bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell cycle kinetics, tissue transglutaminase and programmed cell death (apoptosis)
Authors: El Alaoui S. , Mian S. , Lawry J. , Quash G. , Griffin M.
Published: July 2002

Studies were undertaken on a highly metastatic hamster fibrosarcoma cell line with a view to assessing whether cells entering into apoptosis, measured by counting the number of transglutaminase mediated detergent insoluble envelopes, has any synchrony with a particular phase of the cell cycle. A double exposure of thymidine was used to block cells in early S‐phase. Flow cytometry in combination with [3H]thymidine incorporation into DNA was used to assess the degree of synchrony and progression through the different phases of cell cycle. The apoptotic index was found to be at its maximum in mid‐S‐phase, Measurement of transglutaminase activity in each phase of the cell cycle indicated that the specific activity was also at its greatest during mid S‐phase. The level of enzyme was relatively unchanged throughout the cell cycle indicating that the regulation of transglutaminase activity occurs primarily through effects on catalytic activity rather than enzyme synthesis.
Production of monoclonal antibodies that inhibit ADP‐ribosylation of small GTP‐binding proteins catalyzed by Clostridium botulinum ADP‐ribosyltransferase C3
Authors: Toratani Satoshi , Sekine Nobuyuki , Katada Toshiaki , Yokosawa Hideyoshi
Published: July 2002

Four monoclonal antibodies that inhibited ADP‐ribosylation of 23 kDa protein(s) of ascidian eggs catalyzed by Clostridium botulinum ADP‐ribosyltransferase C3 were produced. They also inhibited C3‐catalyzed ADP‐ribosylation of the 24 kDa protein of rat liver cytosol. By the immunoprecipitation technique, it was found that they recognized small GTP‐binding proteins of ascidian eggs and mammalian brains, but did not interact with the rat brain activator of the ADP‐ribosyltransferase reaction. The antibody can also immunoprecipitate recombinant Rho A irrespective as to whether the Rho A is the GDP‐bound form or the GTPrS‐bound form. Thus the antibodies are novel and useful tools in analyzing the physiological roles of the Rho family of GTP‐binding proteins.
Differential inhibition by cyclosporins of primary‐active ATP‐dependent transporters in the hepatocyte canalicular membrane
Authors: Böhme Matthias , Büchler Markus , Müller Michael , Keppler Dietrich
Published: July 2002

The distinct ATP‐dependent transporters for taurocholate, leukotriene C4, and daunorubicin, studied in rat liver canalicular membrane vesicles, are sensitive to inhibition by cyclosporin A and its non‐immunosuppressive analog PSC 833. K
i values for cyclosporin A were 0.2, 3.4 and 1.5 μM for the transport of taurocholate, leukotriene C4, and daunorubicin, respectively. The corresponding K
i values for PSC 833 were 0.6, 29, and 0.3 μM. Both inhibitors were competitive with respect to the three substrates. The cyclosporins serve as new and potent tools to interfere with different potency with the distinct ATP‐dependent export carriers in the hepatocyte canalicular membrane.
Kinetic and structural characterization of an intermediate in the biomineralization of bacterioferritin
Authors: Le Brun Nick E. , Wilson Michael T. , Andrews Simon C. , Guest John R. , Harrison Pauline M. , Thomson Andrew J. , Moore Geoffrey R.
Published: July 2002

The mechanism by which iron‐storage proteins take up and oxidise iron(II) is not understood. We show by rapid‐kinetic and EPR measurements that iron uptake, in vitro, by a bacterial iron‐storage protein, bacterioferritin, involves at least three kinetically distinguishable phases: phase 1, the binding of Fe(II) ions, probably at a dimeric iron ferroxidase centre; phase 2, oxidation of the Fe(II) dimer and production of mononuclear Fe(III); and phase 3, iron core formation.
Reversible inactivation of endothelial nitric oxide synthase by NG‐nitro‐l‐arginine
Authors: Mayer Bernd , Schmid Martin , Klatt Peter , Schmidt Kurt
Published: July 2002

N
G‐Methyl‐l‐arginine (L‐NMA) and N
G‐nitro‐l‐arginine (L‐NNA) inhibited NO‐induced cGMP accumulation in porcine aortic endothelial cells with half‐maximally effective concentrations of 15 and 3.4 μM, respectively. The effects of both compounds were reversible, but the L‐NNA‐induced inhibition was only reversed by wash‐out in the presence of 1 mM l‐arginine. In short‐term incubations (45 s) of membrane fractions, L‐NMA and L‐NNA exhibited similar potencies to inhibit endothelial NO synthase, but L‐NNA was markedly more potent than L‐NMA after prolonged incubation periods (⩾ 3 min) due to induction of a pronounced, reversible enzyme inactivation.
Cytochrome d axial ligand of the bd‐type terminal quinol oxidase from Escherichia coli
Authors: Tsubaki Motonari , Uno Tadayuki , Hori Hiroshi , Mogi Tatsushi , Nishimura Yoshifumi , Anraku Yasuhiro
Published: July 2002

Using various spectroscopic techniques, we studied the structure of the dioxygen reduction site of the bd‐type terminal quinol oxidase in the aerobic respiratory chain of Escherichia coli. Resonance Raman and FT‐IR spectroscopies identified the v(Fe2+‐CO) and v(C‐O) stretching frequencies at 471 and 1980.7 cm−1, respectively, at the cytochrome d center of the dithionite‐reduced CO‐bound enzyme. The CO ligation in the cytochrome bd complex is considerably different from those of the heme‐copper terminal oxidases. Anaerobic addition of NO to the air‐oxidized enzyme caused an exchange of cytochrome d‐bound dioxygen with NO leading to an appearance of cytochrome d‐NO EPR signal. But there is no superhyperfine structure originating from the cytochrome d proximal 14N ligand in the central resonance of the NO EPR signal. These results suggest that cytochrome d axial ligand of the cytochrome bd complex is likely a histidine residue in an anomalous condition or other than a histidine residue and, therefore, the molecular structure around the dioxygen‐binding site is different from that of the heme‐copper terminal oxidases.
Over‐expression of protein kinase C‐α enhances platelet‐derived growth factor‐ and phorbol ester‐ but not calcium ionophore‐induced formation of prostaglandins in NIH 3T3 fibroblasts
Authors: Finkenzeller Günter , Totzke Frank , Fitzke Edith , Marmé Dieter , Dieter Peter
Published: July 2002

Over‐expression of human protein kinase C‐α in murine NIH 3T3 fibroblasts is associated with an increased platelet‐derived growth factor‐ and phorbol ester‐mediated formation of prostaglandins, whereas the calcium ionophore‐induced release of arachidonic acid metabolites is unaffected; however, the differences of arachidonic acid and prostaglandin formation are much more pronounced with platelet‐derived growth factor than with phorbol ester. Platelet‐derived growth factor induces an identical elevation of intracellular free calcium in control and protein kinase C‐α over‐expressing cells; the phorbol ester has no effect on intracellular free calcium in both cell lines. These results demonstrate that protein kinase C‐α may couple to arachidonic acid cascade in NIH 3T3 fibroblasts.
Flash induced XANES spectroscopy for the Ca‐depleted Mn‐cluster in the photosynthetic O2‐evolving enzyme
Authors: Ono Taka-aki , Noguchi Takumi , Inoue Yorinao , Kusunoki Masami , Yamaguchi Hirotaka , Oyanagi Hiroyuki
Published: July 2002

Flash‐induced changes of the Mn K‐edge absorption spectra have been studied in the oxygen‐evolving complex depleted of Ca. The Mn K‐edge energy for the Ca‐depleted S1‐state was lower by 1.5 eV than that for the normal S1‐state. The K‐edge energy upshifted by 1 eV after one flash, indicative of an oxidation of Mn. After two flashes, the K‐edge was elevated as well by 0.4 eV, and then reached a steady‐state high level after continuous illumination where the K‐edge energy was higher by 0.9 eV than that after one flash. The results indicate that the Mn‐cluster and/or its direct ligand could be oxidized up to two electrons but further events are blocked.
Monoclonal anti‐biotin antibodies simulate avidin m the recognition of biotin
Authors: Bagçi Hasan , Kohen Fortune , Kusçuoglu Unsal , Bayer Edward A. , Wilchek Meir
Published: July 2002

The sequence of the VH gene of a monoclonal anti‐biotin antibody was determined. Biotin‐binding motifs, similar to those in avidin and streptavidin, were identified in complementarity determining regions 2 and 3, suggesting that natural selection of functional motifs may occur in unrelated protein types.
Temporal nature of the promoter and not relative strength determines the expression of an extensively processed protein in a baculovirus system
Authors: Sridhar Padma , Panda Amulya K. , Pal Rahul , Talwar Gursaran P. , Hasnain Seyed E.
Published: July 2002

We demonstrate that the expression of extensively modified and secreted heterologous proteins synthesized in the baculovirus expression vector system (BEVS) depends on the temporal nature of the promoter transcribing the foreign gene. The β subunit of the human chorionic gonadotropin, an extensively modified secretory glycoprotein hormone was expressed under the transcriptional control of the AcNPV basic protein gene promoter (MP) and the polyhedrin gene promoter (POL), respectively. MP, activated late in the infection cycle, is a weaker promoter when compared to the stronger very late POL promoter. Levels of secretion, immunoreactivity and bioactivity of recombinant proteins, βhCGMP and βCGPOL. synthesized under control of the MP and POL promoter were compared. Secretion of βhCGMP was relatively higher. Enzymatic analysis revealed that the synthesized protein was sialylated. Receptor binding assays and testosterone stimulation assays in a mouse Leydig cell system demonstrated that on a unit protein basis, βhCGMP was biologically more active than βhCGPOL.
The ‘non‐exchangeable’ nucleotides of F1‐F0)ATP synthase
Authors: Harris David A.
Published: July 2002

The F1‐Fo ATP synthase bears 6 nucleotide binding sites, only 3 of which turn over during catalysis. The remaining 3 are occupied by slowly exchanging ATP in vivo, although at least 1 molecule is generally lost on isolation of the enzyme in the absence of nucleotide. It is proposed that the function of the slowly exchanging (NC) nucleotides is to participate in catalysis, the terminal phosphate of the bound ATP acting as an acid catalyst in the cleavage/synthesis of the phosphate anhydride bond in the catalytic sites. Such a role has been demonstrated for the bound pyridoxal phosphate moiety in glycogen phosphorylase. Evidence is presented that (i) the NC nucleotide spans the interface between an α subunit and its partner β, interacting near the catalytic binding site on β; (ii) the phosphate moieties of the catalyzed and NC nucleotide are close in space; and (iii) occupation of the NC nucleotide sites promotes ATP hydrolysis by F1 or its subfragments. All of these findings are required by the proposed mechanism. Relationships between phosphorylase and F1 structures are discussed.
Dual relationships of xylitol and alcohol dehydrogenases in families of two protein types
Authors: Persson Bengt , Hallborn Johan , Walfridsson Mats , Hahn-Hägerdal Bärbel , Keränen Sirkka , Penttilä Merja , Jörnvall Hans
Published: July 2002

Xylitol dehydrogenase encoded by gene XYL2 from Pichia stipitis is a member of the medium‐chain alcohol dehydrogenase family, as evidenced by the domain organization and a distant homology (24% residue identity with the human class Iγ1 alcohol dehydrogenase). Much of a loop structure is missing, like in mammalian sorbitol and prokaryotic threonine dehydrogenases, many additional differences occur, and relationships are closest with the sorbitol dehydrogenase, the equivalence of which in P. stipitis may actually be the xylitol dehydrogenase. A second P. stipitis gene, also cloned and corresponding to a xylitol dehydrogenase, is highly different from XYL2, but encodes an enzyme with structural properties typical of the short‐chain dehydrogenase family, which also contains an alcohol dehydrogenase (from Drosophila). Thus, yeast xylitol dehydrogenases, like alcohol and polyol dehydrogenases from other sources, have dual derivations, combining similar enzyme activities in separate protein families. In contrast to the situation with the other enzymes, both forms of xylitol dehydrogenase are present in one organism.
A pH induced two‐dimensional crystal of membrane‐bound Na+,K+‐ATPase of dog kidney
Authors: Tahara Yoshikazu , Ohnishi Shun-ichi , Fujiyoshi Yoshinori , Kimura Yoshiaki , Hayashi Yutaro
Published: July 2002

Two‐dimensional crystals of membrane‐bound Na+,K+‐ATPase were formed in acidic media and their qualities were investigated by electron cryo‐microscopy as well as by conventional electron microscopy. At pH 4.8 in sodium citrate buffer, the best crystallization condition, more than 80% of membranes formed crystals. The high ratio allowed high‐resolution images to be taken by electron cryo‐microscopy. Image processing revealed that they had unique lattice constants (a = 108.7 Å, b = 66.2 Å, γ = 104.2°) and had few defects in the crystalline arrays. The reconstituted Fourier map of the ice‐embedded crystal showed that there are two high contrast parts in one unit cell.
N‐Ethylmaleimide‐sensitive mutant (βVal‐153→Cys) Escherichia coli F1‐ATPase: Cross‐linking of the mutant β subunit with the α subunit
Authors: Iwamoto Atsuko , Orita-Saita Yuji , Maeda Masatomo , Futai Masamitsu
Published: July 2002

A β subunit mutation, βVal‐153→Cys, in the glycine‐rich sequence (phosphate‐binding loop) of Escherichia coli F1 was constructed. Like vacuolar‐type ATPase, the mutant enzyme was inhibited by N‐ethylmaleimide (NEM) and labeled with [14C]NEM. The inhibition and labeling were prevented by ATP. m‐Maleimidobenzoyl‐N‐hydroxysuccinimide (MBS) (3 μM) almost completely inhibited the mutant enzyme, and cross‐linked one pair of α and β subunits. These results suggest that the interaction of the domain near βVal‐153 with the α subunit is essential for catalytic cooperativity of the enzyme and that βVal‐153 is within 10 Å of the α subunit.
Phosphorylation of synapsin I and MARCKS in nerve terminals is mediated by Ca2+ entry via an Aga‐GI sensitive Ca2+ channel which is coupled to glutamate exocytosis
Authors: Coffey Eleanor T. , Sihra Talvinder S. , Nicholls David G. , Pocock Jennifer M.
Published: July 2002

Ca2+ entry is a prerequisite for both exocytosis and the phosphorylation of synapsin I and MARCKS proteins in mammalian cerebrocortical synaptosomes. The novel spider toxin Aga‐GI completely blocks KCl‐evoked glutamate exocytosis but only partially inhibits KCl‐evoked cytoplasmic Ca2+ elevations, thus revealing at least two pathways for KCl‐induced Ca2+ entry. Aga‐GI completely attenuates KCl‐induced phosphorylation of synapsin I and MARCKS proteins. We therefore conclude that both exocytosis and the phosphorylation of synapsin I and MARCKS proteins are specifically coupled to Ca2+ entry via a subset of voltage dependent Ca2+ channels at the nerve terminal which are sensitive to Aga‐GI.
Structural similarity of plant chitinase and lysozymes from animals and phage
Authors: Holm Liisa , Sander Chris
Published: July 2002

A search in the database of known three‐dimensional protein structures with the structure of a plant endochitinase revealed a subtle but unambiguous similarity to lysozymes from animals and phages. An evolutionary connection between plant endochitinases and lysozymes is supported by similar overall topology of fold, overlapping substrate specificities and remarkable conservation of some sequence and architectural detail around the active site. Much of the knowledge about lysozyme can now be extended by analogy to endochitinase. New insights into the mechanism of endochitinase are expected to stimulate genetic engineering studies into plant defense mechanisms against pests and pathogens.
Epidermal growth factor induces acrosomal exocytosis in bovine sperm
Authors: Lax Y. , Rubinstein S. , Breitbart H.
Published: July 2002

At the time of fertilization mammalian spermatozoa undergo a Ca2+‐dependent exocytotic event, which is known as the acrosome reaction (AR). We describe here that EGF‐receptor (EGFR) is localized in the head of bull spermatozoa and that epidermal growth factor (EGF) can induce the occurrence of the AR in its typical dose‐dependent manner. Previously we showed that protein kinase C (PKC) is involved in the cascade leading to AR in bull spermatozoa. Here, we show that PKC is involved in the mechanism in which EGF exerts its effect on AR. These findings together with our results which show inhibition of AR by tyrosine‐phosphorylation inhibitors, indicate that ejaculated bull sperm contain a typical 170‐kDa EGFR which is active in the mechanism leading to AR.
A comparison of demethoxyviridin and wortmannin as inhibitors of phosphatidylinositol 3‐kinase
Authors: Woscholski Ruediger , Kodaki Tsutomu , McKinnon Murray , Waterfield Michael D. , Parker Peter J.
Published: July 2002

The mammalian Ptdlns 3‐kinase is shown to be inhibited by low nanomolar concentrations of demethoxyviridin, an antifungal agent structurally related to wortmannin. The inhibitory potency of both compounds could be observed in purified Ptdlns 3‐kinase whether or not the regulatory subunit (p85α) was present, suggesting that the inhibitors bind to the catalytic subunit (p110) of the Ptdlns 3‐kinase. These inhibitors also show similar potency against the intrinsic p85‐phosphorylating activity of the p110‐kinase. However, the structurally related Ptdlns 3‐kinase from Saccharomyces cerevisae (Vps34p) is not inhibited by either compound. Both inhibitors target the mammalian Ptdlns 3‐kinase in vitro and in vivo, implying that these compounds should be useful in suppressing Ptdlns 3‐kinase in mammalian systems. The inhibitors did not affect the mammalian Ptdlns 4‐kinase, but they are able to inhibit a membrane‐associated Ptdlns 4‐kinase from Schizosacchromyces pombe.
cDNAs encoding spinach stromal and thylakoid‐bound ascorbate peroxidase, differing in the presence or absence of their 3′‐coding regions
Authors: Ishikawa Takahiro , Sakai Kosuke , Yoshimura Kazuya , Takeda Toru , Shigeoka Shigeru
Published: July 2002

Two cDNA clones encoding stromal (SAP28) and thylakoid‐bound (SAP22) ascorbate peroxidase were isolated from a spinach cDNA library constructed by greening cotyledons. The SAP22 and SAP28 contained an open reading frame encoding mature protein of 295 and 345 amino acids with calculated molecular mass of 32 239 Da and 37 710 Da, respectively, preceded by the common transit peptides of 70 amino acid residues. Interestingly, the N‐terminal 364 amino acids of SAP22 were 100% identical with SAP28 except for one C‐terminal amino acid residue (Asp‐365), and the C‐terminal of SAP22, which is the putative transmembrane segment, was 50 amino acids longer than that of SAP28.
Purification and characterization of the cytoplasmic histone acetyltransferase B of maize embryos
Authors: Eberharter Anton , Lechner Thomas , Goralik-Schramel Maria , Loidl Peter
Published: July 2002

From a soluble cellular fraction of maize embryos we purified to apparent homogeneity a cytoplasmic histone acetyltransferase, which matches all criteria for a B‐type enzyme. Using 8 chromatographic steps, we achieved a 6700‐fold purification of an enzymatically active protein with a molecular weight of ∼ 90 kDa. Under denaturing conditions the protein split into 2 components which migrated at 45 and 50 kDa in SDS‐PAGE, suggesting that the native enzyme is a heterodimer. The purified enzyme was characterized in terms of physicochemical and kinetic properties, and substrate specificity. It was specific for histone H4, leading to acetylation of non‐acetylated H4 subspecies into the di‐acetylated state in vitro. Its activity was coincident with the intensity of DNA replication in meristematic cells during embryo germination. We established an electrophoretic system under non‐denaturing conditions for detection of enzyme activity within the gel matrix; in combination with second dimension SDS‐PAGE the procedure allowed the unambiguous identification of histone acetyltransferase, even in crude enzyme preparations.
The polyomavirus major capsid protein VP1 interacts with the nuclear matrix regulatory protein YY1
Authors: Palková Zdena , Španielová Hana , Gottifredi Vanesa , Hollanderová Dana , Forstová Jitka , Amati Paolo
Published: July 2002

Polyomavirus reaches the nucleus in a still encapsidated form, and the viral genome is readily found in association with the nuclear matrix. This association is thought to be essential for viral replication. In order to identify the protein(s) involved in the virus‐nuclear matrix interaction, we focused on the possible roles exerted by the multifunctional cellular nuclear matrix protein Yin Yang 1 (YY1) and by the viral major capsid protein VP1. In the present work we report on the in vivo association between YY1 and VP1. Using the yeast two‐hybrid system we demonstrate that the VP1 and YY1 proteins physically interact through the D‐E region of VP1 and the activation domain of YY1.
Cytokine‐mediated cPLA2 phosphorylation is regulated by multiple MAPK family members
Authors: Geijsen Niels , Dijkers Pascale F , Lammers Jan-Willem J , Koenderman Leo , Coffer Paul J
Published: July 2002

Cytosolic phospholipase A2 (cPLA2) plays a critical role in various neutrophil functions including the generation of leukotrienes and platelet‐activating factor release. Enzyme activity is regulated both by translocation to the membrane in a Ca2+‐dependent manner and serine phosphorylation by members of the mitogen‐activated protein kinase (MAPK) family. In this report, we have investigated the role of granulocyte/macrophage colony‐stimulating factor (GM‐CSF)‐mediated signalling pathways in the regulation of cPLA2. GM‐CSF‐induced cPLA2 phosphorylation was not affected by pharmacological inhibition of p38 MAPK, phosphatidylinositol 3‐kinase or Src. However, inhibition of extracellular signal‐regulated kinase (ERK) MAPK activation resulted in a partial inhibition of cPLA2 phosphorylation, revealed in a slower onset of phosphorylation. A cell line stably transfected with the GM‐CSF receptor was used to further analyze GM‐CSF‐mediated cPLA2 phosphorylation. Mutation of tyrosine residues 577 and 612 resulted in a delayed cPLA2 phosphorylation similar to the pharmacological ERK inhibition. Furthermore, inhibition of p38 MAPK in cells bearing the double mutant βc577/612 completely abrogated GM‐CSF‐induced cPLA2 phosphorylation. We conclude that GM‐CSF can mediate cPLA2 phosphorylation through the redundant activation of both p38 and ERK MAP kinases.
Cleavage of lamin‐like proteins in in vivo and in vitro apoptosis of tobacco protoplasts induced by heat shock
Authors: Chen Hao-Ming , Zhou Jun , Dai Yao-Ren
Published: July 2002

Apoptosis in heat shock‐treated tobacco protoplasts was evidenced by DNA fragmentation, flow cytometric analysis and activation of caspase 3‐like protease. Furthermore, an in vitro apoptosis system was established which reproduced the apoptotic events. Western blotting analysis using an antibody against lamin A and C showed that in both in vivo and in vitro systems lamin‐like proteins were cleaved into a 35‐kDa fragment, and that lamin‐like protein degradation precedes DNA fragmentation. Moreover, we found a 22.8‐fold increase in caspase 6‐like activity in cytosol of heat‐treated protoplasts as compared with the control.
Investigation of the molecular assembly of β‐cell KATP channels
Authors: Mikhailov Michael V , Mikhailova Ellina A , Ashcroft Stephen J.H
Published: July 2002

We have investigated the protein interactions involved in the assembly of pancreatic β‐cell ATP‐sensitive potassium channels. The channels are a heterooligomeric complex of pore‐forming Kir6.2 subunits and sulfonylurea receptor (SUR1) subunits. SUR1 belongs to the ATP binding cassette (ABC) family of proteins and has two nucleotide binding domains (NBD1 and NBD2) and 17 putative transmembrane (TM) sequences. Previously we showed that co‐expression in a baculovirus expression system of two parts of SUR1 divided at Pro1042 between TM12 and 13 leads to restoration of glibenclamide binding activity, whereas expression of either individual N‐ or C‐terminal domain alone gave no glibenclamide binding activity [M.V. Mikhailov and S.J.H. Ashcroft (2000) J. Biol. Chem. 275, 3360–3364]. Here we show that the two half‐molecules formed by division of SUR1 between NBD1 and TM12 or between TM13 and 14 also self‐assemble to give glibenclamide binding activity. However, deletion of NBD1 from the N‐part of SUR1 abolished SUR1 assembly, indicating a critical role for NBD1 in SUR1 assembly. We found that differences in glibenclamide binding activity obtained after co‐expression of different half‐molecules are attributable to different amounts of binding sites, but the binding affinities remained nearly the same. Simultaneous expression of Kir6.2 resulted in enhanced glibenclamide binding activity only when the N‐half of SUR1 included TM12. We conclude that TM12 and 13 are not essential for SUR1 assembly whereas TM12 takes part in SUR1 Kir6.2 interaction. This interaction is specific for Kir 6.2 because no enhancement of glibenclamide binding was observed when half‐molecules were expressed together with Kir4.1. We propose a model of KATP channel organisation based on these data.
Functional characterization of the human high‐affinity choline transporter1
Authors: Okuda Takashi , Haga Tatsuya
Published: July 2002

Na+‐dependent, high‐affinity choline uptake in cholinergic neurons is the rate‐limiting step in acetylcholine synthesis. Here we report the molecular cloning and functional characterization of the human high‐affinity choline transporter (hCHT1). The hCHT1 exhibits significant homology with known members of the Na+‐dependent glucose transporter family, but not with members of the neurotransmitter transporter family. The human CHT1 gene is 25 kb in length with 9 exons and was assigned to chromosome II at position IIq11–12. Northern blot analysis showed that a 5.4 kb hCHT1 transcript was expressed exclusively in tissues containing cholinergic neurons. When expressed in Xenopus oocytes, the human clone induced Na+‐ and Cl−‐dependent, high‐affinity choline uptake, which was sensitive to the specific inhibitor hemicholinium‐3, with a K
i of 1.3 nM. The hCHT1‐mediated choline uptake increased with increasing concentrations of choline, Na+ and Cl−, with EC50 values of 2.0 μM, 76 mM, and 48 mM, and with apparent Hill coefficients of 1, 2.5 and 2.3, respectively.
Chicken neuropeptide Y receptor Y2: structural and pharmacological differences to mammalian Y21
Authors: Salaneck Erik , Holmberg Sara K.S. , Berglund Magnus M. , Boswell Timothy , Larhammar Dan
Published: July 2002

Here we report the molecular cloning of the chicken (Gallus gallus) neuropeptide Y (NPY) receptor Y2, the first non‐mammalian Y2 receptor. It displays 75–80% identity to mammalian Y2 and has a surprisingly divergent cytoplasmic tail. Expression of the receptor protein in a cell line showed that the receptor did not bind the mammalian Y2 selective antagonist BIIE0246. Furthermore, porcine [Leu31, Pro34]NPY, which binds poorly to mammalian Y2, exhibited an unexpectedly high affinity for chicken Y2. In situ hybridisation revealed expression in the hippocampus. Thus, the chicken Y2 receptor exhibits substantial differences with regard to sequence and pharmacological profile in comparison to mammalian Y2 receptors, while the expression pattern in the central nervous system resembles that observed in mammals.
Iron starvation leads to increased expression of Cu/Zn‐superoxide dismutase in Aspergillus
Authors: Oberegger Harald , Zadra Ivo , Schoeser Michelle , Haas Hubertus
Published: July 2002

In a search for iron‐regulated proteins of Aspergillus nidulans and Aspergillus fumigatus a 16‐kDa protein was identified which is about 5‐fold upregulated during iron starvation in both species and which can be approximately 500‐fold enriched by simple one‐step chromatography on Amberlite XAD‐16 resin. N‐terminal protein sequence analysis and cloning of the respective A. nidulans cDNA identified this protein as a Cu/Zn‐superoxide dismutase (SODA). Northern analysis revealed that upregulation of sodA expression occurs at the level of transcript accumulation. This seems to be a specific low iron response and not a general starvation answer since sodA transcript levels do not respond to carbon or nitrogen starvation. In contrast, copper depletion leads to transcriptional downregulation of sodA. Furthermore, sodA expression was found still to be subject to iron regulation in an A. nidulans mutant lacking SREA, a regulator of iron homeostasis, indicating that sodA expression is regulated by an SREA‐independent mechanism. The data presented suggest that SODA plays a protective role under iron deplete conditions.
Structural organization, complete genomic sequences and mutational analyses of the Fukuyama‐type congenital muscular dystrophy gene, fukutin
Authors: Kobayashi Kazuhiro , Sasaki Junko , Kondo-Iida Eri , Fukuda Yoji , Kinoshita Moritoshi , Sunada Yoshihide , Nakamura Yusuke , Toda Tatsushi
Published: July 2002

Fukuyama‐type congenital muscular dystrophy (FCMD) is an autosomal recessive severe muscular dystrophy in combination with cerebral cortical dysplasia. Previously, we identified the gene responsible for FCMD, termed fukutin, through positional cloning. In this study, we have sequenced 131 892 bp of genomic DNA in the region of the fukutin gene on chromosome 9q31 and obtained its complete genomic structure. The fukutin genomic sequence spans approximately 100 kb and is organized into 10 exons (41–6067 bp) and nine introns (1841–21 460 bp). Using these sequence data, we have identified three novel fukutin mutations in FCMD patients. We have also located a putative TATA box in the flanking 5′ region and identified numerous alternatively spliced fukutin mRNA transcripts. Analysis of expressed sequence tag clusters within the region revealed two novel genes upstream of the fukutin gene. These data provide fundamental information to support detailed genetic and functional analyses of the fukutin gene.
Probing the binding sites of exchanged chlorophyll a in LH2 by Raman and site‐selection fluorescence spectroscopies
Authors: Gall Andrew , Robert Bruno , Cogdell Richard J. , Bellissent-Funel Marie-Claire , Fraser Niall J.
Published: July 2002

In this work we have selectively released the 800 nm absorbing bacteriochlorophyll a molecules of the LH2 protein from the photosynthetic bacterium Rhodopseudomonas acidophila, strain 10050, and replaced them with chlorophyll a (Chla). A combination of low‐temperature electronic absorption, resonance Raman and site‐selection fluorescence spectroscopies revealed that the Chla pigments are indeed bound in the B800 binding site; this is the first work that formally proves that such non‐native chlorins can be inserted correctly into LH2.
Effect of side groups on the action of β‐xylosidase from Trichoderma reesei against substituted xylo‐oligosaccharides
Authors: Tenkanen Maija , Luonteri Elina , Teleman Anita
Published: July 2002

The action of β‐xylosidase from Trichoderma reesei against different substituted xylo‐oligosaccharides was studied. The enzyme cleaved off all unsubstituted xylose units from the non‐reducing end of 1,2‐linked uronic acid substituted xylo‐oligosaccharides. Surprisingly, an l‐arabinofuranosyl group linked α‐1,3 to the xylopyranosyl ring was found to protect the β‐1,4‐xylosidic linkage before the substituted xylose unit from being cleaved by the β‐xylosidase. Most probably the 1,3‐linked substituent sterically hinders the hydrolysis. According to the results of the present work, β‐xylosidase of T. reesei is not able to remove all unsubstituted xylose units from the non‐reducing end of substituted xylo‐oligosaccharides, as had been believed previously.
ESTs reveal a multigene family for plant defensins inArabidopsis thaliana
Authors: Epple Petra , Apel Klaus , Bohlmann Holger
Published: July 2002

Plant defensins, formerly named γ‐thionins, are a group of small, cysteine‐rich, basic, and antimicrobial plant proteins. Random sequencing of expressed sequence tags (ESTs) inArabidopsis thalianahas revealed several different plant defensin genes in this plant species which can be grouped into two subfamilies. We have used one EST of each subfamily to study the expression of the corresponding genes inA. thaliana.Pdf2.3is constitutively expressed in seedlings, rosettes, flowers, and siliques and is not inducible in seedlings either by methyl jasmonate, salicylate, ethephon, and silver nitrate or by several different phytopathogenic fungi. The expression of a second gene,Pdf1.2, is in untreated plants only detectable in rosettes. In seedlings, it is inducible by methyl jasmonate, silver nitrate, and different phytopathogenic fungi, notablyFusarium oxysporum f. sp.matthiolae. The regulation ofPdf1.2resembles that of the pathogen‐inducible thionin geneThi2.1.
Ceramide induces apoptosis in PC12 cells
Authors: Hartfield Perry J , Mayne George C , Murray Andrew W
Published: July 2002

The novel lipid second messenger, ceramide, induced apoptosis in PC12 cells as determined morphologically by nuclear appearance and internucleosomal DNA fragmentation. Apoptosis was induced by exogenous C2‐ceramide in a dose‐ and time‐dependent manner. Natural ceramide and C6‐ceramide had a similar effect. This response was specific since the structural analog C2‐dihydroceramide and other related lipids failed to initiate apoptosis. The apoptotic effect of ceramide also depends critically on cell plating density. Furthermore, the peptide inhibitor of interleukin‐1β converting enzyme (ICE)‐like proteases, Z‐VAD.FMK, completely prevented the nuclear changes induced by ceramide, implicating the involvement of ICE‐like protease activation in ceramide‐induced apoptosis in PC12 cells.
Identification of Mel1a melatonin receptors in the human embryonic kidney cell line HEK293: evidence of G protein‐coupled melatonin receptors which do not mediate the inhibition of stimulated cyclic AMP levels
Authors: Conway Shaun , Drew Janice E , Canning Sarah J , Barrett Perry , Jockers Ralf , Strosberg A.Donny , Guardiola-Lemaitre Beatrice , Delagrange Phillipe , Morgan Peter J
Published: July 2002

Binding assays using 2‐[125I]iodomelatonin revealed high‐affinity, guanosine 5′‐O‐(3‐thiotriphosphate) sensitive, melatonin binding sites (B
max 1.1 fmol/mg protein) in the human embryonic kidney cell line HEK293. Competition studies using the selective melatonin receptor antagonist luzindole and RT‐PCR techniques identified these sites as human Mel1a melatonin receptors. Challenge of HEK293 cells with 1 μM melatonin had no effect on forskolin stimulated cyclic AMP levels, whereas in HEK293 cells engineered to stably over‐express the human Mel1a melatonin receptor (B
max>400 fmol/mg protein) melatonin dose‐dependently inhibited stimulated cyclic AMP levels (IC50 7.7 pM). These data may indicate that certain tissues, expressing low levels of G protein‐coupled melatonin receptors, do not display melatonin mediated inhibition of cAMP.
Lysophosphatidic acid as a phospholipid mediator: pathways of synthesis
Authors: Gaits Frédérique , Fourcade Olivier , Le Balle François , Gueguen Geneviève , Gaigé Bernadette , Gassama-Diagne Ama , Fauvel Josette , Salles Jean-Pierre , Mauco Gérard , Simon Marie-Françoise , Chap Hugues
Published: July 2002

From very recent studies, including molecular cloning of cDNA coding for membrane receptors, lysophosphatidic acid (LPA) reached the status of a novel phospholipid mediator with various biological activities. Another strong argument supporting this view was the discovery that LPA is secreted from activated platelets, resulting in its appearance in serum upon blood coagulation. The metabolic pathways as well as the enzymes responsible for LPA production are poorly characterized. However, a survey of literature data indicates some interesting issues which might be used as the basis for further molecular characterization of phospholipases A able to degrade phosphatidic acid.
The arrangement of the transmembrane helices in the secretin receptor family of G‐protein‐coupled receptors
Authors: Donnelly Dan
Published: July 2002

The members of the secretin receptor family of G‐protein‐coupled receptors share no significant sequence similarity to the more familiar rhodopsin‐like family. However, multiple sequence alignment analysis reveals seven hydrophobic regions with significant α‐helical periodicity. Residues that are likely to be buried on the interior of the helical bundle and others that are likely to contact the lipid bilayer are identified. A predicted arrangement of the helical bundle is described in which, by comparison with the arrangement in the rhodopsin family, helices 2 and 7 are more buried within the bundle while helix 3 is more exposed to the lipid bilayer.
Cloning and functional expression of human kynurenine 3‐monooxygenase
Authors: Alberati-Giani Daniela , Cesura Andrea M , Broger Clemens , Warren William D , Röver Stephan , Malherbe Pari
Published: July 2002

Kynurenine 3‐monooxygenase, an NADPH‐dependent flavin monooxygenase, catalyses the hydroxylation of l‐kynurenine to l‐3‐hydroxykynurenine. By hybridization screening using a cDNA probe encoding the entire exon 2 of Drosophila melanogaster kynurenine 3‐monooxygenase, we isolated a 2.0 kb cDNA clone coding for the corresponding human liver enzyme. The deduced amino acid sequence of the human protein consists of 486 amino acids with a predicted molecular mass of 55 762 Da. Transfection of the human cDNA in HEK‐293 cells resulted in the functional expression of the enzyme with kinetic properties similar to those found for the native human protein. RNA blot analysis of human tissues revealed the presence of a major mRNA species of ∼2.0 kb in liver, placenta and kidney.
Continuous hepatocyte growth factor supply prevents lipopolysaccharide‐induced liver injury in rats
Authors: Kaido Toshimi , Yamaoka Shoji , Seto Shin-ichi , Funaki Naomi , Kasamatsu Takayuki , Tanaka Junji , Nakamura Toshikazu , Imamura Masayuki
Published: July 2002

We present a rat model in which continuous supply of hepatocyte growth factor (HGF) prevents liver injury induced by carbon tetrachloride (CCl4) and E. coli 011:B4 lipopolysaccharide (LPS). Rat fibroblasts genetically modified to secrete rat HGF were implanted in syngenic rat spleen 7 days before administration of the hepatotoxins. Rats with HGF‐secreting fibroblasts in the spleen showed a dramatic resistance to CCl4‐ and LPS‐induced liver injury. In the LPS‐induced liver injury model, blood chemical analysis revealed that the increase in serum glutamic oxalacetic transaminase level and the decrease in blood sugar level were remarkably suppressed in rats with HGF‐secreting cells in the spleen. Most importantly, their survival rate was greatly improved compared to other control groups of rats. Thus our results indicate a new role of HGF in liver protection during endotoxemia and convey important clinical implications for developing new therapeutic modalities in the treatment of liver failure caused by endotoxemia.
Tissue‐dependent upregulation of rat uncoupling protein‐2 expression in response to fasting or cold
Authors: Boss Olivier , Samec Sonia , Dulloo Abdul , Seydoux Josiane , Patrick Muzzin  , Giacobino Jean-Paul
Published: July 2002

The control of uncoupling protein‐2 (UCP2) mRNA expression in rat brown adipose tissue (BAT), heart and skeletal muscles was examined. Cold exposure (48 h) increased UCP2 mRNA in BAT, heart and soleus muscle by 2.4‐, 4.3‐ and 2.6‐fold, respectively. Fasting (48 h) had no effect on UCP2 mRNA expression neither in BAT nor in heart, but markedly increased it in skeletal muscles. While the upregulation of UCP2 mRNA in response to cold exposure is in line with a putative uncoupling role for this protein in thermoregulatory thermogenesis, the unexpected upregulation of UCP2 in skeletal muscles in response to fasting seems inconsistent with its role as an uncoupling protein involved in dietary regulation of thermogenesis.
The role of the `Rieske' iron sulfur protein in the hydroquinone oxidation (QP) site of the cytochrome bc1 complex
Authors: Link Thomas A
Published: July 2002

The essential reaction in the widely accepted protonmotive Q‐cycle mechanism of the bc
1 complex is the bifurcation of the electron flow during hydroquinone oxidation at the hydroquinone oxidation (QP) site formed by the `Rieske' iron sulfur protein and by the heme b
L domain of cytochrome b. The `Rieske' [2Fe‐2S] cluster has a unique structure containing two exposed histidine ligands, which are the binding site for quinones. The affinity of the `Rieske' cluster for quinones increases several orders of magnitude upon reduction; this will stabilize semiquinone at the QP site. Based on this affinity change, a reaction scheme is presented which can explain the bifurcation of the electron flow without invoking highly unstable semiquinone species.
Interpretation of the reactivity of peroxidase compounds I and II with phenols by the Marcus equation
Authors: Folkes Lisa K , Candeias Luis P
Published: July 2002

The catalytic cycle of heme peroxidases involves two reactive states, compound I and compound II. Although their reduction potentials at pH 7 are similar, compound I is in general more reactive towards organic substrates than compound II. The different reactivities have until now remained unexplained. In this study, the reactions of compounds I and II of peroxidase from horseradish with phenols were analyzed using the Marcus equation of electron‐transfer. Both reactions exhibit similar reorganization energies, and the different reactivities of the two enzyme states can be ascribed to a higher apparent rate of activationless electron‐transfer in the compound I reactions. This can be attributed to the shorter electron‐tunneling distance on electron‐transfer to the porphyrin radical cation in compound I, compared to electron‐transfer to the iron ion in compound II.
Cooperative thermal transitions of bovine and human apo‐α‐lactalbumins: evidence for a new intermediate state
Authors: Veprintsev Dmitry B , Permyakov Serge E , Permyakov Eugene A , Rogov Vladimir V , Cawthern Kevin M , Berliner Lawrence J
Published: July 2002

The thermal denaturation of bovine and human apo‐α‐lactalbumins at neutral pH has been studied by intrinsic protein fluorescence, circular dichroism (CD), and differential scanning microcalorimetry (DSC) methods. Apo‐α‐lactalbumin possesses a thermal transition with a midpoint about 25–30°C under these conditions (pH 8.1, 10 mM borate, 1 mM EGTA), which is reflected in changes in both fluorescence emission maximum and quantum yield. However, the CD showed a decrease in ellipticity at 270 nm with a midpoint at about 10–15°C, while DSC shows the transition within the region of 15–20°C. The non‐coincidence of transition monitored by different methods suggests the existence of an intermediate state in the course of the thermal denaturation process. This intermediate state is not the classical molten globule state which occurs at higher temperature (i.e. denatured state at these conditions) [D.A. Dolgikh, R.I. Gilmanshin, E.V. Brazhnikov, V.E. Bychkova, G.V. Semisotnov, S.Y. Venyaminov and O.B. Ptitsyn, FEBS Letters, 136 (1981) 311–315] and has physical properties intermediate between the native and molten globule states.
Biochemical characterization, molecular cloning and localization of a putative odorant‐binding protein in the honey bee Apis mellifera L. (Hymenoptera: Apidea)
Authors: Danty E. , Michard-Vanhée C. , Huet J.-C. , Genecque E. , Pernollet J.-C. , Masson C.
Published: July 2002

A honey bee antennal water‐soluble protein, APS2, was purified and characterized as the first Hymenoptera putative odorant‐binding protein. Comparison of its measured M
r (13 695.2±1.6) to that of the corresponding cDNA clone shows it does not undergo any post‐translational modification other than a 19‐residue signal peptide cleavage and formation of three disulfide bridges. These biochemical features are close to those of Lepidoptera odorant‐binding proteins. In situ hybridization experiments demonstrated its specific expression in olfactory areas. Based on its higher expression in the worker than in the drone, ASP2 might be more involved in general odorant than in sex pheromone detection.
Inhibition of HIV‐1 Nef‐induced apoptosis of uninfected human blood cells by serine/threonine protein kinase inhibitors, fasudil hydrochloride and M3
Authors: Okada Harue , Takei Ryouichi , Tashiro Masato
Published: July 2002

The Nef protein of HIV‐1 binds to and induces apoptotic cytolysis of uninfected but activated human peripheral blood mononuclear cells (PBMC) and various cell line cells derived from CD4+ T, CD8+ T and B lymphocytes, macrophages, and neutrophils. The Nef‐induced apoptosis also occurs with blood cells not expressing CD95 (Fas). The Nef‐induced apoptosis as well as Fas‐mediated apoptosis was inhibited by acetyl‐Try‐Val‐Ala‐Asp‐CHO, an IL‐1β converting enzyme (ICE) inhibitor. On the other hand, serine/threonine protein kinase (PK) inhibitors, H‐7, fasudil hydrochloride and M3, inhibited the Nef‐induced apoptosis, and not the Fas‐mediated one, without affecting the cell‐binding activity of Nef and Nef‐binding capacity of the activated cells. Preincubation of the cells with the drugs before being bound by Nef was required for the inhibition of apoptosis. These results suggest that the PK inhibitors specifically act on a cellular protein involved in the upper stream of signal transduction pathway of the Nef‐induced apoptosis, which is different from the Fas‐mediated pathway but meets it upstream of ICE. In addition, the drugs suppressed the cellular activation‐associated cell surface expression of a putative Nef‐binding protein in PBMC, although they had no influence on its expression in cell line cells. These findings suggest the feasibility of clinical use of the PK inhibitors to prevent the development of AIDS by inhibiting the Nef‐induced apoptosis of uninfected blood cells.
A new family of orphan G protein‐coupled receptors predominantly expressed in the brain1
Authors: Valdenaire Olivier , Giller Thomas , Breu Volker , Ardati Ali , Schweizer Anja , Richards J.Grayson
Published: July 2002

The cloning of a cDNA encoding a G protein‐coupled receptor homologous to the endothelin type B receptor, but unable to bind endothelin, was recently reported and termed ETBR‐LP. We report here the isolation of a human cDNA encoding a receptor that is highly related to ETBR‐LP and which was therefore termed ETBR‐LP‐2. Comparison of the two amino acid sequences revealed 68% overall homology and 48% identity. As is the case for ETBR‐LP, the new receptor is strongly expressed in the human central nervous system (e.g. in cerebellar Bergmann glia, cerebral cortex, internal capsule fibers). Membranes of HEK‐293 cells stably expressing ETBR‐LP‐2 did not bind endothelin‐1, endothelin‐2, endothelin‐3, bombesin, cholecystokinin‐8 or gastrin‐releasing peptide.