bluuebunny/crossref_metadata_2025 huggingface dataset:
Local cytokines induce differential expression of matrix metalloproteinases but not their tissue inhibitors in human endometrial fibroblasts
Authors: Singer Christian F. , Marbaix Etienne , Lemoine Pascale , Courtoy Pierre J. , Eeckhout Yves
Published: March 2003

The endometrium is the only human tissue to undergo cyclic breakdown and regeneration. This physiological alternation renders it an advantageous system for studying tissue remodelling. Our previous observations indicate that menstrual endometrial breakdown is initiated by matrix metalloproteinases (MMPs), which are controlled overall by ovarian steroids but are also locally regulated by cytokines. We have therefore compared the effect of several endometrial cytokines on the gene expression of eight MMPs and their tissue inhibitors (TIMP)‐1, ‐2 and ‐3, in primary cultures of human endometrial fibroblasts. Three categories of gene expression were identified: (a) MMP‐13, ‐15 and ‐16 mRNAs were not detected despite stimulation by various cytokines; (b) MMP‐2 and ‐14 as well as TIMP‐1, ‐2 and ‐3 mRNAs were constitutively expressed but not markedly affected by the six cytokines tested; (c) mRNAs for MMP‐1, ‐9 and ‐11 were selectively induced by specific cytokines: insulin‐like growth factor‐II, epidermal growth factor (EGF), platelet derived growth factor (PDGF)‐BB and interleukin (IL)‐6 stimulated MMP‐11 expression; MMP‐1 was induced by EGF, PDGF‐BB, tumour necrosis factor (TNF)α and IL‐1α, which also exerted additive effects. In contrast with MMP‐1 and MMP‐11 gene expression, which was sustained for 48 h, MMP‐9 mRNA was quickly induced by TNFα, but disappeared within 12 h despite continuing stimulation. These results show that several cytokines are able to induce the selective expression of MMPs in cultured human endometrial fibroblasts and are thus good candidates for involvement in local triggering of menstrual tissue breakdown.
Distinct promoters control transmembrane and cytosolic protein tyrosine phosphatase ε expression during macrophage differentiation
Authors: Tanuma Nobuhiro , Nakamura Koji , Kikuchi Kunimi
Published: March 2003

We have recently isolated two cDNAs encoding two forms of transmembrane and cytosolic protein tyrosine phosphatase ε (PTPε). In this study, the 5′ end of the rat PTPε gene was isolated and characterized. Transmembrane PTPε (PTPεM) and cytosolic PTPε (PTPεC) were encoded by a single gene. 5′ RACE analysis and RNase protection assay showed that the mRNA of each PTPε isoform was transcribed from different promoters. The putative promoter regions of two alternative first exons lacked a TATA box, but contained potential recognition sites for several transcription factors. Reverse transcription PCR analysis revealed that PTPεC mRNA was up‐regulated during interleukin 6‐induced differentiation of murine leukemia M1 cells, whereas PTPεM mRNA was down‐regulated. With the use of luciferase as a reporter gene, the promoter activities of the 5′‐flanking regions were examined during phorbol myristate acetate‐induced differentiation of HL‐60 cells. In the differentiated HL‐60 cells, the activity of the PTPεC promoter, but not that of PTPεM, was dramatically elevated. Furthermore, we found that PTPεC mRNA is highly expressed in mouse peritoneal macrophages and enhanced during activation by lipopolysaccharide. These results suggest that the different promoters control expression of PTPε isoforms during the differentiation and/or activation of macrophages.
Kinetic study on the dimer–tetramer interconvertion of glycogen  phosphorylase a
Authors: Wang Zhi‐Xin
Published: March 2003

Kinetic theory of dissociating enzyme systems has been applied to a study of the dimer–tetramer interconversion of glycogen phosphorylase a. All kinetic constants for the dissociating‐associating reaction of phosphorylase a have been determined. The results indicate that (a) the presence of glucose‐1‐phosphate has no influence on either the rate of dissociation or the rate of association, and hence does not shift the dimer–tetramer equilibrium of phosphorylase a; (b) the binding of glycogen to the enzyme decreases the association rate of the dimer to form the tetramer, but has no effect on the dissociation rate of the tetramer; (c) both the dimeric and tetrameric form of phosphorylase a can bind glycogen, but the tetrameric form has a lower affinity for glycogen and is catalytically inactive
Oxidative polymerization of ribonuclease A by lignin peroxidase from Phanerochaete chrysosporium
Authors: Sheng Dawei , Gold Michael H.
Published: March 2003

The mechanism of lignin peroxidase (LiP) was examined using bovine pancreatic ribonuclease A (RNase) as a polymeric lignin model substrate. SDS/PAGE analysis demonstrates that an RNase dimer is the major product of the LiP‐catalyzed oxidation of this protein. Fluorescence spectroscopy and amino acid analyses indicate that RNase dimer formation is due to the LiP‐catalyzed oxidation of Tyr residues to Tyr radicals, followed by intermolecular radical coupling. The LiP‐catalyzed polymerization of RNase is strictly dependent on the presence of veratryl alcohol (VA). In the presence of 100 µm H2O2, relatively low concentrations of RNase and VA, together but not individually, can protect LiP from H2O2 inactivation. The presence of RNase strongly inhibits VA oxidation to veratraldehyde by LiP; whereas the presence of VA does not inhibit RNase oxidation by LiP. Stopped‐flow and rapid‐scan spectroscopy demonstrate that the reduction of LiP compound I (LiPI) to the native enzyme by RNase occurs via two single‐electron steps. At pH 3.0, the reduction of LiPI by RNase obeys second‐order kinetics with a rate constant of 4.7 × 104m–1·s–1, compared to the second‐order VA oxidation rate constant of 3.7 × 105 m–1·s–1. The reduction of LiP compound II (LiPII) by RNase also follows second‐order kinetics with a rate constant of 1.1 × 104 m–1·s–1, compared to the first‐order rate constant for LiPII reduction by VA. When the reductions of LiPI and LiPII are conducted in the presence of both VA and RNase, the rate constants are essentially identical to those obtained with VA alone. These results suggest that VA is oxidized by LiP to its cation radical which, while still in its binding site, oxidizes RNase.
Sequence, heterologous expression and functional characterization of tryparedoxin1 from Crithidia fasciculata
Authors: Guerrero Sergio A. , Flohé Leopold , Kalisz Henryk M. , Montemartini Marisa , Nogoceke Everson , Hecht Hans‐Jürgen , Steinert Peter , Singh Mahavir
Published: March 2003

Tryparedoxin (TXN) has recently been discovered as a constituent of the complex peroxidase system in the trypanosomatid Crithidia fasciculata[Nogoceke et al. (1997) Biol. Chem.378, 827–836] where it catalyzes the reduction of a peroxiredoxin‐type peroxidase by trypanothione. Here we report on the full‐length DNA sequence of the TXN previously isolated from C. fasciculata (TXN1). The deduced amino acid sequence comprises 147 residues and matches with all the peptide sequences of fragments obtained from TXN1. It shares a characteristic sequence motif YFSAxWCPPCR with some thioredoxin‐related proteins of unknown function. This motif is homologous with the CXXC motif, which characterizes the thioredoxin superfamily of proteins and is known to catalyze disulfide reductions. Sequence conservations between TXNs and the typical thioredoxins are restricted to the intimate environment of the CXXC motif and three more remote residues presumed to contribute to the folding pattern of the thioredoxin‐type proteins. The TXNs thus form a distinct molecular clade within the thioredoxin superfamily. TXN1 was expressed in Escherichia coli BL21(DE3)pLysS as a C‐terminally extended and His‐tagged protein, isolated by chelate chromatography and characterized functionally. The recombinant product exhibited a kinetic pattern identical with, and kinetic parameters similar to those of the authentic enzyme in the trypanothione/peroxiredoxin oxidoreductase assay. The recombinant TXN1 can therefore be considered a valuable tool for the screening of specific inhibitors as potential trypanocidal agents.
Probing the role of C‐1 ester group in Naja naja phospholipase  A2–phospholipid interactions using butanetriol‐containing  phosphatidylcholine analogues
Authors: Puri Vishwajeet , Arora Ashish , Gupta Chhitar M.
Published: March 2003

To understand the role of the ester moiety of the sn‐1 acyl chain in phospholipase A2–glycerophospholipid interactions, we introduced an additional methylene residue between the glycerol C1 and C2 carbon atoms of phosphatidylcholines, and then studied the kinetics of hydrolysis and the binding of such butanetriol‐containing phospholipids with Naja naja phospholipase A2. Hydrolysis was monitored by using phospholipids containing a NBD‐labelled sn‐2 acyl chain and binding was ascertained by measuring the protein tryptophan fluorescence. The hydrolysis of butanetriol‐containing phospholipids was invariably slower than that of the glycerol‐containing phospholipids. In addition, the enzyme binding with the substrate was markedly decreased upon replacing the glycerol residue with the 1,3,4‐butanetriol moiety in phosphatidylcholines. These results have been interpreted to suggest that the sn‐1 ester group in glycerophospholipids could play an important role in phospholipase A2–phospholipid interactions.
Comparisons of genomic structures and chromosomal locations of the mouse aldose reductase and aldose reductase‐like genes
Authors: Ho Horace T. B. , Jenkins Nancy A. , Copeland Neal G. , Gilbert Debra J. , Winkles Jeffrey A. , Louie Helena W. Y. , Lee Fuk K. , Chung Stephen S. M. , Chung Sookja K.
Published: March 2003

Aldose reductase (AR), best known as the first enzyme in the polyol pathway of sugar metabolism, has been implicated in a wide variety of physiological functions and in the etiology of diabetic complications. We have determined the structures and chromosomal locations of the mouse AR gene (Aldor1) and of two genes highly homologous to Aldor1: the fibroblast growth factor regulated protein gene (Fgfrp) and the androgen regulated vas deferens protein gene (Avdp). The number of introns and their locations in the mouse Aldor1 gene are identical to those of rat and human AR genes and also to those of Fgfrp and Avdp. Mouse Aldor1 gene was found to be located near the Cald1 (Caldesmon) and Ptn (Pleiotropin) loci at the proximal end of chromosome 6. The closely related genes Fgfrp and Avdp were also mapped in this region of the chromosome, suggesting that these three genes may have arisen by a gene duplication event.
Molecular cloning, cell localization and binding affinity to DNA replication proteins of the p36/LACK protective antigen from Leishmania infantum
Authors: Gonzalez‐Aseguinolaza Gloria , Taladriz Soraya , Marquet Alberto , Larraga Vicente
Published: March 2003

The p36/LACK antigen from Leishmania, an analogue of the receptor for activated protein kinase C (PKC), induces high levels of protection against parasite infection in the BALB/c mouse model. This protection is more than twice as high as that elicited by major parasite antigens such as soluble Leishmania antigen or the main surface protease gp63. We have cloned and purified p36/LACK from Leishmania infantum, the causative agent of visceral leishmaniasis in Europe. This protein belongs to the large family of WD 40 repeat proteins confined to eukaryotes and involved in numerous regulatory functions. Differential solubilization and immunofluorescence experiments indicate that p36/LACK is present close to the kinetoplast disc in the cell cytoplasm, probably bound to multiprotein complexes but not to membrane structures. These complexes probably also include cytoplasm PKC isoforms. The use of a genetically‐encoded peptide library indicates that p36/LACK binds sequences present in several proteins involved in DNA replication and RNA synthesis. The recognition and binding sequences present in vacuolar proteins and at the β‐chain of major histocompatability complex (MHC) class II suggest the involvement of this regulatory protein in the early mechanisms triggering the protective immune response of the host against the parasite infection.
Origin of the integrin‐mediated signal transduction
Authors: Wimmer Wolfram , Perovic Sanja , Kruse Michael , Schröder Heinz C. , Krasko Anatoli , Batel Renato , Müller Werner E. G.
Published: March 2003

Sponges (phylum Porifera) represent the phylogenetically oldest metazoan animals. Recently, from the marine sponge Geodia cydonium a first cDNA encoding a putative integrin receptor molecule was isolated. In the present study basic functional experiments have been conducted to test the hypothesis that in sponges integrin polypeptides also function as adhesion molecules and as outside‐in signaling molecules. The sponge Suberites domuncula has been used for the experiments because from this sponge only has a cell culture been established. Here we report that aggregation factor (AF)‐mediated cell‐cell adhesion is blocked by the RGDS peptide which is known to interact with β integrin. Both RGDS and AF were found to stimulate DNA synthesis within 24 h. The β subunit of the integrin receptor was cloned from S. domuncula; the estimated 91‐kDa molecule comprises the characteristic signatures. Evolutionary conservation of the β integrin was assessed by comparison with corresponding β integrin subunits from evolutionary higher metazoan taxa. Addition of RGDS or of AF to isolated cells of S. domuncula causes a rapid (within 1–2 min) increase in the intracellular Ca2+ concentration which is further augmented in the presence of Ca2+. Furthermore, incubation of the cells with RGDS or AF causes an activation of the GTP‐binding protein Ras. In addition it is shown that after a prolonged incubation of the cells with RGDS and AF the expression of the genes coding for Ras and for calmodulin is upregulated. These results suggest that the integrin receptor functions in the sponge system not only as adhesion molecule but also as a molecule involved in outside‐in signaling.
Bacillus subtilis chorismate mutase is partially diffusion‐controlled
Authors: Mattei Patrizio , Kast Peter , Hilvert Donald
Published: March 2003

The effect of viscosogens on the enzyme‐catalyzed rearrangement of chorismate to prephenate has been studied. The steady‐state parameters kcat and kcat/Km for the monofunctional chorismate mutase from Bacillus subtilis (BsCM) decreased significantly with increasing concentrations of glycerol, whereas the ‘sluggish’ BsCM mutants C75A and C75S were insensitive to changes in microviscosity. The latter results rule out extraneous interactions of the viscosogen as an explanation for the effects observed with the wild‐type enzyme. Additional control experiments show that neither viscosogen‐induced shifts in the pH‐dependence of the enzyme‐catalyzed reaction nor small perturbations of the conformational equilibrium of chorismate can account for the observed effects. Instead, BsCM appears to be limited by substrate binding and product release at low and high substrate concentrations, respectively. Analysis of the kinetic data indicates that diffusive transition states are between 30 and 40% rate‐determining in these concentration regimes; the chemical step must contribute to the remaining kinetic barrier. The relatively low value of the ‘on’ rates for chorismate and prephenate (≈2 × 106m−1·s−1) probably reflects the need for a rare conformation of the enzyme, the ligand, or both for successful binding. Interestingly, the chorismate mutase domain of the bifunctional chorismate mutase‐prephenate dehydratase from Escherichia coli, which has steady‐state kinetic parameters comparable to those of BsCM but has a much less accessible active site, is insensitive to changes in viscosity and the reaction it catalyses is not diffusion‐controlled.
Equilibrium and transient intermediates in folding of human macrophage migration inhibitory factor
Authors: Žerovnik Eva , Janjić Vojko , Francky Andrej , Mozetič‐Francky Bojana
Published: March 2003

Acid, guanidinium‐Cl and urea denaturations of recombinant human macrophage migration inhibitory factor (MIF) were measured using CD and fluorimetry. The acid‐induced denaturation was followed by CD at 200, 222, and 278 nm and by tryptophan fluorescence. All four probes revealed an acid‐denatured state below pH 3 which resembled a typical molten globule. The pH transition is not two‐state as the CD data at 222 nm deviated from all other probes. Urea and guanidinium‐Cl denaturations (pH 7, 25 °C) both gave an apparent ΔGUappH2Oof 31 ± 3 kJ·mol−1when extrapolated to zero denaturant concentration. However, denaturation transitions recorded by fluorescence (at the same protein concentration) occurred at lower urea or guanidinium‐Cl concentrations, consistent with an intermediate in the course of MIF denaturation. CD at 222 nm was not very sensitive to protein concentration (in 10‐fold range) even though size‐exclusion chromatogryphy (SEC) revealed a dimer–monomer dissociation prior to MIF unfolding. Refolding experiments were performed starting from acid, guanidinium‐Cl and urea‐denatured states. The kinetics were multiphasic with at least two folding intermediates. The intrinsic rate constant of the main folding phase was 5.0 ± 0.5 s−1(36.6 °C, pH 7) and its energy of activation 155 ± 12 kJ·mol−1.
Detection of membrane‐bound cytokinin‐binding proteins in Arabidopsis thaliana cells
Authors: Brault Mathias , Caiveau Olivier , Pédron Jacques , Maldiney Régis , Sotta Bruno , Miginiac Emile
Published: March 2003

In order to isolate cytokinin‐binding proteins (CBPs), we have developed new affinity probes constituted of a cytokinin such as zeatin riboside ([9R]Z) conjugated to a carrier protein. These probes were used for detecting CBPs in an ELISA procedure. The efficiency of the cytokinin conjugate in detecting CBPs was controlled with protein model: proteins having an affinity for cytokinin such as the monoclonal anti‐[9R]Z antibodies did bind the cytokinin conjugate whereas proteins unable to bind cytokinin such as bovine serum albumin did not. Using these new affinity probes, we showed that CBPs are present in the membrane fraction of in vitro cultured Arabidopsis thaliana cells. The nature of the protein at the detected binding sites was demonstrated by submitting the microsomal proteins to a proteolytic treatment, which was found to eradicate the binding. Free biologically active cytokinins or monoclonal anti‐[9R]Z antibodies inhibited the binding, thus showing the specificity of the interaction. The detected CBPs were partially solubilized from the membranes with potassium chloride, indicating their peripheral membrane location. The separation by anion exchange chromatography of solubilized microsomal proteins revealed the existence of two different CBPs. They were present at higher levels in cells during the exponential growth phase.
Construction and characterization of a functional mutant of Synechocystis 6803 harbouring a eukaryotic PSII‐H subunit
Authors: Chiaramonte Simona , Giacometti Giorgio M. , Bergantino Elisabetta
Published: March 2003

A Synechocystis 6803 mutant carrying a chimaeric photosystem II (PSII), in which the Zea mays PsbH subunit (7.7 kDa calculated molecular mass) replaces the cyanobacterial copy (7.0 kDa), was constructed. With the exception of the N‐terminal 12 amino acid extension, which has a phosphorylatable threonine, the eukaryotic polypeptide is 78% homologous to its bacterial counterpart. Biochemical characterization of this mutant shows that it expresses the engineered gene correctly and is competent for photoautotrophic growth. Fluorescence analysis and oxygen evolution measurements in the presence of exogenous acceptors indicate that the observed phenotype results from a chimaeric PSII rather than from the absence of function associated with PsbH, suggesting that the heterologous protein is assembled into a functional PSII. Inhibition of oxygen evolution by herbicides belonging to different classes shows that the sensitivity of the mutant PSII is changed only towards phenolic compounds. This result indicates slight conformational modification of the QB/herbicide binding pocket of the D1 polypeptide caused by the bulky PsbH protein in the mutant, and also suggests close structural interaction of the D1 and PsbH subunits in the topological arrangement of PSII.
The structure of the carbohydrate backbone of the core‐lipid A region of the lipopolysaccharide from a clinical isolate of Yersinia enterocolitica O:9
Authors: Müller‐Loennies Sven , Rund Sabine , Ervelä Elise , Skurnik Mikael , Holst Otto
Published: March 2003

Yersinia enterocolitica O:9 strain Ruokola/71‐c‐ΦR1‐37‐R possesses mainly rough‐type lipopolysaccaride (LPS) and smaller amounts of S‐form LPS. Structural analysis of the former is reported here. After deacylation of the LPS, the phosphorylated carbohydrate backbone of the inner core‐lipid A region could be isolated by using high‐performance anion‐exchange chromatography. Its structure was determined by means of compositional and methylation analyses and 1H‐, 13C‐, and 31P‐NMR spectroscopy as:

in which l‐α‐d‐Hep is lglycero‐α‐dmanno‐heptopyranose, d‐α‐d‐Hep is dglycero‐α‐dmanno‐heptopyranose, and Kdo is 3‐deoxy‐dmanno‐oct‐2‐ulopyranosonic acid. All hexoses are pyranoses.
The structural and functional role of lysine residues in the binding domain of cytochrome c in the electron transfer to cytochrome c oxidase
Authors: Döpner Susanne , Hildebrandt Peter , Rosell Federico I. , Mauk A. Grant , von Walter Matthias , Buse Gerhard , Soulimane Tewfik
Published: March 2003

The interactions of yeast iso‐1 cytochrome c with bovine cytochrome c oxidase were studied using cytochrome c variants in which lysines of the binding domain were substituted by alanines. Resonance Raman spectra of the fully oxidized complexes of both proteins reveal structural changes of both the heme c and the hemes a and a3. The structural changes in cytochrome c are the same as those observed upon binding to phospholipid vesicles where the bound protein exists in two conformers, B1 and B2. Whereas the structure of B1 is the same as that of the unbound cytochrome c, the formation of B2 is associated with substantial alterations of the heme pocket. In cytochrome c oxidase, the structural changes in both hemes refer to more subtle perturbations of the immediate protein environment and may be a result of a conformational equilibrium involving two states. These changes are qualitatively different to those observed for cytochrome c oxidase upon poly‐l‐lysine binding. The resonance Raman spectra of the various cytochrome c/cytochrome c oxidase complexes were analyzed quantitatively. The spectroscopic studies were paralleled by steady‐state kinetic measurements of the same protein combinations. The results of the spectra analysis and the kinetic studies were used to determine the stability of the complexes and the conformational equilibria B2/B1 for all cytochrome c variants. The complex stability decreases in the order: wild‐type WT > J72K > K79A > K73A > K87A > J72A > K86A > K73A/K79A (where J is the natural trimethyl lysine). This order is not exhibited by the conformational equilibria. The electrostatic control of state B2 formation does not depend on individual intermolecular salt bridges, but on the charge distribution in a specific region of the front surface of cytochrome c that is defined by the lysyl residues at positions 72, 73 and 79. On the other hand, the conformational changes in cytochrome c oxidase were found to be independent of the identity of the bound cytochrome c variant. The maximum rate constants determined from steady‐state kinetic measurements could be related to the conformational equilibria of the bound cytochrome c using a simple model that assumes that the conformational transitions are faster than product formation. Within this model, the data analysis leads to the conclusion that the interprotein electron transfer rate constant is around two times higher in state B2 than in B1. These results can be interpreted in terms of an increase of the driving force in state B2 as a result of the large negative shift of the reduction potential.
In the Nicotiana sylvestris CMSII mutant, a recombination‐mediated change 5′ to the first exon of the mitochondrial nad1 gene is associated with lack of the NADH:ubiquinone oxidoreductase (complex I) NAD1 subunit
Authors: Gutierres Sophie , Combettes Bruno , De Paepe Rosine , Mirande Marc , Lelandais Christine , Vedel Fernand , Chétrit Philippe
Published: March 2003

We previously reported that the Nicotiana sylvestris CMSII mutant mitochondrial DNA carried a large deletion. Several expressed sequences, most of which are duplicated, and the unique copy of the nad7 gene encoding the NAD7 subunit of the NADH:ubiquinone oxidoreductase complex (complex I) are found in the deletion. Here, we show that the orf87‐nad3‐nad1/A cotranscription unit transcribed from a unique promoter element in the wild‐type, is disrupted in CMSII. Nad3, orf87 and the promoter element are part of the deleted sequence, whilst the nad1/A sequence is present and transcribed from a new promoter brought by the recombination event, as indicated by Northern and primer extension experiments. However, Western analyses of mitochondrial protein fractions and of complex I purified using anti‐NAD9 affinity columns, revealed that NAD1 is lacking in CMSII mitochondria. Our results suggest that translation of nad1 transcripts rather than transcription itself could be altered in the mutant. Consequences of lack of this submit belonging the membrane arm of complex I and thought to contain the ubiquinone‐binding site, are discussed.
Transcriptionally active and inactive mouse β‐globin gene loci are repaired at similar rates after ultraviolet irradiation
Authors: Chakalova Lyubomira , Russev George
Published: March 2003

It has been demonstrated, by Northern blot and in situ hybridization, that the mouse erythroleukaemia cell line F4N‐Sofia constitutively expresses the β‐globin genes. The recently developed quantitative assay for DNA repair has been used to study the overall repair rate in the β‐globin gene domain in this cell line after ultraviolet irradiation and to compare it with the repair rate of the same chromatin domain in mouse Ehrlich ascites tumour cells which do not express the β‐globin genes. The results showed that in both cases the 5′‐end of the domain was repaired preferentially and that the repair rates in the two cell lines were very similar despite the different transcription state of the genes.
The stromal protein large subunit of ribulose‐1,5‐bisphosphate carboxylase is translated by membrane‐bound ribosomes
Authors: Mühlbauer Stefan Klaus , Eichacker Lutz Andreas
Published: March 2003

Translation of the large subunit of ribulose‐1,5‐bisphosphate carboxylase (LSU) was investigated by labeling of isolated barley plastids with [35S]‐methionine. In both chloroplasts and etioplasts, labeling of LSU was severely impaired if plastid membranes were removed from the reaction mixtures. Removal of membrane‐bound polysomes with high salt or puromycin greatly decreased translation of LSU. Pulse‐labeled chloroplast membranes were shown to release LSU if chased with unlabeled methionine in the presence of stroma. Immunoprecipitation detected higher amounts of labeled LSU translation intermediates associated with the membrane fraction than in the soluble fraction. We therefore conclude that, in plastids, membrane‐bound polysomes are required not only for translation of membrane‐intrinsic proteins but also for translation of a soluble protein.
T4 RNA ligase catalyzes the synthesis of dinucleoside polyphosphates
Authors: Atencia Eva Ana , Madrid Olga , Günther Sillero María A. , Sillero Antonio
Published: March 2003

T4 RNA ligase has been shown to synthesize nucleoside and dinucleoside 5′‐polyphosphates by displacement of the AMP from the E‐AMP complex with polyphosphates and nucleoside diphosphates and triphosphates. Displacement of the AMP by tripolyphosphate (P3) was concentration dependent, as measured by SDS/PAGE. When the enzyme was incubated in the presence of 0.02 mm[α‐32P] ATP, synthesis of labeled Ap4A was observed: ATP was acting as both donor (Km, µm) and acceptor (Km, mm) of AMP from the enzyme. Whereas, as previously known, ATP or dATP (but not other nucleotides) were able to form the E‐AMP complex, the specificity of a compound to be acceptor of AMP from the E‐AMP complex was very broad, and with Km values between 1 and 2 mm. In the presence of a low concentration (0.02 mm) of [α‐32P] ATP (enough to form the E‐AMP complex, but only marginally enough to form Ap4A) and 4 mm of the indicated nucleotides or P3, the relative rate of synthesis of the following radioactive (di)nucleotides was observed: Ap4X (from XTP, 100); Ap4dG (from dGTP, 74); Ap4G (from GTP, 49); Ap4dC (from dCTP, 23); Ap4C (from CTP, 9); Ap3A (from ADP, 5); Ap4ddA, (from ddATP, 1); p4A (from P3, 200). The enzyme also synthesized efficiently Ap3A in the presence of 1 mm ATP and 2 mm ADP. The following T4 RNA ligase donors were inhibitors of the synthesis of Ap4G: pCp > pAp > pA2′p.
Accidental cell death and generation of reactive oxygen intermediates in human lymphocytes induced by thionins from Viscum album L.
Authors: Büssing Arndt , Stein Gerburg M. , Wagner Manfred , Wagner Barbara , Schaller Gerhard , Pfüller Uwe , Schietzel Michael
Published: March 2003

The cytotoxic mechanisms of thionins from Viscum album L., the viscotoxins, were investigated in human granulocytes and lymphocytes. The time course of viscotoxin effects indicate accidental cell death, i.e. membrane permeabilization, degradation of cytoplasm and chromatin, swelling of mitochondria with loss of their cristae, and generation of reactive oxygen intermediates within 1–2 h, followed by secondary apoptosis‐associated events. The viscotoxin homologue purothionin from whole‐wheat flour and viscotoxin B, however, did not induce cell death in cultured lymphocytes. Cytotoxicity of cationic and amphipathic viscotoxin was prevented only by cleavage of its disulphide bridges.
Properties and molecular cloning of Ca2+/H+ antiporter in the vacuolar membrane of mung bean
Authors: Ueoka‐Nakanishi Hanayo , Nakanishi Yoichi , Tanaka Yoshiyuki , Maeshima Masayoshi
Published: March 2003

Kinetic and molecular properties of the Ca2+/H+ antiporter in the vacuolar membrane of mung bean hypocotyls were examined and compared with Ca2+‐ATPase. Ca2+ transport activities of both transporters were assayed separately by the filtration method using vacuolar membrane vesicles and 45Ca2+. Ca2+ uptake in the presence of ATP and bafilomycin A1, namely Ca2+‐ATPase, showed a relatively low Vmax (6 nmol·min−1·mg−1 protein) and a low Km for Ca2+. The Ca2+/H+ antiporter activity driven by H+‐pyrophosphatase showed a high Vmax (25 nmol·min−1·mg−1) and a relatively high Km for Ca2+. The cDNA for mung bean Ca2+/H+ antiporter (VCAX1) codes for a 444 amino‐acid polypeptide. Two peptide‐specific antibodies of the antiporter clearly reacted with a 42‐kDa protein from vacuolar membranes and a cell lysate from a Escherichia coli transformant in which VCAX1 was expressed. These observations directly demonstrate that a low‐affinity, high‐capacity Ca2+/H+ antiporter and a high‐affinity Ca2+‐ATPase coexist in the vacuolar membrane. It is likely that the Ca2+/H+ antiporter removes excess Ca2+ in the cytosol to lower the Ca2+ concentration to micromolar levels after stimuli have increased the cytosolic Ca2+ level, the Ca2+‐ATPase then acts to lower the cytosolic Ca2+ level further.
Phenylacetyl‐CoA:acceptor oxidoreductase, a membrane‐bound molybdenum–iron–sulfur enzyme involved in anaerobic metabolism of phenylalanine in the denitrifying bacterium Thauera aromatica
Authors: Rhee Sung‐Keun , Fuchs Georg
Published: March 2003

Phenylacetic acids are common intermediates in the microbial metabolism of various aromatic substrates including phenylalanine. In the denitrifying bacterium Thauera aromatica phenylacetate is oxidized, under anoxic conditions, to the common intermediate benzoyl‐CoA via the intermediates phenylacetyl‐CoA and phenylglyoxylate (benzoylformate). The enzyme that catalyzes the four‐electron oxidation of phenylacetyl‐CoA has been purified from this bacterium and studied. The enzyme preparation catalyzes the reaction phenylacetyl‐CoA + 2 quinone + 2 H2O → phenylglyoxylate + 2 quinone H2 + CoASH. Phenylacetyl‐CoA:acceptor oxidoreductase is a membrane‐bound molybdenum–iron–sulfur protein. The purest preparations contained three subunits of 93, 27, and 26 kDa. Ubiquinone is most likely to act as the electron acceptor, and the oxygen atom introduced into the product is derived from water. The protein preparations contained 0.66 mol Mo, 30 mol Fe, and 25 mol acid‐labile sulfur per mol of native enzyme, assuming a native molecular mass of 280 kDa. Phenylglyoxylyl‐CoA, but not mandelyl‐CoA, was observed as a free intermediate. All enzyme preparations also catalyzed the subsequent hydrolytic release of coenzyme A from phenylglyoxylyl‐CoA but not from phenylacetyl‐CoA. The enzyme is reversibly inactivated by a low concentration of cyanide, but is remarkably stable with respect to oxygen. This new member of the molybdoproteins represents the first example of an enzyme which catalyzes the alpha‐oxidation of a CoA‐activated carboxylic acid without utilizing molecular oxygen.
OP18/stathmin binds near the C‐terminus of tubulin and facilitates GTP binding
Authors: Moreno Francisco José , Bagnat Michel , Lim Filip , Avila Jesús
Published: March 2003

It is has been previously suggested that the protein Op18/stathmin may interact with tubulin via the α‐tubulin subunit [Larsson, N., Marklund, U., Melander Gradin, H., Brattsand, G. & Gullberg, M. (1997) Mol. Cell. Biol.17, 5530–5539]. In this study we have used limited proteolysis and cross‐linking analysis to localize further the stathmin‐binding site on α‐tubulin. Our results indicate that such a binding site is in a region close to the C‐terminus of the molecule comprising residues 307 to the subtilisin‐cleavage site on the α‐tubulin subunit. Based on a recent model of the structure of tubulin [Nogales, E., Wolf, S.G. & Dowing, D.H. (1998) Nature (London)391, 199–203], we found that this region contained the same areas that may be involved in longitudinal contacts of α‐tubulin subunits within the microtubule. We also observed that the binding of stathmin to tubulin can modulate the binding of GTP to tubulin, as a consequence of a conformational change in the β‐tubulin subunit that occurs upon interaction of stathmin with tubulin.
The three‐dimensional structure of Asp189Ser trypsin provides evidence for an inherent structural plasticity of the protease
Authors: Szabó Erika , Böcskei Zsolt , Náray‐Szabó Gábor , Gráf László
Published: March 2003

Trypsin mutant Asp189Ser, first described by Gráf et al. [Gráf, L., Jancsó, Á., Szilágyi, L., Hegyi, G., Pintér, K., Náray‐Szabó, G., Hepp, J., Medzihradszky, K. & Rutter, W.J. (1988) Proc. Natl Acad. Sci. USA85, 4961–4965] has played an important role in recent studies on the structural basis of substrate‐specific catalysis by serine proteases. The present work reports the three‐dimensional structure of this mutant crystallized in unliganded form: the first unliganded rat trypsin structure reported. The X‐ray structure of the Asp189Ser trypsin mutant in complex with bovine pancreatic trypsin inhibitor is already known. The X‐ray structure of free Asp189Ser rat trypsin revealed that the single amino acid mutation at the bottom of the substrate binding pocket of trypsin resulted in extensive structural changes around the mutated site and in dimerization of the mutant, in contrast with the complexed enzyme the structure of which is practically the same as that of wild‐type trypsin. The structural rearrangement in the mutant was shown to be restricted to the activation domain region providing further evidence for the allosteric property of this structural–functional unit of the enzyme. This study supports our view that the plasticity of the activation domain may play an important role in the mechanism of substrate‐specific serine protease action.
Widely different off rates of two closely related cellulose‐binding domains from Trichoderma reesei
Authors: Carrard Géraldine , Linder Markus
Published: March 2003

The filamentous fungus Trichoderma reesei produces two cellobiohydrolases (CBHI and CBHII). These, like most other cellulose‐degrading enzymes, have a modular structure consisting of a catalytic domain linked to a cellulose‐binding domain (CBD). The isolated catalytic domains bind poorly to cellulose and have a much lower activity towards cellulose than the intact enzymes. For the CBDs, no function other than binding to cellulose has been found. We have previously described the reversibility and exchange rate for the binding of the CBD of CBHI to cellulose. In this work, we studied the binding of the CBD of CBHII and showed that it differs markedly from the behaviour of that of CBHI. The apparent binding affinities were similar, but the CBD of CBHII could not be dissociated from cellulose by buffer dilution and did not show a measurable exchange rate. However, desorption could be triggered by shifting the temperature. The CBD of CBHII bound reversibly to chitin. Two variants of the CBHII CBD were made, in which point mutations increased its similarity to the CBD of CBHI. Both variants were found to bind reversibly to cellulose.
Adenosine inhibits the renal plasma‐membrane (Ca2+ + Mg2+)‐ATPase through a pathway sensitive to cholera toxin and sphingosine
Authors: Coka‐Guevara Sandra , Markus Regina P. , Caruso‐Neves Celso , Lopes Aníbal G. , Vieyra Adalberto
Published: March 2003

Adenosine, a potent autacoid produced and released in kidneys, affects nearly all aspects of renal function, and an increase in cytosolic calcium has been implicated in adenosine effects. The aim of this work was to investigate whether adenosine modifies the calcium pump present in basolateral membranes of kidney proximal tubule cells. Adenosine exerts a biphasic influence on (Ca2+ + Mg2+)‐ATPase activity. Inhibition occurs up to 0.1 μm and then gradually disappears as the adenosine concentration increases to 100 µm, an effect mimicked by the adenosine analog N6‐cyclohexyladenosine, which preferentially binds to A1‐type receptors. In contrast, the A2 receptor agonist 5′,N‐ethylcarboxamideadenosine is ineffective. The A1 receptor antagonist 8‐cyclopentyl‐1,3‐dimethylxanthine blocks the inhibitory effect of 0.1 μm adenosine and stimulates (Ca2+ + Mg2+)‐ATPase activity in the presence of 1 mm adenosine, a concentration high enough to occupy the low‐affinity A2 receptors. Inhibition by adenosine increases as medium ATP is lowered to micromolar concentrations, is maintained in the presence of pertussis toxin, and is completely abolished with 0.1 μm cholera toxin or 1 µm sphingosine. The inhibitory effect of adenosine can be reproduced by guanosine 5′‐[γ‐thio]triphosphate, inositol 1,4,5‐trisphosphate or the diacylglycerol analog 12‐O‐tetradecanoylphorbol 13‐acetate. In conjunction with the selectivity for its analogs and for its receptor agonist, the concentration profile of adenosine effects indicates that both inhibitory (A1) and stimulatory (A2) receptors are involved. The results obtained with the toxins indicate that a pathway that is modulated by G‐proteins, involves a phospholipase C and a protein kinase C, and is affected by local variations in adenosine concentrations participates in the regulation of the (Ca2+ + Mg2+)‐ATPase resident in basolateral membranes of kidney proximal tubules.
Characterization of a truncated recombinant form of human membrane type 3 matrix metalloproteinase
Authors: Shimada Taketoshi , Nakamura Hiroyuki , Ohuchi Eiko , Fujii Yutaka , Murakami Yasushi , Sato Hiroshi , Seiki Motoharu , Okada Yasunori
Published: March 2003

Membrane type 3 matrix metalloproteinase (MT3‐MMP), an activator for the zymogen of MMP‐2 (proMMP‐2, or progelatinase A), is known to be expressed in human placenta, brain, lung and rat vascular smooth muscle cells, but information about its biochemical properties is limited. In the present study, we expressed and purified a truncated form of MT3‐MMP lacking the transmembrane and intracytoplasmic domain (ΔMT3) and characterized the enzyme biochemically. ΔMT3 digested type III collagen into characteristic 3/4‐ and 1/4‐fragments by cleaving the Gly781‐Ile782 and Gly784‐Ile785 bonds of α1(III) chains. Although ΔMT3 did not have such an activity against type I collagen, it attacked the Gly4‐Ile5 bond of the triple helical portion of α2(I) chains, leading to removal of the crosslink containing N‐terminal telopeptides. By quantitative analyses of the activities of ΔMT3 and a similar deletion mutant of MT1‐MMP (ΔMT1), ΔMT3 was approximately fivefold more efficient at cleaving type III collagen. ΔMT3 also digested cartilage proteoglycan, gelatin, fibronectin, vitronectin, laminin‐1, α1‐proteinase inhibitor and α2‐macroglobulin into almost identical fragments to those given by ΔMT1, although carboxymethylated transferrin digestion by ΔMT3 generated some extra fragments. The activity of ΔMT3 was inhibited by tissue inhibitor of metalloproteinases‐2 (TIMP‐2) and TIMP‐3 in a 1 : 1 stoichiometry, but not by TIMP‐1. ProMMP‐2 was partially activated by ΔMT3 to give the intermediate form. These results indicate that, like MT1‐MMP, MT3‐MMP exhibits proteolytic activities against a wide range of extracellular matrix molecules. However, differences in the proMMP‐2 activation and tissue distribution suggest that MT3‐MMP and MT1‐MMP play different roles in the pathophysiological digestion of extracellular matrix.
Expression of a higher plant light‐harvesting chlorophyll a/b‐binding protein in Synechocystis sp. PCC 6803
Authors: He Qingfang , Schlich Thomas , Paulsen Harald , Vermaas Wim
Published: March 2003

A chimeric lhcb gene, coding for Lhcb, a higher plant chlorophyll a/b‐binding light‐harvesting complex of photosystem II (LHCII), was constructed using the Synechocystis sp. PCC 6803 psbA3 promoter and a modified lhcb gene from pea. This construct drives synthesis of full‐length, mature Lhcb under the control of the strong psbA3 promoter that usually drives expression of the D1 protein of photosystem II. This chimeric gene was transformed into a photosystem I‐less/chlL−Synechocystis sp. PCC 6803 strain that is unable to synthesize chlorophyll in darkness. In the resulting strain, a high level of lhcb transcript was detected and transcript accumulation was enhanced by addition of exogenous Zn‐chlorophyllide b. The chimeric lhcb gene was translated to produce full‐length Lhcb as demonstrated by pulse‐labeling: a new radioactively labeled band of a size corresponding to full‐length Lhcb was visible on autoradiograms. Using Triton X‐114 phase fractionation, this labeled protein band was found to partition to the phase containing integral membrane proteins, indicating that the pulse‐labeled Lhcb is readily integrated into the membrane. However, Lhcb was rapidly degraded and did not accumulate in thylakoid membranes to levels that were detectable other than by pulse labeling. Upon immunological detection with LHCII antibodies, a small protein (≈ 8 kDa) was found specifically in the lhcb‐containing mutant. We interpret this protein to be a degradation product of the full‐length Lhcb. This fragment was stabilized by supplementing cells with xanthophylls, which incorporated into thylakoid membranes only in the mutant carrying lhcb. The lutein/chlorophyll ratio of thylakoids of this mutant was about 1 : 10. These results indicate that in this cyanobacterial system Lhcb is synthesized, integrated into the membrane, and then degraded to a ≈ 8 kDa fragment that is stabilized by pigment binding and does not require the presence of chlorophyll b.
Identification and characterization of cDNA clones encoding hydroxycinnamoyl‐CoA:tyramine N‐hydroxycinnamoyltransferase from tobacco
Authors: Farmer Mary Jo , Czernic Pierre , Michael Anthony , Negrel Jonathan
Published: March 2003

The sequences of three cDNA clones that include the complete coding region of hydroxycinnamoyl‐CoA:tyramine N‐hydroxycinnamoyltransferase (THT) from tobacco are reported. The three cDNAs were isolated by antibody screening of a cDNA expression library produced from poly(A)+RNA purified from tobacco leaves (Nicotiana tabacum cv. Bottom Special), previously infiltrated with an incompatible strain of Ralstonia solanacearum. The identity of these clones was confirmed by the detection of THT activity in extracts of transformed Escherichia coli and by matching the translated polypeptides with tryptic enzyme sequences. cDNA clones tht4 and tht11 differ only by their 5′ leader and 3′ UTRs and therefore encode the same protein, whereas tht10 and tht11 exhibit 95 and 99% sequence identity at the DNA and deduced amino acid levels, respectively. The three clones encode proteins of 226 amino acids with calculated molecular masses of 26 kDa. The deduced amino acid sequences show no similarity with the sequence of anthranilate hydroxycinnamoyl/benzoyltransferase from Dianthus caryophyllus, the only enzyme exhibiting hydroxycinnamoyltransferase activity to be cloned so far in plants. In contrast, comparison of the THT amino acid sequence with protein sequence databases revealed substantial homology with mammalian diamine acetyltransferases. The THT clones hybridized to a 0.95‐kb mRNA from elicited tobacco cell‐suspension cultures and also to a mRNA of similar size from wound‐healing potato tubers. The messengers for THT were also found to be expressed at relatively high levels in tobacco root tissues. Southern hybridization of tobacco genomic DNA with THT cDNA suggests that several copies of the THT gene occur in the tobacco genome. Inhibition experiments using amino‐acid‐specific reagents demonstrated that both histidyl and cysteyl residues are required for THT activity. In the course of these experiments THT was also found to be inhibited by (2‐hydroxyphenyl) amino sulfinyl acetic acid 1,1‐dimethylethyl ester, an irreversible inhibitor of cinnamyl alcohol dehydrogenase.
Selective targeting of an antioxidant to mitochondria
Authors: Smith Robin A. J. , Porteous Carolyn M. , Coulter Carolyn V. , Murphy Michael P.
Published: March 2003

Mitochondrial oxidative damage contributes significantly to a range of human disorders, including neurodegenerative diseases, ischaemia‐reperfusion injury and ageing‐associated dysfunction. To prevent this damage we have delivered a molecule containing the active antioxidant moiety of vitamin E to mitochondria. This was carried out by covalently coupling the antioxidant moiety to a lipophilic triphenylphosphonium cation. This mitochondrially targeted antioxidant, 2‐[2‐(triphenylphosphonio)ethyl]‐3,4‐dihydro‐2,5,7,8‐tetramethyl‐2H‐1‐benzopyran‐6‐ol bromide (TPPB), accumulated several‐hundred fold within the mitochondrial matrix, driven by the organelle’s large membrane potential. When cells were incubated with micromolar concentrations of TPPB, they accumulated millimolar concentrations within their mitochondria. The amount of TPPB taken up by mitochondria was ≈ 80‐fold greater than endogenous levels of vitamin E. Consequently the targeted derivative of vitamin E protected mitochondrial function from oxidative damage far more effectively than vitamin E itself. The mitochondrially targeted antioxidant TPPB has potential as an antioxidant therapy for disorders involving mitochondrial oxidative damage. It also suggests a new family of mitochondrially targeted antioxidants, redox‐active and pharmacologically active molecules designed to prevent damage or manipulate mitochondrial function.