Negative Regulation of Bone Formation by the Transmembrane Wnt Antagonist Kremen-2

Wnt signalling is a key pathway controlling bone formation in mice and humans. One of the regulators of this pathway is Dkk1, which antagonizes Wnt signalling through the formation of a ternary complex with the transmembrane receptors Krm1/2 and Lrp5/6, thereby blocking the induction of Wnt signalling by the latter ones. Here we show that Kremen-2 (Krm2) is predominantly expressed in bone, and that its osteoblast-specific over-expression in transgenic mice (Col1a1-Krm2) results in severe osteoporosis. Histomorphometric analysis revealed that osteoblast maturation and bone formation are disturbed in Col1a1-Krm2 mice, whereas bone resorption is increased. In line with these findings, primary osteoblasts derived from Col1a1-Krm2 mice display a cell-autonomous differentiation defect, impaired canonical Wnt signalling and decreased production of the osteoclast inhibitory factor Opg. To determine whether the observed effects of Krm2 on bone remodeling are physiologically relevant, we analyzed the skeletal phenotype of 24 weeks old Krm2-deficient mice and observed high bone mass caused by a more than three-fold increase in bone formation. Taken together, these data identify Krm2 as a regulator of bone remodeling and raise the possibility that antagonizing KRM2 might prove beneficial in patients with bone loss disorders.     

Dynamics of the pathomorphological changes in the rat brain in relation to the dose of gamma irradiation

Neurohistological, histochemical, electron-microscopic and biometric techniques were used to study the response of rat brain to irradiation within a wide range of doses. Nerve cells were shown to be highly radioresistant. At the same time, synapses and blood-brain barrier structures were highly radiosensitive. The pathomorphologic changes in different brain areas followed a dose-time function.     

Genetic engineering of the major timothy grass pollen allergen, Phl p 6, to reduce allergenic activity and preserve immunogenicity

On the basis of IgE epitope mapping data, we have produced three allergen fragments comprising aa 1-33, 1-57, and 31-110 of the major timothy grass pollen allergen Phl p 6 aa 1-110 by expression in Escherichia coli and chemical synthesis. Circular dichroism analysis showed that the purified fragments lack the typical alpha-helical fold of the complete allergen. Superposition of the sequences of the fragments onto the three-dimensional allergen structure indicated that the removal of only one of the four helices had led to the destabilization of the alpha helical structure of Phl p 6. The lack of structural fold was accompanied by a strong reduction of IgE reactivity and allergenic activity of the three fragments as determined by basophil histamine release in allergic patients. Each of the three Phl p 6 fragments adsorbed to CFA induced Phl p 6-specific IgG Abs in rabbits. However, immunization of mice with fragments adsorbed to an adjuvant allowed for human use (AluGel-S) showed that only the Phl p 6 aa 31-110 induced Phl p 6-specific IgG Abs. Anti-Phl p 6 IgG Abs induced by vaccination with Phl p 6 aa 31-110 inhibited patients' IgE reactivity to the wild-type allergen as well as Phl p 6-induced basophil degranulation. Our results are of importance for the design of hypoallergenic allergy vaccines. They show that it has to be demonstrated that the hypoallergenic derivative induces a robust IgG response in a formulation that can be used in allergic patients.     

Synthesis of bacteriochlorophyll <Emphasis Type="Italic">a</Emphasis> by the purple nonsulfur bacterium <Emphasis Type="Italic">Rhodobacter capsulatus</Emphasis>

The ability of purple nonsulfur bacteria Rhodobacter capsulatus B10 to synthesize bacteriochlorophyll under phototrophic and dark conditions was studied. The modes for cultivation in the dark with oxygen limitation in a continuous culture at D = 0.1 h^?1 were selected. The yield of biomass reached 20 g/l; the bacteriochlorophyll a output of the process amounted to 16.6 mg/l h^?1.     

Can population genetic structure be predicted from life-history traits?

Population genetic structure is a key parameter in evolutionary biology. Earlier comparative studies have shown that genetic structure depends on species ecological attributes and life-history traits, but species phylogenetic relatedness had not been accounted for. Here we reevaluate the relationships between genetic structure and species traits in seed plants. Each species is characterized by a set of life-history and ecological features as well as by its geographic range size, its heterozygote deficit, and its genetic structure at nuclear and organelle markers to distinguish between pollen- and seed-mediated gene flow. We use both a conventional regression approach and a method that controls for phylogenetic relationships. Once phylogenetic conservatism and covariation among traits are taken into account, genetic structure is shown to be related with only a few synthetic traits, such as mating system for nuclear markers and seed dispersal mode or geographic range size for organelle markers. Along with other studies on invasiveness or rarity, our work illustrates the fact that predicting the fate of species across a broad taxonomic assemblage on the basis of simple traits is rarely possible, a testimony of the highly contingent nature of evolution.     

Evolution of enzymatic activities in the enolase superfamily: L-talarate/galactarate dehydratase from Salmonella typhimurium LT2

We assigned l-talarate dehydratase (TalrD) and galactarate dehydratase (GalrD) functions to a group of orthologous proteins in the mechanistically diverse enolase superfamily, focusing our characterization on the protein encoded by the Salmonella typhimurium LT2 genome (GI:16766982; STM3697). Like the homologous mandelate racemase, l-fuconate dehydratase, and d-tartrate dehydratase, the active site of TalrD/GalrD contains a general acid/base Lys 197 at the end of the second beta-strand in the (beta/alpha)7beta-barrel domain, Asp 226, Glu 252, and Glu 278 as ligands for the essential Mg2+ at the ends of the third, fourth, and fifth beta-strands, a general acid/base His 328-Asp 301 dyad at the ends of the seventh and sixth beta-strands, and an electrophilic Glu 348 at the end of the eighth beta-strand. We discovered the function of STM3697 by screening a library of acid sugars; it catalyzes the efficient dehydration of both l-talarate (kcat = 2.1 s-1, kcat/Km = 9.1 x 10(3) M-1 s-1) and galactarate (kcat = 3.5 s-1, kcat/Km = 1.1 x 10(4) M-1 s-1). Because l-talarate is a previously unknown metabolite, we demonstrated that S. typhimurium LT2 can utilize l-talarate as carbon source. Insertional disruption of the gene encoding STM3697 abolishes this phenotype; this disruption also diminishes, but does not eliminate, the ability of the organism to utilize galactarate as carbon source. The dehydration of l-talarate is accompanied by competing epimerization to galactarate; little epimerization to l-talarate is observed in the dehydration of galactarate. On the basis of (1) structures of the wild type enzyme complexed with l-lyxarohydroxamate, an analogue of the enolate intermediate, and of the K197A mutant complexed with l-glucarate, a substrate for exchange of the alpha-proton, and (2) incorporation of solvent deuterium into galactarate in competition with dehydration, we conclude that Lys 197 functions as the galactarate-specific base and His 328 functions as the l-talarate-specific base. The epimerization of l-talarate to galactarate that competes with dehydration can be rationalized by partitioning of the enolate intermediate between dehydration (departure of the 3-OH group catalyzed by the conjugate acid of His 328) and epimerization (protonation on C2 by the conjugate acid of Lys 197). The promiscuous catalytic activities discovered for STM3697 highlight the evolutionary potential of a "conserved" active site architecture.     

Crystal Structures of the p21-activated kinases PAK4, PAK5, and PAK6 reveal catalytic domain plasticity of active group II PAKs

p21-activated kinases have been classified into two groups based on their domain architecture. Group II PAKs (PAK4-6) regulate a wide variety of cellular functions, and PAK deregulation has been linked to tumor development. Structural comparison of five high-resolution structures comprising all active, monophosphorylated group II catalytic domains revealed a surprising degree of domain plasticity, including a number of catalytically productive and nonproductive conformers. Rearrangements of helix alphaC, a key regulatory element of kinase function, resulted in an additional helical turn at the alphaC N terminus and a distortion of its C terminus, a movement hitherto unseen in protein kinases. The observed structural changes led to the formation of interactions between conserved residues that structurally link the glycine-rich loop, alphaC, and the activation segment and firmly anchor alphaC in an active conformation. Inhibitor screening identified six potent PAK inhibitors from which a tri-substituted purine inhibitor was cocrystallized with PAK4 and PAK5.     

EST sequencing of Onychophora and phylogenomic analysis of Metazoa

Onychophora (velvet worms) represent a small animal taxon considered to be related to Euarthropoda. We have obtained 1873 5' cDNA sequences (expressed sequence tags, ESTs) from the velvet worm Epiperipatus sp., which were assembled into 833 contigs. BLAST similarity searches revealed that 51.9% of the contigs had matches in the protein databases with expectation values lower than 10(-4). Most ESTs had the best hit with proteins from either Chordata or Arthropoda (approximately 40% respectively). The ESTs included sequences of 27 ribosomal proteins. The orthologous sequences from 28 other species of a broad range of phyla were obtained from the databases, including other EST projects. A concatenated amino acid alignment comprising 5021 positions was constructed, which covers 4259 positions when problematic regions were removed. Bayesian and maximum likelihood methods place Epiperipatus within the monophyletic Ecdysozoa (Onychophora, Arthropoda, Tardigrada and Nematoda), but its exact relation to the Euarthropoda remained unresolved. The "Articulata" concept was not supported. Tardigrada and Nematoda formed a well-supported monophylum, suggesting that Tardigrada are actually Cycloneuralia. In agreement with previous studies, we have demonstrated that random sequencing of cDNAs results in sequence information suitable for phylogenomic approaches to resolve metazoan relationships.