bluuebunny/crossref_metadata_2025 huggingface dataset:
Synergistic induction of endothelial tissue factor by tumor necrosis factor and vascular endothelial growth factor: functional analysis of the tumor necrosis factor receptors
Authors: Clauss Matthias , Grell Matthias , Fangmann Carmen , Fiers Walter , Scheurich Peter , Risau Werner
Published: July 2002

Tissue factor expression on the surface of endothelial cells can be induced by tumor necrosis factor (TNF) and vascular endothelial growth factor (VEGF) in a synergistic manner. We have investigated the role of the two different TNF receptors for this synergy. Firstly, stimulation of the 60 kDa TNF receptor (TNFR60) by a mutant of TNF specific for TNFR60 induced responses comparable to wild‐type TNF. In contrast, stimulation of TNFR80 by a TNFR80‐specific TNF mutein did not result in enhancement of tissue factor expression even in the presence of a suboptimal TNFR60 triggering. Secondly, we tested neutralizing TNF receptor antibodies for inhibition of tissue factor synthesis induced by VEGF and TNF. A TNFR60‐specific antibody inhibited tissue factor production over a broad range of TNF concentrations, indicating an essential role of TNFR60 in the TNF/VEGF synergy. In contrast, blocking of TNF binding to TNFR80 strongly inhibited TNF‐induced tissue factor expression at low, but less pronounced at high, TNF concentrations. In conclusion, these data are in agreement with a model in which TNFR80 participates in the synergy between VEGF and low concentrations of soluble TNF by passing the ligand to the signalling TNFR60.
Regulation and function of central nervous system chemokines
Authors: Glabinski A.R. , Tani M. , Aras S. , Stoler M.H. , Tuohy V.K. , Ransohoff R.M.
Published: July 2002

In this paper, we discuss the potential involvement of a new family of cytokines, termed chemokines, in CNS inflammatory pathology. Chemokines are a family of proinflammatory cytokines which are able to stimulate target‐cell‐specific directional migration of leukocytes. Because of this feature, chemokines may be potent mediators of inflammatory processes. We have previously reported observations indicating that chemokines may be involved in the process of lesion formation during autoimmune inflammation within CNS, and, in particular, are likely participants in the process of influx of inflammatory cells into the CNS parenchyma. We observed also that mechanical injury of brain and subsequent post‐traumatic inflammation may in part be mediated by chemokines. Chemokines undoubtedly co‐operate with cell‐associated adhesion molecules during recruitment of leukocytes from blood to CNS. The sequential expression of soluble and membrane‐bound signals for leukocyte migration is an intricate process that can be interrupted by a variety of strategies. Our data suggest that chemokines may represent a promising target for future therapy of inflammatory conditions, including CNS inflammation resulting from varied insults.
An unexpectedly large working stroke from chymotryptic fragments of myosin II
Authors: Molloy J.E. , Kendrick-Jones J. , Veigel C. , Tregear R.T.
Published: July 2002

Recent structural evidence indicates that the light chain domain of the myosin head (LCD) bends on the motor domain (MD) to move actin. Structural models usually assume that the actin‐MD interface remains static and the possibility that part of the myosin working stroke might be produced by rotation about the acto‐myosin interface has been neglected. We have used an optical trap to measure the movement produced by proteolytically shortened single rabbit skeletal muscle myosin heads (S‐1(A1) and S‐1(A2)). The working stroke produced by these shortened heads was more than that which the MD‐LCD bend mechanism predicts from the full‐length (papain) S‐1's working stroke obtained under similar conditions. This result indicates that part of the working stroke may be caused by motor action at the actin‐MD interface.
Phosphorylation of MYPT1 by protein kinase C attenuates interaction with PP1 catalytic subunit and the 20 kDa light chain of myosin
Authors: Tóth Attila , Kiss Enikö , Gergely Pál , Walsh Michael P. , Hartshorne David J. , Erdödi Ferenc
Published: July 2002

The effect of phosphorylation in the N‐terminal region of myosin phosphatase target subunit 1 (MYPT1) on the interactions with protein phosphatase 1 catalytic subunit (PP1c) and with phosphorylated 20 kDa myosin light chain (P‐MLC20) was studied. Protein kinase C (PKC) phosphorylated threonine‐34 (1 mol/mol), the residue preceding the consensus PP1c‐binding motif (35KVKF38) in MYPT11–38, but this did not affect binding of the peptide to PP1c. PKC incorporated 2 mol Pi into MYPT11–296 suggesting a second site of phosphorylation within the ankyrin repeats (residues 40–296). This phosphorylation diminished the stimulatory effect of MYPT11–296 on the P‐MLC20 phosphatase activity of PP1c. Binding of PP1c or P‐MLC20 to phosphorylated MYPT11–296 was also attenuated. It is concluded that phosphorylation of MYPT1 by PKC may therefore result in altered dephosphorylation of myosin.
Two‐electron reduction and one‐electron oxidation of organic hydroperoxides by human myeloperoxidase
Authors: Furtmüller Paul Georg , Burner Ursula , Jantschko Walter , Regelsberger Günther , Obinger Christian
Published: July 2002

The reaction of native myeloperoxidase (MPO) and its redox intermediate compound I with hydrogen peroxide, ethyl hydroperoxide, peroxyacetic acid, t‐butyl hydroperoxide, 3‐chloroperoxybenzoic acid and cumene hydroperoxide was studied by multi‐mixing stopped‐flow techniques. Hydroperoxides are decomposed by MPO by two mechanisms. Firstly, the hydroperoxide undergoes a two‐electron reduction to its corresponding alcohol and heme iron is oxidized to compound I. At pH 7 and 15°C, the rate constant of the reaction between 3‐chloroperoxybenzoic acid and ferric MPO was similar to that with hydrogen peroxide (1.8×107 M−1 s−1 and 1.4×107 M−1 s−1, respectively). With the exception of t‐butyl hydroperoxide, the rates of compound I formation varied between 5.2×105 M−1 s−1 and 2.7×106 M−1 s−1. Secondly, compound I can abstract hydrogen from these peroxides, producing peroxyl radicals and compound II. Compound I reduction is shown to be more than two orders of magnitude slower than compound I formation. Again, with 3‐chloroperoxybenzoic acid this reaction is most effective (6.6×104 M−1 s−1 at pH 7 and 15°C). Both reactions are controlled by the same ionizable group (average pK
a of about 4.0) which has to be in its conjugated base form for reaction.
Cellular retinoic acid binding protein is associated with mitochondria
Authors: Ruff Susan J , Ong David E
Published: July 2002

We report that immunohistochemical staining for cellular retinoic acid‐binding protein (CRABP) was restricted to the cytoplasm of cortical cells in bovine adrenal. In contrast, staining for the similar protein, cellular retinol‐binding protein (CRBP), was found throughout these cells. After transfections of CRABP and CRBP into cultured cells, immunofluorescence analyses again revealed cytoplasmic restriction only for CRABP, with a pronounced punctate appearance. Use of organelle‐specific fluorochromes indicated that CRABP immunofluorescence overlaid exactly with the pattern of the mitochondrial‐specific fluorochrome. Confirmation of this association came with subcellular fractionation of the adrenal cortex. CRABP, but not CRBP, co‐sedimented with the mitochondria, a novel finding for a member of this superfamily of cellular lipid‐binding proteins.
Stimulation of carnitine acylcarnitine translocase activity in heart mitochondria from hyperthyroid rats
Authors: Paradies Giuseppe , Ruggiero Francesca Maria , Petrosillo Giuseppe , Quagliariello Ernesto
Published: July 2002

The effect of hyperthyroidism on fatty acid oxidation and on carnitine‐acylcarnitine translocase activity in rat heart mitochondria has been studied. The rates of palmitoylcarnitine supported respiration as well as the carnitine‐palmitoylcarnitine exchange reaction were both stimulated (approx. 36%) in heart mitochondria from hyperthyroid rats. Kinetic analysis of the carnitine‐carnitine exchange reaction showed that thyroid hormone affects the V
max of this process, while having no effect on the K
m values. The level of cardiolipin was significantly higher (approx. 40%) in heart mitoplasts from hyperthyroid rats than from the control rats. It can be concluded that thyroid hormones produce a stimulation of heart mitochondrial carnitine translocase activity and that the basis of this effect is likely an increase in the cardiolipin content.
The SH3 domain of the tight junction protein ZO‐1 binds to a serine protein kinase that phosphorylates a region C‐terminal to this domain
Authors: Balda Maria S. , Anderson James M. , Matter Karl
Published: July 2002

ZO‐1 is a tight junction phosphoprotein partially homologous to a tumor suppressor in Drosophila. The homologous region contains an SH3 domain with an unidentified function. Using fusion proteins containing the SH3 domain and various N‐ and C‐terminal sequences, we tested for association of a kinase with this protein domain in extracts of MDCK cells. We show that the SH3 domain of ZO‐1 binds a serine protein kinase that phosphorylates a region immediately C‐terminal to the SH3 domain. This kinase associates specifically with the SH3 domain of ZO‐1 and appears to be also associated with junctional complexes extracted from MDCK cells.
Evidence for the involvement of cGMP and protein kinase G in nitric oxide‐induced apoptosis in the pancreatic B‐cell line, HIT‐T15
Authors: Loweth Anne C , Williams Gwyn T , Scarpello John H.B , Morgan Noel G
Published: July 2002

Intracellular production of nitric oxide (NO) is thought to mediate the pancreatic B‐cell‐directed cytotoxicity of cytokines in insulin‐dependent diabetes mellitus, and recent evidence has indicated that this may involve induction of apoptosis. A primary effect of NO is to activate soluble guanylyl cyclase leading to increased cGMP levels and this effect has been demonstrated in pancreatic B‐cells, although no intracellular function has been defined for islet cGMP. Here we demonstrate that the NO donor, GSNO, induces apoptosis in the pancreatic B‐cell line HIT‐T15 in a dose‐ and time‐dependent manner. This response was significantly attenuated by micromolar concentrations of a specific inhibitor of soluble guanylyl cyclase, ODQ, and both 8‐bromo cGMP (100 μM) and dibutyryl cGMP (300 μM) were able to fully relieve this inhibition. In addition, incubation of HIT‐T15 cells with each cGMP analogue directly promoted cell death in the absence of ODQ. KT5823, a potent and highly selective inhibitor of cGMP‐dependent protein kinase (PKG), abolished the induction of cell death in HIT cells in response to either GSNO or cGMP analogues. This effect was dose‐dependent over the concentration range of 10–250 nM. Overall, these data provide evidence that the activation of apoptosis in HIT‐T15 cells by NO donors is secondary to a rise in cGMP and suggest that the pathway controlling cell death involves activation of PKG.
A novel 13 kDa cytoplasmic soluble protein is required for the nucleotide (MgATP) modulation of the Na/Ca exchange in squid nerve fibers
Authors: DiPolo Reinaldo , Berberián Graciela , Delgado Daniel , Rojas Hector , Beaugé Luis
Published: July 2002

The Na/Ca exchange is a highly regulated transport mechanism in which MgATP, a powerful modulatory intracellular substrate, has important implications for its function. As occurs with some preparations, in squid axons, nucleotide regulation is lost after membrane vesicle isolation. This has been a significant obstacle in the biochemical characterization of the MgATP effect. An important clue in solving this long‐standing puzzle is presented in this work by showing that prolonged intracellular dialysis of squid axons produces a complete run down of the MgATP effect. Here we report that a soluble cytoplasmic factor isolated from fresh squid axoplasm and brain reconstitutes the MgATP stimulation of the Na‐gradient‐dependent 45Ca uptake in squid optic nerve membrane vesicles. Partial purification of this factor uncovers the presence of a novel 13 kDa soluble cytoplasmic protein (SCPr) which, when microinjected in ATP de‐regulated dialyzed squid axons, completely restores the MgATP stimulation of Nao‐dependent Ca efflux. We propose that in the squid preparation this SCPr constitutes the link between the nucleotide and target effector: the Na/Ca exchanger itself, or other plasma membrane structures which may secondarily interact with the exchanger.
Muscarinic receptor‐stimulated cytosol‐membrane translocation of RhoA
Authors: Keller Jutta , Schmidt Martina , Hussein Baher , Rümenapp Ulrich , Jakobs Karl H.
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Low‐conductance high selective inositol (1,4,5)‐trisphosphate activated Ca2+ channels in plasma membrane of A431 carcinoma cells
Authors: Kiselyov Kirill I. , Mamin Anton G. , Semyonova Svetlana B. , Mozhayeva Galina N.
Published: July 2002

In many cells, activation of receptors coupled to PIP2 turnover results in Ca2+ release from the intracellular stores accompanied by Ca2+ influx across the PM. It is not well established yet whether Ca2+ influx is activated by IP3 or by an unknown signal generated upon Ca2+ store depletion. We report here a single‐channel study of low‐conductance IP3‐activated channels of very high selectivity for Ca2+ in the PM of A431 carcinoma cells. The channels are strongly potential dependent and sensitive to [Ca2+]i within the physiological range. The data obtained argues for IP3 acting directly on plasma membrane Ca2+ channels.
Mutational analysis of the mouse 5‐HT7 receptor: importance of the third intracellular loop for receptor–G‐protein interaction
Authors: Obosi Louis A , Hen René , Beadle David J , Bermudez Isabel , King Linda A
Published: July 2002

The mouse serotonin (5‐HT) receptor subtype, 5‐HT7, belongs to the family of seven transmembrane G‐protein‐coupled receptors. To identify the structural basis for the coupling of 5‐HT7 receptor to GαS we constructed a number of receptor mutants in which amino acid residues were either substituted or deleted from the second and third intracellular loops. Wild‐type and mutant 5‐HT7 receptors were expressed in insect cells using the baculovirus vectors. Two mutant receptor species, 5‐HT7(E325G) and 5‐HT7(K327S), demonstrated markedly impaired abilities to stimulate adenylyl cyclase. The results suggest the importance of the C‐terminal region of the third intracellular loop in receptor–G‐protein interaction and that specific charged residues, E325 and K327, may play a critical role in this interaction.
NMR characterization of the full‐length recombinant murine prion protein, mPrP(23–231)
Authors: Riek Roland , Hornemann Simone , Wider Gerhard , Glockshuber Rudi , Wüthrich Kurt
Published: July 2002

The recombinant murine prion protein, mPrP(23–231), was expressed in E. coli with uniform 15N‐labeling. NMR experiments showed that the previously determined globular three‐dimensional structure of the C‐terminal domain mPrP(121–231) is preserved in the intact protein, and that the N‐terminal polypeptide segment 23–120 is flexibly disordered. This structural information is based on nearly complete sequence‐specific assignments for the backbone amide nitrogens, amide protons and α‐protons of the polypeptide segment of residues 121–231 in mPrP(23–231). Coincidence of corresponding sequential and medium‐range nuclear Overhauser effects (NOE) showed that the helical secondary structures previously identified in mPrP(121–231) are also present in mPrP(23–231), and near‐identity of corresponding amide nitrogen and amide proton chemical shifts indicates that the three‐dimensional fold of mPrP(121–231) is also preserved in the intact protein. The linewidths in heteronuclear 1H–15N correlation spectra and 15N{1H}‐NOEs showed that the well structured residues 126–230 have correlation times of several nanoseconds, as is typical for small globular proteins, whereas correlation times shorter than 1 nanosecond were observed for all residues of mPrP(23–231) outside of this domain.
Topological isomers of human uroguanylin: interconversion between biologically active and inactive isomers
Authors: Chino Naoyoshi , Kubo Shigeru , Kitani Tetsuya , Yoshida Takuya , Tanabe Ryosuke , Kobayashi Yuji , Nakazato Masamitsu , Kangawa Kenji , Kimura Terutoshi
Published: July 2002

The solution structures of the two compounds of human uroguanylin (I and II), which were generated during disulfide bond forming reaction, were found to be topological isomers by 1H‐nuclear magnetic resonance spectroscopy. These isomers are interconvertible in aqueous media at rates which vary with the pH and temperature of the solution. Because compound I is active in the cGMP producing assay, but compound II is not, this interconversion may be useful for evaluating the activity of human uroguanylin both in vivo and in vitro.
Carrier mediated uptake of dehydroascorbate into higher plant plasma membrane vesicles shows trans‐stimulation
Authors: Horemans Nele , Asard Han , Caubergs Roland Julien
Published: July 2002

The activity of the ascorbate (Asc) carrier of purified Phaseolus plasma membranes is demonstrated to be highly stimulated when membrane vesicles are preloaded with Asc. Asc transport is inhibited by DTT but is not affected by glutathione or ferricyanide, indicating that dehydroascorbate (DHA) is the preferred species for uptake. Asc transport in the loaded vesicles showed saturable kinetics with an apparent affinity constant of 24 μM and maximal uptake rate of 94 pmol/mg/min. Addition of DHA stimulated the efflux of Asc molecules from the loaded vesicles. Together these results suggest the presence of an Asc/DHA exchange mechanism in higher plant plasma membranes.
α1‐Syntrophin has distinct binding sites for actin and calmodulin
Authors: Iwata Yuko , Pan Yan , Yoshida Tomokazu , Hanada Hironori , Shigekawa Munekazu
Published: July 2002

Overlay and co‐sedimentation assays using recombinant α1‐syntrophin proteins revealed that two regions of α1‐syntrophin, i.e. aa 274–315 and 449–505, contain high‐affinity binding sites for F‐actin (K
d 0.16–0.45 μM), although only a single high‐affinity site (K
d 0.35 μM) was detected in the recombinant full‐length syntrophin. We also found that actomyosin fractions prepared from both cardiac and skeletal muscle contain proteins recognized by anti‐syntrophin antibody. These data suggest a novel role for syntrophin as an actin binding protein, which may be important for the function of the dystrophin‐glycoprotein complex or for other cell functions. We also found that α1‐syntrophin binds calmodulin at two distinct sites with high (K
d 15 nM) and low (K
d 0.3 μM) affinity.
Regulation of a plant plasma membrane sucrose transporter by phosphorylation
Authors: Roblin Gabriel , Sakr Soulaiman , Bonmort Janine , Delrot Serge
Published: July 2002

The protein phosphatase inhibitor okadaic acid (OA) either provided directly to sugar beet (Beta vulgaris L.) leaf discs or infiltrated in the leaf blade rapidly inhibited sucrose uptake. Methyl okadaic acid, a biologically inactive analogue of OA, had only a marginal effect on uptake. OA inhibited proton‐motive force‐driven uptake of sucrose into plasma membrane vesicles, without affecting their proton permeability. OA did not significantly affect the amount of sucrose transporters present in the vesicles, as estimated by ELISA with specific antibodies. It is concluded that OA directly inhibits the activity of a H+‐sucrose cotransporter of the plant plasma membrane, likely by maintaining it in a phosphorylated form.
Glucagon‐like peptide‐1 receptor expression in Xenopus oocytes stimulates inositol trisphosphate‐dependent intracellular Ca2+ mobilization
Authors: Gromada Jesper , Anker Charlotte , Bokvist Krister , Knudsen Lotte B , Wahl Philip
Published: July 2002

The signal transduction pathway of the cloned human glucagon‐like peptide‐1 (GLP‐1) receptor was studied in voltage‐clamped Xenopus oocytes. Binding of GLP‐1(7–36)amide was associated with cAMP production, increased [Ca2+]i and activation of Ca2+‐dependent Cl− current. The effect of GLP‐1(7–36)amide reflects intracellular Ca2+ mobilization and was suppressed by injection of the Ca2+ chelator BAPTA and the inositol trisphosphate receptor antagonist heparin. The responses were not mimicked by the adenylate cyclase activator forskolin and unaffected by the protein kinase A (PKA) inhibitor Rp‐cAMPS. We conclude that GLP‐1 receptor expression in Xenopus oocytes evokes inositol trisphosphate‐dependent intracellular Ca2+ mobilization independent of the cAMP/PKA signaling pathway.
Human RanBP3, a group of nuclear RanGTP binding proteins1
Authors: Mueller Liz , Cordes* Volker C , Bischoff F.Ralf , Ponstingl Herwig
Published: July 2002

A group of novel human Ran‐binding proteins, RanBP3, was identified using the yeast two‐hybrid system via Ran‐mediated interaction with the nucleotide exchange factor RCC1. Several open reading frames, representing putative alternatively spliced products, were established by cDNA cloning. Two of them, RanBP3‐a and RanBP3‐b, encode nuclear hydrophilic proteins of 499 and 562 amino acid residues. The sequences contain FXFG motifs, characteristic of a subgroup of nucleoporins, and a C‐terminal domain showing similarity to the Ran‐binding protein RanBP1. These proteins are localized in the nucleus, preferentially bind RanGTP and may be nuclear effectors of the Ran pathway.
LVV‐ and VV‐hemorphins: comparative levels in rat tissues
Authors: Yatskin Oleg N , Philippova Marina M , Blishchenko Elena Yu , Karelin Andrei A , Ivanov Vadim T
Published: July 2002

Screening of hemorphins in extracts of rat lung, brain, heart and spleen was carried out. The threshold for detection of hemorphins was 0.01 nmol for spleen and 0.05 nmol for other tissues. Both the content and the composition of hemorphins differed significantly in the tissues analyzed. Heart and lung extracts were rich in these peptides, the content of the most abundant components reaching 16–44 nmol/g of tissue. In contrast, spleen and brain contained much lower amounts of hemorphins, i.e. about 0.3–2.6 nmol/g of tissue. The most represented hemorphin in lung, heart and brain was VV‐hemorphin‐5, while the content of other members of the hemorphin family depended significantly on the tissue analyzed: lung extract was also rich in LVV‐hemorphin‐5, heart contained similar amounts of LVV‐hemorphin‐7 and LVV‐hemorphin‐5 and brain of LVV‐hemorphin‐6. In contrast, the hemorphin family in spleen was represented mainly by C‐terminally shortened VV‐hemorphins, i.e. VV‐hemorphin‐4 and VV‐hemorphin‐3. The levels of hemorphins in all cases were sufficient to activate the opioid receptors of the respective tissues.
The double‐stranded RNA‐binding domains of Xenopus laevis ADAR1 exhibit different RNA‐binding behaviors
Authors: Brooks Roman , Eckmann Christian R , Jantsch Michael F
Published: July 2002

We have cloned cDNAs encoding two versions of Xenopus double‐stranded RNA adenosine deaminase (ADAR1). Like ADAR1 proteins from other species Xenopus ADAR1 contains three double‐stranded RNA‐binding domains (dsRBDs) which are most likely required for substrate binding and recognition of this RNA‐editing enzyme. Analysis of mammalian ADAR1 identified the third dsRBD in this enzyme as most important for RNA binding. Here we analyzed the three dsRBDs of Xenopus ADAR1 for their in vitro RNA‐binding behavior using two different assays. Northwestern assays identified the second dsRBD in the Xenopus protein as most important for RNA binding while in‐solution assays demonstrated the importance of the third dsRBD for RNA binding. The differences between these two assays are discussed and we suggest that both the second and third dsRBD of Xenopus ADAR1 are important for RNA binding in vivo. We show further that all three dsRBDs can contribute to a cooperative binding effect.
Age‐related increased susceptibility of high‐density lipoproteins (HDL) to in vitro oxidation induced by γ‐radiolysis of water
Authors: Khalil Abdelouahed , Jay-Gerin Jean-Paul , Fülöp Tamàs
Published: July 2002

In the present study, we investigated the age‐related susceptibility of high‐density lipoproteins (HDL) to oxidation. HDL were obtained from healthy, normolipidemic young, middle‐aged and elderly subjects. Oxidation of HDL was induced in vitro by oxygen free radicals generated by water γ‐radiolysis, and followed by the decrease of endogenous vitamin E and the formation of conjugated dienes and thiobarbituric acid‐reactive substances, as well as the alterations of apolipoproteins A‐I/A‐II. The resistance of HDL to oxidation, evaluated by the length of the lag phase, decreased with aging. This increased oxidizability of HDL with aging could have a dramatic impact on the development of atherosclerosis in the elderly population.
Induction of TNF‐α release from human buffy coat cells by Pseudomonas aeruginosa is reduced by lung surfactant protein A
Authors: Hickling T.P. , Sim R.B. , Malhotra R.
Published: July 2002

Lipopolysaccharide (LPS) induction of TNF‐α release is a central event in the pathophysiology of Gram‐negative bacterial septicaemia. Lung surfactant protein A (SP‐A) mediates pathogen/host cell interactions. Binding of SP‐A to Pseudomonas aeruginosa LPS and the effects of SP‐A with LPS or whole bacteria on buffy coat cells were investigated. SP‐A interacts with P. aeruginosa LPS in a concentration and calcium dependent manner, either through the lipid A portion of LPS or through another lectin/carbohydrate interaction. SP‐A decreased TNF‐α secretion induced by bacteria or LPS from buffy coat cells, in a concentration dependent manner.
Conformational changes of the H+‐ATPase from Escherichia coli upon nucleotide binding detected by single molecule fluorescence
Authors: Börsch Michael , Turina Paola , Eggeling Christian , Fries Joachim R , Seidel Claus A.M , Labahn Andreas , Gräber Peter
Published: July 2002

Using a confocal fluorescence microscope with an avalanche photodiode as detector, we studied the fluorescence of the tetramethylrhodamine labeled F1 part of the H+‐ATPase from Escherichia coli, EF1, carrying the γT106‐C mutation [Aggeler, J.A. and Capaldi, R.A. (1992) J. Biol. Chem. 267, 21355–21359] in aqueous solution upon excitation with a mode‐locked argon ion laser at 528 nm. The diffusion of the labeled EF1 through the confocal volume gives rise to photon bursts, which were analyzed with fluorescence correlation spectroscopy, resulting in a diffusion coefficient of 3.3×10−7 cm2 s−1. In the presence of nucleotides the diffusion coefficient increases by about 15%. This effect indicates a change of the shape and/or the volume of the enzyme upon binding of nucleotides, i.e. fluorescence correlation spectroscopy with single EF1 molecules allows the detection of conformational changes.
Isolation of a novel auxin receptor from soluble fractions of rice (Oryza sativa L.) shoots
Authors: Kim Yong-Sam , Kim Donghern , Jung Jin
Published: July 2002

An auxin binding protein (ABP) was isolated from the shoots of rice seedlings and characterized. The ABP was found to be a monomer with a molecular mass of 57 kDa and play a crucial role via auxin binding in regulating H+ translocation activity of the plasma membrane in a typical biphasic manner. The results of binding equilibrium experiments indicate that the ABP binds indole 3‐acetic acid with a high affinity (K
d=1.9×10−8 M), having four primary binding sites for auxin and some secondary sites with low auxin affinities. The ABP appears to have an unambiguous auxin receptor function.
Osmotic changes and ethanol modify TFF gene expression in gastrointestinal cell lines
Authors: Lüdeking Alexander , Fegert Petra , Blin Nikolaus , Gött Peter
Published: July 2002

The gastrointestinal tract is exposed to environmental insult as a result of food intake or in pathological conditions such as diarrhoea, and is therefore protected by the mucus layer. As part of it, trefoil peptides (TFFs) are able to modify the visco‐elastic properties of the mucus, protect against experimental ulceration, and promote repair of the epithelia. We investigated, using transient reporter gene assays and RT‐PCR in the gastric carcinoma cell line MKN45 and colon carcinoma cell lines LS174T and HT29, whether ethanol and osmotic changes can modify transcriptional activity of TFFs. In a mild hypotonic environment (200 mosmol/l) all three TFF genes were up‐regulated by at least a factor of 2. In hypertonic medium (400 mosmol/l), TFF1 and TFF3 were down‐regulated, whereas TFF2 was up‐regulated by elevated concentrations of sodium or chloride in MKN45. Raising the osmolality by ethanol resulted in an up‐regulation of TFF3 in both colon cell lines but not in the gastric cell line. We conclude that alteration in TFF gene expression is a response of gut epithelia to deal with osmotic forces and ethanol.
Role of CFTR and anion exchanger in bicarbonate fluxes in C127 cell lines
Authors: Mastrocola Teresa , Porcelli Anna Maria , Rugolo Michela
Published: July 2002

C127 cell lines transfected with wtCFTR, ΔF508CFTR or vector were employed to determine HCO−
3 fluxes in the presence or absence of functional CFTR, using the pH‐sensitive dye BCECF. Both cytosolic alkalinization and acidification were due to activity of anion exchanger and were similar in the three cell lines, indicating that expression of CFTR did not influence anion exchanger activity. In C127wt cells only, cAMP elevating agents significantly stimulated HCO−
3 fluxes, insensitive to the inhibitor of anion exchanger 4,4′‐diisothiocyanate dihydrostilbene‐2,2′‐disulfonic acid, suggesting that activated CFTR directly mediates both HCO−
3 influx and efflux and therefore can contribute to intracellular and extracellular pH regulation.
Identification of regions in which positive selection may operate in S‐RNase of Rosaceae: Implication for S‐allele‐specific recognition sites in S‐RNase
Authors: Ishimizu Takeshi , Endo Toshinori , Yamaguchi-Kabata Yumi , Nakamura Kazuo T. , Sakiyama Fumio , Norioka Shigemi
Published: July 2002

A stylar S‐RNase is associated with gametophytic self‐incompatibility in the Rosaceae, Solanaceae, and Scrophulariaceae. This S‐RNase is responsible for S‐allele‐specific recognition in the self‐incompatible reaction, but how it functions in specific discrimination is not clear. Window analysis of the numbers of synonymous (dS
) and non‐synonymous (dN
) substitutions in rosaceous S‐RNases detected four regions with an excess of dN
over dS
in which positive selection may operate (PS regions). The topology of the secondary structure of the S‐RNases predicted by the PHD method is very similar to that of fungal RNase Rh whose tertiary structure is known. When the sequences of S‐RNases are aligned with the sequence of RNase Rh based on the predicted secondary structures, the four PS regions correspond to two surface sites on the tertiary structure of RNase Rh. These findings suggest that in S‐RNases the PS regions also form two sites and are candidates for the recognition sites for S‐allele‐specific discrimination.
Ouabain‐sensitive H,K‐ATPase: tissue‐specific expression of the mammalian genes encoding the catalytic α subunit1
Authors: Pestov Nikolay B , Romanova Ludmila G , Korneenko Tatyana V , Egorov Maxim V , Kostina Maria B , Sverdlov Vladimir E , Askari Amir , Shakhparonov Mikhail I , Modyanov Nikolai N
Published: July 2002

Human ATP1AL1 and corresponding genes of other mammals encode the catalytic α subunit of a non‐gastric ouabain‐sensitive H,K‐ATPases, the ion pump presumably involved in maintenance of potassium homeostasis. The tissue specificity of the expression of these genes in different species has not been analyzed in detail. Here we report comparative RT‐PCR screening of mouse, rat, rabbit, human, and dog tissues. Significant expression levels were observed in the skin, kidney and distal colon of all species (with the exception of the human colon). Analysis of rat urogenital organs also revealed strong expression in coagulating and preputial glands. Relatively lower expression levels were detected in many other tissues including brain, placenta and lung. In rabbit brain the expression was found to be specific to choroid plexus and cortex. Prominent similarity of tissue‐specific expression patterns indicates that animal and human non‐gastric H,K‐ATPases are indeed products of homologous genes. This is also consistent with the high sequence similarity of non‐gastric H,K‐ATPases (including partial sequences of hitherto unknown cDNAs for mouse and dog proteins).
Regulation of voltage‐dependent K+ channels by methionine oxidation: effect of nitric oxide and vitamin C
Authors: Ciorba Matthew A. , Heinemann Stefan H. , Weissbach Herbert , Brot Nathan , Hoshi Toshinori
Published: July 2002

Methionine oxidation is known to alter functional properties of a transient A‐type potassium channel expressed in Xenopus oocytes. We show here that nitric oxide (NO) slows down the K+ channel inactivation time course by oxidizing a critical methionine residue in the inactivation ball domain of the channel protein. We also demonstrate that the channel protein is protected from methionine oxidation by the enzyme methionine sulfoxide reductase and the antioxidant vitamin C.
Regulation of human hsp90α gene expression
Authors: Zhang Shu-ling , Yu Jun , Cheng Xiao-kuan , Ding Li , Heng Feng-yan , Wu Ning-hua , Shen Yu-fei
Published: July 2002

Mammalian HSP90α and HSP90β are encoded by two individual genes. On the basis of the upstream sequences of the human hsp90α gene, GenBank accession number U25822, we have constructed CAT reporter plasmids driven by individual fragments of the hsp90α gene. We found that (1) the proximal heat shock element complex located at −96/−60 enhances hsp90α promoter expression; (2) heat shock induction depends upon the coexistence of distal heat shock element at −1031/−1022 and the proximal heat shock element complex of the hsp90α gene; (3) unlike hsp90β, downstream sequences of the transcription start site inhibit hsp90α expression. We conclude that the regulatory mechanisms for the expression of hsp90α and hsp90β genes are different.
Activation of recombinant human SK4 channels by metal cations
Authors: Cao Ying-Jun , Houamed Khaled M.
Published: July 2002

The effects of metal cations on the activation of recombinant human SK4 (also known as hIK1 or hKCa4) channels, expressed in HEK 293 cells, were tested using patch clamp recording. Of the nine metals tested, cobalt, iron, magnesium, and zinc did not activate the SK4 channels when applied, at concentrations up to 100 μM, to the inside of SK4 channel‐expressing membrane patches. Barium, cadmium, calcium, lead, and strontium activated SK4 channels in a concentration‐dependent manner. The rank order of potency was at Ca2+>Pb2+>Cd2+>Sr2+>Ba2+.
Identification in the NK1 tachykinin receptor of a domain involved in recognition of neurokinin A and septide but not of substance P
Authors: Wijkhuisen Anne , Sagot Marie-Astrid , Frobert Yveline , Créminon Christophe , Grassi Jacques , Boquet Didier , Couraud Jean-Yves
Published: July 2002

The three mammalian tachykinins, substance P (SP), neurokinin A (NKA) and neurokinin B (NKB), exert their physiological effects through specific receptors, NK1, NK2 and NK3, respectively. However, homologous binding studies have recently demonstrated that, contrary to the generally accepted belief, NKA could bind NK1 receptor with high affinity (Hastrup and Schwartz, 1996). Using COS‐7 cells expressing the human NK1 receptor, we show that two simultaneous point mutations (E193L and V195R) in a restricted five amino acid sequence (the (193–197) region), selected because of its hydropathic complementarity with the common C‐terminal extremity of tachykinins, abolish both the high‐affinity binding and highly potent biological activity of NKA, without affecting those of SP. In addition, the same mutations also suppressed the high functional activity of septide, a synthetic SP atypical agonist ([pGlu6‐Pro9] SP 6–11). These results suggest that the (193–197) region, located at the end of the second extracellular loop of the receptor, could be part of a common high‐affinity binding domain for both NKA and septide, distinct from the SP binding site.
Apoptosis induced by growth factor withdrawal in fibroblasts overproducing fructose 2,6‐bisphosphate
Authors: Durante Paula , Gueuning Marie-Agnès , Darville Martine I , Hue Louis , Rousseau Guy G
Published: July 2002

Fructose 2,6‐bisphosphate is a potent endogenous stimulator of glycolysis. A high aerobic glycolytic rate often correlates with increased cell proliferation. To investigate this relationship, we have produced clonal cell lines of Rat‐1 fibroblasts that stably express transgenes coding for 6‐phosphofructo‐2‐kinase, which catalyzes the synthesis of fructose 2,6‐bisphosphate, or for fructose 2,6‐bisphosphatase, which catalyzes its degradation. While serum deprivation in culture reduced the growth rate of control cells, it caused apoptosis in cells overproducing fructose 2,6‐bisphosphate. Apoptosis was inhibited by 5‐amino‐4‐imidazolecarboxamide riboside, suggesting that 5′‐AMP‐activated protein kinase interferes with this phenomenon.
N‐glycans are not the signal for apical sorting of corticosteroid binding globulin in MDCK cells
Authors: Larsen Jakob E. , Avvakumov George V. , Hammond Geoffrey L. , Vogel Lotte K.
Published: July 2002

It has been suggested that N‐glycans act as a general sorting signal for secretory proteins in MDCK cells [Scheiffele et al. (1995) Nature 378, 96–98]. Human corticosteroid binding globulin contains six consensus sites for N‐glycosylation and is known to be secreted to the apical side of MDCK cells. Our results show that wild‐type corticosteroid binding globulin is N‐glycosylated when it is recombinantly expressed in MDCK cells. Six mutants, each lacking one of the N‐glycosylation sites, and a mutant lacking all six N‐glycosylation sites were also secreted to the apical side of MDCK cells in a polarized manner. Thus, the N‐glycans on corticosteroid binding globulin do not act as an apical sorting signal in MDCK cells.
N‐glycans are not a universal signal for apical sorting of secretory proteins
Authors: Su Tao , Cariappa Rohit , Stanley Keith
Published: July 2002

In MDCK cells, N‐glycans have been shown to determine the sorting of secretory proteins and membrane proteins to the apical domain in the absence of a dominant basolateral targeting signal. We have examined the sorting of endogenous proteins in ECV304 cells in the presence and absence of tunicamycin, an inhibitor of N‐linked glycosylation. A prominent apically secreted protein of 71 kDa was not N‐glycosylated and continued to be secreted apically in the presence of tunicamycin. In contrast, other endogenous proteins that were N‐glycosylated were secreted preferentially into the basolateral medium or without polarity. When rat growth hormone was expressed in MDCK and ECV304 cells, we observed 65 and 94% of the secretion to the basolateral medium, respectively. Introduction of a single N‐glycan caused 83% of the growth hormone to be secreted at the apical surface in MDCK cells but had no significant effect on the polarity of secretion of growth hormone in ECV304 cells. These results indicate that not all cell lines recognise N‐glycans as a signal for apical sorting and raises the possibility of using ECV304 cells as a model system for analysis of apical sorting molecules.
Establishment of an embryonic stem (ES) cell line derived from a non‐obese diabetic (NOD) mouse: in vivo differentiation into lymphocytes and potential for germ line transmission
Authors: Nagafuchi Seiho , Katsuta Hitoshi , Kogawa Kazuhiko , Akashi Tomoyuki , Kondo Shiori , Sakai Yumiko , Tsukiyama Tadasuke , Kitamura Daisuke , Niho Yoshiyuki , Watanabe Takeshi
Published: July 2002

A non‐obese diabetic (NOD) mouse‐derived embryonic stem (ES) cell line has been stably maintained in an undifferentiated state with a characteristic ES cell‐like morphology, expressing the stem cell marker alkaline phosphatase, and displaying a normal diploid karyotype. After injecting the NOD‐ES cells into blastocysts, chimeric mice were obtained. Small but significant numbers of lymphocytes expressed the NOD‐derived MHC allele. When a chimeric mouse was mated with C57BL/6 mice, an agouti mouse was obtained, having the NOD‐derived H‐2 I‐Aβ
g7 haplotype. Thus, an NOD‐ES cell line which can differentiate into lymphocytes with potential for germ line transmission was successfully established.
Hypoxic upregulation of tyrosine hydroxylase gene expression is paralleled, but not induced, by increased generation of reactive oxygen species in PC12 cells
Authors: Höhler B. , Lange B. , Holzapfel B. , Goldenberg A. , Hänze J. , Sell A. , Testan H. , Möller W. , Kummer W.
Published: July 2002

Oxygen sensing was investigated in rat pheochromocytoma PC12 cells. They respond to hypoxia with an increased intracellular generation of reactive oxygen species (ROS), measured by oxidation of dihydrorhodamine 123. This increase is abolished by intracellular superoxide scavenging by Mn(III)‐tetrakis(1‐methyl‐4‐pyridyl)‐porphyrin, and reduced or absent in the presence of the flavoprotein/complex I inhibitors, diphenyleneiodonium and rotenone. The same inhibitors, but neither intra‐ nor extracellular (superoxide dismutase) superoxide scavenging, abolish the hypoxia‐induced increase in tyrosine hydroxylase (TH) gene expression. Thus, ROS production increases in PC12 cells during hypoxia, but this is not the cause of hypoxic TH mRNA upregulation that involves a flavoprotein.
Transient Ca2+‐dependent activation of ERK1 and ERK2 in cytotoxic responses induced by maitotoxin in breast cancer cells
Authors: Malaguti Claudia , Yasumoto Takeshi , Paolo Rossini Gian
Published: July 2002

Treatment of MCF‐7 breast cancer cells with the marine toxin maitotoxin (MTX) induces cell death. The cytotoxic effects are clearly detectable within 2–4 h after cell treatment with 10−10–10−9 M concentrations of MTX. The response was found to depend on extracellular Ca2+, inasmuch as cell death was prevented when culture dishes received MTX, following addition of EGTA. MTX caused transient phosphorylation of extracellular signal‐regulated kinase isoforms 1 and 2 (ERK1 and ERK2) mitogen‐activated protein kinase isoforms in MCF‐7 cells, which was maximal 15 min after toxin addition to culture vessels. The effect was dependent on influx of extracellular Ca2+, as it was abolished by EGTA, and was induced by ionophores, such as A23187 and ionomycin. Our findings show that signaling pathways involving Ca2+ ions may cause activation of ERK1 and ERK2 in cell death responses.
Specific loss of chondromodulin‐I gene expression in chondrosarcoma and the suppression of tumor angiogenesis and growth by its recombinant protein in vivo
Authors: Hayami Tadashi , Shukunami Chisa , Mitsui Kaori , Endo Naoto , Tokunaga Kunihiko , Kondo Jun , Takahashi Hideaki E. , Hiraki Yuji
Published: July 2002

Chondromodulin‐I (ChM‐I) was previously identified as an angiogenesis inhibitor in cartilage. Here, we demonstrated that the level of ChM‐I transcripts was substantially reduced to 100 or even less in the lower‐grade chondrosarcomas, in articular cartilage or other benign cartilage tumors. We implanted human chondrosarcoma OUMS‐27 cells into nude mice that reproducibly produced tumors with cartilaginous matrix. Tumor‐induced angiogenesis was evident when the tumors were excised 30 days after implantation. However, the local administration of recombinant human ChM‐I almost completely blocked vascular invasion and tumor growth in vivo. Moreover, ChM‐I also inhibited the growth of HT‐29 colon adenocarcinoma in vivo, implying its therapeutic potential for solid tumors.
On stage single cell identification of rat spermatogenic cells
Authors: Reyes Juan G , Diaz Alvaro , Osses Nelson , Opazo Carlos , Benos Dale J
Published: July 2002

Summry— The study of spermatogenic cell physiology has been hindered by the absence of unbiased methods of identification of cells upon which single cell techniques are being applied. In this work, we have used histochemical techniques, digital videoimaging, quantification of chromatin‐bound DNA probes, and measurements of cell diameter to identify single spermatogenic cells at different periods of development. Our criteria of identification permit the definition of four developmental stages of spermatogenesis on which to perform single cell analyses: spermatogonia B/preleptotene spermatocytes, leptotene/zygotene spermatocytes, pachytene spermatocytes, and round spermatids. The use of voltage‐sensitive dyes and Ca2+‐sensitive dyes does not interfere with the estimations of DNA content. The estimations of DNA content of spermatogenic cells can be performed both with near‐UV exciged dyes (H33342) and long wavelength‐excited dyes (ethidium bromide), allowing the use of a wide range of physiological and immunocytochemical fluorescent probes to study the spermatogenic process.
Developmental localization of GAP‐43 and olfactory marker protein in rat olfactory bulb transplants
Authors: Kott Jon N. , Westrum Lesnick E.
Published: July 2002

In an effort to identify and understand the laminar disorganization that occurs in the transplanted (TX) rat olfactory bulb (OB), we examined the development of fiber systems within these TX OBs. One antibody for olfactory marker protein (OMP) was used to identify axons of mature olfactory receptor neurons (ONs) and a second antibody, for a growth‐associated protein (GAP‐43), provided a marker for all extending or immature fibers. Donor OBs were taken from fetuses on embryonic days 14 or 15 (sperm‐positive day is zero) and TX directly into the cavity produced by removal of an OB in 1‐day‐old hosts of the same strain. After survival times of I and 2 weeks and at maturity, adjacent 8 pm paraffin sections from the TX material were examined for OMP and GAP‐43 reactivity.

Fiber bundles, reactive for OMP, were found within the TX by 1 week post‐TX, indicating rapid reinnervation of the donor OB by ONs. The appearance of OMP reactivity gradually shifted from tightly packed, well‐defined fiber bundles at 1 week post‐TX to a diffuse reticulated pattern of individual fibers emerging from bundles at maturity. The OMP‐reactive fiber bundles of the TX OB also contained GAP‐43‐reactive fibers, but GAP‐43 reactivity also extended to other (OMP‐negative) bundles and fields. Reactivity for GAP‐43 in the TX OB was nearly ubiquitous at 2 weeks post‐TX but, as development progressed (in both the TX and normal OB), such reactivity gradually decreased. Thus, while maturation in sensory afferent fiber systems in the TX OB may be delayed, it eventually follows a pattern similar to that in the normal OB, suggesting that factors other than the timing of fiber extension may be responsible for the laminar disorganization of the TX OB.