Identification of the Mycobacterium marinum Apa antigen O-mannosylation sites reveals important glycosylation variability with the M. tuberculosis Apa homologue

The 45/47kDa Apa, an immuno-dominant antigen secreted by Mycobacterium tuberculosis is O-mannosylated at multiple sites. Glycosylation of Apa plays a key role in colonization and invasion of the host cells by M. tuberculosis through interactions of Apa with the host immune system C-type lectins. Mycobacterium marinum (M.ma) a fish pathogen, phylogenetically close to M. tuberculosis, induces a granulomatous response with features similar to those described for M. tuberculosis in human. Although M.ma possesses an Apa homologue, its glycosylation status is unknown, and whether this represents a crucial element in the pathophysiology induced by M.ma remains to be addressed. To this aim, we have identified two concanavalin A-reactive 45/47kDa proteins from M.ma, which have been further purified by a two-step anion exchange chromatography process. Advanced liquid chromatography-nanoESI mass spectrometry-based proteomic analyses of peptides, derived from either tryptic digestion alone or in combination with the Asp-N endoproteinase, established that M.ma Apa possesses up to seven distinct O-mannosylated sites with mainly single mannose substitutions, which can be further extended at the Ser/Thr/Pro rich region near the N-terminus. This opens the way to further studies focussing on the involvement and biological functions of Apa O-mannosylation using the M.ma/zebrafish model.     

Chemoenzymatic synthesis and antiviral evaluation of conformationally constrained and 3'-methyl-branched carbanucleosides using both enantiomers of the same building block

Starting from both enantiomers of a readily available building block, a straightforward enantioselective approach to constrained 3'-methyl-2',3'-alpha-oxirane-fused and 3'-methyl-3',4'-alpha-oxirane-fused carbanucleosides bearing different purine base analogues is described. The title compounds were evaluated as potential antiviral agents against important viruses. None of the new compounds had significant antiviral activity at a concentration of 100 microg/mL, which was the highest concentration tested.     

Genetic and phenotypic studies of the dark-like mutant mouse

The dark-like (dal) mutant mouse has a pleiotropic phenotype that includes dark dorsal hairs and reproductive degeneration. Their pigmentation phenotype is similar to Attractin (Atrn) mutants, which also develop vacuoles throughout the brain. In further characterizing the testicular degeneration of dal mutant males, we found that they had reduced serum testosterone and developed vacuoles in their testes. Genetic crosses placed dal upstream of the melanocortin 1 receptor (Mc1r) and downstream of agouti, although dal suppressed the effect of agouti on pigmentation but not body weight. Atrn(mg-3J) and dal showed additive effects on pigmentation, testicular vacuolation, and spongiform neurodegeneration, but transgenic overexpression of Attractin-like-1 (Atrnl1), which compensates for loss of ATRN, did not rescue dal mutant phenotypes. Our results suggest dal and Atrn function in the same pathway and that identification of the dal gene will provide insight into molecular mechanisms of vacuolation in multiple cell types.     

A PTS EII mutant library in Group A Streptococcus identifies a promiscuous man‐family PTS transporter influencing SLS‐mediated hemolysis

The Group A Streptococcus (GAS, Streptococcus pyogenes) is a Gram‐positive human pathogen that must adapt to unique host environments in order to survive. Links between sugar metabolism and virulence have been demonstrated in GAS, where mutants in the phosphoenolpyruvate‐dependent phosphotransferase system (PTS) exhibited Streptolysin S (SLS)‐mediated hemolysis during exponential growth. This early onset hemolysis correlated with an increased lesion size and severity in a murine soft tissue infection model when compared with parental M1T1 MGAS5005. To identify the PTS components responsible for this phenotype, we insertionally inactivated the 14 annotated PTS EIIC‐encoding genes in the GAS MGAS5005 genome and subjected this library to metabolic and hemolysis assays to functionally characterize each EIIC. It was found that a few EIIs had a very limited influence on PTS sugar metabolism, whereas others were fairly promiscuous. The mannose‐specific EII locus, encoded by manLMN, was expressed as a mannose‐inducible operon that exhibited the most influence on PTS sugar metabolism, including mannose. Importantly, components of the mannose‐specific EII also acted to prevent the early onset of SLS‐mediated hemolysis. Interestingly, these roles were not identical in two different M1T1 GAS strains, highlighting the possible versatility of the PTS to adapt to strain‐specific needs.     

Id2 reverses cell cycle arrest induced by {gamma}-irradiation in human HaCaT keratinocytes

Id2 plays a key role in epithelial cells, regulating differentiation, the cell cycle, and proliferation. Because human skin constantly renews itself and is the first target of irradiation, it is of primary interest to evaluate whether such a gene may be regulated in keratinocytes exposed to ionizing radiation. We show here that Id2 is induced in response to gamma-irradiation and have investigated the consequence of this regulation on cell fate. Using RNA interference, we observed that Id2 extinction significantly reduces cell growth in human keratinocytes through the control of the G(1)-S transition of the cell cycle. We have investigated whether the impact of Id2 on the cell cycle may have a physiological role on the cell's ability to cope with radiative stress. Indeed, when Id2 is down-regulated through interfering RNA, cells are more sensitive to irradiation. Conversely, when Id2 is overexpressed, this somehow protects the cell. We propose that Id2 favors reentering the cell cycle after radiation-induced cell cycle arrest to permit the recovery of keratinocytes exposed to ionizing radiation.     

First passage time problem for a drifted Ornstein-Uhlenbeck process

We consider a continuous stochastic process defined as a drifted Ornstein-Uhlenbeck, for which the first passage time is of interest. The process being non-homogeneous, the first passage time probability density function cannot be found analytically, but numerical methods enable to find its estimate. Estimating the first passage time implies solving an unsteady convection-diffusion equation, with variable coefficients, and we use an implicit Euler scheme to solve it. This work is applied to simulated data, and the continuous process is inspired from recent work on biological marker modelling for HIV-positive patients. The first passage time probability density function can be useful to compare the marker progression in different groups. Numerical results show that the first passage time is highly dependent from the process perturbation, and is then more relevant than methods not considering the stochastic process directly to compare the progression.     

Patterns of protein oxidation in Arabidopsis seeds and during germination

Increased cellular levels of reactive oxygen species are known to occur during seed development and germination, but the consequences in terms of protein degradation are poorly characterized. In this work, protein carbonylation, which is an irreversible oxidation process leading to a loss of function of the modified proteins, has been analyzed by a proteomic approach during the first stages of Arabidopsis (Arabidopsis thaliana) seed germination. In the dry mature seeds, the legumin-type globulins (12S cruciferins) were the major targets. However, the acidic alpha-cruciferin subunits were carbonylated to a much higher extent than the basic (beta) ones, consistent with a model in which the beta-subunits are buried within the cruciferin molecules and the alpha-subunits are more exposed to the outside. During imbibition, various carbonylated proteins accumulated. This oxidation damage was not evenly distributed among seed proteins and targeted specific proteins as glycolytic enzymes, mitochondrial ATP synthase, chloroplastic ribulose bisphosphate carboxylase large chain, aldose reductase, methionine synthase, translation factors, and several molecular chaperones. Although accumulation of carbonylated proteins is usually considered in the context of aging in a variety of model systems, this was clearly not the case for the Arabidopsis seeds since they germinated at a high rate and yielded vigorous plantlets. The results indicate that the observed specific changes in protein carbonylation patterns are probably required for counteracting and/or utilizing the production of reactive oxygen species caused by recovery of metabolic activity in the germinating seeds.     

Allozyme polymorphism and interspecific relationships in the Common starling (Sturnus vulgaris) and Spotless starling (S. unicolor) (Aves: Sturnidae)

This paper reports a genetic study of Sturnus vulgaris and S. unicolor (Sturnidae), two similar bird species which have recently become sympatric in north-eastern Spain. Seven enzyme systems (alcohol dehydrogenase, esterase, glutamate dehydrogenase, glucose-6-phosphate dehydrogenase, leucin aminopeptidase, L-lactate dehydrogenase and superoxide dismutase) were analyzed, resolving 22 presumed genetic loci with 28 alleles; 27 of them were found in both species. Gene flow was estimated to be number of migrants per generation (Nm) = 6, and genetic identities among populations were high, ranging from I = 0.96 to I = 0.99. On the whole, the results demonstrate that these Sturnus populations share a gene pool, showing slight genetic differences between the two starling speices.     

Production and secretion of a heterologous protein by turnip hairy roots with superiority over tobacco hairy roots

A fully contained and efficient heterologous protein production system was designed using Brassica rapa rapa (turnip) hairy roots. Two expression cassettes containing a cauliflower mosaic virus (CaMV) 35S promoter with a duplicated enhancer region, an Arabidopsis thaliana sequence encoding a signal peptide and the CaMV polyadenylation signal were constructed. One cassette was used to express the green fluorescent protein (GFP)-encoding gene in hairy roots grown in flasks. A stable and fast-growing hairy root line secreted GFP at >120 mg/l culture medium. GFP represented 60 % of the total soluble proteins in the culture medium. Turnip hairy roots retained sustainable growth and stable GFP production over 3 years. These results were superior to those obtained using tobacco hairy roots.     

Site-specific attachment of polyethylene glycol-like oligomers to proteins and peptides

Modification of proteins with polymers is a viable method to tune protein properties, e.g., to render them more water-soluble by using hydrophilic polymers. We have utilized precision-length, polyethylene glycol-based oligomers carrying a thioester moiety in transthioesterification and native chemical ligation reactions with internal and N-terminal cysteine residues in proteins and peptides. These reactions lead to uniquely modified proteins with an increased solubility in chaotrope- and detergent-free aqueous systems. Polymer modification of internal cysteines is fully reversible and allows generation of stable protein-polymer conjugates for enzymatic manipulations as demonstrated by proteolytic cleavage of a protein construct that was only soluble in buffers incompatible with protease activity before polymer modification. The permanent polymer modification of a Rab protein at its N-terminal cysteine produced a fully active Rab variant that was efficiently prenylated. Thus, PEGylation of prenylated proteins might be a viable route to increase water solubility of such proteins in order to carry out experiments in detergent- and lipid-free systems.