Solution NMR structure of an immunodominant epitope of myelin basic protein. Conformational dependence on environment of an intrinsically unstructured protein

Using solution NMR spectroscopy, three-dimensional structures have been obtained for an 18-residue synthetic polypeptide fragment of 18.5 kDa myelin basic protein (MBP, human residues Q81-T98) under three conditions emulating the protein's natural environment in the myelin membrane to varying degrees: (a) an aqueous solution (100 mM KCl pH 6.5), (b) a mixture of trifluoroethanol (TFE-d2) and water (30 : 70% v/v), and (c) a dispersion of 100 mM dodecylphosphocholine (DPC-d38, 1 : 100 protein/lipid molar ratio) micelles. This polypeptide sequence is highly conserved in MBP from mammals, amphibians, and birds, and comprises a major immunodominant epitope (human residues N83-T92) in the autoimmune disease multiple sclerosis. In the polypeptide fragment, this epitope forms a stable, amphipathic, alpha helix under organic and membrane-mimetic conditions, but has only a partially helical conformation in aqueous solution. These results are consistent with recent molecular dynamics simulations that showed this segment to have a propensity to form a transient alpha helix in aqueous solution, and with electron paramagnetic resonance (EPR) experiments that suggested a alpha-helical structure when bound to a membrane [I. R. Bates, J. B. Feix, J. M. Boggs & G. Harauz (2004) J Biol Chem, 279, 5757-5764]. The high sensitivity of the epitope structure to its environment is characteristic of intrinsically unstructured proteins, like MBP, and reflects its association with diverse ligands such as lipids and other proteins.     

Genetic evidence that the acyl coenzyme A binding protein AcbA and the serine protease/ABC transporter TagA function together in Dictyostelium discoideum cell differentiation

The acyl coenzyme A (CoA) binding protein AcbA is cleaved to form a peptide (SDF-2) that coordinates spore encapsulation during the morphogenesis of Dictyostelium discoideum fruiting bodies. We present genetic evidence that the misspecification of cell types seen in mutants of the serine protease/ABC transporter TagA results from the loss of normal interactions with AcbA. Developmental phenotypes resulting from aberrant expression of the TagA protease domain, such as the formation of supernumerary tips on aggregates and the production of excess prestalk cells, are suppressed by null mutations in the acbA gene. Phenotypes resulting from the deletion of tagA, such as overexpression of the prestalk gene ecmB and the misexpression of the prespore gene cotB in stalk cells, are also observed in acbA mutants. Moreover, tagA- mutants fail to produce SDF-2 during fruiting body morphogenesis but are able to do so if they are stimulated with exogenous SDF-2. These results indicate that the developmental program depends on TagA and AcbA working in concert with each other during cell type differentiation and suggest that TagA is required for normal SDF-2 signaling during spore encapsulation.     

Differential expression of sarcoplasmic proteins in four heterogeneous ovine skeletal muscles

Fiber-type distribution is known to vary widely within and between muscles according to differences in muscle functions. 2-DE and MALDI-MS were used to investigate the molecular basis of muscle fiber type-related variability. We compared four lamb skeletal muscles with heterogeneous fiber-type composition that are relatively rich in fast-twitch fiber types, i.e., the semimembranosus, vastus medialis, longissimus dorsi, and tensor fasciae latae (TL). Our results clearly showed that none of the glycolytic metabolism enzymes detected, including TL which was most strongly glycolytic, made intermuscular differentiation possible. Muscle differentiation was based on the differential expression of proteins involved in oxidative metabolism, including not only citric acid cycle enzymes but also other classes of proteins with functions related to oxidative metabolism, oxidative stress, and probably to higher protein turnover. Detected proteins were involved in transport (carbonate dehydratase, myoglobin, fatty acid-binding protein), repair of misfolding damage (heat shock protein (HSP) 60 kDa, HSP-27 kDa, alpha-crystallin beta subunit, DJ1, stress-induced phosphoprotein), detoxification or degradation of impaired proteins (GST-Pi, aldehyde dehydrogenase, peroxiredoxin, ubiquitin), and protein synthesis (tRNA-synthetase). The fractionating method led to the detection of proteins involved in different functions related to oxidative metabolism that have not previously been shown concomitancy.     

Synthesis of 3'-deoxy-3'-C-methyl nucleoside derivatives

2',3'-Dideoxy-3'-C-methyl nucleosides bearing the five naturally occurring nucleic acid bases were synthesized. Additionally, the 3'-deoxy-3'-C-methyl nucleoside analogues bearing 5-aminoimidazole-4-carboxamide as well as 1,2,4-triazole-3-carboxamide moieties were prepared. The synthesis of the corresponding 2',3'-dideoxy-3'-C-methyl triazole derivative was also accomplished. The dideoxynucleoside derivatives were prepared by radical deoxygenation from their 3'-deoxy-3'-C-methyl parent ribonucleosides. When evaluated for their antiviral activity in cell culture experiments, none of these compounds showed any significant antiviral activity.     

Sustained neurochemical plasticity in central terminals of mouse DRG neurons following colitis

Sensitization of dorsal root ganglia (DRG) neurons is an important mechanism underlying the expression of chronic abdominal pain caused by intestinal inflammation. Most studies have focused on changes in the peripheral terminals of DRG neurons in the inflamed intestine but recent evidence suggests that the sprouting of central nerve terminals in the dorsal horn is also important. Therefore, we examine the time course and reversibility of changes in the distribution of immunoreactivity for substance P (SP), a marker of the central terminals of DRG neurons, in the spinal cord during and following dextran sulphate sodium (DSS)-induced colitis in mice. Acute and chronic treatment with DSS significantly increased SP immunoreactivity in thoracic and lumbosacral spinal cord segments. This increase developed over several weeks and was evident in both the superficial laminae of the dorsal horn and in lamina X. These increases persisted for 5 weeks following cessation of both the acute and chronic models. The increase in SP immunoreactivity was not observed in segments of the cervical spinal cord, which were not innervated by the axons of colonic afferent neurons. DRG neurons dissociated following acute DSS-colitis exhibited increased neurite sprouting compared with neurons dissociated from control mice. These data suggest significant colitis-induced enhancements in neuropeptide expression in DRG neuron central terminals. Such neurotransmitter plasticity persists beyond the period of active inflammation and might contribute to a sustained increase in nociceptive signaling following the resolution of inflammation.     

Potential of fecal microbiota for early-stage detection of colorectal cancer

Several bacterial species have been implicated in the development of colorectal carcinoma (CRC), but CRC-associated changes of fecal microbiota and their potential for cancer screening remain to be explored. Here, we used metagenomic sequencing of fecal samples to identify taxonomic markers that distinguished CRC patients from tumor-free controls in a study population of 156 participants. Accuracy of metagenomic CRC detection was similar to the standard fecal occult blood test (FOBT) and when both approaches were combined, sensitivity improved > 45% relative to the FOBT, while maintaining its specificity. Accuracy of metagenomic CRC detection did not differ significantly between early- and late-stage cancer and could be validated in independent patient and control populations (N = 335) from different countries. CRC-associated changes in the fecal microbiome at least partially reflected microbial community composition at the tumor itself, indicating that observed gene pool differences may reveal tumor-related host–microbe interactions. Indeed, we deduced a metabolic shift from fiber degradation in controls to utilization of host carbohydrates and amino acids in CRC patients, accompanied by an increase of lipopolysaccharide metabolism.     

Impact of active transport and transpiration on nickel and cadmium accumulation in the leaves of the Ni-hyperaccumulator Leptoplax emarginata: a biophysical approach

Aims

The primary aim of this study was to investigate the impact of active nickel and cadmium transport, transpiration and shoot biomass production on Ni and Cd accumulation in the leaves of the Ni-hyperaccumulator Leptoplax emarginata. A secondary objective was to observe the effects of various concentrations of nickel and cadmium in solutions on the plant growth and ecophysiological characteristics of these plants. Finally, the study sought to identify possible nickel and cadmium concentration gradients in solution as a function of the root distance.

Methods

The Intact Plant Transpiration Stream Concentration Factor (TSCF=xylem/solution solute concentration ratio) was determined for both Ni and Cd and for the selected intact transpiring Ni-hyperaccumulator Leptoplax emarginata, cultivated on two contrasting fertilized and Ni-Cd-contaminated sandy porous media (rhizotrons with central root compartments, linked to Mariotte tubes operated at ?1?kPa). IPTSCF_Ni and IPTSCF_Cd were calculated as the ratios between the hyperaccumulator plant’s nickel or cadmium mass in the leaves and the nickel or cadmium concentration in solution by the volume of water transpired during the period of culture. Plant growth characteristics and gas exchanges were also recorded.

Results

IPTSCF values were much greater than 1 (IPTSCF_Ni?=?5.2?±?0.9 and IPTSCF_Cd?=?4.4?±?0.6) whatever the amount of available Ni and Cd. This characterized a predominantly active plant metal uptake. Moreover, biological regulation was reported: plant growth and transpiration were significantly lower for hyperaccumulator plants cultivated in sand which was rich in available Ni and Cd, than for hyperaccumulator plants cultivated in topsoil, poor in available Ni and Cd. In the soil rhizosphere, capillary flow was related to transpiration and a depletion pattern was developed for Ni and sometimes for Cd.

Conclusions

Overall, the Intact Plant Transpiration Stream Concentration Factor appeared to be a relevant metal bioconcentration factor taking into account the predominant type of metal transport from roots to leaves, plant growth and transpiration coupling and metal availability. IPTSCF_Ni and IPTSCF_Cd values were much greater than 1 and similar whatever the amount of available Ni and Cd. This characterized a predominantly active plant combining Ni and Cd uptake and biological regulations dependent of the Ni and Cd concentrations in solution.