Conservation priorities of genetic diversity in domesticated metapopulations: a study in taurine cattle breeds

We estimated neutral diversity of 21 European cattle breeds with 105 microsatellites. Nine of them resembled unselected Balkan Buša strains with diffuse breeding barriers and the 12 others were strongly differentiated, isolated breeds. Because of the impact of neutral genetic diversity on long-term population adaptive capacity, we discuss the long-term outcome of different conservation priorities in a subdivided metapopulation of the investigated cattle breeds. The optimal contribution to a pool of total genetic diversity allocated more than 95% of long-term relevant neutral diversity to virtually unselected strains of the Balkan Buša, while the maximization of total variance preferred inbred breeds. Current artificial selection methods, such as genomic selection sped up and a recovery of underestimated traits becomes quickly impossible. We emphasize that currently neutral and even deleterious alleles might be required for future genotypes in sustainable and efficient livestock breeding and production systems of a 21st century. We provide cumulative evidences that long-term survival relies on genetic complexity and complexity relies on allelic diversity. Our results suggest that virtually unselected, nonuniform strains harbor a crucial proportion of neutral diversity and should be conserved with high global priority. As one example, we suggest a cooperative maintenance of the nondifferentiated, highly fragmented, and fast vanishing metapopulation of Balkan Buša.     

Right atrial pacemaker lead thrombosis incidentally detected by transesophageal echocardiography

In a 62-year-old man with permanent atrial fibrillation and recurrent stroke, a large right atrial thrombus attached to a permanent pacemaker lead was incidentally identified by transesophageal echocardiography. Surgical treatment, recommended because of the large dimensions of the mass, was refused by the patient, and thrombus was successfully dissolved by anticoagulant treatment. Pathogenesis of pacemaker lead thrombosis, clinical implications, diagnostic and therapeutic options are discussed.     

Amphiphile-induced tubular budding of the bilayer membrane

Amphiphile-induced tubular budding of the erythrocyte membrane was studied using transmission electron microscopy. No chiral patterns of the intramembraneous particles were found, either on the cylindrical buds, or on the tubular nanoexovesicles. In agreement with these observations, the tubular budding may be explained by in-plane ordering of anisotropic membrane inclusions in the buds where the difference between the principal membrane curvatures is very large. In contrast to previously reported theories, no direct external mechanical force is needed to explain tubular budding of the bilayer membrane.     

Recombinant human cathepsin X is a carboxymonopeptidase only: a comparison with cathepsins B and L

The S1 and S2 subsite specificity of recombinant human cathepsins X was studied using fluorescence resonance energy transfer (FRET) peptides with the general sequences Abz-Phe-Xaa-Lys(Dnp)-OH and Abz-Xaa-Arg-Lys(Dnp)-OH, respectively (Abz=ortho-aminobenzoic acid and Dnp=2,4-dinitrophenyl; Xaa=various amino acids). Cathepsin X cleaved all substrates exclusively as a carboxymonopeptidase and exhibited broad specificity. For comparison, these peptides were also assayed with cathepsins B and L. Cathepsin L hydrolyzed the majority of them with similar or higher catalytic efficiency than cathepsin X, acting as an endopeptidase mimicking a carboxymonopeptidase (pseudo-carboxymonopeptidase). In contrast, cathepsin B exhibited poor catalytic efficiency with these substrates, acting as a carboxydipeptidase or an endopeptidase. The S1' subsite of cathepsin X was mapped with the peptide series Abz-Phe-Arg-Xaa-OH and the enzyme preferentially hydrolyzed substrates with hydrophobic residues in the P1' position.     

Fulminant meningococcal sepsis in a young child--a case report

We are presenting a case of isolated fulminant meningococcal sepsis with two and a half year old child. Initial symptoms were obscure and common to many medical conditions, but also previously described as symptoms of meningococcal sepsis. Unrecognizing the seriousness of the condition child died at home, within few hours after examination and discharge from the hospital. Autopsy and microbiological findings unquestionably proved that the child died from septic shock caused by fulminant meningococcal sepsis.     

Fine-mapping and candidate gene analysis of bovine spinal muscular atrophy

Bovine spinal muscular atrophy (SMA), an autosomal recessive neurodegenerative disease, has been mapped at moderate resolution to the distal part of Chromosome 24. In this article we confirm this location and fine-map the SMA locus to an interval of approximately 0.8 cM at the very distal end of BTA24. Despite remarkable similarity to human SMA, the causative gene SMN can be excluded in bovine SMA. However, the interval where the disease now has been mapped contains BCL2, like SMN an antiapoptotic factor, and shown to bind to SMN. Moreover, knockout mice lacking the BCL2 gene show rapid motor neuron degeneration with early postnatal onset, as observed in bovine SMA. A comparative cattle/human map of the distal end of BTA24, based on the emerging bovine genome sequencing data, shows conserved synteny to HSA18 with hints of a segmental duplication and pericentic inversion just after the last available bovine marker DIK4971. This synteny lets us conclude that SMA is in immediate vicinity of the telomere. Candidate gene analysis of BCL2, however, excludes most of this gene, except its promoter region, and draws attention to the neighboring gene VPS4B, part of the endosomal protein-sorting machinery ESCRT-III which is involved in several neurodegenerative diseases. Stefan Krebs and Ivica Medugorac contributed equally to this work and agreed to be considered as first authors.     

Site-specific mutagenesis of the histidine precursor of diphthamide in the human elongation factor-2 gene confers resistance to diphtheria toxin

Protein synthesis elongation factor 2 (EF-2) from eukaryotes contains a conserved post-translationally modified histidine residue known as diphthamide. Diphthamide is a unique site of ADP-ribosylation by diphtheria toxin (DT), which is responsible for cell killing. In this report, we describe the construction of DT-resistant HeLa cell lines by engineering the toxin-resistant form of its specific substrate, protein elongation factor-2. Using site-specific mutagenesis of the histidine precursor of diphthamide, the histidine residue of codon 715 in human EF-2 cDNA was substituted with one of four amino acid residue codons: leucine, methionine, asparagine or glutamine. Mutant EF-2s were subcloned into a pCMVexSVneo expression vector, transfected into HeLa cells, and DT-resistant cell clones were isolated. The protective effect of mutant EF-2s against cell killing by DT, after exposing all four mutant strains derived from HeLa cells to different concentrations of the toxin (5-20 ng/mL) was demonstrated by: (1) the normal morphological appearance of the cells; (2) their unaffected or slightly slower growth rates; (3) their undisturbed electrophoretic DNA profiles whose integrity was virtually preserved. Mutant cell strains showed also considerable levels of resistance to very high concentrations of DT, in that they maintained slower but consistent rates of cell growth. It was hence concluded that despite its strict conservation and unique modification, the diphthamide histidine appears not to be essential to the function of human EF-2 in protein synthesis. In addition, DT-resistant HeLa cell clones should prove valuable hosts for various DT gene-containing vectors that express the toxin intracellularly.     

Mouth cavity base defect treated with biosynthetic graft

The use of in vitro prepared biosynthetic grafts can considerably improve the patient’s quality of life. This work reports on the use of an autologous graft prepared from a patient’s preputial cells cultivated on biodegradable polymeric membrane. Coladerm membrane is based on the chemically modified polyelectrolyte complex of atelocollagen and hyaluronan. The graft was used to cover a defect in the mouth cavity base and tongue after reconstruction surgery performed at this site in the past. The presented clinical case showed that the autologous biosynthetic graft prepared from foreskin cells can be successfully used for covering of medium-size defects in mouth cavity base resulting in the regeneration of target mouth structures with significant improvement of patient’s quality of life.     

Botulinum toxin's axonal transport from periphery to the spinal cord

Axonal transport of enzymatically active botulinum toxin A (BTX-A) from periphery to the CNS has been described in facial and trigeminal nerve, leading to cleavage of synaptosomal-associated protein 25 (SNAP-25) in central nuclei. Aim of present study was to examine the existence of axonal transport of peripherally applied BTX-A to spinal cord via sciatic nerve. We employed BTX-A-cleaved SNAP-25 immunohistochemistry of lumbar spinal cord after intramuscular and subcutaneous hind limb injections, and intraneural BTX-A sciatic nerve injections. Truncated SNAP-25 in ipsilateral spinal cord ventral horns and dorsal horns appeared after single peripheral BTX-A administrations, even at low intramuscular dose applied (5 U/kg). Cleaved SNAP-25 appearance in the spinal cord after BTX-A injection into the sciatic nerve was prevented by proximal intrasciatic injection of colchicine (5 mM, 2 μl). Cleaved SNAP-25 in ventral horn, using choline-acetyltransferase (ChAT) double labeling, was localized within cholinergic neurons. These results extend the recent findings on BTX-A retrograde axonal transport in facial and trigeminal nerve. Appearance of truncated SNAP-25 in spinal cord following low-dose peripheral BTX-A suggest that the axonal transport of BTX-A occurs commonly following peripheral application.