New perspectives for pharmacological chaperoning treatment in methylmalonic aciduria cblB type

Methylmalonic aciduria cblB type (MMA cblB) is caused by the impairment of ATP:cob(I)alamin adenosyltransferase (ATR), the enzyme responsible for the synthesis of adenosylcobalamin (AdoCbl) from cob(I)alamin. No definitive treatment is available for patients with this condition and novel therapeutic strategies are therefore much needed. Recently, we described a proof-of-concept regarding the use of pharmacological chaperones as a treatment. This work describes the effect of two potential pharmacological chaperones - compound V (N-{[(4-chlorophenyl)carbamothioyl]amino}-2-phenylacetamide) and compound VI (4-(4-(4-fluorophenyl)-5-methyl-1H-pyrazol-3-yl)benzene-1,3-diol) - on six ATR mutants, including the most common, p.Arg186Trp. Comprehensive functional analysis identified destabilizing (p.Arg186Gln, p.Arg190Cys, p.Arg190His, p.Arg191Gln and p.Glu193Lys) and oligomerization (p.Arg186Trp and p.Arg191Gln) mutations. In a cellular model overexpressing the destabilizing/oligomerization mutations, compounds V and VI had a positive effect on the stability and activity of all ATR variants. When provided in combination with hydroxocobalamin a more positive effect was obtained than with the compounds alone, even in mutations previously described as B₁₂ non-responsive. In addition, a normal oligomerization profile was recovered after treatment of the p.Arg186Trp mutant with both compounds. These promising results confirm MMA cblB type as a conformational disorder and hence, pharmacological chaperones as a new therapeutic option alone or in combination with hydroxocobalamin for many patients with MMA cblB.     

Protoplast regeneration and fusion in Cucumis: melon × cucumber

The aim of this investigation was to develop a protocol to be used in asymmetric protoplast fusions with breeding material of melon and cucumber. Efficient methods for plant regeneration from unfused protoplasts of commercial melon lines were established and are reported. Ploidy levels of explant material and plants, regenerated from protoplasts were analyzed. Electrofusion was carried out between melon protoplasts and irradiated and non-irradiated donor protoplasts of cucumber. Although initial plating efficiencies of heterokaryons were high, development stopped after a few divisions. In control experiments, shoots were regenerated at high frequencies. In only two fusion experiments, development continued to the callus stage, but further development was not observed. When analyzed with PCR using arbitrary primers, the majority of these calli DNA were identical to the melon DNA. However, a few of the examined calli, although being mainly homologous to melon, were observed to have new bands corresponding to bands specific for cucumber. Due to sexual incompatibility, successful hybridization between cucumber and melon was never obtained by sexual crosses. We suggest that the failure to regenerate plants in our fused material could be explained partially by an analogous somatic incompatibility reaction.Abbreviations BA benzyl adenine - 2,4-d 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - FITC fluorescein isothiocyanate - IAA indoleacetic acid - MS Murashige & Skoog - NAA naphtalene acetic acid - PEG polyethylene glycol - PE plating efficiency - RAPD random amplified polymorphic DNA     

Fast Mapping of Global Protein Folding States by Multivariate NMR: A GPS for Proteins

To obtain insight into the functions of proteins and their specific roles, it is important to establish efficient procedures for exploring the states that encapsulate their conformational space. Global Protein folding State mapping by multivariate NMR (GPS NMR) is a powerful high-throughput method that provides such an overview. GPS NMR exploits the unique ability of NMR to simultaneously record signals from individual hydrogen atoms in complex macromolecular systems and of multivariate analysis to describe spectral variations from these by a few variables for establishment of, and positioning in, protein-folding state maps. The method is fast, sensitive, and robust, and it works without isotope-labelling. The unique capabilities of GPS NMR to identify different folding states and to compare different unfolding processes are demonstrated by mapping of the equilibrium folding space of bovine α-lactalbumin in the presence of the anionic surfactant sodium dodecyl sulfate, SDS, and compare these with other surfactants, acid, denaturants and heat.     

Human phenotypically distinct TGFBI corneal dystrophies are linked to the stability of the fourth FAS1 domain of TGFBIp

Mutations in the human TGFBI gene encoding TGFBIp have been linked to protein deposits in the cornea leading to visual impairment. The protein consists of an N-terminal Cys-rich EMI domain and four consecutive fasciclin 1 (FAS1) domains. We have compared the stabilities of wild-type (WT) human TGFBIp and six mutants known to produce phenotypically distinct deposits in the cornea. Amino acid substitutions in the first FAS1 (FAS1-1) domain (R124H, R124L, and R124C) did not alter the stability. However, substitutions within the fourth FAS1 (FAS1-4) domain (A546T, R555Q, and R555W) affected the overall stability of intact TGFBIp revealing the following stability ranking R555W>WT>R555Q>A546T. Significantly, the stability ranking of the isolated FAS1-4 domains mirrored the behavior of the intact protein. In addition, it was linked to the aggregation propensity as the least stable mutant (A546T) forms amyloid fibrils while the more stable variants generate non-amyloid amorphous deposits in vivo. Significantly, the data suggested that both an increase and a decrease in the stability of FAS1-4 may unleash a disease mechanism. In contrast, amino acid substitutions in FAS1-1 did not affect the stability of the intact TGFBIp suggesting that molecular the mechanism of disease differs depending on the FAS1 domain carrying the mutation.     

Public Perceptions of the Attractiveness of Restored Nature

Ecological restoration efforts often encounter public resistance. Recreational visitors resist imposition of restoration efforts they fear may result in a visually unattractive area. Public support is, however, essential for restoration efforts on public lands. This study seeks insight into hiker perceptions of perceived attractiveness of nature before and after efforts to restore exotic conifer plantations to native communities containing bog and wet forest communities. Visitors (N = 247) to a Dutch National Park sorted 32 photographs depicting landscapes before and after restoration efforts. Findings show that the most attractive landscape types (bog and wet forest communities containing visible water) are results of restoration efforts and the least attractive landscape types (young deciduous and coniferous forest) are representative of traditional nature before restoration. However, the “middle category” consists of landscape types existing both before and after restoration efforts. Visitors value old coniferous and old deciduous forests as much as products of restoration that lack water. These perceptions are unrelated to either visitor characteristics or the provision of information to visitors explaining restoration goals. The continued existence of resistance to restoration strategies despite their effect on perceived landscape attractiveness implies that the experience of nature has more than only visual dimensions. We expect that more acceptable results of restoration efforts will emerge from the active engagement of the public before restoration practices take place in processes that specifically address feelings of attachment and resistance to change.     

The three-dimensional structure of CsmA: a small antenna protein from the green sulfur bacterium Chlorobium tepidum

The structure of the chlorosome baseplate protein CsmA from Chlorobium tepidum in a 1:1 chloroform:methanol solution was determined using liquid-state NMR spectroscopy. The data reveal that the 59-residue protein is predominantly alpha-helical with a long helical domain extending from residues V6 to L36, containing a putative bacteriochlorophyll a binding domain, and a short helix in the C-terminal part extending from residues M41 to G49. These elements are compatible with a model of CsmA having the long N-terminal alpha-helical stretch immersed into the lipid monolayer confining the chlorosome and the short C-terminal helix protruding outwards, thus available for interaction with the Fenna-Matthews-Olson antenna protein.     

Complete mitochondrial genomes of eleven extinct or possibly extinct bird species

Natural history museum collections represent a vast source of ancient and historical DNA samples from extinct taxa that can be utilized by high‐throughput sequencing tools to reveal novel genetic and phylogenetic information about them. Here, we report on the successful sequencing of complete mitochondrial genome sequences (mitogenomes) from eleven extinct bird species, using de novo assembly of short sequences derived from toepad samples of degraded DNA from museum specimens. For two species (the Passenger Pigeon Ectopistes migratorius and the South Island Piopio Turnagra capensis), whole mitogenomes were already available from recent studies, whereas for five others (the Great Auk Pinguinis impennis, the Imperial Woodpecker Campehilus imperialis, the Huia Heteralocha acutirostris, the Kauai Oo Moho braccathus and the South Island Kokako Callaeas cinereus), there were partial mitochondrial sequences available for comparison. For all seven species, we found sequence similarities of >98%. For the remaining four species (the Kamao Myadestes myadestinus, the Paradise Parrot Psephotellus pulcherrimus, the Ou Psittirostra psittacea and the Lesser Akialoa Akialoa obscura), there was no sequence information available for comparison, so we conducted blast searches and phylogenetic analyses to determine their phylogenetic positions and identify their closest extant relatives. These mitogenomes will be valuable for future analyses of avian phylogenetics and illustrate the importance of museum collections as repositories for genomics resources.