Glycosyl phosphatidylinositol myristoylation in African trypanosomes. New intermediates in the pathway for fatty acid remodeling

Glycosyl phosphatidylinositol (GPI) anchors in the bloodstream form of Trypanosoma brucei are unusual in that their two fatty acids are myristate. The myristates are added in the final stages of GPI biosynthesis in a remodeling reaction. Remodeling occurs first at the sn-2 position of glycerol, involving removal of a longer fatty acid and subsequent attachment of myristate. The second myristate is then incorporated into the sn-1 position, but the mechanism has been unclear due to the unavailability of a reliable cell-free system supporting complete remodeling. Here, we first refined the cell-free system (by removing Mn(2+) ions), thereby allowing efficient production of the dimyristoylated GPI precursor. Using this improved system, we made three new discoveries concerning the pathway for fatty acid remodeling. First, we discovered a monomyristoylated GPI (known as glycolipid theta') as an intermediate involved in remodeling at the sn-1 position. Second, we found an alternative pathway for production of glycolipid theta, the first lyso intermediate in remodeling. The alternative pathway involves an inositol-acylated GPI known as glycolipid lyso-C'. Finally, we found that there is significant breakdown of GPIs during remodeling in the cell-free system, and we speculate that this breakdown has a regulatory role in GPI biosynthesis.     

Nutrient concentration in shape Sphagna at increased N-deposition rates and raised atmospheric CO2 concentrations

Sphagnum fuscum, S. magellanicum, S. angustifolium and S. warnstorfii were treated with N deposition rates (0, 10, 30 and 100 kg ha^-1 a^-1) and with four atmospheric CO₂ concentrations (350, 700, 1000 and 2000 ppm) in greenhouse for 71–120 days. Thereafter, concentrations of total N, P, K, Ca and Mg in the capitulae of the Sphagna were determined. The response of each species to N deposition was related to ecological differences. With increasing N deposition treatments, moss N concentrations increased and higher N:P-ratios were found, the increase being especially clear at the highest N load. Sphagnum fuscum, which occupies ombrotrophic habitats, was the most affected by the increased nitrogen load and as a consequence the other elements were decreased. Oligotrophic S. magellanicum, wide nutrient status tolerant S. angustifolium and meso-eutrophic S. warnstorfii tolerated better increased N deposition, though there were increased concentrations of Ca and Mg in S. warnstorfii and Mg in S. magellanicum. Nitrogen and P concentrations decreased with raised CO₂ concentrations, except for S. magellanicum. This seems to be the first time this kind of response in nutrient concentrations to enhanced CO₂ concentration has been shown to exist in bryophytes. The concentration of K clearly decreased in S. fuscum as did the concentration of Mg in the other Sphagna with increasing CO₂. Sphagnum angustifolium and S. magellanicum, which are the less specialized species, were the least affected by the CO₂ treatments.     

Sickness Absence from Work among Persons with New Physician-Diagnosed Carpal Tunnel Syndrome: A Population-Based Matched-Cohort Study

Background

Carpal tunnel syndrome is common among employed persons. Data on sickness absence from work in relation to carpal tunnel syndrome have been usually based on self-report and derived from clinical or occupational populations. We aimed to determine sickness absence among persons with physician-diagnosed carpal tunnel syndrome as compared to the general population.

Methods

In Skåne region in Sweden we identified all subjects, aged 17–57 years, with new physician-made diagnosis of carpal tunnel syndrome during 5 years (2004–2008). For each subject we randomly sampled, from the general population, 4 matched reference subjects without carpal tunnel syndrome; the two cohorts comprised 5456 and 21,667 subjects, respectively (73% women; mean age 43 years). We retrieved social insurance register data on all sickness absence periods longer than 2 weeks from 12 months before to 24 months after diagnosis. Of those with carpal tunnel syndrome 2111 women (53%) and 710 men (48%) underwent surgery within 24 months of diagnosis. We compared all-cause sickness absence and analyzed sickness absence in conjunction with diagnosis and surgery.

Results

Mean number of all-cause sickness absence days per each 30-day period from 12 months before to 24 months after diagnosis was significantly higher in the carpal tunnel syndrome than in the reference cohort. A new sickness absence period longer than 2 weeks in conjunction with diagnosis was recorded in 12% of the women (n = 492) and 11% of the men (n = 170) and with surgery in 53% (n = 1121) and 58% (n = 408) of the surgically treated, respectively; median duration in conjunction with surgery was 35 days (IQR 27–45) for women and 41 days (IQR 28–50) for men.

Conclusions

Persons with physician-diagnosed carpal tunnel syndrome have substantially more sickness absence from work than age and sex-matched persons from the general population from1 year before to 2 years after diagnosis. Gender differences were small.     

B-function expression in the flower center underlies the homeotic phenotype of Lacandonia schismatica (Triuridaceae)

Spontaneous homeotic transformations have been described in natural populations of both plants and animals, but little is known about the molecular-genetic mechanisms underlying these processes in plants. In the ABC model of floral organ identity in Arabidopsis thaliana, the B- and C-functions are necessary for stamen morphogenesis, and C alone is required for carpel identity. We provide ABC model-based molecular-genetic evidence that explains the unique inside-out homeotic floral organ arrangement of the monocotyledonous mycoheterotroph species Lacandonia schismatica (Triuridaceae) from Mexico. Whereas a quarter million flowering plant species bear central carpels surrounded by stamens, L. schismatica stamens occur in the center of the flower and are surrounded by carpels. The simplest explanation for this is that the B-function is displaced toward the flower center. Our analyses of the spatio-temporal pattern of B- and C-function gene expression are consistent with this hypothesis. The hypothesis is further supported by conservation between the B-function genes of L. schismatica and Arabidopsis, as the former are able to rescue stamens in Arabidopsis transgenic complementation lines, and Ls-AP3 and Ls-PI are able to interact with each other and with the corresponding Arabidopsis B-function proteins in yeast. Thus, relatively simple molecular modifications may underlie important morphological shifts in natural populations of extant plant taxa.     

Immobilization and bone structure in humans

Long-term immobilization is known to result in substantial bone loss. The present review examined the existing evidence for deterioration of bone structure during long-term disuse in humans. Paralysis due to spinal cord injury, long-term exposure to microgravity in space or tightly restricted mobility during bed rest provide reasonable models to assess the influence of immobilization on bone structure. Expectedly, the duration of immobilisation was the major determinant of bone loss, but irrespective of whether the skeletal unloading was due to irrecoverable paralysis, long-term spaceflight or bed rest, the mean pattern of structural deterioration of bone, mainly manifest as substantial cortical thinning and trabecular bone loss, was quite similar. However, skeletal responses to disuse can be highly variable between individuals. Apparently the relative decline in individual’s bone loading in relation to loading prior to immobilization accounts for inter-individual variation.     

Investigation of Chlamydiaceae in semen and cauda epididymidis and seroprevalence of Chlamydophila abortus in breeding bulls

Background

Reproductive disorders associated with chlamydial infection have been reported worldwide in cattle and there are indications of potential venereal transmission.

Methods

Semen samples from 21 dairy bulls and cauda epididymidis tissue samples from 43 beef bulls were analysed for chlamydial agent by real-time polymerase chain reaction (PCR) including an internal amplification control (mimic). Additionally, presence of antibodies against Chlamydophila (Cp.) abortus among the bulls was investigated with the commercial Pourquier^® ELISA Cp. abortus serum verification kit.

Results

No chlamydial agent was detected by PCR in either the semen samples or in the tissue samples. Additionally, no antibodies against Cp. abortus were detected.

Conclusions

The results suggest that Cp. abortus is very rare, or absent in Swedish bulls and thus the risk for venereal transmission of chlamydial infection through their semen is low. However, because Chlamydophila spp. infection rates seem to differ throughout the world, it is essential to clarify the relative importance of transmission of the infection through semen on cattle fertility.     

Sensitive ELISA detection of amyloid-beta protofibrils in biological samples

Amyloid-beta (Abeta) protofibrils are known intermediates of the in vitro Abeta aggregation process and the protofibrillogenic Arctic mutation (APPE693G) provides clinical support for a pathogenic role of Abeta protofibrils in Alzheimer's disease (AD). To verify their in vivo relevance and to establish a quantitative Abeta protofibril immunoassay, Abeta conformation dependent monoclonal antibodies were generated. One of these antibodies, mAb158 (IgG2a), was used in a sandwich ELISA to specifically detect picomolar concentrations of Abeta protofibrils without interference from Abeta monomers or the amyloid precursor protein (APP). The specificity and biological significance of this ELISA was demonstrated using cell cultures and transgenic mouse models expressing human APP containing the Swedish mutation (APPKN670/671ML), or the Swedish and Arctic mutation in combination. The mAb158 sandwich ELISA analysis revealed presence of Abeta protofibrils in both cell and animal models, proving that Abeta protofibrils are formed not only in vitro, but also in vivo. Furthermore, elevated Abeta protofibril levels in the Arctic-Swedish samples emphasize the usefulness of the Arctic mutation as a model of enhanced protofibril formation. This assay provides a novel tool for investigating the role of Abeta protofibrils in AD and has the potential of becoming an important diagnostic assay.     

Identification of amino acid residues at the active site of endosialidase that dissociate the polysialic acid binding and cleaving activities in Escherichia coli K1 bacteriophages

Endosialidase (endo-N-acetylneuraminidase) is a tailspike enzyme of bacteriophages specific for human pathogenic Escherichia coli K1, which specifically recognizes and degrades polySia (polysialic acid). polySia is also a polysaccharide of the capsules of other meningitis- and sepsis-causing bacteria, and a post-translational modification of the NCAM (neural cell-adhesion molecule). We have cloned and sequenced three spontaneously mutated endosialidases of the PK1A bacteriophage and one of the PK1E bacteriophage which display lost or residual enzyme activity but retain the binding activity to polySia. Single to triple amino acid substitutions were identified, and back-mutation constructs indicated that single substitutions accounted for only partial reduction of enzymic activity. A homology-based structural model of endosialidase revealed that all substituted amino acid residues localize to the active site of the enzyme. The results reveal the importance of non-catalytic amino acid residues for the enzymatic activity. The results reveal the molecular background for the dissociation of the polySia binding and cleaving activities of endosialidase and for the evolvement of 'host range' mutants of E. coli K1 bacteriophages.     

SNP discovery by mismatch-targeting of Mu transposition

Single nucleotide polymorphisms (SNPs) represent a valuable resource for the mapping of human disease genes and induced mutations in model organisms. SNPs may become the markers of choice also for population ecology and evolutionary studies, but their isolation for non-model organisms with unsequenced genomes is often difficult. Here, we describe a rapid and cost-effective strategy to isolate SNPs that exploits the property of the bacteriophage Mu transposition machinery to target mismatched DNA sites and thereby to effectively detect polymorphic loci. To demonstrate the methodology, we isolated 164 SNPs from the unsequenced genome of the Glanville fritillary butterfly (Melitaea cinxia), a much-studied species in population biology, and we validated 24 of them. The strategy involves standard molecular biology techniques as well as undemanding MuA transposase-catalyzed in vitro transposition reactions, and it is applicable to any organism.     

Impacts of nutrient enrichment and sediment on phytoplankton community structure in the northern Baltic Sea

A three-week mesocosm experiment was conducted in order to study the effects of bottom sediment and nutrient enrichment on phytoplankton and zooplankton community structure in the Archipelago Sea, northern Baltic Sea. The transparent polyethylene enclosures included the whole water column and varied in volume from 30 to 40 m³. There were two types of enclosures: some with natural sediment as a bottom and others with a plastic bottom. The experiment was a 2 × 2 factorial design with presence of sediment and nutrient enrichment as treatment factors. Both the sediment presence and nutrient enrichment significantly increased water nutrient concentrations and the rate of primary production. However, external nutrient enrichment and the presence of sediment stimulated the growth of different phytoplankton groups, indicating that the effect of sediment was not related to nutrient fluxes alone, but involved more complex interactions. External nutrient enrichment was primarily channelled to picoplanktonic cyanobacteria, the biomass of which increased four- to fivefold due to enrichment. The presence of sediment increased the biomass of cryptophytes, chrysophytes and prasinophytes, but decreased the biomass of N₂-fixing cyanobacteria. Zooplankton biomass increased during the experiment, but was not affected by the treatments. The study shows that sediment plays a significant role in phytoplankton dynamics, underlining the importance of including sediment in shallow-water mesocosm experiments. Handling editor: J. Padisak