bluuebunny/crossref_metadata_2025_split huggingface dataset:
In vitro translation in a cell‐free system from Trypanosoma brucei yields glycosylated and glycosylphosphatidylinositol‐anchored proteins
Authors: Duszenko Michael , Kang Xuedong , Böhme Ulrike , Hömke Rolf , Lehner Manfred 
Published: March 2003

African trypanosomes escape many cellular and unspecific immune reactions by the expression of a protective barrier formed from a repertoire of several hundred genes encoding immunologically distinct variant surface glycoproteins (VSGs). All mature VSGs are glycosylphosphatidylionositol‐anchored and N‐glycosylated.To study trypanosome‐specific post‐translational modifications of VSG, a cell‐free system capable of in vitro translation, translocation into the rough endoplasmic reticulum, N‐glycosylation and glycosylphosphatidylinositol‐anchor addition was established using lysates of the bloodstream form of Trypanosoma brucei. Monitoring protein synthesis by [35S]methionine incorporation, labeled protein bands were readily detected by fluorography following SDS/PAGE. Appearance of these bands increased during a time‐course of 45 min and was sensitive to cycloheximide but not chloramphenicol treatment. Efficiency of this system, in terms of incorporation of radiolabeled amino acids into newly formed proteins, is similar to reticulocyte lysates. The system does not, however, allow initiation of protein synthesis.Depending on the clone used, immunoprecipitation revealed one or two newly formed VSG bands. Upon digestion with N‐glycosidase F these bands resulted in a single band of a lower apparent molecular mass, indicating that newly synthesized VSG underwent translocation and glycosylation in the cell‐free system. Biotinylation of VSG and a combination of precipitation with immobilized avidin and detection of VSG using antibodies specific for clones and cross‐reacting determinants revealed that newly formed VSG contained the glycosylphosphatidylinositol anchor.
Detection of modifications in the glucose metabolism induced by genetic mutations in Saccharomyces cerevisiae by 13C‐ and 1H‐NMR spectroscopy
Authors: Herve Martine , Buffin‐Meyer Bénédicte , Bouet Françoise , Tran‐Dinh Son 
Published: March 2003

NMR spectroscopy may offer a suitable technique to characterize the glucose metabolism in response to genetic mutations in cells. The effects of various genetic modifications in Saccharomyces cerevisiae yeast were investigated using 13C‐ and 1H‐NMR spectroscopy associated with biochemical techniques. Cells were incubated with [1‐13C]glucose in order to study glucose consumption and the formation of various end‐products (ethanol, trehalose, glycerol, glutamate and amino acids) as a function of time.Two types of genetic modifications were studied in S. cerevisiae. A genetic modification deleted the N‐terminal part of the TFC7 protein which is the smallest subunit (tau55) of the TFIIIC transcription factor. One secondary effect of this mutation was a large deletion of mitochondrial DNA giving the rho‐phenotype. The other genetic modification corresponded to the disruption of the HUF gene; the mutated cells were rho+ like the reference strain. Both mutations increase the glycolysis rate and glycerol synthesis and decrease trehalose production. The most modified cells, which contain both TFC7 deletion and HUF gene disruption, utilize glucose in the most extreme manner as in these cells the largest production of the two glycolytic products (ethanol and glycerol) and the smallest trehalose formation occur. The HUF gene disruption serves as a positive modulator of glycolysis and respiration. However, the TFC7 deletion, associated with the phenotype rho‐, induces the most damage in the cellular function, dramatically altering the behaviour of the Krebs cycle. The cycle becomes blocked at the level of 2‐oxoglutarate, detected by a characteristic pattern of the 13C‐NMR glutamate spectra. These NMR spectra corroborate the phenotypic data, the rho‐phenotype corresponding to deletions of mitochondria DNA which block all mitochondria protein synthesis and render the cells unable to derive energy from respiration. Moreover, as a consequence of the Krebs cycle blocking, alanine formation is also observed.
Novel processive and nonprocessive glycosyltransferases from Staphylococcus aureus and Arabidopsis thaliana synthesize glycoglycerolipids, glycophospholipids, glycosphingolipids and glycosylsterols
Authors: Jorasch Petra , Warnecke Dirk C. , Lindner Buko , Zähringer Ulrich , Heinz Ernst 
Published: March 2003

A processive diacylglycerol glucosyltransferase has recently been identified from Bacillus subtilis[Jorasch, P., Wolter, F.P., Zähringer, U., and Heinz, E. (1998) Mol. Microbiol.29, 419–430]. Now we report the cloning and characterization of two other genes coding for diacylglycerol glycosyltransferases from Staphylococcus aureus and Arabidopsis thaliana; only the S. aureus enzyme shows processivity similar to the B. subtilis enzyme. Both glycosyltransferases characterized in this work show unexpected acceptor specificities.We describe the isolation of the ugt106B1 gene (GenBank accession number Y14370) from the genomic DNA of S. aureus and the ugt81A1 cDNA (GenBank accession number AL031004) from A. thaliana by PCR. After cloning and expression of S. aureus Ugt106B1 in Escherichia coli, SDS/PAGE of total cell extracts showed strong expression of a protein having the predicted size of 44 kDa. Thin‐layer chromatographic analysis of the lipids extracted from the transformed E. coli cells revealed several new glycolipids and phosphoglycolipids not present in the controls. These lipids were purified from lipid extracts of E. coli cells expressing the S. aureus gene and identified by NMR and mass spectrometry as 1,2‐diacyl‐3‐[O‐β‐d‐glucopyranosyl]‐sn‐glycerol, 1,2‐diacyl‐3‐[O‐β‐d‐glucopyranosyl‐(1→6)‐O‐β‐d‐glucopyrano‐syl]‐sn‐glycerol, 1,2‐diacyl‐3‐[O‐β‐d‐glucopyranosyl‐(1→6)‐O‐β‐d‐glucopyranosyl‐(1→6)‐O‐β‐d‐glucopyranosyl]‐sn‐glycerol, sn‐3′‐[O‐β‐d‐glucopyranosyl]‐phosphatidylglycerol and sn‐3′‐[O‐(6′″‐O‐acyl)‐β‐d‐glucopyranosyl‐(1′′′→6′′)‐O‐β‐d‐glucopyranosyl]‐sn‐2′‐acyl‐phospha‐tidylglycerol. A 1,2‐diacyl‐3‐[O‐β‐d‐galactopyranosyl]‐sn‐glycerol was isolated from extracts of E. coli cells expressing the ugt81A1 cDNA from A. thaliana.The enzymatic activities expected to catalyze the synthesis of these compounds were confirmed by in vitro assays with radioactive substrates. Experiments with several of the above described glycolipids as 14C‐labeled sugar acceptors and unlabeled UDP‐glucose as glucose donor, suggest that the ugt106B1 gene codes for a processive UDP‐glucose:1,2‐diacylglycerol‐3‐β‐d‐glucosyltransferase, whereas ugt81A1 codes for a nonprocessive diacylglycerol galactosyltransferase. As shown in additional assays with different lipophilic acceptors, both enzymes use diacylglycerol and ceramide, but Ugt106B1 also accepts glucosyl ceramide as well as cholesterol and cholesterol glucoside as sugar acceptors.
Organization‐dependent effects of toxic bivalent ions
Authors: Liliom Károly , Wágner Gábor , Pácz Anita , Cascante Marta , Kovács János , Ovádi Judit 
Published: March 2003

The effects of bivalent ions on tubulin dynamics and the upper phase of glycolysis were investigated at different organization levels in vitro. Cu2+, Cd2+, Hg2+ and CrO2–4 inhibit the tubulin polymerization at an IC50 of 14–24 µm with high cooperativity and also induce microtubule disassembly. The apparent binding constants of the ions to tubulin, estimated by fluorescence quenching, vary between 6 and 28 µm. BIAcore measurements for tubulin–tubulin interaction suggest that the presence of Cu2+ affects neither koff nor kon, but the amount of the bound tubulin. While the inhibitory effect of Cu2+ on tubulin polymerization is partially abolished by cross‐linking of microtubules with substoichiometric amounts of phosphofructokinase or decoration of tubules with cytosolic proteins, in the presence of kinase but not with cytosolic proteins the tubules are resistant to CrO42–. No inhibitory effect of Cu2+ or CrO2–4 on microtubule assembly was detected in the MAP‐containing cytosolic fraction. Electron microscopy revealed that tubules assembled in the presence of Cu2+ or CrO2–4 ions contain aggregates of thread‐like oligomers that are less conspicuous in the presence of cytosolic proteins. Cu2+, Cd2+, and Hg2+ inhibit the glycolytic flux in the cytosolic fraction characterized at equilibrium by an IC50 of 10–14 µm with high cooperativity. Tubulin diminishes the inhibitory effect of the cations. These data indicate that the responses elicited by the bivalent ions are highly dependent on the supramolecular organization of the systems.
A fluorescence‐quenched chitopentaose for the study of endo‐chitinases and chitobiosidases
Authors: Cottaz Sylvain , Brasme Bernard , Driguez Hugues 
Published: March 2003

A new fluorogenic substrate displaying intramolecular fluorescence energy transfer (FRET) has been synthetized from NI,NII,NIII,NIV‐tetra‐acetyl‐chitopentaose. Two molecules, a fluorophore (5‐(2‐aminoethyl) amino‐1‐naphtalene‐sulfonic acid; EDANS) and a quenching group (dimethylaminophenylazophenyl; DAB) were chemically introduced on to the chitopentaose, one at each end. Among eight enzymes tested, only endo‐chitinase and chitobiosidase activities could be specifically assayed by monitoring the variation of fluorescence after enzymatic hydrolysis of this substrate. Chitobiases and N‐acetyl‐β‐glucosaminidases are not active on the compound, the presence of a bulky chromogenic group at the 2 position of the nonreducing end of the subtrate preventing the binding and thus hydrolysis by these two exo‐enzymes. The observation that chitobiosidases are able to hydrolyse a chitooligosaccharide functionalized on both extremities demonstrates the possibility of an endo‐action for this class of chitinases, which are generally classified as exo‐enzymes. This fluorogenic chitooligosaccharide should prove to be very useful for the detection and the convenient assay of chitinolytic activities at nanomolar concentrations.
Shifting the NAD/NADP preference in class 3 aldehyde dehydrogenase
Authors: Perozich John , Kuo Ingrid , Wang B. C. , Boesch Josette S. , Lindahl Ronald , Hempel John 
Published: March 2003

Among pyridine‐nucleotide‐dependent oxidoreductases, the class 3 family of aldehyde dehydrogenases (ALDHs) is unusual in its ability to function with either NAD or NADP. This is all the more surprising because an acidic residue, Glu140, coordinates the adenine ribose 2′ hydroxyl. In many NAD‐dependent dehydrogenases a similarly placed carboxylate is thought to be responsible for exclusion of NADP. The corresponding residue in most (≈ 71%) sequences in the ALDH extended family is also Glu, and most of these are NAD‐specific enzymes. Site‐directed mutagenesis was performed on this residue in rat class 3 ALDH. Our results indicate that this residue contributes to tighter binding of NAD in the native enzyme, but suggest that additional factors must contribute to the ability to utilize NADP. Mutagenesis of an adjacent basic residue (Lys137) indicates that it is even more essential for binding both coenzymes, consistent with its conservation in nearly all ALDHs (> 98%).
Specificities of functionally expressed chalcone and acridone synthases from Ruta graveolens
Authors: Springob Karin , Lukačin Richard , Ernwein Claudia , Gröning Inga , Matern Ulrich 
Published: March 2003

The common rue, Ruta graveolens L., expresses two types of closely related polyketide synthases that condense three malonyl‐CoAs with N‐methylanthraniloyl‐CoA or 4‐coumaroyl‐CoA to produce acridone alkaloids and flavonoid pigments, respectively. Two acridone synthase cDNAs (ACS1 and ACS2) have been cloned from Ruta cell cultures, and we report now the cloning of three chalcone synthase cDNAs (CHS1 to CHS3) from immature Ruta flowers. The coding regions of these three cDNAs differ only marginally, and the translated polypeptides show about 90% identity with the CHSs from Citrus sinensis but less than 75% with the Ruta endogeneous ACSs. CHS1 was functionally expressed in Eschericha coli and its substrate specificity compared with those of the recombinant ACS1 and ACS2. 4‐Coumaroyl‐CoA was the preferred starter substrate for CHS1, but cinnamoyl‐CoA and caffeoyl‐CoA were also turned over at significant rates. However, N‐methylanthraniloyl‐CoA was not accepted. In contrast, highly active preparations of recombinant ACS1 or ACS2 showed low, albeit significant, CHS side activities with 4‐coumaroyl‐CoA, which on average reached 16% (ACS1) and 12% (ACS2) of the maximal activity determined with N‐methylanthraniloyl‐CoA as the starter substrate, while the conversion of cinnamoyl‐CoA was negligible with both ACSs. The condensation mechanism of the acridone ring system differs from that of chalcone/flavanone formation. Nevertheless, our results suggest that very minor changes in the sequences of Ruta CHS genes are sufficient to also accommodate the formation of acridone alkaloids, which will be investigated further by site‐directed mutagenesis.
A folding variant of human α‐lactalbumin induces mitochondrial permeability transition in isolated mitochondria
Authors: Köhler Camilla , Gogvadze Vladimir , Håkansson Anders , Svanborg Catharina , Orrenius Sten , Zhivotovsky Boris 
Published: March 2003

A human milk fraction containing multimeric α‐lactalbumin (MAL) is able to kill cells via apoptosis. MAL is a protein complex of a folding variant of α‐lactalbumin and lipids. Previous results have shown that upon treatment of transformed cells, MAL localizes to the mitochondria and cytochrome c is released into the cytosol. This is followed by activation of the caspase cascade. In this study, we further investigated the involvement of mitochondria in apoptosis induced by the folding variant of α‐lactalbumin. Addition of MAL to isolated rat liver mitochondria induced a loss of the mitochondrial membrane potential (ΔΨm), mitochondrial swelling and the release of cytochrome c. These changes were Ca2+‐dependent and were prevented by cyclosporin A, an inhibitor of mitochondrial permeability transition. MAL also increased the rate of state 4 respiration in isolated mitochondria by exerting an uncoupling effect. This effect was due to the presence of fatty acids in the MAL complex because it was abolished completely by BSA. BSA delayed, but failed to prevent, mitochondrial swelling as well as dissipation of ΔΨm, indicating that the fatty acid content of MAL facilitated, rather than caused, these effects. Similar results were obtained with HAMLET (human α‐lactalbumin made lethal to tumour cells), which is native α‐lactalbumin converted in vitro to the apoptosis‐inducing folding variant of the protein in complex with oleic acid. Our findings demonstrate that a folding variant of α‐lactalbumin induces mitochondrial permeability transition with subsequent cytochrome c release, which in transformed cells may lead to activation of the caspase cascade and apoptotic death.
Isolation and structural characterization of an R‐form lipopolysaccharide from Yersinia enterocolitica serotype O:8
Authors: Oertelt Clemens , Lindner Buko , Skurnik Mikael , Holst Otto 
Published: March 2003

The lipopolysaccharide (LPS) of strain 8081‐c‐R2, a spontaneous R‐mutant of Yersinia enterocolitica serotype O:8, was isolated using extraction with phenol/chloroform/light petroleum. Its compositional analysis indicated the presence of d‐GlcN, d‐Glc, lglycerodmanno‐ and dglycerodmanno‐heptose, 3‐deoxy‐dmanno‐oct‐2‐ulosonic acid (Kdo) and phosphate. From deacylated LPS obtained after successive treatment with hydrazine and potassium hydroxide, three oligosaccharides (1–3) were isolated using high‐performance anion‐exchange chromatography, the structures of which were determined by compositional analysis and one‐ and two‐dimensional NMR spectroscopy asin which all sugars are pyranoses, and R and R′ represent β‐d‐Glc (in 1 and 2) and β‐d‐GlcN (in 1 only), respectively. d‐α‐d‐Hep is dglycero‐α‐dmanno‐heptose, l‐α‐d‐Hep is lglycero‐α‐dmanno‐heptose, Kdo is 3‐deoxy‐dmanno‐oct‐2‐ulosonic acid, and P is phosphate. The liberated lipid A was analyzed by compositional analyses and MALDI‐TOF MS. Its β‐d‐GlcN4P‐(1→6)‐α‐d‐GlcN‐1→P backbone is mainly tetra‐acylated with two amide‐ and one ester‐linked (at O3 of the reducing GlcN) (R)‐3‐hydroxytetradecanoic acid residues, and one tetradecanoic acid that is attached to the 3‐OH group of the amide‐linked (R)‐3‐hydroxytetradecanoic acid of the nonreducing GlcN. Additionally, small amounts of tri‐ and hexa‐acylated lipid A species occur.
Histamine prevents polyamine accumulation in mouse C57.1 mast cell cultures
Authors: Fajardo Ignacio , Urdiales José L. , Paz José C. , Chavarría Teresa , Sánchez‐Jiménez Francisca , Medina Miguel A. 
Published: March 2003

The effects of histamine on polyamine uptake and metabolism was studied in a mouse mast cell line (C57.1), as a cell model in which both biogenic amines are important for maintaining cell function and viability. Results obtained after incubations with exogenous histamine indicated that histamine prevents polyamine accumulation by affecting polyamine uptake. A plasma membrane transport system for polyamines has been also studied in mast cells. It seems to be a Na+‐dependent uptake with high affinity for both spermine and spermidine and lower affinity for putrescine and agmatine. Polyamine uptake was reduced in both cells treated with exogenous histamine and histamine‐preloaded cells. However, ornithine decarboxylase activity and cell proliferation were not affected by histamine. Incubation with histamine enhanced the spermidine/spermine acetyl transferase induction caused by N1‐ethyl‐N11‐[(cyclopropyl)methyl]‐4,8‐diazaundecane, suggesting that polyamine acetylation could be another mechanism by which histamine prevents polyamine accumulation in C57.1 mast cells.
Inhibitory sequences in the N‐terminus of the double‐stranded‐RNA‐dependent protein kinase, PKR, are important for regulating phosphorylation of eukaryotic initiation factor 2α (eIF2α)
Authors: Vattem Krishna M. , Staschke Kirk A. , Zhu Shuhao , Wek Ronald C. 
Published: March 2003

During viral infection, phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) by the interferon‐induced RNA‐dependent protein kinase, PKR, leads to inhibition of translation initiation and viral proliferation. Activation of PKR is mediated by association of virally encoded double‐stranded RNAs (dsRNAs) with two dsRNA binding domains (dsRBDs) located in the N‐terminus of PKR. To better understand the molecular mechanisms regulating PKR, we characterized the activities of wild‐type and mutant versions of human PKR expressed and purified from yeast. The catalytic rate of eIF2α phosphorylation by our purified PKR was increased in response to dsRNA, but not single‐stranded RNA or DNA, consistent with the properties previously described for PKR purified from mammalian sources. While both dsRBD1 and dsRBD2 were required for activation of PKR by dsRNA, only deletion of dsRBD1 severely reduced the basal eIF2α kinase activity. Removal of as few as 25 residues at the C‐terminal junction of dsRBD2 dramatically increased eIF2α kinase activity and characterization of larger deletions that included dsRBD1 demonstrated that removal of these negative‐acting sequences could bypass the dsRBD1 requirement for in vitro phosphorylation of eIF2α. Heparin, a known in vitro activator of PKR, enhanced eIF2α phosphorylation by PKR mutants lacking their entire N‐terminal sequences, including the dsRBDs. The results indicate that induction of PKR activity is mediated by multiple mechanisms, one of which involves release of inhibition by negative‐acting sequences in PKR.
Thermodynamic analysis of binding of p‐substituted benzamidines to trypsin
Authors: Talhout Reinskje , Engberts Jan B. F. N. 
Published: March 2003

Understanding the structural basis of inhibitor–enzyme interactions, important for the design of new drugs, requires a complete thermodynamic characterization of the binding process as well as a description of the structure of the complex. In this paper, the binding of p‐substituted benzamidinium derivatives to the structurally well‐characterized serine proteinase bovine pancreatic trypsin has been studied using isothermal titration calorimetry. These experiments have permitted a complete characterization of the temperature dependence of the inhibitor‐binding thermodynamics. At 25 °C, both the enthalpy and entropy of binding are favourable for all studied derivatives, but this is only true for a relatively narrow temperature range. As binding is characterized by a negative change in heat capacity, the process is characterized by enthalpy–entropy compensation, resulting in a change of the net thermodynamic driving force for association from entropic to enthalpic with increasing temperature. These phenomena are not unusual when hydrophobic forces play an important role. The trend in the relative binding potencies can, to a significant extent, be attributed to the electron‐donating/withdrawing character of the substituent at the para position, as shown by the Hammett

plot for the different inhibitors; the more polar the p‐substituted benzamidine, the less potent it will be as a trypsin inhibitor. This behaviour might result from a bulk solvation effect, meaning that the more polar, lower potency inhibitors will be more stabilized in water than the less polar, higher potency inhibitors.
Diversity of O‐linked glycosylation patterns between species
Authors: Mourad Rabih , Morelle Willy , Neveu André , Strecker Gérard 
Published: March 2003

Amphibia egg jelly coats are formed by components secreted along the oviduct. These secretion products overlay the oocytes as they pass along the different oviducal portions. Mucin type glycoproteins are the major constituents of the egg jelly coats. In this study, the O‐linked carbohydrate chains of the jelly coats surrounding the eggs of Rana ridibunda were released by alkaline borohydride treatment. Fractionation of the mixture of O‐linked oligosaccharide‐alditols was achieved by a combination of chromatographic techniques including gel‐permeation chromatography, ion‐exchange chromatography and high‐performance liquid chromatography using an amino‐bonded silica column. The primary structures of these O‐glycans were determined by one‐dimensional and two‐dimensional 1H‐NMR spectroscopy and matrix‐assisted laser‐desorption‐ionization‐time‐of flight mass spectrometry. 25 oligosaccharide structures, possessing a core consisting of Gal(β1–3)GalNAc‐ol with or without branching through a GlcNAc residue linked (β1–6) to the GalNAc residue (core type 2 or core type 1, respectively) are described. The most representative antennae are: HSO3(6)[Fuc(α1‐3)]GlcNAc; Gal(β1‐2)Gal; Gal(β1‐2)Gal(α1‐3)[Fuc(α1‐2)]Gal; GlcA(β1‐3)‐Gal(β1‐3)[Fuc(α1‐2)]Gal; GalNAc(α1‐4)Gal(β1‐4)Gal; Gal(β1‐3)GalNAc(α1‐4)Gal(β1‐4)Gal and GlcA(β1‐3)Gal(β1‐3)GalNAc. These results confirm the species‐specific O‐glycosylation of Amphibia oviducal mucins. The significance of this observation should be linked to a symbiotic role of carbohydrates involved in host–parasite interactions.