Fast and efficient synthesis of a novel homologous series of L-fucosylated trisaccharides using the Helix pomatia alpha-(1-->2)-L-galactosyltransferase

The alpha-(1-->2)-L-galactosyltransferase from the albumen gland of the vineyard snail Helix pomatia exhibits high alpha-(1-->2)-L-fucosyltransferase activity and can be used to transfer L-fucose from GDP-L-fucose to terminal, non-reducing D-galactose residues of an oligosaccharide, thus providing facile access to a range of H-antigen-containing oligosaccharides. The enzymatic glycosylation was applied here on a milligram scale to a series of disaccharide acceptor substrates. Apparently the site of interglycosidic linkage between the terminal and subterminal acceptor sugar units is of little or no consequence. The homologous series of trisaccharides thus produced were fully characterised by NMR analysis of their peracetates.     

Requirements for Cu(A) and Cu-S center assembly of nitrous oxide reductase deduced from complete periplasmic enzyme maturation in the nondenitrifier Pseudomonas putida

Bacterial nitrous oxide (N(2)O) reductase is the terminal oxidoreductase of a respiratory process that generates dinitrogen from N(2)O. To attain its functional state, the enzyme is subjected to a maturation process which involves the protein-driven synthesis of a unique copper-sulfur cluster and metallation of the binuclear Cu(A) site in the periplasm. There are seven putative maturation factors, encoded by nosA, nosD, nosF, nosY, nosL, nosX, and sco. We wanted to determine the indispensable proteins by expressing nos genes from Pseudomonas stutzeri in the nondenitrifying organism Pseudomonas putida. An in silico study of denitrifying bacteria revealed that nosL, nosX (or a homologous gene, apbE), and sco, but not nosA, coexist consistently with the N(2)O reductase structural gene and other maturation genes. Nevertheless, we found that expression of only three maturation factors (periplasmic protein NosD, cytoplasmic NosF ATPase, and the six-helix integral membrane protein NosY) together with nosRZ in trans was sufficient to produce catalytically active holo-N(2)O reductase in the nondenitrifying background. We suggest that these obligatory factors are required for Cu-S center assembly. Using a mutational approach with P. stutzeri, we also studied NosA, the Cu-containing outer membrane protein previously thought to have Cu insertase function, and ScoP, a putative membrane-anchored chaperone for Cu(A) metallation. Both of these were found to be dispensable elements for N(2)O reductase biosynthesis. Our experimental and in silico data were integrated in a model of N(2)O reductase maturation.     

Process formation of podocytes: morphogenetic activity of microtubules and regulation by protein serine/threonine phosphatase PP2A

Podocytes possess major processes containing microtubules (MTs) and intermediate filaments and foot processes containing actin filaments (AFs) as core cytoskeletal elements. Although the importance of these cytoskeletal elements for maintaining podocyte processes was previously shown, so far no data are available concerning the developmental regulation of podocyte process formation. A conditionally immortalized mouse podocyte cell line, which can be induced to develop processes similar to those found in vivo, was treated with various reagents to disrupt cytoskeletal elements or to inhibit protein phosphatases. MTs colocalized with vimentin intermediate filaments but not with AFs. After AF disassembly, major processes were maintained, whereas after depolymerization of MTs, podocytes lost their processes, rounded up, and maintained only actin-based peripheral projections. Suppression of MT elongation by nanomolar vinblastine or inhibition of serine/threonine phosphatase PP2A with okadaic acid abolished process formation. PP2A was expressed in undifferentiated but not in differentiated podocytes. One- and two-dimensional western blot analyses revealed a dose-dependent increase in serine/threonine phosphorylation after okadaic acid treatment. Hence, morphogenetic activity of MTs induces podocyte process formation via serine/threonine protein dephosphorylation by PP2A. These results may open new avenues for understanding the signaling mechanism underlying podocyte cytoskeleton alterations during development and in glomerular diseases.     

Complexes trans-[RuCl(2)(nic)(4)] and trans-[RuCl(2)(i-nic)(4)] as free radical scavengers

This study evaluates the action of the new ruthenium complexes trans-RuCl(2)(nic)(4)] (I) and trans-[RuCl(2)(i-nic)(4)] (II) as free radical scavengers. In our experiments, both compounds acted as scavengers of superoxide anion (O(2)*(-)), hydroxyl radicals (HO*) and nitrogen monoxide (formally known as 'nitric oxide'; NO*). In addition, complexes I and II potentiated the release of NO* from S-nitroso-N-acetyl-DL-penicilamine (SNAP), a NO* donor. Complex II, but not I, also decreased the nitrite levels in culture media of activated macrophages. A hypsochromic shift of lambda(max) and a significant change in half-wave potential (E(1/2)) was observed when NO* was added to the Complex II. Thiobarbituric reactive substance (TBARS) levels were significantly reduced in rats treated for 1 week with Complex II plus tert-butylhydroperoxide, when compared to rats treated only with tert-butylhydroperoxide. None of the complexes showed cytotoxicity. These findings support the suggestion that the new ruthenium complexes, especially trans-[RuCl(2)(i-nic)(4)] or its derivatives, might provide potential therapeutic benefits in disorders where reactive nitrogen (RNS) or oxygen (ROS) species are involved.     

Cyclo19,31[D-Cys19]-uPA19-31 is a potent competitive antagonist of the interaction of urokinase-type plasminogen activator with its receptor (CD87)

Urokinase-type plasminogen activator (uPA) represents a central molecule in pericellular proteolysis and is implicated in a variety of physiological and pathophysiological processes such as tissue remodelling, wound healing, tumor invasion, and metastasis. uPA binds with high affinity to a specific cell surface receptor, uPAR (CD87), via a well defined sequence within the N-terminal region of uPA (uPA19-31). This interaction directs the proteolytic activity of uPA to the cell surface which represents an important step in tumor cell proliferation, invasion, and metastasis. Due to its fundamental role in these processes, the uPA/uPAR-system has emerged as a novel target for tumor therapy. Previously, we have identified a synthetic, cyclic, uPA-derived peptide, cyclo19,31uPA19-31, as a lead structure for the development of low molecular weight uPA-analogues, capable of blocking uPA/uPAR-interaction [Burgle et al., Biol. Chem. 378 (1997), 231-237]. We now searched for peptide variants of cyclo19,31uPA19-31 with elevated affinities for uPAR binding. Among other tasks, we performed a systematic D-amino acid scan of uPA19-31, in which each of the 13 L-amino acids was individually substituted by the corresponding D-amino acid. This led to the identification of cyclo19,31[D-Cys19]-uPA19-31 as a potent inhibitor of uPA/uPAR-interaction, displaying only a 20 to 40-fold lower binding capacity as compared to the naturally occurring uPAR-ligands uPA and its amino-terminal fragment. Cyclo19,31[D-Cys19]-uPA19-31 not only blocks binding of uPA to uPAR but is also capable of efficiently displacing uPAR-bound uPA from the cell surface and to inhibit uPA-mediated, tumor cell-associated plasminogen activation and fibrin degradation. Thus, cyclo19,31[D-Cys19]-uPA19-31 represents a promising therapeutic agent to significantly affect the tumor-associated uPA/uPAR-system.     

Role of protein phosphatases in hypoxic preconditioning

To find a protein kinase C (PKC)-independent preconditioning mechanism, hypoxic preconditioning (HP; i.e., 10-min anoxia and 10-min reoxygenation) was applied to isolated rat hearts before 60-min global ischemia. HP led to improved recovery of developed pressure and reduced end-diastolic pressure in the left ventricle during reperfusion. Protection was unaffected by the PKC inhibitor bisindolylmaleimide (BIM; 1 micromol/l). It was abolished by the inhibitor of protein phosphatases 1 and 2A cantharidin (20 or 5 micromol/l) and partially enhanced by the inhibitor of protein phosphatase 2A okadaic acid (5 nmol/l). In adult rat cardiomyocytes treated with BIM and exposed to 60-min simulated ischemia (anoxia, extracellular pH 6.4), HP led to attenuation of anoxic Na(+)/Ca(2+) overload and of hypercontracture, which developed on reoxygenation. This protection was prevented by treatment with cantharidin but not with okadaic acid. In conclusion, HP exerts PKC-independent protection on ischemic-reperfused rat hearts and cardiomyocytes. Protein phosphatase 1 seems a mediator of this protective mechanism.     

Genetic diversity in apricot revealed by AFLP markers: species and cultivar comparisons

The genetic diversity of apricot ( Prunus armeniaca; 2n = 16) was studied using AFLP markers. Forty seven apricot cultivars were selected from the following geographic regions: Europe, North America, North Africa, Turkey, Iran and China. Five EcoRI- MseI AFLP primer combinations revealed 416 legible bands, of which 379 were polymorphic markers. A similarity matrix was prepared using the simple matching coefficient of similarity. A UPGMA dendrogram demonstrated a gradient of decreasing genetic diversity of varieties from the former USSR to Southern Europe. This is coherent with the historical dissemination of apricot from its center of origin in Asia. The American cultivars were intermediate demonstrating a different genetic base than the European and/or Mediterranean cultivars. Euclidean distances from the first ten Factorial Component Analysis coordinate axes were used to generate a tree using the Ward algorithm. The results of these analyses were evaluated based on the known geographic origins and agronomic characteristics of the cultivars studied. Four cultivar groups were identified: Diversification, Geographically Adaptable, Continental Europe and Mediterranean Basin. To evaluate the relationship of the common apricot with some closely related species, one or two accessions of the following related species or sub-species from within the section Armeniaca were included in the analysis: Prunus armeniaca var. ansu, Prunus mume, Prunus brigantiaca, Prunus dasycarpa, and Prunus holosericea. A Neighbour Joining dendrogram was made using the similarity matrix. The P. holosericea accession fell well within the cultivar group, thus supporting its classification as a variant of P. armeniaca. The P. armeniaca var. ansu accession was sister to the common apricot cluster with a bootstrap value of 96%. P. mume was farther removed. P. brigantiaca was the most-distant from the common apricots. P. dasycarpa was intermediate between P. brigantiaca and P. mume, in accord with its plum-apricot hybrid origin. The results have a direct application for the selection of new breeding progenitors.     

A Dilution Technique For The Direct Measurement Of Viral Production: A Comparison In Stratified And Tidally Mixed Coastal Waters

The abundance of heterotrophic bacteria and viruses, as well as rates of viral production and virus-mediated mortality, were measured in Discovery Passage and the Strait of Georgia (British Columbia, Canada) along a gradient of tidal mixing ranging from well mixed to stratified. The abundances of bacteria and viruses were approximately 10(6) and 10(7) mL(-1), respectively, independent of mixing regime. Viral production estimates, monitored by a dilution technique, demonstrated that new viruses were produced at rates of 10(6) and 10(7) mL(-1)h(-1) across the different mixing regimes. Using an estimated burst size of 50 viruses per lytic event, ca. 19 to 27% of the standing stock of bacteria at the stratified stations and 46 to 137% at the deep-mixed stations were removed by viruses. The results suggest that mixing of stratified waters during tidal exchange enhances virus-mediated bacterial lysis. Consequently, viral lysis recycled a greater proportion of the organic carbon required for bacterial growth under non-steady-state compared to steady-state conditions.     

Oxidative damage increases with age in a canine model of human brain aging

We assayed levels of lipid peroxidation, protein carbonyl formation, glutamine synthetase (GS) activity and both oxidized and reduced glutathione to study the link between oxidative damage, aging and beta-amyloid (Abeta) in the canine brain. The aged canine brain, a model of human brain aging, naturally develops extensive diffuse deposits of human-type Abeta. Abeta was measured in immunostained prefrontal cortex from 19 beagle dogs (4-15 years). Increased malondialdehyde (MDA), which indicates increased lipid peroxidation, was observed in the prefrontal cortex and serum but not in cerebrospinal fluid (CSF). Oxidative damage to proteins (carbonyl formation) also increased in brain. An age-dependent decline in GS activity, an enzyme vulnerable to oxidative damage, and in the level of glutathione (GSH) was observed in the prefrontal cortex. MDA level in serum correlated with MDA accumulation in the prefrontal cortex. Although 11/19 animals exhibited Abeta, the extent of deposition did not correlate with any of the oxidative damage measures, suggesting that each form of neuropathology accumulates in parallel with age. This evidence of widespread oxidative damage and Abeta deposition is further justification for using the canine model for studying human brain aging and neurodegenerative diseases.