bluuebunny/crossref_metadata_2025 huggingface dataset:
Correlation between energy‐dependent tritium incorporation into CF1 and light‐induced protonation of thylakoid membranes
Authors: Zolotareva E.K. , Opanasenko V.K. , Zakharov S.D. , Kuzmina V.P.
Published: July 2002

DCCD effects on proton titration of thylakoid membranes and light‐induced tritium incorporation in CF1 were compared. The pool of ionogenic groups exposed in the light (about 0.22 μmol · mg Chl−1 in the pH range 7–9) and tritiation of CF1 were both blocked by DCCD. The results are discussed with reference to the role of the stage of proton (tritium) translocation through the H+ channel of ATP‐synthase in these processes.
Benzodiazepine agonists protect a histidine residue from modification by diethyl pyrocarbonate whereas propyl β‐carboline does not
Authors: Lambolez B. , Rossier J.
Published: July 2002

The pH sensitivity of benzodiazepine binding suggests that a histidine residue may be present in, or close to the benzodiazepine binding site. This was confirmed by the selective modification of histidine residues using diethyl pyrocarbonate which was found to block both benzodiazepine and β‐carboline binding. In order to assess whether this histidine residue is located in or adjacent to the benzodiazepine and β‐carboline binding sites, experiments were performed using either benzodiazepine or β‐carboline to protect against diethyl pyrocarbonate treatment. It was found that benzodiazepine agonists, but not propyl β‐carboline protect the benzodiazepine binding sites from diethyl pyrocarbonate modification.
Identifying subunits of ATP synthase TF0·F1 in contact with phospholipid head groups α‐Subunits are labelled selectively by a new photoreactive phospholipid designed for hydrophilic photolabelling
Authors: Gao Zhan , Bäuerlein Edmund
Published: July 2002

A new phospholipid photolabel was introduced by modifying 1,2‐dipalmitoylphosphatidylethanolamine with 4‐azidosalicylate to 1,2‐dipalmitoyl‐sn‐glycero‐3‐phospho‐N‐(4‐azido‐2‐hydroxybenzoyl)ethanolamine (ASA‐PE), which could be radioiodinated easily to 125 I‐ASA‐PE. The ATP synthase TF0·F1 of the thermophilic bacterium PS3 was reconstituted with soybean phospholipids forming proteoliposomes with high ATP‐32 Pi exchange activity. These proteoliposomes were incubated with 125I‐ASA‐PE to provide its selective incorporation into the outside of the phospholipid bilayer. Upon illumination with ultraviolet light α‐subunits of TF1 were predominantly labelled.
Mitochondria catalyze the reduction of NAD by reduced methylviologen
Authors: Nagata Shinichi , Günther Helmut , Bader Johann , Simon Helmut
Published: July 2002

Mitochondria from beef heart and yeast catalyze the reduction of NAD to NADH at the expense of reduced methylviologen (MV+). Based on protein the specific activity of mitochondria for this reaction is about 10–20‐times higher than the consumption of oxygen in the presence of succinate or NADH. In 2H2O buffer (4S)‐[4‐2H]NADH is formed in high enantiomeric excess if the reduced methylviologen is electrochemically regenerated.
The refined 2.3 Å crystal structure of human leukocyte elastase in a complex with a valine chloromethyl ketone inhibitor
Authors: An-Zhi Wei , Mayr Irmgard , Bode Wolfram
Published: July 2002

The stoichiometric complex formed between human leukocyte elastase and a synthetic MeO‐Suc‐Ala‐Ala‐Pro‐Val chloromethyl ketone inhibitor was co‐crystallized and its X‐ray structure determined, using Patterson search methods. Its structure has been crystallographically refined to a final R value of 0.145 (8.0 and 2.3 Å). The enzyme structure is very similar to that recently observed in a complex formed with the ovomucoid third domain from turkey [(1986) EMBO J. 5,2453–2458]. The rms deviation of all α‐carbon atoms is 0.32 Å. The peptidic inhibitor is bound in a similar overall conformation as the ovomucoid binding segment. Covalent bonds are formed between Val‐P1 of the inhibitor and His‐57 NE2 and Ser‐195 OG of the enzyme. The carbonyl carbon is tetrahedrally deformed to a hemiketal. The valine side chain is arranged in the S1 pocket in the g
− conformation.
Effect of truncated glucagon‐like peptide 1 on cAMP in rat gastric glands and HGT‐1 human gastric cancer cells
Authors: Hansen A.B. , Gespach C.P. , Rosselin G.E. , Holst J.J.
Published: July 2002

We tested the truncated 7–37 glucagon‐like peptide 1 (TGLP‐1), a naturally occurring porcine intestinal peptide, and other members of the glucagon family, including pancreatic glucagon (G‐29), GLP‐1 and GLP‐2 for their ability to activate the cAMP generating system in rat gastric glands and HGT‐1 human gastric cancer cells. In rat fundic glands, TGLP‐1 was about 100 times more potent (EC50 = 2.8 × 10−9M) than GLP‐1 of G‐29, and 10 times more potent than G‐29 in the HGT‐1 cell line. Our results support the notion that TGLP‐1 plays a direct role in the regulation of acid secretion in rat and human gastric mucosa.
Two adjacent cysteine residues in the C‐terminal cytoplasmic fragment of bovine rhodopsin are palmitylated
Authors: Ovchinnikov Yu.A. , Abdulaev N.G. , Bogachuk A.S.
Published: July 2002

Covalent coupling of bovine rhodopsin to CPG‐thiol glass was used for separation of CNBr peptides. It is shown that cysteine residues 322 and 323 in the C‐terminal cytoplasmic fragment of rhodopsin are modified with palmitic acid.
The fast‐twitch muscle calsequestrin isoform predominates in rabbit slow‐twitch soleus muscle
Authors: Fliegel Larry , Leberer Ekkehard , Green N.Michael , MacLennan David H.
Published: July 2002

The major form of calsequestrin in rabbit slow‐twitch soleus muscle is shown to be identical to that isolated and cloned from rabbit fast‐twitch muscle on the following bases: identity of cDNAs cloned from mRNAs from the two muscle sources; equivalent hybridization of a fast‐twitch calsequestrin cDNA probe to mRNAs isolated from fast‐twitch and slow‐twitch muscles; identity of the 23 amino‐terminal amino acids; strong binding of 45Ca2+ in a gel overlay of slow muscle sarcoplasmic reticulum protein to a band at the level of the fast‐twitch calsequestrin isoform and only weak binding at the level of the cardiac isoform. No evidence was obtained for developmentally regulated alternative splicing of the calsequestrin transcript in mature slow or fast‐twitch muscle.
Binding of mRNA by an oligopeptide containing an evolutionarily conserved sequence from RNA binding proteins
Authors: Schwemmle Martin , Görlach Matthias , Bader Michael , Sarre Thomas F. , Hilse Kurt
Published: July 2002

Several proteins with an affinity to RNA contain a conserved sequence of 8 amino acids which is postulated as being important for RNA binding. An oligopeptide of 11 amino acids containing this sequence is shown to bind 32P‐labelled globin mRNA in a filter binding assay. High concentrations of heparin compete for this binding. 10 other peptides with different sequences do not exhibit affinity to RNA in this assay. These results support the relevance of the conserved peptide sequence in the binding of proteins to RNA.
Terbutryn resistance in a purple bacterium can induce sensitivity toward the plant herbicide DCMU
Authors: Sinning I. , Michel H. , Mathis P. , Rutherford A.W.
Published: July 2002

Herbicide‐resistant mutant; Reaction center, Photosynthetic; Purple bacteria; Diuron; Terbutryn
Vanadyl‐induced Fenton‐like reaction in RNA
Authors: Carmichael Alasdair J.
Published: July 2002

Vanadyl (VO2+) complexee to RNA reacts with hydrogen peroxide in a Fenton‐like manner producing hydroxyl radicals ('OH). The hydroxyl radicals can be spin trapped with 5,5‐dimethyl‐1‐pyrroline‐1‐oxide (DMPO) forming the DMPO‐OH spin adduct. In addition, in the presence of ethanol the formation of the hydroxyethyl radical adduct of DMPO (DMPO‐ETOH) confirms the production of hydroxyl radicals by the RNA/ VO2+ complex. When the reaction between the RNA/VO2+ complex and H2O2 is carried out in the presence of the spin trap 2‐methyl‐2‐nitrosopropane (MNP), radicals produced in the reaction of .OH with RNA are trapped. Base hydrolysis of the MNP‐RNA adducts (pH 12) followed by a reduction in the pH to pH 7 after hydrolysis is complete, yields an MNP adduct with a well‐resolved ESR spectrum identical to the ESR spectrum obtained from analogous experiments with poly U. The ESR spectrum consists of a triplet of sextets (a
N = 1.48 mT, a
N
β = 0.25 mT and a
H
β = 0.14 mT), indicating that the unpaired nitroxide electron interacts with the nuclei of a β‐nitrogen and β‐hydrogen. The results suggest that the .OH generated in the RNA/VO2+ reaction with H2O2 add to the C(5) carbon of uracil forming a C(6) carbon centered radical. This radical is subsequently spin trapped by MNP.
A rev/β‐galactosidase fusion protein binds in vitro transcripts spanning the rev‐responsive element of human immunodeficiency virus type 1 (HIV‐1)
Authors: Aepinus Christian , Voll Reinhard , Bröker Michael , Fleckenstein Bernhard
Published: July 2002

The rev protein of human immunodeficiency virus type 1 (HIV‐1), a phosphoprotein of 20 kDa apparent molecular mass, is essential to target the mRNA for virion polypeptides into the cytoplasm. This effect is mediated by a specific RNA stretch (rev‐responsive element = RRE) localized within a 3′‐terminal segment of the mRNA encoding virion proteins. We present evidence that rev expressed as a β‐galactosidase fusion protein in E. coli forms a complex with in vitro transcripts containing the RRE; it can be precipitated by monoclonal antibodies with rev or β‐galactosidasespecificity. In addition, specific binding of rev protein to RNA could be demonstrated by Northwestern blotting.
Analysis of the binding of P‐chlorophenyl‐methoxybenzyl‐ketoxime (CPMB‐Oxime) to mitochondrial cytochrome c reductase
Authors: Brandt Ulrich , von Jagow Gebhard
Published: July 2002

Cytochrome c reductase is inhibited by p‐chlorophenyl‐methoxybenzyl‐ketoxime (CPMB‐oxime). CPMB‐oxime induces a red‐shift of the reduced spectrum of cytochrome b. The inhibitor blocks the oxidation of ubihydroquinone at the Qp center of this enzyme in a non‐competitive way. The binding stoichiometry equals one inhibitor molecule per Qp center. The apparent k
d in a red‐shift assay was 6.9 ± 0.6 μM. All binding characteristics analysed in this study were very similar to those of the E‐β‐methoxyacrylate inhibitors, although the chemical structure is different from these inhibitors. This result is interpreted as a support for the inhibitory mechanism based on the model of a ‘catalytic switch’ proposed recently for the E‐β‐methoxyacrylate inhibitors (MOA‐inhibitors (Brandt and von Jagow, Eur. J. Biochem. (1991) 195, 163‐170).
cDNA cloning and expression of a hamster α‐thrombin receptor coupled to Ca2+ mobilization
Authors: Rasmussen Ulla B. , Vouret-Craviari Valérie , Jallat Sophie , Schlesinger Yasmin , Pagès Gilles , Pavirani Andrea , Lecocq Jean-Pierre , Pouysségur Jacques , Van Obberghen-Schilling Ellen
Published: July 2002

The serine protease α‐thrombin (thrombin) potently stimulates G‐protein‐coupled signaling pathways and DNA synthesis in CCL39 hamster lung fibroblasts. To clone a thrombin receptor cDNA, selective amplification of mRNA sequences displaying homology to the transmembrane domains of G‐protein‐coupled receptor genes was performed by polymerase chain reaction. Using reverse transcribed poly(A)+ RNA from CCL39 cells and degenerate primers corresponding to conserved regions of several phospholipase C‐coupled receptors, three novel putative receptor sequences were identified. One corresponds to an mRNA transcript of 3.4 kb in CCL39 cells and a relatively abundant cDNA. Microinjection of RNA transcribed in vitro from this cDNA in Xenopus oocytes leads to the expression of a functional thrombin receptor. The hamster thrombin receptor consists of 427 amino acid residues with 8 hydrophobic domains, including one at the extreme N‐terminus that is likely to represent a signal peptide. A thrombin consensus cleavage site is present in the N‐terminal extracellular region of the receptor sequence followed by a negatively charged cluster of residues present in a number of proteins that interact with the anion‐binding exosite of thrombin.
Substrate antagonism in the kinetic mechanism of E. coli phosphofructokinase‐1
Authors: Deville-Bonne Dominique , Laine Romuald , Garel Jean-Renaud
Published: July 2002

In the presence of its allosteric activator GDP, the major phosphofructokinase‐1 from Escherichia coli K12 follows Michaelis—Menten kinetics. The kinetic behavior observed at steady‐state using different concentrations of the substrates ATP and fructose‐6‐phosphate and the pattern of inhibition by the substrate analogs adenylyl‐(β,γ‐methylene)‐diphosphonate and D‐arabinose‐5‐phosphate are consistent with a random sequential mechanism in rapid equilibrium, rather than with an ordered binding as was suggested earlier. However, ATP and fructose‐6‐phosphate do not bind independently to the same active site, since the apparent affinity for one substrate is decreased about 20‐fold when the other substrate is already bound. The antagonism between ATP and fructose‐6‐phosphate shows that a negative interaction occurs during the reaction with E. coli phosphofructokinase‐1 which must be considered in addition to its allosteric properties.
Isolation and primary structure of a novel chromogranin A‐derived peptide, WE‐14, from a human midgut carcinoid tumour
Authors: Curry W.J. , Shaw C. , Johnston C.F. , Thim L. , Buchanan K.D.
Published: July 2002

The primary structure of a novel human chromogranin A‐derived tetradecapeptide, WE‐14, possessing N‐terminal tryptophanyl(W) and C‐terminal glutamyl (E) residues was isolated from a hepatic metastasis of an human ileal carcinoid tumour. Human and bovine WE‐14 are structurally identical, while rat, mouse and porcine analogues exhibit 93 % homology. WE‐14 is flanked by paired basic residues (KR) in all known chromogranin A sequences.
New intramolecularly quenched fluorogenic peptide substrates for the study of the kinetic specificity of papain
Authors: Garcia-Echeverria Carlos , Rich Daniel H.
Published: July 2002

A series of new substrates for determining the catalytic activity of cysteine proteinases is described. The rate of hydrolysis by papain was monitored by a fluorescence continuous assay based on internal resonance energy transfer using 5‐[(2‐aminoethyl)amino]naphtalene‐1‐sulfonic acid (EDANS) and 4‐(4‐dimethylaminophenylazo)benzoic acid (DABCYL) as fluorescent donor and quenching acceptor, respectively, in peptides with the general structure: DABCYL‐Lys‐Phe‐Gly‐Xxx‐Ala‐Ala‐EDANS. The substrates were used to evaluate the effect of amino acid structure in the Sl1 position on the kinetic parameters for papain catalyzed hydrolysis.
Structure and expression of Xenopus prohormone convertase PC2
Authors: Braks Joanna A.M. , Guldemond Karin C.W. , van Riel Maarten C.H.M. , Coenen Anton J.M. , Martens Gerard J.M.
Published: July 2002

The multifunctional prohormone, proopiomelanocortin (POMC), is processed in the melanotrope cells of the pituitary pars intermedia at pairs of basic amino acid residues to give a number of peptides, including α‐melanophore‐stimulating hormone (α‐MSH). This hormone causes skin darkening in amphibians during background adaptation. Here we report the complete structure of Xenopus laevis prohormone convertase PC2, the enzyme thought to be responsible for processing of POMC to α‐MSH. A comparative structural analysis revealed an overall amino acid sequence identity of 85–87% between Xenopus PC2 and its mammalian counterparts, with the lowest degree of identity in the signal peptide sequence (28–36%) and the region amino‐terminal to the catalytic domain (59–60%). The occurrence of a second, structurally different PC2 protein reflects the expression of two Xenopus PC2 genes. The expression pattern of PC2 in the Xenopus pituitary gland of black‐ and white‐adapted animals was found to be similar to that of POMC, namely high expression in active melanotrope cells of black animals. This observation is in line with a physiological role for PC2 in processing POMC to α‐MSH.
Effect of the d‐Phe2 residue on molecular conformation of an endogenous neuropeptide achatin‐I Comparison of X‐ray crystal structures of achatin‐I (H‐Gly‐d‐Phe‐Ala‐Asp‐OH) and achatin‐II (H‐Gly‐Phe‐Ala‐Asp‐OH)
Authors: Ishida Toshimasa , In Yasuko , Inoue Masatoshi , Yasuda-Kamatani Yoshimi , Minakata Hiroyuki , Iwashita Takashi , Nomoto Kyosuke
Published: July 2002

The molecular conformation of achatin‐II neutral form (H‐Gly‐Phe‐Ala‐Asp‐OH), an endogenous peptide from the Achatina fulica ganglia, was elucidated by X‐ray crystal analysis. The molecule takes an extended β‐pleated structure stabilized by 5 intermolecular hydrogen bonds with the antiparallely arranged molecules. This is in contrast with the turn conformation of a neuroactive achatin‐I (H‐Gly‐d‐Phe‐Ala‐Asp‐OH) [(1992) FEBS Lett. 276, 95–97]. The conformational comparison of both of the molecules makes clear the structural role which d‐Phe residue of achatin‐I plays in forming a definite active form.
Zymosan induces selective release of arachidonic acid from rabbit alveolar macrophages via stimulation of a β‐glucan receptor
Authors: Daum Timothy , Rohrbach Michael S.
Published: July 2002

Zymosan, which is composed primarily of α‐mannan and β‐glucan polymers, is a well recognized activator of macrophages. The type receptor by which unopsonized zymosan induces arachidonic acid release was investigated. It was found that particulate β‐glucan and zymosan stimulated an identical dose‐dependent release of arachidonic acid. This release of arachidonic acid by zymosan was blocked by soluble β‐glucans whereas soluble mannan had no effect. This inhibition was not due to a general toxic effect of the soluble β‐glucans as they had no effect on calcium ionophore‐induced release of arachidonic acid. β‐glucan‐induced fatty acid release from these cells was shown to be fairly specific for arachidonic acid. These data reveal that zymosan stimulates the specific release of arachidonic acid from rabbit alveolar macrophages, at least in part, via a β‐glucan receptor.
The murine NPY‐1 receptor gene Structure and delineation of tissue‐specific expression
Authors: Eva Carola , Oberto Alessandra , Sprengel Rolf , Genazzani Enrico
Published: July 2002

The murine gene for the NPY‐1 receptor subtype for neuropeptide Y was characterized by DNA sequencing and expression studies. It comprises three exons with a 6,400 bp 5′‐untranslated and a 80 bp internal intronic sequence. The 5′‐flanking region of this gene lacks TATA or CCAAT consensus sequences in the proximity to the multiple transcription initiation sites. A 1,300 bp genomic fragment of the 5′‐flanking region drives the expression of the lacZ reporter gene in NG108‐15 cells and primary cultured neurons but not in glial and human embryonic kidney cells. In addition, it contains consensus sequences for various transcription factors including cAMP‐ and glucocorticoid‐responsive elements.
Structure of three acidic O‐linked carbohydrate chains of porcine zona pellucida glycoproteins
Authors: Hokke Comelis H. , Damm Jan B.L. , Kamerling Johannis P. , Vliegenthart Johannes F.G.
Published: July 2002

Structural analysis by ID and 2D 1H NMR spectroscopy of three acidic O‐linked oligosaccharide alditols, released from porcine zona pellucida glycoproteins by alkaline borohydride treatment, afforded the following structures: Galβl‐4(6SO4‐)GlcNAcβl‐3Galβl‐4GlcNAcβ1‐3Galβ1‐3GalNAc‐ol Neu5Gcα2‐3Gal/91 ‐4(6SO4‐)GlcNAqβl ‐3Ga1βl ‐4GlcNAcβl ‐3Galβl ‐3GalNAc‐ol Neu5Acα2‐3Galβl ‐4(6SO4‐)GlcNAcβl ‐3Galβl ‐4GlcNAcβl ‐3Galβl‐3GalNAc‐ol These oligosaccharides are the smallest compounds that contain the structural elements which are present in the acidic, high‐molecular mass O‐linked carbohydrate chains of porcine zona pellucida glycoproteins.
Carcinogenic antioxidants
Authors: Wiseman Helen , Halliwell Barry
Published: July 2002

The synthetic oestrogens diethylstilboestrol, hexoestrol and 17 α‐ethynyloestradiol are known to be carcinogenic, yet they all exert antioxidant properties in vitro in that they are good inhibitors of iron ion‐dependent lipid peroxidation. In rat liver microsomes incubated with Fe(III)‐ascorbate or Fe(III)‐ADP/NADPH and also in ox‐brain phospholipid liposomes incubated with Fe(III)‐ascorbate; the overall order of effectiveness of the compounds tested as inhibitors of lipid peroxidation was diethylstilboestrol ⪢ hexoestrol ⪢ 17 α‐ethynyloestradiol ⪢ 4‐hydroxytamoxifen ⪢ 17 β‐oestradiol ⪢ tamoxifen. Compounds acting as antioxidants towards lipids may also exert pro‐oxidant effects towards other molecules such as DNA and thus must never be assumed to be safe for human use.
Secondary structure of globular proteins at the early and the final stages in protein folding
Authors: Kuwajima Kunihiro , Semisotnov Gennady V , Finkelstein Alexei V , Sugai Shintaro , Ptitsyn Oleg B
Published: July 2002

The ellipticities for an early transient intermediate in refolding observed by kinetic circular dichroism measurements at 220–225 nm for 14 different proteins are summarized, and the ellipticity values are compared with those for the final native proteins and also with the ellipticities expected from a physical theory of protein and polypeptide secondary structure. The results show that a substantial part of the protein secondary structure is in general formed in the earliest detectable intermediate in refolding and that the ellipticities in both the native and the intermediate states are consistent with the physical theory of protein secondary structure.
The specificity of the S1' subsite of cysteine proteases
Authors: Ménard Robert , Carmona Euridice , Plouffe Céline , Brömme Dieter , Konishi Yasuo , Lefebvre Jean , Storer Andrew C.
Published: July 2002

The specificity of the S'1 subsite of the cysteine proteases cathepsin B, L, S and papain has been investigated using a series of intramolecularly quenched fluorogenic substrates (Dansyl‐Phe‐Arg‐AA‐Trp‐Ala) where the P'1 amino acid (AA) has been varied. Taken individually, each enzyme displays a relatively broad S'1 subsite specificity and this subsite cannot be considered as a primary site of specificity. Notable differences do exist however between the various proteases. Cathepsin B prefers large hydrophobic residues in the P'1 position of a substrate while cathepsin L has an opposite trend, favoring amino acids with small (Ala, Ser) or long but non‐branched (Asn, Gin, Lys) side chains. Cathepsin S and papain display a somewhat broader S'1 subsite specificity.
Cloned human brain nitric oxide synthase is highly expressed in skeletal muscle
Authors: Nakane Masaki , Schmidt Harald H.H.W. , Pollock Jennifer S. , Förstermann Ulrich , Murad Ferid
Published: July 2002

Complementary DNA clones corresponding to human brain nitric oxide (NO) synthase have been isolated. The deduced amino acid sequence revealed an overall identity with rat brain NO synthase of about 93% and contained all suggested consensus sites for binding of the co‐factors. The cDNA transfected COS‐1 cells showed significant NO synthase activity with the typical co‐factor requirements. Unexpectedly, messenger RNA levels of this isoform of NO synthase was more abundant in human skeletal muscle than human brain. Moreover, we detected high NO synthase activity and the expressed protein in human skeletal muscle by Western blot analysis, indicating a possible novel function of NO in skeletal muscle.
Processing of Kex2 pro‐region at two interchangeable cleavage sites
Authors: Germain Doris , Thomas David Y. , Boileau Guy
Published: July 2002

In Saccharomyces cerevisiae, the Kex2 endoprotease (Kex2p) is required for the proteolytic maturation of α‐pheromone and also for the removal of its own pro‐region. Kex2p is specific for pairs of basic amino acid residues. Two putative processing sites are present in the pro‐region of Kexlp. We have expressed processing site mutants of Kex2p and assayed the production of active Kex2p. Mutations affecting either putative cleavage site do not alter the activity. However, mutations affecting both sites led to a reduction in both Kex2 activity and the amount of protein. These results suggest that removal of Kex2p pro‐peptide is required for the production of a stable enzyme and can occur at either processing site.
Stabilization of Bacillus stearothermophilus neutral protease by introduction of prolines
Authors: Hardy F. , Vriend G. , Veltman O.R. , van der Vinne B. , Venema G. , Eijsink V.G.H.
Published: July 2002

The thermostability of neutral proteases has been shown to depend on autolysis which presumably occurs in flexible regions of the protein. In an attempt to rigidify such a region in the neutral protease of Bacillus stearothermophilus, residues in the solvent‐exposed 63–69 loop were replaced by proline. The mutations caused large positive (Ser‐65 → Pro, Ala‐69 → Pro) or negative (Thr‐63 → Pro, Tyr‐66 → Pro) changes in thermostability, which were explained on the basis of molecular modelling of the mutant proteins. The data show that the introduction of prolines at carefully selected positions in the protein can be a powerful method for stabilization.
Isolation and characterization of PSII core complexes from a brown alga, Laminaria saccharina
Authors: Douady Dominique , Rousseau Bernard , Berkaloff Claire
Published: July 2002

PSII‐enriched particles, active for DCIP‐reduction, were prepared from Laminaria saccharina chloroplasts, and PSII core complexes were further purified by ion‐exchange chromatography. They contained several polypeptides, four of them cross‐reacting with antibodies raised against CP47, CP43, D1 and D2 of green plants. A second chromatography was required to separate: (i) a core antenna, composed of 51 kDa polypeptide subunits, binding 11 β‐carotene, 4 chlorophyll (Chl) c and 7 fucoxanthin for 100 Chl a, and reacting with CP47 antibodies; and (ii) a reaction center complex consisting of two main polypeptides of 34 and 36 kDa. The pigment stoichiometry was of 5 Chl a and 0.5 β‐carotene for 2 pheophytin a. The 34 and 36 kDa components cross‐reacted with anti‐D1 and anti‐D2 antibodies, respectively. The presence of cytochrome b‐559 was substantiated by spectrophotometry.
The organisation of the multigene family which encodes the major cell surface protein, pMGA, of Mycoplasma gallisepticum
Authors: Markham P.F. , Glew M.D. , Sykes J.E. , Bowden T.R. , Pollocks T.D. , Browning G.F. , Whithear K.G. , Walker I.D.
Published: July 2002

The genome of the avian pathogen Mycoplasma gallisepticum contains a number of related genes for putative adhesion molecules (pMGA). Cloning and sequence analysis of several pMGA genes suggested that all of them might be transcriptionally and translationally functional. Analysis of the gene sequence encoding the sole pMGA variant expressed in vitro in the S6 strain (pMGA1.1) revealed no unambiguous feature that could account for its unique expression. It is estimated that the pMGA gene family may contain up to 50 members, and its possible role is discussed herein.
Inhibition by N‐acetyl‐l‐cysteine of interleukin‐6 mRNA induction and activation of NFκB by tumor necrosis factor α in a mouse fibroblastic cell line, Balb/3T3
Authors: Shibanuma Motoko , Kuroki Toshio , Nose Kiyoshi
Published: July 2002

Redox‐based modulation plays a role in transcriptional control of gene expression. In the present study, we investigated the possible role of reactive oxygen species in the induction of interleukin‐6 (IL‐6) mRNA and in increases in NFκB binding activity by tumor necrosis factor (TNF) α using a mouse fibroblastic cell line, Balb/3T3. Expression of IL‐6 mRNA is known to be dependent upon NFκB that binds to the 5′‐flanking region of the IL‐6 gene. We found that: (i) TNFα increased IL‐6 mRNA levels and this increase was inhibited by N‐acetyl‐l‐cysteine (NAC), a scavenger of reactive oxygen species. (ii) NFκB binding activity in this cell line was also increased by TNFα, and the increase was inhibited in the presence of NAC. (iii) The treatment of cells with low doses of hydrogen peroxide increased the NFκB binding activity. (iv) Expression of a reporter gene in which the chloramphenicol acetyltransferase (CAT) gene was under the control of NFκB binding sites was induced by hydrogen peroxide. These results suggest that the induction of IL‐6 mRNA is regulated by a mechanism involving reactive oxygen species and that NFκB, whose activity is sensitive to the cellular redox state, plays an important role in this induction in a fibroblastic cell line, Balb/3T3, stimulated with TNFα.
Processing and secretion of rat α1‐microglobulin‐bikunin expressed in eukaryotic cell lines
Authors: Bratt Tomas , Cedervall Tommy , Åkerström Bo
Published: July 2002

The precursor protein α1‐microglobulin‐bikunin was cleaved to the same degree whether expressed in CHO cells or in mutated CHO cells, RPE.40 cells, suggested to lack a functional form of the intracellular protease furin. Thus, α1‐microglobulin‐bikunin probably is not cleaved in vivo by furin. However, simultaneous overexpression of the precursor and furin in COS, CHO and RPE.40 cells increased the cleavage, suggesting that compartmentalisation and concentrations of protease and precursor are important for the cleavage, besides the in vitro specificity. Expression of α1‐microglobulin and bikunin alone gave different protein patterns on SDS‐PAGE as compared to expression of the precursor and subsequent cleavage, suggesting that the precursor protein is important for the post‐translational handling of α1‐microglobulin and bikunin.
Localization of hormone‐sensitive lipase to rat Sertoli cells and its expression in developing and degenerating testes
Authors: Holst Lena Stenson , Hoffmann Anna Maria , Mulder Hindrik , Sundler Frank , Holm Cecilia , Bergh Anders , Fredrikson Gudrun
Published: July 2002

Using in situ hybridization, hormone‐sensitive lipase was found to be expressed in a stage‐dependent manner in Sertoli cells of rat testis. No expression was found in Leydig cells but expression in spermatids could not be excluded. These results suggest a role for hormone‐sensitive lipase in the metabolism of lipid droplets in Sertoli cells, in contrast to its previously proposed function in steroid biosynthesis. The expression of testicular hormone‐sensitive lipase mRNA and protein, both larger in size compared to other tissues, coincided with the onset of spermatogenesis and was dependent on scrotal localization of the testis, suggesting a temperature‐dependent, pretranslational regulation of expression.
Molecular cloning and characterisation of a putative pectin methylesterase cDNA in Arabidopsis thaliana (L.)
Authors: Richard Luc , Qin Li-Xian , Gadal Pierre , Goldberg Renée
Published: July 2002

Pectin methylesterase (PME) is a cell wall enzyme that catalyses the de‐esterification of pectins leading to fundamental changes which confer new properties to the micro‐environment of each cell. In order to elucidate the meaning of PME‐mediated changes of pectin in the time course of cell differentiation, we attempted to study the regulation of PME genes in Arabidopsis thaliana. In this report, the first full cDNA sequence showing sequence similarities with other PME genes characterised so far in other plant species has been isolated from an Arabidopsis shoot cDNA library. This ATPME1 cDNA is 1,970 bp long and contains an open reading frame encoding a protein of 64,1 kDa and a basic pI of 8.7 as predicted from the nucleotide sequence. Northern blot analyses denoted changes in the expression level of the ATPME1 mRNA according to plant organs. High mRNA levels were found in young developing organs such as cauline leaves while they were significantly lower in rosette leaves, stems and inflorescences, and almost undetectable in roots. Beside this molecular approach, isoelectrofocusing analyses revealed the occurrence of three PME isoforms in Arabidopsis. Two PME isoforms with pI values of 4.9 and 9.1 were found throughout the plant, but at a higher level in the root, while an other PME isoform with a pI of 5.7 was essentially detected in the inflorescence. The relationship between our observations and the data reported for other plant species is discussed.
Biochemical approaches of increasing thermostability of β‐amylase from Bacillus megaterium B6
Authors: Ray Rina Rani , Jana Subhas Chandra , Nanda Geeta
Published: July 2002

Studies on the irreversible thermoinactivation of β‐amylase from Bacillus megaterium B6 exposed to 60°C revealed that the deactivation mechanism probably results from the oxidation of thiols present at the active site of the enzyme. Several attempts were made to increase its thermostability, which indicated that Mn2+ played a key role in determining thermostability and partially reactivating the inactivated enzyme. Immobilization of β‐amylase through gel‐entrapment and covalent crosslinking brought about a remarkable increase in thermotolerance with about a 14‐fold increase in catalytic half‐life.
Receptor‐mediated endocytosis of plasminogen activators and activator/inhibitor complexes
Authors: Andreasen Peter A. , Sottrup-Jensen Lars , Kjøller Lars , Nykjær Anders , Moestrup Søren K. , Petersen Claus Munch , Gliemann Jørgen
Published: July 2002

Recent findings have elucidated the mechanism for clearance from the extracellular space of the two types of plasminogen activators, urokinase‐type plasminogen activator (u‐PA) and tissue‐type plasminogen activator (t‐PA), and their type‐1 inhibitor (PAI‐1). Activator/PAI‐1 complexes and uncomplexed t‐PA bind to the multiligand receptors α2 macroglubulin receptor/low density lipoprotein receptor‐related protein (α2MR) and epithelial glycoprotein 330 (gp330). These receptors mediate endocytosis and degradation of u‐PA/PAI‐1 complex bound to the glycosyl phosphatidyl inositol‐anchored urokinase receptor (u‐PAR) on cell surfaces, and participate, in cooperation with other receptors, in hepatic clearance of activator/PAI‐1 complexes and uncomplexed t‐PA from blood plasma. The α2MR‐ and gp330‐mediated endocytosis of a ligand (u‐PA/PAI‐1 complex) initially bound to another receptor (u‐PAR) is a novel kind of interaction between membrane receptors. Binding to α2MR and gp330 is a novel kind of molecular recognition of serine proteinases and serpins.
Serpins from wheat grain
Authors: Rosenkrands Ida , Hejgaard Jørn , Rasmussen Søren K. , Bjørn Søren E.
Published: July 2002

Wheat serpin genes have been identified by Southern blot hybridization with three distinct barley protein Z probes. Immunoblot analysis with a monoclonal antibody towards barley protein Z confirmed expression of related M
r ~ 40 kDa proteins in wheat grain. The wheat serpins were extracted under reducing conditions and separated from β‐amylase and other seed proteins by thiophilic adsorption and anion‐exchange chromatography. One molecular form possessing chymotrypsin inhibitory activity was isolated in a reactive site cleaved form on a chymotrypsin affinity column. N‐terminal amino acid sequences of a CNBr fragment and of the C‐terminal peptide from the cleaved inhibitor (Mr 4574 ± 4 Da) verified homology with barley protein Z and mammalian serpins. The native inhibitory serpin was demonstrated to form an SDS‐stable complex with α‐chymotrypsin.
Histidine‐118 of elongation factor Tu: its role in aminoacyl‐tRNA binding and regulation of the GTPase activity
Authors: Jonák Jiři , Anborgh Pieter H. , Parmeggiani Andrea
Published: July 2002

The function of His118 in elongation factor (EF)‐Tu from Escherichia coli was investigated by its substitution with glycine. The substitution had a differential effect on individual functions of the protein. The affinity for aminoacyl (aa)‐tRNA and the intrinsic GTPase activity of the mutant EF‐Tu were decreased whereas the response of its GTPase center to aa‐tRNA was strongly increased. These results suggest that the region around His118 is involved in the binding of aa‐tRNA and in the transmission of a turn‐off signal generated by the interaction with aa‐tRNA and directed to the GTPase center of EF‐Tu.
Nuclear DNA strand breaks during ethanol‐induced oxidative stress in rat brain
Authors: Renis M. , Calabrese V. , Russo A. , Calderone A. , Barcellona M.L. , Rizza V.
Published: July 2002

Free radical‐mediated oxidative damage has been implicated in the pathophysiological mechanisms of apoptosis. In this study we report that statistically significant strand breaks were induced primarily in the hippocampus and cerebellum during chronic, and not acute, ethanol treatment. Damage to DNA observed in hippocampus and cerebellum was also correlated with significant modification in the activities of mitochondrial respiratory complexes I and IV and with a significant increase in lipid peroxidation products. This finding lends support to the fact that hippocampus and cerebellum are brain areas particularly vulnerable to redox changes induced by alcohol intoxication, suggesting lower threshold levels of ethanol tolerance.
Impact of maternal HIV‐1 infection on perinatal outcome
Authors: Kumar R.M. , Uduman S.A. , Khurranna A.K.
Published: July 2002

Objectives: To study the impact of HIV‐1 infection on pregnancy and maternal and early fetal outcome. Method: From January 1992 to January 1993, 160 HIV‐1 seropositive women and 164 HIV‐1 seronegative age‐and parity‐matched pregnant tribal women from Manipur, India, were recruited into a prospective study. Mother and infant were followed until 6 weeks postpartum. Results: Nine percent (15/160) of subjects had AIDS (CDC IV), 38% (60/160) were symptomatic (CDC III) and 53% (85/160) were asymptomatic (CDC I/II). Symptomatic (CDC III/IV) HIV‐1 infection is associated with a significantly increased rate of miscarriage, low birth weight, intrauterine fetal death and preterm delivery. Perinatal, infant and maternal deaths were limited to symptomatic women. HIV‐1 infected women were significantly younger than their HIV‐1 negative counterparts both in age and age at sexual debut. Placental membrane inflammation was significantly higher in the seropositive group and this correlated well with a higher risk of preterm delivery and postpartum endometritis. Asymptomatic HIV‐1 infection was not associated with adverse pregnancy outcome. Conclusion: Symptomatic (CDC III/IV) HIV‐1 infection in Indian tribal women is associated with adverse maternal and fetal outcome.
Emergent and elective cervical cerclage for cervical incompetence
Authors: Wu M.Y. , Yang Y.S. , Huang S.C. , Lee T.Y. , Ho H.N.
Published: July 2002

Objective: The clinical outcomes of emergent and elective McDonald cerclage including cases of prolapsed amniotic sac were compared. Method: Forty‐eight patients undergoing elective cervical cerclage suture and 21 patients receiving emergent cervical cerclage including eight cases of prolapsed fetal membranes in the vagina were retrospectively analyzed. Results: Prolongation of pregnancy (145 ± 27 vs. 58 ± 56 days, P < 0.001), delay of delivery (37.0 ± 3.1 vs. 28.3 ± 7.8 gestational weeks, P < 0.001), higher birth weight (3033 ± 751 vs. 1410 ± 1139 g, P = 0.001), better fetal salvage rate (98 vs. 52%, P < 0.05), and fewer lower Apgar scores (score < 7: 4/48 vs. 13/21at 1 min, P = 0.000; 2/48 vs.10/21 at 5 min, P = 0.000) were attained in the elective group compared with those in the emergent group. The clinical outcomes were not significantly different between those with and those without prolapsed fetal membranes, but this may have been due to the small sample size. Conclusion: In addition to elective cervical cerclage, we recommended this procedure in emergency cases even in those with a prolapsed amniotic sac.
Differential regulation of astrocyte TNF‐α expression by the cytokines TGF‐β, IL‐6 and IL‐10
Authors: Benveniste Etty N. , Tang Li Ping , Law Robert M.
Published: July 2002

In this study, we demonstrate that transforming growth factor‐beta (TGF‐β), interleukin‐10 (IL‐10) and interleukin‐6 (IL‐6) inhibit tumor necrosis factor‐alpha expression by primary rat astrocytes. Treatment of astrocytes with TGF‐β alone had no effect on TNF‐α expression, however, TGF‐β suppressed induction on TNF‐α expression at both the protein and mRNA level. In contrast, IL‐10 and IL‐6 both inhibited TNF‐α protein expression by astrocytes, but had no effect on mRNA levels. The extent of IL‐6 mediated inhibition was greatest when astrocytes were pretreated with IL‐6 for 12–24 hr, then exposed to the inducing stimuli, while IL‐10 was an effective inhibitor even when added simultaneously with the inducing stimuli. Collectively, these data indicate that TGF‐β, IL‐6 and IL‐10 are all capable of inhibiting TNF‐α expression by astrocytes, although these immunosuppressive cytokines act at different levels of gene expression; i.e. TGF‐β at the transcriptional level and IL‐10/IL‐6 at the translational level. These results indicate that TGF‐β, IL‐6 and IL‐10 are important regulators of cytokine production by astrocytes under inflammatory conditions in the brain, and can contribute to controlling the production of detrimental cytokines such as TNF‐α.
Signal transduction pathways in oligodendrocytes: Role of tumor necrosis factor‐α
Authors: Soliven B. , Szuchet S.
Published: July 2002

We have used a combination of electrophysiological and biochemical approaches to investigate the effects and the mechanisms of action of tumor necrosis factor‐α (TNF‐α) on cultured oligodendrocytes (OLGs). Our studies have led to the following conclusions: (1) prolonged exposure of mature ovine OLGs to TNF‐α leads to inhibition of process extension, membrane depolarization and a decrease in the amplitudes of both inwardly rectifying and outward K+currents; (2) brief exposure of OLGs to TNF‐α does not elicit membrane depolarization or consistent changes in cytosolic Ca2+levels; (3) incubation of OLGs with TNF‐α for 1 hr results in inhibition of phosphorylation of myelin basic protein and 2′, 3′‐cyclic nucleotide phosphohydrolase. Ceramides, which have been shown to be effectors of TNF‐α, are ineffective in inhibiting phosphorylation, whereas sphingomyelinase mimicks TNF‐α in this action. These observations suggest that other products of sphingomyelin hydrolysis may be the mediator(s) of TNF‐α effect on protein phosphorylation. We have thus demonstrated that TNF‐α can perturb the functions of OLGs via modulation of ion channels and of protein phosphorylation without necessarily inducing cell death. It is conceivable that modulation of ion channels and protein phosphorylation constitutes effective mechanisms for the participation of cytokines in signal transduction during myelination, demyelination and remyelination.
PC12 CELL AGGREGATION AND NEURITE GROWTH IN GELS OF COLLAGEN, LAMININ AND FIBRONECTIN
Authors: Baldwin Samuel P. , Krewson Christine E. , Saltzman W. Mark
Published: July 2002

PC12 cells form aggregates when suspended within three‐dimensional, self‐assembled, type I collagen gels; these aggregates increase in size over time. In addition, when the cells are cultured in the presence of nerve growth factor, they express neurites, which extend through the three‐dimensional matrix. In this report, the roles of fibronectin, laminin and nerve growth factor in PC12 cell aggregation and neurite growth following suspension in collagen matrices were evaluated. Single cells and small clusters of cells were suspended in collagen gels; the kinetics of aggregation were determined by measurement of the projected area of each aggregate, and neurite lengths were determined by measurement of end‐to‐end distance. Fibronectin and laminin inhibited the aggregation of PC12 cells at 50 μg/ml, and fibronectin, but not laminin, inhibited the growth of neurites at 100 μg/ml. In the absence of serum, the aggregation of cells cultured with nerve growth factor was almost completely inhibited, but the average neurite length was unaffected. In the presence of nerve growth factor, the extent of cell aggregation could not be explained simply by an increase in cell number, suggesting the presence of two separate mechanisms for aggregate growth: one dependent on cell motility and another dependent on cell proliferation.
Semi‐synthetic Rab proteins as tools for studying intermolecular interactions
Authors: Iakovenko Andrei , Rostkova Elena , Merzlyak Ekaterina , Hillebrand Alexandra M. , Thomä Nicolas H. , Goody Roger S. , Alexandrov Kirill
Published: July 2002

Rab GTPases play a key role in the regulation of membrane traffic. Posttranslational geranylgeranylation is critical for their biological activity and is conferred by a Rab geranylgeranyl transferase (RabGGTase). To study the interactions between Rab proteins and RabGGTase, we used in vitro ligation methodology to generate a fluorescent semi‐synthetic Rab7 protein. The obtained protein was functionally active and was used to demonstrate a micromolar affinity interaction of Rab7 with the RabGGTase in the absence of Rab escort protein (REP). This finding is consistent with an earlier proposed model according to which RabGGTase possesses two independent weak binding sites for REP and Rab proteins.
The Antennapedia peptide penetratin translocates across lipid bilayers – the first direct observation
Authors: Thorén Per E.G. , Persson Daniel , Karlsson Mattias , Nordén Bengt
Published: July 2002

The potential use of polypeptides and oligonucleotides for therapeutical purposes has been questioned because of their inherently poor cellular uptake. However, the 16‐mer oligopeptide penetratin, derived from the homeodomain of Antennapedia, has been reported to enter cells readily via a non‐endocytotic and receptor‐ and transporter‐independent pathway, even when conjugated to large hydrophilic molecules. We here present the first study where penetratin is shown to traverse a pure lipid bilayer. The results support the idea that the uptake mechanism involves only the interaction of the peptide with the membrane lipids. Furthermore, we conclude that the translocation does not involve pore formation.
Interaction between β‐amyloid and lens αB‐crystallin
Authors: Liang Jack J.-N
Published: July 2002

In Alzheimer's disease, β‐amyloid peptides (βA1–40 and βA1–42) are deposited on the brain cell surfaces as neurotoxic plaques. Some reports indicate that small heat shock proteins, Hsp27 and αB‐crystallin, colocalize in the plaques, but their functions are not known. Interaction between βA and αB‐crystallin must be determined in order to understand the role of αB‐crystallin in βA fibril formation. We used a pyrene (Pyr)‐labeled βA1–40 in a fluorescence energy transfer experiment. Upon incubation together at 37°C, energy transfer between Trp of αB‐crystallin and Pyr of Pyr‐labeled βA was observed, indicating that βA participated in subunit exchange of αB‐crystallin, which promoted fibril formation.
An NMR‐based metabonomic approach to investigate the biochemical consequences of genetic strain differences: application to the C57BL10J and Alpk:ApfCD mouse
Authors: Gavaghan Claire L , Holmes Elaine , Lenz Eva , Wilson Ian D , Nicholson Jeremy K
Published: July 2002

As the human genome sequencing projects near completion, there is an active search for technologies that can provide insights into the genetic basis for physiological variation and interpreting gene expression in terms of phenotype at the whole organism level in order to understand the pathophysiology of disease. We present a novel metabonomic approach to the investigation of genetic influences on metabolic balance and metabolite excretion patterns in two phenotypically normal mouse models (C57BL10J and Alpk:ApfCD). Chemometric techniques were applied to optimise recovery of biochemical information from complex 1H NMR urine spectra and to determine metabolic biomarker differences between the two strains. Differences were observed in tricarboxylic acid cycle intermediates and methylamine pathway activity. We suggest here a new ‘metabotype’ concept, which will be of value in relating quantitative physiological and biochemical data to both phenotypic and genetic variation in animals and man.