bluuebunny/crossref_metadata_2025_split huggingface dataset:
Chemical and antigenic structure of the O‐polysaccharide of the lipopolysaccharides from two Acinetobacter haemolyticus strains differing only in the anomeric configuration of one glycosyl residue in their O‐antigens
Authors: Pantophlet Ralph , Haseley Simon R. , Vinogradov Evgeny V. , Brade Lore , Holst Otto , Brade Helmut 
Published: March 2003

In a previous study [Pantophlet, R., Brade, L., Dijkshoorn, L., and Brade, H. (1998) J. Clin. Microbiol.36, 1245–1250] the O‐polysaccharide of the lipopolysaccharides (LPS) from Acinetobacter haemolyticus strains 57 and 61 exhibited indistinguishable banding‐patterns following Western blot and immunostaining with homologous or heterologous rabbit antiserum. In this report, the molecular basis for the observed cross‐reactivity was elucidated, by determining the chemical structure of the polysaccharides by compositional analysis and NMR spectroscopy. The structures are:for strain 57, andfor strain 61 [GulpNAcA, 2‐acetamido‐2‐deoxy‐gulopyranosyluronic acid; ManpNAcA, 2‐acetamido‐2‐deoxy‐mannopyranosyluronic acid; QuipN4N, 2,4‐diamino‐2,4,6‐trideoxy‐glucopyranose; acyl (S)‐3‐hydroxybutyryl], thus, differing only in the anomeric configuration of the QuipN4N residue. The antigenic structures were determined by generating murine monoclonal antibodies, which were characterized by Western blot using LPS as antigen, by ELISA using LPS and de‐O‐acylated LPS as solid‐phase antigens, and by ELISA inhibition studies using LPS, polysaccharide, and de‐O‐acylated LPS as inhibitors. Of the four antibodies selected, two were specific for the respective LPS moieties and two were cross‐reactive. All antibodies were found to require the presence of the O‐acetyl group for reactivity.
A superoxide dismutase from the archaeon Sulfolobus solfataricus is an extracellular enzyme and prevents the deactivation by superoxide of cell‐bound proteins
Authors: Cannio Raffaele , D’Angelo Anna , Rossi Mosè , Bartolucci Simonetta 
Published: March 2003

An oxygen‐induced iron superoxide dismutase was found in the culture fluid of the thermoacidophilic crenarchaeon Sulfolobus solfataricus during growth on glucose‐rich media.This protein was also identified as being associated with the cell‐surface, with the amount of the released and cell‐bound protein fractions depending on the growth phase of the cells. The steady decrease in cell‐associated superoxide dismutase during continued growth correlated with the increase of free superoxide dismutase in the medium. Both enzyme fractions were purified to homogeneity and found to be active with different catalytic efficiency, with the released superoxide dismutase showing a fourfold lower specific activity.Characterization in comparison with the cytosolic superoxide dismutase revealed identical N‐terminal sequences, electrophoretic mobility, isoelectric point, and molecular mass for all three differently located enzymes.In order to clarify the physiological role of the cell‐associated superoxide dismutase, the prevention of cell‐bound protein deactivation by oxyradicals was also investigated. Glucose dehydrogenase, which was chosen as a model enzyme, was demonstrated to be located on the cell surface and to be inactivated by potassium superoxide by in vivo assays. The direct protective effect of superoxide dismutase on glucose dehydrogenase was demonstrated by in vitro assays on the free released enzyme.Similarly, the prevention of deactivation by potassium superoxide was also demonstrated for the integral membrane protein succinate dehydrogenase by intact cell assay.Superoxide dismutase added to cells was shown to moderately reduce the critical damaging peroxidation and hence play a major role in maintaining the integrity of the outer cell envelope components.
High expression of the human hepatocarcinoma‐intestine‐pancreas/pancreatic‐associated protein (HIPPAP) gene in the mammary gland of lactating transgenic mice
Authors: Christa Laurence , Pauloin Alain , Simon Marie‐Thérèse , Stinnakre Marie‐Georges , Fontaine Marie‐Louise , Delpal Serge , Ollivier‐Bousquet Michèle , Bréchot Christian , Devinoy Eve 
Published: March 2003

The human hepatocarcinoma‐intestine‐pancreas/pancreatic‐associated protein (HIPPAP) gene was previously identified because of its increased expression in primary liver cancers and during the acute phase of pancreatitis. In normal tissues, HIP/PAP is expressed both in endocrine and exocrine cells of the intestine and pancreas. HIP/PAP is a lactose binding C‐type lectin which acts as an adhesion molecule for rat hepatocytes.The aim of the work was to study the HIP/PAP secretory pathway and to produce high levels of HIP/PAP in the milk of lactating transgenic mice. In view of its lactose C‐type lectin properties, we have studied the consequences of the expression of HIP/PAP on mammary epithelial cells.In homozygous mice, production reached 11.2 mg·mL−1 of milk. High levels of soluble and pure HIP/PAP (18.6 mg) were purified from 29 mL of milk. The purified protein was sequenced and the N‐terminal amino acid of the mature HIP/PAP was identified as Glu27, thus localizing the site of cleavage of the signal peptide. The HIP/PAP transgene was only expressed in the mammary gland of lactating transgenic mice. HIP/PAP was detected by immunofluorescence in the whole gland, but labelling was heterogeneous between alveolar clusters, with strongly positive sparse cells. Using immuno electron microscopy, HIP/PAP was observed in all the compartments of the secretory pathway within the mammary epithelial cells.We provide evidence that HIP/PAP is secreted through the Golgi pathway. However, the number of distended Golgi saccules was increased when compared to that found in wild‐type mouse mammary cells. These modifications could be related to HIP/PAP C‐type lectin specific properties.
31P‐NMR and 13C‐NMR studies of mannose metabolism in Plesiomonas shigelloides
Authors: Rager Marie N. , Binet Marie R. B. , Ionescu Gabriel , Bouvet Odile M. M. 
Published: March 2003

The metabolism of mannose was examined in resting cells in vivo using 13C‐NMR and 31P‐NMR spectroscopy, in cell‐free extracts in vitro using 31P‐NMR spectroscopy, and by enzyme assays. Plesiomonas shigelloides was shown to transport mannose by a phosphoenolpyruvate‐dependent phosphotransferase system producing mannose 6‐phosphate. However, a toxic effect was observed when P. shigelloides was grown in the presence of mannose.Investigation of mannose metabolism using in vivo13C NMR showed mannose 6‐phosphate accumulation without further metabolism. In contrast, glucose was quickly metabolized under the same conditions to lactate, ethanol, acetate and succinate. Extracts of P. shigelloides exhibited no mannose‐6‐phosphate isomerase activity whereas the key enzyme of the Embden–Meyerhof pathway (6‐phosphofructokinase) was found. This result explains the mannose 6‐phosphate accumulation observed in cells grown on mannose. The levels of phosphoenolpyruvate and Pi were estimated by in vivo31P‐NMR spectroscopy. The intracellular concentrations of phosphoenolpyruvate and Pi were relatively constant in both starved cells and mannose‐metabolizing cells. In glucose‐metabolizing cells, the phosphoenolpyruvate concentration was lower, and about 80% of the Pi was used during the first 10 min. It thus appears that the toxic effect of mannose on growth is not due to energy depletion but probably to a toxic effect of mannose 6‐phosphate.
The iron–sulfur clusters in 2‐hydroxyglutaryl‐CoA dehydratase from Acidaminococcus fermentans
Authors: Hans Marcus , Buckel Wolfgang , Bill Eckhard 
Published: March 2003

The reversible dehydration of (R)‐2‐hydroxyglutaryl‐CoA to (E)‐glutaconyl‐CoA is catalysed by the combined action of two oxygen‐sensitive enzymes from Acidaminococcus fermentans, the homodimeric component A (2 × 27 kDa) and the heterodimeric component D (45 and 50 kDa). Component A was purified to homogeneity (specific activity 25–30 s−1) using streptavidin‐tag affinity chromatography. In the presence of 5 mm MgCl2 and 1 mm ADP or ATP, component A could be stabilized and stored for 4–5 days at 4 °C without loss of activity. The purification of component D from A. fermentans was also improved as indicated by the 1.5‐fold higher specific activity (15 s−1). The content of 1.0 riboflavin 5′‐phosphate (FMN) per heterodimer could be confirmed, whereas in contrast to an earlier report only trace amounts of riboflavin (< 0.1) could be detected. Each active component contains an oxygen sensitive diamagnetic [4Fe−4S]2+ cluster as revealed by UV‐visible, EPR and Mössbauer spectroscopy. Reduction of the [4Fe−4S]2+ cluster in component A with dithionite yields a paramagnetic [4Fe−4S]1+ cluster with the unusual electron spin ground state S = 3/2 as indicated by strong absorption type EPR signals at high g values, g = 4–6. Spin‐Hamiltonian simulations of the EPR spectra and of magnetic Mössbauer spectra were performed to determine the zero field splitting (ZFS) parameters of the cluster and the 57Fe hyperfine interaction parameters. The electronic properties of the [4Fe−4S]2+,1+ clusters of component A are similar to those of the nitrogenase iron protein in which a [4Fe−4S]2+ cluster bridges the two subunits of the homodimeric protein. Under air component A looses its activity within seconds due to irreversible degradation of its [4Fe−4S]2+ cluster to a [2Fe−2S]2+ cluster. The [4Fe−4S]2+ cluster of component D could not be reduced to a [4Fe−4S]1+ cluster, even with excess of Ti(III)citrate or dithionite. Exposure to oxic conditions slowly converts the diamagnetic [4Fe−4S]2+ cluster of component D to a paramagnetic [3Fe−4S]+ cluster concomitant with loss of activity (30% within 24 h at 4 °C).
Transport and metabolism of agmatine in rat hepatocyte cultures
Authors: Cabella Claudia , Gardini Giulia , Corpillo Davide , Testore Giovanni , Bedino Stefano , Solinas Sandro P. , Cravanzola Carlo , Vargiu Cristina , Grillo Maria A. , Colombatto Sebastiano 
Published: March 2003

Rat hepatocytes in culture take up [14C]‐agmatine by both a high‐affinity transport system [KM = 0.03 mm; Vmax = 30 pmol·min·(mg protein)−1] and a low‐affinity system. The high‐affinity system also transports putrescine, but not cationic amino acids such as arginine, and the polyamines spermidine and spermine. The rate of agmatine uptake is increased in cells deprived of polyamines with difluoromethylornithine. Of the agmatine taken up, 10% is transformed into polyamines and 50% is transformed into 4‐guanidinobutyrate, as demonstrated by HPLC and MS. Inhibition by aminoguanidine and pargyline shows that this is due to diamine oxidase and an aldehyde dehydrogenase. 14C‐4‐aminobutyrate is also accumulated in the presence of an inhibitor of 4‐aminobutyrate transaminase.
Protein‐glutaminase from Chryseobacterium proteolyticum, an enzyme that deamidates glutaminyl residues in proteins
Authors: Yamaguchi Shotaro , Jeenes David J. , Archer David B. 
Published: March 2003

A novel protein‐deamidating enzyme was purified to homogeneity from Chryseobacterium proteolyticum and the gene encoding it was cloned. The enzyme is a monomer with a pI of 10.0, a measured Mr of ≈ 20 000 and a calculated Mr of 19 860. Extensive comparison with Streptoverticillium transglutaminase showed that the protein‐deamidating enzyme lacked transglutaminase activity in terms of hydroxamate‐formation between benzyloxycarbonyl‐Gln‐Gly and hydroxylamine, or monodansylcadaverine incorporation into casein. The enzyme deamidated the two glutaminyl residues in the oxidized insulin A chain and deamidated both casein and the oxidized insulin B chain with higher catalytic efficiencies (kcat/Km) than with short peptides. The enzyme was active against several proteins, including insoluble wheat gluten, but did not deamidate asparaginyl residues in peptides, free glutamine or other amides. The enzyme was therefore named protein‐glutaminase (EC 3.5.1). The gene encoding the protein was cloned and, when expressed in Escherichia coli, the protein product had protein‐glutaminase activity and cross‐reacted with antiserum raised against the purified enzyme. The protein‐glutaminase was shown to be expressed as a prepro‐protein with a putative signal peptide of 21 amino acids and a pro‐sequence of 114 amino acids. The amino‐acid sequence had no obvious homology to any published sequence and is therefore a novel protein‐glutaminase.
Regulation of expression of terminal oxidases in Paracoccus denitrificans
Authors: Otten Marijke F. , Stork David M. , Reijnders Willem N. M. , Westerhoff Hans V. , Van Spanning Rob J. M. 
Published: March 2003

In order to study the induction of terminal oxidases in Paracoccus denitrificans, their promoters were fused to the lacZ reporter gene and analysed in the wild‐type strain, in an FnrP‐negative mutant, in a cytochrome bc1‐negative mutant, and in six single or double oxidase‐negative mutant strains. The strains were grown under aerobic, semi‐aerobic, and denitrifying conditions. The oxygen‐sensing transcriptional‐regulatory protein FnrP negatively regulated the activity of the qox promoter, which controls expression of the ba3‐type quinol oxidase, while it positively regulated the activity of the cco promoter, which controls expression of the cbb3‐type cytochrome c oxidase. The ctaDII and ctaC promoters, which control the expression of the aa3‐type cytochrome c oxidase subunits I and II, respectively, were not regulated by FnrP. The activities of the latter two promoters, however, did decrease with decreasing oxygen concentrations in the growth medium, suggesting that an additional oxygen‐sensing mechanism exists that regulates transcription of ctaDII and ctaC. Apparently, the intracellular oxygen concentration (as sensed by FnrP) was not the only signal to which the oxidase promoters responded. At given extracellular oxygen status, both the qox and the cco promoters responded to mutations in terminal oxidase genes, whereas the ctaDII and ctaC promoters did not. The change of electron distribution through the respiratory network, resulting from elimination of one or more oxidase genes, may have changed intracellular signals that affect the activities of the qox and cco promoters. On the other hand, the re‐routing of electron distribution in the respiratory mutants hardly affected the oxygen consumption rate as compared to that of the wild‐type. This suggests that the mutants adapted their respiratory network in such a way that they were able to consume oxygen at a rate similar to that of the wild‐type strain.
Pfnek‐1, a NIMA‐related kinase from the human malaria parasite Plasmodium falciparum
Authors: Dorin Dominique , Le Roch Karine , Sallicandro Pina , Alano Pietro , Parzy Daniel , Poullet Patrick , Meijer Laurent , Doerig Christian 
Published: March 2003

We have cloned Pfnek‐1, a gene encoding a novel protein kinase from the human malaria parasite Plasmodium falciparum. This enzyme displays maximal homology to the never‐in‐mitosis/Aspergillus (NIMA)/NIMA‐like kinase (Nek) family of protein kinases, whose members are involved in eukaryotic cell division processes. Similar to other P. falciparum protein kinases and many enzymes of the NIMA/Nek family, Pfnek‐1 possesses a large C‐terminal extension in addition to the catalytic domain. Bacterially expressed recombinant Pfnek‐1 protein is able to autophosphorylate and phosphorylate a panel of protein substrates with a specificity that is similar to that displayed by other members of the NIMA/Nek family. However, the FXXT motif usually found in NIMA/Nek protein kinases is substituted in Pfnek‐1 by a SMAHS motif, which is reminiscent of a MAP/ERK kinase (MEK) activation site. Mutational analysis indicates that only one of the serine residues in this motif is essential for Pfnek‐1 kinase activity in vitro. We show (a) that recombinant Pfnek‐1 is able to specifically phosphorylate Pfmap‐2, an atypical P. falciparum MAPK homologue, in vitro, and (b) that coincubation of Pfnek‐1 and Pfmap‐2 results in a synergistic increase in exogenous substrate labelling. This suggests that Pfnek‐1 may be involved in the modulation of MAPK pathway output in malaria parasites. Finally, we demonstrate that recombinant Pfnek‐1 can be used in inhibition assays to monitor the effect of kinase inhibitors, which opens the way to the screening of chemical libraries aimed at identifying potential new antimalarials.
Photoactivatable α‐conotoxins reveal contacts with all subunits as well as antagonist‐induced rearrangements in the Torpedo californica acetylcholine receptor
Authors: Kasheverov Igor , Rozhkova Anna , Zhmak Maxim , Utkin Yuri , Ivanov Vadim , Tsetlin Victor I 
Published: March 2003

Azidobenzoyl (AzBz) and benzoylbenzoyl (BzBz) derivatives of α‐conotoxin MI and l‐benzoylphenylalanine (Bpa) analogs of α‐conotoxin GI were synthesized. All these compounds, similarly to native α‐conotoxins, completely displaced the radioiodinated MI or GI from the membrane‐bound nicotinic acetylcholine receptor (AChR) of Torpedo californica. However, the GI(Bpa11) analog was considerably less potent than GI in competing with radioiodinated α‐bungarotoxin (αBgt). Irradiation of iodinated AzBz derivatives bound to AChR resulted in labeling of all AChR subunits. The BzBz and Bpa derivatives gave lower levels of specific cross‐linking but considerable labeling at additional sites that was enhanced, rather than suppressed, by an excess of native α‐conotoxins or αBgt. Both equilibrium binding of benzophenone‐derivatized α‐conotoxins and their cross‐linking could be totally abolished by physostigmine. The results obtained demonstrate that (a) specific binding sites for α‐conotoxins and αBgt are overlapping but not identical, (b) each of the AChR subunits can be labeled with photoactivatable α‐conotoxins and (c) enhancement of benzophenone‐derivatized α‐conotoxins cross‐linking at additional (physostigmine‐related) sites by αBgt or GI indicates that these antagonists induce structural alterations in the AChR outside their binding sites.
Concurrent maltodextrin and cellodextrin synthesis by Fibrobacter succinogenes S85 as identified by 2D NMR spectroscopy
Authors: Matulova Maria , Delort Anne‐Marie , Nouaille Régis , Gaudet Geneviève , Forano Evelyne 
Published: March 2003

1D and 2D NMR experiments were used to analyse the synthesis of various metabolites by resting cells of Fibrobacter succinogenes S85 when incubated with [1‐13C]glucose, in both extracellular and cellular media. Besides the expected glycogen, succinate, acetate, glucose‐1‐P and glucose‐6‐P, maltodextrins and cellodextrins were detected. Maltodextrins were excreted into the external medium. They were found to have linear structures with a maximum degree of polymerization (DP) of about 6 or 7 units. Cellodextrins were located in the cells (cytoplasm and/or periplasm), and their DP was ≤ 4. Both labelled (1‐13C and 6‐13C) and unlabelled maltodextrins and cellodextrins were detected, showing the contribution of carbohydrate cycling in F. succinogenes, including the reversal of glycolysis and the futile cycle of glycogen. The mechanisms of these oligosaccharide syntheses are discussed.
Structural basis for the inhibitory efficacy of efavirenz (DMP‐266), MSC194 and PNU142721 towards the HIV‐1 RT K103N mutant
Authors: Lindberg Jimmy , Sigurðsson Snævar , Löwgren Seved , O. Andersson Hans , Sahlberg Christer , Noréen Rolf , Fridborg Kerstin , Zhang Hong , Unge Torsten 
Published: March 2003

The K103N substitution is a frequently observed HIV‐1 RT mutation in patients who do not respond to combination‐therapy. The drugs Efavirenz, MSC194 and PNU142721 belong to the recent generation of NNRTIs characterized by an improved resistance profile to the most common single point mutations within HIV‐1 RT, including the K103N mutation.In the present study we present structural observations from Efavirenz in complex with wild‐type protein and the K103N mutant and PNU142721 and MSC194 in complex with the K103N mutant. The structures unanimously indicate that the K103N substitution induces only minor positional adjustments of the three inhibitors and the residues lining the binding pocket. Thus, compared to the corresponding wild‐type structures, these inhibitors bind to the mutant in a conservative mode rather than through major rearrangements. The structures implicate that the reduced inhibitory efficacy should be attributed to the changes in the chemical environment in the vicinity of the substituted N103 residue. This is supported by changes in hydrophobic and electrostatic interactions to the inhibitors between wild‐type and K103N mutant complexes. These potent inhibitors accommodate to the K103N mutation by forming new interactions to the N103 side chain. Our results are consistent with the proposal by Hsiou et al. [Hsiou, Y., Ding, J., Das, K., Clark, A.D. Jr, Boyer, P.L., Lewi, P., Janssen, P.A., Kleim, J.P., Rosner, M., Hughes, S.H. & Arnold, E. (2001) J. Mol. Biol.309, 437–445] that inhibitors with good activity against the K103N mutant would be expected to have favorable interactions with the mutant asparagines side chain, thereby compensating for resistance caused by stabilization of the mutant enzyme due to a hydrogen‐bond network involving the N103 and Y188 side chains.
The Mechanism of Replacement of Red Squirrels by Grey Squirrels: A Test of the Interference Competition Hypothesis
Authors: Wauters Luc A. , Gurnell John 
Published: March 2003

Introduced American grey squirrels have replaced native red squirrels in most of the range currently occupied in Britain and northern Italy. The mechanisms of the replacement are not yet fully understood. We restated the commonly cited Interference Competition Hypothesis (ICH) that grey squirrels interfere with the behaviour of red squirrels in three possible ways: 1. by direct aggressive interactions; 2. by interrupting red squirrel mating‐chases; or 3. by forcing red squirrels to actively avoid areas intensively used by grey squirrels. We compared the activity pattern, behaviour and reproductive performance of red squirrels in two study areas in northern Italy, one with only red squirrels (control area C1), the other with both species (experimental area E1). The following predictions were tested: 1. the total time spent in both intraspecific and interspecific interactions by red squirrels increases in the experimental area; 2. most interspecific interactions are aggressive, with grey squirrels being the dominant species; 3. the proportion of breeding female red squirrels that are unsuccessful at weaning offspring increases in area E1; 4. grey squirrels take part and interfere with red squirrel mating‐chases, and thereby decrease the reproductive output of red squirrel females; 5. the activity pattern of red squirrels in the mixed‐species area is shifted with respect to that in the control area to the hours of the day during which grey squirrels show little activity; and 6. red squirrels will shift their home range (or at least their core‐area) when grey squirrel densities increase to avoid interspecific core‐area overlap.Our results supported only the first prediction of the ICH: they failed to support all the other predictions. Moreover, the increase in the percentage of active time red squirrels spent interacting with other squirrels in the experimental study area was very small (only 1–2 min/day). Red squirrels did not avoid the woodland patches most intensively used by grey squirrels and the interspecific core‐area overlap was similar to red squirrel intraspecific core‐area overlap. This suggested that red squirrels avoided spatial overlap with grey squirrels in a similar manner as with conspecifics and that an increase in grey squirrel numbers will augment the intensity of resource competition. We therefore conclude that our results do not lend support to the Interference Competition Hypothesis and that interference competition by grey squirrels cannot explain the large‐scale replacement of red by grey squirrels that has occurred in Britain and in Piedmont.