bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of calcium and arachidonate metabolism in acetylated‐low‐density‐lipoprotein‐stimulated tumor‐necrosis‐factor‐α production by rat peritoneal macrophages
Authors: Pollaud‐Chérion Cécile , Vandaele Jean , Quartulli Fabio , Séguélas Marie‐Hélène , Decerprit Jacques , Pipy Bernard
Published: March 2003

We show that lipopolysaccharide‐free actetylated low‐density lipoprotein (LDL), but not native LDL, stimulates tumor‐necrosis factor‐α (TNF‐α) secretion by rat peritoneal macrophages and the signal‐transduction pathways involved. The role of the scavenger receptor (SR) in this response was suggested by the absence of an effect induced by native LDL, signal coupling involving pertussis‐toxin‐dependent guanine‐nucleotide‐binding regulatory (G) protein, and the complete inhibition of this response by SR ligands [poly(I) and dextran sulfate]. Acetylated LDL induces rapid Ca2+ release from inositol‐phosphate‐sensitive Ca2+ stores mediated by pertussis‐sensitive G proteins and a sustained Ca2+ rise mediated by Ca2+ influx and by Ca2+ release from ryanodine‐sensitive Ca2+ stores. Acetylated LDL‐induced Ca2+ influx and TNF‐α production were abolished by inhibitors of phospholipase C (U73122) and phospholipase A2 (bromophenacyl bromide), but were not affected by an inhibitor of protein kinase C (calphostine C). Therefore, Ca2+ influx induced by acetylated LDL is dependent on Ca2+ store depletion. Arachidonate released by acetylated LDL acts as a second messenger to activate TNF‐α secretion via Ca2+ influx. While the Ca2+ signal was not modified by an inhibitor of protein tyrosine kinases (PTK; herbimycin A), this inhibitor completely blocked TNF‐α production, suggesting the involvement of PTK downstream of the Ca2+ signal. These results suggest that a sustained elevation of intracellular Ca2+, mediated through Ca2+ influx via the phospholipase‐A2‐dependent pathway, is essential for induction of TNF‐α secretion. The type of SR class involved in these pathways remains to be identified.
Tyrosine phosphorylation of the muscle‐specific kinase is exclusively induced by acetylcholine receptor‐aggregating agrin fragments
Authors: Hopf Carsten , Hoch Werner
Published: March 2003

During formation of the neuromuscular junction, the basal membrane protein agrin initiates the aggregation of acetylcholine receptors (AChR) on the surface of myotubes. A muscle‐specific kinase (MuSK) becomes phosphorylated upon incubation with agrin, although it does not bind to agrin on its own. Utilizing MuSK‐specific antibodies, we demonstrate that the ability of different splicing variants and truncation fragments of agrin to trigger MuSK phosphorylation and AChR aggregation are correlated. Only agrin forms which are potent inducers of AChR‐clustering are able to trigger the phosphorylation of MuSK. Picomolar concentrations of agrin are already sufficient to induce MuSK phosphorylation. Similar amounts are necessary for the aggregation of AChRs as well as their phosphorylation on a tyrosine residue. The complete overlap of specificities for MuSK phosphorylation and AChR aggregation suggests that only binding of agrin to a MuSK‐containing receptor complex is responsible for the initiation of AChR aggregation. In contrast, interactions of agrin with binding proteins on the muscle surface harbouring different specifities such as α‐dystroglycan do not seem to be necessary for this process.
Structural characterisation of N‐linked and O‐linked oligosaccharides derived from interferon‐α2b and interferon‐α14c produced by Sendai‐virus‐induced human peripheral blood leukocytes
Authors: Nyman Tuula A. , Kalkkinen Nisse , Tölö Hannele , Helin Jari
Published: March 2003

We have previously isolated and partially characterised the components of a highly purified interferon‐α (IFN‐α) preparation produced by Sendai‐virus‐induced human peripheral blood leukocytes. Nine IFN‐α species were identified, and two of these were found to be glycosylated [Nyman, T. A., Tölö, H., Parkkinen, J. & Kalkkinen, N. (1998) Identification of nine interferon‐α subtypes produced by Sendai‐virus‐induced human peripheral blood leukocytes, Biochem. J. 329, 295−302]. Here, we isolated the N‐linked oligosaccharides of IFN‐α14c and the O‐linked chains of IFN‐α2b, and the glycans were characterised by electrospray tandem mass spectrometry and by specific glycosidase digestions monitored by matrix‐assisted laser desorption ionisation time of flight mass spectrometry. The IFN‐α14c N‐glycans were shown to exhibit core‐fucosylated biantennary glycans, with about 10 % carrying an additional α1,3‐linked fucose unit at the antennae. The IFN‐α2b was shown to carry about 50 % core type‐1 disialyltetrasaccharides, 30 % core type‐1 monosialyltrisaccharides and 20 % core type‐2 monosialylpentasaccharides.
Several weak signals in the cytosolic and transmembrane domains of the interleukin‐2‐receptor β chain allow for its efficient endocytosis
Authors: Subtil Agathe , Dautry‐Varsat Alice
Published: March 2003

Plasma membrane receptors are retrieved continually from the cell surface by endocytosis and transported to intracellular organelles. They are internalized at various rates depending on their ability to interact with endocytic structures of the plasma membrane. The interleukin‐2‐receptor β chain is endocytosed constitutively and efficiently. Here we show that different motifs in its cytosolic tail promote entry in an additive way, each of them acting as a weak internalization signal. The transmembrane domain of β also participates in endocytosis. In conclusion, several weak endocytic determinants can be responsible for the rapid internalization of a membrane protein.
Regulation of translation initiation factors by signal transduction
Authors: Kleijn Miranda , Scheper Gert C. , Voorma Harry O. , Thomas Adri A. M.
Published: March 2003

Regulation of eukaryotic translation initiation is a process that requires collaboration between multiple proteins. The cap‐binding factor eukaryotic initiation factor (eIF)4E, its binding protein 4E‐BP1, and the guanine‐nucleotide‐exchange factor eIF2B play important roles in the regulation of the rate of protein synthesis. This review describes the regulation of the activity of these three proteins and the signal‐transduction pathways involved therein.
The formate dehydrogenase‐cytochrome c553 complex from Desulfovibrio vulgaris Hildenborough
Authors: Sebban‐Kreuzer Corinne , Dolla Alain , Guerlesquin Françoise
Published: March 2003

The electron transfer between formate dehydrogenase and cytochrome c553 from the anaerobic bacteria Desulfovibrio vulgaris Hildenborough has been investigated. Parameters of the electron transfer kinetics are reported. The ionic strength dependence of the complex formation has been evidenced. Two mutants of cytochrome c553 have been obtained using site‐directed mutagenesis with the substitutions K62E and K62E,K63E. According to one‐dimensional and two‐dimensional NMR analysis, the two variants were found to have the same folding pattern as that of the wild‐type cytochrome. The replacements of the lysine residues by acidic groups have important effects on the affinity between the two oxidoreduction partners. K62 and K63 are essential for recognition between the formate dehydrogenase and the cytochrome c553. Previous structural studies of cytochrome c553 have demonstrated the involvement of the polypeptide chain in the modulation of the particular low oxidoreduction potential of this cytochrome. The present study provides evidence that, during the evolution of cytochromes from the anaerobic metabolism to aerobic respiration and photosynthesis, the electrostatic distribution at the recognised encounter surface around the heme is highly conserved in all cytochromes.
Transcriptional regulation of the non‐specific cross‐reacting antigen gene, a member of the carcinoembryonic antigen gene family up‐regulated in colorectal carcinomas
Authors: Koops M. Derek , Thompson John , Zimmermann Wolfgang , Stanners Clifford P.
Published: March 2003

Human non‐specific cross‐reacting antigen (NCA), a close relative of the tumor marker human carcinoembryonic antigen (CEA), is also an in vitro homotypic intercellular adhesion molecule capable of inhibiting differentiation when expressed ectopically by myoblasts. Moreover, NCA appears to be overexpressed at the transcriptional level to a greater extent and more frequently in colorectal carcinomas than CEA. This study examines the transcriptional control mechanisms responsible for orchestrating NCA expression. The region within 284 bp upstream of the translational start site of the NCA gene was found to be capable of directing high levels of expression in functional promoter assays. Footprinting experiments identified three cis‐acting elements and mobility‐shift assays revealed that the first of these elements is bound by the upstream stimulating factors USF1 and USF2 while the other two are bound by the stimulatory proteins Sp1 and Sp3. No cis‐acting elements corresponding to CEA footprint FP4 or the silencer CEA FP5 were detected in the NCA promoter, which may contribute to the differential expression of NCA versus CEA in tumorigenesis.
DNA‐binding properties of the tandem HMG boxes of high‐mobility‐group protein 1 (HMG1)
Authors: Grasser Klaus D. , Teo Soo‐Hwang , Lee Keng‐Boon , Broadhurst R. William , Rees Christine , Hardman Colin H. , Thomas Jean O.
Published: March 2003

High‐mobility‐group protein 1 (HMG1) is a conserved chromosomal protein with two homologous DNA‐binding HMG‐box domains, A and B, linked by a short basic region to an acidic carboxy‐terminal tail. NMR spectroscopy on the free didomain (AB) shows that the two HMG boxes do not interact. The didomain has a higher affinity for all DNA substrates tested than single HMG‐box domains and has a significantly higher ability to distort DNA by bending and supercoiling. The interaction of the didomain with DNA is stabilized by the presence of the basic region (≈20 residues, 9 of which are Lys) that links the second HMG box to the acidic tail in intact HMG1; this may be, at least in part, why this region also enhances supercoiling of relaxed circular DNA by the didomain and circularization of short DNA fragments (in the presence of ligase). Competition assays suggest significantly different structure‐specific preferences of single and tandem HMG boxes for four‐way junction and supercoiled plasmid DNA. Binding to supercoiled DNA appears to be promoted by protein oligomerization, which is pronounced for the didomains. Electron microscopy suggests that the oligomers are globular aggregates, associated with DNA looping. One box versus two (or several) is likely to be an important determinant of the properties of (non‐sequence specific) HMG‐box proteins.
In vitro activation of pro‐phenol‐oxidase by two kinds of pro‐phenol‐oxidase‐activating factors isolated from hemolymph of coleopteran, Holotrichia diomphalia larvae
Authors: Lee So Young , Kwon Tae Hyuk , Hyun Ji Hoon , Choi Jae Sue , Kawabata Shun‐Ichiro , Iwanaga Sadaaki , Lee Bok Luel
Published: March 2003

Previously, we purified and characterized a pro‐phenol‐oxidase (pro‐PO) of 79 kDa from coleopteran insect, Holotrichia diomphalia larvae [Kwon et al. (1997) Mol. Cells 7, 90−97]. Here, we describe the identification of two pro‐PO‐activating factors (PPAF), named PPAF‐I and PPAF‐II, directly involved in the activation of the isolated pro‐PO. When pro‐PO was incubated with either PPAF‐I or PPAF‐II, no phenol oxidase activity was observed. However, incubation of pro‐PO with both PPAF‐I and PPAF‐II specifically exhibited phenol oxidase activity. The purified PPAF‐I with a molecular mass of 33 kDa on SDS/PAGE had characteristics of a serine protease. It exhibited amidase activity against fluorogenic peptide substrates, tert‐butoxycarbonyl‐phenylalanyl‐seryl‐arginyl‐4‐methylcoumaryl‐7‐amide being the best among the substrates examined. The activity was completely inhibited by 0.02 mM p‐nitrophenyl‐p′‐guanidinobenzoate    HCl and diisopropylflurophosphate. The NH2‐terminal sequence of PPAF‐I had significant sequence similarity to those of serine proteases. On the other hand, the purified PPAF‐II had a molecular mass of 40 kDa on SDS/PAGE and 400 kDa determined by gel filtration, indicating an oligomeric protein. The NH2‐terminal sequence of PPAF‐II showed no similarity to known proteins. PPAF‐II exhibited no amidase activity against the fluorogenic substrates. Reconstitution experiments and immunoblotting analysis using affinity‐purified antibody against pro‐PO demonstrated that PPAF‐I first cleaves the intact pro‐PO to an intermediate of 76 kDa with no phenol oxidase activity, and then, PPAF‐I converts the intermediate to the active phenol oxidase of 60 kDa in the presence of PPAF‐II. These results indicate that the activation of pro‐PO system in hemolymph of H. diomphalia larvae is accomplished by at least two activating factors, a serine protease and a protein cofactor.
Oligonucleotide facilitators enable a hammerhead ribozyme to cleave long RNA substrates with multiple‐turnover activity
Authors: Jankowsky Eckhard , Schwenzer Bernd
Published: March 2003

Trans‐acting hammerhead ribozymes are usually efficient in cleaving short RNA model substrates under both single‐turnover and multiple‐turnover conditions. In contrast, when long RNAs are the substrates, the cleavage efficiency of these ribozymes decreases, including a loss of multiple‐turnover activity in many cases. Since target substrates for potential therapeutical purposes are mostly long RNAs, a multiple‐turnover cleavage of long RNAs would essentially increase the efficiency of hammerhead ribozymes. Therefore, we explored if oligonucleotide facilitators, capable of enhancing multiple‐turnover activity with short substrates, can also affect or cause multiple turnover with long substrates. We examined the effects of 12‐base and 24‐base oligonucleotide facilitators on the multiple‐turnover activity with substrates of different length containing 39‐, 452‐ and 942‐base sequences of the human tissue factor (HTF) mRNA. In the absence of facilitator, the ribozyme cleaved only the 39‐base substrate with multiple‐turnover activity, but not the long 452‐base and 942‐base substrates. However, facilitator addition enabled the ribozyme to cleave even the 452‐base and the 942‐base substrates with multiple‐turnover activity. All facilitators tested showed a remarkable activating effect with the long substrates.

The data demonstrate that a hammerhead ribozyme which, by itself, can only act as a single‐turnover catalyst with long substrates, can be switched by facilitators into a multiple‐turnover catalyst. Thus, the inactivation of long target RNAs in multiple‐turnover reactions may be achieved by addition of oligonucleotide facilitators.
Molecular characterization of cyanophycin synthetase, the enzyme catalyzing the biosynthesis of the cyanobacterial reserve material multi‐ L‐arginyl‐poly‐	L‐aspartate (cyanophycin)
Authors: Ziegler Karl , Diener Annette , Herpin Carine , Richter Ralf , Deutzmann Rainer , Lockau Wolfgang
Published: March 2003

Cyanophycin (multi‐ L‐arginyl‐poly‐
L‐aspartate), a water‐insoluble reserve polymer of cyanobacteria, is a product of nonribosomal peptide synthesis. The purification of cyanophycin synthetase of the cyanobacterium Anabaena variabilis is described. In sodium dodecylsulfate/polyacrylamide gel electrophoresis, the enzyme preparation shows one band with an apparent molecular mass of 100 kDa. The native enzyme has an apparent molecular mass of approximately 230 kDa, as determined by size‐exclusion chromatography, suggesting that the active form is a homodimer. During catalysis, ATP is converted to ADP. The gene coding for cyanophycin synthetase has been identified in the sequenced genome of Synechocystis sp. PCC 6803. The C‐terminal 60 % of the deduced amino acid sequence of cyanophycin synthetase show sequence similarity to enzymes of the superfamily of ligases involved in the biosynthesis of murein and of folyl‐poly(γ‐glutamate). Cells of Escherichia coli harbouring the gene on a plasmid express active synthetase and accumulate cyanophycin‐like material. The results prove that a single enzyme catalyzes the de novo synthesis of cyanophycin.
Role of the actin cytoskeleton in regulating the outer phosphatidylethanolamine levels in yeast plasma membrane
Authors: Dixit Bharat L. , Gupta Chhitar M.
Published: March 2003

Transbilayer phosphatidylethanolamine (PtdEtn) movements in the plasma membrane of Saccharomyces cerevisiae are regulated by an ATP‐dependent, protein‐mediated process(es). To examine whether this process is influenced by the actin cytoskeleton, we have studied the PtdEtn translocation in S. cerevisiae cells after treatment with microfilament disrupting and microtubule‐disrupting agents. PtdEtn translocation was studied by measuring the external PtdEtn levels, using fluorescamine as the external membrane probe, in the ATP‐depleted, ATP‐depleted and repleted, and N‐ethylmaleimide‐treated cells. The microfilaments and microtubules were disrupted by treatment with various cytochalasins and colchicine (or benomyl) respectively PtdEtn translocation became abnormal in the cytochalasin‐treated cells but not in cells that were treated with microtubule‐disrupting agents, such as colchicine or benomyl. These results have been interpreted to suggest that the actin cytoskeleton is involved in regulating the PtdEtn translocase activity in the yeast cell plasma membrane.
Calcitriol and transforming growth factor‐β upregulate 5‐lipoxygenase mRNA expression by increasing gene transcription and mRNA maturation
Authors: Härle Damaris , Rådmark Olof , Samuelsson Bengt , Steinhilber Dieter
Published: March 2003

Differentiation of the human monocytic cell line Mono Mac 6 with calcitriol plus transforming growth factor‐β (TGFβ) strongly induces 5‐lipoxygenase (5‐LO) mRNA and protein expression. The mechanism of 5‐LO mRNA induction by these agents was investigated. Analysis of mature 5‐LO mRNA by reverse‐transcriptase‐PCR gave a 42‐fold induction which was not due to alterations in 5‐LO half life and which was only in part due to an induction of gene transcription. There was an up to fivefold increase in 5‐LO primary transcripts by TGFβ and calcitriol, which could be inhibited by cycloheximide. No significant effects on 5‐LO transcription were observed with TGFβ or calcitriol alone. However, treatment of the cells with either calcitriol or TGFβ and addition of the corresponding second inducer lead to an about fourfold induction of primary transcript levels. Addition of cycloheximide together with the second inducer inhibited only the TGFβ but not the calcitriol effects, which indicated that there is a direct stimulation of 5‐LO transcription by calcitriol in the presence of TGFβ‐induced proteins.

In order to investigate the effects of TGFβ/calcitriol on 5‐LO transcript, elongation and maturation, the relative changes in immature and mature 5‐LO RNA species were analyzed by reverse‐transcription‐PCR. Analysis of exons 1−5 indicated an about threefold induction of 5‐LO transcripts by calcitriol/TGFβ, respectively. However, when exons 6−14 were determined, more pronounced increments were found (3.6−12‐fold). Selective analysis of polyadenylated and spliced 5‐LO mRNA species gave a 42‐fold induction. The effects of both TGFβ and calcitriol on transcript elongation and maturation were inhibited by cycloheximide.

Our results show that induction of 5‐LO mRNA by calcitriol and TGFβ is due to a modest increase in 5‐LO gene transcription and to the stimulation of transcript elongation and maturation.
Clusterin overexpression in rat pancreas during the acute phase of pancreatitis and pancreatic development
Authors: Calvo Ezequiel L. , Mallo Gustavo V. , Fiedler Fritz , Malka David , Vaccaro Maria I. , Keim Volker , Morisset Jean , Dagorn Jean‐Charles , Iovanna Juan L.
Published: March 2003

Molecular mechanisms associated with apoptosis in pancreas remain largely unknown. Clusterin mRNA is induced in several tissues in response to most apoptotic stimuli. In these tissues, clusterin has an antiapoptotic activity. The aim of this work was to test whether clusterin, which is not expressed in normal pancreas, was induced in pancreas during pancreatitis and pancreatic development. Clusterin mRNA levels were strongly increased 6 h after pancreatitis induction. Maximal expression happened between 24−48 h and decreased progressively to undetectable levels at day 5. Clusterin mRNA was expressed with similar intensity in oedematous caerulein‐induced pancreatitis and in response to various degrees of necrohaemorrhagic taurocholate‐induced pancreatitis, indicating a maximal gene activity in all types of pancreatitis; in situ hybridization showed that the acinar cells and some ducts expressed clusterin mRNA. A single band of about 35−38 kDa was detected by western blot in pancreatic homogenates and in pancreatic juice from rats with acute pancreatitis, but not from control rats. Clusterin mRNA expression was strong in late fetal life and remains high until day 11 post‐partum, then decreased progressively with a minimum from 35 to 90 days post‐partum. Clusterin mRNA levels were strongly induced in pancreatic acinar AR4‐2J cells in response to various apoptotic stimuli (i.e., cycloheximide, staurosporine, ceramide and H2O2) but not with interleukin (IL)‐1, IL‐4 or IL‐6 or heat shock, which do not induce apoptosis in AR4‐2J cells. In conclusion, we demonstrated that clusterin is synthesized and released by the pancreas. Its strong expression during acute pancreatitis suggests its involvement in the pancreatic response to injury. Clusterin is also induced during pancreatic development. Because these situations are associated with apoptosis and clusterin was shown to protect against apoptosis, we speculate that clusterin could be involved in the control of acinar cell apoptosis.
Purification and characterization of an alcohol dehydrogenase from the Antarctic psychrophile Moraxella sp. TAE123
Authors: Tsigos Iason , Velonia Kelly , Smonou Ioulia , Bouriotis Vassilis
Published: March 2003

An NAD+‐dependent alcohol dehydrogenase (ADH) of the Antarctic psychrophile Moraxella sp. TAE123 was purified to homogeneity with an overall yield of 16.7  % and further characterized. The native enzyme had an apparent molecular mass of 240 kDa and consisted of four identical 52‐kDa subunits. The pI of the enzyme was determined to be 5.5, while its optimum pH is 7.5. The enzyme contained 1 zinc atom/subunit and exhibited a remarkable thermal lability.

Moraxella sp. TAE123 ADH exhibited a wide range of substrate specificity similar to its mammalian counterparts and in contrast to other microbial ADHs. It oxidized mainly primary and secondary aliphatic alcohols. The highest reaction rate was observed when ethanol was used as substrate. A gradual decrease in rate was observed by increasing the length and branching of the carbon chain of the alcohol. This enzyme oxidized effectively large bulky alcohols, such as diphenylmethanol. Reduction of aldehydes and ketones was also observed.

N‐terminal amino acid sequence analysis of the enzyme did not reveal any similarity with the amino termini of all other ADHs, while an unexpected significant similarity was observed with the amino terminal sequence of four prokaryotic aldehyde dehydrogenases.
Characterization of three transcriptional repressor sites within the 3′ untranslated region of the rat serine protease inhibitor 2.3 gene
Authors: Paul Conception , Simar‐Blanchet Anne‐Emmanuelle , Ro Hyo‐Sung , Le Cam Alphonse
Published: March 2003

The activity of the rat serine protease inhibitor 2.3 gene (spi 2.3) is controlled by several positive promoter elements [Simar‐Blanchet, A.‐E., Paul, C., Mercier, L. & Le Cam, A. (1996) Eur. J. Biochem. 236, 638−648] and a negative element located in the 3′ untranslated gene region (3′ UTR) [Le Cam, A. & Legraverend, C. (1996) Eur. J. Biochem. 231, 620−627]. In the present studies, we dissected the 348‐bp spi 2.3 3′ UTR silencer to precisely define repressor sites and look for specifically interacting proteins. Three short elements referred to as A (nucleotides 1751−1776 in the cDNA), B (nucleotides 1812−1827) and C (nucleotides 1958−1974) sites repressed transcription from the homologous spi 2.3 promoter as well as from a heterologous minimal promoter containing the spi GAGA box enhancer. All three sites harbor a (TTTC) motif whose mutation affected silencer activity that was also dependent on flanking sequences. Those sites share the (TTTC) motif and a CCAAT/enhancer‐binding‐protein(C/EBP)−binding site with a fatty‐acid‐binding‐protein gene promoter element shown to interact specifically with a transcriptional repressor [He, G. P., Muise, A., Wu Li, A. & Ro, H.‐S. (1995) Nature 378, 92−96]. This repressor is however unlikely to mediate spi 2.3 3′ UTR silencer action since it was not detected in rat hepatocytes.

In vitro footprinting of the spi 2.3 3′ UTR silencer region revealed a strong interaction with liver nuclear proteins. Among the six identified footprints, three of them (F‐II, FIII and F‐IV) bound C/EBPs and mapped in regions harboring the repressor function. Binding of C/EBPs to all three spi 2.3 3′ UTR repressor sites, although rather weak, was confirmed by electrophoretic mobility shift assays that otherwise failed to reveal specific interactions with other liver nuclear proteins in vitro. However, none of the most largely liver expressed C/EBP species (i.e. α, β and δ) activated the spi 2.3 3′ UTR silencer function in NIH 3T3 cells, suggesting that binding of those transcription factors did not mediate the transcriptional repression.
Occurrence, overexpression and partial purification of the protein (majastridin) corresponding to the URF6 gene of the Rhodobacter blasticus atp operon
Authors: Brosché Mikael , Kalbina Irina , Arnfelt Mikael , Benito Gisela , Karlsson B. Göran , Strid Åke
Published: March 2003

Antibodies were produced against two antigenic peptides of a protein, which was named majastridin, corresponding to the URF6 gene of the Rhodobacter blasticus atp operon [Tybulewicz, V. L. J., Falk, G. & Walker, J. E. (1984) J. Mol. Biol. 179, 185−214]. A protein band of the expected size is labelled by immunoblotting in Western blots containing the cytosolic fractions from Rb. blasticus and Paracoccus denitrificans but not from Escherichia coli or Rhodospirillum rubrum. Although the protein is present during the entire life cycle of a Rb. blasticus culture, it is most abundant early during the stationary phase. Plasmid constructs of the URF6 gene for overexpression in E. coli were made. These constructs were designed to obtain proteins both with and without His‐tagging. In both cases, a protein product was visible in induced cells. The His‐tagged protein was purified to 85 % on a Ni column and, further, to at least 95 % by anion‐exchange chromatography. By N‐terminal sequencing of the His‐tagged protein, its identity was confirmed.
31P‐NMR spectroscopy of human and Paracoccus denitrificans electron transfer flavoproteins, and 13C‐ and 15N‐NMR spectroscopy of human electron transfer flavoprotein in the oxidised and reduced states
Authors: Griffin Kurt J. , Degala Gregory D. , Eisenreich Wolfgang , Müller Franz , Bacher Adelbert , Frerman Frank E.
Published: March 2003

Human and Paracoccus denitrificans wild‐type electron transfer flavoproteins have been investigated by 31P‐NMR in the oxidised and reduced states. The 31P chemical shifts of the diphosphate moiety of the protein‐bound FAD were similar in the proteins and were independent of the redox state. The chemical shifts were remarkably similar to those of ferredoxin−NADP+ reductase and, to a lesser degree, with those of NADPH−cytochrome P‐450 reductase.

The wild‐type human electron transfer apoprotein was reconstituted with [2,4a‐13C2]FAD, [4,10a‐13C2]FAD, or [U‐15N4]FAD. The reconstituted proteins were studied by 13C‐ and 15N‐NMR techniques in the oxidised and reduced states. The chemical shifts were compared with those of free flavin in aqueous solution or in chloroform, and those of flavoproteins published in the literature.

In the oxidised state, strong hydrogen bonds exist between residues of the apoprotein and C(2)O and N(5) of FAD. The N(1) atom is also hydrogen bonded and, as shown by X‐ray data, involves the C′(4)‐OH group of FAD. The sp2 hybridisation of N(10) is small compared to other flavoproteins.

In the reduced state, there are strong hydrogen bonds involving C(2)O and N(5) of FAD. The N(1) atom is ionised as observed also in other flavoproteins when investigated by NMR. The intramolecular hydrogen bond between the C′(4)‐OH group and the N(1) atom of FAD is maintained in the reduced state, suggesting an involvement in the stabilisation of a certain configuration of the diphosphate group of protein‐bound FAD in both redox states. The N(10) atom in the reduced protein is highly sp3 hybridised in comparison to those of other flavoproteins.
Nearest‐neighbor analysis of a Photosystem II complex from Marchantia polymorpha L. (liverwort), which contains reaction center and antenna proteins
Authors: Harrer Roswitha , Bassi Roberto , Testi Maria Grazia , Schäfer Christian
Published: March 2003

A new photosystem II preparation was isolated from Marchantia polymorpha thylakoids upon solubilization with dodecyl β‐
D‐maltoside and glycerol gradient ultracentrifugation. Its protein composition was analyzed, and all tested polypeptides from the core complex, the oxygen‐evolving enhancer and the light‐harvesting complex (LHC) could be detected. The only component severely depleted compared with the grana membrane preparation was the psbS gene product. This complex was subjected to chemical cross‐linking using the cleavable homobifunctional cross‐linker dithiobis(sulfosuccinimidylpropionate). The overall pattern of cross‐linking‐products was analyzed by diagonal electrophoresis, where the cross‐linking agent was cleaved by reduction of the disulfide bond between the first and second dimensions of the gel, followed by immunoblotting. Many cross‐linking products were characterized and these data used in order to identify protein masses revealed by electron microscopy [Boekema, E. J., Hankamer, B., Bald, D., Kruip, J., Nield, J., Boonstra, A. F., Barber, J. & Rögner, M. (1995) Proc. Natl Acad. Sci. USA 92, 175−179]. lt is concluded that the core proteins CP43 and CP47 are located at opposite sides of the D1‐D2‐cytochrome b559 complex. Minor CAB proteins were found to interact with core complex subunits (CP29, CP26) and LHCII (CP26), supporting the view that these proteins could interface the major LHCII with the reaction center.
Expression of collagenase/gelatinase activity from basement‐membrane fibronectin
Authors: Boudjennah Laziz , Dalet‐Fumeron Véronique , Pagano Maurice
Published: March 2003

We have isolated the collagenase/gelatinase activity of fibronectin from a bovine lens capsule hydrolysate, using heparin‐agarose, gelatin‐agarose, immunopurification with polyclonal antibodies directed against bovine plasma fibronectin, and immunopurification with a monoclonal antibody directed against the extra‐domain A of cellular fibronectin. The expression of collagenase/gelatinase activity by the purified fibronectin fragment was dependent on the incubation time at 37 +C and the addition of gelatin to the purified sample. Under these conditions, the purified fibronectin fragment exhibited collagenase/gelatinase activity, as measured by means of gelatin zymography and the intramolecularly quenched fluorogenic substrate of collagenases (7‐methoxycoumarin‐4‐yl)‐acetylprolylleucylglycylleucyl‐[3‐(2,4‐dinitrophenyl)‐ L‐2,3‐diaminopropionyl]‐alanylarginylamide. This activity was due to proteins of 47 kDa and 37 kDa, as indicated by the gelatin‐zymography pattern. When the processing was analyzed, by means of SDS/PAGE under reducing conditions, purified starting material of 66 kDa and 55 kDa was observed, and molecular masses of 45, 30 and 27 kDa were found for the processed samples. Under these conditions, the processing was more significant when a substrate, i.e the fluorogenic peptide or gelatin, was added to the processing mixture. An inhibition‐profile study showed a zinc‐dependent collagenase activity. Using the 45‐kDa chymotryptic fragment from human plasma fibronectin, which contains the collagen‐binding site, the same results were obtained. These results allow us to define a thiol‐dependent zinc metalloproteinase expressed after limited proteolysis of both basement membrane and plasma fibronectins. This proteinase contains a collagen‐binding domain, a zinc‐binding sequence, and a cysteine involved in catalysis. This enzyme is a member of the thimet family of zinc metalloproteinases.
Synthesis and release of ATP by soluble mitochondrial F1 in complex with its inhibitor protein during dimethylsulfoxide‐water transitions
Authors: Tuena de Gómez‐Puyou Marietta , Sandoval Francisca , García José J. , Gómez‐Puyou Armando
Published: March 2003

Soluble mitochondrial F1 and F1 in complex with the natural ATPase inhibitor protein (F1‐IP) catalyze the spontaneous synthesis of [γ‐32P]ATP from medium [32P]phosphate and enzyme‐bound ADP when incubated in media with dimethylsulfoxide (Me2SO); under these conditions, the synthesized [γ‐32P]ATP is not released into the media, it remains tightly bound to the enzymes [Gómez‐Puyou, A., Tuena de Gómez‐Puyou, M. & de Meis, L. (1986) Eur. J. Biochem. 159, 133−140]. Some of the characteristics of the synthesized [γ‐32P]ATP were studied in F1 and F1‐IP (ATPase activities of 70 and 1−3 μmol  min−1 mg−1, respectively). In Me2SO media, γ‐phosphate of synthesized ATP in F1 or F1‐IP exchanges with medium phosphate. From the rates of the exchange reaction, the half‐times for hydrolysis of the synthesized ATP in F1 and F1‐IP were calculated : 45 min and 58 min for F1 and F1‐IP, respectively. The course that synthesized [γ‐32P]ATP follows after dilution of the Me2SO synthetic mixture with aqueous buffer was determined. After dilution, the half‐life of synthesized ATP in F1 was less than 1 min. In F1‐IP, ATP was also hydrolyzed, but at significantly lower rates. In F1‐IP, dilution also produced release of the synthesized [γ‐32P]ATP. This was assayed by the accessibility of [γ‐32P]ATP to hexokinase. About 25 % of [γ‐32P]ATP synthesized in F1‐IP, but not in F1, was released into the media after dilution with aqueous buffer that contained 20 mM phosphate. Release of tightly bound ATP required the binding energy of phosphate and solvation of F1‐IP, however, the particular kinetics of F1‐IP were also central for medium ATP synthesis in the absence of electrochemical H+ gradients.
Structure and properties in solution of PsaD, an extrinsic polypeptide of photosystem I
Authors: Xia Zhicheng , Broadhurst R. William , Laue Ernest D. , Bryant Donald A. , Golbeck John H. , Bendall Derek S.
Published: March 2003

PsaD is a small, extrinsic polypeptide located on the stromal side (cytoplasmic side in cyanobacteria) of the photosystem I reaction centre complex. The gene from the cyanobacterium Nostoc sp. PCC 8009 was expressed in Escherichia coli and the structure of the recovered protein in solution investigated. Size‐exclusion chromatography, dynamic light scattering and measurement of 15N transverse relaxation times showed that the protein is a stable dimer in solution, whereas in the reaction centre complex it is a monomer. NMR experiments showed that the dimer is symmetrical and that there are at least two domains, one structured and the remainder unstructured. The structured domain contains a small amount of β‐sheet. Three‐dimensional heteronuclear NMR spectra of [13C, 15N]PsaD showed that the structured domain is associated with the central part of the sequence while the N‐ and C‐terminal regions are mobile. Evidence was obtained for a shift in equilibrium between two slightly different conformational states at about pH 6, and the protein was shown to bind to PsaE preferentially at neutral pH. Addition of trifluoroethanol was shown to induce the formation of a small amount of α‐helix, and the form present in 30 % trifluoroethanol appears to be more closely related to the in situ structure, which has been reported to contain one short helix in crystals [Schubert, W.‐D., Klukas, O., Krauss, N., Saenger, W., Fromme, P. & Witt, H. T. (1997) J. Mol. Biol. 272, 741−769]. The significance of these findings for the assembly of the complex is discussed.
The recombinant Klebsiella pneumoniae outer membrane protein OmpA has carrier properties for conjugated antigenic peptides
Authors: Haeuw Jean‐François , Rauly Isabelle , Zanna Laurence , Libon Christine , Andreoni Christine , Nguyen Thien Ngoc , Baussant Thierry , Bonnefoy Jean‐Yves , Beck Alain
Published: March 2003

Klebsiella pneumoniae OmpA, the 40‐kDa major protein of the outer membrane, was cloned and expressed in Escherichia coli. The recombinant protein was produced intracellularly in E. coli as inclusion bodies. Fusion of a short peptide to the N‐terminus of native P40 facilitated high‐level expression of the recombinant protein. Purified recombinant P40 was analyzed to verify purity and structural integrity. The molecular mass of purified recombinant P40 determined by electrospray mass spectrometry was 37 061 Da, in agreement with the theoretical mass deduced from the DNA sequence. Specific proliferation of recombinant‐P40‐primed murine lymph node cells in response to recombinant P40 stimulation in vitro indicated the presence of a T‐cell epitope on recombinant P40. The induction of high serum antibody titers to a synthetic peptide derived from the attachment protein G of the respiratory syncytial virus when chemically coupled to recombinant P40 indicated that the protein had potent carrier properties.
The ubiquityl‐calmodulin synthetase system from rabbit reticulocytes : isolation of the calmodulin‐binding second component and enzymatic properties
Authors: Majetschak Matthias , Laub Markus , Klocke Christoph , Steppuhn Johannes A. , Jennissen Herbert P.
Published: March 2003

Ubiquitin−calmodulin ligase (uCaM synthetase; EC6.3.2.21), which has been detected in all tissues so far examined, catalyzes the Ca2+‐dependent reversible synthesis of ubiquityl‐calmodulin which is not directed to degradation by the ATP‐dependent 26‐S protease [Laub, M. & Jennissen, H. P. (1997) Biochim. Biophys. Acta 1357, 173−191]. As has been shown in the preceding paper in this journal, the uCaM synthetase holosystem can be separated into two essential protein components : uCaM Syn‐F1, a ubiquitin‐binding protein belonging to the ubiquitin‐activating enzyme family (E1) and uCaM Syn‐F2 which bestows the reaction specificity leading to the covalent modification of calmodulin with ubiquitin. UCaM Syn‐F2, which binds to calmodulin−Sepharose in a Ca2+‐dependent manner, has been purified over 3500‐fold in seven steps from rabbit reticulocytes and has a native molecular mass of ≈620 kDa. It binds calmodulin with a Km of 5 μM and to uCaM Syn‐F1, i.e. ubiquitin‐activating enzyme (E1), with a Km of 3 nM. The maximal specific activity obtained in enriched uCaM Syn‐F2 is 6−8 pkat/mg. The pH optimum of uCaM synthetase lies at pH 8.5. In kinetic experiments the Km values for 125I‐ubiquitin and ATP/Mg2+ were determined to be 8 μM and 16 nM, respectively, for the uCaM synthetase holosystem. The existence of a third separable protein component of uCaM synthetase, as is the case in E1, E2, E3 systems, is very unlikely since affinity chromatography on calmodulin−Sepharose, two ion‐exchange chromatography steps and finally a gel‐filtration step failed to indicate any additional protein component essential for synthetase activity. We therefore propose a two‐component model for uCaM synthetase. This model is also supported by simple hyperbolic velocity curves in kinetic experiments based on the variation of these two components. The data suggests that uCaM Syn‐F2 is neither an E2 nor an E3 but evidently combines the properties of both, making the Ca2+‐dependent uCaM synthetase the member of a group of two‐component ubiquitin ligase systems.
Transport of amino acid aryl amides by the intestinal H+/peptide cotransport system, PEPT1
Authors: Börner Volker , Fei You‐Jun , Hartrodt Bianka , Ganapathy Vadivel , Leibach Frederick H. , Neubert Klaus , Brandsch Matthias
Published: March 2003

Transport of amino acid aryl amides by the intestinal H+/peptide symporter (PEPT1) was studied in Caco‐2 cells and in Xenopus laevis oocytes expressing human PEPT1. Several amino acid amides were able to inhibit the uptake of [14C]glycylsarcosine in Caco‐2 cells. Ala‐4‐nitroanilide (Ki = 0.08 mM), Phe‐4‐nitroanilide (Ki = 0.09 mM) and Ala‐4‐phenylanilide (Ki = 0.03 mM) were accepted as substrates with equal or higher affinity than natural Ala‐Xaa dipeptides. Ala‐anilide (Ki = 2.9 mM), Ala‐7‐amido‐4‐methylcoumarin (Ki = 0.2 mM), Ala‐4‐chloroanilide (Ki = 0.3 mM) and Ala‐4‐methylanilide (Ki = 0.3 mM) were also recognized by PEPT1 as substrates. In contrast, alanine, Ala‐amide, Phe‐amide, Ala‐methyl ester, Ala‐4‐nitrobenzyl ester and Ala‐methylamide were not recognized (Ki > 20 mM). In X. laevis oocytes, transport of Ala‐4‐nitroanilide, Ala‐7‐amido‐4‐methylcoumarin, Ala‐4‐methylanilide and Ala‐anilide was associated with transfer of positive charge and the currents were saturable with respect to substrate concentration (K0.5 values : 0.1, 0.2, 0.8 and 3.1 mM, respectively). The currents induced by Ala‐4‐methylanilide were saturable with respect to the substrate concentration and influenced by the membrane potential. The affinity of the transporter for Ala‐4‐methylanilide was also found to be influenced by the membrane potential. We conclude that the intestinal H+/peptide cotransport system PEPT1 accepts amino acid aryl amides as substrates.
Characterization of promoter regions involved in high expression of KlCYC1
Authors: Ramil Elvira , Freire‐Picos M. Angeles , Cerdán M. Esperanza
Published: March 2003

Functional analysis of the KlCYC1 promoter reveals that sequences located upstream to those already published [Freire‐Picos, M. A., Rodríguez‐Torres, A. M., Ramil, E., Cerdán, M. E., Breuning, K. D., Hollenberg, C. P. & Zitomer, R. S. (1993) Sequence of a cytochrome c from Kluyveromyces lactis and its upstream region, Yeast9, 201−204] and extending from positions −780 to −371 are important for maintaining high levels of expression, although this region contains both negative and positive elements. A consensus sequence for interaction with KlCpf1p is present at position −492, into the negative site, and specific protein binding to KlCpf1p has been demonstrated. Deletion of the sequences from positions −413 to −338 diminishes KlCYC1 transcription; protein binding to two sequences included in this activator region is detected and several points of evidence indicate that the complex observed is different from the Hap2/3/4/5p complex. Binding of KlCpf1p and the activator complex to the promoter is constitutive in different carbon sources. Although the promoter contains CCAAT boxes, directed mutagenesis has revealed that they are not related to the moderate de‐repression observed in glycerol media.
Modified glycosylation of cellobiohydrolase I from a high cellulase‐producing mutant strain of Trichoderma reesei
Authors: Harrison Mathew J. , Nouwens Amanda S. , Jardine Daniel R. , Zachara Natasha E. , Gooley Andrew A. , Nevalainen Helena , Packer Nicolle H.
Published: March 2003

Cellobiohydrolase I is an industrially important exocellulase secreted in high yields by the filamentous fungus Trichoderma reesei. The nature and effect of glycosylation of CBHI and other cellulolytic enzymes is largely unknown, although many other structural and mechanistic aspects of cellulolytic enzymes are well characterised. Using a combination of liquid chromatography, electrospray mass spectrometry, solid‐phase Edman degradation, and monosaccharide analysis we have identified every site of glycosylation of CBHI from a high cellulase‐producing mutant strain of T. reesei, ALKO2877, and characterised each site in terms of its modifying carbohydrate and site‐specific heterogeneity. The catalytic core domain comprises three N‐linked glycans which each consist of a single N‐acetylglucosamine residue. Within the glycopeptide linker domain, all eight threonines are variably glycosylated with between at least one, and up to three, mannose residues per site. All serines in this domain are at least partially glycosylated with a single mannose residue. This linker region has also been shown to be sulfated by a combination of ion chromatography and collision‐induced dissociation electrospray mass spectrometry. The sulfate is probably mannose‐linked. The biological significance of N‐linked single N‐acetylglucosamine in the catalytic core, and mannose sulfation in the linker region, is not known.
Studies on the Zn‐containing S14 ribosomal protein from Thermus thermophilus
Authors: Tsiboli Paraskevi , Triantafillidou Dimitra , Franceschi François , Choli‐Papadopoulou Theodora
Published: March 2003

The S14 ribosomal protein from the thermophilic organism Thermus thermophilus, which contains a zinc‐finger‐like motif, namely ‐C‐X2‐C‐X12‐C‐X2‐C‐ [Tsiboli, P. & Choli, T. (1995) Biol. Chem. Hoppe‐Seyler 376, 127−130], has been overproduced, purified and investigated for its zinc content. According to atomic absorption experiments, the protein contains zinc at a molar ratio of one. Denaturation experiments with simultaneous use of denaturing and chelating agents (guanidine hydrochloride and EDTA), as well as renaturation experiments, have shown both that Zn is strongly bound to the protein and with 1 : 1 Zn/protein stoicheiometry. These findings provide very strong evidence in support of the participation of the zinc‐finger motif and the Zn in the formation of a zinc‐finger domain.
Interferon‐γ variants with deletions in the AB surface loop
Authors: Waschütza Gero , Dengler Uwe , Villmann Carmen , Böttinger Heiner , Otto Bernd
Published: March 2003

The receptor‐binding AB loop of recombinant human interferon‐γ (IFN‐γ) has multiple contacts with the extracellular part of the IFN‐γ receptor α chain (IFN‐γRα). We explored the possible length of truncated AB loops and their conformations by molecular modelling. Deletions of two amino acids at the tip of the loop were tolerated in the model without van der Waals collisions of the AB loop with helix F. Based on these modelling results, two deletion mutants were constructed by overlap‐extension PCR mutagenesis : des‐(A23, D24)‐IFN‐γ and des‐(N25, G26)‐IFN‐γ. Both mutations were tolerated by the folding pattern of recombinant human IFN‐γ, as proved by CD spectroscopy. The stability of both mutants against cosolvent‐induced unfolding was equal to that of wild‐type IFN‐γ. In contrast to the biophysical similarities of wild‐type and mutant IFN‐γ proteins, the biological activities of both mutants dropped significantly. Antiviral activity and human leucocyte antigen (HLA)‐DR induction of des‐(N25, G26)‐IFN‐γ was 10 % that of wild‐type activity. des‐(A23, D24)‐IFN‐γ had only 1 % remaining activity. Receptor‐binding experiments confirmed that both deletions had a negative influence on the affinity of recombinant human IFN‐γ to its cellular receptor. We conclude from this combined molecular modelling and mutagenesis experiments, that the reduced flexibility of the truncated AB loop abrogates the possibility of the formation of a 310 helix in the receptor‐bound state as observed in the X‐ray structure of the IFN‐γRα−IFN‐γ complex.