The structure of the O-polysaccharide from the lipopolysaccharide of Providencia alcalifaciens O36 containing 3-deoxy-d-manno-oct-2-ulosonic acid

An oligosaccharide that corresponds to the repeating unit of the O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O36. Structural studies of the oligosaccharide and O-deacylated lipopolysaccharide were performed using sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including 2D (1)H,(1)H COSY, TOCSY, ROESY, and H-detected (1)H,(13)C HSQC and HMBC experiments. It was found that the O-polysaccharide is built up of linear trisaccharide repeating units containing 2-acetamido-2-deoxyglucose, 6-deoxy-l-talose (l-6dTal), and 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) and has the following structure. [structure: see text]     

Structure of the O-polysaccharide and serological cross-reactivity of the lipopolysaccharide of Providencia alcalifaciens O32 containing N-acetylisomuramic acid

The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O32 and studied by sugar and methylation analyses, solvolysis with triflic acid, 1H and 13C NMR spectroscopy, including two-dimensional 1H,1H COSY, TOCSY, ROESY, H-detected 1H,13C HSQC and HMBC experiments. It was found that the polysaccharide has a branched tetrasaccharide repeating unit containing 2-acetamido-3-O-[(S)-1-carboxyethyl]-2-deoxy-D-glucose (D-GlcNAc3Slac, N-acetylisomuramic acid) with the following structure: [STRUCTURE: SEE TEXT]. Serological studies with O-antisera showed antigenic relationships between P. alcalifaciens O32 and O29 as well as several other Providencia and Proteus strains sharing putative epitopes on the O-polysaccharides.     

Sex-specific expression of a HOX gene associated with rapid morphological evolution

Animal diversity is shaped by the origin and diversification of new morphological structures. Many examples of evolutionary innovations are provided by male-specific traits involved in mating and sexual selection. The origin of new sex-specific characters requires the evolution of new regulatory interactions between sex-determining genes and genes that control spatial patterning and cell differentiation. Here, we show that sex-specific regulation of the HOX gene Sex combs reduced (Scr) is associated with the origin and evolution of the Drosophila sex comb — a novel and rapidly diversifying male-specific organ. In species that primitively lack sex combs, Scr expression shows little spatial modulation, whereas in species that have sex combs, Scr is upregulated in the presumptive sex comb region and is frequently sexually dimorphic. Phylogenetic analysis shows that sex-specific regulation of Scr has been gained and lost multiple times in Drosophila evolution and correlates with convergent origin of similar sex comb morphologies in several independent lineages. Some of these transitions occurred on microevolutionary timescales, indicating that HOX gene expression can evolve with surprising ease. This is the first example of a sex-specific regulation of a HOX gene contributing to the development and evolution of a secondary sexual trait.     

Heregulin-induced epigenetic regulation of the utrophin-A promoter

Utrophin is the autosomal homolog of dystrophin, the product of the Duchenne's muscular dystrophy (DMD) locus. Utrophin is of therapeutic interest since its over-expression can compensate dystrophin's absence. Utrophin is enriched at neuromuscular junctions due to heregulin-mediated utrophin-A promoter activation. We demonstrate that heregulin activated MSK1/2 and phosphorylated histone H3 at serine 10 in cultured C2C12 muscle cells, in an ERK-dependent manner. MSK1/2 inhibition suppressed heregulin-mediated utrophin-A activation. MSK1 over-expression potentiated heregulin-mediated utrophin-A activation and chromatin remodeling at the utrophin-A promoter. These results identify MSK1/2 as key effectors modulating utrophin-A expression as well as identify novel targets for DMD therapy.     

Asymmetry of syringomycin E channel studied by polymer partitioning

To probe the size of the ion channel formed by Pseudomonas syringae lipodepsipeptide syringomycin E, we use the partial blockage of ion current by penetrating poly(ethylene glycol)s. Earlier experiments with symmetric application of these polymers yielded a radius estimate of approximately 1 nm. Now, motivated by the asymmetric non-ohmic current-voltage curves reported for this channel, we explore its structural asymmetry. We gauge this asymmetry by studying the channel conductance after one-sided addition of differently sized poly(ethylene glycol)s. We find that small polymers added to the cis-side of the membrane (the side of lipodepsipeptide addition) reduce channel conductance much less than do the same polymers added to the trans-side. We interpret our results to suggest that the water-filled pore of the channel is conical with cis- and trans-radii differing by a factor of 2-3 and that the smaller cis-radius is in the 0.25-0.35 nm range. In symmetric, two-sided addition, polymers entering the pore from the larger opening dominate blockage.     

Functional analysis of gene duplications in Saccharomyces cerevisiae

Gene duplication can occur on two scales: whole-genome duplications (WGD) and smaller-scale duplications (SSD) involving individual genes or genomic segments. Duplication may result in functionally redundant genes or diverge in function through neofunctionalization or subfunctionalization. The effect of duplication scale on functional evolution has not yet been explored, probably due to the lack of global knowledge of protein function and different times of duplication events. To address this question, we used integrated Bayesian analysis of diverse functional genomic data to accurately evaluate the extent of functional similarity and divergence between paralogs on a global scale. We found that paralogs resulting from the whole-genome duplication are more likely to share interaction partners and biological functions than smaller-scale duplicates, independent of sequence similarity. In addition, WGD paralogs show lower frequency of essential genes and higher synthetic lethality rate, but instead diverge more in expression pattern and upstream regulatory region. Thus, our analysis demonstrates that WGD paralogs generally have similar compensatory functions but diverging expression patterns, suggesting a potential of distinct evolutionary scenarios for paralogs that arose through different duplication mechanisms. Furthermore, by identifying these functional disparities between the two types of duplicates, we reconcile previous disputes on the relationship between sequence divergence and expression divergence or essentiality.     

Site-specific conformational studies of prion protein (PrP) amyloid fibrils revealed two cooperative folding domains within amyloid structure

Despite the ability of most proteins to form amyloid, very little is know about amyloid fibril structures and the factors that govern their stability. Using amyloid fibrils produced from full-length prion protein (PrP), we describe a reliable approach for determining both site-specific and global conformational stability of the fibrillar form. To measure site-specific stability, we produced six variants of PrP by replacing the residues at positions 88, 98, 127, 144, 196, and 230 with cysteine, labeled the new cysteines with the fluorescent dye acrylodan, and investigated their conformational status within the amyloid form in guanidine hydrochloride-induced denaturation experiments. We found that the fibrils labeled at positions 127, 144, 196, and 230 displayed cooperative unfolding and showed a very high C1/2 value similar to that observed for the global unfolding of the amyloid structure. The unfolding at residue 98 was also cooperative; however, it showed a C1/2 value substantially lower than that of global unfolding, whereas the unfolding of fibrils labeled at residue 88 was non-cooperative. These data illustrate that there are at least two independent cooperative folding domains within the amyloid structure of the full-length PrP. In addition, kinetic experiments revealed only a partial overlap between the region that constituted the fibrillar cross-beta core and the regions that were involved in nucleation. This result illustrates that separate PrP regions accounted for the nucleation and for the formation of the conformationally most stable fibrillar core.     

Co-transcribed genes for long chain polyunsaturated fatty acid biosynthesis in the protozoon Perkinsus marinus include a plant-like FAE1 3-ketoacyl coenzyme A synthase

The marine parasitic protozoon Perkinus marinus synthesizes the polyunsaturated fatty acid arachidonic acid via the unusual alternative Delta8 pathway in which elongation of C18 fatty acids generates substrate for two sequential desaturations. Here we have shown that genes encoding the three P. marinus activities responsible for arachidonic acid biosynthesis (C18 Delta9-elongating activity, C20 Delta8 desaturase, C20 Delta5 desaturase) are genomically clustered and co-transcribed as an operon. The acyl elongation reaction, which underpins this pathway, is catalyzed by a FAE1 (fatty acid elongation 1)-like 3-ketoacyl-CoA synthase class of condensing enzyme previously only reported in higher plants and algae. This is the first example of an elongating activity involved in the biosynthesis of a polyunsaturated fatty acid that is not a member of the ELO/SUR4 family. The P. marinus FAE1-like elongating activity is sensitive to the herbicide flufenacet, similar to some higher plant 3-ketoacyl-CoA synthases, but unable to rescue the yeast elo2Delta/elo3Delta mutant consistent with a role in the elongation of polyunsaturated fatty acids. P. marinus represents a key organism in the taxonomic separation of the single-celled eukaryotes collectively known as the alveolates, and our data imply a lineage in which ancestral acquisition of plant-like genes, such as FAE1-like 3-ketoacyl-CoA synthases, occurred via endosymbiosis. The P. marinus FAE1-like elongating activity is also indicative of the independent evolution of the alternative Delta8 pathway, distinct from ELO/SUR4-dependent examples.     

Application of cellulose-based self-assembled tri-enzyme system in a pseudo-reagent-less biosensor for biogenic catecholamine detection

Amorphous cellulose was used as a specific carrier for the deposition of self-assembled multienzyme complexes capable of catalyzing coupled reactions. Naturally glycosylated fungal cellobiohydrolases (CBHs) of glycosyl hydrolase families 6 and 7 were specifically deposited onto the cellulose surface through their family I cellulose-binding modules (CBM). Naturally glycosylated fungal laccase was then deposited onto the preformed glycoprotein layer pretreated by ConA, through the interaction of mannosyl moieties of fungal glycoproteins with the multivalent lectin. The formation of a cellulase-ConA-laccase composite was proven by direct and indirect determination of activity of immobilized laccase. In the absence of cellulases and ConA, no laccase deposition onto the cellulose surface was observed. Finally, basidiomycetous cellobiose dehydrogenase (CDH) was deposited onto the cellulose surface through the specific interaction of its FAD domain with cellulose. The obtained paste was applied onto the surface of a Clark-type oxygen electrode and covered with a dialysis membrane. In the presence of traces of catechol or dopamine as mediators, the obtained immobilized multienzyme composite was capable of the coupled oxidation of cellulose by dissolved oxygen, thus providing the basis for a sensitive assay of the mediator. Swollen amorphous cellulose plays three different roles in the obtained biosensor as: (i) a gelforming matrix that captures the analyte and its oxidized intermediate, (ii) a specific carrier for protein self-assembly, and (iii) a source of excess substrate for a pseudo-reagent-less assay with signal amplification. The detection limit of such a tri-enzyme biosensor is 50-100 nM dopamine.