Plasmepsin II, an acidic hemoglobinase from the Plasmodium falciparum food vacuole, is active at neutral pH on the host erythrocyte membrane skeleton.

Plasmepsin II, an aspartic protease from the human intraerythrocytic parasite Plasmodium falciparum, is involved in degradation of the host cell hemoglobin within the acidic food vacuole of the parasite. Previous characterization of enzymatic activities from Plasmodium soluble extracts, responsible for in vitro hydrolysis of erythrocyte spectrin, had shown that the hydrolysis process occurred at pH 5.0 and involved aspartic protease(s) cleaving mainly within the SH3 motif of the spectrin alpha-subunit. Therefore, we used a recombinant construct of the erythroid SH3 motif as substrate to investigate the involvement of plasmepsins in spectrin hydrolysis. Using specific anti-plasmepsin II antibodies in Western blotting experiments, plasmepsin II was detected in chromatographic fractions enriched in the parasite SH3 hydrolase activity. Involvement of plasmepsin II in hydrolysis was demonstrated by mass spectrometry identification of cleavage sites in the SH3 motif, upon hydrolysis by Plasmodium extract enzymatic activity, and by recombinant plasmepsin II. Furthermore, recombinant plasmepsin II digested native spectrin at pH 6.8, either purified or situated in erythrocyte ghosts. Additional degradation of actin and protein 4.1 from ghosts was observed. Specific antibodies were used in confocal imaging of schizont-infected erythrocytes to localize plasmepsin II in mature stages of the parasite development cycle; antibodies clearly labeled the periphery of the parasites. Taken together, these results strongly suggest that, in addition to hemoglobin degradation, plasmepsin II might be involved in cytoskeleton cleavage of infected erythrocytes.     

Bundles of Spider Silk,Braided into Sutures,Resist Basic Cyclic Tests: Potential Use for Flexor Tendon Repair

Repair success for injuries to the flexor tendon in the hand is often limited by the in vivo behaviour of the suture used for repair. Common problems associated with the choice of suture material include increased risk of infection, foreign body reactions, and inappropriate mechanical responses, particularly decreases in mechanical properties over time. Improved suture materials are therefore needed. As high-performance materials with excellent tensile strength, spider silk fibres are an extremely promising candidate for use in surgical sutures. However, the mechanical behaviour of sutures comprised of individual silk fibres braided together has not been thoroughly investigated. In the present study, we characterise the maximum tensile strength, stress, strain, elastic modulus, and fatigue response of silk sutures produced using different braiding methods to investigate the influence of braiding on the tensile properties of the sutures. The mechanical properties of conventional surgical sutures are also characterised to assess whether silk offers any advantages over conventional suture materials. The results demonstrate that braiding single spider silk fibres together produces strong sutures with excellent fatigue behaviour; the braided silk sutures exhibited tensile strengths comparable to those of conventional sutures and no loss of strength over 1000 fatigue cycles. In addition, the braiding technique had a significant influence on the tensile properties of the braided silk sutures. These results suggest that braided spider silk could be suitable for use as sutures in flexor tendon repair, providing similar tensile behaviour and improved fatigue properties compared with conventional suture materials.     

Structural characterization by tandem mass spectrometry of the posttranslational polyglycylation of tubulin.

Polyglycylation is a posttranslational modification specific to tubulin. This modification was originally identified in highly stable microtubules from Paramecium cilia. As many as 34 posttranslationally added glycine residues have been located in the C-terminal domains of Paramecium alpha- and beta-tubulin. In this study, post source decay matrix-assisted laser desorption/ionization mass spectrometry (PSD MALDI MS) and electrospray ionization on a hybrid quadrupole orthogonal time-of-flight tandem mass spectrometer (ESI Q-TOF MS/MS) were both used to demonstrate that a single molecule of beta-tubulin, from either dynamic cytoplasmic microtubules or stable axonemal microtubules, can be glycylated on each of the last four C-terminal glutamate residues Glu437, Glu438, Glu439, and Glu441 in the sequence 427DATAEEEGEFEEEGEQ442. In both dynamic and stable microtubules the most abundant beta-tubulin isoform contains six posttranslationally added glycine residues: two glycine residues on both Glu437 and Glu438 and one glycine residue on both Glu439 and Glu441. The number and relative abundance of glycylated isoforms of beta-tubulin in both cytoplasmic and axonemal microtubules were compared by MALDI MS.1 The abundance of the major glycylated isoforms in axonemal tubulin decreases regularly with glycylation levels from 6 to 19 whereas it drops abruptly in cytoplasmic tubulin with glycylation levels from 6 to 9. However, the polyglycine chains are similarly distributed on the four C-terminal glutamate residues of cytoplasmic and axonemal tubulin. The polyglycylation results in bulky C-terminal domains with negatively charged surfaces, all surrounding the microtubular structure.     

Complement C3 and C5 Deficiency Affects Fracture Healing

There is increasing evidence that complement may play a role in bone development. Our previous studies demonstrated that the key complement receptor C5aR was strongly expressed in the fracture callus not only by immune cells but also by bone cells and chondroblasts, indicating a function in bone repair. To further elucidate the role of complement in bone healing, this study investigated fracture healing in mice in the absence of the key complement molecules C3 and C5. C3^-/- and C5^-/- as well as the corresponding wildtype mice received a standardized femur osteotomy, which was stabilized using an external fixator. Fracture healing was investigated after 7 and 21 days using histological, micro-computed tomography and biomechanical measurements. In the early phase of fracture healing, reduced callus area (C3^-/-: -25%, p=0.02; C5^-/-: -20% p=0.052) and newly formed bone (C3^-/-: -38%, p=0.01; C5^-/-: -52%, p=0.009) was found in both C3- and C5-deficient mice. After 21 days, healing was successful in the absence of C3, whereas in C5-deficient mice fracture repair was significantly reduced, which was confirmed by a reduced bending stiffness (-45%; p=0.029) and a smaller callus volume (-17%; p=0.039). We further demonstrated that C5a was activated in C3^-/- mice, suggesting cleavage via extrinsic pathways. Our results suggest that the activation of the terminal complement cascade in particular may be crucial for successful fracture healing.     

Methyl chloride isotopic signatures from Irish forest soils and a comparison between abiotic and biogenic methyl halide soil fluxes

Forest soils demonstrate in a microcosm the difficulties that are faced in quantifying methyl halide budgets. Carbon isotopic analyses have been proposed as a potential tool to address these concerns and in this study we have measured significant enrichment of the methyl chloride ¹³C/¹²C isotopic ratio (from ?40.2 ± 0.8‰ to ?33.4 ± 7.4‰) after 9 min chamber emplacement on local Irish forest soils. This enrichment occurred independent of direction of methyl chloride fluxes. Measurements from soil cores in a flow‐through system (FTS) are comparable with chamber‐based isotopic measurements and indicate that methyl chloride produced abiotically from organic soil horizons has an isotopic ¹³C signature of ?53 ± 49‰, significantly less depleted than previously reported. Average net methyl chloride, methyl bromide and methyl iodide fluxes from soils (77.8 ± 2.1, 1.25 ± 3.63 and 0.35 ± 2.00 μg MeX m^?2 day^?1, respectively) are in line with previously reported values; however, a better understanding of spatial and temporal variability is needed for budget quantification. Methyl halide fluxes from FTS soil cores demonstrate that, on a per gram basis, most consumption occurs through biologically driven processes in the O horizon, with progressively smaller contributions in deeper horizons. Sporadic biogenic production was observed in shallow soil horizons only. Abiotic production was at most one‐tenth the net biological reaction rate in the O horizon and did not appear to be significantly different from zero in lower horizons. Modelled emissions based upon observed and reported rates for production, consumption and diffusion within the soil atmosphere system are unable to replicate all observed isotopic signatures from chamber fluxes.     

Genetic structure, habitat fragmentation and bottlenecks in Danish bank voles (Clethrionomys glareolus)

Tissue-samples from 161 bank voles (Clethrionomys glareolus) collected in three forests (five sampling localities) situated in eastern Jutland (Denmark) were analysed by nine microsatellite loci. The genetic diversity found within the populations was high (H_e=0.753–0.806).Bank voles have specific habitat requirements favouring woodlots, hedgerows and deciduous forests as their prime living area. Hence, a natural or human-induced fragmentation of the forest may cause a sub-structuring of the populations and thereby a restriction of dispersal among populations. The sub-structuring indicated by the observed significant genetic differentiation among the five geographically distinct localities (F_st =0.033, P<0.05) could either result from habitat fragmentation or a combination of home range behaviour and different tree composition in the forests. A road situated between two adjacent forests was not found to exert any barrier effect to the gene flow of bank voles. In one out of five localities investigated, genetic evidence for a recent bottleneck-like situation was found. Bank voles are known to exhibit sometimes huge density fluctuations not only from year to year but also from season to season. The bottleneck-like situation found could therefore be due to the low number of individuals during the low-density phase.     

Mammography screening in the county of Fyn. November 1993-December 1999

This report covers the outcome of the first three invitation rounds of the organised mammography screening programme in the county of Fyn. The programme started in November 1993, and the third invitation round ended on 31 December 1999. The screening takes place either at a special clinic located at University Hospital Odense or in a mobile unit. Women living in and around the city of Odense are examined at the clinic (about 55%), while the rest are examined in the mobile unit. Two-view mammography is used at the first screening. Women with dense breast tissue will continue to have two-view mammography (about 60%), whereas the rest will have singleview mammography at the subsequent screens. All screening images are exposed at the mammography-screening clinic and evaluated with double reading in the clinic. The programme targets women aged 50-69, except those undergoing treatment for breast cancer or going for regular check-ups following breast cancer. Based on the updated population register, the IT-Centre of the county of Fyn issues the invitations. Invited are all women aged 50-69 and living in the county of Fyn when their general practitioners' patients are invited. During the first 3 invitation rounds, 136,079 screening tests were made. Of these, 129,375 tests were made in the women aged 50-69 targeted by the programme. In addition, 6682 screening tests were made in women aged 70 and above, and 22 screening tests were made in women below the age of 50. As a consequence of the mammography screening 2657 assessments were made, 1145 women had surgery, 782 women were diagnosed with invasive breast cancer, and 109 women were diagnosed with ductal carcinoma in situ. A participation rate for the first invitation round was calculated immediately after the end of the round based on the number of participants divided by the number of women invited. This percentage was 88%. Invitation data are, however, not stored. It is therefore not possible now to calculate the participation rates in previous invitation rounds based on the same method. We have therefore chosen to calculate the participation rate as the coverage, i.e. the number of participants divided by the average number of women in the county of Fyn during a given invitation round. Calculated in this way, 84% participated in the first round, 84% in the second round, and 82% in the third round. It should be remembered that these figures do not take into account that some women are not invited because they 1) were undergoing current treatment for breast cancer or going for regular check-ups following breast cancer, or 2) did not participate in the previous round (and never actively informed the programme that they wanted an invitation to the next invitation round), relevant only for the second and third invitation round. For the second and third invitation rounds, the programme only invited women who participated in the previous invitation round, asked the clinic for an invitation, or entered the target population since the last invitation round. Therefore the participation rate in the second invitation round among actually invited women will be close to 94%, as 94% of those participating in the first round came for the second round. For the third invitation round, the participation rate among actually invited women will be close to 96%, as 96% of those participating in the first and second rounds came for the third round. One per cent of the participants in the first invitation round were diagnosed with invasive breast cancer or ductal carcinoma in situ. The detection rate was 0.5% in both the second and third invitation rounds. Ductal carcinoma in situ cases constituted 14% of the detected cases in the first and second rounds, and 10% in the third round. The percentage of invasive breast cancer 10 mm of less was 38%, 31%, and 32%, respectively, and 68%, 74%, and 73%, respectively, were node-negative. The screening programme of the county of Fyn fulfilled all the quality assessment parameters specified by the European guidelines on breast cancer screening, except two. The proportionate interval cancer rate was higher than specified in the guidelines, probably mainly due to the fact that the Fyn programme operates without early recalls. The proportion of stage II+ cancers was higher than specified in the guidelines, which seems, however, to be due to inconsistency between some of the performance indicators in the European guidelines. This analysis of the outcome from the first three invitation rounds of the mammography screening programme in the county of Fyn thus showed that it is a programme of high quality with a favourable profile of the prognostic indicators. The screening programme is hopefully well on its way to reducing breast cancer mortality in the county of Fyn.     

Utilizing Nanobody Technology to Target Non-Immunodominant Domains of VAR2CSA

Placental malaria is a major health problem for both pregnant women and their fetuses in malaria endemic regions. It is triggered by the accumulation of Plasmodium falciparum-infected erythrocytes (IE) in the intervillous spaces of the placenta and is associated with foetal growth restriction and maternal anemia. IE accumulation is supported by the binding of the parasite-expressed protein VAR2CSA to placental chondroitin sulfate A (CSA). Defining specific CSA-binding epitopes of VAR2CSA, against which to target the immune response, is essential for the development of a vaccine aimed at blocking IE adhesion. However, the development of a VAR2CSA adhesion-blocking vaccine remains challenging due to (i) the large size of VAR2CSA and (ii) the extensive immune selection for polymorphisms and thereby non-neutralizing B-cell epitopes. Camelid heavy-chain-only antibodies (HcAbs) are known to target epitopes that are less immunogenic to classical IgG and, due to their small size and protruding antigen-binding loop, able to reach and recognize cryptic, conformational epitopes which are inaccessible to conventional antibodies. The variable heavy chain (VHH) domain is the antigen-binding site of camelid HcAbs, the so called Nanobody, which represents the smallest known (15 kDa) intact, native antigen-binding fragment. In this study, we have used the Nanobody technology, an approach new to malaria research, to generate small and functional antibody fragments recognizing unique epitopes broadly distributed on VAR2CSA.     

Duplication and transposition of the<Emphasis Type="Italic"> NF1</Emphasis> pseudogene regions on chromosomes 2, 14, and 22

Numerous NF1 pseudogenes have been identified in the human genome. Those in 2q21, 14q11, and 22q11 form a subset with a similar genomic organization and a high sequence homology. We have studied, by polymerase chain reaction and fluorescence in situ hybridization, the extent of homology of the regions surrounding these NF1 pseudogenes. Our analyses have demonstrated that a fragment of at least 640 kb is homologous between the three regions. Based on previous studies and these new findings, we propose a model for the spreading of the NF1 pseudogene-containing regions. A fragment of approximately 640 kb was first duplicated in chromosome region 2q21 and transposed to 14q11. Subsequently, this fragment was duplicated in 14q11 and transposed to 22q11. A part of the 640-kb fragment in 14q11, with a length of about 430 kb, was further duplicated to a variable extent in 14q11. In addition, we have identified sequences that may facilitate the duplication and transposition of the 640-kb and 430-kb fragments.