Structural modeling of two plant UDP-dependent sugar-sugar glycosyltransferases reveals a conserved glutamic acid residue that is a hallmark for sugar acceptor recognition.

Glycosylation is one of the common modifications of plant metabolites, playing a major role in the chemical/biological diversity of a wide range of compounds. Plant metabolite glycosylation is catalyzed almost exclusively by glycosyltransferases, mainly by Uridine-diphosphate dependent Glycosyltransferases (UGTs). Several X-ray structures have been determined for primary glycosyltransferases, however, little is known regarding structure-function aspects of sugar-sugar/branch-forming O-linked UGTs (SBGTs) that catalyze the transfer of a sugar from the UDP-sugar donor to an acceptor sugar moiety of a previously glycosylated metabolite substrate. In this study we developed novel insights into the structural basis for SBGT catalytic activity by modelling the 3d-structures of two enzymes; a rhamnosyl-transferase Cs1,6RhaT - that catalyzes rhamnosylation of flavonoid-3-glucosides and flavonoid-7-glucosides and a UGT94D1 - that catalyzes glucosylation of (+)-Sesaminol 2-O-ß-d-glucoside at the C6 of the primary sugar moiety. Based on these structural models and docking studies a glutamate (E290 or E268 in Cs1,6RhaT or UGT94D1, respectively) and a tryptophan (W28 or W15 in Cs1,6RhaT or UGT94D1, respectively) appear to interact with the sugar acceptor and are suggested to be important for the recognition of the sugar-moiety of the acceptor-substrate. Functional analysis of substitution mutants for the glutamate and tryptophan residues in Cs1,6RhaT further support their role in determining sugar-sugar/branch-forming GT specificity. Phylogenetic analysis of the UGT family in plants demonstrates that the glutamic-acid residue is a hallmark of SBGTs that is entirely absent from the corresponding position in primary UGTs.

POSEIDON - Bringing Assistive Technology to People with Down Syndrome: Results of a Three Year European Project.

The POSEIDON project aimed to increase the independence and autonomy of people with Down syndrome with the help of technical assistants. It followed a user-centered approach by involving people with Down syndrome and their parents, carers etc. A requirement analysis was the first step of the project. It became clear that people with Down syndrome especially need support in the areas of time management, mobility and money handling. Different applications were developed which were tested and evaluated in two field tests in three countries. Results indicate that POSEIDON can help to overcome daily challenges and that it can increase the autonomy and independence of people with Down syndrome.

Inhibition of a multiproduct terpene synthase from Medicago truncatula by 3-bromoprenyl diphosphates.

The multiproduct sesquiterpene synthase MtTPS5 from Medicago truncatula catalyzes the conversion of farnesyl diphosphate (FDP) into a complex mixture of 27 terpenoids. 3-Bromo substrate analogues of geranyl diphosphate (3-BrGDP) and farnesyl diphosphate (3-BrFDP) were evaluated as substrates of MTPS5 enzyme. Kinetic studies demonstrated that these compounds were highly potent competitive inhibitors of the MtTPS5 enzyme with fast binding and slow reversibility. Since there is a lack of knowledge about the crystal structure of multiproduct terpene synthases, these molecules might be ideal candidates for obtaining a co-crystal structure with multiproduct terpene synthases. Due to the structural and mechanistic similarity between various terpene synthases we expect these 3-bromo isoprenoids to be ideal probes for crystal structure studies.

Glandular ß-glucosidases in juvenile Chrysomelina leaf beetles support the evolution of a host-plant-dependent chemical defense.

Plant-feeding insects are spread across the entire plant kingdom. Because they chew externally on leaves, leaf beetle of the subtribe Chrysomelina sensu stricto are constantly exposed to life-threatening predators and parasitoids. To counter these pressures, the juveniles repel their enemies by displaying glandular secretions that contain defensive compounds. These repellents can be produced either de novo (iridoids) or by using plant-derived precursors. The autonomous production of iridoids pre-dates the evolution of phytochemical-based defense strategies. Both strategies include hydrolysis of the secreted non-toxic glycosides in the defensive exudates. By combining in vitro as well as in vivo experiments, we show that iridoid de novo producing as well as sequestering species rely on secreted ß-glucosidases to cleave the pre-toxins. Our phylogenetic analyses support a common origin of chrysomeline ß-glucosidases. The kinetic parameters of these ß-glucosidases demonstrated substrate selectivity which reflects the adaptation of Chrysomelina sensu stricto to the chemistry of their hosts during the course of evolution. However, the functional studies also showed that the broad substrate selectivity allows building a chemical defense, which is dependent on the host plant, but does not lead to an "evolutionary dead end".

Molecular models and mutational analyses of plant specifier proteins suggest active site residues and reaction mechanism.

As components of the glucosinolate-myrosinase system, specifier proteins contribute to the diversity of chemical defenses that have evolved in plants of the Brassicales order as a protection against herbivores and pathogens. Glucosinolates are thioglucosides that are stored separately from their hydrolytic enzymes, myrosinases, in plant tissue. Upon tissue disruption, glucosinolates are hydrolyzed by myrosinases yielding instable aglucones that rearrange to form defensive isothiocyanates. In the presence of specifier proteins, other products, namely simple nitriles, epithionitriles and organic thiocyanates, can be formed instead of isothiocyanates depending on the glucosinolate side chain structure and the type of specifier protein. The biochemical role of specifier proteins is largely unresolved. We have used two thiocyanate-forming proteins and one epithiospecifier protein with different substrate/product specificities to develop molecular models that, in conjunction with mutational analyses, allow us to propose an active site and docking arrangements with glucosinolate aglucones that may explain some of the differences in specifier protein specificities. Furthermore, quantum-mechanical calculations support a reaction mechanism for benzylthiocyanate formation including a catalytic role of the TFP involved. These results may serve as a basis for further theoretical and experimental investigations of the mechanisms of glucosinolate breakdown that will also help to better understand the evolution of specifier proteins from ancestral proteins with functions outside glucosinolate metabolism.

Molecular and structural basis of metabolic diversity mediated by prenyldiphosphate converting enzymes.

General thermodynamic calculations using the semiempiric PM3 method have led to the conclusion that prenyldiphosphate converting enzymes require at least one divalent metal cation for the activation and cleavage of the diphosphate-prenyl ester bond, or they must provide structural elements for the efficient stabilization of the intermediate prenyl cation. The most important common structural features, which guide the product specificity in both terpene synthases and aromatic prenyl transferases are aromatic amino acid side chains, which stabilize prenyl cations by cation-pi interactions. In the case of aromatic prenyl transferases, a proton abstraction from the phenolic hydroxyl group of the second substrate will enhance the electron density in the phenolic ortho-position at which initial prenylation of the aromatic compound usually occurs. A model of the structure of the integral transmembrane-bound aromatic prenyl transferase UbiA was developed, which currently represents the first structural insight into this group of prenylating enzymes with a fold different from most other aromatic prenyl transferases. Based on this model, the structure-activity relationships and mechanistic aspects of related proteins, for example those of Lithospermum erythrorhizon or the enzyme AuaA from Stigmatella aurantiaca involved in the aurachin biosynthesis, were elucidated. The high similarity of this group of aromatic prenyltransferases to 5-epi-aristolochene synthase is an indication of an evolutionary relationship with terpene synthases (cyclases). This is further supported by the conserved DxxxD motif found in both protein families. In contrast, there is no such relationship to the aromatic prenyl transferases with an ABBA-fold, such as NphB, or to any other known family of prenyl converting enzymes. Therefore, it is possible that these two groups might have different evolutionary ancestors.

Absence of lymphatics at the bone-implant interface: implications for periprosthetic osteolysis.

BACKGROUND: Wear particles, found at the bone-implant interface surrounding a loose prosthesis, are commonly phagocytosed by macrophages. Wear particles and wear particle-containing macrophages are also found in regional lymph nodes draining arthroplasty tissues. The means by which wear particles are transported from arthroplasty tissues to lymph nodes is uncertain, as the presence or absence of lymphatic vessels in periprosthetic tissues has not been established. METHODS: We determined immunophenotypic expression of LYVE-1 and podoplanin, two highly specific lymphatic endothelial cell markers, in the hip arthroplasty pseudocapsule surrounding the false joint and the bone-implant interface of the femoral and acetabular pseu-domembrane. RESULTS: LYVE-1+/podoplanin+ lymphatic vessels were not identified in the pseudomembrane but were found in the pseudocapsule. Normal bone did not contain lymphatic vessels. INTERPRETATION: Our findings suggest that the wear particles shed at the bone-implant interface are not transported to draining lymph nodes by lymphatics directly from the pseudomembrane, but via the pseudocapsule. The absence of a lymphatic clearance mechanism may contribute to accumulation of wear particles at the bone-implant interface and promote periprosthetic osteolysis through stimulation of osteoclast formation and activity.

Coating of titanium implants with type-I collagen.

PURPOSE: Type-I collagen, the major structural protein in bone, has beneficial properties regarding bone regeneration. Little is known about the potential effects of collagen coating on orthopedic implants. METHODS: 3 to 6 microg/cm2 of lyophilized type-I collagen was absorbed on titanium rods. Six coated and uncoated pins of 0.9 mm diameter were inserted into the tibia of adult male Wistar rats for 1, 2, 4, 7, 14, and 28 days. Specimens were embedded in methacrylate-based Technovit 9100N resin. From one portion cutting and grinding sections were obtained. The implant was removed from the other half that was depolymerized, sectioned, and mounted for immunohistochemistry. RESULTS: At day 4, the interface around the collagen-coated implants displayed a granulation tissue with higher numbers of cathepsin D-positive mononucleated cells compared to the uncoated implants (p<0.05). Active osteoblasts, reactive for osteopontin, were increased around the collagen-coated pins at day 4 and 7 (p<0.01). After 28 days of implantation, direct bone contact averaged 74.9% around the collagen-coated implants and 62.1% around uncoated implants (NS). The amount of newly formed bone averaged 76.3% around the collagen-coated pins and 67.8% around the uncoated pins (NS). The histomorphometric findings were confirmed by SRmicroCT in two specimens. CONCLUSIONS: The earlier observation of mononuclear phagocytozing cells and the earlier and higher expression of bone-specific matrix proteins suggest an increased early bone remodeling around titanium pins through collagen coating. A tendency towards increased bone formation was observed around the coated implants.

Collagen type I increases bone remodelling around hydroxyapatite implants in the rat tibia.

The early interface reaction of cancellous bone to a nanocrystalline hydroxyapatite (HA) cement containing 3 wt% collagen type I (HA/Coll) with a setting under physiological temperature and pH was observed using immunohistochemical techniques. Pure HA served as a control. Cylinders with a diameter of 2 mm were implanted into the proximal tibia of 72 adult Wistar rats. Histological sections of 6 animals were prepared after 1, 2, 4, 6, 14 and 28 days. First, osteoblast-like cells as well as a marked reaction for osteonectin, osteopontin and its ligand CD44 were observed as early as 2 days after implantation at the interface around HA/Coll implants. Further, reactivity for ED1 and cathepsin D, both markers for phagocytotic cells, appeared earlier and stronger around HA/Coll. In cell counts, a significantly higher average number of ED1- and cathepsin D-positive phagocytotic cells was observed around the HA/Coll implants on days 6 (p < 0.01), 14 and 28 (p < 0.05). The number of osteopontin-positive cells was significantly higher around HA/Coll implants at days 6 and 14 (p < 0.05). Two weeks after the implantation, first islands of newly formed woven bone were observed around the HA/Coll implant, but not around the control implant. The amount of direct bone contact after 28 days averaged 28% around pure HA and 51% around HA/Coll implants (p < 0.05). While both implants displayed a good osteoconductivity, a higher bone remodelling activity was observed around collagen-containing HA implants compared to pure HA implants. It appears that the addition of collagen to HA implants can enhance both phagocytotic and osteogenic processes. This may result in an earlier acceptance and better osseointegration of the HA/Coll implants into the surrounding tissue.

Immunohistochemical demonstration of -(carboxymethyl)lysine protein adducts in normal and osteoarthritic cartilage.

N(epsilon)-(carboxymethyl)lysine (CML) is an advanced glycation end product formed by non-enzymatic glycation and oxidation of proteins. The distribution pattern of CML-modified proteins in normal and osteoarthritic (OA) cartilage was investigated using specific antibodies. In healthy articular cartilage, immunoreactivity for CML was preferably found in the extracellular matrix (ECM) of the superficial layer. In OA samples, CML immunoreactivity was not restricted to the ECM of the superficial layer. Interestingly, OA chondrocytes showed a remarkable cytoplasmic immunoreactivity for CML. With the help of a western blot analysis CML-modified proteins between 68 and 39 kDa could be demonstrated in OA cartilage samples. These results suggest that the accumulation of CML adducts contributes to the matrix damage in osteoarthritis. Therefore, the inhibition of CML accumulation may represent an effective therapeutic strategy to prevent severe OA cartilage injury.