Microtubule alteration is an early cellular reaction to the metabolic challenge in ischemic cardiomyocytes

Cytoskeleton damage, particularly microtubule (MT) alterations, may play an important role in the pathogenesis of ischemia-induced myocardial injury. However, this disorganization has been scarcely confirmed in the cellular context. We evaluated MT network disassembly in myoblast cell line H9c2 and in neonatal rat cardiomyocytes in an in vitro substrate-free hypoxia model of simulated ischemia (SI). After different duration of SI from 30 up to 180 min, the cells were fixed and the microtubule network was revealed by immunocytochemistry. The microtubule alterations were quantified using a house-developed image analysis program. Additionally, the tubulin fraction were extracted and quantified by Western blotting. The cell respiration, the release of cellular LDH and the cell viability were evaluated at the same periods. An early MT disassembly was observed after 60 min of SI. The decrease in MT fluorescence intensity at 60 and 90 min was correlated with a microtubule disassembly. Conversely, SI-induced significant LDH release (35%) and decrease in cell viability (34%) occurred after 120 min only. These results suggest that the simulated ischemia-induced changes in MT network should not be considered as an ultrastructural hallmark of the cell injury and could rather be an early ultrastructural correlate of the cellular reaction to the metabolic challenge.     

Fine mapping of the Schnyder’s crystalline corneal dystrophy locus

Schnyders crystalline corneal dystrophy (SCCD) is a rare autosomal dominant eye disease with a spectrum of clinical manifestations that may include bilateral corneal clouding, arcus lipoides, and anterior corneal crystalline cholesterol deposition. We have previously performed a genome-wide linkage analysis on two large Swede-Finn families and mapped the SCCD locus to a 16-cM interval between markers D1S2633 and D1S228 on chromosome 1p36. We have collected 11 additional families from Finland, Germany, Turkey, and USA to narrow the critical region for SCCD. Here, we have used haplotype analysis with densely spaced microsatellite markers in a total of 13 families to refine the candidate interval. A common disease haplotype was observed among the four Swede-Finn families indicating the presence of a founder effect. Recombination results from all 13 families refined the SCCD locus to 2.32 Mbp between markers D1S1160 and D1S1635. Within this interval, identity-by-state was present in all 13 families for two markers D1S244 and D1S3153, further refining the candidate region to 1.58 Mbp.     

Phenotypic variation of Pseudomonas brassicacearum as a plant root-colonization strategy

Pseudomonas brassicacearum was isolated as a major root-colonizing population from Arabidopsis thaliana. The strain NFM421 of P. brassicacearum undergoes phenotypic variation during A. thaliana and Brassica napus root colonization in vitro as well as in soil, resulting in different colony appearance on agar surfaces. Bacteria forming translucent colonies (phase II cells) essentially were localized at the surface of young roots and root tips, whereas wild-type cells (phase I cells) were localized at the basal part of roots. The ability of phase II cells to spread and colonize new sites on root surface correlates with over-production of flagellin as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of surface proteins and microsequencing. Moreover, phase II cells showed a higher ability to swim and to swarm on semisolid agar medium. Phase I and phase II cells of P. brassicacearum NFM421 were tagged genetically with green fluorescent protein and red fluorescent protein. Confocal scanning laser microscopy was used to localize phase II cells on secondary roots and root tips of A. thaliana, whereas phase I cells essentially were localized at the basal part of roots. These experiments were conducted in vitro and in soil. Phenotypic variation on plant roots is likely to be a colonization strategy that may explain the high colonization power of P. brassicacearum.     

Discrimination between lumenal and cytosolic sites of deglycosylation in endoplasmic reticulum-associated degradation of glycoproteins by using benzyl mannose in CHO cell lines

Recent studies demonstrated that deglycosylation step is a prerequisite for endoplasmic reticulum (ER)-associated degradation of misfolded glycoproteins. Here, we report the advantages of using benzyl mannose during pulse-chase experiments to study the subcellular location of the deglycosylation step in Chinese hamster ovary (CHO) cell lines. Benzyl mannose inhibited both the ER-to-cytosol transport of oligomannosides and the trimming of cytosolic-labeled oligomannosides by the cytosolic mannosidase in vivo. We pointed out the occurrence of two subcellular sites of deglycosylation. The first one is located in the ER lumen, and led to the formation of Man8GlcNAc2 (isomer B) in wild-type CHO cell line and Man4GlcNAc2 in Man-P-Dol-deficient cell line. The second one was revealed in CHO mutant cell lines for which a high rate of glycoprotein degradation was required. It occurred in the cytosol and led to the liberation of oligosaccharides species with one GlcNAc residue and with a pattern similar to the one bound onto glycoproteins. The cytosolic deglycosylation site was not specific for CHO mutant cell lines, since we demonstrated the occurrence of cytosolic pathway when the formation of truncated glycans was induced in wild-type cells.     

Detection of mustelid herpesvirus-1 infected European badgers (Meles meles) in the British Isles

The aim of this study was to assess the frequency of mustelid herpesvirus-1 (MusHV-1) infection in free-ranging badgers (Meles meles) in the British Isles. A polymerase chain reaction assay was developed that detected MusHV-1 DNA in 95% (18/19) and 100% (10/10) of anticoagulant-treated blood samples collected from free-ranging badgers sampled in the southwest of England and the Republic of Ireland, respectively. An indirect immunoassay was also developed to detect MusHV-1-specific immunoglobulin-G in serum samples. Using an arbitrary cutoff of twice the optical density obtained with a virus-negative preparation, 32.7% (36/110) of sera sampled from badgers were positive. The conclusion drawn from these data is that infection with MusHV-1 is common among free-ranging badgers in the British Isles.     

NMR of Redox Proteins of Plants, Yeasts and Photosynthetic Bacteria

NMR spectroscopy has evolved dramatically over the past 15 years, establishing a new, reliable methodology for studying biomacromolecules at atomic resolution. The three-dimensional structure and dynamics of a biomolecule or a biomolecular complex is only one of the main types of information available using NMR. The spectral assignment to the specific nuclei of a biostructure is a very precise reflection of their electronic environment. Any change in this environment due to a structural change, the binding of a ligand or the redox state of a redox cofactor, will be very sensitively reported by changes in the different NMR parameters. The capabilities of the NMR method are currently expanding dramatically and it is turning into a powerful means to study biosystems dynamically in exchange between different conformations, exchanging ligands, transient complexes, or the activation/inhibition of regulated enzymes. We review here several NMR studies that have appeared during the past 5 or 6 years in the field of redox proteins of plants, yeasts and photosynthetic bacteria. These new results illustrate the recent biomolecular NMR evolution and provide new physiological models for understanding the different types of electron transfer, including glutaredoxins, thioredoxins and their dependent enzymes, the ferredoxin-NADP oxidoreductase complex, flavodoxins, the plastocyanin-cytochrome f complex, and cytochromes c.