Cloning, expression and characterization of the murine orthologue of SBF2, the gene mutated in Charcot-Marie-Tooth disease type 4B2

Autosomal recessive hereditary motor and sensory neuropathy (HMSN) or Charcot-Marie-Tooth disease (CMT) is a clinically and genetically heterogeneous disorder of the peripheral nervous system. The clinical picture includes progressive distal weakness and atrophy, foot deformities, and distal sensory loss. For autosomal recessive CMT type 4B2 one locus was mapped to chromosome 11p15. Recently, mutations in SET binding factor 2 (SBF2), were identified as cause of CMT4B2. SBF2 is a member of the pseudo-phosphatase branch of myotubularins and all disease-associated mutations known to date lead to shortened or truncated proteins, also implicating loss-of-function. Here, we describe the molecular cloning and the expression pattern of Sbf2. The mRNA spans around 8 kb, and the protein shares high amino acid identity compared to the human protein suggesting a conserved function. Sbf2 is encoded by 40 exons on murine chromosome 7. In situ hybridization, Northern blots and RT-analysis revealed a very broad pattern of Sbf2 expression. Overexpressed epitope tagged Sbf2 showed cytoplasmic distribution. Taken together, this study provides information about the mRNA expression and subcellular localization of Sbf2 and as such helps in further understanding its function in development and disease.     

Loading of the knee joint during activities of daily living measured in vivo in five subjects

Detailed knowledge about loading of the knee joint is essential for preclinical testing of implants, validation of musculoskeletal models and biomechanical understanding of the knee joint. The contact forces and moments acting on the tibial component were therefore measured in 5 subjects in vivo by an instrumented knee implant during various activities of daily living.Average peak resultant forces, in percent of body weight, were highest during stair descending (346% BW), followed by stair ascending (316% BW), level walking (261% BW), one legged stance (259% BW), knee bending (253% BW), standing up (246% BW), sitting down (225% BW) and two legged stance (107% BW). Peak shear forces were about 10–20 times smaller than the axial force. Resultant forces acted almost vertically on the tibial plateau even during high flexion. Highest moments acted in the frontal plane with a typical peak to peak range ?2.91% BWm (adduction moment) to 1.61% BWm (abduction moment) throughout all activities. Peak flexion/extension moments ranged between ?0.44% BWm (extension moment) and 3.16% BWm (flexion moment). Peak external/internal torques lay between ?1.1% BWm (internal torque) and 0.53% BWm (external torque).The knee joint is highly loaded during daily life. In general, resultant contact forces during dynamic activities were lower than the ones predicted by many mathematical models, but lay in a similar range as measured in vivo by others. Some of the observed load components were much higher than those currently applied when testing knee implants.     

Site Mapping and Characterization of O-Glycan Structures on α-Dystroglycan Isolated from Rabbit Skeletal Muscle

The main extracellular matrix binding component of the dystrophin-glycoprotein complex, α-dystroglycan (α-DG), which was originally isolated from rabbit skeletal muscle, is an extensively O-glycosylated protein. Previous studies have shown α-DG to be modified by both O-GalNAc- and O-mannose-initiated glycan structures. O-Mannosylation, which accounts for up to 30% of the reported O-linked structures in certain tissues, has been rarely observed on mammalian proteins. Mutations in multiple genes encoding defined or putative glycosyltransferases involved in O-mannosylation are causal for various forms of congenital muscular dystrophy. Here, we explore the glycosylation of purified rabbit skeletal muscle α-DG in detail. Using tandem mass spectrometry approaches, we identify 4 O-mannose-initiated and 17 O-GalNAc-initiated structures on α-DG isolated from rabbit skeletal muscle. Additionally, we demonstrate the use of tandem mass spectrometry-based workflows to directly analyze glycopeptides generated from the purified protein. By combining glycomics and tandem mass spectrometry analysis of 91 glycopeptides from α-DG, we were able to assign 21 different residues as being modified by O-glycosylation with differing degrees of microheterogeneity; 9 sites of O-mannosylation and 14 sites of O-GalNAcylation were observed with only two sites definitively exhibiting occupancy by either type of glycan. The distribution of identified sites of O-mannosylation suggests a limited role for local primary sequence in dictating sites of attachment.     

Advanced fertility diagnosis in stallion semen using transmission electron microscopy

Routine semen analysis of stallions is based on light microscopy (LM). However, there are still a number of animals that are subfertile or even infertile not being identified with conventional semen analysis. The objective of this study was to investigate the suitability of transmission electron microscopy (TEM) for advanced fertility diagnosis in stallion. We examined ejaculates of 46 stallions with known fertility. Animals were divided into three different groups: group 1, fertile stallions (pregnant mares> or =70%, n=29); group 2, subfertile stallions (pregnant mares 10-69%, n=14); group 3, infertile stallions (pregnant mares<10%, n=3). Ejaculates were collected in spring 2002. Conventional semen analysis (volume, sperm concentration, motility, live:dead ratio and percentage of morphologically normal sperm) was immediately performed after semen collection. Ultrastructural analysis included the evaluation of 200 acrosomes, heads, midpieces and cross-sections of tails as well as 100 longitudinal sections of tails from every ejaculate. Using LM, we found a significant increase of morphological deviations from 24.5% (x ) in group 1 to 34.5% in group 2 and 73.5% in group 3. Using TEM, we found a significant increase of detached acrosomes from 6.1% in group 1 to 7.6% in group 2 and 21.4% in group 3. Deviations in tubule pattern were also increased (but not significant) from 2.7% in fertile and 2.8% in subfertile to 11.4% in infertile stallions as well as multiple tails from 1.9% in fertile to 2.0% in subfertile and 8.9% in infertile. Our data indicate that TEM is suitable for advanced fertility diagnostic in stallions, giving a connection between fertility and morphology. It suggests that the most likely reason for sub- and infertility in stallion in case of increased LM pathomorphology of semen are acrosomal alterations, especially detached acrosomes.     

M line-deficient titin causes cardiac lethality through impaired maturation of the sarcomere

Titin, the largest protein known to date, has been linked to sarcomere assembly and function through its elastic adaptor and signaling domains. Titin's M-line region contains a unique kinase domain that has been proposed to regulate sarcomere assembly via its substrate titin cap (T-cap). In this study, we use a titin M line-deficient mouse to show that the initial assembly of the sarcomere does not depend on titin's M-line region or the phosphorylation of T-cap by the titin kinase. Rather, titin's M-line region is required to form a continuous titin filament and to provide mechanical stability of the embryonic sarcomere. Even without titin integrating into the M band, sarcomeres show proper spacing and alignment of Z discs and M bands but fail to grow laterally and ultimately disassemble. The comparison of disassembly in the developing and mature knockout sarcomere suggests diverse functions for titin's M line in embryonic development and the adult heart that not only involve the differential expression of titin isoforms but also of titin-binding proteins.     

Cytochrome c-d regulates developmental apoptosis in the Drosophila retina

The role of cytochrome c (Cyt c) in caspase activation has largely been established from mammalian cell-culture studies, but much remains to be learned about its physiological relevance in situ. The role of Cyt c in invertebrates has been subject to considerable controversy. The Drosophila genome contains distinct cyt c genes: cyt c-p and cyt c-d. Loss of cyt c-p function causes embryonic lethality owing to a requirement of the gene for mitochondrial respiration. By contrast, cyt c-d mutants are viable but male sterile. Here, we show that cyt c-d regulates developmental apoptosis in the pupal eye. cyt c-d mutant retinas show a profound delay in the apoptosis of superfluous interommatidial cells and perimeter ommatidial cells. Furthermore, there is no apoptosis in mutant retinal tissues for the Drosophila homologues of apoptotic protease-activating factor 1 (Ark) and caspase 9 (Dronc). In addition, we found that cyt c-d--as with ark and dronc-regulates scutellar bristle number, which is known to depend on caspase activity. Collectively, our results indicate a role of Cyt c in caspase regulation of Drosophila somatic cells.     

Phylogenetic analysis of the tenascin gene family: evidence of origin early in the chordate lineage

Background  

Tenascins are a family of glycoproteins found primarily in the extracellular matrix of embryos where they help to regulate cell proliferation, adhesion and migration. In order to learn more about their origins and relationships to each other, as well as to clarify the nomenclature used to describe them, the tenascin genes of the urochordate Ciona intestinalis, the pufferfish Tetraodon nigroviridis and Takifugu rubripes and the frog Xenopus tropicalis were identified and their gene organization and predicted protein products compared with the previously characterized tenascins of amniotes.     

A novel surface protein of Trichomonas vaginalis is regulated independently by low iron and contact with vaginal epithelial cells

Background  

Trichomonosis caused by Trichomonas vaginalis is the number one, non-viral sexually transmitted disease (STD) that affects more than 250 million people worldwide. Immunoglobulin A (IgA) has been implicated in resistance to mucosal infections by pathogens. No reports are available of IgA-reactive proteins and the role, if any, of this class of antibody in the control of this STD. The availability of an IgA monoclonal antibody (mAb) immunoreactive to trichomonads by whole cell (WC)-ELISA prompted us to characterize the IgA-reactive protein of T. vaginalis.     

The C-terminal fragment of axon guidance molecule Slit3 binds heparin and neutralizes heparin's anticoagulant activity

Slit3 is a large molecule with multiple domains and belongs to axon guidance families. To date, the biological functions of Slit3 are still largely unknown. Our recent study demonstrated that the N-terminal fragment of Slit3 is a novel angiogenic factor. In this study, we examined the biological function of the C-terminal fragment of human Slit3 (HSCF). The HSCF showed a high-affinity binding to heparin. The binding appeared to be heparin/heparan sulfate-specific and depends on the size, the degree of sulfation, the presence of N- and 6-O-sulfates and carboxyl moiety of the polysaccharide. Functional studies observed that HSCF inhibited antithrombin binding to heparin and neutralized the antifactor IIa and Xa activities of heparin and the antifactor IIa activity of low-molecular-weight heparin (LMWH). Thromboelastography analysis observed that HSCF reversed heparin's anticoagulation in global plasma coagulation. Taken together, these observations demonstrate that HSCF is a novel heparin-binding protein that potently neutralizes heparin's anticoagulation activity. This study reveals a potential for HSCF to be developed as a new antidote to treat overdosing of both heparin and LMWH in clinical applications.     

Zugang zur Artenvielfalt im Schulunterricht

Mapping a songbird fauna with pupils How to induce pupils aged 13 to 14 years to successfully map the songbird fauna of a city park? The mapping project took place within 1.5 hours on an April morning. Pupils were organized as small working groups using maps, binoculars, and field guides produced by themselves. The project had been prepared by a 6 lesson course. Results were later discussed with nature conservancy officials. Pupils having participated in the project displayed increased knowledge of birds and motivation to observe them.