bluuebunny/crossref_metadata_2025_split huggingface dataset:
Study of polynucleotide conformation by resolution‐enhanced ultraviolet spectroscopy
Authors: GARRIGA Pere , GARCIA‐QUINTANA David , MANYOSA Joan 
Published: March 2005

Self‐deconvolution and the fourth derivative of ultraviolet absorption spectra have been used to study stacked single‐stranded and double‐helix structures of different cytosine‐containing polynucleotides for the first time. These compounds were studied under different solution conditions (pH and organic solvents) and at low temperatures. The red shift of the lower band (B2u band plus possibly some n→π* transition) of the absorption spectra in the cytosine‐containing polynucleotides and the appearance of new peaks in the deconvoluted and derivative spectra in the 280–310 nm region are attributed mainly to cytosine‐cytosine stacking interactions. In particular, the fourth‐derivative peaks at wavelengths higher than 290 nm can be associated to coupling of electronic transitions of cytosine bases. The nature of the electronic transitions producing the absorption bands which are resolved in the aforementioned fourth‐derivative peaks is discussed.It is concluded that the resolution‐enhancement techniques used in this work, i.e. self‐deconvolution and fourth derivative, complement each other and are useful methods to study structural changes of single‐stranded and double‐stranded polynucleotides allowing, at the same time, more information to be obtained about specific stacking interactions than classical absorption spectrophotometry.
Inhibition of chloroplast ATPase by the K+ channel blocker α‐dendrotoxin
Authors: SCHULENBURG Peter , SCHWARZ Martin , WAGNER Richard 
Published: March 2005

Possible structural and functional similarities between the channel part, CF0, of chloroplast ATPase (CF0CF1) and ion channels permeable to monovalent cations were investigated using high‐affinity toxins mainly targeted against voltage‐sensitive K+ channels. In particular, the effect of the K+‐channel blocker α‐dendrotoxin and the crude scorpion venom of Leiurus quinquestriatus hebraeus (LQ venom) on ATP synthesis in thylakoid membranes and in CF0CF1‐containing liposomes was characterised.α‐dendrotoxin (Ki∼ 5.05 μM) and the LQ venom (Ki∼ 1.55 μg/ml) specifically inhibited ATP synthesis in thylakoid membranes and in CF0CF1‐containing liposomes. Our results show that α‐dendrotoxin and peptides of the LQ venom with an apparent molecular mass of about 4.0 kDa, probably isoforms of charybdotoxin, specifically bind to CF0CF1. This binding was reversible and induced a high leak conductance for H+ through CF0. The Ca2+‐dependent ATPase activity of the isolated soluble part of CF0CF1 (CF1) was completely inhibited by 1 μM α‐dendrotoxin, while the crude LQ venom, at concentrations up to 10 μg/ml, had no affect on ATPase activity. The concentration dependence of the inhibition by α‐dendrotoxin indicates that approximately 2 mol α‐dendrotoxin bind/mol CF0CF1 and 1 mol α‐dendrotoxin/mol CF1. Known inhibitors of H+‐flow‐through CF0 acted in the presence of α‐dendrotoxin synergistically. Dicyclohexylcarbodiimide and venturicidin, in contrast to their known effect of blocking H+‐flow‐through CF0, increased the leak conductance through CF0 in the presence of α‐dendrotoxin drastically. This uncoupling effect indicates that their normal mode of blocking is a secondary effect. Binding of the inhibitors to their respective sites apparently does not affect the proton pathway in CF0, but induces a conformation which closes the channel part for H+.Protein sequence comparison between the known binding site of charybdotoxin in the shaker K+ channel from Drosophila [MacKinnon, R. & Heginbotham, L. (1990) Neuron 5, 767–771] and the choroplast ATPase showed that subunit III reveals a significant similarity (64%) in parts of its sequence (Gln28–Leu53) to the helix 5 and helix 6 (S5‐S6) linker region (Ala413–Cys462; the charybdotoxin‐binding site) of the shaker K+ channel. According to secondary‐structure predictions, the homologous sequences in subunit III and the shaker K+ channel represent putative hydrophilic loops connecting two transmembrane α‐helices. Apparently the shaker K+ channel and subunit III share significant topological features in these hydrophilic loops which may be part of the respective channel entrance.
The human rhabdomyosarcoma cell line A204 lays down a highly insoluble matrix composed mainly of α1 type‐XI and α2 type‐V collagen chains
Authors: KLEMAN Jean‐Philippe , HARTMANN Daniel J. , RAMIREZ Francesco , van der REST Michel 
Published: March 2005

The biosynthesis of collagen by the A204 cell line was examined using polyclonal antibodies raised against collagen type V and type XI. The study of the pepsin‐digested collagen showed that it is composed mainly of α1(XI) and α2(V) collagen chains in an apparent 2:1 ratio, suggesting the formation of heterotypic molecules [α1(XI)]2α2(V). The existence of this chain stoichiometry was further demonstrated by immunoprecipitation of the molecule with an antibody recognizing α2(V) but not α1(XI) collagen chains.Electron microscopy analyses of 24‐h cultures showed that this matrix is composed of thin fibrils, that can be decorated with immunogold‐labelled anti‐(type‐V collagen) IgG, but not with anti‐(type‐XI collagen) IgG. The collagen matrix laid down by A204 cells is highly insoluble. In the presence of β‐aminopropionitrile, an inhibitor of lysyl oxidase, only a small proportion of intact collagen could be extracted without proteolytic treatment. Immunoblotting of intact medium collagen from cultures performed in the presence of β‐aminopropionitrile showed four distinct bands with each antibody. The migration of the bands, stained with anti‐(type‐V collagen) IgG, had apparent molecular masses of 127, 149, 161 and 198 kDa (compared to globular standards) while the bands stained with anti‐(type‐XI collagen) IgG had apparent masses of 145, 182, 207 and 225 kDa.These data indicate that type‐V and type‐XI collagen chains can assemble in heterotypic isoforms. In this system, the synthesized isoforms are able to aggregate into a highly cohesive matrix and they undergo a proteolytic processing closely similar to that of other fibrillar collagens.
Subunits VIIa,b,c of human cytochrome c oxidase
Authors: VAN KUILENBURG André B. P. , VAN BEEUMEN Jozef J. , VAN DER MEER Niek M. , MUIJSERS Anton O. 
Published: March 2005

The N‐terminal amino acid sequences and the electrophoretic mobilities of the subunits VIIa, VIIb and VIIc of cytochrome c oxidase purified from human heart were investigated and compared with those from human skeletal muscle and from bovine heart. In purified human heart cytochrome c oxidase, both so‐called ‘heart‐type’ and ‘liver‐type’ isoforms of subunit VIIa were found. The first 30 residues of the N‐terminal amino acid sequences of these ‘heart‐type’ and ‘liver‐type’ subunits VIIa showed nine differences. The two isoforms of subunit VIIa in human heart were present in almost equal amounts, in contrast to the situation in skeletal muscle, where the ‘heart‐type’ subunit VIIa was predominant. Therefore, our results imply that in human heart a cytochrome c oxidase isoform pattern is present that differs from that found in skeletal muscle.Subunits VIIb and VIIc purified from human heart oxidase proved to be very similar to their bovine heart counterparts. Our direct demonstration of the presence of subunit VIIb, the sequence of which has only recently been identified in the bovine heart enzyme, suggests that human cytochrome c oxidase also contains 13 subunits. We found no evidence for the presence of different isoforms of subunit VIIc in cytochrome c oxidase from human heart and skeletal muscle.We observed clear differencs in the electrophoretic mobility of the subunits VIIa,b,c between bovine and human cytochrome c oxidase. On Tricine/glycerol/SDS/polyacrylamide gels the ‘heart‐type’ and ‘liver‐type’ subunits VIIa present in human heart cytochrome c oxidase migrated with almost the same electrophoretic mobility. Subunit VIIb migrated only slightly faster than subunit VIIa, whereas VIIc proved to have the highest electrophoretic mobility on Tricine/SDS/glycerol/polyacrylamide gels. Our findings may have implications for the elucidation of certair tissue‐specific cytochrome c oxidase deficiencies in man.
Inhibition of proton‐translocating transhydrogenase from photosynthetic bacteria by N,N′‐dicyclohexylcarbodiimide
Authors: PALMER Tracy , WILLIAMS Ross , COTTON Nick P. J. , THOMAS Christopher M. , JACKSON J. Baz 
Published: March 2005

The effects of N,N′‐dicyclohexylcarbodiimide [(cHxN)2C] on the proton‐translocating enzyme, NAD(P) H+‐transhydrogenase (H+‐Thase), from two species of phototrophic bacteria have been investigated. The polypeptides of H+‐Thase from Rhodobacter capsulatus are membrane‐associated, requiring detergent to maintain solubility. The enzyme from Rhodospirillum rubrum, however, has a water soluble polypeptide (Ths) and a membrane‐associated component (Thm) which, separately, have no activity but which can be fully reconstituted to give functional complex.Two observations suggest that (cHxN)2C inhibited H+‐Thase from both species by modification either close to or at the NADP(H)‐binding site on the enzyme: (a) the presence of NADP+ or NADPH caused increased inhibition by (cHxN)2C and (b) after treatment of the purified enzyme from Rb. capsulatus with (cHxN)2C, the release of NADP+ became rate‐limiting, as evidenced by a stimulated rate of NADPH‐dependent reduction of acetylpyridine adenine dinucleotide by NADH. Experiments in which Ths and Thm from R. rubrum were separately treated with (cHxN)2C then reconstituted with the complementary, untreated component revealed that the NADP(H)‐enhanced modification by (cHxN)2C was confined to Thm.In contrast to some experiments with mitochondrial H+‐Thase [Wakabayashi, S. & Hatefi, Y. (1987) Biochem. Int. 15, 667–675], there was no protective effect of either NAD+ or NADH on the inhibition by (cHxN)2C of enzyme from photosynthetic bacteria. However, amino acid sequence analysis of proteolytic fragments of Ths revealed that the NAD(H)‐protectable, (cHxN)2C‐reactive glutamate residue in mitochondrial H+‐Thase might be replaced by glutamine in R. rubrum.
Interaction of myelin basic protein with artificial membranes
Authors: ter BEEST Martin B. A. , HOEKSTRA Dick 
Published: March 2005

The interaction of myelin basic protein (MBP) with large unilamellar vesicles, composed of phosphatidylserine (PtdSer), phosphatidylserine/phosphatidylcholine (PtdSer/Ole2GroPCho) and phosphatidylcholine/cholesterol (Ole2GroPCho/cholesterol) was examined. Binding of MBP to the bilayers as well as the kinetics of this process were determined by a resonance energy transfer procedure. The ability of the protein to aggregate the vesicles subsequently was monitored continuously by absorbance measurements. The interaction was further characterized by determining the ability of MBP to induce membrane perturbations, as reflected by release of aqueous vesicle contents, and lipid mixing.The results demonstrate that Ole2GroPCho inhibits, while PtdSer and cholesterol strongly facilitate MBP‐induced membrane aggregation. Furthermore, binding of MBP to vesicles and the subsequent aggregation event are separate processes, i. e. the extent of binding does not necessarily retlect the aggregation susceptibility. Overall, aggregation appears to the rate‐limiting step. Interaction of MBP with PtdSer bilayers results in a limited degree of lipid mixing, which is accompanied by extensive release of vesicle contents. For all other compositions, no lipid mixing occurs, while cholesterol effectively prevents release of vesicle contents. pH‐dependent experiments indicate distinct mechanisms to be operative in MBP‐induced aggregation of PtdSer and Ole2GroPCho/cholesterol bilayers. At neutral pH, protein‐protein interactions appear relevant, while at acidic pH intervesicular bridges, established by monomers that may cause aggregation of PtdSer vesicles, but not of Ole2GroPCho/cholesterol vesicles. The observation that divalent cations reverse MBP‐induced vesicle aggregation may have physiological relevance.
Overexpression of trypanosomal triosephosphate isomerase in Escherichia coli and characterisation of a dimer‐interface mutant
Authors: BORCHERT Torben V. , PRATT Kathryn , ZEELEN Johan Ph. , CALLENS Mia , NOBLE Martin E. M. , OPPERDOES Fred R. , MICHELS Paul A. M. , WIERENGA Rik K. 
Published: March 2005

In this paper, the successful expression of trypanosomal triosephosphate isomerase (TIM) from Trypanosoma brucei brucei to high yield in Escherichia coli, using a T7‐polymerase‐based expression system, is described. Overexpressed trypanosomal TIM is fully active. The measured physicochemical properties of this recombinant TIM and TIM purified from trypanosomes are indistinguishable. Crystals of recombinant TIM have been grown in the presence of 2.4 M ammonium sulphate under the same conditions as for trypanosomally expressed TIM. The recombinant TIM crystal structure has been refined at 0.23 nm resolution; no differences were detected between this structure and the original crystal structure.A TIM mutant was made in which a unique dimer‐interface histidine residue (His47) was changed into an asparagine. This variant ([H47N]TIM) could be expressed and purified to homogeneity by a procedure which was somewhat different from the purification of recombinant wild‐type TIM. It is shown that the [H47N]TIM dimer is considerably less stable than wild‐type trypanosomal TIM. The catalytic activity of [H47N]TIM is concentration dependent. The dilution‐dependent inactivation is reversile. His47 is involved in a water‐mediated hydrogen bond with Asp385 of the other subunit. The lower stability of the [H47N]TIM dimer implies that this water‐mediated hydrogen bond is important for the stability of the TIM dimer.
Specificity of the binding domain of glucoamylase 1
Authors: BELSHAW Nigel J. , WILLIAMSON Gary 
Published: March 2005

Glucoamylase 1 from Aspergillus niger hydrolyses granular starch at an increased rate due to the presence of a C‐terminal starch‐binding domain. This domain was isolated and shown to bind to the malto‐oligosaccharides Glc2 to Glc11 with a stoichiometry of 1 mol ligand/mol protein. The affinity for these ligands increased with increasing degree of polymerisation until Glc9, above which no further increase was observed. We suggest that this indicates that for maximum affinity the substrate should be able to form a helical conformation, which mimics the conformation of amylose in granular starch. We propose a model of how the complex between the malto‐oligosaccharides and the binding domain is formed and indicate how this affects the differences in binding modes for soluble and insoluble substrates.Glucono‐1,5‐lactone interacts with the binding domain at a different site to the malto‐oligosaccharides allowing the formation of a ternary complex between the binding domain, a malto‐oligosaccharide and glucono‐1,5‐lactone. The binding domain also binds to linear α‐1,6‐linked glucose digosaccharides (dextran), but with much lower affinity than for α‐1,4‐linked glucose. This ligand appears to interact with the binding domain at both binding sites, i. e. at the site to which the malto‐oligosaccharides bind and also at the site to which glucono‐1,5‐lactone binds. The relevance of the results to the mechanism of action of other polysaccharide‐hydrolysing enzymes containing both a catalytic and a binding domain is discussed.
Role of sugar chains in the in‐vitro activity of recombinant human interleukin 5
Authors: KODAMA Shiho , TSUJIMOTO Masafumi , TSURUOKA Nobuo , SUGO Tsukasa , ENDO Tamao , KOBATA Akira 
Published: March 2005

The structures of O‐linked sugar chains of recombinant human interleukin 5 (rhIL‐5) produced by Chinese hamster ovary cells were determined employing high‐performance anion‐exchange column chromatography under high pH conditions. The core sequence was elucidated to be Galβ1→3‐GalNAc by its susceptibility to endo‐α‐N‐acetylgalactosaminidase and sialic acid linkages were determined using sialidase of different specificities.To investigate the role of sugar chains in the in‐vitro activity of rhIL‐5, it was digested with various glycosidases. While removal of N‐linked sugar chains resulted in 2.8‐fold increase of the activity, de‐O‐glycosylated rhIL‐5 showed 10‐fold higher activity than intact rhIL‐5, suggesting that the presence of O‐linked sugar chains suppresses the activity more effectively than that of N‐linked chains. While incubation of de‐N‐glycosylated rhIL‐5 at 70°C for 30 min decreased the activity, intact and de‐O‐glycosylated rhIL‐5 lost little activity, suggesting that N‐linked sugar chains contributed to the thermostability of the molecule.
Cloning of the fourth functional gene for Protein Phosphatase 1 in Drosophila melanogaster from its chromosomal location
Authors: DOMBRÁDI Viktor , MANN David J. , SAUNDERS Robert D. C. , COHEN Patricia T. W. 
Published: March 2005

Complementary DNA encoding a catalytic subunit of protein phosphatase 1, PP1 87B, hybridises at four positions (87B, 9C, 13C and 96A) to Drosophila melanogaster polytene chromosomes, three of which are known to be expressed [Dombrádi, V., Axton, J. M., Brewis, N. D., Da Cruz e Silva, E. F., Alphey, L. & Cohen, P. T. W. (1990) Eur. J. Biochem. 194, 739–745]. The fourth gene has been isolated by screening a genomic library of cosmid clones, representing division 13 of the X‐chromosome of D. melanogaster, with a PP1 87B probe. This library was constructed as part of the Drosophila genome mapping project [Sidén‐Kiamos, I., Saunders, R. D. C., Spanos, L., Majerus, T., Trenear, J., Savakis, C., Louis, C., Glover, D. M., Ashburner, M. & Kafatos, F. C. (1990) Nucleic Acids Res. 18, 6261–6270]. The 5′ non‐coding region of the isolated gene hybridised to cytological position 13C1–2. By combining reverse transcription and the polymerase chain reaction, the gene was shown to be expressed at a very low level.The PP1 13C gene encodes a protein of 302 amino acids with a predicted molecular mass of 34.5 kDa. It shows 85–94% amino acid identity to the other three protein phosphatase 1 catalytic subunits (PP1 87B, PP1 96A and PP1 9C) described previously, being most closely related to the isoform PP1 87B, which is involved in the control of chromosome separation at cell division and the regulation of chromosome condensation at interphase.
Phosphorylation and activation of the arachidonate‐mobilizing phospholipase A2 in macrophages in response to bacteria
Authors: SVENSSON Ulf , HOUWELING Martin , HOLST Elisabet , SUNDLER Roger 
Published: March 2005

The role of potential target enzymes in the protein‐kinase‐C‐independent eicosanoid response triggered by certain bacteria in murine peritoneal macrophages [Svensson, U., Holst, E. & Sundler, R. (1991) Eur. J. Biochem. 202, 699–705] has been investigated. The eicosanoid response was found to be due to an increase in the mobilization of arachidonate rather than to inhibition of arachidonate esterification or activation of the cyclooxygenase pathway and to be accompanied by a persistent increase in the activity of the arachidonate‐mobilizing phospholipase A2 (PLA2−85). Also, down‐regulation of protein‐kinase C by prolonged treatment with 4β‐phorbol 12‐myristate 13‐acetate did not reduce the bacterial activation of PLA2‐85. The increase in activity of PLA2−85, like the increase in eicosanoid formation, showed a lag period of approximately 10 min.Furthermore, exposure of 32P‐labeled macrophages to either bacteria (Gardnerella vaginalis) or the protein‐phosphatase inhibitor okadaic acid caused an increase in the phosphorylation of PLA2−85. Okadaic acid (0.5 μM), which itself caused arachidonate mobilization and activation of PLA2−85 after a lag period of approximately 45 min, greatly promoted the response to bacteria even at earlier time points. This study provides strong evidence that the eicosanoid response to bacteria in macrophages occurs via a protein‐kinase‐C‐independent activation of PLA2−85 and that this activation is due to an increase in the phosphorylation of the enzyme.
Stimulatory and inhibitory guanine‐nucleotide‐binding regulatory protein involvement in stimulation of arachidonic‐acid release by N‐formyl‐methionyl‐leucyl‐phenylalanine and platelet‐activating factor from guinea‐pig alveolar macrophages
Authors: LEVISTRE Romuald , MASLIAH Joëlle , BEREZIAT Gilbert 
Published: March 2005

The involvement of guanine‐nucleotide‐binding regulatory proteins (G proteins) in the regulation of arachidonic‐acid release induced by N‐formyl‐methionyl‐leucyl‐phenylalanine (fMet‐Leu‐Phe) or platelet‐activating factor (PAF) was examined in guinea‐pig alveolar macrophages. We report that maximal release of arachidonic acid in permeabilized cells requires the simultaneous addition of the agonist (fMet‐Leu‐Phe or PAF) and of GTP (or GTP[S]). Prior treatment of cells with increasing concentrations of pertussis toxin induces a parallel decrease of arachidonic‐acid release and of the labeling of a 40‐kDa protein in membranes incubated with [32P]NAD and pertussis toxin. fMet‐Leu‐Phe, but not PAF, allows the ADP‐ribosylation of a 40‐KDa protein by cholera toxin in the presence of Mg2+. This effect is prevented by guanyl nucleotides and by prior treatment with pertussis toxin. The 40‐kDa protein ADP‐ribosylated seems to be αi1 and/or αi2. Stimulation of GTPase activity by fMet‐Leu‐Phe and PAF has the same amplitude and is completely inhibited by pertussis toxin, but only in part by cholera toxin. Prior treatment of alveolar macrophages with cholera toxin, which ADP‐ribosylates Gs, inhibits PAF‐stimulated and fMet‐Leu‐Phe‐stimulated arachidonic‐acid release to the same extent, via a cAMP‐protein‐kinase‐A cascade. The decreased responsiveness of alveolar macrophages previously treated with cholera toxin to fMet‐Leu‐Phe and PAF is associated with a strong increase of in‐vitro [32P]NAD labeling of Gi proteins either by pertussis or by cholera toxin. This effect is mimicked by prior treatment of the cells with dibutyryl cAMP and okadaic acid, a protein‐phosphatase inhibitor, suggesting the involvement of protein‐kinase A in this process.In conclusion, our results demonstrate that fMet‐Leu‐Phe and PAF receptors interact differently with Gi1/2 proteins in guinea‐pig alveolar macrophages. Gi1/2 proteins are a possible target of the cross‐regulation of arachidonic‐acid release by a Gs‐mediated pathway.