bluuebunny/crossref_metadata_2025 huggingface dataset:
Temperature dependent chaperone‐like activity of alpha‐crystallin
Authors: Raman B. , Ramakrishna T. , Mohan Rao Ch.
Published: April 2003

Alpha‐crystallin, a multimeric protein present in the eye lens, is known to have chaperone‐like activity in preventing the aggregation of enzymes and other crystallins. We have studied the chaperone‐like activity of this protein towards the aggregation of insulin B chain, induced by reducing the interchain disulphide bond with dithiothreitol. At room temperature, there is no detectable protection (at a 1:1 (w/w) ratio of insulin: α‐crystallin) against the aggregation of insulin B chain by α‐crystallin, whereas it completely prevents this aggregation at 40°C. We have monitored the temperature dependence of the protection of aggregation by α‐crystallin; the protection increases sharply above 30°C and reaches almost 100% by 41°C. Probing the hydrophobic surfaces of α‐crystallin with the hydrophobic fluorphore 8‐anilino‐1 naphthalene sulfonate suggests that the hydrophobic surfaces of α‐crystallin are exposed to a greater extent above 30°C. A complete prevention of the aggregation is achieved at 27.6°C by increasing the concentration of α‐crystallin by more than 8 fold. Similar temperature dependent chaperone‐like activity of α‐crystallin is observed towards the aggregation of zetacrystallin, an enzyme crystallin from guinea pig. We have earlier shown that α‐crystallin exposes hydrophobic surface(s) at temperatures above 30°C. These results support our earlier hypothesis [Raman, B. and Rao, Ch.M. (1994) J. Biol. Chem. 269, 27264–27268] that the chaperone‐like activity of α‐crystallin is more pronounced in its structurally perturbed state.
A novel kDa form of subtilisin cleaved actin: structural and functional consequences of cleavage between Ser234 and Ser235
Authors: Vahdat Arash , Miller Carl , Phillips Martin , Muhlrad Andras , Reisler Emil
Published: April 2003

A new 27/16 kDa form of cleaved actin was prepared by subtilisin cleavage between Ser234 and Ser235 of F(MgADP)‐actin complexed with BeFx. The cleavage had little effect on actin‐actin interactions as probed in polymerization measurements and by electron microscopy. In circular dichroism melting experiments the thermostability of F‐actin was reduced by about 10°C by this cleavage. The in vitro motility and V
max, but not K
m, of actomyosin ATPase were decreased by about 20% upon 27/16 kDa cleavage of F‐actin. The binding of tropomyosin to actin was unchanged by this modification.
Studies on human porin: XIII. The type‐1 VDAC ‘Porin 31HL’ biotinylated at the plasmalemma of Trypan blue excluding human B lymphocytes
Authors: Jakob Christian , Götz Hilde , Hellmann Thea , Hellmann Klaus Peter , Reymann Susanne , Flörke Harald , Thinnes Friedrich P. , Hilschmann Norbert
Published: April 2003

In 1989, we demonstrated for the first time the expression of the VDAC ‘Porin 31HL’ in the plasmalemma of human B lymphocytes, then giving first evidence of a multi‐topological expression of VDAC. Meanwhile, data from this and other laboratories support our proposal of a multi‐compartment distribution of the channel in mammalian tissues. Here, by biotinylation of plasmalemma‐integrated proteins of proven living B lymphocytes, followed by two‐dimensional electrophoresis, immuno‐ and streptavidin affinity blotting, we show that part of the channel molecules can be labelled at the outer membrane of the cells. Thus, by a relevant approach our results invalidate objections concerning putative cross‐reactivity of anti‐human Type‐1 porin antibodies with non‐VDAC proteins at the outer cell membrane.
Structure‐activity study of a laminin α1 chain active peptide segment Ile‐Lys‐Val‐Ala‐Val (IKVAV)
Authors: Nomizu Motoyoshi , Weeks Benjamin S. , Weston Christi A. , Kim Woo Hoo , Kleinman Hynda K. , Yamada Yoshihiko
Published: April 2003

The IKVAV sequence, one of the most potent active sites of laminin‐1, has been shown to promote cell adhesion, neurite outgrowth, and tumor growth. Here we have determined the structural requirements of the IKVAV sequence for cell attachment and neurite outgrowth using various 12‐mer synthetic peptide analogs. All‐l‐ and all‐d‐IKVAV peptides showed cell attachment and neurite outgrowth activities. In contrast, all‐l‐ and all‐d‐reverse‐sequence peptides were not active. Some of the analogs, in which the lysine and isoleucine residues of the IKVAV peptide were substituted with different amino acids, promoted cell attachment, but none of the analog peptides showed neurite outgrowth activity comparable to that of the IKVAV peptide. These results suggest that the lysine and isoleucine residues are critical for the biological functions of the IKVAV peptide.
Composite control of cell function: metabolic pathways behaving as single control units
Authors: Kholodenko Boris N. , Schuster Stefan , Rohwer Johann M. , Cascante Marta , Westerhoff Hans V.
Published: April 2003

This paper shows that under some conditions the control exerted by a part of a metabolic network (a pathway) on a flux or concentration in any other part can be described through a single (overall) control coefficient. This has the following implications: (i) the relative contributions of a pathway enzyme to the regulation of the pathway (output) flux and of any flux or concentration outside are identical; therefore, the control analysis of the pathway ‘in isolation’ allows one to determine the control exerted by any pathway enzyme on the rest of the cell by estimation of the control efficient of just one, arbitrarily chosen enzyme; (ii) the relative control of any two metabolic variables outside the pathway (measured as the ratio of the control coefficients over these two variables outside) is the same for all pathway enzymes. These properties allow one to substitute effectively a pathway by a single (super)reaction and make it possible to consider such a pathway as a metabolic unit within the cellular enzyme network.
S15261 antagonises amylin‐induced impaired glucose tolerance
Authors: Espinal J. , Lacour F. , Berger S. , Duhault J.
Published: April 2003

Amylin has been postulated to antagonise or inhibit the action of insulin in peripheral rat tissues and thus contribute to, or be responsible for, the development of insulin resistance. We have recently reported that S15261 is a compound capable of increasing insulin sensitivity in ageing insulin resistant rats. In order to assess whether S15261 had any effects on amylin induced insulin resistance we used a model where amylin causes an impairement in glucose tolerance in an acute manner, by means of an intraportal infusion of the hormone in normal rats. We report here that S15261 can antagonise this amylin‐induced impaired glucose tolerance.
Biochemical characterization of recombinant yeast PPZ1, a protein phosphatase involved in salt tolerance
Authors: Posas Francesc , Bollen Mathieu , Stalmans Willy , Ariño Joaquín
Published: April 2003

The Saccharomyces cerevisiae gene PPZ1 codes for a 692‐residues protein that shows in its carboxyl‐terminal half about 60% identity with the catalytic subunit of mammalian and yeast protein phosphatase‐1 and that is involved in salt homeostasis. T The complete PPZ1 protein has been succesfully expressed as a soluble glutathione‐S‐transferase fusion protein. The recombinant protein, after purification by a single affinity chromatography step, displayed phosphatase activity towards a number of substrates, including myelin basic protein, histone 2A and casein, but was ineffective in dephosphorylating glycogen phosphorylase. It was also active towards p‐nitrophenylphosphate. The activity was severalfold increased by the presence of Mn2+ ions and by limited trypsinolysis. The enzyme was inhibited by okadaic acid and microcystin‐LR at concentrations comparable to what is found for type 1 protein phosphatase although it was much less sensitive to inhibitor‐2. The recombinant protein was phosphorylated in vitro by cAMP‐dependent protein kinase, protein kinase C and casein kinase‐2. Phosphorylation affected preferentially sites located in the amino‐terminal half of the protein and did not alter the activity of the phosphatase.
ERK2/p42 MAP kinase stimulates both autonomous and SRF‐dependent DNA binding by Elk‐1
Authors: Sharrocks Andrew D.
Published: April 2003

A ternary complex comprised of SRF, ternary complex factor (TCF) and the c‐fos SRE is the target of several extracellular signal regulated pathways. Phosphorylation of the TCF Elk‐1 is a key event in the activation of this complex. We demonstrate that ERK2/p42 phosphorylation of Elk‐1 stimulates its recruitment into ternary complexes with SRF. Moreover, phosphorylation of Elk‐1 also stimulates its autonomous SRF‐independent binding to high affinity binding sites. Thus part of the effect of ERK2/p42 phosphorylation is to stimulate DNA‐binding by the ETS DNA‐binding domain of Elk‐1.
The mode of actions of glyceraldehyde‐3‐phosphate dehydrogenase identified as an immunoglobulin production stimulating factor
Authors: Sugahara Takuya , Shirahata Sanetaka , Sasaki Takeshi , Murakami Hiroki
Published: April 2003

Immunoglobulin production stimulating factor‐IIα, which enhances immunoglobulin production of human and mouse hybridomas was purified from cell lysate of human Burkitt's lymphoma, Namalwa cells, and identified as glyceraldehyde‐3‐phosphate dehydrogenase. The enhancement of immunoglobulin production with this enzyme was not linked with its enzymatic activity. The enzyme enhanced immunoglobulin productivity of transcription‐suppressed hybridomas, but did not enhance that of translation‐suppressed hybridomas. From these results, it is suggested that this enzyme takes part in the post‐translational control or the enhancement of translation activity to stimulate immunoglobulin production of hybridomas.
Electron microscopic evidence for a mucin‐like region in chick muscle α‐dystroglycan
Authors: Brancaccio Andrea , Schulthess Therese , Gesemann Matthias , Engel Jürgen
Published: April 2003

α‐Dystroglycan has been isolated from chicken cardiac muscle and its molecular weight was estimated to be ≈135 kDa. The avian protein interacts with murine Engelbreth‐Holm‐Swarm (EHS) tumor laminin via interaction with the C‐terminal LG4 and LG5 domains (fragment E3) of the laminin α‐chain. This laminin binding is calcium‐dependent and can be competed by heparin. Electron microscopy investigation on the shape of α‐dystroglycan suggests that the core protein consists of two roughly globular domains connected by a segment which most likely corresponds to a mucin‐like central region also predicted by sequence analysis on mammalian isoforms. This segment may act as a spacer in the dystrophin‐associated glycoproteins complex exposing the N‐terminal domain of α‐dystroglycan to laminin in the extracellular space.
Rapid turnover of tryptophan hydroxylase in serotonin producing cells: demonstration of ATP‐dependent proteolytic degradation
Authors: Hasegawa Hiroyuki , Kojima Masayo , Oguro Kazuya , Nakanishi Nobuo
Published: April 2003

A rapid and continuous proteolysis of tryptophan hydroxylase was demonstrated with two mast cell lines derived from rat basophilic leukemia cells (RBL2H3) and mouse mastocytoma (FMA3). Under conditions in which protein biosynthesis was arrested by administration of cycloheximide, the decay profile of tryptophan hydroxylase protein was traced by Western blot analysis. Incorporation of [35S]methionine and the chase experiment performed without interfering with the metabolic stage also showed that tryptophan hydroxylase has been cleaved rapidly. The half life of the enzyme was 11–15 min in RBL2H3 cells and 40–60 min in FMA3 cells, and the process was demonstrated to be dependent on intracellular ATP.
Evidence that conformational changes upon the transition of the native to the modified form of vitronectin are not limited to the heparin binding domain
Authors: Seiffert Dietmar
Published: April 2003

Vitronectin (Vn) exists in vivo in at least two different conformational states, the native and the modified form, and these forms have different ligand binding properties. To characterize the molecular events associated with this conformational flexibility, modified Vn was analyzed by competitive ELISA using a panel of conformationally sensitive antibodies with known epitopes. These studies provided evidence for major molecular rearrangements upon the transition from the native to the modified form that are not limited to the C‐terminal heparin binding domain, but also occur in the N‐terminal part of the molecule.
Topological properties of two cubic phases of a phospholipid : cholesterol: diacylglycerol aqueous system and their possible implications in the phospholipase C‐induced liposome fusion
Authors: Nieva J.L. , Alonso A. , Basáñez G. , Goñi F.M. , Gulik A. , Vargas R. , Luzzati V.
Published: April 2003

Water dispersions of phospholipid: cholesterol: diacylglycerol may, under certain conditions, originate either the lipid‐ and water‐permeable Q224 cubic phase, or the lipid‐permeable but water‐impermeable Q227 cubic phase. These results are discussed within the framework of the phospholipase C‐induced fusion of liposomes [Nieva et al. (1993) Biochemistry 32, 1054]. It is suggested that the cubic phases Q224 and Q227 represnt two classes of lipid organization, one promoting, the other hindering the mixing of aqueous contents that is characteristic of membrane fusion. In this context, inverted micelles appear to be the end point of the fusion process, rather than fusion intermediates.
Simultaneous measurements of Ca2+ in the intracellular stores and the cytosol of hepatocytes during hormone‐induced Ca2+ oscillations
Authors: Chatton Jean-Yves , Liu Haiyan , Stucki Jörg W.
Published: April 2003

Simultaneous Ca2+ measurements in the cytosol and intracellular stores (IS) of rat hepatocytes were performed using two Ca2+‐sensitive probes (Fluo‐3 and Mag‐fura‐2), and combined whole‐cell patch clamp and fluorescence microscopy. A steady‐state Ca2+ concentration of ∼630 μM was estimated in the IS. α1‐Adrenergic stimulation induced periodic elevations of cytosolic Ca2+ and parallel synchronized transient declines in the IS. Subsequent application of the intracellular Ca2+‐pump inhibitor thapsigargin resulted in a release of Ca2+ from the IS to reach a level of Ca2+ depletion much lower than the lowest transient decline observed during the oscillations.
Labeling of a cysteine in the cardiotonic glycoside binding site by the steroid derivative HDMA
Authors: Antolovic R. , Schoner W. , Geering K. , Canessa C. , Rossier B.C. , Horisberger J.-D.
Published: April 2003

The digoxigenin derivative N‐hydroxysuccinimidyl digoxigenin‐3‐O‐methylcarbonylϵ‐aminocaproate (HDMA) has been shown to covalently label the ouabain binding site of the Na,K‐ATPase ϵ subunit [Antolovic et al. (1995) Eur. J. Biochem. 227, 61–67]. In the present study we observed both, labeling and inactivation of the activity, of wild type Na,K‐ATPase overexpressed in Xenopus oocyte. In contrast, no significant inhibition and no labeling could be detected when a Cys‐113 of the first transmembrane segment was mutated to serine, although the affinity of this mutant for digoxigenin or HDMA measured in acute inhibition experiments was similar to the wild type. This indicates that after docking of its genin moiety, HDMA can form a thioester bond with Cys‐113.
ADP‐ribosylation of Rho enhances actin polymerization‐coupled shape oscillations in human neutrophils
Authors: Ehrengruber Markus U. , Boquet Patrice , Coates Thomas D. , Deranleau David A.
Published: April 2003

Stimulated neutrophils exhibit coordinated sinusoidal oscillations in filamentous actin content and cellular shape. We investigated the effect of inhibition of the small G protein Rho on neutrophil actin polymerization, shape changes and oscillations using a genetically engineered toxin that enters cells and selectively ADP‐ribosylates endogenous Rho. This treatment increased the amplitudes and frequencies of shape oscillations and duration of the oscillating transient. However, it had no effect on the initial actin polymerization and shape changes induced by N‐formyl‐Met‐Leu‐Phe. Regulation of these oscillations may be important for the control of neutrophil motility.
A mutation in the 5′ untranslated region of the human α‐galactosidase A gene in high‐activity variants inhibits specific protein binding
Authors: Saifudeen Zubaida , Desnick Robert J. , Ehrlich Melanie
Published: April 2003

Recently, normal individuals were identified who had high levels of plasma α‐galactosidase A activity and a G to A transition in the 5′ untranslated (5′ UT) region of the α‐galactosidase A gene. Electrophoretic mobility shift assays revealed that the wild‐type sequence at the site of this mutation complexed with specific nuclear proteins. A standard NF‐κB site competed with the 5′ UT site for formation of these DNA‐protein complexes. Complex formation was inhibited by the transition mutation. Therefore, the wild‐type site might down‐modulate expression of the α‐galactosidase A gene from this 5′ untranslated region, which includes a previously described protein‐binding site for another family of sequence‐specific DNA‐binding proteins, methylated DNA‐binding protein.
The 64 kDa polypeptide of spinach may not be the LHCII kinase, but a lumen‐located polyphenol oxidase
Authors: Sokolenko Anna , Fulgosi Hrvoje , Gal Alma , Altschmied Lothar , Ohad Itzhak , Herrmann Reinhold G.
Published: April 2003

Phosphorylation of chlorophyll a/b‐binding proteins of the of photosystem II light‐harvesting assembly controls the energy distribution between the two photosystems as well as the turnover of thylakoid membrane proteins. The LHCII kinase, suggested to be a 64 kDa protein, is light‐regulated by a mechanism involving reduction of plastoquinone and the participation of the cytochrome b
6/f complex. A cDNA encoding that protein has been isolated from a lambda gt11‐based library made from spinach polyadenylated RNA using a two‐step strategy involving screening by polyclonal monospecific antisera and plaque hybridization. The protein of 73.1 kDa molecular mass represents a precursor which contains a bipartite transit peptide of 101 amino acid residues (11.0 kDa) that directs the protein into the thylakoid lumen. It can be phosphorylated in vitro, and exhibits significant homology to plant polyphenol oxidases, not to kinases. The gene was therefore designated PpoA. Reinvestigation of components in the molecular mass range of 50–70 kDa disclosed five additional proteins which can accompany kinase‐active cytochrome b
6/f, photosystem II and AMS [1] preparations. Four of them can be phosphorylated in vitro; two with apparent molecular masses of 53 and 66 kDa are capable of phosphorylation, and represent new, yet unidentified proteins.
The first observation of O2− generation at real time in vivo from non‐Kupffer sinusoidal cells in perfused rat liver during acute ethanol intoxication
Authors: Nakano Minoru , Kikuyama Masataka , Hasegawa Tadashi , Ito Takashi , Sakurai Kazushi , Hiraishi Katsuya , Hashimura Etsuro , Adachi Masakazu
Published: April 2003

Infusion of ethanol or phorbol myristate acetate (PMA) into the perfused rat liver immediately produces O2
− which was detected directly by infusion of a Cypridina luciferin analogue, MCLA as a chemiluminescence reagent. The MCLA photon emission was inhibitable by SOD. Generation of O2
− in the liver was further verified by nitroblue tetrazolium, formazan precipitate formation. Ethanol‐induced O2
− generation was unaffected by gadolinium chloride (GdCl3), an inhibitor of kupffer cells, while PMA induced O2
− generation was completely abolished by GdCl3, Since PMA is a known stimulator of phagocytic cells including Kupffer cells, the results indicate, for the first time that ethanol stimulates a non‐Kupffer cell population, probably liver sinusoid endothelial cell to produce O2
−
Inhibition of aromatase expression by a psoralen‐linked triplex‐forming oligonucleotide targeted to a coding sequence
Authors: Macaulay V.M. , Bates P.J. , McLean M.J. , Rowlands M.G. , Jenkins T.C. , Ashworth A. , Neidle S.
Published: April 2003

The cytochrome P450 enzyme aromatase (P450arom) is an important target in breast cancer treatment. We have designed a 20‐base pyrimidine oligodeoxynucleotide (ODN) which forms a sequence‐specific triple helix (triplex) with a purine‐rich tract in the P450arom coding sequence. The psoralen‐linked ODN (Pso20T) formed photo‐induced cross‐linked products with target double‐stranded DNA. Cross‐linked adducts formed in vitro between ODNs and P450arom expression constructs were used to transfect COS and human MCF‐7 breast cancer cells. Levels of aromatase transcripts and enzyme activity were significantly lower in cultures transfected with Pso20T‐treated cDNA relative to controls. Pso20T had a lesser inhibitory effect on aromatase expression from a mutant P450arom construct, consistent with predicted effects of the mutations on triplex formation. These results are compatible with triplex‐mediated interruption of transcription within intact cells.
Mutations in the NKXD consensus element indicate that GTP binds to the γ‐subunit of translation initiation factor eIF2
Authors: Naranda Tatjana , Sirangelo Ivana , Fabbri Bradon J. , Hershey John W.B.
Published: April 2003

Initiation factor eIF2 binds GTP and promotes the binding of methionyl‐tRNA to ribosomes. Biochemical and sequence evidence suggests that the GTP might bind to either the β‐ or γ‐subunit of eIF2. Mutations were made in the NKXD consensus elements found in both subunits and individual mutant forms were overexpressed in transiently transfected COS‐1 cells. The effect on the translational efficiency of a reporter mRNA for dihydrofolate reductase was monitored. Mutations in the γ‐subunit cause severe repression of protein synthesis, whereas those in the β‐subunit are only mildly inhibitory. The results support the view that GTP binds exclusively to the γ‐subunit.
Molecular cloning of the cDNA encoding a novel protein disulfide isomerase‐related protein (PDIR)
Authors: Hayano Toshiya , Kikuchi Masakazu
Published: April 2003

We isolated the cDNA of a novel protein disulfide isomerase (PDI)‐related protein, designated PDIR, from a human placental cDNA library. Deduced from its nucleotide sequence, PDIR has the three CXXC‐like motifs (Cys‐Ser‐Met‐Cys, Cys‐Gly‐His‐Cys and Cys‐Pro‐His‐Cys), which are found in proteins within the PDI superfamily and are responsible for oxidoreductase activity. PDIR has a hydrophobic stretch at its amino terminus, which may serve as a signal sequence, and the putative endoplasmic reticulum (ER) retention signal ‘Lys‐Glu‐Glu‐Leu’ at its carboxy terminus, indicating that PDIR is an ER resident protein. Northern blots showed that PDIR is preferentially expressed in cells actively secreting proteins and that the expression of PDIR is stress‐inducible. These results suggested that PDIR has oxidoreductase activity of disulfide bonds against polypeptides and that it acts as a catalyst of protein folding in the lumen of the ER.
Involvement of protein kinase in Δ12‐prostaglandin J2‐induced expression of rat heme oxygenase‐1 gene
Authors: Negishi Manabu , Odani Noriko , Koizumi Tomonobu , Takahashi Senye , Ichikawa Atsushi
Published: April 2003

We recently identified the cis‐regulatory element and its specific nuclear binding factors for Δ
12‐prostaglandin (PG) J2‐induced expression of the rat heme oxygenase, HO‐1 [Koizumi, T., Odani, N., Okuyama, T., Ichikawa, A. and Negishi, M. (1995)J. Biol. Chem. 270, in press]. Here we further examined the molecular mechanism underlying the Δ
12‐PGJ2‐induced HO‐1 gene expression. Protein kinase inhibitors, 2‐aminopurine and staurosporine, suppressed the Δ
12‐PGJ2‐induced HO‐1 mRNA and the nuclear protein binding to the Δ
12‐PGJ2‐responsivecis‐regulatory element in rat basophilic leukemia cells. Furthermore, the nuclear protein binding to the element was suppressed by in vitro phosphatase treatment of the nuclear proteins from Δ
12‐PGJ2‐treated cells. These findings suggest that Δ
12‐PGJ2 induces the expression of the HO‐1 gene through phosphorylation of the nuclear proteins which bind to the Δ
12‐PGJ2‐responsive element.
Identification of atypical (non‐AT1, non‐AT2) angiotensin binding sites with high affinity for angiotensin I on IEC‐18 rat intestinal epithelial cells
Authors: Smith Roger D.
Published: April 2003

Specific high‐affinity (Kd = 3.4 nM) binding sites for 125I‐labelled angiotensin I ([125I]Ang I) were identified on an epithelial cell line (IEC‐18) derived from the rat small intestine. The sites, which also have high affinity for Ang II, are insensitive to both AT1‐ and AT2‐specific angiotensin receptor antagonists, The rank order of potency with which various angiotensin peptides inhibited[125I]Ang I binding to the cells (Ang I ≥ Ang II > Ang(1–7) > [Sar1,Ile8]‐Ang II > Ang(3–8) > Ang III) also distinguishes these sites from AT1 and AT2 angiotensin receptors.
N‐Glycosylation‐defective receptor for erythropoietin can transduce the ligand‐induced cell proliferation signal
Authors: Nagao Masaya , Morishita Emi , Hanai Yasumitsu , Kobayashi Kazuhira , Sasaki Ryuzo
Published: April 2003

Erythropoietin receptor (EPOR) contains a single N‐linked sugar in an extracellular domain. It has been suggested that an erythroleukemia cell line with high sensitivity to EPO expresses a high molecular mass form of EPOR, which appears to be a highly N‐glycosylated form responsible for EPO‐mediated signal transduction [Sawyer and Hankins (1993) Proc. Natl. Acad. Sci. USA 90, 6849–6853]. To examine the role of the N‐linked sugar chain, we prepared EPO‐dependent cell lines expressing the wild‐type EPOR and N‐glycosylation‐defective EPOR. There was little difference in the expression of EPOR on the cell surface, EPO binding kinetics, and EPO‐induced cell proliferation between the clones expressing the mutant EPOR and those expressing the wild‐type EPOR.
Molecular cloning and functional characterization of a GABA/betaine transporter from human kidney
Authors: Rasola Andrea , Galietta Luis J.V. , Barone Virginia , Romeo Giovanni , Bagnasco Serena
Published: April 2003

The human homologue of the canine GABA/betaine transporter (BGT‐1) was isolated from a kidney inner medulla cDNA library. The coding sequence predicts a 614 amino acids protein with the typical features of neurotransmitter transporter family. The gene maps to chromosome 12p13 and, in addition to kidney, is also expressed in brain, liver, heart, skeletal muscle, and placenta. Functional studies reveal a K
m = 20 μM for GABA transport and a coupling to Na+ and Cl− with a stoichiometry 3 Na+:2 Cl−:1 GABA. At 500 μM the GABA transport was inhibited by various compounds with the following potency order: quinidine > verapamil > phloretin > betaine.
A new mutation in the pufL gene responsible for the terbutryn resistance phenotype in Rubrivivax gelatinosus
Authors: Ouchane S. , Picaud M. , Astier C.
Published: April 2003

Rubrivivax gelatinosus is a facultative phototrophic non‐sulfur bacterium belonging to the β subclass of the purple bacteria. A terbutryn‐resistant mutant of R. gelatinosus has been isolated and characterized. Increased resistance levels to terbutryn (300‐fold), atrazine (6‐fold) and o‐phenanthroline (3‐fold) were observed for the mutant compared with wild type. Sequence analysis of the mutant revealed a new mutation in the pufL gene coding for the L subunit of the reaction centre (RC) at codon 192 leading to an amino‐acid substitution from Gly in the wild type to Asp in the mutant. This substitution is located in the D helix of the L subunit, suggesting an interaction between terbutryn and this part of the polypeptide in the RC of R. gelatinosus. This is the first report of a mutation leading to herbicide resistance and affecting the D helix in purple bacteria. Furthermore R. gelatinosus wild type is highly sensitive to o‐phenanthroline compared with other purple bacteria (Rhodobacter capsulatus and Rhodobacter sphaeroides). Sequence comparison of the L subunit from six purple bacteria in which o‐phenanthroline sensitivity was measured suggests that SerL226 might be responsible for this phenotype.
Electron cryomicroscopy of two‐dimensional crystals of the H+‐ATPase from chloroplasts
Authors: Böttcher Bettina , Gräber Peter , Boekema Egbert J. , Lücken Uwe
Published: April 2003

The H+‐ATPase from spinach chloroplasts was isolated and purified. Two‐dimensional crystals were obtained from the protein/lipid/detergent micelles by treatment with phospholipase and simultaneous removal of detergent and fatty acids by Biobeads. The resulting two‐dimensionally ordered arrays were investigated by electron cryomicroscopy. The ordered arrays showed top view projections of CF0F1. The images were analysed by correlation averaging. In this view CF0F1 has dimensions of 11.4 × 9 nm. The average view shows a strongly asymmetric molecule, in contrast to the rather hexagonal features of CF1, previously analyzed from two‐dimensional arrays. It is concluded that this is due either to an asymmetric structure and positioning of CF0 relative to CF1 or to a rearrangement of CF1 subunits induced by binding of CF0 to CF1.
Cloning, expression and characterization of human thioltransferase (glutaredoxin) in E. coli
Authors: Chrestensen Carol A. , Eckman Christopher B. , Starke David W. , Mieyal John J.
Published: April 2003

PCR primers were designed from the known amino acid (aa) sequence for human red blood cell thioltransferase (hRBC TTase) and the known cDNA sequence for pig liver TTase (82% homologous) and used to amplify thioltransferase from a pool of human brain cDNAs. The PCR product was inserted into the pKK233‐2 expression vector. The DNA sequence of the insert agreed with the aa sequence. High level expression of the enzyme was accomplished in E. coli, and Western blot analysis confirmed its identity. Recombinant TTase displayed catalytic properties indistinguishable from natural hRBC TTase.
Thrombopoietin, c‐Mpl ligand, induces tyrosine phosphorylation of Tyk2, JAK2, and STAT3, and enhances agonists‐induced aggregation in platelets in vitro
Authors: Ezumi Yasuharu , Takayama Hiroshi , Okuma Minoru
Published: April 2003

We investigated in vitro effects of recombinant human thrombopoietin (TPO), or c‐Mpl ligand, on human platelets. TPO induced rapid dose‐dependent tyrosine phosphorylation of several proteins. We identified Janus tyrosine kinases, Tyk2 and JAK2, and a member of STAT (signal transducers and activators of transcription) family, STAT3, as the tyrosine‐phosphorylated proteins in response to TPO. TPO by itself did not cause platelet aggregation and shape change, but augmented ADP‐induced aggregation in a dose‐dependent manner. Acetylsalicylic acid inhibited the secondary aggregation enhanced by TPO, but not the TPO‐induced potentiation of the primary aggregation. TPO modulates platelet activation possibly through protein‐tyrosine phosphorylation.
Bimodal distribution of proteinase 3 (PR3) surface expression reflects a constitutive heterogeneity in the polymorphonuclear neutrophil pool
Authors: Halbwachs-Mecarelli L. , Bessou G. , Lesavre P. , Lopez S. , Witko-Sarsat V.
Published: April 2003

Proteinase 3, which is known as an intracellular serine protease of neutrophils, was detected at the surface of a subpopulation of freshly isolated PMN. The proportion of PR3‐positive and ‐negative PMN, observed by flow cytometry with anti‐PR3 mAbs or ANCA autoantibodies, varies among individuals but is extremely stable for each individual over prolonged time periods. After PMN degranulation by FMLP with cyt. B, membrane PR3 expression increases but the proportion of low and high PR3‐expressing cells remains stable. The existence of a subset of PMN which spontaneously expresses PR3 and varies among individuals, may be relevant to the pathogenesis of anti‐PR3 ANCA autoantibody‐related vasculitis.
Stimulation of catecholamine secretion from adrenal chromaffin cells by 14‐3‐3 proteins is due to reorganisation of the cortical actin network
Authors: Roth Dagmar , Burgoyne Robert D.
Published: April 2003

Catecholamine release from digitonin‐permeabilized adrenal chromaffin cells is increased by exogenous 14‐3‐3 proteins. In order to determine how 14‐3‐3 proteins stimulate exocytosis their effect on the cortical actin network was examined. Increased amounts of β and γ isoforms of 14‐3‐3 proteins were associated with the Triton‐insoluble cytoskeleton of chromaffin cells following incubation with exogenous 14‐3‐3 proteins. The stimulation of catecholamine release by 14‐3‐3 proteins was abolished by prior incubation with the actin filament stabilising drug phalloidin. Rhodamine phalloidin staining showed that the cortical actin network was disassembled and actin reorganised into intracellular foci following treatment with 14‐3‐3 proteins. These data suggest that 14‐3‐3 proteins enhance catecholamine release in permeabilized chromaffin cells by reorganisation of the cortical actin barrier to allow increased availability of secretory vesicles for exocytosis.
Modification of a specific tyrosine enables tracing of the end‐to‐end distance during apomyoglobin folding
Authors: Rischel Christian , Poulsen Flemming M.
Published: April 2003

In order to follow the overall geometry of the apomyoglobin molecule during folding, we have converted a specific tyrosine residue into 3‐nitro‐tyrosine. The specificity of the modification was verified by proteolytic cleavage of the modified protein and mass spectroscopy of the resulting fragments. By measuring the energy transfer from the tryptophanyl side‐chains to the modified residue the average end‐to‐end distance can be followed. The experiment shows that after initiation of folding the N‐ and C‐termini are rapidly brought into proximity, possibly to a near‐native distance.
Proopiomelanocortin, corticotropin releasing hormone and corticotropin releasing hormone receptor genes are expressed in human skin
Authors: Slominski A. , Ermak G. , Hwang J. , Chakraborty A. , Mazurkiewicz J.E. , Mihm M.
Published: April 2003

Evidence is provided that human skin, the largest body organ exposed to multiple stressors, expresses proopiomelanocortin (POMC), corticotropin releasing hormone (CRH) and CRH‐receptor (CRHR) genes in vivo. In vitro studies show that POMC and CRHR mRNAs are transcribed in melanocytes, cells derived from the neural crest, and in keratinocytes, cells derived from the ectoderm. CRH mRNA is transcribed in cultured melanocytes but not in keratinocytes. It is proposed that an equivalent of the ‘hypothalamus‐pituitary axis’ composed of the CRH‐CRHR‐POMC loop is conserved in mammalian skin.
Interaction of cryptophycin 1 with tubulin and microtubules
Authors: Kerksiek Kristen , Mejillano Marisan R. , Schwartz Robert E. , Georg Gunda I. , Himes Richard H.
Published: April 2003

The cryptophycins are newly discovered antimitotic agents isolated from the cyanobacterium Nostoc. Previous studies using cultured cells demonstrated that microtubules are the target of these compounds. We have studied the interaction of cryptophycin 1 with tubulin and microtubules in vitro. Cryptophycin 1 is an effective inhibitor of tubulin polymerization, causes tubulin to aggregate, and depolymerizes microtubules to linear polymers somewhat similar to the spiral‐like structures produced by the Vinca alkaloids. Cryptophycin 1 also inhibits vinblastine binding to tubulin but not colchicine binding. Thus, it appears that the cryptophycins may bind to the Vinca site in tubulin or to a site that overlaps with the Vinca site.
Phorbol ester‐induced suppression of leukotriene C4 synthase activity in human granulocytes
Authors: Sjölinder Mikael , Tornhamre Susanne , Werga Petra , Edenius Charlotte , Lindgren Jan Åke
Published: April 2003

The effect of the protein kinase C activator, phorbol 12‐myristate 13‐acetate (PMA), on the metabolism of exogenous leukotriene (LT)A4 in human granulocytes was investigated. After incubation with LTA4 decreased levels of LTC4 but not LTB4 were observed in granulocyte suspensions pretreated with PMA. This finding could in part be ascribed to oxidative metabolism of LTC4, since PMA induced a rapid degradation of exogenously added LTC4. After blocking of LTC4 metabolism with the H2O2 scavenger catalase, a PMA‐provoked suppression of the conversion of LTA4 to LTC4 was observed, indicating PKC‐dependent regulation of LTC4 synthase activity. This effect, as well as PMA‐induced degradation of LTC4 was presented by specific protein kinase C inhibitors.
Cloning and characterisation of the rabbit 5‐HT1Dα and 5‐HT1Dβ receptors
Authors: Harwood Gail , Lockyer Michael , Giles Heather , Fairweather Neil
Published: April 2003

The genes encoding the rabbit 5HT1Dα and 5HT1Dβ receptors have been cloned. The deduced amino acid sequence of these receptors shows 91–92% amino acid sequence identity with their human homologues, and similar high sequence identity with homologues from other species. The receptors were transiently expressed in COS‐7 cells and exhibit a pharmacological profile closely resembling their human homologues, including a higher affinity of ketanserin for the 5‐HT1Dα subtype. However, sumatriptan had a lower affinity for both the rabbit receptors compared to their human counterparts. This may be accounted for by differences between the primary amino acid sequences of these species homologues.
Mapping the residues of protein kinase CK2 α subunit responsible for responsiveness to polyanionic inhibitors
Authors: Vaglio Philippe , Sarno Stefania , Marin Oriano , Meggio Flavio , Issinger Olaf-G. , Pinna Lorenzo A.
Published: April 2003

The quadruple mutation of the whole basic cluster, K74KKK77 conserved in the catalytic subunits of protein kinase CK2 and implicated in substrate recognition, not only abolishes inhibition by heparin but even induces with some peptide substrates an up to 5‐fold stimulation by heparin in the 0.5–5 μg/ml concentration range. Two other mutants defective in substrate recognition, R191,195K198A and K79R80K83A, display either a 100‐fold reduction or no alteration at all in heparin inhibition, respectively. In contrast sensitivity to heparin inhibition is increased 30‐fold by a single mutation affecting Arg‐228 while it is not altered by a triple mutation in the small insert of subdomain XI (mutant R278K279R280A). The effect of the same mutations on inhibition by pseudosubstrate EEEEEYEEEEEEE is different, the mutant displaying the most reduced sensitivity being R191,195K198A, followed by K74‐77A and K79R80K83A; the other mutants are almost indistinguishable from CK2 wild type. Substantial reduction of inhibition by poly(Glu,Tyr)4:1 is only observable with mutant R191,195K198A, whereas R228A is significantly more sensitive to inhibition. These data show that the mode of inhibition of CK2 by polyanionic compounds occurs through substantially different mechanisms involving residues that are variably concerned with substrate recognition.
Detection of vanadyl‐nitrogen interaction in organs of the vanadyl‐treated rat: electron spin echo envelope modulation study
Authors: Fukui Kouichi , Ohya-Nishiguchi Hiroaki , Nakai Masami , Sakurai Hiromu , Kamada Hitoshi
Published: April 2003

ESEEM spectroscopy was applied for the first time to organs of an animal, viz. the kidney and liver of the rat treated with vanadyl sulfate. The aim of this study is to investigate the in vivo coordination structure of vanadyl ions administrated, and to gain information concerning the insulin‐mimic activity of vanadium. ESEEM measurements for kidney and liver performed at 77 K have established nitrogen coordination to a certain percentage of vanadyl ion in the organs. The rotios of nitrogen‐coordinating vanadyl ion were estimated as 70–80% in the liver, and 50–55% in the kidney. Isotropic portions of the 14N HFC were estimated as |A
iso| ∼ 5.0 MHz for liver, and ∼ 5.2 MHz for kidney, indicating that the coordinating nitrogen is an amino nitrogen. Coordination of the Lys ϵ‐amine or the N‐terminal α‐amine of a protein or (a peptide) to vanadyl ion in vivo is suggested.
Inhibition of the skeletal muscle ryanodine receptor calcium release channel by nitric oxide
Authors: Mészáros LászlóG. , Minarovic Igor , Zahradnikova Alexandra
Published: April 2003

NO donors were found to reduce the rate of Ca2+ release from isolated skeletal muscle sarcoplasmic reticulum (SR) and the open probability of single ryanodine receptor Ca2+ release channels (RyRCs) in planar lipid bilayers, and these effects were prevented by the NO quencher hemoglobin and reversed by 2‐mercaptoethanol. Ca2+ release assessed in skeletal muscle homogenates was also reduced by NO that was generated in situ from l‐arginine by endogenous, nitro‐l‐arginine methylester‐sensitive NO‐synthase. The effect of NO on the RyRC might explain NO‐induced depression of contractile force in striated muscles and, since both RyRC isoforms and NOS isoenzymes are ubiquitous, may represent a wide‐spread feedback mechanism in Ca2+ signaling; i.e. Ca‐dependent activation of NO production and NO‐evoked reduction of Ca2+ release from intracellular Ca2+ stores.
Stability study of Rhodobacter capsulatus ferrocytochrome c2 wild‐type and site‐directed mutants using hydrogen/deuterium exchange monitored by electrospray ionization mass spectrometry
Authors: Jaquinod Michel , Guy Philippe , Halgand Frederic , Caffrey Michael , Fitch John , Cusanovich Michael , Forest Eric
Published: April 2003

To estimate the stability of Rhodobacter capsulatus ferrocytochrome c
2 wild‐type and site‐directed mutants, charge state distributions and hydrogen/deuterium exchange rates were monitored by electrospray ionization mass spectrometry. The relative stability of the mutants was observed with the order: V11 insert > Y75F > wild‐type = K32E > K12D = K14E ≥ K52E > K14E/K32E > W67Y > P35A > 157N > G34S. (A preliminary account has been presented for mutants G34S and P35A [Jaquinod et al. (1995) Rapid Commun. Mass Spectrom. 9, 1135–1140].) This approach is shown to be a useful tool for rapid characterization of mutational effects on protein conformation.
Activation of SRC family kinases in human neutrophils. Evidence that p58c‐frg and p53/56lyn redistributed to a Triton X‐100‐insoluble cytoskeletal fraction, also enriched in the caveolar protein Caveolin, display an enhanced kinase activity
Authors: Seng Rong Yan  , Fumagalli Laura , Berton Giorgio
Published: April 2003

Protein tyrosine phosphorylation is one of the signals involved in stimulation of neutrophil (PMN) functions. We found that phorbol myristate acetate (PMA) activates the src family tyrosine kinases p58c−fgr and p53/56lyn in suspended PMNs. Moreover, we found that up to about 20% of p58c−fgr and p53/56lyn redistribute to a Triton X‐100‐insoluble fraction after PMA stimulation, and it is this fraction of the two kinases which displays an increased activity. These changes of p58c−fgr and p53/56lyn distribution and activity correlated with tyrosine phosphorylation of endogenous substrates. In fact, in PMA‐stimulated PMNs tyrosine phosphorylated proteins are mostly recovered in a Triton‐insoluble cell fraction. To separate cytoskeletal from caveolar structures, which both display Triton X‐100‐insolubility, we used the detergent n‐octyl ß‐d‐glucopyranoside (OGP) which solubilises components of caveolae. We found that the caveolae marker protein, caveolin, as well as the cytoskeletal protein α‐actinin and p58c−fgr and p53/56lyn, is insoluble in OGP. These findings suggest that PMA stimulation promotes the formation of multimolecular complexes containing cytoskeletal proteins, caveolin‐containing structures and src family protein tyrosine kinases. Moreover, they show that p58c−fgr and p53/56lyn associated with this multimolecular complex display an enhanced kinase activity.
The membrane anchor of mammalian brain acetylcholinesterase consists of a single glycosylated protein of 22 kDa
Authors: Boschetti N. , Brodbeck U.
Published: April 2003

Mammalian brain acetylcholinesterase (AChE; EC 3.1.1.7) is membrane‐bound through a structural subunit of about 20 kDa. So far little is known about this anchor because it is only detectable after hydrophobic labelling. In the present study we demonstrate that the two bands migrating around 20 kDa on SDS‐PAGE derive from the same protein containing the same N‐terminal amino acid sequence. The difference in their mobility is due to different N‐glycosidation. Radioalkylation of cysteine residues reveals that the anchor contains just the two cysteine residues involved in binding the catalytic subunits.
Partially folded structure of monomeric bovine β‐lactoglobulin
Authors: Molinari Henriette , Ragona Laura , Varani Luca , Musco Giovanna , Consonni Roberto , Zetta Lucia , Monaco Hugo L.
Published: April 2003

Bovine β‐LG (β‐lactoglobulin) has been studied under a variety of solution conditions by one‐ and two‐dimensional NMR spectroscopy. At highly acidic pH (pH = 2) and low ionic strength the protein is present in a monomeric form, exhibiting a highly structured β‐sheet core and less ordered regions as evidenced by both CD data and the NOESY spectra. Marginal protection was observed for most of the amide protons as a result of high conformational mobility. This structural state of β‐LG may be considered as an attractive model for a partially folded structure occurring late in the folding process of the protein.
A new method for identifying the amino acid attached to a particular RNA in the cell
Authors: Suzuki Tsutomu , Ueda Takuya , Watanabe Kimitsuna
Published: April 2003

To investigate the function of tRNAs or any other aminoacylable RNAs in vivo, it is important to be able to estimate the amounts and species of aminoacylated RNAs in living cells. We have developed a method of analyzing amino acids attached to particular tRNAs obtained from cells. After the ester bond between the amino acid and the 3′‐adenosine moiety of a specific aminoacyl‐tRNA is stabilized by acetylation of the amino acid with [14C]acetic anhydride, the aminoacyl‐tRNA can be fished out with a solid‐phase‐attached DNA probe. The 14C‐labeled acetylamino acid is then released from the thus purified acetyl‐aminoacyl‐tRNAs by alkaline treatment and detected by TLC analysis.
Protein kinase C inhibitors enhance G‐protein induced phospholipase A2 activation in intact human platelets
Authors: Iorio Paolo , Gresele Paolo , Stasi Mario , Nucciarelli Fabio , Vezza Roberta , Nenci Giuseppe G. , Goracci Gianfrancesco
Published: April 2003

Washed intact human platelets were prelabelled with [3H]arachidonic acid ([3H]AA) and stimulated with thrombin or with AIF4
−, a known unspecific activator of G‐proteins. Both stimuli induced the liberation of [3H]AA, the release of β‐thromboglobulin (β‐TG) and platelet aggregation. PMA did not induce liberation of [3H]AA although it induced β‐TG release and aggregation; preincubation with PMA did not modify significantly the amounts of [3H]AA and β‐TG released by thrombin or AlF4
−. Different inhibitors of PKC (staurosporine, H‐7 and calphostin C) increased the release of [3H]AA and inhibited β‐TG release and aggregation induced by AlF4
− but they had no effect when platelets were stimulated with thrombin (0.5 U/ml). Calphostin C was able to release [3H]AA by itself without inducing aggregation or β‐TG release. Okadaic acid (a serine/threonine phosphoprotein phosphatase inhibitor) greatly inhibited the release of [3H]AA, β‐TG and aggregation in AlF4
−‐stimulated platelets. These results indicate the presence of a G‐protein mediated mechanism for the activation of a platelet phospholipase A2 which is negatively affected by a protein kinase, sensible to putative inhibitors of protein kinase C, and it is activated by a protein phosphatase, sensible to okadaic acid.