bluuebunny/crossref_metadata_2025 huggingface dataset:
‘Rinrei’, a brassinosteroid‐deficient dwarf mutant of faba bean (Vicia faba L.)
Authors: Fukuta Naoko , Fujioka Shozo , Takatsuto Suguru , Yoshida Shigeo , Fukuta Yoshimichi , Nakayama Masayoshi
Published: June 2004

A dwarf mutant of broad bean (Vicia faba L.), the variety Rinrei, has been created by γ‐ray irradiation. Rinrei is characterized by dark green leaves and by reduced plant length, internode and petiole length, shoot weight, and number of branches. Genetic analysis of hybrids between Rinrei and two wild‐type lines indicated that these characteristics are controlled by a single recessive gene. The phenotype of Rinrei was restored to that of the wild type by application of brassinolide, but not by GA3. Qualitative and quantitative analysis by gas chromatography–mass spectrometry indicated that 24‐methylenecholesterol and isofucosterol accumulated in Rinrei to levels more than 30 times higher than in the wild type. In contrast, Rinrei had lower than wild‐type levels of campesterol, sitosterol and brassinosteroids. Therefore, Rinrei is a brassinosteroid‐deficient mutant defective in sterol C‐24 reduction. The gene was tentatively designated as brassinosteroid deficient dwarf 1, bdd1, which seems to be a homologue of Arabidopsis dwf1 (dim, cbb1) and pea lkb.
Effects of ecdysteroids on Chlorella vulgaris
Authors: Bajguz Andrzej , Dinan Laurence
Published: June 2004

The effects of three ecdysteroids, 20‐hydroxyecdysone (20E), 2‐deoxy‐20‐hydroxyecdysone (2d20E) and 20‐hydroxyecdysone 22‐acetate (20E22Ac), on growth and the levels of cellular components in Chlorella vulgaris Beijerinck (Trebouxiophyceae) are reported and compared with data previously reported for ecdysone (E; Bajguz A and Koronka A, Plant Physiol Biochem 39: 707–715, 2001). All three 20‐hydroxyecdysteroids stimulate growth of C. vulgaris cells over a wide concentration range (10−16 to 10−7 M). Optimal stimulation is observed at 10−9 M with each ecdysteroid. High concentrations (>10−6 M) are cytotoxic. The potency ranking of the ecdysteroids is 20E > 20E22Ac > 2d20E > E. Levels per cell of DNA, RNA, protein, sugars, organic and inorganic phosphorus, chlorophylls a and b and phaeophytins a and b are all stimulated by ecdysteroid treatment when compared with the untreated control cells. Possible modes of action of ecdysteroids on C. vulgaris cells are discussed.
Structural and genomic organization, cDNA characterization and expression analysis of the urate oxidase gene from chickpea (Cicer arietinum)†
Authors: Redondo‐Nevado José , Aguilar Miguel , Caballero José L. , Pineda Manuel
Published: June 2004

A complete cDNA and a genomic DNA fragment coding for urate oxidase (urate: oxygen oxidoreductase, EC 1.7.3.3) from chickpea (Cicer arietinum L.) were isolated and characterized. The 1032 bp cDNA (CAUR1) contains a complete open reading frame that encodes a 308 amino acid protein with a predicted size of 34.06 kDa and a pI of 9.38. This protein shows a strong similarity with other uricases present in the databases. Heterologous expression in Escherichia coli showed urate oxidase activity in crude extracts from induced cells, demonstrating that CAUR1 encodes a complete and functional uricase enzyme. Kinetic properties of the recombinant enzyme were similar to those of the native uricase purified from chickpea leaves. The genomic organization of the chickpea uricase gene (CAUR1) remarkably resembles that of the soybean (Glycine max L.) uricase gene, presenting eight exons and seven introns. Within the 5′‐flanking region of the chickpea gene, nucleotide sequences matching consensus motifs of soybean nodule NAT2‐nuclear factors and common bean (Phaseolus vulgaris L.) proteins (PNF1) are present. It demonstrates that a single copy of the uricase gene is present in the chickpea genome and that the gene is expressed not only in nodules, but also in leaves and roots.
5′‐Deoxyisopentenyladenosine and other cytokinins in culture filtrates of the bacterium Pantoea agglomerans
Authors: Omer Zahra Saad , Björkman Per‐Olof , Nicander Björn , Tillberg Elisabeth , Gerhardson Berndt
Published: June 2004

A Pantoea agglomerans isolate from barley seeds, selected for inducing growth promotion in tomato test plants, was found to excrete several hormones of the cytokinin class into its culture medium. In addition to isopentenyladenine, isopentenyladenosine and the 2‐methylthiol derivates of these, an unknown compound with affinity to anticytokinin antibodies was also isolated. Mass spectroscopy indicated the structure of this to be a deoxyisopentenyladenosine. The structure of 9‐(5′‐deoxy‐β‐d‐ribofuranosyl)‐6‐(3‐methyl‐2‐butenylamino)purine was verified after synthesis of standards and analysis with GC–MS. The synthesized 5′‐deoxyisopentenyl‐adenosine showed activity in the Amaranthus bioassay, specific for cytokinins. To our knowledge this is the first report of a naturally occurring cytokinin containing 5′‐deoxyribose.
Low temperature effects on ubiquinone content, respiration rates and lipid peroxidation levels of etiolated seedlings of two differentially chilling‐sensitive species
Authors: Munro Kathleen D. , Hodges D. Mark , DeLong John M. , Forney Charles F. , Kristie David N.
Published: June 2004

Ubiquinone functions primarily in the electron transport chain of the mitochondria of plants and animals. Secondary roles in plant tissue, such as antioxidant activity, have also been proposed. The effect of low temperature exposure on etiolated seedling embryonic axes of two differentially chilling‐sensitive species, mung bean (Vigna radiata L.) (chilling‐sensitive) and pea (Pisum sativum L. cv. Lincoln) (chilling‐tolerant) with respect to respiration rate, lipid peroxidation and ubiquinone content was examined. Whole seedlings (embryonic axis and cotyledon) of both species were exposed to control temperatures (20°C) (6 days) or an acclimatory low temperature treatment of 10°C (3 days) followed by exposure at 5°C (3 days). Measurements were initiated 3 days after seedlings had reached 50% germination (D0). Prior to measurements the cotyledons were removed and only the embryonic axis was used in these experiments. Ubiquinol (UQH2), ubiquinone (UQ) and total ubiquinone (UQtot) content decreased in mung bean in response to the temperature treatment and UQH2 and UQtot remained stable in the more chilling‐tolerant pea. The reduction of the total Q‐pool was approximately 85–92%, suggesting a high degree of saturation of the respiration pathways. Respiration declined and the RQ ratio increased in both species in response to low temperature. Cytochrome c oxidase (COX) (EC 1.9.3.1) activity was higher in pea than in mung bean but decreased during low temperature exposure in both species. Considering that levels of MDA (lipid peroxidation) did not increase in either species in response to chilling, decreased levels of UQH2 and UQ observed in chilling‐sensitive mung bean may indicate that these compounds were damaged prior to other membrane lipids during low temperature treatment and rendered undetectable.
The Proteolytic Cleavage of Protein Kinase C Isotypes, Which Generates Kinase and Regulatory Fragments, Correlates with Fas‐Mediated and 12‐O‐Tetradecanoyl‐Phorbol‐13‐Acetate‐Induced Apoptosis
Authors: Mizuno Keiko , Noda Kumi , Araki Tamao , Imaoka Tomomi , Kobayashi Yuko , Akita Yoshiko , Shimonaka Motoyuki , Kishi Shuji , Ohno Shigeo
Published: June 2004

Protein kinase C (PKC) has been implicated in signaling induced by diverse sets of stimuli regulating growth, differentiation, and apoptosis. The present study focused on the fate of PKC isotype proteins during Fas‐mediated apoptosis of human leukemic cell lines. Among the PKC isotypes expressed in different cell types, such as Jurkat, HPB‐ALL, U937, and HL60, all the nPKC isotypes including nPKCδ, nPKCε, and nPKCθ, but not cPKCα and βII and aPKCζ (n, c, and a represent novel, conventional and atypical, respectively), showed limited proteolytic cleavage during Fas‐mediated apoptosis. The limited proteolysis of nPKC isotypes means the disappearance of the intact protein band concomitant with the appearance of two fragments, most likely containing the kinase and regulatory domains, in contrast to the so‐called down‐regulation known for both cPKC and nPKC isotypes following exposure to stimuli such as 12‐O‐tetradecanoyl‐phorbol 13‐acetate (TPA). The time course of Fas‐mediated apoptosis in Jurkat cells parallels that of the activation of a 32‐kDa cysteine protease (CPP32)‐like protease and also closely parallels the proteolytic cleavage of nPKC isotypes. A peptide inhibitor of the CPP32‐like protease, Ac‐DEVD‐CHO, blocked the proteolytic cleavage of nPKC isotypes as well as apoptosis mediated by Fas. Transfection of recombinant protein coding for the catalytic fragment of nPKCδ to COS1 cells resulted in the apoptotic morphology of cells and nuclei.

The effect of TPA on apoptosis depends on the cell type. TPA significantly suppressed Fas‐mediated apoptosis in Jurkat, whereas TPA alone caused apoptosis in HPB‐ALL, U937, and HL60, only slight apoptosis in Jurkat. The proteolytic fragmentation of nPKC isotypes again closely correlated with the degree of apoptosis even in apoptosis induced by TPA. Separation of TPA‐treated cells into apoptotic and non‐apoptotic differentiating cells revealed that the proteolytic fragmentation of nPKC isotypes occurs only in apoptotic cells and, in adherent differentiating cells, nPKC isotypes as well as cPKCα were down‐regulated without the generation of nPKC fragments. These results are consistent with the idea that nPKC isotypes meet two different fates, down‐regulation and proteolytic cleavage generating kinase and regulatory fragments, and that the proteolytic cleavage of nPKC isotypes is a step in the signaling pathway involved in Fas‐mediated and TPA‐induced apoptosis.
Cell‐Growth Regulation of the Hamster Dihydrofolate Reductase Gene Promoter by Transcription Factor Sp1
Authors: Noé Véronique , Chen Chao , Alemany Cristina , Nicolás Marta , Caragol Isabel , Chasin Lawrence A. , Ciudad Carlos J.
Published: June 2004

The dihydrofolate reductase (DHFR) gene (dhfr) promoter contains as‐acting elements for the transcription factors Spl and E2F. Given the ability of Spl to activate the dhfr promoter, we have evaluated the contribution of Sp1 to the cell‐growth regulation of the dhfr gene. Using gel‐mobility assays performed with DNA probes from the minimal promoter of the hamster dhfr gene and nuclear extracts from cultured hamster cells (CHO K1) we show that the binding of Sp1 to the dhfr promoter is cell‐growth‐phase regulated. Accordingly, dhfr transcription and mRNA levels in K1 cells increase upon serum stimulation. Cytological detection of Sp1 by immunofluorescence reveals a decrease of this protein in the process leading to the G0 state, and an increase upon serum stimulation of quiescent cells. These results were confirmed by western blot analysis. It is concluded that Sp1 progressively binds to the hamster dhfr promoter after stimulation of cell proliferation, which can account for the transcriptional regulation of the dhfr gene during the cell cycle. The role of Sp1 in the specific control of dhfr during the cell cycle was confirmed in vivo using cell lines derived from dhfr‐negative. cells transfected with dhfr plasmids carrying either the wild‐type or mutated Sp1‐binding or E2F‐binding sites in the dhfr minimal promoter.
The Recombinant Product of theChryptomonasφ Plastid GenehlpAis an Architectural Hu‐Like Protein that Promotes the Assembly of Complex Nucleoprotein Structures
Authors: Crasser Klaus D. , Ritt Christoph , Krieg Marion , Fernández Silvia , Alonso Juan C. , Grimm Rudi
Published: June 2004

The HlpA protein which is encoded by thehlpAgene in the plastid genome of the cryptomonad algaChryptomonas (I)is structurally related to the non‐sequence‐specific DNA‐binding and DNA‐bending HU family of chromatin‐associated proteins. The expression of the HlpA protein complements the mutant phenotype ofBacillus subtiliscells impaired in the Hbsu protein (B. subtilisHU), as measured by the resistance of the cells to methylmethane sulphonate. To analyse the interactions of HlpA with DNA, we expressed the protein in Escherichia coli and purified it to homogeneity. HlpA interacts preferentially with four‐way junction DNA or DNA minicircles, when compared with linear DNA, recognising DNA structure. HlpA andE. coliHU display comparable affinities for all types of DNA tested; however, HlpA exhibits a stronger tendency to self‐associate in the presence of DNA. Accordingly, HlpA oligomerises more readily than HU in protein crosslinking experiments. In the presence of topoisomerase I, HlpA constrains negative superhelical turns in closed circular plasmid DNA. The HlpA protein mediates the joining of distant recombination sites into a complex nucleoprotein structure, as judged by β‐mediated site‐specific recombination. The results presented provide evidence that HlpA is a functional plastid equivalent of nuclear and mitochondrial HMG1‐like proteins and bacterial HU proteins.
P‐Glycoprotein‐Mediated Hoechst 33342 Transport Out of the Lipid Bilayer
Authors: Shapiro Adam B. , Corder Adam B. , Ling Victor
Published: June 2004

High‐level expression of P‐glycoprotein, a 170‐kDa mammalian plasma membrane ATPase, is the cause of an important and widespread form of cancer multidrug resistance. P‐glycoprotein reduces cellular accumulation of an enormous variety of lipophilic compounds. The basis for this broad substrate specificity is not well understood. We explored this issue by measuring the kinetics of transport of the lipophilic P‐glycoprotein substrate Hoechst 33342 by P‐glycoprotein‐rich plasma membrane vesicles from CHRB30 cells. Hoechst 33342 is fluorescent when bound to the membrane, but not when in the aqueous medium, allowing movement of the dye out of the membrane to be quantitated by fluorescence intensity. The initial specific rate of transport was directly proportional to the amount of Hoechst 33342 in the lipid phase and inversely proportional to the concentration in the aqueous phase. This demonstrates that P‐glycoprotein removes Hoechst 33342 from the lipid membrane, where it concentrates due to its hydrophobicity. Because the membrane concentration of hydrophobic P‐glycoprotein substrates is high, it may be that P‐glycoprotein need not recognize them with high affinity. Transport of hydrophobic substrates out of the lipid bilayer instead of the cytoplasm thus helps to explain the broad substrate specificity of P‐glycoprotein.
Positively Cooperative Sites for Drug Transport by P‐Glycoprotein with Distinct Drug Specificities
Authors: Shapiro Adam B. , Ling Victor
Published: June 2004

In this paper, we show that P‐glycoprotein contains two distinct sites for drug binding and transport, and that, unexpectedly, these sites interact in a positively cooperative manner. The kinetics of transport of rhodamine 123 and Hoechst 33342 in isolated P‐glycoprotein‐rich plasma membrane vesicles from Chinese hamster ovary CHRB30 cells were followed by continuous fluorescence monitoring. Each substrate stimulated P‐glycoprotein‐mediated transport of the other. Colchicine and quercetin stimulated rhodamine 123 transport and inhibited Hoechst 33342 transport. In contrast, anthracyclines such as daunorubicin and doxorubicin stimulated Hoechst 33342 transport and inhibited rhodamine 123 transport. Vinblastine, actinomycin D, and etoposide inhibited transport of both dyes. The results are consistent with a functional model of P‐glycoprotein containing at least two positively cooperative sites (H site and R site) for drug binding and transport. This model is consistent with earlier observations of competitive and non‐competitive effects of P‐glycoprotein substrates and chemosensitizers. Such a two‐site model may be fundamental to multidrug transport by P‐glycoprotein, and it may be a feature common to other ATP‐dependent transporters belonging to the ATP‐binding cassette superfamily.
Localization of Heparin Binding Activity in Recombinant Laminin G Domain
Authors: Sung Uhna , O'rear Julian J. , Yurchenco Peter D.
Published: June 2004

Basement membrane laminin (laminin‐1) is a multidomain glycoprotein that interacts with itself, heparin and cells. The interaction with heparin/heparan sulfate proteglycans is thought to be important for the architectural formation of basement membranes and adhesion to cells. The major heparin binding site has been known to reside in the long arm globular domain (G domain). The G domain is in turn subdivided into five subdomains (G1–G5). In order to localize the heparin binding regions further, recombinant G domains (rG and rG5) were expressed in Sf9 insect cells using baculovirus expression vector. By the limited proteolysis of recombinant G domains followed by either heparin affinity HPLC or overlay with radiolabeled heparin, the relative affinity of each subdomain to heparin was assigned as G1>G2 = G4>G5>G3, such that G1 bound strongly and G3 not at all. Since the activity in G1–G3 is cryptic in intact laminin long arm [Sung, U., O'Rear, J. J. & Yurchenco, P. D. (1993) J. Cell Biol. 123, 1255–1268], the active heparin binding site of G domain appears to be located in G4 and proximal G5.
Cloning of an Unusual Natriuretic Peptide from the South American Coral Snake Micrurus corallinus
Authors: Ho Paulo Lee , Soares Marcelo Bento , Maack Thomas , Gimenez Ignacio , Puorto Giuseppe , Furtado Maria de Fátima Domingues , Raw Isaias
Published: June 2004

In the course of cloning abundant cDNAs from the South American coral snake Micrurus corallinus venom gland, we characterized a cDNA coding for a putative natriuretic peptide. All the natural natriuretic peptides described so far, possess a ring structure composed of 17 amino acids formed through an S‐S bridge which is extended at the N‐terminus by few to several amino acids and may be extended at the C‐terminus, usually 4–7 amino acids. In contrast, the M. corallinus natriuretic peptide presents several distinct features: (a) the proform of the deduced natriuretic peptide displays an unusual C‐terminus extension. This implies that the mature peptide has a long C‐terminal tail or it is further extensively processed to result in the mature natriuretic peptide with the expected 4–7 amino‐acid extension, (b) the deduced natriuretic peptide presents an unusual internal Cys within the ring structure. This raises the possibility of natriuretic peptides with a smaller ring structure, (c) the putative natriuretic peptide is flanked by two homologous peptides of unknown function. In addition, an analogous peptide was synthesized and assayed on perfused rat kidney, showing a dose‐dependent response in urinary volume and sodium excretion. Moreover, northern‐blot studies showed that M. corallinus natriuretic peptide transcripts were highly expressed in venom glands, but they were not detectable in other tissues like heart and brain, suggesting a main role for this M. corallinus natriuretic peptide in the venom gland or in the envenomation by this coral snake's bite.
Metabolism of Tentoxin by Hepatic Cytochrome P‐450 3A Isozymes
Authors: Delaforge Marcel , Andre François , Jaouen Maryse , Dolgos Hugues , Benech Henri , Gomis Jean‐Marie , Noel Jean‐Pierre , Cavelier Florine , Verducci Jean , Aubagnac Jean‐Louis , Liebermann Bernd
Published: June 2004

The interaction between rat and human liver cytochrome P‐450 with tentoxin, a natural phytotoxic cyclotetrapeptide having chlorotic properties, was studied by difference ultraviolet visible spectroscopy. Tentoxin interacted with rat liver microsomes and the difference spectrum was characteristic of binding to a protein site close to the heme. The intensity of this spectrum was clearly dependent on the amounts of P‐450 3A in the microsomes and was optimal in dexamethasone‐treated rat microsomes. Tentoxin exhibited a high affinity for P‐450 3A (Ks approximately 10 μM). Similar results were observed with human P‐450 isozymes expressed in yeast. Only P‐450 3A4 and 3A5 were able to give spectral interactions with tentoxin.

Liver microsomes from rats pretreated with dexamethasone, a specific inducer of P‐450 3A, were found to be particularly active for the oxidation of tentoxin, which occurs mainly on its Ala(Me) function leading to demethylation. Yeast‐expressed P‐450 3A also exhibited high activity to metabolize tentoxin.

The metabolites were identified by their ultraviolet and mass spectra in fast atom bombardment and collision‐activated dissociation modes. In addition to the major N‐demethylated metabolite, other hydroxylated metabolites were formed.

Preliminary analysis showed that as tentoxin, some metabolites were still efficient chloroplast ATPase inhibitors, while at least one of them exhibited even at low concentration stimulatory effects.
Primary Structure of 21 Novel Monoantennary and Diantennary N‐Linked Carbohydrate Chains from αD‐Hemocyanin of Helix Pomatia
Authors: Lommerse Jos P. M. , Thomas‐Oates Jane E. , Gielens Constant , Préaux Gisèle , Kamerling Johannis P. , Vliegenthart Johannes F. G.
Published: June 2004

The primary structures of 21 novel monoantennary and diantennary N‐glycans of the glycoprotein αD‐hemocyanin (αD‐Hc) of Helix pomatia have been determined. Outer oligosaccharide fragments (antennae) were released from the glycoprotein by Smith degradation of an αD‐Hc pronase digest. The major antenna, obtained following HPLC fractionation on Lichrosorb‐NH2, was characterized using 1H‐NMR spectroscopy, fast‐atom‐bombardment mass spectrometry, and linkage analysis, and corresponds to a pentasaccharide fragment. The intact carbohydrate chains of αD‐Hc were released with peptide‐N4‐(N‐acetyl‐β‐glucosaminy1)asparagine amidase‐F digestion, separated from the protein on Bio‐Gel P‐100, and subfractionated on Bio‐Gel P‐4. A portion of subfractions was reduced with sodium borodeuteride, and the non‐reduced and reduced samples were further fractionated on CarboPac PA‐1, Lichrosorb‐NH2/Lichrosphere‐NH2, and/or Lichrosphere‐C18. Purified oligosaccharides and oligosaccharide‐alditols were analyzed using 500/600‐MHz 1H‐NMR spectroscopy. In total, four novel types of antenna were identified, namely,
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which are all attached to 0–2 of αMan residues of the trimannosyl‐N,N′‐diacetylchitobiose core element, which is generally β‐1,2‐xylosylated and α‐1,6‐fucosylated, Man(α1‐6)[Man(α1‐3)][±Xyl(β1‐2)]Man(β1‐4)GlcNAc(β1‐4) [±Fuc(α1‐6)]GlcNAc.
Ordered Cell Cycle Phase Perturbations in Chinese Hamster Ovary Cells Treated with an S‐Adenosylmethionine Decarboxylase Inhibitor
Authors: Fredlund Jan O. , Oredsson Stina M.
Published: June 2004

The polyamines putrescine, spermidine, and spermine are needed for normal cell cycle progression and polyamine‐depleted cells cease to proliferate. We have investigated cell cycle perturbations in Chinese hamster ovary cells seeded in the presence of 4‐amidinoindan‐1‐one 2’‐amidinohydrazone (CGP 48664), a potent inhibitor of S‐adenosylmethionine decarboxylase, an enzyme which is essential for the synthesis of spermidine and spermine. At 9 h and at 1, 2, and 3 days after seeding, cells were labelled with the thymidine analog bromodeoxyuridine (BrdUrd) for 30 min, after which the BrdUrd‐containing medium was removed and the cells were allowed to progress through the cell cycle in BrdUrd‐free medium before sampling (post‐labelling time). Using flow cytometry, coupled with an indirect immunofluorenscence technique, utilizing monoclonal anti‐BrdUrd and secondary fluorescein‐isothiocyanate‐conjugated antibodies, and the DNA stain propidium iodide, cellular BrdUrd and DNA contents were quantified. By investigating the movement of BrdUrd‐nonlabelled G, cells into S phase during the post‐labelling time, a measure of the G1/S transition was obtained. The time of appearence in G1 of BrdUrd‐labelled cells which had divided was used to monitor the length of the G2+M phase. A measure of the S phase length (DNA synthesis time) was obtained by monitoring the progression of BrdUrd‐labelled cells through S phase. CGP 48664‐induced spermine depletion significantly increased the length of the S phase already 9 h after seeding cells in the presence of the inhibitor. No effects on the G1/S transition or on the length of the G2+M phase were observed until 2 days after seeding.
Regulation of the Flavin Redox Potential by Flavin‐Binding Antibodies
Authors: Bruggeman Yvonne E. , Honegger Annemarie , Kreuwel Huub , Visser Antonie J. W. G. , Laane Colja , Schots Arjen , Hilhorst Riet
Published: June 2004

Single‐chain Fv antibody fragments binding different flavin forms [10‐(5'‐carboxybutyl‐)flavin (Flox) and 10‐(5'‐carboxybutyl)‐1,5‐dihydroflavin (Flred)] have been generated from an antibody phage‐display library to study how a protein environment regulates the redox potential, starting from a protein other than a natural flavoprotein. These ‘flavobodies’ are characterized by time‐resolved and steady‐state fluorescence spectroscopy, by competitive ELISA methods (mapping of the antigen‐binding site), and by molecular modelling. The three‐dimensional models of the antigen‐binding sites are consistent with the experimental results. Binding of anti‐Flred 5 to flavin increases the redox potential, mainly due to an Arg residue interacting with the flavin N1. Thus anti‐Flred 5 shows an ‘oxidase‐like’ redox‐potential behaviour, confirming the idea that positively charged residues in the vicinity of N1 increase the redox potential. The results obtained with anti‐Flox, which do not resemble a natural flavoprotein, show that when the pyrimidine‐like nucleus of the flavin is not involved in binding, the redox potential is not significantly affected. These results are in contrast to those obtained with chicken riboflavin‐binding protein.
Tumor‐Necrosis Factor‐α Modulates Mitogen‐Activated Protein Kinase Activity of Epidermal‐Growth‐Factor‐Stimulated MCF‐7 Breast Cancer Cells
Authors: Flury Nathalie , Eppenberger Urs , Mueller Heinz
Published: June 2004

Tumor‐necrosis factor(TNF)‐α inhibited in a dose‐dependent fashion the proliferation of epidermal‐growth‐factor(EGF)‐stimulated MCF‐7 breast cancer cells with an IC50 of 0.25 nM. A comparable TNF‐α‐mediated inhibition of p42/44 mitogen‐activated protein (MAP) kinase activity was observed in 10 nM EGF‐stimulated cells. The MAP kinase activity dropped 50% within 3 min of TNF‐α (1 nM) addition to EGF‐stimulated MCF‐7 cells. EOF and TNF‐a, when added independently, led to a transient stimulation of MAP kinase activity with maximal activations within 6–8 min and 1–2 min, respectively. These observations suggest that MAP kinase activity in EGF‐stimulated MCF‐7 cells is modulated by the growth‐inhibitory receptor pathways of TNF‐α. Phosphorylation measurements on western blots determined the involvement of several individual MAP kinases, namely p42/44 MAP kinases, p38 MAP kinase and c‐Jun N2‐terminal kinase 1 (JNK1), in EGF and TNF‐α‐induced signalling. Phosphorylation of p42 and p38 MAP kinases only was observed after treatment with either TNF‐α or EGF. A combination of both ligands inhibited p42 and p38 MAP kinase phosphorylation in MCF‐7 cells. In contrast, no JNK1 phosphorylation was detected in these cells. Simultaneous addition of okadaic acid, a potent inhibitor of phosphatases 1 and 2A, blocked the decay of EGF‐stimulated MAP kinase activity over 40 min. TNF‐α added to EGF‐stimulated and okadaic‐acid‐treated cells increased the MAP kinase activity twofold within 1 min. Similarly, okadaic acid treatment partly reverted the TNF‐α‐inhibited growth of MCF‐7 cells. These experiments suggest that phosphatases are involved in the rapid shut‐down by TNF‐α of p42 MAP kinase activity.
Ultraviolet‐B‐Radiation‐Induced Changes in Nicotinamide and Glutathione Metabolism and Gene Expression in Plants
Authors: Kalbin Georgi , Ohlsson Anna B. , Berglund Torkel , Rydström Jan , Strid Åke
Published: June 2004

Pea (Pisum sativum L. cv. Greenfeast) plants were exposed to supplementary ultraviolet‐B (UV‐B) radiation (biologically effective dose rates normalised to 300 nm, UV‐BBE300: 0.18, 0.32 or 1.4 W m−2). Leaf nicotinamide, trigonelline, GSHtot (total glutathione) and GSSG (oxidised glutathione) levels remained unchanged after exposure to the lowest dose rates. 1.4 W m−2 UV‐BBE,300: gave rise to 60‐fold and 4.5‐fold increases in GSSG and GSHtot, respectively. 3.5‐fold and 9.5‐fold increases were found in nicotinamide and trigonelline, respectively, cab (Chlorophyll‐a/b‐binding protein) transcript levels decreased and CHS (chalcone synthase) and PAL (phenylalanine ammonia‐lyase) mRNA increased after shorter UV‐B exposures (hours) to the higher dose rate of UV‐B, and after exposure to the intermediate dose rate. CHS and PAL mRNAs also increased after prolonged exposure to the lowest dose rate. cah transcripts completely disappeared, whereas CHS and PAL mRNA levels rose by 60‐fold and 17‐fold, respectively, after 12 h exposure at the highest dose rate and 12 h of development. Our results indicate that nicotinamide or trigonelline do not function as signalling compounds for CHS and PAL gene expression. Elevated nicotinamide and trigonelline levels occur in response to UV‐B, but only at UV‐B doses high enough to cause oxidative stress.
Amino Acid Sequence, Binding Properties and Evolutionary Relationships of the Basic Liver Fatty‐Acid‐Binding Protein from the Catfish Rhamdia sapo
Authors: DI Pietro Santiago M. , Dell'angelica Esteban C. , Veerkamp Jacques H. , Sterin‐Speziale Norma , Santomé José A.
Published: June 2004

The complete amino acid sequence of a basic liver fatty acid‐binding protein (L‐FABP) from catfish (Rhamdia sapo) was determined. Alignment of sequences shows that it has more similarity to chicken basic L‐FABP than to mammalian L‐FABP. The phylogenetic analysis suggests that basic L‐FABP from catfish, chicken and iguana diverged from the mammalian protein before the fish‐tetrapod divergence, thus implying that the two types are encoded by different genes. Supporting this conclusion, a 14‐kDa protein, structurally closely related to mammalian L‐FABP, was isolated from catfish intestine, indicating the presence of the two genes in the same species. The catfish basic L‐FABP binds only one fatty acid/molecule, while mammalian L‐FABP bind two. The former has more affinity for trans‐parinaric acid than for cis‐parinaric acid, in constrat to the latter proteins.
Design and NMR Study of an Immobile DNA Four‐Way Junction Containing 38 Nucleotides
Authors: Overmars Franc J. J. , Lanzotti Virginia , Galeone Aldo , Pepe Antonietta , Mayol Luciano , Pikkemaat Jeroen A. , Altona Cornelis
Published: June 2004

The DNA Holliday junction is a central intermediate in genetic recombination. We have designed and synthesized a DNA oligomer, J1a, as a model compound for the Holliday junction suitable to be studied by NMR spectroscopy and future molecular modelling. The design was based on a 46‐base oligomer, J4, previously studied by Pikkemaat, J. A., van den Elst, H., van Boom, J. H. & Altona, C. [Biochemistry 33, 14896–14907 (1994)], including the propensity to undergo a self‐folding process to give a four‐way junction in which three of the four arms are capped with a hairpin loop. J1a, however, is considerably shortened by eight bases and thus contains only 38 residues which significantly facilitates the proton resonance assignments. The base sequence at the branch point is identical to that in J4. 1H‐NMR data clearly point to the presence of three hairpin loops in J1a and show that the double‐helical arms adopt the B‐DNA form. Quasicontinuous pairwise stacking between helical arms to give a single preferred stacked X‐conformation is evident. The extent of folding into this stacked conformation is strongly dependent upon the magnesium concentration. Full Watson‐Crick base pairing at the branch point is completely preserved. The A/D‐stacking preference of the small junction is the same as that exhibited by J4.
Cell Adhesion Properties of Hemolin, an Insect Immune Protein in the Ig Superfamily
Authors: Bettencourt Raul , Lanz‐Mendoza Humberto , Lindquist Katarina Roxström , Faye Ingrid
Published: June 2004

The isolation of antibacterial peptides from the giant silkmoth Hyalophora cecropia has opened the area of animal antibiotics [Boman, H. G. (1991) Cell 65, 205–207] and the study of insect immune genes has revealed striking similarities to many immune response genes in mammals [Hultmark, D. (1994) Nature 267, 116–117]. However, the molecules and mechanisms behind primordial immune recognition are not understood. One candidate for one such recognition molecule is hemolin, a 48‐kDa immunoglobulin‐related protein first isolated from H. cecropia, where it is up‐regulated upon infection and secreted into the hemolymph. Hemolin was shown to bind to bacteria and to hemocytes, giving rise to changes in hemocyte adhesiveness and intracellular phosphorylation patterns [Faye, I. & Kanost, M.(1997) in Molecular mechanisms of immune responses in insects (Brey P. T. & Hultmark D., eds) Chapman and Hall, London]. In the present publication, we give evidence for the presence of a 52‐kDa membrane form of hemolin on hemocytes, based on flow‐activated cell sorting and membrane protein extractions. In addition we reveal calcium‐dependent homophilic binding properties of hemolin, using hemolin‐coated microspheres. When biotinylated recombinant hemolin was allowed to bind to hemocyte membranes, higher molecular‐mass complexes were formed. Furthermore, we used immunological methods and Northern‐blot analysis to demonstrate the presence of hemolin in embryos and retinal discs, suggesting that hemolin is expressed in several tissues at different developmental stages. These results show novel cell adhesion features of hemolin, corroborating its multifunctional character with putative roles in cellular and humoral immunity and in development.
Transport of Activated Fatty Acids by the Peroxisomal ATP‐binding‐cassette Transporter Pxa2 in a Semi‐Intact Yeast Cell System
Authors: Verleur Nicolette , Hettema Ewald H. , Van Roermund Carlo W. T. , Tabak Henk F. , Wanders Ronald J. A.
Published: June 2004

In the yeast Saccharomyces cerevisiae, fatty acid β‐oxidation is restricted to peroxisomes. Previous studies have shown two possible routes by which fatty acids enter the peroxisome. The first route involves transport of medium‐chain fatty acids across the peroxisomal membrane as free fatty acids, followed by activation within the peroxisome by Faa2p, an acyl‐CoA synthetase. The second route involves transport of long‐chain fatty acids. Long‐chain fatty acids enter the peroxisome via a route that involves activation in the extraperoxisomal space, followed by transport across the peroxisomal membrane. It has been suggested that this transport is dependent upon the peroxisomal ATP‐binding‐cassette transporters Pxa1p and Pxa2p. In this paper we investigated whether Pxa2p is directly responsible for the transport of C18:1‐CoA, a long‐chain acyl‐CoA ester. Using protoplasts in which the plasma membrane has been selectively permeabilised by digitonin, we show that C18:1‐CoA, but not C8:0‐CoA, enters the peroxisome via Pxa2p, in an ATP‐dependent fashion. The results obtained may contribute to the elucidation of the primary defect in the human disease X‐linked adrenoleukodystrophy.
The Glycosomal ATP‐Dependent Phosphofructokinase of Trypanosoma Brucei must have Evolved from an Ancestral Pyrophosphate‐Dependent Enzyme
Authors: Michels Paul A. M. , Chevalier Nathalie , Opperdoes Fred R. , Rider Mark H. , Rigden Daniel J.
Published: June 2004

Trypanosoma brucei contains an ATP‐dependent phosphofructokinase (PFK), located in its glycosomes, which are peroxisome‐like organelles sequestering the majority of its glycolytic enzymes. In this paper, we report the cloning and sequencing of the single‐copy gene encoding this enzyme. Its amino‐acid sequence is more similar to pyrophosphate (PPi)‐dependent PFKs than to other ATP‐dependent PFKs. A phylogenetic analysis suggests that the enzyme must have been derived from a PPi‐dependent ancestral PFK, which changed its phospho‐donor specificity during evolution. The enzyme is no longer capable of using PPi as phospho substrate, nor can it catalyze the reverse reaction as PPi‐PFKs generally can. Moreover, the presence of a high pyrophosphatase activity in the cell renders it unlikely that PPi can function as free‐energy source in present‐day trypanosomes. It remains to be determined which mutations were responsible for the change in phospho‐substrate specificity of the trypanosomatid PFK. As a result of its particular evolutionary history, the T. brucei PFK shows many structural differences, even at the active site, when compared with other ATP‐dependent PFKs. These differences offer great potential for the structure‐based design of trypanocidal drugs.
Three‐Dimensional Solution Structure of Human Angiogenin Determined by 1H,15N‐NMR Spectroscopy — Characterisation of Histidine Protonation States and Pka Values
Authors: Lequin Olivier , Thüring Harald , Robin Michel , Lallemand Jean‐Yves
Published: June 2004

Human angiogenin is a member of the pancreatic ribonuclease superfamily that induces blood vessel formation. Its three‐dimensional solution structure has been determined to high resolution by heteronuclear NMR spectroscopy. 30 structures were calculated, based on a total of 1441 assigned NOE correlations, 64 coupling constants and 50 hydrogen bonds. The backbone atomic rms difference from the mean coordinates is 0.067 ± 0.012 nm and 0.13 nm from the previously determined crystal structure. The side‐chain of Gln117 was found to obstruct the active site as observed in the crystal state. There was no evidence of an alternative open form of angiogenin, although two sets of chemical shifts were observed for some residues, mainly around the active site and in the C‐terminal segment. The topology of the ribonucleolytic active site is described with a particular emphasis on the conformation and protonation of active‐site His residues. The side‐chain of Hisll4 adopts two main conformations in solution. In contrast to pancreatic ribonuclease A, Hisl3 was shown to be more basic than Hisll4, with pKa values of 6.65 and 6.05 respectively. The His47 residue is located in an environment very resistant to protonation with a pKa lower than 4.
The Inhibition of Cathepsin S by its Propeptide — Specificity and Mechanism of Action
Authors: Maubach Gunter , Schilling Klaus , Rommerskirch Winfried , Wenz Ingrid , Schultz Joachim E. , Weber Ekkehard , Wiederanders Bernd
Published: June 2004

The interaction of human recombinant full‐length cathepsin S propeptide (amino acids 16–114) with mature cysteine proteinases was studied with respect to selectivity and pH dependence. The inhibitory capacity was tested towards mature human recombinant cathepsin S, purified cathepsin L from rat and Paramecium tetraurelia, rat cathepsin B, human cathepsin H, and papain. The propeptide of cathepsin S strongly inhibited cathepsin S (Ki= 0.27 nM) and the two cathepsin L species (Kt= 0.36 nM) at neutral pH. Papain, and to a minor extent cathepsin H, hydrolyzed the propeptide of cathepsin S, leading to competition with the hydrolysis of the fluorogenic substrates in the respective assays. Cathepsin B activity was nearly unaffected up to micromolar propeptide concentrations in the assay. The inhibition of cath‐epsin‐L‐like peptidases was diminished with decreasing pH, probably due to dramatic changes in the conformation of the propeptide. This assumption was supported by far‐ultraviolet CD spectroscopy and by the finding of rapid hydrolysis of the cathepsin S propeptide by cathepsin L at pH values less than 5.5.
Comparative Study of Chicken and Human Parathyroid Hormone‐(1–34)‐Peptides in Solution with SDS
Authors: Kanaori Kenji , Takai Michihiro , Nosaka Atsuko Y.
Published: June 2004

Molecular conformations of chicken [cPTH‐(1–34)] and human [hPTH‐(1–34)] parathyroid hormone fragments in aqueous solutions with various concentrations of SDS were investigated by CD, fluorescence and NMR spectroscopy techniques. In the presence of SDS, chicken and human PTH‐(1–34) adopt an α‐helical structure making up 32–38% of all the peptide amino acids. The process of the α‐helical formation of these two fragments is considerably different. The CD spectral change of hPTH‐(1–34) was characteristic of a monotonous increase in the negative peak at 222 nm with increasing SDS concentrations. However, for cPTH‐(1–34) a β‐turn is formed first, followed by α‐helix formation upon an increase in SDS concentrations. The change of the tryptophan fluorescence spectra of cPTH‐(1–34) is well correlated with the changes in CD spectra, suggesting that the side chain of Trp23 is involved in the conformational change from random coil to α‐helix via β‐turn. The three‐dimensional structure of cPTH‐(1–34) with SDS micelle was elucidated by 1H‐NMR at pH 3.8 and 300 K, with the combined use of distance geometry and restrained molecular dynamics calculations. NMR results indicated that it contains two helices encompassing residues 7–12 and 24–30, respectively. The C‐terminal helix in the residue range of 24–30 is amphiphilic, which is stabilized by the hydrophobic interactions among Trp23, Leu24 and Lys27.
Anthocyanin 5‐Aromatic Acyltransferase from Gentiana Triflora
Authors: Fujiwara Hiroyuki , Tanaka Yoshikazu , Fukui Yuko , Nakao Masahiro , Ashikari Toshihiko , Kusumi Takaaki
Published: June 2004

Acylation with hydroxycinnamic acids stabilizes anthocyanins and makes their colour bluer (bathochromic shift). We purified to homogeneity one acylation enzyme, hydroxycinnamoyl‐CoA:anthocyanidin 3,5‐diglucoside 5‐O‐glucoside‐6′″‐O‐hydroxycinnamoyltransferase, from blue petals of Gentiana triflora. It is a single polypeptide protein of 52 kDa with a pi of 4.6. It catalyzes the transfer of the p‐coumaric acid and caffeic acid from their CoA esters to the 5‐glucosyl moiety of anthocyanidin 3,5‐diglucosides but could not use malonyl‐CoA as an acyl donor. Neither anthocyanidin 3‐monoglucoside nor anthocyanins aromatically acylated at the 3‐glucosyl moiety could be acylated by this enzyme. Aromatic acylation of anthocyanidin 3,5‐diglucoside by this enzyme caused a bathochromic shift and increased pigment stability in neutral to weakly basic pH. Other anthocyanins from the petals of G. triflora were isolated and their structures were determined by fast‐atom‐bombardment MS and NMR. The biosynthetic pathway of genti‐odelphin, a diacylated anthocyanin accumulating in G. triflora petals, is proposed on the basis of these results.
SP‐22 is a Thioredoxin‐Dependent Peroxide Reductase in Mitochondria
Authors: Watabe Shoji , Hiroi Tomoko , Yamamoto Yoshimi , Fujioka Yoko , Hasegawa Hiroyuki , Yago Nagasumi , Takahashp Susumu Y.
Published: June 2004

SP‐22 is a mitochondrial antioxidant protein in bovine adrenal cortex. The protein is homologous to thioredoxin peroxidase and other antioxidant proteins. It protects radical‐sensitive enzymes from oxidative damage by a radical‐generating system (Fe2+/dithiothreitol) in the presence of a small amount of serum. In this study we purified a second mitochondrial protein with M, 11 777, which cooperates with SP‐22 to protect glutamine synthetase and other proteins from Fe2+/dithiothreitol‐mediated damage. Without SP22, the protein had no protecting activity. We determined amino acid and nucleotide sequences of the protein and its cDNA, respectively, and found that it was a protein of the thioredoxin family. The protein, designated as mt‐Trx (mitochondrial thioredoxin), had a presequence composed of 59 amino acids that seemed to be a mitochondrial targeting signal.

Mitochondrial extract prepared from adrenal cortex contained NADPH‐dependent 5,5’dithiobis(2‐ni‐trobenzoic acid) (Nbs2) reductase activity. The enzyme was thought to have thioredoxin reductase activity, since the Nbs2‐reducing activity was stimulated by mt‐Trx. We partially purified the Nbs2 reductase from bovine adrenocortical mitochondria. In the presence of the partially purified reductase, mt‐Trx, and NADPH, SP‐22 showed the activity to protect oxyhemoglobin against ascorbate‐induced damage. Furthermore, with the three protein components (Nbs2 reductase, mt‐Trx, and SP‐22) NADPH was oxidized in the presence of hydrogen peroxide or tert‐butyl hydroperoxide. The oxidation of NADPH was concomitant with the disappearance of an equimolar amount of hydrogen peroxide. Without any one of the protein components no hemoglobin‐protecting and peroxide‐dependent NADPH‐oxidizing activities were observed. From these results we concluded that SP‐22 is thioredoxin‐dependent peroxide reductase or so‐called thioredoxin peroxidase in mitochondria from the adrenal cortex.
Detergent‐Mediated Reconstitution of a Glycosyl‐Phosphatidylinositol‐Protein into Liposomes
Authors: Angrand Muriel , Briolay Anne , Ronzon Frédéric , Roux Bernard
Published: June 2004

A three‐step detergent‐mediated reconstitution has been applied to the incorporation of a glycosyl‐phosphatidylinositol–protein into liposomes. The protein studied was alkaline phosphatase from bovine intestine. Liposomes prepared by dialysis were treated with various amounts of two detergents, either n‐octyl β‐d‐glucoside or Triton X‐100. At different steps of the solubilization process, protein was added and the detergent was removed by hydrophobic resins. The most efficient reconstitutions were obtained with an octyl glucoside concentration corresponding to the onset of liposome solubilization and with a Triton X‐100 concentration leading to partial solubilization of the liposomes. The involvement of the glycosyl‐phosphatidylinositol anchor in alkaline phosphatase reconstitution was demonstrated by the inability of phosphoinositol‐specific phospholipase‐C‐hydrolysed alkaline phosphatase to incorporate into liposomes. Between 70–85% of the protein associated with liposomes were anchored in the outer leaflet of the bilayer, oriented towards the outside of the liposome. The remainder was trapped within the lumen of the liposomes.
Influence of Mutations in Hexose‐Transporter Genes on Glucose Repression in Kluyveromyces Lactis
Authors: Weirich Jorg , Goffrini Paola , Kuger Petra , Ferrero Iliana , Breunig Karin D.
Published: June 2004

The variability of Kluyveromyces lactis strains in sensitivity to glucose is correlated with genetic differences in Kluyveromyces hexose transporter (KHT) genes. The glucose sensitive strain JA6 was shown to contain an additional gene, KHT2, not found in strains that are less sensitive. KHT2 is tandemly arranged with KHT1 which is identical to the low‐affinity transporter gene RAG1, except for the C‐terminus. Sequence analysis indicated that most of KHT2 had been lost by a recombination event between KHT1 and KHT2 generating the chimeric gene RAG1. Recombination between KHT1 and KHT2 was also found in mutants of JA6 selected as 2‐deoxyglucose resistant colonies. These mutants, like khtl kht2 double mutants were unable to grow on glucose when respiration was blocked (Rag− phenotype) and glucose repression was strongly reduced, kht1 or kht2 single mutants of JA6 were Rag+ but still an influence of the kht mutations on glucose repression was detectable. Repression was not affected in a Rag− mutant deleted for the phosphoglucose isomerase gene suggesting that the influence of transporter genes on repression is not caused by a reduction of the glycolytic flux. The data rather suggest that sensitivity to glucose repression is dependent on the rate of glucose uptake.