Proper management and exploitation of the Barents Sea king crab stock require better understanding of the spatial dynamics at different scales. This study examines the small-scale movement patterns of seven adult male crabs tracked for a period of up to one month from mid July to mid August at 150 m depth in a semi-enclosed fjord on the Russo-Norwegian border. The crabs were tagged with acoustic transmitters and their movements monitored with an acoustic positioning system. Low walking speeds (<0.01 m s⁻¹) were most frequent but the crabs could move at a maximum speed of 0.15 m s⁻¹ and walk an actual distance of up to 270 m over a period of one hour. However, the crabs usually moved within a relatively restricted area with mean hourly longest rectilinear distance varying from 26 to 64 m. The crabs alternated between periods of low and high activity, which could reflect feeding in and movements between food patches. The lack of a diel activity rhythm may be due to high light levels during the polar summer night, or a chemically mediated food search strategy.     

N- and O-methylation of sphingomyelin markedly affects its membrane properties and interactions with cholesterol

We have prepared palmitoyl sphingomyelin (PSM) analogs in which either the 2-NH was methylated to NMe, the 3-OH was methylated to OMe, or both were methylated simultaneously. The aim of the study was to determine how such modifications in the membrane interfacial region of the molecules affected interlipid interactions in bilayer membranes. Measuring DPH anisotropy in vesicle membranes prepared from the SM analogs, we observed that methylation decreased gel-phase stability and increased fluid phase disorder, when compared to PSM. Methylation of the 2-NH had the largest effect on gel-phase instability (T(m) was lowered by ~7°C). Atomistic molecular dynamics simulations showed that fluid phase bilayers with methylated SM analogs were more expanded but thinner compared to PSM bilayers. It was further revealed that 3-OH methylation dramatically attenuated hydrogen bonding also via the amide nitrogen, whereas 2-NH methylation did not similarly affect hydrogen bonding via the 3-OH. The interactions of sterols with the methylated SM analogs were markedly affected. 3-OH methylation almost completely eliminated the capacity of the SM analog to form sterol-enriched ordered domains, whereas the 2-NH methylated SM analog formed sterol-enriched domains but these were less thermostable (and thus less ordered) than the domains formed by PSM. Cholestatrienol affinity to bilayers containing methylated SM analogs was also markedly reduced as compared to its affinity for bilayers containing PSM. Molecular dynamics simulations revealed further that cholesterol's bilayer location was deeper in PSM bilayers as compared to the location in bilayers made from methylated SM analogs. This study shows that the interfacial properties of SMs are very important for interlipid interactions and the formation of laterally ordered domains in complex bilayers.     

Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) S18Y polymorphism in Alzheimer's disease

Alzheimer's disease (AD) is characterized by protein aggregates, i.e. senile plaques and neurofibrillary tangles. The ubiquitin-proteasome system has been proposed a role in proteolytic removal of these protein aggregates. Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is a de-ubiquitinating enzyme with important functions in recycling of ubiquitin. The S18Y polymorphism of the UCHL1 gene confers protection against Parkinson's disease. In this study, the genotype and allele frequencies of the UCHL1 S18Y polymorphism were investigated in 452 AD patients and 234 control subjects, recruited from four memory clinics in Sweden. Using a binary logistic regression model including UCHL1 allele A and APOE ε4 allele positivity, age and sex as covariates with AD diagnosis as dependent variable, an adjusted OR of 0.82 ([95% CI 0.55-1.24], P = 0.35) was obtained for a positive UCHL1 allele A carrier status. The present study thus do not support a protective effect of the UCHL1 S18Y polymorphism against AD.