bluuebunny/crossref_metadata_2025 huggingface dataset:
Modification of the electron‐transfer sites of Pseudomonas aeruginosa azurin by site‐directed mutagenesis
Authors: Pascher Torbjörn , Bergström Jörgen , Malmström Bo G. , Vänngård Tore , Lundberg Lennart G.
Published: July 2002

Site‐directed mutagenesis of the structural gene for azurin from Pseudomonas aeruginosa has been used to prepare azurins in which amino acid residues in two separate electron‐transfer sites have been changed: His‐35‐Lys and Glu‐91‐Gln at one site and Phe‐114‐Ala at the other. The chargetransfer band and the EPR spectrum are the same as in the wild‐type protein in the first two mutants, whereas in the Phe‐114‐Ala azurin, the optical band is shifted downwards by 7 nm and the copper hyperfine splitting is decreased by 4·10−4/cm. This protein also shows an increase of 20–40 mV in the reduction potential compared to the other azurins. The potentials of all four azurins decrease with increasing pH in phosphate but not in zwitterionic buffers with high ionic strength. The rate constant for electron exchange with cytochrome c
551 is unchanged compared to the wild‐type protein in the Phe‐114‐Ala azurin, but is increased in the other two mutant proteins. The results suggest that Glu‐91 is not important for the interaction with cytochrome c
551 and that His‐35 plays no critical role in the electron transfer to the copper site.
Addition of ATP increases the apparent molecular mass of the multicatalytic proteinase, ingensin
Authors: Ishiura Shoichi , Nomura Yasuhiro , Tsukahara Toshifumi , Sugita Hideo
Published: July 2002

Multicatalytic proteinase; Isoenzyme; ATP; (Rat liver)
Agonist binding to purified glycine receptor reconstituted into giant liposomes elicits two types of chloride currents
Authors: Riquelme Gloria , Morato Esperanza , López Elena , Ruiz-Gómez Ana , Ferragut José A. , González Ros José M. , Mayor Frederico
Published: July 2002

Using ‘inside‐out’ membrane patches obtained from reconstituted giant liposomes containing purified glycine receptor from rat spinal cord, we have detected chloride currents elicited in response to the presence of the agonists glycine or β‐alanine. Regardless of the agonist employed, two different patterns of single channel currents could be detected, which differ in their main conductance, complexity of substates and opening frequency. In agreement with the expectations of glycine receptor heterogeneity suggested recently at the mRNA and cDNA level, our results indicate the existence of functionally different glycine receptors in the adult rat spinal cord.
A semi‐empirical method for prediction of antigenic determinants on protein antigens
Authors: Kolaskar A.S. , Tongaonkar Prasad C.
Published: July 2002

Analysis of data from experimentally determined antigenic sites on proteins has revealed that the hydrophobic residues Cys, Leu and Val, if they occur on the surface of a protein, are more likely to be a part of antigenic sites. A semi‐empirical method which makes use of physicochemical properties of amino acid residues and their frequencies of occurrence in experimentally known segmental epitopes was developed to predict antigenic determinants on proteins. Application of this method to a large number of proteins has shown that our method can predict antigenic determinants with about 75% accuracy which is better than most of the known methods. This method is based on a single parameter and thus very simple to use.
Interactions between Salmonella typhimurium lipopolysaccharide and the antimicrobial peptide, magainin 2 amide
Authors: Rana Fazale R. , Sultany Catherine M. , Blazyk Jack
Published: July 2002

Effects of magainin 2 amide on the phase behavior of Salmonella typhimurium lipopolysaccharide were characterized by FT‐IR spectroscopy. This antimicrobial cationic peptide disorders the lipopolysaccharide at molecular ratios of lipopolysaccharide to magainin greater than 4, and can induce a temperature‐dependent structural reorientation. The nature of the five phosphate groups of lipopolysaccharide was determined by 31P NMR spectroscopy. At pH 7.4, the net charge on the phosphates is −7. Lipopolysaccharide undoubtedly plays an important role in modulating the interactions of magainin with the gram‐negative cell envelope and may act as a molecular sponge to protect the plasma membrane.
Structure of the DNA binding wing of the gene‐V encoded single‐stranded DNA binding protein of the filamentous bacteriophage M13
Authors: van Duynhoven J.P.M. , Folkers P.J.M. , Stassen A.P.M. , Harmsen B.J.M. , Konings R.N.H. , Hilbers C.W.
Published: July 2002

The structure in solution of a β‐loop in mutant Y41H of the single‐stranded DNA binding protein encoded by gene‐V of the filamentous phage M13 has been elucidated using 2‐dimensional 1H‐nuclear magnetic resonance techniques. Furthermore, these studies enabled us to demonstrate that an identical structural element is present in wild‐type gene‐V‐protein and that this element intimately is involved in the binding of gene‐V‐protein to single‐stranded DNA. It is shown that the structure of the DNA binding wing deviates from that proposed for the same amino acid sequence on the basis of X‐ray diffraction data. The structure is, however, identical to that of the DNA binding wing present in the single‐stranded DNA binding protein encoded by the genome of the evolutionary distantly related filamentous phage IKe. The latter observations support our current view that in the binding of these proteins to single‐stranded DNA a common structural motif is involved.
Interferon‐γ activates the cleavage of double‐stranded RNA by bovine seminal ribonuclease
Authors: Schein Catherine H. , Haugg Monika , Benner Steven A.
Published: July 2002

Bovine seminal ribonuclease (BS‐RNase), a dimeric homologue of RNase A, cleaves both single‐ and double‐stranded RNA and inhibits the growth of tumor cells. Its catalytic activity against double‐stranded RNA, either homopolymeric ([3H]polyA/polyU) or mixed sequence, is enhanced by bovine or human recombinant interferon‐γ (IFN‐γ). Activation is seen with as little as 4–10 interferon units per assay. Enhancing the degradation of double‐stranded RNA, an intermediate in the growth cycle of many viruses, could contribute to IFN‐γ's ability to control cell growth and induce an antiviral state.
Prokaryotic 20β‐hydroxysteroid dehydrogenase is an enzyme of the ‘short‐chain, non‐metalloenzyme’ alcohol dehydrogenase type
Authors: Marekov Lyuben , Krook Maria , Jörnvall Hans
Published: July 2002

The primary structure of 20β‐hydroxysteroid dehydrogenase from Streptomyces hydrogenans was determined after FPLC purification of a commercial preparation. Peptides obtained from different proteolytic cleavages were purified by reverse phase HPLC. The 255‐residue structure deduced was found to be distantly homologous to those of Drosophila alcohol dehydrogenase and several other dehydrogenases, establishing that prokaryotic 20β‐hydroxysteroid dehydrogenase as a member of the ‘short‐chain alcohol dehydrogenase family’. With the enzymes characterized, the identity is greatest (31–34%) towards 4 other prokaryotic dehydrogenases, but the family also includes mammalian steroid and prostaglandin dehydrogenases. These enzymes are low in Cys and have a strictly conserved Tyr residue that appears to be important.
Zn2+‐induced deprotonation of a peptide nitrogen in angiotensin I
Authors: Arnold Alan P. , Stanley Darren M. , Collins J.Grant
Published: July 2002

The interaction of Zn2+ with angiotensin I, a decapeptide containing two histidyl residues, has been studied by 1H‐NMR spectroscopy in both water and dimethylsulfoxide. When Zn2+ is added to the peptide in dimethylsulfoxide, binding occurs by coordination of the imidazole rings of both histidines to the metal‐ion, enabling the deprotonation of the Phe peptide nitrogen.
Melatonin binding proteins identified in the rat brain by affinity labeling
Authors: Laudon Moshe , Zisapel Nava
Published: July 2002

N‐Bromoacetyl‐2‐iodo‐5‐methoxytryptamine (BIM), a novel derivative of the biologically active melatonin analog, 2‐iodomelatonin, was prepared and used to identify melatonin binding proteins in rat brain synaptosomes. Incubation of the synaptosomes with BIM resulted in a time and concentration dependent, irreversible inhibition of 2‐[125I]iodomelatonin binding. In parallel, the radioactive form of BIM, N‐bromoacetyl‐2‐[125I]iodo‐5‐methoxytryptamine [125I]BIM) became incorporated into the synaptosomes. The incorporation of [125I]BIM was inhibited by BIM, 2‐iodomelatonin and melatonin but not by 5‐methoxytryptamine or N‐acetyl serotonin. [125I]BIM became covalently attached to three polypeptides with apparent molecular weight values of 92, 55 and 45 kDa; the labeling of all three proteins was markedly inhibited by melatonin. These results indicate that the 92, 55 and 45 kDa polypeptides are melatonin binding proteins.
ADP/ATP translocator is essential only for anaerobic growth of yeast Saccharomyces cerevisiae
Authors: Drgoň T. , Šabová L. , Nelson N. , Kolarov J.
Published: July 2002

All three genes (AAC1,AAC2 and AAC3) encoding the mitochondrial ADP/ATP translocator, were inactivated in a haploid yeast strain by a gene disruption technique. The triple mutant was still able to grow on fermentable carbon sources but only in the presence of oxygen. Under aerobic conditions neither translocator‐protein nor carrier‐mediated transport was detected in all mutants in which the AAC2 gene was disrupted. It was further shown that a functional AAC genes product is essential only for anaerobic growth of Saccharomyces cerevisiae but not for growth under derepressed conditions. Under anaerobic conditions a non‐detectable amount of AAC3 gene product is sufficient to ensure the cell growth and multiplication.
The amino acid sequence of AQN‐3, a carbohydrate‐binding protein isolated from boar sperm Location of disulphide bridges
Authors: Sanz Libia , Calvete Juan J. , Mann Karlheinz , Schäfer Wolfram , Schmid Erich R. , Töpfer-Petersen Edda
Published: July 2002

Gamete recognition and adhesion are essential steps in fertilization. Among others, carbohydrate‐binding proteins on the sperm surface have been recognized to play a central role in the initial interaction of the male gamete with components of the zona pellucida of the homologous investing oocyte. We have isolated several members of a carbohydrate‐ and zona pellucida‐binding protein family from ejaculated sperm. Here we report the biological origin and structural characterization of AQN‐3, a component of this carbohydrate‐binding family. The molecular weight of purified AQN‐3 was determined by plasma desorption mass spectrometry. The protein was chemically and enzymatically degraded, the proteolytic fragments isolated and characterized by N‐terminal sequencing and fast atom bombardment mass spectrometry. In this manner we established the complete amino acid sequence of AQN‐3 and the location of its two disulphide bonds. No analogous protein sequence could be found in the MIPS protein sequence data bank, indicating that AQN‐3 may belong to a novel mammalian carbohydrate‐binding protein family.
Regions in the promoter of the yeast FBP1 gene implicated in catabolite repression may bind the product of the regulatory gene MIG1
Authors: Mercado Juan J. , Vincent Olivier , Gancedo Juana M.
Published: July 2002

We have identified in the promoter of the yeast FBP1 gene two sites able to bind nuclear proteins. These sites have a nucleotide sequence strongly similar to that of sites which bind the regulatory protein MIG1 in the promoters of GAL4 and SUC2. Deletions performed in the FBP1 promoter showed that one of the sites contributes to catabolite repression of this gene. In the same promoter, another region was identified with a strong effect on the catabolite repression of FBP1. In this region a sequence similar to the consensus for the binding site of the MIG1 protein was also present.
Identification of the active‐site serine in human pancreatic lipase by chemical modification with tetrahydrolipstatin
Authors: Lüthi-Peng Qiaoqian , Märki Hans Peter , Hadváry Paul
Published: July 2002

A chemical modification approach was used in this study to identify the active site serine residue of human pancreatic lipase. Purified human pancreatic lipase was covalently modified by incubation with [3H], [14C]tetrahydrolipstatin (THL), a potent inhibitor of pancreatic lipase. The radiolabeled lipase was digested with thermolysin, and the peptides were separated by HPLC. A single THL‐peptide‐adduct was obtained which was identical to that obtained earlier from porcine pancreatic lipase. This pentapeptide with the sequence VIGHS is covalently bound to a THL molecule via the side chain hydroxyl group of the serine unit corresponding to Ser‐152 of the lipase. The selective cleavage of the THL‐serine bond by mild acid treatment resulted in the formation of the δ‐lactone Ro 40–444] in high yield and clearly proves that THL is attached via an ester bond and with retention of stereochemistry at all chiral centers to the side chain hydroxyl group of Ser‐152 of the lipase. The results obtained for human pancreatic lipase corroborate the inhibition mechanism of THL found on the porcine enzyme, and are in full agreement with the identification of the Ser‐152…His‐263…Asp‐176 catalytic triad in the X‐ray structure of human pancreatic lipase.
A signal peptide with a proline next to the cleavage site inhibits leader peptidase when present in a sec‐independent protein
Authors: Nilsson IngMarie , von Heijne Gunnar
Published: July 2002

Proline residues are rarely found in the three most C‐terminal positions of bacterial signal peptides, and have never been found in position +1 immediately following the cleavage site. It was recently shown that a Pro+1 mutation in the E. coli maltose binding protein precursor not only prevents cleavage of the signal peptide but also inhibits the leader peptidase enzyme, resulting in cessation of cell growth (Barkocy‐Gallagher, G.A. and Bassford, P.J. (1992) J. Biol. Chem. (in press)). Since maltose binding protein is dependent on the sec machinery for translocation across the inner membrane, it was not clear if this ‘Pro+1’ effect was restricted to sec‐dependent proteins, or whether it applies also to proteins that do not require the sec functions for translocation. We now present data suggesting that the striking phenotypic effects of Pro+1 mutations can be elicited also by sec‐independent proteins.
Functional role of TTF‐1 binding sites in bovine thyroglobulin promoter
Authors: Javaux Françoise , Bertaux Fabien , Donda Alena , Francis-Lang Helen , Vassart Gilbert , DiLauro Roberto , Christophe Daniel
Published: July 2002

We have studied the binding of purified TTF‐1 on the bovine thyroglobulin gene promoter. DNase I footprinting experiments revealed three binding sites which corresponded in location to the A, B and C sites found in the rat thyroglobulin promoter. Mutants in the A and C regions showing reduced binding of TTF‐1, also exhibited largely decreased promoter activity in transient expression experiments in primary‐cultured dog thyrocytes. Two mutants in the B site that exhibited a reduced capacity to bind TTF‐1 also displayed a drastically affected transcriptional activity in transient assays. As in the rat, sites A and C only are critical for promoter activity, these results suggest that full occupancy of the B site is required for thyroglobulin promoter activity in the cow only.
Ankyrin‐like proteins of variola and vaccinia viruses
Authors: Shchelkunov Sergei N. , Blinov Vladimir M. , Sandakhchiev Lev S.
Published: July 2002

Computer analysis of full coding sequences of variola major virus strain India‐1967 genome and vaccinia virus strain Copenhagen genome have been carried out. A wide set of proteins containing ankyrin‐like repeats have been identified for both viruses. Only three proteins of this family of the studied viruses are highly homologous. The rest of the proteins are different. The possible role of such proteins in determination of virus tissue tropism is discussed.
Two‐dimensional 1H NMR spectra of ferricytochrome c551 from Pseudomonas aeruginosa
Authors: Moratal José María , Donaire Antonio , Salgado Jesús , Jiménez Hernias R. , Castells Josep , Piccioli Mario
Published: July 2002

The full assignment of 1H NMR signals of heme proton resonances of ferricytochrome c
551 from Pseudomonas aeruginosa has been performed by means of 2D NMR experiments. This technique allows the complete and unequivocal assignment of all heme resonances, including methylene resonances of the propionic groups, directly implicated in the pH dependence of the redox properties of cytochrome c
551.
Association of annexin V with mitochondria
Authors: Sun Jiuru , Bird Catherina H. , Salem Hatem H. , Bird Phillip
Published: July 2002

Annexin V is an intracellular protein recently shown to be localized to nucleoli and cytosol. In this study we show that cytosolic annexin V is associated with mitochondria. To assess the nature of the annexin V‐mitochondrial interaction, an annexin V binding activity was partially purified from placental cytosol by annexin V‐affinity chromatography. Five polypeptides in the eluate appeared to be associated with annexin V, with a predominant species of 27 kDa. Antibodies to the 27 kDa polypeptide recognised mitochondria but not nucleoli. We conclude that annexin V interacts with a 27 kDa nutochondrial polypeptide that is possibly part of a larger complex.
Petunia p34cdc2 protein kinase activity in G2/M cells obtained with a reversible cell cycle inhibitor, mimosine
Authors: Perennes Claudette , Qin Li-Xian , Glab Nathalie , Bergounioux Catherine
Published: July 2002

Protoplasts isolated from petunia leaf mesophyll are non‐cycling cells mostly with 2C content. Cells regenerating from protoplast culture enter mitosis after 48 h. This experimental model is used to relate p34cd2 kinase activity to cell cycle phase. Our results show that the histone Hl phosphorylation, and hence p34cdc2 kinase activity, peaks with G2+ early M cell cycle phase. However, a trace kinase activity was already present when most cells were entering S phase. To obtain a maximum of cells in G1+S phases, the protoplast culture was treated with the rare amino acid, mimosine. Mimosine blocked plant cells derived from protoplast culture both at g1 and in early and mid S phase. Despite the increased G1+S level, p34cdc2 kinase activity did not increase. This suggests that the trace activity appearing when the majority of cells are entering S does not correspond to any putative P34cdc2 activation at G1/S transition but to the activation of the minor 4C population initially present in the leaf: the hypothesis remains that p34cdc2 kinase activity is solely related to G2+M phase in petunia.
Selective cleavage of 28S rRNA variable regions V3 and V13 in myeloid leukemia cell apoptosis
Authors: Houge Gunnar , Døskeland Stein O. , Bøe Roald , Lanotte Michel
Published: July 2002

Vigorous apoptosis is induced 3–4 hours after activation of cAMP‐dependent protein kinase I in the rat myeloid leukemia cell line IPC‐81 [J. Cell. Physiol. 146 (1991) 73‐80]. We will report a novel feature of apoptosis in these cells: a selective and temporarily ordered cleavage within the two largest 28S ribosomal RNA variable regions (V3 and V13). The cleavage of 28S rRNA coincided with internucleosomal DNA fragmentation and cessation of cellular protein synthesis. The implication of 28S variable regions as targets in apoptosis is a clue to the function of these so far apparently superfluous parts of eukaryotic ribosomes.
The effect of annexin IV and VI on the fluidity of phosphatidylserine/phosphatidylcholine bilayers studied with the use of 5‐deoxylstearate spin label
Authors: Sobota Andrzej , Bandorowicz Joanna , Jezierski Adam , Sikorski Aleksander F.
Published: July 2002

An effect of annexin IV and VI on the fluidity of phosphatidylserine/phosphatidylcholine (PS/PC) membranes was studied by spin labeling technique with the use of 5‐doxylstearic acid. It was found that calcium ions at micromolar concentrations induced a marked decrease in the order parameter of PS/PC membranes. This effect was completely abolished by both annexins. The influence of annexins on the mobility of fatty acid chains in the studied region in PE/PC membranes was insignificant.
Cyclosporin A suppression of uncoupling in liver mitochondria of ground squirrel during arousal from hibernation
Authors: Brustovetsky N.N. , Egorova M.V. , Gnutov D.Yu. , Mokhova E.N. , Skulachev V.P.
Published: July 2002

Energy coupling parameters were studied in liver mitochondria of ground squirrel during arousal from hibernation. It was found that such mitochondria become uncoupled during incubation with phosphate in a salt medium. The uncoupling was revealed by respiration rate increase and membrane potential decrease in the presence of oligomycin. Both effects were reversed by addition of cyclosporin A. Under the same in vitro conditions, mitochondria from aroused (active) animals showed no uncoupling but could be uncoupled by addition of palmitate in the cyclosporin A‐sensitive fashion. It is proposed that formation of cyclosporin A‐sensitive pores can be involved in urgent heat production in arousing hibernators.
Nucleoside analogues previously found to be inactive against HIV may be activated by simple chemical phosphorylation
Authors: McGuigan Christopher , Kinchington Derek , Wang Meng Fang , Nicholls Simon R. , Nickson Caleb , Galpin Sarah , Jeffries Donald J. , O'Connor Timothy J.
Published: July 2002

Nucleoside analogues previously found to be inactive against the human immunodeficiency virus (HIV) may be activated by simple chemical derivatisation. As part of our effort to deliver masked phosphates inside living cells we have discovered that certain phosphate triester derivatives of inactive nucleoside analogues become inhibitors of HIV replication. This discovery underlies the importance of the masked phosphate approach, and has significant implications for the future design of chemotherapeutic nucleoside analogues. If highly modified nucleoside analogues may be active without the intervention of nucleoside kinase enzymes, major advantage may accrue in terms of low toxicity and enhanced selectivity. Moreover, the increased structural freedom may have implications for dealing with the emergence of resistance. The concept herein described as ‘kinase bypass’ may thus stimulate the discovery of a new generation of antiviral agents.
Expression of synaptotagmin and syntaxin associated with N‐type calcium channels in small cell lung cancer
Authors: David Pascale , El Far Oussama , Martin-Mouto Nicole , Poupon Marie France , Takahashi Masami , Seagar Michael J.
Published: July 2002

The presence of synaptic proteins involved in excitation/secretion coupling was examined in ten small cell lung cancer lines. N‐Type calcium channels (ω)‐conotoxin receptors), synaptotagmin (p65) and syntaxin (HPC‐1) were detected in eight. Co‐immunoprecipitation experiments indicated that syntaxin can form a complex with synaptotagmin and calcium channels. The expression of synaptotagmin in small cell lung cancer may elicit an autoimmune response that reduces transmitter release at the nerve terminal.
Characterization of the paramagnetic iron‐containing redox centres of Thiosphaera pantotropha periplasmic nitrate reductase
Authors: Breton Jacques , Berks Ben C. , Reilly Ann , Thomson Andrew J. , Ferguson Stuart J. , Richardson David J.
Published: July 2002

Electron paramagnetic resonance spectroscopy signals attributable to low‐spin haem c in the oxidised protein and [4Fe–4S]1+ in the dithionite‐reduced protein were identified, at low temperature, in Thiosphaera pantotropha periplasmic nitrate reductase. Spin integration of these signals as well as elemental analysis suggest a stoichiometry of 1.3–1.6 c‐haem and 1 [4Fe–4S] cluster per enzyme molecule. The E
m (at pH 7.4) of the [4F–4S]2+,1+ couple, −160 mV, means that it is unlikely to be physiologically reducible. Peptide sequences from the 90 kDa subunit indicate that the enzyme is a member of the family of molybdopterin guanine dinucleotide‐binding polypeptides, the majority of which possess a putative [4Fe–4S] cluster binding sequence and thus may also bind a (low potential) iron—sulphur cluster.
Calorimetric studies of the energetics of protein—DNA interactions in the E. coli methionine repressor (MetJ) system
Authors: Cooper Alan , McAlpine Alan , Stockley Peter G.
Published: July 2002

Calorimetric measurements of binding of a specific DNA fragment and S‐adenosyl methionine (SAM) co‐repressor molecules to the E. coli methionine repressor (MetJ) show significant differences in the energetics of binary and ternary protein‐DNA complexes. Formation of the MetJ: SAM: DNA ternary complex is significantly more exothermic (ΔH ⋍ −99 kJ·mol−1) than either MetJ:DNA or MetJ:SAM binary complexes alone (ΔH ⋍ −10 kJ·mol−1 each). The protein is also significantly more stable to unfolding (ΔT
m ⋍ 5.4°C) when bound to DNA. These observations suggest that binding of SAM to the protein‐DNA complex leads to a significant reduction in dynamic flexibility of the ternary complex, with considerable entropy‐enthalpy compensation, not necessarily involving any overall conformational change.
PHF‐tau from Alzheimer's brain comprises four species on SDS‐PAGE which can be mimicked by in vitro phosphorylation of human brain tau by glycogen synthase kinase‐3β
Authors: Mulot S.F.C. , Hughes K. , Woodgett J.R. , Anderton B.H. , Hanger D.P.
Published: July 2002

Extensive in vitro phosphorylation of a purified preparation of control human brain tau consistently produces four rather than, as previously believed, three tau species on SDS‐PAGE. The species thus generated are shifted on SDS‐PAGE to positions that match those of PHF‐tau isolated from Alzheimer's disease brain. A mixture of recombinant human tau isoforms phosphorylated by GSK‐3β gave similar results to those obtained with control human brain tau. In vitro phosphorylation of the individual recombinant isoforms by GSK‐3β showed that the four bands of PHF‐tau are likely to consist of isoforms 3R,0 alone; 4R,0 with 3R,29; 4R,29 with 3R,58 and 4R,58 alone.
Levels of normal and abnormally phosphorylated tau in different cellular and regional compartments of Alzheimer disease and control brains
Authors: Khatoon Sabiha , Grundke-Iqbal Inge , Iqbal Khalid
Published: July 2002

Microtubule associated protein tau is abnormally phosphorylated in Alzheimer disease (AD) brain. In the present study we investigated (i) whether tau is axonal or both axonal and somatodendritic, (ii) whether tau is a marker of Alzheimer neurofibrillary pathology, and (iii) whether the levels of tau in the cytosol (100,000 × g supernate) from AD brain are altered. Frozen autopsied tissue from 20 AD, 17 normal aged and 15 neurological control cases obtained 3–8 h postmortem were analyzed. Levels of normal, total, and abnormally phosphorylated tau were determined by a radioimmunoslot‐blot assay using mAb Tau‐1 as the primary antibody. Both frontal gray matter homogenate and cytosol from normal brains had 30–45% higher levels of normal tau than the corresponding fractions from the white matter. In AD frontal and temporal cortices, the total tau levels were 6‐ to 7‐fold higher than in cerebellar cortex (P < 0.01 and P < 0.02). Furthermore, tau levels of cerebellar cortex, an area of the brain unaffected with Alzheimer neurofibrillary changes, were indistinguishable between AD and control groups. The levels of normal tau in cytosol from both frontal gray and white matters in AD were reduced by approximately 40% (P < 0.05). The levels of total tau in AD frontal and temporal cortex were 4‐to 5‐fold higher than in the corresponding tissue from control cases (P < 0.01) and this increase was in the form of abnormally phosphorylated tau. These studies suggest (i) that there is probably at least as much tau in the somatodendritic compartment as in the axonal compartment, (ii) that the abnormally phosphorylated tau is a biochemical marker of the neurofibrillary pathology in AD, and (iii) that the levels of normal tau are significantly reduced in the 100,000 × g brain supernate from AD cases.
Yta10p, a member of a novel ATPase family in yeast, is essential for mitochondrial function
Authors: Tauer Reimund , Mannhaupt Gertrud , Schnall Ralf , Pajic Alexander , Langer Thomas , Feldmann Horst
Published: July 2002

The yeast gene, YTA10, encodes a member of a novel family of putative ATPases. Yta10p, as deduced from the nucleotide sequence, is 761 amino acids in length (predicted molecular mass 84.5 kDa). The amino acid sequence of Yta10p exhibits high similarity to two other yeast proteins, Yta11 and Yta12, and to E coli FtsH. Several features of Yta10p are compatible with its localization in mitochondria. We report here that Yta10p is a yeast mitochondrial protein and that import is dependent on a membrane potential and accompanied by processing to a protein of approximately 73 kDa. Disruption of YTA10 leads to a nuclear petite phenotype and to a loss of respiratory competence, as shown by spectrophotometric measurement of the activities of respiratory complexes I–III and IV, respectively. These findings together with the high similarity of Yta10p to several ATP‐dependent proteases suggest that Yta10p is a mitochondrial component involved, directly or indirectly, in the correct assembly and/or maintenance of active respiratory complexes.
Identification of a fatty acyl responsive regulator (FarR) in Escherichia coli
Authors: Quail Michael A. , Dempsey Clare E. , Guest John R.
Published: July 2002

FarR (formerly P30) has been identified as a fatty acid and fatty acyl‐CoA responsive DNA‐binding protein. It is encoded by the farR gene (g30) in the citric acid cycle gene cluster of E. coli (gltA‐sdhCDAB‐sucABCD‐farR). The amplified FarR protein specifically bound to the farR promoter (PfarR
) and exhibited weak binding to the citrate synthase and lipoamide dehydrogenase promoters. Binding at PfarR
was abolished by long‐chain fatty acids and their CoA thioesters. In DNaseI footprints, FarR binding at PfaR
protected two sites each characterised by two related 10‐bp direct repeats. It is suggested that farR autoregulates farR expression and may modulate citric acid cycle expression in response to long‐chain fatty acids.
The molten globule is a third thermodynamical state of protein molecules
Authors: Ptitsyn O.B , Uversky V.N
Published: July 2002

Analysis of published data on conformational transitions in relatively small proteins shows that the slopes of these transitions are proportional to the protein molecular weight. It is true both for transitions from the native (N) to the unfolded (U) states (when protein denaturation is coupled to its unfolding) and for transitions from the native to the molten globule (MG) states and from the molten globule to the unfolded state (when protein denaturation is decoupled from protein unfolding). This is precisely the behaviour predicted by thermodynamics for first order phase transitions (‘all‐or‐none’ transitions) in small systems. It follows that N → U, N → MG and MG → U transitions in proteins are all of the ‘all‐or‐none’ type. Thus the molten globule state of protein molecules is separated by an ‘all‐or‐none’ transition both from the native and the unfolded state, i.e. the molten globule state is a third thermodynamic state of protein molecules in addition to the two previously established states ‐ the native and the unfolded.
Ultrafast chlorophyllb‐chlorophyllaexcitation energy transfer in the isolated light harvesting complex, LHC II, of green plants
Authors: Pålsson Lars Olof , Spangfort Michael D. , Gulbinas Vidmantas , Gillbro Tomas
Published: July 2002

The excitation energy transfer between chlorophyllb(Chlb) and chlorophylla(Chla) in the isolated trimeric chlorophyll‐a/b‐binding protein complex of spinach photosystem 2 (LHC II) has been studied by femtosecond spectroscopy. In the main absorption band of Chlbthe ground state recovery consists of two components of 0.5 ps and 2.0 ps, respectively. Also in the Chlaabsorption band, at 665 nm, the ground state recovery is essentially bi‐exponential. In this case is, however, the fastest relaxation lifetime is a 2.0 ps component followed by a slower component with a lifetime in the order of 10–20 ps. In the Chlbabsorption band a more or less constant anisotropy ofr= 0.2 was observed during the 3 ps the system was monitored. In the Chlaabsorption band there was, however, a relaxation of the anisiotropy fromr= 0.3 to a quasi steady state level ofr= 0.18 in about 1 ps. Since the 0.5 ps component is only seen upon selective excitation of Chlbwe assign this component to the energy transfer between Chlband Chla. The other components most likely represents redistribution processes of energy among spectrally different forms of Chla. The energy transfer process between Chlband Chlacan well be explained by the Förster mechanism which also gives a calculated distance of 13 Å between interacting chromophores. The organisation of chlorophylls in LHC II is discussed in view of the recent crystal structure data (1991) Nature 350, 130].
Induction of aromatase in stromal vascular cells from human breast adipose tissue depends on cortisol and growth factors
Authors: Schmidt Martin , Löffler Georg
Published: July 2002

The regulation of aromatase (estrogen synthase) activity of cultured stromal vascular cells from human breast adipose tissue by cortisol, db‐cAMP and growth factors was studied m a serum‐free culture system. While PDGF‐BB alone inhibited the effect of db‐cAMP on aromatase induction, it stimulated aromatase activity in the presence of cortisol with or without db‐cAMP. In the presence of 1μM insulin consistently higher aromatase activities were found as compared to 1 nM insulin. In contrast to PDGF‐BB, bFGF led to an increase of aromatase activity only in the presence of both cortisol and db‐cAMP.
Two different respiratory Rieske proteins are expressed in the extreme thermoacidophilic crenarchaeon Sulfolobus acidocaldarius: cloning and sequencing of their genes
Authors: Schmidt C.L. , Anemüller S. , Schäfer G.
Published: July 2002

We have isolated two genes encoding Rieske iron sulfur proteins from the genomic DNA of the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius (DSM 639). One of the genes, named soxL, codes for the previously isolated novel Rieske‐I protein [1]. The second gene (soxF) [2] codes for the Rieske‐II protein associated with the second terminal oxidase of Sulfolobus [3]. Both proteins exhibit only 24% identical residues. The Rieske‐I protein shows a number of unusual features. (i) The distance between the two cluster binding sites is significantly larger than in all known proteins. (ii) An unexpected Pro → Asp exchange in one of the cluster binding sites. (iii) It shows some resemblance to the mitochondrial and plastidic Rieske proteins insofar as the soxL gene codes for a pre‐sequence which is no longer present in the mature Rieske‐I protein. Both proteins cluster together on a separate branch of the phylogenetic tree. To our knowledge this is the first proven case of two significantly different Rieske proteins in a prokaryote.
Two Sox9 messenger RNA isoforms: isolation of cDNAs and their expression during gonadal development in the frog Rana rugosa
Authors: Takase Minoru , Noguchi Satoru , Nakamura Masahisa
Published: July 2002

Sox is a family of SRY‐related testis‐determining genes. We have isolated two different mRNA isoforms of the frog Sox9 gene from adult frog testis cDNAs. One form (Sox9 α) encodes a 482 amino acid protein containing the HMG box, whereas the other form (Sox9 β), which completely lacks the HMG box, is a truncated 265 amino acid protein of Sox9 α. Sox9 α is 82% similar to mouse, 86% to chicken, and 77% to trout Sox9 at the amino acid level. Sox9 expression was up‐regulated in embryos after stage 16, and was seen in both developing testes and ovaries. The size of Sox9 transcripts was determined to be 7.8 knt by Northern blot analysis. In addition, Sox9 α expression was found prominently in the testis and brain among various tissues of adult frogs examined, and was considerably higher than Sox9 β. The fact that Sox9 is expressed in both sexes suggests that this gene is involved in gonadal development of male and female frogs. This is dissimilar to the pattern in birds and mammals, in which Sox9 expression is male‐specific.
The salicylate metabolite gentisic acid, but not the parent drug, inhibits glucose autoxidation‐mediated atherogenic modification of low density lipoprotein
Authors: Exner Markus , Hermann Marcela , Hofbauer Roland , Kapiotis Stylianos , Speiser Wolfgang , Held Irmtraud , Seelos Christian , Gmeiner Bernhard M.K
Published: July 2002

Oxidation of low density lipoprotein (LDL) by glucose‐derived radicals may play a role in the aetiology of atherosclerosis in diabetes. Salicylate was shown to scavenge certain radicals. In the present study, aspirin, salicylate and its metabolites 2,5‐ and 2,3‐dihydroxybenzoic acid (DHBA) were tested for their ability to impair LDL oxidation by glucose. Only the DHBA derivatives, when present during LDL modification, inhibited LDL oxidation and the increase in endothelial tissue factor synthesis induced by glucose oxidised LDL. The LDL glycation reaction was not affected by DHBA. The antioxidative action of DHBA may be attributed to free radical scavenging and/or chelation of transition metal ions catalysing glucose autoxidation.
Covalently bound flavin in the NqrB and NqrC subunits of Na+‐translocating NADH‐quinone reductase from Vibrio alginolyticus
Authors: Nakayama Yuji , Yasui Mikako , Sugahara Kazuya , Hayashi Maki , Unemoto Tsutomu
Published: July 2002

Na+‐translocating NADH‐quinone reductase (NQR) from the marine bacterium Vibrio alginolyticus is composed of six subunits (NqrA to NqrF). On SDS–PAGE of the purified complex, NqrB and NqrC subunits were found to give yellow–green fluorescent bands under UV illumination. Both the NqrB and NqrC, electroeluted from the gel, had an absorption maximum at 448 nm, and the fluorescence excitation maxima at 365 and 448 nm and the emission maximum at 514 nm. The electroeluted NqrB and NqrC, respectively, were identified from their N‐terminal amino acid sequences. These results clearly indicated that the NqrB and NqrC subunits have covalently bound flavins. The two subunits were digested by protease and then the fluorescent peptide fragments were separated by a reversed‐phase high performance liquid chromatography. N‐Terminal amino acid sequence analyses of the fluorescent peptides revealed that the flavin is linked to Thr‐235 in the NqrB and Thr‐223 in the NqrC subunits. This is the first example that the flavin is linked to a threonine residue. The amino acid sequence around the flavin‐linked threonine was well conserved between NqrB and NqrC. Identification of the flavin group is in progress.
Identification of organic anion transporting polypeptide 4 (Oatp4) as a major full‐length isoform of the liver‐specific transporter‐1 (rlst‐1) in rat liver
Authors: Cattori Valentino , Hagenbuch Bruno , Hagenbuch Niels , Stieger Bruno , Ha Riem , Winterhalter Kaspar E. , Meier Peter J.
Published: July 2002

A novel organic anion transporting polypeptide (Oatp)41 was isolated from rat liver that is 35 amino acids longer than the reported rat liver specific organic anion transporter (rlst)‐1 and exhibits a 64% amino acid sequence identity with the human OATP‐C (LST‐1/OATP2; gene symbol SLC21A6). When expressed in Xenopus laevis oocytes, Oatp4 (Slc21a10) mediated polyspecific uptake of a variety of organic anions including taurocholate (K
m∼27 μM), bromosulfophthalein (K
m∼1.1 μM) and steroid conjugates. Based on nuclease protection analysis Oatp4 appears to be the predominant transcript in rat liver indicating that rlst‐1 plays a minor role in overall hepatic organic anion uptake.
Glycoprotein folding in the endoplasmic reticulum: a tale of three chaperones?
Authors: High Stephen , Lecomte Fabienne J.L , Russell Sarah J , Abell Benjamin M , Oliver Jason D
Published: July 2002

The endoplasmic reticulum (ER) is a major site of protein synthesis and its inside, or lumen, is a major site of protein folding. The lumen of the ER contains many folding factors and molecular chaperones, which facilitate protein folding by increasing both the rate and the efficiency of this process. Amongst the many ER folding factors, there are three components that specifically modulate the folding glycoproteins bearing N‐linked carbohydrate side chains. These components are calnexin, calreticulin and ERp57, and this review focuses on the molecular basis for their capacity to influence glycoprotein folding.
The pre‐transmembrane region of the human immunodeficiency virus type‐1 glycoprotein: a novel fusogenic sequence
Authors: Suárez Tatiana , Nir Shlomo , Goñi Félix M. , Saéz-Cirión Asier , Nieva José L.
Published: July 2002

We have investigated membrane interactions and perturbations induced by NH2‐DKWASLWNWFNITNWLWYIK‐COOH (HIVc), representing the membrane interface‐partitioning region that precedes the transmembrane anchor of the human immunodeficiency virus type‐1 gp41 fusion protein. The HIVc peptide bound with high affinity to electrically neutral vesicles composed of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine and cholesterol (molar ratio, 1:1:1), and induced vesicle leakage and lipid mixing. Infrared spectra suggest that these effects were promoted by membrane‐associated peptides adopting an α‐helical conformation. A sequence representing a defective gp41 phenotype unable to mediate both cell–cell fusion and virus entry, was equally unable to induce vesicle fusion, and adopted a non‐helical conformation in the membrane. We conclude that membrane perturbation and adoption of the α‐helical conformation by this gp41 region might be functionally meaningful.
PKB/Akt interacts with inosine‐5′ monophosphate dehydrogenase through its pleckstrin homology domain
Authors: Ingley Evan , Hemmings Brian A.
Published: July 2002

The pleckstrin homology (PH) domain of the protooncogenic serine/threonine protein kinase PKB/Akt can bind phosphoinositides. A yeast‐based two‐hybrid system was employed which identified inosine‐5′ monophosphate dehydrogenase (IMPDH) type II as specifically interacting with PKB/Akts PH domain. IMPDH catalyzes the rate‐limiting step of de novo guanosine‐triphosphate (GTP) biosynthesis. Using purified fusion proteins, PKB/Akts PH domain and IMPDH associated in vitro and this association moderately activated IMPDH. Purified PKB/Akt also associated with IMPDH in vitro. We could specifically pull‐down PKB/Akt or IMPDH from mammalian cell lysates using glutathione‐S‐transferase (GST)–IMPDH or GST–PH domain fusion proteins, respectively. Additionally, PKB/Akt and IMPDH could be co‐immunoprecipitated from COS cell lysates and active PKB/Akt could phosphorylate IMPDH in vitro. These results implicate PKB/Akt in the regulation of GTP biosynthesis through its interaction with IMPDH, which is involved in providing the GTP pool used by signal transducing G‐proteins.
The bulk of UCP3 expressed in yeast cells is incompetent for a nucleotide regulated H+ transport
Authors: Heidkaemper Dörthe , Winkler Edith , Müller Veronika , Frischmuth Karina , Liu Quingyun , Caskey Thomas , Klingenberg Martin
Published: July 2002

The impact of uncoupling protein (UCP) 1, UCP3 and UCP3s expressed in yeast on oxidative phosphorylation, membrane potential and H+ transport is determined. Intracellular ATP synthesis is inhibited by UCP3, much more than by UCP1, while similar levels of UCP3 and UCP1 exist in the mitochondrial fractions. Measurements of membrane potential and H+ efflux in isolated mitochondria show that, different from UCP1, with UCP3 and UCP3s there is a priori a preponderant uncoupling not inhibited by GDP. The results are interpreted to show that UCP3 and UCP3s in yeast mitochondria are in a deranged state causing uncontrolled uncoupling, which does not represent their physiological function.
Three new nuclear genes, sigD, sigE and sigF, encoding putative plastid RNA polymerase σ factors in Arabidopsis thaliana
Authors: Fujiwara Makoto , Nagashima Akitomo , Kanamaru Kengo , Tanaka Kan , Takahashi Hideo
Published: July 2002

Three new nuclear genes (sigD, sigE and sigF) of Arabidopsis thaliana, encoding putative plastid RNA polymerase σ factors, were identified and analyzed. Phylogenetic analysis revealed that higher plant σ factors fell into at least four distinct subgroups within a diverse protein family. In addition, Arabidopsis sig genes contained conserved chromosomal intron sites, indicating that these genes arose by DNA duplication events during plant evolution. Transcript analyses revealed two alternatively spliced transcripts generated from the sigD region, one of which is predicted to encode a σ protein lacking the carboxy‐terminal regions 3 and 4. Finally, the amino‐terminal sequence of the sigF gene product was shown to function as a plastid‐targeting signal using green fluorescent protein fusions.
CEO1, a new protein from Arabidopsis thaliana, protects yeast against oxidative damage1
Authors: Belles-Boix Enric , Babiychuk Elena , Van Montagu Marc , Inzé Dirk , Kushnir Sergei
Published: July 2002

The Saccharomyces cerevisiae strain WYT, deficient in the YAP1 transcription factor, was used in a molecular screen to identify genes from Arabidopsis thaliana that could overcome the oxidative stress‐sensitive phenotype of these yeast cells. A cDNA named CEO1 increased the tolerance to oxidative damage caused by tert‐butylhydroperoxide of both the Yap1− mutant and the wild‐type yeast. Additionally, in Yap1− yeast, CEO1 also induced cross‐tolerance to oxidative damage caused by hydrogen peroxide and diamide. CEO1 was assigned as being part of a small gene family that, until now, is exclusively restricted to plants. In Arabidopsis, CEO1 was produced in all organs, especially in roots and stems. By using the yeast two‐hybrid system, proteins that specifically interact with CEO1 in yeast were identified, and putative DNA‐binding proteins were consistently recovered.
A new family of small, palmitoylated, membrane‐associated proteins, characterized by the presence of a cysteine‐rich hydrophobic motif
Authors: Cools Jan , Mentens Nicole , Marynen Peter
Published: July 2002

We recently cloned the CHIC2 gene (previously BTL) by virtue of its involvement in a chromosomal translocation t(4;12)(q11;p13) occurring in acute myeloid leukemias. In this study we show that CHIC2 is a member of a highly conserved family of proteins characterized by the presence of a striking cysteine‐rich hydrophobic (CHIC) motif. Our data illustrate that cysteines in this central CHIC motif are palmitoylated and that CHIC2 is associated with vesicular structures and the plasma membrane. The CHIC proteins thus resemble the cysteine string proteins, which function in regulated exocytosis.
Receptor accessory folding helper enzymes: the functional role of peptidyl prolyl cis/trans isomerases
Authors: Schiene-Fischer Cordelia , Yu Chao
Published: July 2002

Receptor accessory peptidyl prolyl cis/trans isomerases (PPIases) of the FKBP and cyclophilin types form receptor heterocomplexes with different stabilities. PPIases have been found to associate with other receptor heterocomplex constituents via either proline‐directed active sites or additional domains of the enzymes. The single‐domain PPIases FKBP12 and FKBP12.6 are shown to interact with receptor protein kinases and calcium channels at their active sites. In contrast, heterooligomeric nuclear receptors contain multi‐domain PPIases like FKBP51, FKBP52 or cyclophilin 40 that directly interact with the chaperone hsp90 via the tetratricopeptide repeat modules of the folding helper enzymes. PPIases play a critical role in the functional arrangement of components within receptor heterocomplexes.