bluuebunny/crossref_metadata_2025 huggingface dataset:
A tandem repeat of the SPKK peptide motif induces Ψ‐type DNA structures at alternating AT sequences
Authors: Bailly Fabrice , Bailly Christian , Colson Pierre , Houssier Claude , Hénichart Jean-Pierre
Published: July 2002

The interaction between a tandem repeat of the SPKK peptide motif and calf thymus DNA or several polynucleotides has been investigated by circular dichroism. The octapeptide SPKKSPKK does not induce any important changes in the CD spectra of the polynucleotides poly(dG) · poly(dC), poly(dG‐dC) · poly(dG‐dC) and poly(dA) · poly(dT) while the spectrum of calf thymus DNA is slightly modified. Binding of this basic peptide to the alternating copolymer poly(dA‐dT) · poly(dA‐dT) results in a marked Ψ‐type condensation in a manner similar to that induced by the entire C‐terminal domain of histone H1.
The unique properties of glutamate receptor channels
Authors: Sprengel R. , Seeburg P.H.
Published: July 2002

Rapid excitatory neurotransmission in the central nervous system (CNS) is mediated predominantly by synaptically released l‐glutamate which activates cation selective channels with different kinetic and ion conductance properties. Studies with cloned glutamate receptor channels helped delineate the functional properties of channels defined in subunit composition. Moreover, molecular studies have revealed novel genetic mechanisms controlling the expression of important structural channel determinants.
In vitro characterization of Ro 46‐2005, a novel synthetic non‐peptide endothelin antagonist of ETA and ETB receptors
Authors: Breu Volker , Löffler Bernd-Michael , Clozel Martine
Published: July 2002

Ro 46‐2005 is a new synthetic non‐peptide endothelin (ET) receptor antagonist. In binding experiments, Ro 46‐2005 proved to be equipotent (IC50 200–500 nM) for inhibition of [125I]ET‐1 binding on the two known ET receptor subtypes (ETA and ETB). Scatchard analysis was consistent with a competitive binding mode. Ro 46‐2005 also inhibited the functional consequences of ET‐1 stimulation: the ET‐1‐induced release of arachidonic acid from rat mesangial cells was inhibited with an IC50 of 1.8 μM.
Ubiquitin found in the archaebacterium Thermoplasma acidophilum
Authors: Wolf Stefan , Lottspeich Friedrich , Baumeister Wolfgang
Published: July 2002

Systematic N‐terminal sequencing of the low molecular weight proteins from Thermoplasma acidophilum separated by two‐dimensional polyacrylamide get electrophoresis led to the discovery of a polypeptide with an apparent M
r of 4.5 kDa identical as its first 18 amino acid residues to human ubiquitin. The occurrence of ubiquitin and proteasomes in an archaebacterium strongly suggests that ATP‐ubiquitin‐dependent proteolysis is a cellular function that developed early in evolution.
Proteolytically cleaved mutant antithrombin‐Hamilton has high stability to denaturation characteristic of wild type inhibitor serpins
Authors: Wright H.Tonie , Blajcliman Morris A.
Published: July 2002

The serpin family of proteins consists primarily of proteinase inhibitors which form tight complexes with target proteinases. Inhibitor serpins are cleaved by proteinase and undergo a large conformational change in which the polypeptide segment terminating at the target reactive site, at which cleavage takes place, inserts itself as an additional strand, s4A, in the center of a preexisting β‐sheet. This change in conformation increases the stability towards denaturation of the cleaved serpin relative to the native uncleaved form. Mutant serpins with single amino acid changes in the s4A strand have been identified, and in most cases these are proteinase substrates but not inhibitors. We have measured the stability to denaturation of one of these non‐inhibitor substrate mutants, antithrombin‐Hamilton, which has an Ala → Thr change at position P12 in strand s4A. We find that it undergoes the transformation to the more stable form which is observed for inhibitor serpins, from which we conclude that the Ala → Thr change in antithrombin‐Hamilton does not prevent insertion of s4A into β‐sheet A in the cleaved form.
Crystal structure of the cyanide‐inhibited Xenopus laevis Cu,Zn superoxide dismutase at 98 K
Authors: Carugo Kristina Djinovic , Battistoni Andrea , Carrì Maria Teresa , Polticelli Fabio , Desideri Alessandro , Rotilio Giuseppe , Coda Alessandro , Bolognesi Martino
Published: July 2002

The crystal structure of cyanide‐inhibited X. laevis Cu,Zn superoxide dismutase has been studied and refined based on diffraction data collected at 98 K. The final R‐factor for the 27,299 reflections in the 10.0‐1.7 Å resolution range is 0.170. The cyanide anion, which is a competitive inhibitor expected to mimic the superoxide binding mode, binds directly to the active site copper atom, replacing the coordinated water molecule. Moreover, the anion establishes a strong electrostatic interaction with the guanidinium group of the conserved active site residue Arg141. The coordination sphere of Cu2+ is partly altered with respect to the uninhibited enzyme: a displacement of 0.41 Å in subunit A, and 0.27 Å in subunit B of the dimeric enzyme is observed for the Cu2+ ions. Only two ligands in the Cu2+ coordination sphere (His46 and His118) are significantly affected by cyanide binding, whereas virtually no rearrangement of the Zn2+ ligands is reported.
Carboxyl‐terminal truncation of apolipoproteinB‐100 inhibits lipoprotein(a) particle formation
Authors: Gabel Brent , Yao Zemin , McLeod Roger S. , Young Stephen G. , Koschinsky Marlys L.
Published: July 2002

Recombinant expression systems for both apo(a) and apoB were used to identify sequences in apoB which are required for Lp(a) formation. Incubation of a [35S]Cys‐labelled 17‐kringle form of apo(a) with supernatants from rat hepatoma (McA‐RH7777) cells expressing apoB‐88, apoB‐94 and apoB‐100 resulted in covalent r‐Lp(a) formation only with apoB‐100. Additionally, apoB‐86 present in the LDL of a hypobetalipoproteinemic subject did not associate with a 12‐kringle form of recombinant apo(a) to form r‐Lp(a) complexes. Our data suggest that sequences within the C‐terminal 6% of apoB‐100 are essential for Lp(a) assembly.
Stimulation of tyrosine‐ and serine‐phosphorylation of focal adhesion kinase in mouse 3T3 cells by fibronectin and fibroblast growth factor
Authors: Hatai Mika , Hashi Hidetaka , Mogi Akira , Soga Hisae , Yokota Jun , Yaoi Yoshihito
Published: July 2002

Phosphorylation of both tyrosine and serine residues of focal adhesion kinase (FAK) was stimulated by the adhesion of BALB/c mouse 3T3 cells to fibronectin, but phosphorylation of threonine was not detectable. Acidic and basic fibroblast growth factors also stimulated the phosphorylation of serine and tyrosine of FAK in cells adhered to poly‐l‐lysine, but epidermal growth factor and platelet‐derived growth factor did not. A fusion protein of fibronectin and basic fibroblast growth factor effectively induced the phosphorylation of FAK. Phosphorylation of FAK in the rat myoblast L‐6 cell line, which lacks fibroblast growth factor receptors, was not stimulated by fibroblast growth factors, suggesting that the interaction of fibroblast growth factors with their receptors might cause the phosphorylation of FAK.
Crystallisation and crystallographic investigations of cod alcohol dehydrogenase class I and class III enzymes
Authors: Ramaswamy S. , El-Ahmad Mustafa , Danielsson Olle , Jörnvall Hans , Eklund Hans
Published: July 2002

Cod liver alcohol dehydrogenase of class‐hybrid properties has been crystallized as an NAD+—pyrazole complex in the monoclinic space group P21 with cell dimensions a = 103.3 Å, b = 47.4 Å, c = 80.7 Å, β = 104.6°, and with one dimer in the asymmetric unit. The position of the dimer molecule in the crystal was determined by molecular replacement methods at 3.0 Å resolution. The successful search model was the poly‐alanine structure of the horse enzyme. Side chains were then replaced according to the amino acid sequence of the cod enzyme, and the structure has been refined at 2.8 Å to an R‐factor of 0.26. Cod liver class III alcohol dehydrogenase crystallizes in the monoclinic space group C2 with cell dimensions a = 127.5 Å, b = 76.6 Å, c = 93.4 Å, β = 99.4° and with probably one dimer in the asymmetric unit.
Vitamin D influences gap junctional communication in C3H/10T 1/2 murine fibroblast cells
Authors: Stahl Wilhelm , Nicolai Swantje , Hanusch Michael , Sies Helmut
Published: July 2002

Vitamin D3, cholecalciferol, induces cell—cell communication via gap junctions in murine fibroblasts (C3H/10T 1/2 cells) at concentrations between 0.01 and 1.0 μM, as assayed by the dye transfer method using Lucifer yellow CH. The extent of induction is similar to that obtained with the positive controls, canthaxanthin or retinoic acid, applied at 10 and 1 μM, respectively. Vitamin D2 also induces cell—cell communication. At elevated concentrations of vitamin D3 (5 μM) there is a suppression of gap junctional communication, reversible upon exposure to all‐trans retinoic acid (1 μM) after removal of vitamin D3 from the medium. Conversely, communication between cells prestimulated with retinoic acid (1 μM) rapidly decreases when the retinoid is replaced by vitamin D3 (5 μM). The data demonstrate a role for vitamin D in the regulation of intercellular communication. This novel property of vitamin D may contribute to the antiproliferative effects of vitamin D exhibited in some types of cancer.
The protective function of the xanthophyll cycle in photosynthesis
Authors: Sarry Jean-Emmanuel , Montillet Jean-Luc , Sauvaire Yves , Havaux Michel
Published: July 2002

The rapid conversion of the carotenoid violaxanthin to zeaxanthin via antheraxanthin (xanthophyll cycle) in potato leaves exposed at 23°C to a strong white light of 2000 μE·M−2·S−1was associated with a slight inhibition of photosynthetic electron transport (as estimated from chlorophyll fluorescence measurements) and a low lipid peroxidation (as estimated from ethane measurements). When the xanthophyll cycle was blocked by dithiothreitol (3 mM) or low temperature (3°C), photoinhibition of electron transport was exacerbated and pronounced lipid peroxidation occurred concomitantly. Accumulation of zeaxanthin and antheraxanthin in potato leaves by a non‐photoinhibitory light treatment at 23°C (900 μE·m−2·s−1for 1 h) considerably reduced the level of lipid peroxidation during subsequent light stress at 3°C. The presented results indicate that one of the functions of the xanthophyll cycle could be the protection of thylakoid membranes against lipid peroxidation, suggesting that zeaxanthin and antheraxanthin synthesized in strong light are present as free pigments in the membrane lipid bilayer.
Thermal recovery of iodopsin from its meta I‐intermediate
Authors: Imamoto Yasushi , Imai Hiroo , Yoshizawa Tôru , Shichida Yoshinori
Published: July 2002

The thermal reaction of meta I‐intermediate of iodopsin (metaiodopsin I), a chicken red‐sensitive cone pigment, was studied by low‐temperature spectrophotometry at −20°C. Irradiation of iodopsin at −20°C produced metaiodopsin I, whose absorption maximum was at about 470 nm. An incubation of metaiodopsin I at −20 °C resulted in a conversion to metaiodopsin II having absorption maximum at about 380 nm, as well as a concurrent formation of a red‐shifted product stable at room temperature. Since the absorption spectrum and photo‐reactivity of the red‐shifted product were identical with those of iodopsin, the red‐shifted product should be iodopsin. Thus a part of metaiodopsin I can revert to iodopsin by the thermal reaction unlike metarhodopsin I.
Isolation and characterization of multiple isoforms of the rat urokinase receptor in osteoblasts
Authors: Rabbani S.A. , Rajwans N. , Achbarou A. , Murthy K.K. , Goltzman D.
Published: July 2002

A rat urokinase receptor (uPAR) cDNA fragment was amplified by RT‐PCR from RNA of the rat osteoblastic cell line CFK‐1. Using this DNA species as a hybridization probe two rat uPAR cDNAs were isolated from a CFK‐1 cDNA library. These two cDNAs encode an identical uPAR protein except for a single base mutation which results in the substitution of cysteine to serine at amino acid 71 in one variant. PCR analysis of rat genomic DNA revealed the presence of an additional uPAR arising from alternate splicing which is expressed in a variety of tissues. These studies provide the tools for examining uPAR function in fibrinolysis, tumor invasion and metastasis in the rat and for identifying the mechanism of species specificity in uPA actions.
Antisense retinoic acid receptor γ‐1 oligonucleotide enhances chondrogenesis of mouse limb mesenchymal cells in vitro
Authors: Motoyama Jun , Eto Kazuhiro
Published: July 2002

Retinoic acid receptor (RAR) γ gene is expressed in the precartilaginous cells during chondrogenesis in mouse embryos, but the role of the gene products is still unclear. To examine the role during chondrogenesis, we isolated mesenchymal cells from the limb bud of mouse embryos and exposed them to antisense RAR γ‐1 oligodeoxynucleotide in micromass culture. The antisense oligodeoxynucleotide inhibited RAR γ‐1 protein expression and enhanced chondrogenesis in the exposed cells. These results suggest that the complex of RAR γ‐1 protein and its ligand RA acts as a suppressor of the chondrogenesis in the limb development.
Monoclonal non‐specific suppressor factor (MNSF) inhibits the IL4 secretion by bone marrow‐derived mast cell (BMMC)
Authors: Nakamura Morihiko , Xavier Ricardo M. , Tanigawa Yoshinori
Published: July 2002

We investigated whether monoclonal non‐specific suppressor factor (MNSF) has an effect on the IL4 secretion by BMMC sensitized with anti‐DNP IgE monoclonal antibody. The sensitized BMMC showed a maximum increase of IL4 secretion at 10 antigen activation. When MNSF (10 U) was added, it remarkably suppressed the IL4 secretion (82 ± 3%). MNSF also suppressed the IL4 secretion by the mast cell line, CFTL‐15, but not CFTL‐12. Northern blot analysis showed that, when the same amounts of MNSF were added, IL4 mRNA expression was decreased, suggesting that the suppression results from the inhibition of a transcript.
Iron‐sulphur clusters as genetic regulatory switches: the bifunctional iron regulatory protein‐1
Authors: Paraskeva Efrosyni , Hentze Matthias W.
Published: July 2002

In the eighties, iron regulatory protein‐1 (IRP‐1) was iDAntified as a cytoplasmic mRNA‐binding protein that regulates vertebrate cell iron metabolism. More recently, IRP‐1 was found to represent the functional cytoplasmic homologue of mitochondrial aconitase, a citric acid cycle enzyme. Its two functions are mutually exclusive and DApend on the status of an Fe‐S cluster: the (cluster‐less) apoIRP‐1 binds to RNA, while the incorporation of a cubane 4Fe‐4S cluster is required for enzymatic activity. Cellular signals including iron levels, nitric oxiDA and oxidative stress can regulate between the two activities posttranslationally and reversibly via the Fe‐ cluster. Recent reports suggest that other regulatory proteins may be controlled by similar mechanisms.
Molecular biology of transmissible spongiform encephalopathies
Authors: Weissmann Charles
Published: July 2002

The prion, the transmissible agent that causes spongiform encephalopathies such as scrapie, bovine spongiform encephalopathy (BSE) and Creutzfeldt‐Jakob disease, is believed to be DAvoid of nucleic acid and iDAntical with PrPSc, a modified form of the normal host protein PrPC which is encoDAd by the single copy gene Prnp. The ‘protein only’ hypothesis proposes that PrPSc, when introduced into a normal host, causes the conversion of PrPC into PrPSc; it therefore predicts that an animal DAvoid of PrPC should be resistant to prion diseases. We generated homozygous Prnpolo
(‘PrP knockout’) mice and showed that, after inoculation with prions, they remained free of scrapie for at least 2 years while wild‐type controls all died within 6 months. There was no propagation of prions in the Prnpolo
animals. Surprisingly, heterozygous Prnp

ol+ mice, which express PrPC at about half the normal level, also showed enhanced resistance to scrapie disease DAspite high levels of infectious agent and PrPSc in the brain early on. After introduction of murine PrP transgenes Prnpolo
mice became highly susceptible to mouse but not to hamster prions, while the insertion of Syrian hamster PrP transgenes rendered them susceptible to hamster but to a much lesser extent to mouse prions. These complementation experiments paved the way to the application of reverse genetics. We have prepared animals transgenic for genes encoding PrP with amino terminal DAletions of various lengths and have found that PrP lacking 48 amino proximal amino acids, which comprise four of the five octa repeats of PrP, is still biologically active.
Fusion of reconstituted influenza virus envelopes with liposomes mediated by streptavidin/biotin interactions
Authors: Schoen Pieter , Leserman Lee , Wilschut Jan
Published: July 2002

Reconstituted influenza virus envelopes (virosomes) containing the viral hemagglutinin (HA) represent an efficient fusogenic cellular delivery system. By interaction of HA with its natural receptors, sialylated lipids (gangliosides) or proteins, virosomes bind to cells and, following endocytic uptake, deliver their contents to the cytosol through fusion from within acidic endosomes. Here, we show that binding to sialic acid is not necessary for fusion. In the presence of streptavidin, virosomes containing a biotinylated lipid fused with liposomes lacking sialic acid if these liposomes also had a biotinylated lipid in their membranes. Moreover, fusion characteristics corresponded well with fusion of virosomes with ganglioside‐containing liposomes.
Cyclodextrins as templates for the presentation of protease inhibitors
Authors: Schaschke N. , Musiol H.-J. , Assfalg-Machleidt I. , Machleidt W. , Rudolph-Böhner S. , Moroder L.
Published: July 2002

Mono(6‐succinylamido‐6‐deoxy)‐β‐cyclodextrin was synthesized by classical carbohydrate chemistry and used as a template mono‐functionalized with the linear, fully flexible 4C‐spacer carboxylate for covalent linkage of the calpain inhibitor leucyl‐leucyl‐norleucinal. Spectroscopic analyses of the conjugate do not support a self‐inclusion of part of the hydrophobic peptide tail, but confirm its intra‐ or intermolecular interaction with the template moiety that leads to full water solubility. The inhibitory potency of the β‐cyclodextrin/peptide aldehyde construct was compared with that of the parent Ac‐Leu‐Leu‐Nle‐H against cathepsin B and calpain. Despite the large size of the template the inhibition of cathepsin B was only slightly reduced in full agreement with the X‐ray structure of this enzyme which shows full accessibility of the S‐subsites. For this enzyme the 4C‐spacer is apparently sufficient to guarantee optimal interaction of the peptide tail with the binding cleft. Conversely, for μ‐calpain a significantly decreased inhibitory potency was obtained with the conjugate suggesting steric interference of the template in the binding process. These results show that the beneficial properties of the cyclodextrin template can be retained in conjugates with bioactive peptides if attention is paid to optimize in each case the size and nature of the spacer for optimal recognition of the grafted biomolecule.
Morphological and GABA‐immunoreactive development of the embryonic chick telencephalon
Authors: Lundgren Per , Mattsson Mats‐olof , Johansson Lenore , Ottersen Ole Petter , Sellström Åke
Published: July 2002

The development of neurons utilizing γ‐aminobutyric acid (GABAergic neurons) in prosencephalon and telencephalon from chicken embryonic days 4–14 (E4–E14) was studied by means of immunohistochemistry. Furthermore, routine histology and transmission electron microscopy, respectively, were performed in order to study the morphological development in the designated area. The main finding is that development of GABAergic neurons in the chick telencephalon is rapid; the GABA neurons are appearing in bulk at day 8, being “overexpressed” at days 10–11, decreasing in numbers thereafter and achieving mature morphology on day 14, which is considerably faster than in the rodent.

Morphological analysis revealed that the prosencephalon mainly consisted of a thin layer of undifferentiated neuroblasts in the E4 embryo. By E6, the prosencephalon had increased in thickness and occasional cells outside the neuroepithelium showed a more mature morphology with a few cells weakly staining positive for GABA. At E8, the prospective granular and subventricular layers had developed. At E14, the appearance of the telencephalon is approximating that of the adult since both ependymal cells and morphologically mature neurons can be seen.
The HIV‐1 gp120 inhibits the binding of adenosine deaminase to CD26 by a mechanism modulated by CD4 and CXCR4 expression
Authors: Blanco Julià , Valenzuela Agustı́n , Herrera Carolina , Lluı́s Carmen , Hovanessian Ara G. , Franco Rafael
Published: July 2002

HIV‐1 external envelope glycoprotein gp120 inhibits adenosine deaminase (ADA) binding to its cell surface receptor in lymphocytes, CD26, by a mechanism that does not require the gp120–CD4 interaction. To further characterize this mechanism, we studied ADA binding to murine clones stably expressing human CD26 and/or human CD4, and transiently expressing human CXCR4. In this heterologous model, we show that both recombinant gp120 and viral particles from the X4 HIV‐1 isolate IIIB inhibited the binding of ADA to wild‐type or catalytically inactive forms of CD26. In cells lacking human CXCR4 expression, this gp120‐mediated inhibition of ADA binding to human CD26 was completely dependent on the expression of human CD4. In contrast, when cells were transfected with human CXCR4 the inhibitory effect of gp120 was significantly enhanced and was not blocked by anti‐CD4 antibodies. These data suggest that the interaction of gp120 with CD4 or CXCR4 is required for efficient inhibition of ADA binding to CD26, although in the presence of CXCR4 the interaction of gp120 with CD4 may be dispensable.
Gene expression data analysis
Authors: Brazma Alvis , Vilo Jaak
Published: July 2002

Microarrays are one of the latest breakthroughs in experimental molecular biology, which allow monitoring of gene expression for tens of thousands of genes in parallel and are already producing huge amounts of valuable data. Analysis and handling of such data is becoming one of the major bottlenecks in the utilization of the technology. The raw microarray data are images, which have to be transformed into gene expression matrices – tables where rows represent genes, columns represent various samples such as tissues or experimental conditions, and numbers in each cell characterize the expression level of the particular gene in the particular sample. These matrices have to be analyzed further, if any knowledge about the underlying biological processes is to be extracted. In this paper we concentrate on discussing bioinformatics methods used for such analysis. We briefly discuss supervised and unsupervised data analysis and its applications, such as predicting gene function classes and cancer classification. Then we discuss how the gene expression matrix can be used to predict putative regulatory signals in the genome sequences. In conclusion we discuss some possible future directions.
Evidence that the substrate backbone conformation is critical to phosphorylation by p42 MAP kinase
Authors: Weiwad Matthias , Küllertz Gerhard , Schutkowski Mike , Fischer Gunter
Published: July 2002

The effect of prolyl bond isomers on the substrate recognition capabilities of various endoproteases may be investigated in a reaction where both cis/trans isomers co‐exist. Here we address the question of whether enzyme reactions at the side chain of an amino acid preceding proline proceed through an isomer specific pathway. The proline‐directed p42 mitogen‐activated protein kinase (ERK2) was used to phosphorylate the serine side chain in Pro‐Arg‐Ser‐Pro‐Phe‐4‐nitroanilide under conditions where different amounts of cis prolyl isomer of the substrate were present. Initial phosphorylation rates were calculated ranging between zero at 100% cis isomer and around 60 pM/min at the equilibrium content of 83.5% trans isomer. In the presence of the peptidyl‐prolyl cis/trans isomerase human hFKBP12 (500 nM), cis/trans isomerization proceeds rapidly, permitting the maximal phosphorylation rate to be observed in the dead time of the experiment. Results show that correct signature sequences are not sufficient to render potential substrates reactive to proline‐directed enzymatic phosphorylations, but that the conformational state of the peptide bond following serine (threonine) is a critical determinant. Therefore, catalysis by peptidyl‐prolyl cis/trans isomerases may add a new level of control to intracellular protein phosphorylations.
The rat hepatoma cell line H4‐II‐E‐C3 expresses high activities of the high‐affinity glutamate transporter GLT‐1A
Authors: Pollard Matthew , McGivan John
Published: July 2002

The rate of uptake of aspartate into the rat hepatoma cell line H4‐II‐E‐C3 is very much higher than that exhibited by normal rat hepatocytes. Using an RT‐PCR‐based strategy, a glutamate transporter resembling mouse liver GLT‐1A has been cloned from H4‐II‐E‐C3 cells. Northern blotting confirmed that relatively high levels of mRNA for GLT‐1A are expressed in hepatoma cells compared with negligible levels in rat hepatocytes. To our knowledge, this is the first report of the cloning of a high‐affinity glutamate transporter from a transformed cell line and also the first demonstration of functional expression of GLT‐1 outside the central nervous system.
NMR analysis of secondary structure and dynamics of a recombinant peptide from the N‐terminal region of human erythroid α‐spectrin
Authors: Park Sunghyouk , Johnson Michael E. , Fung L.W.-M.
Published: July 2002

We have studied the nuclear magnetic resonance solution secondary structure of the N‐terminal region in human erythroid α‐spectrin using a recombinant model peptide of α‐spectrin consisting of residues 1–156. Pulsed field gradient diffusion coefficient measurements show that the model peptide exists as a monomer under the solution conditions used. The first 20 residues are in a random coil conformation, followed by a helix of 25 residues and then a random coil segment before the next helix. The random coil nature of this linker was confirmed by the presence of fast internal motion from15N relaxation measurements. The second, third and fourth helices are thought to form the triple helical bundle structural domain, consistent with previous studies. Our study shows that the N‐terminal region of α‐spectrin prior to the first structural domain forms a well behaved helix without its β‐spectrin partner.
Changes in the thermal unfolding of p‐phenylenedimaleimide‐modified myosin subfragment 1 induced by its ‘weak’ binding to F‐actin
Authors: Kaspieva Olga V. , Nikolaeva Olga P. , Orlov Victor N. , Ponomarev Michael A. , Drachev Vladimir A. , Levitsky Dmitrii I.
Published: July 2002

Differential scanning calorimetry (DSC) was used to analyze the thermal unfolding of myosin subfragment 1 (S1) with the SH1 (Cys‐707) and SH2 (Cys‐697) groups cross‐linked by N,N′‐p‐phenylenedimaleimide (pPDM‐S1). It has been shown that F‐actin affects the thermal unfolding of pPDM‐S1 only at very low ionic strength, when some part of pPDM‐S1 binds weakly to F‐actin, but not at higher ionic strength (200 mM KCl). The weak binding of pPDM‐S1 to F‐actin shifted the thermal transition of pPDM‐S1 by about 5°C to a higher temperature. This actin‐induced increase in thermal stability of pPDM‐S1 was similar to that observed with ‘strong’ binding of unmodified S1 to F‐actin. Our results show that actin‐induced structural changes revealed by DSC in the myosin head occur not only upon strong binding but also on weak binding of the head to F‐actin, thus suggesting that these changes may occur before the power‐stroke and play an important role in the motor function of the head.
Elongation factor‐2 kinase: immunological evidence for the existence of tissue‐specific isoforms
Authors: Hait William N. , Ward Michael D. , Trakht Ilya N. , Ryazanov Alexey G.
Published: July 2002

eEF‐2 kinase is a ubiquitous Ca2+/calmodulin‐dependent protein kinase that is specific for protein synthesis elongation factor‐2 (eEF‐2). This study describes an improved procedure for the purification of eEF‐2 kinase from rabbit reticulocyte lysate. The eEF‐2 kinase preparation was used to raise polyclonal antibodies, which immunoprecipitated eEF‐2 kinase protein and activity from rabbit reticulocyte lysate. The antibodies recognized a single 103 kDa band in extracts from several cell lines including NIH 3T3, PC12, C6 glioma, HeLa, and MCF‐7 breast carcinoma. However, there was no immunoreactivity in extracts of rabbit or bovine liver or rabbit kidney despite the presence of abundant eEF‐2 kinase activity in these tissues. Exposure of PC12 cells to nerve growth factor (NGF) resulted in rapid down‐regulation of eEF‐2 kinase activity and a decrease in immunoreactivity. After 24 h of incubation with NGF, the activity of the kinase recovered to 80% of initial values. In contrast, the immunoreactivity of eEF‐2 kinase continued to decrease. These data suggest that tissue‐specific isoforms of eEF‐2 kinase may exist and that these isoforms may be regulated by growth factors.
Increasing cAMP antagonizes hypertrophic response to angiotensin II without affecting Ras and MAP kinase activation in vascular smooth muscle cells
Authors: Takahashi Tomosaburo , Kawahara Yasuhiro , Okuda Masanori , Yokoyama Mitsuhiro
Published: July 2002

Angiotensin II (Ang II), a potent hypertrophic factor for vascular smooth muscle cells (VSMC), induces activation of the ras proto‐oncogene product (Ras) and mitogen‐activated protein (MAP) kinases, and tyrosine phosphorylation of a focal adhesion‐associated protein, paxillin. Forskolin, a direct activator of adenylate cyclase, and dibutyryl cAMP (Bt2 cAMP), a membrane permeable cAMP analogue, potently inhibited Ang II‐stimulated protein synthesis. However, they did not inhibit Ang II‐induced activation of Ras and MAP kinases. Although both forskolin and Bt2 cAMP potently reduced background tyrosine phosphorylation of paxillin, they allowed Ang II to induce the same reaction. These results indicate that increasing cAMP antagonizes the hypertrophic response to Ang II without affecting Ras and MAP kinase activation in VSMC and suggest that it does not interrupt signaling from the Ang II receptor to focal adhesions.
Molecular cloning, expression and tissue distribution of a chicken angiotensin II receptor
Authors: Kempf Hervé , Moullec Jean-Michel le , Corvol Pierre , Gasc Jean-Marie
Published: July 2002

A cDNA encoding a chicken angiotensin II receptor from adrenal gland was isolated to serve as a molecular tool to study the role of AngII in avian embryonic development. This cDNA, sharing a high homology with another avian receptor (turkey), encodes a protein of 359 amino acids with 75% sequence identity with the mammalian type 1 receptor. Transient xpression has revealed pharmacological properties distinct from mammalian receptors and a functional coupling leading to the increase in inositol phosphate production. The AngII receptor mRNA is expressed in classical target organs for AngII (adrenal gland, heart, kidney) and, interestingly, in endothelial cells where it may mediate the peculiar vasorelaxation effect of AngII in the chicken.
Crystallization and preliminary X‐ray diffraction analysis of proline iminopeptidase from Xanthomonas campestris pv. citri
Authors: Medrano F.J , Alonso J , Garcı́a J.L , Bode W , Gomis-Rüth F.X
Published: July 2002

Proline iminopeptidase from Xanthomonas campestris pv. citri, displaying no significant sequence homology to any protein previously analyzed by X‐ray crystallography, has been crystallized using the vapour diffusion method. Two different orthorhombic crystal forms (space group C222 and I222) were obtained from a solution containing NaCl or polyethylene glycol monomethyl ether (MW 5000) as precipitating agent for the native and lanthanum‐derivatized protein, respectively. Complete diffraction data sets have been collected up to 2.6 Å (native) and 3.0 Å (lanthanum derivative) resolution. Cell dimensions are a=147.2 Å, b=167.8 Å, and c=85.6 Å (C222) and a=146.7 Å, b=167.7 Å, and c=171.4 Å (I222), respectively. Considerations of the possible values of V
m and analysis of the self‐rotation function of the native crystals account for the presence of one dimer per asymmetric unit, whereas a tetramer probably would occupy the smallest crystallographically independent crystal portion in the lanthanum‐derivatized protein crystals.
nmd, a novel gene differentially expressed in human melanoma cell lines, encodes a new atypical member of the enzyme family of lipases
Authors: van Groningen Jan J.M. , Egmond Maarten R. , Bloemers Henri P.J. , Swart Guido W.M.
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Fetal cells in the peripheral blood of pregnant women express thymidine kinase: a new marker for detection
Authors: Hengstschläger Markus , Bernaschek Gerhard
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Differential translational initiation of lbp mRNA is caused by a 5′ upstream open reading frame1
Authors: Mittag Maria , Eckerskorn Christoph , Strupat Kerstin , Hastings J.Woodland
Published: July 2002

Expression of the luciferin‐binding protein (LBP) from Gonyaulax polyedra is regulated by the circadian clock at the translational level. Here we report that in vitro translation of lbp mRNA results in the synthesis of two LBP variants of different sizes, which is shown to be due to translational initiation at different in‐frame AUG codons on lbp mRNA. Differential initiation is caused by a small open reading frame (ORF, situated in the 5′ untranslated region of lbp mRNA), which gives rise to a leaky scanning mechanism. In Gonyaulax, only one of these variants, which is produced by initiation from the first AUG of the lbp ORF, exhibits a circadian rhythm and is far more abundant during night phase.
Stromal and thylakoid‐bound ascorbate peroxidases are produced by alternative splicing in pumpkin1
Authors: Mano Shoji , Yamaguchi Katsushi , Hayashi Makoto , Nishimura Mikio
Published: July 2002

A cDNA encoding stromal ascorbate peroxidase (sAPX) was isolated using poly(A)+ RNA of pumpkin cotyledons by RT‐PCR. The cDNA encodes a polypeptide with 372 amino acids and shares complete sequence identity with pumpkin thylakoid‐bound ascorbate peroxidase (tAPX), except for the deletion of a putative membrane spanning region located in the carboxy domain of tAPX. Southern blot hybridization and analysis of intron structure indicated that mRNAs for sAPX and tAPX, whose suborganellar localizations in chloroplasts are different, are produced by alternative splicing. Immunoblot analysis showed that the accumulation of sAPX and tAPX was differently regulated during germination and subsequent greening of pumpkin cotyledons.
Thyroid hormone‐induced expression of the ADP/ATP carrier and its effect on fatty acid‐induced uncoupling of oxidative phosphorylation
Authors: Schönfeld Peter , Wiêckowski Mariusz R , Wojtczak Lech
Published: July 2002

Liver mitochondria from rats made hypothyroid by administration of 2‐mercapto‐1‐methylimidazole were less sensitive to the uncoupling effect of myristic acid, as measured by the increase of resting state respiration, than mitochondria from euthyroid animals, whereas subsequent administration to the animals of triiodothyronine (`hyperthyroidism') resulted in an increased uncoupling action of myristate. `Hyperthyroidism' also resulted in doubling of the carboxyatractyloside‐sensitive portion of the myristate‐stimulated respiration. Parallel to this was a twofold increase of the mitochondrial content of the ADP/ATP carrier protein and an over threefold increase of its activity. The uncoupling effect of phytanic acid was less sensitive to carboxyatractyloside and was increased in the hyperthyroid state to a smaller extent than in the case of myristate. These results provide further support to the thesis [Skulachev, V.P., FEBS Lett. 294 (1991) 158–162] that the ADP/ATP carrier is involved in the mechanism of the uncoupling effect of long‐chain fatty acids.
Spontaneous spectral changes of the reduced cytochrome bd
Authors: Azarkina Natalya , Borisov Vitaliy , Konstantinov Alexander A
Published: July 2002

Reduction of the membrane‐bound cytochrome bd from Bacillus subtilis, Escherichia coli and Azotobacter vinelandii as well as of the purified enzyme from E. coli was followed by secondary absorption changes on a time scale of tens of minutes. The difference absorption spectra of these changes resembled those induced by CO binding with heme d
2+ indicating interaction of the heme with an endogenous π‐acceptor ligand. The spontaneous spectral changes were prevented and reversed by CO binding with the reduced cytochrome bd. Bonding of heme d iron to an endogenous protein ligand at the sixth axial position upon reduction is proposed and several possible mechanisms of such a process are considered.
The 5′‐untranslated region of the human muscle acylphosphatase mRNA has an inhibitory effect on protein expression
Authors: Fiaschi Tania , Marzocchini Riccardo , Raugei Giovanni , Veggi Daniele , Chiarugi Paola , Ramponi Giampietro
Published: July 2002

The cDNA of the human muscle type acylphosphatase was isolated and characterized. The mRNA presents a very long 5′‐untranslated region, covering the first half of the molecule: 175 bases of this part were cloned and prediction of the possible secondary structure showed that a very stable stem‐loop structure could be formed in that region. Moreover, an additional AUG triplet was found upstream of the start codon of the protein, defining an open reading frame of 60 codons which overlapped that of acylphosphatase. The possible regulatory effect on translation of this part of the mRNA molecule was studied by means of transient transfection experiments: a 10‐fold decrease in the expression of a reporter protein and a dramatic decrease in the corresponding mRNA was observed, due to the presence of the 5′‐untranslated region of acylphosphatase mRNA. Mutagenesis of the upstream AUG triplet eliminated mRNA instability, leading to the hypothesis that the product of the upstream open reading frame could play a role in this mechanism.
Vascular types I and II transforming growth factor‐beta receptor expression: differential dependency on tyrosine kinases during induction by TGF‐β
Authors: Ward Michael R , Agrotis Alex , Jennings Garry , Bobik Alex
Published: July 2002

Recent evidence indicates that the type II transforming growth factor‐β (TGF‐β) receptor (TβRII) is a serine‐threonine‐tyrosine kinase. However, the significance of its tyrosine kinase is unclear. We investigated in vascular smooth muscle cells the effects of tyrosine kinase inhibition on the expression of TGF‐β receptor types I (ALK‐5) and II (TβRII) mRNA, induced by TGF‐β
1. TGF‐β
1 elevated ALK‐5 mRNA levels 5‐fold; essentially similar TGF‐β
1‐dependent elevations were observed with growth factors, PDGF‐BB and FGF‐2. The tyrosine kinase inhibitor genistein abolished these TGF‐β
1 and growth factor responses. TGF‐β
1 also elevated TβRII mRNA levels which were not inhibited by genistein. We conclude that tyrosine kinases participate in defining how cells respond to TGF‐β.
Shared determinants of receptor binding for subtype selective, and dual endothelin‐angiotensin antagonists on the AT1 angiotensin II receptor
Authors: Dascal David , Nirula Vaneet , Lawus Kimberly , Yoo Sung-Eun , Walsh Thomas F , Sandberg Kathryn
Published: July 2002

Site‐directed interspecies amino acid exchange was used to compare the binding determinants of a novel dual endothelial‐angiotensin receptor ligand, L‐746,072, with type‐1 angiotensin receptor (AT1) selective antagonists on AT receptors expressed in COS cells. These studies suggest that residues on AT receptors which are non‐conserved between amphibian and mammalian species play a greater role in subtype selective ligand recognition than for dual receptor ligands. These data also support the hypothesis that a common non‐peptide binding site exists within transmembrane domains on peptidergic receptors.
An Arabidopsis cDNA encodes an apparent polyprotein of two non‐identical thylakoid membrane proteins that are associated with photosystem II and homologous to algal ycf32 open reading frames
Authors: Mant Alexandra , Robinson Colin
Published: July 2002

We have characterised an Arabidopsis thaliana cDNA homologous to the ycf32 open reading frames present in the Synechocystis genome and the plastid genomes of several eukaryotic algae. The predicted protein is also homologous to a novel protein reported to be associated with photosystem II. The protein is synthesised as a 23 kDa precursor with an N‐terminal presequence that appears to be bipartite in structure, and the protein is targeted into the thylakoid membrane of pea chloroplasts. Although the Ycf32 presequence contains an apparent signal peptide, we find that this protein is not imported by either of the standard Sec‐ or ΔpH‐dependent pathways. The mature protein is also unusual in two respects. First, there are two distinct, non‐identical copies of typical single‐span Ycf32 sequences in the Arabidopsis sequence, separated by an additional hydrophobic region. Secondly, the imported protein runs as a doublet of 6 kDa and 7 kDa polypeptides whereas the mature protein is predicted to be 14 kDa. We speculate that the protein undergoes further maturation once inserted into the thylakoid membrane to yield two separate Ycf32‐like polypeptides.
Proteasome function is dispensable under normal but not under heat shock conditions in Thermoplasma acidophilum
Authors: Ruepp Andreas , Eckerskorn Christoph , Bogyo Matthew , Baumeister Wolfgang
Published: July 2002

Hitherto the biology of proteolysis in prokaryotes, particularly in archaea, is only poorly understood. We have used the tri‐peptide vinyl sulfone inhibitor carboxybenzyl‐leucyl‐leucyl‐leucine vinyl sulfone (Z‐L3VS) to study the in vivo function of proteasomes in Thermoplasma acidophilum. Z‐L3VS is a potent inhibitor of the Thermoplasma proteasome and is capable of modifying 75 to 80% of the proteasomal β‐subunits in cell cultures. Inhibition of proteasomes has only marginal effects under normal growth conditions. Under heat shock conditions, however, the effects of proteasome inhibition are much more severe, to the extent of complete cell growth arrest. These data suggest that other proteolytic systems may exist that can compensate for the loss of proteasome function in T. acidophilum.
Further assignment of resonances in 1H NMR spectra of cerebrospinal fluid (CSF)
Authors: Lutz Norbert W , Maillet Serge , Nicoli François , Viout Patrick , Cozzone Patrick J
Published: July 2002

A number of previously unidentified 1H NMR signals detected in CSF spectra of patients with various neurological and metabolic diseases are assigned to metabolites, drugs and drug excipients. Two‐dimensional 1H NMR spectroscopy (COSY and J‐resolved) is employed to resolve resonances which are hidden by superimposed peaks in one‐dimensional spectra. Assignments obtained by making use of 2‐D techniques, and of a 1‐D 1H NMR data base created for ca. 150 authentic compounds, enable us to clarify the nature of complex signal patterns found in crowded spectral regions of CSF such as the aliphatic methyl region at ca. 1.0 ppm.
Scavenging of peroxynitrite by a phenolic/peroxidase system prevents oxidative damage to DNA1
Authors: Grace Stephen C , Salgo Maria G , Pryor William A
Published: July 2002

We examined the ability of horseradish peroxidase (HRP), an analog of human myeloperoxidase, to protect DNA against oxidative damage caused by peroxynitrite in the presence of chlorogenic acid (CGA), a naturally occurring polyphenol. Chlorogenic acid inhibits the formation of single strand breaks in supercoiled pBR322 DNA by acting as a scavenger of peroxynitrite. Horseradish peroxidase markedly enhances the extent of DNA protection by catalyzing the decomposition of peroxynitrite in the presence of CGA. Horseradish peroxidase alone does not inhibit peroxynitrite‐induced DNA strand breaks, indicating that CGA is required as an electron donor to regenerate the active enzyme. The apparent second order rate constant for the HRP‐mediated oxidation of CGA in the presence of peroxynitrite at pH 6.9 is 3.4×107 M−1 s−1. This high rate suggests that CGA and other dietary polyphenols might efficiently scavenge peroxynitrite in peroxidase‐containing systems in vivo.
Reconstituted adenine nucleotide translocase forms a channel for small molecules comparable to the mitochondrial permeability transition pore
Authors: Rück Alexander , Dolder Max , Wallimann Theo , Brdiczka Dieter
Published: July 2002

Highly purified adenylate translocase (ANT) from rat heart mitochondria was functionally reconstituted as ATP/ADP exchange carrier in asolectin/cardiolipin vesicles. The ANT preparations used were free of porin, cyclophilin D, and Bax as analysed immunologically and by activity measurements. After pre‐loading the ANT‐containing proteoliposomes with ATP, malate or AMP, a gradual release of the trapped compounds by increasing the external Ca2+concentrations could be demonstrated.N‐Methyl‐Val‐4‐cyclosporin did not inhibit the Ca2+dependent release of internal substances from ANT liposomes. This inhibitor was found to be specific for the mitochondrial permeability transition pore (MTP) in intact mitochondria or reconstituted MTP‐like protein complexes (e.g. hexokinase, porin, ANT complex). However, ADP in concentrations >20 μM inhibited the liberation of internal compounds, while in contrast, atractyloside (30 μM) and HgCl2(5 μM) both induced permeability of the ANT‐containing liposomes resulting in a release of trapped substances. These results strongly suggest that ANT itself is capable to adopt a pore‐like structure under conditions known to induce the permeability transition in mitochondria.
Adducin in essential hypertension
Authors: Manunta Paolo , Barlassina Cristina , Bianchi Giuseppe
Published: July 2002

However adducin polymorphisms account for only a portion of hypertension both in humans and rats. Therefore additive or epistatic interactions with other genes involved in renal sodium handling need to be studied.
Expression of Arabidopsis response regulator homologs is induced by cytokinins and nitrate
Authors: Taniguchi Mitsutaka , Kiba Takatoshi , Sakakibara Hitoshi , Ueguchi Chiharu , Mizuno Takeshi , Sugiyama Tatsuo
Published: July 2002

We examined cytokinin and nitrate responsiveness in gene expression of five distinct response regulator homologs (ARR3–ARR7) in the leaves of nitrogen‐starved Arabidopsis plants. The transcripts accumulated after spraying the shoots with t‐zeatin. The induction of accumulation was highly specific for cytokinins. The transcripts also accumulated by supply of nitrate to the culture medium. These findings suggest that ARRs are involved in inorganic nitrogen signal transduction mediated by cytokinin as in the case of ZmCip1, a response regulator homolog recently identified in maize.
The 5′‐AMP‐activated protein kinase inhibits the transcriptional stimulation by glucose in liver cells, acting through the glucose response complex
Authors: Leclerc Isabelle , Kahn Axel , Doiron Bruno
Published: July 2002

5‐Amino‐4‐imidazolecarboxamide riboside (AICAR) is known to stimulate rat liver 5′‐AMP‐activated protein kinase (AMPK). AMPK is the mammalian homologue of Snf1p in yeast, involved in derepression of glucose‐repressed genes. We used AICAR to test if AMPK could also play a role in the regulation of glucose‐dependent genes in mammalian cells. At a concentration which induces phosphorylation‐dependent inactivation of HMG‐CoA reductase, AICAR blocked glucose activation of three glucose responsive genes, namely L‐type pyruvate kinase (L‐PK), Spot 14 and fatty acid synthase genes in primary cultured hepatocytes, but was without any action on glucose phosphorylation to glucose 6‐phosphate and on expression of PEPCK, albumin and β‐actin genes. AICAR was also found to inhibit activation of the L‐PK gene promoter by glucose in transiently transfected hepatoma cells. Therefore our results suggest that AMPK is probably involved in the glucose signal pathway regulating gene expression in the liver.