bluuebunny/crossref_metadata_2025_split huggingface dataset:
Carnitine palmitoyltransferase II specificity towards β‐oxidation intermediates
Authors: Ventura Fátima V. , Ijlst Lodewijk , Ruiter Jos , Ofman Rob , Costa Catarina G. , Jakobs Cornelis , Duran Marinus , De Almeida Isabel Tavares , Bieber Loran L. , Wanders Ronald J. A. 
Published: March 2003

Using isolated rat liver mitochondria, in the absence or presence of malonyl‐CoA (an inhibitor of carnitine palmitoyltransferase I), we have found that carnitine palmitoyltransferase II (CPT II) is active with palmitoyl‐CoA as well as with its β‐oxidation intermediates. A partially purified CPT II fraction from rat liver mitochondria was shown to be able to convert 3‐hydroxypalmitoyl‐CoA to 3‐hydroxypalmitoylcarnitine, which could be identified by fast‐atom‐bombardment mass spectrometry. This apparent broad specificity of CPT II was further evaluated by kinetic studies using purified CPT II. It was found that CPT II readily accepts 3‐oxopalmitoyl‐CoA, palmitoyl‐CoA, 3‐hydroxypalmitoyl‐CoA and 2,3‐unsaturated palmitoyl‐CoA as substrates with decreasing order of affinity. The apparent Vmax values found for the first three compounds were of the same order of magnitude; the 2,3‐unsaturated acyl‐CoA was the poorest substrate. Kinetic studies with purified CPT II showed 3‐hydroxypalmitoyl‐CoA to have the lowest K0.5 value (20 ± 6 μM) of all the CoA esters studied; the highest K0.5 value (65 ± 17 μM) was found for the 3‐oxo intermediate.These findings support the hypothesis that CPT II is involved in the export of toxic long‐chain acyl‐CoA esters from the mitochondria by first converting them into the corresponding carnitine esters, followed by transport out of the mitochondria and subsequently out of the cell.
Control by ATP and ADP of voltage‐induced mammalian‐cell‐membrane permeabilization, gene transfer and resulting expression
Authors: Rols Marie‐Pierre , Delteil Christine , Golzio Muriel , Teissié Justin 
Published: March 2003

We have permeabilized and transfected mammalian cells by transient alteration of their native transmembrane electrical potential difference. Chinese hamster ovary cells were chosen as a model in order to study the electropermeabilization and electrotransfection processes. Propidium iodide was used to monitor permeabilization. A plasmid carrying the β‐galactosidase gene was used to follow direct gene transfer and expression by determining transient expression of the electrotransfered activity at the single‐cell level. The effect of nucleotides on cell permeabilization and transfection was studied by altering the cytosolic ATP and ADP contents of cells either during the pulsation or during the period following it.Permeabilization and transfection are not regulated in the same way by the ATP and ADP levels. The permeabilization efficiency remains unaffected. Cell viability and the transfection yield are dramatically affected. While ADP is involved in the step of DNA transfer across the electropermeabilized plasma membrane, ATP controls other steps (cytoplasmic DNA migration towards the nucleus, expression). Our results prove, firstly, that membrane resealing is required but is not sufficient to preserve cell viability and, secondly, that transfection is a cell‐mediated process and not only an electrophoretic step driven by the external field.
Biosynthesis of vitamin B12 in anaerobic bacteria
Authors: Schulze Bettina , Vogler Bernhard , Renz Paul 
Published: March 2003

In anaerobic bacteria 5‐hydroxybenzimidazole and 5‐hydroxy‐6‐methylbenzimidazole are precursors of the 5,6‐dimethylbenzimidazole moiety of vitamin B12. In order to elucidate the pathway from these bases to vitamin B12, experiments on the transformation of 5‐hydroxy‐6‐methylbenzimidazole, of 5‐hydroxy‐6‐methylbenzimidazole‐α‐
D‐ribofuranoside, of 5‐hydroxybenzimidazolylcobamide and of 5‐hydroxy‐6‐methylbenzimidazolylcobamide into vitamin B12 were carried out.The vitamin B12 synthesized by the anaerobe Eubacterium limosum in the presence of 5‐hydroxy‐6‐methylbenzimidazole and
L‐[methyl‐13C]methionine was subjected to NMR spectroscopy. It revealed that the methyl group at C5 of the 5,6‐dimethylbenzimidazole moiety was 13C labeled, whereas the methyl group at C6 was unlabeled. This shows that the transformation of 5‐hydroxy‐6‐methylbenzimidazole into the base moiety of vitamin B12 occurs regiospecifically.5‐Hydroxy‐6‐methylbenzimidazole‐α‐ D‐ribofuranoside as well as 5‐hydroxybenzimidazolylcobamide and 5‐hydroxy‐6‐methylbenzimidazolylcobamide were also transformed into vitamin B12 by E. limosum. When 5‐hydroxy‐6‐methylbenzimidazolylcobamide 13C labeled at C2 of the base part and 14C labeled in the ribose was used for this experiment, the vitamin B12 obtained from this cobamide was 13C and 14C labeled in the same positions. This demonstrates that the α‐glycosidic bond of the precursor cobamide is not split during the formation of vitamin B12.It can be deduced from these results that the precursor bases are transformed regiospecifically into their α‐nucleotides, and partially into their cobamides. The α‐nucleotides are then transformed into αribazole‐5′‐phosphate and, subsequently, into vitamin B12. Most likely the cobamides are degraded to the α‐nucleotides before being used for the biosynthesis of vitamin B12. A pathway for the latter process is suggested.
Growth‐hormone‐receptor and cytokine‐receptor‐family signaling
Authors: Moutoussamy Soraya , Kelly Paul A. , Finidori Joëlle 
Published: March 2003

Structure of growth‐hormone receptor and the class I type of cytokine receptors : common structural features; cytokine‐receptor isoforms; oligomerization of receptor components initiates cytokine signalling.Role of the Jak kinases in mediating specific functions of growth‐hormone receptor and cytokine receptors.Role of signal transducer and activator of transcription (Stat) proteins in growth hormone and cytokine functions.Other pathways activated by cytokine receptors : the mitogen‐activated protein kinase pathway; insulin‐receptor substrates 1 and 2 and phosphatidylinositol‐3‐kinase pathways; the Src pathways and other tyrosine kinase pathways; the phospholipase C/protein kinase C/Ca2+ pathways.Regulation of growth‐hormone receptor and cytokine receptor signaling : binding sites, internalization and ubiquitination; phosphatases and Janus kinase/Stat inhibitors.Conclusions and future prospects.
Probing the inhibitor‐binding site of aldose reductase with site‐directed mutagenesis
Authors: Hohman Thomas C. , El‐Kabbani Ossama , Malamas Michael S. , Lai Kehdih , Putilina Tatiana , McGowan Michelle H. , Wane Yi‐Qun , Carper Deborah A. 
Published: March 2003

Aldose reductase (AR) has been implicated in the etiology of the secondary complications of diabetes, and enzyme inhibitors have been proposed as therapeutic agents. While effectively preventing the development of diabetic complications in animals, results from clinical studies of AR inhibitors have been disappointing, possibly due to poor potency in man. To assist in the design of more potent and specific inhibitors, crystallographic studies have attempted to identify enzyme‐inhibitor interactions. Resolution of crystal complexes has suggested that the inhibitors bind to the enzyme active site and are held in place through hydrogen bonding and van der Waals interactions formed within two hydrophobic pockets. To confirm and extend these findings we quantified inhibitor activity with single, site‐directed, mutant, human AR enzymes in which the apolar active‐site residues tryptophan 20, −79, −111 and phenylalanine 115 were replaced with alanine or tyrosine, decreasing the potential for van der Waals interactions.Consistent with molecular models, the inhibitory activity of Tolrestat, Sorbinil and Zopolrestat decreased 800−2000‐fold when tested with the mutant enzyme in which Trp20 was replaced with alanine. Further, alanine substitution for Trp111 decreased Zopolrestat's activity 400‐fold, while mutations to Trp79 and Phe115 had little effect on the activity of any of the inhibitors. The alanine mutation at Trp111 had no effect on Tolrestat's activity but decreased the activity of Sorbinil by about 1000‐fold. These latter effects were unanticipated based on the number of non‐bonded interactions between the inhibitors, Tolrestat and Sorbinil, and Trp20 and Trp111 that have been identified in the crystal structures. In spite of these unexpected findings, our results are consistent with the hypothesis that AR inhibitors occupy the enzyme active site and that hydrophobic interactions between the enzyme and inhibitor contribute to inhibitor binding stability.
Quinoprotein ethanol dehydrogenase of Pseudomonas aeruginosa is a homodimer
Authors: Diehl Annette , v. Wintzingerode Friedrich , Görisch Helmut 
Published: March 2003

The gene coding for the periplasmic quinoprotein ethanol dehydrogenase of Pseudomonas aeruginosa ATCC 17933 was cloned and sequenced. The deduced amino acid sequence contained a signal peptide of 34 residues and the major protein of 589 amino acids showed high similarities to pyrroloquinoline‐quinone‐dependent periplasmic and membrane‐bound dehydrogenases acting on alcohols, glucose and quinate or shikimate.It was demonstrated by alignment with the amino acid sequence of the large subunit of the quinoprotein methanol dehydrogenase from Methylobacterium extorquens, whose X‐ray structure is known, that the amino acid residues involved in the binding of pyrroloquinoline quinone and Ca2+ at the active site are conserved in the quinoprotein ethanol dehydrogenase of P. aeruginosa. Also, the glycine/tryptophan docking motifs involved in stabilizing the superbarrel structure of the quinoprotein methanol dehydrogenase of M. extorquens were conserved. The known sequences of pyrroloquinoline‐quinone‐dependent dehydrogenases were used to derive new, more specific sequence motifs for detecting members of this family of enzymes.Despite the sequence similarity between the large α subunit of quinoprotein methanol dehydrogenase from M. extorquens and the quinoprotein ethanol dehydrogenase from P. aeruginosa, the two enzyme systems were quite different. In the presence of the prosthetic group, pyrroloquinoline quinone expression of the Pseudomonas gene encoding the 60‐kDa subunit of quinoprotein ethanol dehydrogenase in Escherichia coli resulted in formation of active enzyme. The formation of active quinoprotein methanol dehydrogenase, however, is known to require, in addition to the large α subunit, the expression of a small β subunit, and helper proteins [Lidstrom, M. E. (1995) Genetics of bacterial quinoproteins, Methods Enzymol. 258, 217−227].
Active site mutants of pyruvate decarboxylase from Zymomonas mobilis
Authors: Pohl Martina , Siegert P. , Mesch K. , Bruhn H. , Grötzinger J. 
Published: March 2003

The homotetrameric pyruvate decarboxylase (PDC) from Zymomonas mobilis requires the cofactors thiamin diphosphate and Mg2+ for catalytic activity. We have investigated the role of various amino acid residues in the direct environment of the active site. The role of residue E473 in the catalytic activity and stability of the enzyme was probed by several mutations. All mutant enzymes were either inactive or failed to give any recombinant protein. The close interaction of E473 and N482, which can be deduced from the X‐ray structure, has been probed by mutagenesis of N482 to D. This mutation has a significant influence especially on the carboligation reaction of PDC, whereas the binding of the cofactors and the thermostability were not affected. These data suggest a specific interaction of N482 and E473 which is essential for coordinating the second aldehyde molecule during carboligation.Three hydrophobic residues (L112, I472 and I476) in the vicinity of the active centre have been investigated with respect to their potential influence on the transition states during catalysis. In contrast to L112, I472 and I476 influence the decarboxylation and carboligation reactions. The enlarged substrate‐binding site of PDCI472A allows the decarboxylation of longer aliphatic 2‐keto acids (C4‐C6) as well as aromatic 2‐keto acids besides pyruvate. Carboligations using PDCI472A as a catalyst yielded 2‐hydroxypropiophenone, benzoin and phenylacetylcarbinol. The enantioselectivity of PAC formation is impaired by mutations of both I472 and I476. The stereochemistry is most significantly affected with the mutant enzyme PDCI476E, which catalyses predominantly the synthesis of (S)‐phenylacetylcarbinol.
Structural characterisation of a rhamnan and a fucorhamnan, both present in the lipopolysaccharide of Burkholderia vietnamiensis strain LMG 10926
Authors: Gaur Deepa , Galbraith Lesley , Wilkinson Stephen G. 
Published: March 2003

The polymeric fraction isolated after mild acid hydrolysis of the lipopolysaccharide (LPS) from Burkholderia vietnamiensis strain LMG 10926 contained
L‐rhamnose (Rha) and
D‐fucose (Fuc). From NMR studies supported by the results of methylation analysis and Smith degradation, it could be inferred that the material was probably a mixture of two glycans. One component was a linear rhamnan with a trisaccharide repeating unit (1); the other was a branched fucorhamnan with a tetrasaccharide repeating unit (2). The presence of two distinct polymeric fractions in LPS is a common feature for Burkholderia species.←4)‐α‐ L‐Rhap‐(1←2)‐α‐
L‐Rhap‐(1←3)‐β‐
L‐Rhap‐(1←   1  α‐ L‐Rhap1↓2←3)‐α‐ D‐Fucp‐(1←3)‐α‐
D‐Fucp‐(1←3)‐α‐
L‐Rhap‐(1←   2
FhuF, an iron‐regulated protein of Escherichia coli with a new type of [2Fe‐2S] center
Authors: Müller Kerstin , Matzanke Berthold F. , Schünemann Volker , Trautwein Alfred X. , Hantke Klaus 
Published: March 2003

We previously used fhuF as a sensitive reporter gene of the iron status of Escherichia coli. In this report, the fhuF gene was identified as open reading frame f262b at 99.2 min on the genome sequence map of E. coli K‐12. The FhuF protein was labeled with a His‐tag and then purified to electrophoretic homogeneity. Based on sulfur determinations and Mössbauer and EPR spectroscopy, FhuF was identified as a [2Fe‐2S] protein. The g values (gx = 1.886, gy = 1.961, gz = 1.994) and some of the Mössbauer parameters of FhuF obtained [oxidized protein as isolated : ΔEQ,4.2K = 0.474 mm s−1; Fe3+ (reduced protein) : ΔEQ = 0.978 mm s−1] are not typical of common [2Fe‐2S] proteins and indicate that FhuF has unusual structural properties.The primary sequence of FhuF does not show any sequence similiarities to known [2Fe‐2S] proteins. By site‐directed mutagenesis, each of the six cysteines of FhuF was replaced by serine. EPR of the six reduced mutant proteins revealed that the terminal cysteine residues 244, 245, 256, and 259 form the [2Fe‐2S]Cys4 cluster. Mutants having the Cys‐to‐Ser replacement at positions 244, 245, 256, or 259 did not complement a fhuF mutant. The motif Cys‐Cys‐Xaa10‐Cys‐Xaa2‐Cys in FhuF differs considerably from the motif Cys‐Xaa2‐Cys‐Xaa9−15‐Cys‐Xaa2‐Cys found in other [2Fe‐2S] proteins. The unusual Cys‐Cys terminal group of the cluster may explain the atypical EPR and Mössbauer spectroscopic properties of the FhuF protein; possibly the tetrahedral symmetry at the ferric ion site is distorted. The phenotype of fhuF mutants and the structural features of the FhuF protein suggest that FhuF is involved in the reduction of ferric iron in cytoplasmic ferrioxamine B.
Thermotoga maritima maltosyltransferase, a novel type of maltodextrin glycosyltransferase acting on starch and malto‐oligosaccharides
Authors: Meissner Harald , Liebl Wolfgang 
Published: March 2003

A novel enzyme acting on starch and malto‐oligosaccharides was identified and characterised. The non‐hydrolytic enzyme, designated maltosyltransferase (MTase), of the hyperthermophilic bacterium Thermotoga maritima MSB8 disproportionates malto‐oligosaccharides via glycosyl transfer reactions. The enzyme has a unique transfer specificity strictly confined to the transfer of maltosyl units. Incubation of MTase with starch or its constituents, i.e. amylose and amylopectin, led to the formation of a set of multiples of maltose (i.e. maltose, maltotetraose, maltohexaose etc.). Malto‐oligosaccharides with a degree of polymerization (DP) X were disproportionated to products with a DP of X ± 2n (with X  3 and n = 0, 1, 2, ...). Maximum activity in a 10‐min assay was recorded at pH 6.5 and 85−90 +C. The enzyme displayed extraordinary resistance to thermal inactivation. For example, at 90, 85, and 70 +C (pH 6.5, 0.34 mg ml−1 protein), MTase half‐lives of about 2.5 h, 17 h, and 21 days, respectively, were recorded.The gene for MTase, designated mmtA, was isolated from a gene library of T. maritima strain MSB8. Analysis of the MTase primary structure as deduced from the nucleotide sequence of mmtA revealed that the enzyme is not closely related to known protein sequences. However, low‐level local similarity between MTase and the α‐amylase enzyme family (glycosyl hydrolase family 13) was detected, including conserved acidic residues essential for catalysis. Therefore, MTase should be assigned to this family. Based on detailed sequence analyses and comparison with amylolytic enzymes of known crystal structure we propose that MTase contains a (β/α)8‐fold as the core supersecondary structure which is typical for the α‐amylase family. On the other hand, MTase is unique in that it lacks several residues highly conserved throughout this family. Also, MTase possesses an extraordinarily large domain B (a domain typical for the α‐amylase family, inserted between β‐strand 3 and α‐helix 3 of the (β/α)8‐barrel fold).
Structure and serological specificity of a new acidic O‐specific  polysaccharide of Proteus vulgaris O45
Authors: Bartodziejska Beata , Shashkov Alexander S. , Torzewska Agnieszka , Grachev Aleksei A. , Ziolkowski Andrzej , Paramonov Nikolay A. , Rozalski Antoni , Knirel Yuriy A. 
Published: March 2003

The following structure of the O‐specific polysaccharide (OPS) of Proteus vulgaris O45 lipopolysaccharide (LPS) was established using 1H‐ and 13C‐NMR spectroscopy, including two‐dimensional NOESY and H‐detected 1H, 13C heteronuclear multiple‐quantum coherence (HMQC) experiments: Immunochemical studies, using rabbit polyclonal anti‐P. vulgaris O45 serum and LPS, OPS and Smith‐degraded OPS of P. vulgaris O45, showed the importance of β‐d‐GlcA in manifesting the serological specificity of the O‐antigen studied.
All three IκB isoforms and most Rel family members are stably associated with the IκB kinase 1/2 complex
Authors: Heilker Ralf , Freuler Felix , Pulfer Ruth , Di Padova Franco , Eder Jörg 
Published: March 2003

Nuclear factor kappa B (NF‐κB) is an important transcription factor for the genes of many pro‐inflammatory proteins and is strongly activated by the cytokines interleukin‐1 and tumor necrosis factor (TNF)α under various pathological conditions. In nonstimulated cells, NF‐κB is present in the cytosol where it is complexed to its inhibitor IκB. Activation of NF‐κB depends on the signal‐induced phosphorylation of IκB by specific IκB kinases which initiates the inhibitor's conjugation to ubiquitin and subsequent degradation by the proteasome. We used both TNF‐stimulated and okadaic‐acid‐stimulated HeLa cells to purify three biochemically distinct kinase activities targeting one or both of the two serines (S32 and S36) in IκBα which induce its rapid degradation upon cytokine stimulation. All three activities correspond to known IκB kinases: the mitogen‐activated 90 kDa ribosomal S6 kinase (p90rsk1), the IκB kinase 1/2 complex (IKK1/2) and casein kinase II (CK II). However, we found that only one of the activities, namely the IKK1/2 complex, exists as a pre‐assembled kinase–substrate complex in which the IKKs are directly or indirectly associated with several NF‐κB‐related and IκB‐related proteins: RelA, RelB, cRel, p100, p105, IκBα, IκBβ and IκBε. The existence of stable kinase–substrate complexes, the presence of all three known IκB isoforms in these complexes and our observation that the IKK complex is capable of phosphorylating IκBα‐, IκBβ‐ and IκBε‐derived peptides at the respective degradation‐relevant serines suggests that the IKK complex exerts a broad regulatory role for the activation of different NF‐κB species. In contrast to previous studies, which locate CK II phosphorylation sites exclusively to the C‐terminal PEST sequence of IκBα, we observed efficient phosphorylation of serine 32 in IκBα by the purified endogenous CK II complex. Therefore, both p90rsk1 and CK II have the same preference for phosphorylating only one of the two serines which are relevant for inducible degradation.
Preferential uptake and accumulation of oxidized vitamin C by THP‐1 monocytic cells
Authors: Laggner Hildegard , Besau Veronika , Goldenberg Hans 
Published: March 2003

THP‐1 cells preferentially accumulate vitamin C in its oxidized form. The uptake displays first‐order kinetics and leads to a build‐up of an outward concentration gradient which is stable in the absence of extracellular vitamin. The transport is faster than reduction by extracellular glutathione or by added cytosolic extract, and glutathione‐depleted cells show the same uptake rates as control cells. In addition, energy depletion or oxidation of intracellular sulfhydryls does not inhibit accumulation of ascorbate. The accumulation, however, always occurs in the reduced form. The affinity for dehydroascorbate is lower (Km 450 µm vs 60 µm) than for reduced ascorbate, but the maximal rate is more than 30 times higher (581 compared to 19 pmol·min−1 per 106 cells), and it is independent of sodium, whereas the uptake of ascorbate is not. The sodium gradient also allows accumulation of reduced ascorbate. Inhibitors of glucose transport by the GLUT‐1 transporter also inhibit uptake of dehydroascorbate (DHA), but there are some inconsistencies, because the Ki‐values are higher than reported for the isolated transporter and one inhibitor (deoxyglucose) is noncompetitive. The preferential uptake of the dehydro‐form of the vitamin may be useful for situations where this short‐lived metabolite is formed by oxidation in the environment.
Structural studies of the O‐specific polysaccharide of Hafnia alvei strain PCM 1207 lipopolysaccharide
Authors: Jachymek Wojciech , Czaja Jolanta , Niedziela Tomasz , Lugowski Czeslaw , Kenne Lennart 
Published: March 2003

The structure of the O‐specific side‐chain of the Hafnia alvei strain PCM 1207 lipopolysaccharide (LPS) has been investigated. Methylation analysis, partial acid hydrolysis, matrix‐assisted laser‐desorption ionization time‐of‐flight (MALDI‐TOF) MS, fast atom bombardment (FAB)‐MS/MS and 1H‐ and 13C‐NMR spectroscopy were the principal methods used. Glycerol phosphate was identified as a constituent in the polysaccharide and the following structure of a pentasaccharide repeating unit was established:The polysaccharide is partially (≈ 10%) substituted with O‐acetyl groups. The lipopolysaccharide was also subjected to high resolution magic angle spinning (HR‐MAS) NMR analysis, which showed both the signals of the O‐specific polysaccharide as well as several signals from unsubstituted core oligosaccharides. This confirmed the presence of the described structure in the native LPS.