bluuebunny/crossref_metadata_2025 huggingface dataset:
Two excited states in aequorin bioluminescence induced by tryptophan modification
Authors: Ohmiya Yoshihiro , Ohashi Mamoru , Tsuji Frederick I.
Published: July 2002

The Ca2+‐activated photoprotein, aequorin, contains six tryptophan residues and has a bioluminescence emission maximum at 465 nm. On converting the six tryptophan residues to phenylalanine, the mutant aequorins exhibited varied luminescence activities and spectra, but one mutant, with tryptophan‐86 replaced by phenylalanine, gave a bimodal emission spectrum, with maxima at 455 nm and 400 nm. This result suggests that tryptophan‐86 may be importantly involved in the generation of the product excited state during aequorin bioluminescence.
Nerve growth factor rapidly regulates VGF gene transcription through cycloheximide sensitive and insensitive pathways
Authors: Baybis Marianna , Salton Stephen R.J.
Published: July 2002

Nerve growth factor (NGF) initiates and maintains a regulatory cascade, involving gene induction, which results in the neuronal differentiation of PC12 cells. The VGF gene encodes one of the most rapidly induced neuronal mRNAs identified in NGF‐treated PC12 cells (Science, 229 (1985) 393–395; Mol. Cell Biol., 11 (1991) 2335‐2349]. In this communication we show that NGF‐treatment for 60–90 min maximally increases VGF gene transcription by 12‐ to 14‐fold. VGF mRNA half‐life was found to substantially dccrease in PC12 cells treated with NGF for 9–25 h. Partial inhibition of VGF gene transcription and superinduction of cytoplasmic VGF mRNA levels in the presence of both NGF and cycloheximide suggests that the VGF gene may be regulated through multiple pathways, some of which can be activated in the presence of protein synthesis inhibitors as are the immediate early genes, while others require newly synthesized proteins.
Glycine‐glutamate interactions at the NMDA receptor: role of cysteine residues
Authors: Laube Bodo , Kuryatov Alexander , Kuhse Jochen , Betz Heinrich
Published: July 2002

The NMDA subtype of ionotropic glutamate receptors requires occupation by both l‐glutamate and the co‐agonist glycine for efficient channel opening. To elucidate the role of disulfide bridges for the allosteric interaction of these agonists we mutated the cysteine residues in the ligand‐binding NMDAR1 (NR1 orζ) subunit of the rodent NMDA receptor and co‐expressed the resulting mutants with the NR2B (ϵ2) subunit in Xenopus oocytes. Most of the cysteine substitutions had no effect on agonist responses. However, replacement of cysteines 402 and 418 by alanine largely abolished the potentiation of glutamate currents by glycine. These cysteine residues in the putative extracellular domain of the NR1 subunit may form a disulfide bridge important for agonist interaction.
Assignment of the backbone 1H and 15N NMR resonances and secondary structure characterization of barstar
Authors: Lubienski Michael J. , Bycroft Mark , Jones David N.M. , Fersht Alan R.
Published: July 2002

Barstar, a polypeptide inhibitor of ribonucleases, has been studied by 2D and 3D NMR techniques using uniformly 15N‐labeled protein. Backbone (15NH‐CαH‐CβH) resonances were assigned for all but 5 of the 89 residues. Dihedral angle and deuterium exchange studies were used in conjunction with medium range inter‐proton NOEs to characterize the secondary structure of barstar. The protein is composed of four α‐helices and three short stretches of extended strand. By further analysis of the NOE data three of the helices were found to be parallel to each other with the single disulphide bond linking the second and fourth helices at their C‐terminal ends.
Expression of native dimers of bovine seminal ribonuclease in a eukaryotic cell system
Authors: Russo Nello , De Nigris Mena , Di Donato Alberto , D'Alessio Giuseppe
Published: July 2002

Bovine seminal ribonuclease, a uniquely dimeric pancreatic‐like RNase, with its dimeric structure stabilized by two intersubunit disulfides, and endowed with special, i.e. non‐catalytic, actions (antitumor, immunosuppressive, antispermatogenic), was stably expressed in Chinese hamster ovary cells. The recombinant protein, secreted in the culture medium as a correctly folded dimeric enzyme, was purified to homogeneity and found to be fully active both in its catalytic and antitumor activities.
Amino acid sequence and thermostability of xylanase A from schizophyllum commune
Authors: Oku T. , Roy C. , Watson D.C. , Wakarchuk W. , Campbell R. , Yaguchi M. , Jurasek L. , Paice M.G.
Published: July 2002

The amino acid sequence (197 residues) of xylanase A from the fungus, Schizophyllum commune, was determined by automated analysis of peptides from proteolytic and acid cleavage. The sequence is similar to two Trichoderma xylanases (approximately 56% identical amino acids), but also shows at least 40% identities with xylanases from Bacillus subtilis, B. pumilus and B. circulans. The conserved regions of the enzyme contain only two glutamic acid residues which implicates their possible involvement in catalysis. The disulfide bond in xylanase A is not conserved in this family. In spite of this, the B. subtilis xylanase was found to be more thermostable than xylanase A.
Evidence for stacking interactions between 5‐mercurated polyuridylic acid and HIV‐1 p7 nucleocapsid protein obtained by phosphorescence and optically detected magnetic resonance (ODMR)
Authors: Lam Wai-Chung , Maki August H. , Casas-Finet José R. , Erickson John W. , Sowder Raymond C. , Henderson Louis E.
Published: July 2002

The photoexcited triplet state of Trp‐37 in the C‐terminal zinc finger of the HIV‐1 p7 nucleocapsid protein was used as a probe of p7 interactions with the heavy atom‐derivatized RNA homopolymer, poly‐5‐mercuriuridylic acid (5‐HgU). Binding of p7 to 5‐HgU (Hg blocked with 2‐mercaptoethanol) produces an external heavy atom effect (HAE) on Trp‐37 characterized by fluorescence quenching, reduction of the phosphorescence lifetime by three orders of magnitude, and the appearance of the D + E phosphorescence‐detected ODMR signal, absent in unperturbed Trp, but induced by a HAE. The details of the HAE are consistent with out‐of‐plane van der Waals contact of Hg with the indole chromophore of Trp‐37. Steric requirements suggest further that the Trp‐RNA contact occurs via an aromatic stacking interaction.
Zonation of glucokinase in rat liver changes during postnatal development
Authors: Kirchner Gabi , Harbers Matthias , Bünsch Almuth , Seitz Hans-Joachim , Höppner Wolfgang
Published: July 2002

In the liver many metabolic pathways are preferentially localized in different zones of the acinus. It is assumed that this zonation allows an efficient adaptation to different states of nutrition, because alternative pathways can be regulated independently. It is reported that the rate limiting enzyme for the glycolytic pathway, glucokinase (EC 2.7.1.2), is predominantly located in the pericentral zone. The gene expression of glucokinase is induced to a maximum level after a carbohydrate‐rich diet. In starved or diabetic rats glucokinase gene expression is barely detectable. In postnatal development glucokinase is induced to significant levels only from day 14 onwards. The distribution of the glucokinase protein in the rat liver lobule in the first 4 weeks of postnatal life was investigated by immunohistochemistry and compared to the distribution observed in adult rats. In adult rats considerably high levels of glucokinase are measureable as shown by immunoblotting utilizing a monospecific antibody and a photometric assay of glucokinase enzyme activity, respectively. Immunohistochemically the hepatic glucokinase protein is detected in the perivenous area. During postnatal development, the quantities of hepatic glucokinase protein and glucokinase enzyme activity start to increase significantly from day 15 onwards. Subsequently, glucokinase levels rise further until day 29. In contrast to the results obtained by immunoblotting, glucokinase is already detectable in some liver cells in sections from 6‐day‐old rats by immunohistochemistry. The liver lobule structure at this age is not completely developed, therefore it is not possible to definitely assign these cells to periportal or pericentral areas. At day 10 post partum the number of glucokinase expressing cells, which appear to be localized preferentially in the periportal zone, increases. In agreement with the immunoblotting, an immense increase in glucokinase activity was observed at day 14. The periportal zonation, clearly detectable at this time, remains stable until day 24. In sections from 29‐day‐old rats the periportal zonation begins to change into a more homogeneous pattern with a slight preference for periportal areas. The observed appearance of the periportal zonation of glucokinase during neonatal development is obviously in contrast to the perivenous expression of glucokinase in adult rats.
Characterization of a membrane‐associated, receptor and G‐protein responsive phosphoinositide‐specific phospholipase C from avian erythrocytes
Authors: Ruiz-Larrea Fernanda , Berrie Christopher P.
Published: July 2002

We describe the reconstitution and purification of a membrane‐associated phosphoinositide‐specific phospholipase C (PIC) from turkey erythrocyte ghosts. This PIC is responsive to a G‐protein coupled to P2y purinergic receptors which are expressed in turkey erythrocytes. Reconstitution is achieved by adding partially purified PIC to [3H]inositol‐prelabelled turkey erythrocyte membranes depleted of their endogenous PIC (acceptor membranes). PIC activity is associated with a 52 kDa polypeptide on SDS‐polyacrylamide gel electrophoresis. Addition of a 307‐fold purified enzyme to the acceptor membranes has no effect on basal PIC activity, but markedly increases the response to GTPγS and P2y‐purinergic receptor activation.
Purification of a novel factor which binds to the mouse α2 (I) collagen promoter
Authors: Hatamochi Atsushi , de Crombrugghe Benoit , Krieg Thomas
Published: July 2002

We have identified and purified a DNA binding protein which specifically binds to a segment of the mouse α2 (I) collagen promoter between −420 and −399 bp upstream of the start of transcription. Purification included heparin‐agarose and sequence‐specific DNA‐affinity chromatography, followed by SDS‐PAGE and renaturation of the DNA binding activity after elution from SDS‐polyacrylamide gel. The DNA binding activity resides in two species of 42 kDa and 40 kDa, respectively. The levels of DNA binding activity of this factor, which has been tentatively designated as ColFl, are considerably higher in nuclear extracts of NIH‐3T3 fibroblasts than in nuclear extracts from epidermal cells, lymphoid cells and transformed NIH‐3T3 fibroblasts.
Functions of interleukin‐8 are mediated through thiol group(s) of IL‐8 receptor in human polymorphonuclear neutrophils
Authors: Dutta Supriti , Ali Esahak , Samanta Ajoy K.
Published: July 2002

Interleukin‐8, a neutrophi] chemotactic agent causes excessive accumulation of the cells in a number of inflammatory diseases. The activity has been shown to be mediated through a specific functional receptor present on the surface of neutrophils. No information is available about the amino acids constituting the IL‐8 binding domain of the receptor. Treatment of neutrophils with 5,5'‐dithio‐bis(2‐nitrobenzoic acid), a thiol‐specific modifier, at the concentrations of 0.4 mM and 1 mM reduced IL‐8 binding ability and IL‐8‐induced migration of the cells by 45% and 65%, respectively. Dithiothreitol could regenerate the binding capacity and the ligand could protect the receptor from the effect of the reagent. All the evidence suggests that one or more critical thiol residues are located in the IL‐8 binding site of the receptor which are indispensible for normal functions of IL‐8.
Functional expression of 5‐HT1c receptor cDNA in COS 7 cells and its influence on protein kinase C
Authors: Lutz E.M. , Mitchell R. , Johnson M.S. , MacEwan D.
Published: July 2002

Two subtypes of receptors for serotonin (5‐hydroxytryptamine; 5‐HT) are known to stimulate inositol (l,4,5)‐trisphosphate production, the 5‐HT1c and 5‐HT2 receptors. In this study we investigated the ability of 5‐HT1c receptors, transiently expressed in COS 7 cells, to functionally interact with protein kinase C‐α, the indigenous (phorbol ester‐responsive) isoform of the enzyme in those cells. Serotonin caused translocation of the [3H]phorbol 12,13‐dibutyrate (PDBu) binding site of PKC‐α from the cytosolic to the membrane fraction in a Ca2+‐dependent manner which was prevented by the 5‐HT1c receptor antagonist mianserin. The lipid activators of PKC, PDBu and 1,2‐dioctanoyl‐sn‐glycerol (DOG) also caused translocation, but through a mechanism apparently quite independent of Ca2+
Site‐directed mutagenesis in hemoglobin
Authors: Vallone B. , Vecchini P. , Cavalli V. , Brunori M.
Published: July 2002

The role of selected amino acid residues in the monomer‐monomer contacts of Hb A has been studied by site‐directed mutagenesis of the α chain bearing substitutions in the subunit surfaces. Mutation α 38Thr → Trp induced a stabilization of tetrameric Hb‐CO with a decrease of the K
d for the equilibrium α2β2 ⇌ 2αβ, but had no effect on ligand binding. Mutation α40Thr→Arg resulted in a complete loss of cooperativity in ligand binding. Mutation α103His→Val had no noticeable effect. We also studied the behaviour of isolated, mutated α chains with respect to self association: compared to wt α chains, mutant α38Thr→Trp showed stabilization of the dimeric state and (at high protein concentration) a detectable amount of tetramers. Mutant α103His→Val showed only a minor stabilization of the α2 dimer.
Purification and properties of a 155 kDa bone marrow‐derived glycoprotein enhancing the activity of granulocyte‐macrophage colony stimulating factor (GM‐CSF)
Authors: Katlinsky Anton V. , Petrov Rem V. , Mikhailova Augusta A. , Laerum Ole Didrik
Published: July 2002

A protein has been purified from pig bone marrow which enhances the activity of exogenously added granulocyte‐macrophage colony stimulating factor (GM‐CSF) on mouse myelopoiEtic colony formation in vitro. The substance alone did not exhibit any colony stimulating activity. The active fraction was isolated from the medium of pig bone marrow suspension cultures during 20–24 h. Purification to homogeneity was then performed by subsequent HPLC and SDS‐PAGE. The GM‐CSF enhancing protein was identified as a 155 kDa glycoprotein.
Early induction of Na+‐dependent uridine uptake in the regenerating rat liver
Authors: Ruiz-Montasell Bonaventura , Martinez-Mas Joan Vicenç , Enrich Carlos , Casado F.Javier , Felipe Antonio , Pastor-Anglada Marçal
Published: July 2002

Na+‐dependent uridine transport into liver plasma membrane vesicles from partially hepatectomized and sham‐operated rats was studied. Preparations purified 6 h after 70% hepatectomy exhibited an increased V
max of uridine uptake (3.7 vs. 1.4 pmol/mg prot/3 s) without any change in Km (6μM). Incubation of the vesicles in the presence of monensin decreased uridine uptake although the differences between both experimental groups remained identical. It is concluded that uridine transport is induced early after partial hepatectomy by a mechanism which does not involve changes in the transmembrane Na+ gradient. This is the first evidence in favor of modulation of nucleoside transport into liver cells.
Indoleacetic acid pretreatment of tobacco plants in vivo increases the in vitro sensitivity to auxin of the plasma membrane H+‐ATPase from leaves and modifies the polypeptide composition of the membrane
Authors: Santoni Véronique , Vansuyt Gérard , Rossignol Michel
Published: July 2002

The sensitivity to auxin of the H+‐ATPase‐mediated proton translocation was investigated in vitro using purified plasma membrane vesicles from tobacco leaves. In vivo pretreatment of tobacco plants with auxin promotes a 100‐fold increase of the in vitro sensitivity to auxin. This effect is specific for biologically active auxins and is dose‐ and time‐dependent. In addition, pretreatment with auxin induces the accumulation of several polypeptides in the plasma membrane. These polypeptides constitute the first set of hormone‐responsive polypeptides evidenced in plant membranes.
Na+‐driven ATP synthesis in Methanobacterium thermoautotrophicum and its differentiation from H+‐driven ATP synthesis by rhodamine 6G
Authors: Šmigáň Peter , Majerník Alan , Greksák Miloslav
Published: July 2002

Rhodamine 6G (3 μM) effectively inhibited ΔpH‐driven ATP synthesis in Methanobacterium thermoautotrophicum while ΔpNa‐driven ATP synthesis was not affected by it. Rhodamine 6G inhibited Mg2+‐stimulated ATPase activity of membrane vesicles prepared from these cells but the ATPase catalytic sector detached from the membrane was insensitive to this inhibitor. Methanogenesis‐driven ATP synthesis at pH 6.8 of cells grown in the presence of 50 mM NaCl was inhibited by rhodamine 6G both in the presence of 5 mM and 50 mM NaCl. On the other hand, the methanogenesis‐driven ATP synthesis at pH 8.0 of cells grown in the presence of 50 mM NaCl was slightly inhibited by rhodamine 6G in the presence of 5 mM NaCl and was not inhibited at all in the presence of 50 mM NaCl. The growth experiments have shown that cells of Methanobacterium thermoautotrophicum can grow under alkaline conditions even in the presence of rhodamine 6G and of high NaCl concentration when the growth media were inoculated with the cells which had been grown in the presence of 50 mM NaCl. These results indicate that sodium‐motive force‐driven ATP synthase in Methanobacterium thermoautotrophicum operates effectively in alkaline conditions and it might be the sole ATP synthesizing system when the proton‐motive force‐supported ATP synthesis is inhibited by rhodamine 6G.
Erythrocyte band 3 protein strongly interacts with phosphoinositides
Authors: Hanicak Andreas , Maretzki Dieter , Reimann Barbara , Pap Eward , Visser Antonie J.W.G. , Wirtz Karel W.A. , Schubert Dieter
Published: July 2002

85% of the phosphorus coisolated with band 3 protein during separation of the intrinsic proteins of the human erythrocyte membrane by zonal electrophoresis in high concentrations of acetic acid was found to be derived from phosphoinositides, mainly phosphatidylinositol 4,5‐bisphosphate. When native band 3 protein and pyrene‐labelled phospholipids were present in micelles of the nonionic detergent nonaethyleneglycol lauryl ether, strong resonance energy transfer was observed between the tryptophan residues and phosphatidylinositol 4,5‐bisphosphate and, to a smaller degree, phosphatidylinositol 4‐phosphate. We conclude that band 3 protein strongly interacts with phosphoinositides, in particular with phosphatidylinositol 4,5‐bisphosphate.
Injection of a K+ channel (Kv1.3) cRNA in fertilized eggs leads to functional expression in cultured myotomal muscle cells from Xenopus embryos
Authors: Honoré Eric , Guillemare Eric , Lesage Florian , Barhanin Jacques , Lazdunski Michel
Published: July 2002

The synthetic cRNA encoding for the major T lymphocyte K+ channel (Kv1.3) was injected into Xenopus fertilized eggs. Somites from embryos of stage 20–22 (about 40 h post‐fertilization at 19°C) were dissociated and myotomal muscle cells were cultured in vitro for 2 days. The whole cell configuration of the tight seal patch‐clamp technique was used to record K+ channel activity in cultured myocytes. These myocytes have two endogenous delayed‐rectifiers (sustained and transient) and an inward‐rectifier K+ currents, all of which are insensitive to the scorpion toxin charybdotoxin. Cultured myocytes dissociated from embryos injected with the Kv1.3 cRNA expressed the exogenous Kv1.3 channel. The Kv1.3 channel was identified by its physiological (a very low recovery from inactivation) and its pharmacological properties (a high sensitivity to charybdotoxin). This work demonstrates that Xenopus cultured myotomal muscle cells represent a very efficient and practical assay system for the functional expression of cloned ion channels.
Efficient interaction of recA protein with fluorescent dye‐labeled oligonucleotides
Authors: Volodin Alexander A. , Smirnova Helen A. , Bocharova Tatjana N.
Published: July 2002

Some fluorescein derivatives attached to the 5′‐end of oligonucleotides stimulate recA protein—oligonucleotide binding. The complex formation at near stoichiometric DNA/protein ratios is demonstrated for 18‐bases‐long oligonucleotides. The complexes with dye‐labeled oligonucleotides are shown to be active in the reaction of homologous strand exchange. The strand exchange reaction in the presence of adenosine‐5′‐O‐(3‐thiotriphosphate) proceeds with the formation of a stable complex of recA protein with the double stranded oligonucleotide, which is a product of the strand exchange. The displaced single‐stranded oligonucleotide is shown to be bound weakly.
Crystal structure of domain A of Thermus flavus 5S rRNA and the contribution of water molecules to its structure
Authors: Betzel Ch. , Lorenz S. , Fürste J.P. , Bald R. , Zhang M. , Schneider Th.R. , Wilson K.S. , Erdmann V.A.
Published: July 2002

This is the first high resolution crystal structure of an RNA molecule made by solid phase chemical synthesis and representing a natural RNA. The structure of the domain A of Thermus flavus ribosomal 5S RNA is refined to R = 18% at 2.4 Å including 159 solvent molecules. Most of the 2′‐hydroxyl groups as well as the phosphate oxygens are involved either in specific hydrogen bonds in intermolecular contacts or to solvent molecules. The two U‐G and G‐U base‐pairs are stabilized by H‐bonds supplied via three water molecules to compensate for the lack of base‐pair hydrogen bonds. The structure shows for the first time in detail the importance of highly ordered internal water in stablizing an RNA structure.
Neomycin: A novel potent blocker of communication between T‐tubule and sarcoplasmic reticulum
Authors: Yano Masafumi , El-Hayek Roque , Antoniu Bozena , Ikemoto Noriaki
Published: July 2002

Ca2+ release from the sarcoplasmic reticulum (SR) was induced in isolated triads by direct stimulation of the SR moiety by polylysine, or stimulation via chemical depolarization of the transverse tubule (T‐tubule) moiety. Polylysine‐induced release was blocked by neomycin with an IC50 (the concentration for half‐maximal inhibition) of O.3, μM. However, the IC50 for neomycin block of depolarization‐induced Ca2+ release sharply decreased in a voltage‐dependent fashion, and it was 5.3 nM at a maximal extent of T‐tubule depolarization. These results suggest that the high affinity binding of neomycin to the triad leads to the specific blocking of the signal transmission from T‐tubule to SR.
Modification of a metal ligand in carbonic anhydrase: Crystal structure of His94→Glu human isozyme II
Authors: Xue Yafeng , Jonsson Bengt-Harald , Liljas Anders , Lindskog Sven
Published: July 2002

One of the zinc ligands in human carbonic anhydrase II, His94, has been replaced with glutamic acid by site‐directed mutagenesis. The mutation leads to a less stable zinc binding site and to significant non‐local perturbations of the protein structure. The crystals are composed of a mixture of holo‐ and apoenzyme, and the side chain of Glu94 has two conformations. In the holoenzyme, Glu94 coordinates to the metal ion and is hydrogen bonded to Gln92. In the apo form, Glu94 is hydrogen bonded to Asn67. The mutation has resulted in a 500‐fold decrease of the catalyzed rate of CO2 hydration (k
cat/K
m).
Peroxide‐induced spectral perturbations of the 280‐nm absorption band of cytochrome c oxidase
Authors: Larsen Randy W.
Published: July 2002

It is now widely believed that the first two electrons transferred to the dioxygen reduction site in cytochrome c oxidase (CcO) are not coupled to proton translocation. The activation of the pump cycle correlates with the binding of dioxygen to the binuclear center. In order to investigate conformational changes in CcO associated with the formation of dixoygen intermediates during the catalytic cycle of CcO, the effects of hydrogen peroxide binding to CcO have been examined using UV optical absorption and second derivative techniques. Our data indicates that in the presence low concentrations of H2O2 (2:1 molar ratio) an initial CcO‐peroxide species is formed in which the 280‐nm absorption band is red shifted. This red shift occurs prior to spectral changes associated with H2O2 binding to cytochrome a
3. Upon addition of higher concentrations of H2O2 (⪢10 equivalents of H2O2 per equivalent of CcO) oxidized CcO is converted to F‐state enzyme with no corresponding shift at 280 nm. It is suggested that H2O2 initially binds to Cu2+
B resulting in a conformational change in the enzyme giving rise to a red‐shifted 280 nm band. The absence of any conformational changes in F‐state enzyme is consistent with the lack of bridging interactions with Cu2+
B in this intermediate.
Expression of type II thyroxine 5′‐deiodinase from rat Harderian gland in Xenopus laevis oocytes
Authors: Garcia-Macias Juan F. , Molinero Patrocinio , Guerrero Juan M. , Osuna Carmen
Published: July 2002

The presence of isoenzymes mediating the conversion of thyroxine to 3,5,3′‐triiodothyronine has been studied according to characteristic kinetics and physiological regulation. In this paper, we report the expression of type II 5′‐deiodinase (5′D) activity in oocytes of Xenopus laevis. Oocytes injected with total RNA extracted from rat Harderian gland, and then incubated up to five days demonstrated a progressive increase in 5′D activity, reaching a maximal value at 24 h; then, 5′D activity remained almost stable for an additional period of four days. Characteristics of the enzyme activity expressed by oocytes included its inhibition by iopanoic acid, but not by propylthiouracil, and its increase during β‐adrenergic agonist treatment and hypothyroidism. The expressed activity manifests characteristics typical of the type II isoenzyme. Deiodinating activity in oocytes also exhibited diurnal variations. In this study, 5′D activity expressed in oocytes exhibited low values when animals were killed during the day, and high values when animals were killed at night. Maximal values were reached 3–4 h before the nocturnal peak of 5′D activity in Harderian gland crude homogenates. Results suggest that the in vivo activation of 5′D by isoproterenol, hypothyroidism, or dark exposure may be caused by an increase in the synthesis and/or maturation of the RNA expressing the enzyme.
Carbohydrate induced modulation of cell membrane I. Interaction of sialic acid with peripheral blood lymphocytes: A spin label study
Authors: Hajela Krishnan , Kayestha Rajeev , Pradeep Kumar G.
Published: July 2002

Sialic acid is now known to serve as ligand for lymphocyte lectins known as selectins. Although its role as a ligand for adhesion molecules has been studied extensively, no studies have been performed to determine the physiological changes in lymphocytes after binding of sialic acid to lymphocyte lectin. We report for the first time that interaction of lymphocytes with sialic acid severely restricts the rotational mobility of the cell surface proteins as well as membrane lipids, as studied by EPR spectroscopy using spin probes. Binding of mucin totally immobilizes the lymphocyte membrane. Surprisingly the binding of sialic acid or mucin also immobilized the aqueous probe TEMPO, indicating an appreciable increase in cytoplasmic viscosity.
Alteration of the substrate specificity of human lysozyme by site‐specific intermolecular cross‐linking
Authors: Muraki Michiro , Jigami Yoshifumi , Harata Kazuaki
Published: July 2002

Human lysozyme dimers were prepared by the intermolecular cross‐linking of the monomer that contained the mutation of either Arg41 to Cys or Ala73 to Cys with a divalent maleimide compound. Among the three kinds of possible dimers only R41C–R41C dimer, in which the two catalytic clefts can come close to each other due to the proximity of the conjugation site to the active sites, turned out to be 2.3 times more specific to a polymer substrate, ethylene glycol chitin, as compared to an oligomer substrate, PNP‐(GlcNAc)5. The result indicates that it is possible to alter the substrate specificity of an enzyme by artificially controlling the orientation of the active sites.
Germinating conidiospores of Aspergillus amino acid auxotrophs are hypersensitive to heat shock, oxidative stress and DNA damage
Authors: Donnelly E. , Barnett Y.A. , McCullough W.
Published: July 2002

Germinating conidiospores (conidia) of Aspergillus nidulans amino acid‐requiring strains are hypersensitive to heat, oxidative stress, UV radiation and chemical mutagens when compared with other strains. They also showed an increased mutation rate. Sensitivity to stress conditions has been correlated with an abnormal RAS/cAMP pathway in mutants of S. cerevisiae. We suggest that the RAS/cAMP pathway is defective in germinating conidia of Aspergillus amino acid auxotrophs and that this is responsible for suppressing DNA repair and conferring sensitivity to oxidative stress and heat shock.
Biochemical characterization of glucosaminylmuramyldipeptide binding sites of murine macrophages
Authors: Golovina T.N. , Sumaroka M.V. , Samokhvalova L.V. , Shebzukhov Yu.V , Andronova T.M. , Nesmeyanov V.A.
Published: July 2002

By using radioligand analysis, murine peritoneal macrophages were shown to express several hundred high‐affinity cell surface GMDP‐binding sites (K
a 350 pM). Photoaffinity labeling followed by SDS‐PAGE enabled us to identify 32–34 and 38 kDa proteins inside these cells that bound GMDP specifically.
Precursor‐mediated opening of translocation pores in chloroplast envelopes
Authors: Bulychev Alexander , Pilon Marinus , Dassen Hans , van't Hof Ron , Vredenberg Wim , de Kruijff Ben
Published: July 2002

Direct electrical measurements on native chloroplast envelopes reveal that a full‐length chloroplast precursor protein causes an increase in the conductivity of the envelope membranes, due to its transit sequence. The conductivity is not influenced by a truncated precursor protein incapable of efficient translocation, suggesting precursor‐mediated opening of translocation pores in chloroplast envelopes.
Induction of phagocytic activity of M1 cells by an inhibitor of vacuolar FT‐ATPase, bafilomycin A1
Authors: Kinoshita Kuninori , Hidaka Hiroyoshi , Ohkuma Shoji
Published: July 2002

Bafilomycin a1, a selective inhibitor of vacuolar H+‐ATPase, time‐and dose‐dependently induced the differentiation of M1 cells, a murine myeloid leukemic cell line, into macrophage‐like cells as revealed by the phagocytosis of polystyrene latex particles. This differentiation was inhibited not only by actinomycin D and cycloheximide but also by ST‐638 (an inhibitor of tyrosine kinase). However, it was affected neither by K‐252a (an inhibitor of C‐kinase) nor by H‐89 (an inhibitor of A‐kinase), in contrast to the M1 cell differentiation induced by leukemia inhibitory factor (LIF). Okadaic acid inhibited both the bafilomycin A1‐induced and LIF‐induced differentiation of M1 cells.
Phosphoinositide 3‐phosphatase segregates from phosphatidylinositol 3‐kinase in EGF‐stimulated A431 cells and fails to in vitro hydrolyse phosphatidylinositol(3,4,5)trisphosphate
Authors: Payrastre Bernard , Gironcel Daisy , Plantavid Monique , Mauco Gérard , Breton Monique , Chap Hugues
Published: July 2002

Beside 4‐ and 5‐phosphatases playing a role in the interconversion between the D‐3 phosphorylated polyphosphoinositides, the only enzyme described so far to be responsible for a phosphomonoesterasic activity on the D‐3 position of inositol lipids is a specific 3‐phosphatase that hydrolyzes PtdIns(3)P in NIH 3T3 cells. We report here the presence of a potent 3‐phosphatase activity in different cell types. This activity is detected both in cytosol and membranes of A431 cells and is inhibited by VO4
3− and Zn2+. Interestingly, the cytosolic activity from A431 cells selectively hydrolyzes in vitro PtdIns(3)P and PtdIns(3,4)P2, whereas PtdIns(3,4,5)P3 remains a very poor substrate under the same conditions. Finally, assays of phosphatidylinositol 3‐kinase and 3‐phosphatase activities in the pool of phosphotyrosine‐containing proteins isolated from EGF‐stimulated A431 cells suggest a compartmentation of these two antagonistic activities during cell activation.
Superoxide production by cytochrome b559
Authors: Koshkin Vasilij , Pick Edgar
Published: July 2002

Purified cytochrome b
559 relipidated with either a mixture of phosphatidylcholine and phosphatidic acid or with phosphatidylcholine only exhibits high and low superoxide (O2) producing ability, respectively, in the absence of cytosolic activators [Koshkin, V. and Pick, E. (1993) FEBS Lett. 327, 57‐62]. This system was used as a model for the study of the mechanism of NADPH oxidase activation. It is shown that, depending on the composition of the phospholipid environment, cytochrome b
559 binds FAD with high or low affinity, this being accompanied by changes in flavin absorbance and fluorescence. High affinity binding of FAD to cytochrome b
559 relipidated with phosphatidylcholine combined with phosphatidic acid is associated with an enhanced NADPH‐driven O2
− producing capacity. A kinetic study of O2
− production by cytochrome b
559 reflavinated under stoichiometric FAD binding conditions revealed an FAD/heme ratio of 1:2. A further kinetic study of O2 production by high‐ and low‐activity relipidated and reflavinated eytochrome b
559, at varying substrate concentrations, and the determination of steady‐state difference spectra of such preparations, reduced by NADPH, indicated that O2
− production is activated by facilitation of electron transfer from NADPH to FAD rather than by an enhancement of NADPH binding.
Actinic light density dependence of the O intermediate of the photocycle of bacteriorhodopsin
Authors: Luchian Tudor , Tokaji Zsolt , Dancsházy Zsolt
Published: July 2002

The O intermediate of the photocycle of bacteriorhodopsin (BR) was studied by absorption kinetic measurements at different actinic light densities. With increasing exciting flash intensity, the relative yield of O slightly increases, while that of Mf strongly decreases at the expense of Ms. Kinetic calculations and the optical anisotropy of O show that O can be formed only from Mf although Mf and O have different light intensity dependences. In order to resolve the apparent contradiction, a phenomenologically new cooperative regulatory mechanism seems to be necessary.
Growth inhibition by Abl requires an interplay of its SH2 and tyrosine kinase domains
Authors: Mattioni Tiziana , Mayer Bruce J. , Picard Didier
Published: July 2002

Overexpression of c‐Abl tyrosine kinase can be growth inhibitory in certain fibroblast cell lines. Using a series of conditional chimeras between Abl and Src, we have now further dissected the Abl protein to determine which domains are required for this function. We found that growth inhibition, unlike transformation by oncogenic forms of Abl, is dependent on the presence of the cognate SH2 and tyrosine kinase domains. Since growth inhibition correlates with low tyrosine kinase activity, it may involve highly specific interactions of target proteins with both domains without the processivity of phosphorylation associated with oncogenic Abl.
Fructose 2,6‐bisphosphate induces irreversible transitions in cell‐free extracts of rat liver
Authors: Frenzel Jochen , Schellenberger Wolfgang , Eschrich Klaus
Published: July 2002

The effect of fructose 2,6‐bisphosphate on the dynamics of the 6‐phosphofructo‐1‐kinase/fructose‐1,6‐bisphos‐phatase cycle is investigated in a cell‐free extract of rat liver under steady‐state conditions. Bistability emerges on the basis of the reciprocal allosteric modulation of 6‐phosphofructo‐1‐kinase and fructose‐1,6‐bisphosphatase. Under conditions of bistability fructose 2,6‐bisphosphate may cause transitions between alternative steady states. However, in contrast to what is frequently observed in bistable systems, within a broad range of experimental conditions these transitions proceed irreversibly from states with high ATP to states characterized by low ATP.
Purification and characterization of the membrane‐bound nitrate reductase isoenzymes of Bradyrhizobium japonicum
Authors: Fernández-López Manuel , Olivares José , Bedmar Eulogio J.
Published: July 2002

Two respiratory membrane‐bound nitrate reductase (NR) isoenzymes, NRI and NRII, have been purified for the first time from one single microorganism. Triton X‐100‐solubilized NRs were purified by a three‐step procedure of differential centrifugation, Q‐Sepharose chromatography, and gel filtration on Sephacryl S‐300. Both isoenzymes were purified to homogeneity by the criteria of NR activity staining in polyacrylamide gels run under non‐denaturating conditions and coincident staining of the protein band by silver nitrate. NRI is composed of three subunits of 116 kDa, 68 kDa, and 56 kDa, whereas NRII is composed of four subunits of 116 kDa, 68 kDa, 59 kDa, and 56 kDa. The 116‐kDa subunit of NRI and the 59‐kDa subunit of NRII exhibited immunological cross‐reactivity with the respiratory NR of Pseudomonas stutzeri strain ZoBell.
The structure of the lantibiotic lacticin 481 produced by Lactococcus lactis: location of the thioether bridges
Authors: van den Hooven Henno W. , Lagerwerf Fija M. , Heerma Wigger , Haverkamp Johan , Piard Jean-Christophe , Hilbers Cornelis W. , Siezen Roland J. , Kuipers Oscar P. , Rollema Harry S.
Published: July 2002

The lantibiotic lacticin 481 is a bacteriocin produced by Lactococcus lactis ssp. lactis. This polypeptide contains 27 amino acids, including the unusual residues dehydrobutyrine and the thioether‐bridging lanthionine and 3‐methyllanthionine. Lacticin 481 belongs to a structurally distinct group of lantibiotics, which also include streptococcin A‐FF22, salivaricin A and variacin. Here we report the first complete structure of this type of lantibiotic. The exact location of the thioether bridges in lacticin 481 was determined by a combination of peptide chemistry, mass spectrometry and NMR spectroscopy, showing connections between residues 9 and 14, 11 and 25, and 18 and 26.
Lipoxygenase activity in heart cells
Authors: Breitbart Eyal , Sofer Yossi , Shainberg Asher , Grossman Shlomo
Published: July 2002

Arachidonic acid (AA) metabolism via the lipoxygenase (LOX) pathway in rat hearts and in cultured rat cardiomyocytes was investigated using 1‐[14C]AA. LOX activity was detected in the microsomal fraction, in the high speed supernatant prepared from rat hearts and in rat cardiomyocyte supernatant. LOX products from all fractions comigrated in thin layer chromatography as 12‐hydroxyeicosatetraenoic acid (12‐HETE) and 15‐HETE. Enzyme linked immunosorbent assay for 12‐HETE showed its formation by the microsomal fraction, the ammonium sulfate (AS) pellet, and by rat cardiomyocyte supernatant, while radioimmunoassay for 15‐HETE showed its formation only by the AS pellet. The properties of LOX in each fraction are reported here.
Immunochemical identification of the pssA gene product as phosphatidylserine synthase I of Chinese hamster ovary cells
Authors: Saito Kyoko , Kuge Osamu , Akamatsu Yuzuru , Nishijima Masahiro
Published: July 2002

We have previously shown that a Chinese hamster ovary (CHO) cell mutant defective in phosphatidylserine synthase I recovers the enzyme activity on transfection with a pssA cDNA clone isolated from the parental CHO‐K1. The resultant transfectant, CDT‐1, exhibited about 20‐fold higher specific activity of the enzyme in the membrane fraction than CHO‐K1 cells. Polyclonal antibodies against two peptides of the predicted pssA product cross‐reacted with a membrane protein having an apparent molecular mass of 42 kDa, which was overproduced in CDT‐1 cells. By immunoprecipitation with the antibody, phosphatidylserine synthase I activity as well as the 42‐kDa protein was eliminated from solubilized membrane proteins of CDT‐1 cells. Both the enzyme activity and the 42‐kDa protein of CHO‐K1 cells were enriched in the mitochondria‐associated membrane fraction and the microsome fraction, but neither was enriched in the mitochondria fraction or the cytosol fraction. These results suggest that the pssA gene encodes phosphatidylserine synthase I.
Astrocyte survival in the absence of exogenous substrate: Comparison of immature and mature cells
Authors: Hertz L. , Yager J.Y. , Juurlink B.H.J.
Published: July 2002

Astrocyte cultures prepared from newborn mouse neopallium were grown for either one or three weeks (representing, respectively, immature and mature astrocytes) and then exposed to deprivation of substrate (glucose and amino acids) for up to 48 hr. Cultures which had been deprived of metabolic substrates for either 24, 30, 36 or 48 hr were examined for lactate dehydrogenase efflux into the medium (an indicator of cell death) and ATP content. Significant cell death in mature astrocytes began after 30 hr of incubation in the substrate‐deprived medium, a time when ATP had fallen to ∼10% of its initial value. Immature astrocytes survived on a substrate‐free medium for 48 hr before there was any indication at all of cell death, and this corresponded to a time when ATP values had fallen to 5% of the initial values. These findings are compared to previous observations during simulated ischemia (substrate deprivation plus anoxia) when (1) there was a faster cell death and (2) cell death occurred at higher ATP levels.
Steroid induced rare bipolar mood disorder
Authors: Mazumdar PK , Najib MA , Varma SL
Published: July 2002

A patient with multiple psychosomatic disorder developed a steroid induced rare bipolar mood disorder (both mania and depression). The “unmasking effect” of steroids and a positive family history of psychiatric disorder as a possible risk factor, hitherto undocumented, is suggested in steroid induced psychosis.
Verapamil treatment in clozapine-induced sleep-related enuresis: a case report
Authors: Pojurovsky M , Schneidman M , Mark M , Weizman A
Published: July 2002

Enuresis is an embarrassing rare side effect of clozapine treatment. Using single-blind placebo-control design, the antienuretic activity of the calcium channel blocker verapamil (up to 80 mg/day per os, at 21.00 hours) was evaluated in a schizophrenic patient with comorbid obsessive-compulsive disorder (OCD) who developed nocturnal functional enuresis during clozapine treatment. Verapamil (80 mg/day) displayed antienuretic activity. No correlation between the bradycardiac effect and the antienuretic activity of verapamil was detected.
ATP synthesis at 100°C by an ATPase purified from the hyperthermophilic archaeon Pyrodictium abyssi
Authors: Dirmeier R , Hauska G , Stetter K.O
Published: July 2002

The chemolithoautotrophic archaeon Pyrodictium abyssi isolate TAG 11 lives close to 100°C and gains energy by sulfur respiration, with hydrogen as electron donor. From the membranes of this hyperthermophile, an ATPase complex was isolated. The purified enzyme consists of six major polypeptides, the 67, 51, 41, 26 and 22 kDa subunits composing the AF1 headpiece, and the 7 kDa proteolipid of the AF0 component. The headpiece of the enzyme restored the formation of ATP during sulfur respiration in membrane vesicles from which it had been removed by low salt treatment. Characteristics of the reconstituted activity suggest that the same enzyme is responsible for ATP formation in untreated membranes. ATP formation was neither sensitive to ionophores and uncouplers, nor to dicyclohexyl carbodiimide, but depended on closed vesicles. Both ATPase activity (up to 2 μmol per min and mg protein) as well as ATP formation (up to 0.4 μmol per min and mg membrane protein) were highest at 100°C. A P/e2 ratio of close to one can be estimated for sulfur respiration with hydrogen. In addition to ATP, autoradiographic detection revealed the formation of high quantities of 33Pi‐labeled ADP and of another compound not identified so far.
Purification and characterization of bovine cone arrestin (cArr)
Authors: Maeda Tadao , Ohguro Hiroshi , Sohma Hitoshi , Kuroki Yoshio , Wada Hiroshi , Okisaka Shigekuni , Murakami Akira
Published: July 2002

To elucidate the quenching mechanism of phototransduction in vertebrate cone photoreceptors, a cDNA clone encoding cone specific arrestin (cArr) was isolated from a bovine retinal cDNA library using a human cArr cDNA probe. Affinity‐purified anti‐peptide antibody specific to cArr was prepared. Immunohistochemical staining displayed specific labeling of cArr in cone photoreceptors and immunoblotting identified a 46 kDa protein band. We purified cArr from bovine retinas by sequential column chromatography using DEAE‐cellulose, gel filtration and mono Q columns. Binding studies revealed no binding of cArr to rhodopsin regardless of whether it was bleached and/or phosphorylated. cArr also failed to bind to heparin‐Sepharose under conditions which rod arrestin (rArr) bound to the column. The present data suggest that cArr may play a role in the quenching of phototransduction in cone photoreceptors and that its activity therein is different to that of rArr.
The important role of residue F268 in ligand binding by LXRβ
Authors: Urban Frank , Cavazos Gabriel , Dunbar James , Tan Bing , Escher Pascal , Tafuri Sherrie , Wang Minghan
Published: July 2002

Liver X receptors (LXRs) are nuclear receptors that regulate the metabolism of cholesterol and bile acids. Despite information on the specificity of their natural ligands, oxysterols, relatively little is known about the ligand binding site in LXRs. The helix 3 region in the ligand binding domain (LBD) of peroxisome proliferator‐activated receptors (PPARs) has been implicated in ligand entry. Sequence alignment of LXRs, farnesoid X receptor (FXR), and PPARs identified the corresponding helix 3 region in the LXRβ LBD. Residues F268 and T272, which are conserved in all the aligned sequences and only in LXRs and FXR, respectively, were replaced with alanine. The effects of these mutations on ligand binding and receptor activation were examined using an in vitro ligand binding assay and a cell based reporter assay, respectively. The LXRβ mutant F268A did not bind ligand. In contrast, conversion of T272 to alanine has no effect on ligand binding. By transiently expressing a chimeric receptor containing Escherichia coli tetracycline repressor (TetR) and LXRβ LBD and a reporter with a TetR binding site, we show that mutant F268A lost the ability to activate transcription of the reporter, whereas mutant T272A still has an activity similar to that of the wild‐type LXRβ. These data, consistent with the findings in the in vitro ligand binding assay and our 3D modeling, are the first study that identifies a residue critical for ligand binding in LXRβ.
Conserved gene structure and transcription factor sites in the human and mouse deoxycytidine kinase genes1
Authors: Johansson Magnus , Norda Ameli , Karlsson Anna
Published: July 2002

Deoxycytidine kinase (dCK) phosphorylates several anti‐cancer and anti‐viral nucleoside analogs. The enzyme is predominantly expressed in lymphoid tissues regulated by an unknown mechanism. We have cloned and sequenced the 20 kbp mouse dCK gene and ≈1.7 kbp of the 5′ flanking regions of both the human and mouse dCK genes. Five major inter‐species conserved motifs were identified in the 5′ region including the transcription initiation region, an SP1 site and two closely located putative octamer transcription factor sites. Luciferase reporter experiments showed that the human dCK 5′ region efficiently initiated transcription but no tissue regulatory element could be identified.