Effect of metal ions on sucrose synthase from rice grains--a study on enzyme inhibition and enzyme topography

The inhibition of the plant glycosyltransferase sucrose synthasefrom rice grains by free metal ions was studied. Decreasingsucrose synthase activities in the order of metal ions (Cu²⁺>> Zn²⁺ Ni²⁺ > Fe²⁺; 15·4% residual activitywith 30 µM Cu²⁺) as well as inhibition by diethyl pyrocarbonate(27% residual activity at pH 7·2 and 43 µM diethylpyrocarbonate) provided evidence that histidyl residues areimportant for sucrose synthase activity. Chelated metal ions,due to the geometric restriction of the reagent, gave a lesspronounced inhibitory effect (11·7% residual activitywith 100 µM Cu²⁺), but suggested that surface-accessiblehistidine residues are probably involved. Inhibition of sucrosesynthase could always be prevented by metal ion scavengers [ethylenediaminetetra-aceticacid (EDTA), dithiothreitol (DTT), mercaptoethanol, reducedglutathione, imidazole and histidine]. Sucrose synthase inhibitedby free and chelated Cu²⁺, respectively, could be partly (60%)reactivated by EDTA. These results led to a topographical analysisof histidines on the surface of the homotetrameric protein byimmobilized metal ion chromatography (IMAC). From the orderby which sucrose synthase was bound to immobilized chelatedmetal ions in the presence of 1 mM imidazole (Cu²⁺ > Ni²⁺> Zn²⁺ = Co²⁺), it could be concluded that the enzyme hasat least 5–7 surface-accessible histidines. Sucrose synthasecould not be eluted from a Cu²⁺ column by an increasing imidazolegradient. These results are of particular interest for the furtherpurification of sucrose synthase(s), as well as for the evaluationof cloning and expression strategies using poly-hLstidine tails. glycosyltransferase IMAC nucleotide sugars sucrose synthase     

The effect of acetyl-coenzyme A on phosphate-activated glutaminase from pig kidney and brain

Phosphate-activated glutaminase (EC 3.5.1.2; l-glutamine amidohydrolase) purified from pig kidney and brain is activated by CoA and short-chain acyl-CoA derivatives. Acetyl-CoA is the most powerful activator (K(A) about 0.2mm). Acetyl-CoA is maximally effective in the absence of other activating anions such as phosphate and citrate, and at low glutamine concentrations. The negative co-operative substrate activation observed at pH7 becomes more pronounced in the presence of acetyl-CoA. Similarly to phosphate, acetyl-CoA produces at high protein concentrations a different type of activation, which is time-dependent, depends on protein concentration and is accompanied by an increase in the sedimentation coefficient. Acetyl-CoA, phosphate and citrate appear to have binding sites in common. No significant difference was observed between kidney and brain phosphate-activated glutaminase.     

Enzymatic synthesis of UDP-galactose on a gram scale

Uridine 5′-diphospho-- -galactose (UDP-Gal) was synthesized on a gram scale from uridine 5′-diphospho-- -glucose and - -galactose 1-phosphate using the enzymes galactose-1-phosphate uridyltransferase (EC 2.7.7.12), phosphoglucomutase (EC 2.7.5.1) and glucose-6-phosphate dehydrogenase (EC 1.1.1.27). The synthesis was performed in a repetitive batch mode in which the enzymes, some of which are expensive, were used in 16 subsequent batches without any loss of enzyme activity. The space time yield of the synthesis was 7.1 g/l d. The overall yield of the synthesis amounted to 40% and 1.1 gram of pure UDP-Gal was obtained.     

Dendroecological investigation of the vitality of Common Beech (Fagus sylvatica L.) in mixed mountain forests of the Northern Alps (South Bavaria)

Time series of radial growth and crown condition were used to assess the vitality of Common Beech (Fagus sylvatica L.) in mixed mountain forests of the Bavarian Alps. The investigation plots are located in three altitudinal belts at the mountains Kranzhorn and Laber. Sites at Kranzhorn are permanent monitoring plots since 1985, where beech show a high and, especially since 1996, at high altitudes increasing degree of crown transparency. No significant and ecological sensible relationship was found neither between crown condition data and weather, nor between crown condition data and water balance parameters. Tree-ring analyses revealed repeated growth depressions during the last decades starting at the end of the 1970s. Their intensity and duration increased with altitude. Climate–growth analyses carried out for the period 1931–2003 point to a temperature dependence of growth. Cool and short vegetation periods and especially late frost events appear as growth-limiting factors. Even in dry and warm years no serious growth-relevant water deficiency occurred. Neither weather fluctuations and extreme events, evaluated by different methods, nor age- or site-related differences provided sufficient explanations for growth disturbances and vitality loss in the last decades. Hence, further investigations are necessary to explain vitality loss, damage and destabilization of Common Beech in mixed mountain forest of Southern Germany. At Kranzhorn, strong relations between radial growth and defoliation were found, indicating the importance of long-term observation and monitoring data for these purposes.     

Apoptosome-independent activation of the lysosomal cell death pathway by caspase-9

The apoptosome, a heptameric complex of Apaf-1, cytochrome c, and caspase-9, has been considered indispensable for the activation of caspase-9 during apoptosis. By using a large panel of genetically modified murine embryonic fibroblasts, we show here that, in response to tumor necrosis factor (TNF), caspase-8 cleaves and activates caspase-9 in an apoptosome-independent manner. Interestingly, caspase-8-cleaved caspase-9 induced lysosomal membrane permeabilization but failed to activate the effector caspases whereas apoptosome-dependent activation of caspase-9 could trigger both events. Consistent with the ability of TNF to activate the intrinsic apoptosis pathway and the caspase-9-dependent lysosomal cell death pathway in parallel, their individual inhibition conferred only a modest delay in TNF-induced cell death whereas simultaneous inhibition of both pathways was required to achieve protection comparable to that observed in caspase-9-deficient cells. Taken together, the findings indicate that caspase-9 plays a dual role in cell death signaling, as an activator of effector caspases and lysosomal membrane permeabilization.     

Chemo-enzymatic synthesis of poly-<Emphasis Type="Italic">N</Emphasis>-acetyllactosamine (poly-LacNAc) structures and their characterization for CGL2-galectin-mediated binding of ECM glycoproteins to biomaterial surfaces

Poly-N-acetyllactosamine (poly-LacNAc) structures have been identified as important ligands for galectin-mediated cell adhesion to extra-cellular matrix (ECM) proteins. We here present the biofunctionalization of surfaces with poly-LacNAc structures and subsequent binding of ECM glycoproteins. First, we synthesized β-GlcNAc glycosides carrying a linker for controlled coupling onto chemically functionalized surfaces. Then we produced poly-LacNAc structures with defined lengths using human β1,4-galactosyltransferase-1 and β1,3-N-acetylglucosaminyltransferase from Helicobacter pylori. These compounds were also used for kinetic characterization of glycosyltransferases and lectin binding assays. A mixture of poly-LacNAc-structures covalently coupled to functionalized microtiter plates were identified for best binding to our model galectin His₆CGL2. We further demonstrate for the first time that these poly-LacNAc surfaces are suitable for further galectin-mediated binding of the ECM glycoproteins laminin and fibronectin. This new technology should facilitate cell adhesion to biofunctionalized surfaces by imitating the natural ECM microenvironment. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

2-Aminobenzimidazoles as potent Aurora kinase inhibitors

This Letter describes the discovery and key structure–activity relationship (SAR) of a series of 2-aminobenzimidazoles as potent Aurora kinase inhibitors. 2-Aminobenzimidazole serves as a bioisostere of the biaryl urea residue of SNS-314 (1c), which is a potent Aurora kinase inhibitor and entered clinical testing in patients with solid tumors. Compared to SNS-314, this series of compounds offers better aqueous solubility while retaining comparable in vitro potency in biochemical and cell-based assays; in particular, 6m has also demonstrated a comparable mouse iv PK profile to SNS-314.     

Enzymatic synthesis of nucleotide sugars

The present review gives a survey on the biosynthetic pathways of nucleotide sugars which are important for the in vitro synthesis of mammalian glycoconjugates. With respect to the use of these enzymes in glycotechnology the availability as recombinant enzymes from different sources, the large-scale synthesis of nucleotide sugars and their in situ regeneration in combination with glycosyltransferases are summarized and evaluated.     

Human acute lymphoblastic leukemia (ALL) blasts as accessory cells during T-cell activation: differences between patients in costimulatory capacity affect proliferative responsiveness and cytokine release by activated T cells

The ability of acute lymphoblastic leukemia (ALL) blasts to mediate costimulatory signals during T-lymphocyte activation was investigated in an experimental model in which monoclonal T-cell populations were stimulated with standardized activation signals (anti-CD3 and anti-CD28 monoclonal antibodies; phytohemagglutinin, PHA). Leukemia cells from 12 consecutive ALL patients with high peripheral blood blast counts were studied. Proliferative T-cell responses were detected for a majority of these patients when irradiated leukemia blasts were used as accessory cells during activation. T-cell cytokine release was also observed for most patients when using nonirradiated ALL accessory cells. Low or undetectable cytokine levels were usually observed for CD8+ clones, whereas the CD4+ clones often showed a broad cytokine response with release of interleukin-2 (IL-2), IL-4, IL-10, IL-13 and interferon gamma(IFN-gamma) in the presence of the ALL accessory cells. ALL blasts were also able to function as allostimulatory cells for normal peripheral blood mononuclear responder cells. However, both T-cell proliferation and cytokine release showed a wide variation between ALL patients. The accessory cell function of ALL blasts showed no correlation with the release of immunomodulatory mediators (IL-2, IL-10, IL-15) or the expression of any single adhesion/costimulatory membrane molecule (CD54, CD58, CD80, CD86) by the blasts. We conclude that for a majority of patients, native ALL blasts can mediate costimulatory signals needed for accessory cell-dependent T-cell activation, but differences in costimulatory capacity between ALL patients affects both the proliferative responsiveness and cytokine release by activated T cells.     

Combined preparative enzymatic synthesis of dTDP-6-deoxy-4-keto-D-glucose from dTDP and sucrose

dTDP–6–deoxy–4–keto–D–glucose (1), the common intermediate in the biosyntheses of the mainfold deoxysugars, was synthesized on a gram–scale by the combination of sucrose synthase and dTDP–D–glucose 4,6–dehydratase in a fed batch, starting the reaction with dTDP. This process allowed a dTDP conversion with a 100% rate. An easy and efficient three–step purification with anion–exchange chromatography and gel filtration gave 1.1 g of 1 in an overall yield of 73%. This work realizes a first step for an economic access to activated deoxysugars.