bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of gene expression of macrophage‐colony stimulating factor in human fibroblasts by the acute phase response mediators interleukin (IL)‐1β, tumor necrosis factor‐α and IL‐6
Authors: Mantovani Luisa , Henschler Reinhard , Brach Marion A. , Mertelsmann Roland H. , Herrmann Friedhelm
Published: July 2002

Fibroblasts constitute a major element of the bone marrow stroma. They play a pivotal role in blood cell development by providing the scaffolding required for cellular organization and tissue cohesion and by producing soluble molecules including colony stimulating factors (CSFs) and various interleukins regulating hematopoiesis. Our data demonstrate that the acute phase response mediators interleukin (IL)‐1 β, tumor necrosis factor (TNF)‐α and IL‐6 which are abundantly produced by activated monocytes, enhance levels of macrophage‐colony stimulating factor (M‐CSF) in fibroblasts by both transcriptional and post‐transcriptional mechanisms. The action of these proteins to induce M‐CSF transcript levels was dependent on synthesis of new proteins and was not mediated by protein kinase C (PKC) stimulation as depletion of cellular PKC pools by prolonged exposure of fibroblasts to phorbolester TPA did not prevent factor induced synthesis of M‐CSF transcripts. However, blockade of PKC by the isoquinoline sulfonamide derivative H7 and thus inhibition of phosphorylation was associated with augmentation of the fibroblasts response to TNF‐α and IL‐6.
Tyr‐426 of the Escherichia coli asparaginyl‐tRNA synthetase, an amino acid in a C‐terminal concerved motif, is involved in ATP binding
Authors: Anselme Jocelyne , Härtlein Michael
Published: July 2002

Sequence comparisons of the E. Coli asparaginyl‐tRNA synthetase (NRSEC) with aminoacyl‐tRNA synthetase sequences of class II enzymes show significant homologies with aspartyl‐ and lysyl‐tRNA synthetases. Three conserved regions were found, one of which is located in the C‐terminal part of the NRSEC sequence. Site‐directed mutagenesis was performed in this conserved region. A single point mutation Tyr‐426→Ser results in a 15‐fold increase in the K
m for ATP, while all the other kinetic parameters remain unchanged. The replacement of this Tyr‐426 by a Phe does not affect the behavior of the enzyme. These data indicate that Tyr‐426 is part of the ATP binding site.
Improving the thermostability of the neutral protease of Bacillus stearothermophilus by replacing a buried asparagine by leucine
Authors: Eijsink Vincent G.H. , van der Zee Rob , van den Burg Bertus , Vriend Gerrit , Venema Gerard
Published: July 2002

Amino acids buried in the hydrophobic interior of a protein with polar side chain atoms, which are not involved in hydrogen bonding or electrostatic interactions, have an adverse effect on protein stability. Replacing such residues by hydrophobic ones may render a protein more stable. Asparagine 241, which is buried in the neutral protease of Bacillus stearothermophilus, was replaced by leucine by side‐directed mutagenesis. This mutation increased the stability of the protein by 0.7 ± 0.1 degree.
Protein phosphorylation in isolated nuclei from etiolated Avena seedlings Effects of red/far‐red light and cholera toxin
Authors: Romero Luis C. , Biswal Basanti , Song Pill-Soon
Published: July 2002

We have studied the phosphorylation/dephosphorylation of several nuclear proteins in isolated nuclei from etiolated Avena seedlings as a function of red/far‐red light. The effect of stimulatory (ADP‐ribosylation by cholera toxin) or inhibitory (GDPβS) conditions for GTP‐binding proteins was also studied. Red or far‐red light enhanced the phosphorylation level of 2 nuclear proteins with molecular masses of 75 and 60 kDa. The phosphorylation pattern was affected by the addition of cholera toxin or GDPβS to the isolated nuclei. At least 2 proteins with molecular masses of 24 and 75 kDa cross‐reacted by Westerrs blot with GTP‐binding protein antibodies.
rig encodes ribosomal protein S15 The primary structure of mammalian ribosomal protein S15
Authors: Kitagawa Motoo , Takasawa Shin , Kikuchi Norihisa , Itoh Takako , Teraoka Hiroshi , Yamamoto Hiroshi , Okamoto Hiroshi
Published: July 2002

rig, a gene originally isolated from a rat insulinoma cDNA library, codes for a basic 145 amino acid protein [(1986) Diabetes 35, 1178–1180]. Here we show that the immunoreactivity to a monocional antibody against the deduced rig protein and the translation product of rig mRNA comigrated with ribosomal protein S15. The amino acid sequence of ribosomal protein S15 purified from rat liver coincided with that deduced from the nucleotide sequence of rig mRNA, but there were indications that the initiator methionine was removed and the succeeding alanyl residue was monoacetylated. From these results, we conclude that the product of rig is ribosomal protein S15.
Hormone specific regulation of natural killer cells by cortisol Direct inactivation of the cytotoxic function of cloned human NK cells without an effect on cellular proliferation
Authors: Callewaert Denis M. , Moudgil Virinder K. , Radcliff Gilbert , Waite Ross
Published: July 2002

Corticosteroids have previously been reported to partially inhibit the natural cytotoxic activity of peripheral blood lymphocytes. However, since only a few percent of peripheral lymphocytes are natural killer (NK) cells, it has not been possible to determine whether corticosteroids directly inhibit NK cells or mediate this effect via other cell types. This report documents direct functional inactivation, but unimpeded proliferation, of cloned human NK cells by subphysiologic levels of cortisol. In contrast, high concentrations of testosterone, progesterone or estradiol had no significant effect on proliferation or cytotoxic activity of the cloned NK cells. The kinetics of inhibition of NK function by cortisol are consistent with a transcription‐dependent mechanism.
Genes encoding peroxisomal enzymes are not necessarily assigned on the same chromosome of an n‐alkane‐utilizable yeast Candida tropicalis
Authors: Fukuda Yuki , Atomi Haruyuki , Kurihara Tatsuo , Hikida Masaki , Ueda Mitsuyoshi , Tanaka Atsuo
Published: July 2002

We have resolved eight chromosomal bands from an n‐alkane‐assimilating yeast, Candida tropicalis pK 233, by using contour‐clamped homogeneous electric field gel electrophoresis (CHEF). From the results of hybridization of DNA probes of yeast peroxisomal enzymes—catalase, acyl‐CoA oxidase, carnitine acetyltransferase, isocitrate lyase, malate synthase, acetoacetyl‐CoA thiolase, and 3‐ketoacyl‐CoA thiolase — to Southern transfers of CHEF gels, these genes were proven not necessarily to be located on the same chromosome. This fact shows that the genes encoding the enzymes tested were not distributed to be cistronic, although simultaneous and inducible synthesis of peroxisomal enzymes occurred in harmony with the proliferation of peroxisomes, suggesting that their co‐ordinated expression might be mainly regulated by certain trans‐acting factors.
Multisite serine phosphorylation of the insulin and IGF‐I receptors in transfected cells
Authors: Pillay T.S. , Whittaker J. , Lammers R. , Ullrich A. , Siddle K.
Published: July 2002

Serine phosphorylation of insulin/IGF‐I receptors in transfected fibroblasts was analysed by peptide mapping, PMA stimulated the phosphorylation of 5 distinct insulin receptor phosphopeptides: a single major phosphothreonine peptide containing Thr‐1348, one major and 3 minor phosphoserine peptides. The major insulin‐stimulated phosphoserine peptides were the same as those after PMA, with the exception of 2 minor phosphoserine peptides. PMA stimulated phosphorylation of a single major IGF‐I receptor phosphoserine peptide which was phosphorylated to a lesser extent after IGF‐I. We conclude that insulin/IGF‐I and PMA stimulate phosphorylation of the same sites, but differ in the extents of phosphorylation.
2D 1H NMR studies of oxidized 2(Fe4S4) ferredoxin from Clostridium pasteurianum
Authors: Bertini Ivano , Briganti Fabrizio , Luchinat Claudio , Messori Luigi , Monnanni Roberto , Scozzafava Andrea , Vallini Giovanni
Published: July 2002

Oxidized ferredoxin from Clostridium pastetirianum, containing two Fe4S4 clusters, has been investigated using 2D 1H NMR spectroscopy at 600 MHz. 2D NMR experiments allowed complete assignment of the sixteen isotropically shifted signals corresponding to the β‐CH2 protons of the eight metal coordinated cysteines. Geminal connectivities of Cys β‐CH2 protons were identified through magnitude COSY experiments and confirmed through 2D NOESY experiments. A few additional signals could be assigned to the corresponding α‐CH protons. The importance of 2D experiments to achieve firm assignments of isotropically shifted signals in paramagnetic metalloproteins is stressed.
A modified Kex2 enzyme retained in the endoplasmic reticulum prevents disulfide‐linked dimerisation of recombinant human insulin‐like growth factor‐1 secreted from yeast
Authors: Chaudhuri Bhabatosh , Stephan Christine
Published: July 2002

The majority of the recombinant human insulin‐like growth factor‐1 (IGF1) molecules, secreted from yeast using the prepro sequence of the prepro‐α‐factor, are not active monomers but inactive, disulfide‐linked dimers. The prepro sequence of the prepro‐α‐factor, usually referred to as the α‐factor leader (αFL), consists of a pre or signal sequence and a proregion. After signal sequence removal during translocation into the endoplasmic reticulum (ER) the proregion is still attached to IGF1 when it folds to acquire a tertiary structure. Mature IGF1 is released only in a late Golgi compartment by the membrane‐bound endoprotease Kex2p. We find that co‐expression of a novel ER‐retained Kex‐2p variant, soluble Kex2pHDEL, can prevent intermolecular disulfide bond formation between two IGF1 molecules, implying that the presence of the proregion during the folding of IGF1 in the ER could be a reason for disulfide‐linked dimerisation. This result indicates that the proregion of the αFL may have a role in the folding of some heterologous proteins in yeast, and that the ER‐retained Kex2p mutant could be used as a convenient tool to study the cellular function of the proregions present naturally in various eucaryotic precursor proteins.
Do G protein subunits associate via a three‐stranded coiled coil?
Authors: Lupas Andrei N. , Lupas Janice M. , Stock Jeffry B.
Published: July 2002

We used a computer‐based prediction algorithm to identify probable coiled‐coil segments at the N‐termini of G protein α, β and γ subunits. This result indicates that G protein trimers may form via a three‐stranded coiled coil. Previous biochemical results had shown that the N‐termini of α and β are involved in subunit interactions. Here we present a structural model for the N‐terminal domain of βγ and a hypothesis for the reversible association of α to βγ.
Chromogranin A processing in sympathetic neurons and release of chromogranin A fragments from sheep spleen
Authors: Miserez Bernard , Annaert Wim , Dillen Lieve , Aunis Dominique , De Potter Werner
Published: July 2002

Chromogranin A (CGA) has been localized to the large dense cored vesicles (LDV) of sympathetic neurons. SDS‐PAGE and immunoblotting of soluble LDV proteins from ox and dog adrenergic neuronal cell bodies, axons and nerve terminals, revealed an increasing number of CGA‐immunoreactive forms, consistent with proteolytic processing during axonal transport. Splenic nerve electrical stimulation (10 Hz, 2 min) revealed that, apart from CGA. these CGA‐processing products are released from the sheep spleen. The secretion of CGA‐derived fragments from sympathetic neurons might suggest a role in the regulation of synaptic transmission.
Expression of a functional α‐macroglobulin receptor binding domain in Escherichia coli
Authors: Salvesen Guy , Quan Long T. , Enghild Jan J. , Snipas Scott , Fey Georg H. , Pizzo Salvatore V.
Published: July 2002

We have expressed receptor‐binding domains of human α2‐macroglobulin in Escherichia coli. Expression levels of both recombinants were quite high, but the human one was insoluble, probably forming inclusion bodies. The rat domain, which lacks the human disulfide, was produced in a soluble form and readily purified by two simple chromatographic steps. Purified recombinant rat α1‐macroglobulin receptor‐binding domain was fully functional in binding to the α‐macroglobulin receptor on human fibroblasts. This 142 residue domain should serve as an excellent template for analyzing the structural requirements for α‐macroglobulin receptor ligation and dissecting the varied biological functions resulting from such ligation.
The in situ degradation of ceramide, a potential lipid mediator, is not completely impaired in Farber disease
Authors: Levade Thierry , Tempesta Marie-Caroline , Salvayre Robert
Published: July 2002

The time course of degradation of a radiolabelled natural ceramide has been studied in intact, living lymphoid cells and skin fibroblasts from normal individuals and from patients affected with Farber disease, an inborn disorder of ceramide metabolism due to deficient activity of lysosomal ceramidase. The hydrolysis of ceramide in lysosomes was selectively followed by examining the turnover of an LDL‐associated radioactive sphingomyelin. This permitted to estimate accurately the effective lysosomal ceramidase activity and to demonstrate: (i) a very active catabolism of ceramide in normal cells; and (ii) the absence of a complete block of ceramide degradation in Farber cells. The possible implication of ceramide as a lipid mediator of the pathogenesis of Farber disease is discussed.
Identification of the gene encoding the activator of(R)‐2‐hydroxyglutaryl‐CoA dehydratase from Acidaminococcus fermentans by gene expression in Escherichia coli
Authors: Bendrat K. , Müller U. , Klees A.-G. , Buckel W.
Published: July 2002

(R)‐2‐Hydroxyglutaryl‐CoA dehydratase (HGDA/B) from Acidaminococcus fermentans requires an activator protein for activity. This activator (HGDC) has not yet been purified from its natural source due to its low concentration combined with an extreme sensitivity towards oxygen. Gene expression in Escherichia coli identified an open reading frame (780 bp) as the gene encoding HGDC. Dehydratase activity was stimulated at least tenfold by cell‐free extracts of E. coli cells transformed with a plasmid carrying hgdC. On the chromosome the hgdC gene is located just before hgdA and hgdB.
A G‐protein‐coupled 130 kDa phospholipase C isozyme, PLC‐β4, from the particulate fraction of bovine cerebellum
Authors: Min Do Sik , Kim Yong , Lee Young Han , Suh Pann-Ghill , Ryu Sung Ho
Published: July 2002

A 130 kDa PLC isozyme was purified from the particulate fraction of bovine cerebellum. This PLC was recognized by a polyclonal antiserum generated against the purified 97 kDa PLC‐β4. Reconstitution of the purified 130 kDa PLC with the membranes of C6 Bu‐1 cells in the presence of GTPγS or Alp4
− resulted in PLC activation as well as the association of PLC with the membranes. Both the association and activation were revoked when the membrane was washed with 2 M KC1. The 97 kDa PLC‐β4 did not associate with membranes. These data suggest that the 130 kDa PLC is the intact form of PLC‐β4 the activity of which is likely to be regulated by a G‐protein on the membrane.
An enhancer unit of L‐type pyruvate kinase gene is responsible for transcriptional stimulation by dietary fructose as well as glucose in transgenic mice
Authors: Noguchi Tamio , Okabe Masaru , Wang Ziyuan , Yamada Kazuya , Imai Enyu , Tanaka Takehiko
Published: July 2002

We produced three lines of transgenic mice containing the 5' flanking region of the L‐type pyruvate kinase gene from nucleotides −189 to +37, which includes an enhancer unit and TATA box as functional elements, linked to the chloramphenicol acetyltransferase gene. Since transgene expression was stimulated by both dietary fructose and glucose in a tissue‐dependent manner, we suggest that this unit is responsive to both stimuli.
A novel β‐N‐acetylglucosaminidase activity in hog gastric mucosal microsomes: Preferential hydrolysis of terminal GlcNAcß1‐3 linkages in GlcNAcß1‐3(GlcNAcß1‐6)Galß1‐GlcNAc, but GlcNAcß1‐6 linkages in GlcNAcß1‐3(GlcNAcß1‐6)Gal
Authors: Helin Jari , Seppo Antti , Leppänen Anne , Penttilä Leena , Maaheimo Hannu , Niemelä Ritva , Lauri Sari , Renkonen Ossi
Published: July 2002

Hog gastric mucosal microsomes contain β‐N‐acetylgrucosaminidase activity which cleaves GlcNAcß1‐3(GlcNAcß1‐6)Galß1‐4GlcNAc at the terminal GlcNAcß1‐3Gal linkage faster than at the GlcNAcß1‐6Gal bond, producing mainly GlcNAcß1‐6Galß1‐4GlcNAc. In a marked contrast, GlcNAcß1‐3(GlcNAcß1‐6)Gal is cleaved primarily at the GlcNAcß1‐6Gal bond, while partial hydrolysis of GlcNAcß1‐3(GlcNAcß1‐6)Gal ß1‐4Glc reveals similar rates of cleavage for the (1–3) and (1–6) linkages. Our data support the notion that the terminal ß1,6‐linked GlcNAc unit of GlcNAcß1‐3(GlcNAcß1‐6)Galß1‐4GlcNAc may interact with the reducing end GlcNAc unit intramolecularly in water solution.
Evidence for the existence of hyaluronectin‐binding proteins in the plasma membranes
Authors: Gupta Sanjay , Deb Tushar Baran , Datta Kasturi
Published: July 2002

This report documents for the first time the existence of specific binding proteins for hyaluronic acid binding protein (hyaluronectin) in the plasma membranes of normal and transformed cells. Firstly, we showed the specific binding of hyaluronic acid binding protein to the cell surface of normal rat heart fibroblasts (NRHF) by saturation and competition methods using 125I‐labeled hyaluronic acid binding protein and calculated the binding dissociation constant (0.43 × 10−13 M). In order to identify hyaluronectin‐binding protein on the cell surface, plasma membranes isolated from rat brain, liver and flbrosarcoma were separated by SDS‐PAGE and transferred to nitrocellulose paper by electroblotting. Incubation of the transferred membrane proteins with 125I‐labeled hyaluronectin in the presence of non‐ionic as well as ionic detergents revealed two prominent bands of approximate molecular mass of 37 kDa and 40 kDa in brain, liver and flbrosarcoma. The specificity of the binding [125I]hyaluronectin to 37‐kDa and 40‐kDa membrane proteins was further confirmed, as the intensity of the bands was reduced in the presence of a 20‐fold excess of unlabeled hyaluronectin. We discuss our observations on hyaluronectin‐binding membrane proteins in the context of hyaluronectin‐mediated cellular functions.
Molecular cloning and developmental expression of human cardiac troponin T
Authors: Mesnard Laurence , Samson Françoise , Espinasse Isabelle , Durane Jacques , Neveux Jean-Yves , Mercadier Jean-Jacques
Published: July 2002

We have isolated a full‐size cDNA coding for cardiac troponin T (cTnT) from a human adult heart library, using a slow skeletal TnT probe. This cDNA detected a 1.2 kb mRNA in fetal and post‐natal human heart, the amount of which increased during ontogenic development. Interestingly, a similar transcript was coexpressed in fetal skeletal muscle, together with the 0.9 kb slow skeletal muscle mRNA, and its expression was down‐regulated during further development.
Chaperonins dependent increase of Cu,Zn Superoxide dismutase production in Escherichia coli
Authors: Battistoni Andrea , Carrí M.Teresa , Steinkühler Christian , Rotilio Giuseppe
Published: July 2002

Over‐expression of the chaperonins GroEL and GroES significantly suppressed the temperature‐dependent pattern of expression of Cu,Zn Superoxide dismutases in Escherichia coli and increased the yield of active enzyme. The results obtained indicate that chaperonins prevent degradation of metal‐deficient enzyme molecules. GroEL was shown to form a complex with unfolded Cu,Zn Superoxide dismutase in vitro, confirming that GroEL can interact with β‐stranded proteins.
Purification and characterization of the head‐activator receptor from a multi‐headed mutant of chlorohydra viridissima
Authors: Christians Stefan , Neubauer Karl-Heinz , Ulrich Henning
Published: July 2002

In hydra the neuropeptide head activator (HA) is responsible for head‐specific growth and differentiation processes. The effects of HA are mediated by high and low affinity receptors on hydra cells. In the current study HA receptors from a multi‐headed mutant of Chlorohydra viridissima were solubilized from the membrane fraction using 1% Triton X‐100 and 2.5 M urea. Scatchard analysis showed that the solubilized receptor had a K
d of 1.55 × 10−9 M, indicating the low affinity subtype of the HA receptor. The solubilized receptor was purified by DMAE chromatography and subsequent affinity chromatography to homogeneity. SDS‐PAGE revealed a single protein band with a molecular mass of 96 ± 4 kDa. The native receptor eluted during gel filtration as a 113 kDa protein, and focussed with an isoelectric point of 4.8.
β‐Sheet topology A new system of nomenclature
Authors: Flower Darren R.
Published: July 2002

The topology of a protein β‐sheet, the relationship between the sequential ordering of strands and their connectedness in space, is an important and well studied feature of protein structures. The prevalent nomenclature for describing β‐sheet topologies is based on following a path through the sequence order of strands and noting their separation in space. Although powerful, this approach can be usefully complemented by a notation based on following a path through the connectedness of neighbouring strands and noting sequence separation. This leads in turn to a short hand expression of sheet topology, based on a method for describing the covalent structure of small molecules, which is able to express concisely the complex non‐linear topological relationships of β‐sheets, including bifurcations and closed structures, in a clear and natural manner. Using this novel system of notation it is possible to follow simultaneously the sequence and hydrogen bonded connectedness of strands within the topology of a sheet.
Purification and characterization of the 210‐amino acid recombinant basic fibroblast growth factor form (FGF‐2)
Authors: Patry Véronique , Bugler Béatrix , Amalric François , Promé Jean-Claude , Prats Hervé
Published: July 2002

Four forms of basic fibroblast growth factor (bFGF or FGF‐2), using one AUG (155 amino acids) and three upstream CUG (210, 201 and 196 amino acids) start codons, were synthesized through an alternative use of initiation codons. The 210‐amino acid form of FGF‐2 (210FGF‐2) was expressed in a plasmid vector under the control of a bacteriophage T7 RNA polymerase promoter system in Escherichia coli. Characterization of the purified protein was performed by electrospray mass spectrometry and Edman degradation. The recombinant 210FGF‐2 produced in E. coli had a mitogenic activity similar to the 146‐amino acid form extracted from tissues.
Identification and hypotensive activity of proadrenomedullin N‐terminal 20 peptide (PAMP)
Authors: Kitamura Kazuo , Kangawa Kenji , Ishiyama Yuichiro , Washimine Hisanori , Ichiki Yoshinari , Kawamoto Mari , Minamino Naoto , Matsuo Hisayuki , Eto Tanenao
Published: July 2002

Proadrenomedullin N‐terminal 20 peptide (PAMP) is a candidate for a novel biologically active peptide processed from an adrenomedullin precursor. Using a radioimmunoassay for human PAMP, major and minor immunoreactive PAMPs were purified from porcine adrenal medulla and complete amino acid sequences were determined. The major immunoreactive peptide was PAMP itself with an amidated carboxy terminus. The minor one was determined to be PAMP[5–20]. An intravenous bolus injection of human PAMP in anesthetized rats caused a rapid and strong hypotensive effect in a dose dependent manner. The present data indicate that PAMP is an endogenous biologically active peptide which is processed from adrenomedullin precursor.
Alterations at the 3′‐CCA end of Escherichia coli and Thermus thermophilus tRNAPhe do not abolish their acceptor activity
Authors: Moor N.A. , Repkova M.N. , Yamkovoy V.I. , Lavrik O.I.
Published: July 2002

The 3′‐CCA end of tRNAPhe from Escherichia coli and Thermus thermophilus was changed to AAA, CCC, UUU and UUA by the stepwise degradation procedure of the 3′‐CCA end of tRNAPhe followed by the ligation with oligoribotrinucleotides. Substrate activity of tRNAUPhe
UUA and tRNAPhe
CCC in tRNA aminoacylation was shown. tRNAPhe
AAA is a bad substrate for E. coli and Th. thermophilus phenylalanyl‐tRNA synthetases. tRNAPhe
UUU has no detectable activity in tRNA aminoacylation. Therefore the nature of the 3′‐end of tRNAPhe plays an important role in tRNA binding and its substrate efficiency. Nevertheless the CCA sequence at the 3′‐end of tRNAPhe does not seem to be an absolute requirement for tRNA aminoacylation.
Specificities of the two center N inhibitors of mitochondial bc1 complex, antimycin and funiculosin: strong involvement of cytochrome b‐asparagine‐208 in funiculosin binding
Authors: Brasseur Gaël , Brivet-Chevillotte Paule
Published: July 2002

Funiculosin, a center N inhibitor of the bc
1 complex, induces a blue‐shift in the cytochrome b spectrum. A thermosensitive revertant [Coppee, J.Y. et al., J. Biol. Chem. 269 (1994) 4221–4226] isolated from a cytochrome b respiratory‐deficient mutant, exhibits a red‐shift instead of the blue‐shift retained in the original mutant and shows resistance to this inhibitor. Replacing cytochrome b‐Asparagine‐208 by Lysine in this revertant, keeping the original mutation S206L, leads, when mitochondria are incubated at non‐permissive temperature, to complete loss of bc
1 complex activity and funiculosin‐binding, while the antimycin‐binding is conserved. These data suggest some inhibitor site specificity and close proximity between the funiculosin‐binding site and the catalytic center N domain (QN).
Immunoreactive adrenomedullin in human plasma
Authors: Kitamura Kazuo , Ichiki Yoshinari , Tanaka Miho , Kawamoto Mari , Emura Junji , Sakakibara Shumpei , Kangawa Kenji , Matsuo Hisayuki , Eto Tanenao
Published: July 2002

A specific and sensitive radioimmunoassay for adrenomedullin has been developed. Half‐maximal inhibition of binding of radioiodinated adrenomedullin was observed at 4 tool/tube. The radioimmunoassay recognized the entire adrenomedullin molecule and has little crossreactivity with adrenomedullin fragment peptides. Adrenomedullin‐like immunoreactivity was found to circulate in human plasma at considerable concentration (3.3 ± 0.39 ). The immunoreactivity of adrenomedullin was eluted at almost the same position as synthetic adrenomedullin on gel‐filtration chromatography and reverse‐phase high‐performance liquid chromatography, suggesting that circulating adrenomedullin recognized by the present radioimmunoassay is identical or very similar to authentic adrenomedullin. Plasma immunoreactive adrenomedullin significantly increased in patients with hypertension, with a progressive rise proportionate to disease severity.
Structure of protein kinase CK2: Dimerization of the human β‐subunit
Authors: Boldyreff Brigitte , Mietens Ulrike , Issinger Olaf-Georg
Published: July 2002

Protein kinase CK2 has been shown to be elevated in all so far investigated solid tumors and its catalytic subunit has been shown to serve as an oncogene product. CK2 is a hetero‐tetrameric serine‐threonine kinase composed of two catalytic (α and/or α′) and two regulatory β‐subunits. Using the two‐hybrid system we could show that the α‐ or α′‐subunits of CK2 can interact with the β‐subunits of CK2, but not with other α‐ or α′‐subunits. By comparison, the β‐subunit of CK2 can interact with another β‐subunit. Important amino acids for successful dimerization of the β‐subunit were localized between amino acid residues 156 and 165. Furthermore, we identified residues between amino acid 170 and 180 which antagonize the dimerization.
Investigation of oxidation state‐dependent conformational changes in Desulfovibrio vulgaris Hildenborough cytochrome C553 by two‐dimensional 1H‐NMR spectra
Authors: Blanchard Laurence , Blackledge Martin J. , Marion Dominique , Guerlesquin Françoise
Published: July 2002

Two‐dimensional nuclear magnetic resonance spectroscopy (2D‐NMR) was used to assign the proton resonances of ferricytochrome C
553 from Desulfovibrio vulgaris Hildenborough. The spin systems of 76 out of 79 amino acids were identified by J‐correlation spectroscopy (COSY and HOHAHA) in H2O and D2O and correlated by nuclear Overhauser effect spectroscopy (NOESY). The proton chemical shifts are compared in both oxidized and reduced states of the protein at 23°C and pH 5.9. Chemical shift variations between reduced and oxidized states are due to the paramagnetic contribution. Medium and longrange nOe demonstrate the lack of major changes between the two redox states. NMR data provide evidence that in this low oxidoreduction potential cytochrome, the oxidized state is more rigid than the reduced state.
Physiological correlation between glycyrrhizin, glycyrrhizin‐binding lipoxygenase and casein kinase II
Authors: Shimoyama Yoshihito , Ohtaka Hisayuki , Nagata Nobuyuki , Munakata Hiroshi , Hayashi Norio , Ohtsuki Kenzo
Published: July 2002

By means of glycyrrhizin (GL)‐affinity column chromatography, a GL‐binding lipoxygenase (gbLOX) was selectively purified from the partially purified soybean LOX‐1 fraction. Polypeptide analysis of the purified gbLOX by SDS‐PAGE detected two distinct polypeptides (p96 and p94), which were identical to LOX‐3 as determined by their partial N‐terminal amino acid sequences. Moreover, it was found that (i) phosphorylation of gpLOX by casein kinase II (CK‐II) is significantly stimulated by 3 μM GL, but inhibited by 30 μM GL or 10 μM oGA; and (ii) gbLOX activity is enhanced when the enzyme is phosphorylated by CK‐II in the presence of 3 μM GL. These results suggest that (i) CK‐II is a kinase responsible for the activation of gbLOX through its specific phosphorylation; and (ii) GL is one of the regulatory substances for specific phosphorylation of gbLOX (LOX‐3) by CK‐II in plant cells.
Analysis of the role of basic‐helix‐loop‐helix transcription factors in the development of neural lineages in the mouse
Authors: Guillemot François
Published: July 2002

Summry— The development of neural lineages is a complex process which is being actively investigated in both vertebrate and invertebrate models. A detailed genetic analysis of sensory organ development in Drosophila has revealed the contribution of numerous genes which fonction to progressively to specify the fate of neural precursor cells. Many of these genes have mammalian homologs which are also expressed in the developing nervous system, among which two families of genes encoding transcription factors of the helix‐loop‐helix class. The two mouse Mash genes are homologs of the Drosophila achaete‐scute genes, which are positive regulators of neural precursor development. The five mouse HES genes are homologs of the Drosophila hairy and Enhancer of split genes, which act as negative regulators in this process. We have generated a null mutation in the mouse Mash1 gene by homologous recombination in embryonic stem cells. Animals homozygous for this mutation die at birth, and show severe losses in olfactory and autonomic neurons. In both lineages, the mutation appears to affect the development of neuronal precursors. We have also started a genetic analysis of the mouse HES genes to study their function during neurogenesis and the possibility that they regulate the activity of Mash1 and other positive regulators.
Production of recombinant soluble human integrin α4β1
Authors: Clark Katherine , Newham Peter , Burrows Louise , Askari Janet A. , Humphries Martin J.
Published: July 2002

Integrin α4β1 is a major leukocyte adhesion receptor that is a key target for the development of anti‐inflammatory therapeutics. With the dual long‐term goals of developing a reagent for use in high‐throughput inhibitor screening assays and for crystallisation trials and subsequent structure determination, we have generated a recombinant soluble α4β1 receptor. Both subunits were truncated prior to the transmembrane domains by site‐directed mutagenesis and expressed using baculovirus infection of insect cells. The molecular weights of the recombinant subunits were as expected for post‐translationally unmodified protein. In addition, as observed for the native subunit, a proportion of the α4 subunit was proteolytically processed into two fragments. ELISA and solid phase ligand‐binding assays were performed to investigate the folding and functionality of the soluble integrin. The data suggest that the receptor was correctly folded and that it bound recombinant ligands with similar kinetics to the native molecule.
The adenovirus‐2 E1B‐55K protein interacts with a mSin3A/histone deacetylase 1 complex
Authors: Punga Tanel , Akusjärvi Göran
Published: July 2002

The adenovirus E1B‐55K protein is a multifunctional phosphoprotein that regulates nuclear to cytoplasmic export of host cell and viral mRNAs during lytic viral growth. E1B‐55K also blocks apoptosis by binding and functionally inactivating the human tumor suppressor protein p53. Here, we show that E1B‐55K interacts with histone deacetylase 1 (HDAC1) and the transcriptional corepressor protein mSin3A, both in the adenovirus‐transformed 293 cell line and during a lytic adenovirus infection. Furthermore, we show that the central amino acids 156–261 in E1B‐55K are necessary for efficient HDAC1 interaction. Importantly, the E1B‐55K/mSin3A/HDAC1 complex is also enzymatically active, catalyzing deacetylation of a histone substrate peptide. Collectively, our results suggest that E1B‐55K interaction with mSin3A/HDAC1 containing complexes may be significant for one or several of the multiple activities ascribed to this protein.
Iron(II) induces changes in the conformation of mammalian mitochondrial DNA resulting in a reduction of its transcriptional rate
Authors: Ası́n Jordi , Pérez-Martos Acisclo , Fernández-Silva Patricio , Montoya Julio , Andreu Antoni L.
Published: July 2002

Living isolated mitochondria incubated with iron(II) show a major alteration in mitochondrial DNA (mtDNA) conformational forms as assessed by Southern blot analysis of undigested mtDNA. In the presence of iron(II), form I is transformed into form III in a dose‐dependent manner. This alteration in mtDNA conformation shows a strong correlation with a decrease in the mtDNA transcription rate (r=0.965, P<0.002), suggesting that iron(II) load results in double‐strand breaks and unwinding of mtDNA, which, in turn, is unable to maintain its normal transcriptional rate.
Unique features of HIV‐1 Rev protein phosphorylation by protein kinase CK2 (‘casein kinase‐2’)
Authors: Marin Oriano , Sarno Stefania , Boschetti Marco , Pagano Mario A , Meggio Flavio , Ciminale Vincenzo , D'Agostino Donna M , Pinna Lorenzo A
Published: July 2002

The HIV‐1 Rev transactivator is phosphorylated in vitro by protein kinase CK2 at two residues, Ser‐5 and Ser‐8; these sites are also phosphorylated in vivo. Here we show that the mechanism by which CK2 phosphorylates Rev is unique in several respects, notably: (i) it is fully dependent on the regulatory, β‐subunit of CK2; (ii) it relies on the integrity of an acidic stretch of CK2β which down‐regulates the phosphorylation of other substrates; (iii) it is inhibited in a dose‐dependent manner by polyamines and other polycationic effectors that normally stimulate CK2 activity. In contrast, a peptide corresponding to the amino‐terminal 26 amino acids of Rev, including the phosphoacceptor site, is readily phosphorylated by the catalytic subunit of CK2 even in the absence of the β‐subunit. These data, in conjunction with the observation that two functionally inactive derivatives of Rev with mutations in its helix‐loop‐helix motif are refractory to phosphorylation, indicate the phosphorylation of Rev by CK2 relies on conformational features of distinct regions that are also required for the transactivator's biological activity.
Mitochondrial copper metabolism in yeast: interaction between Sco1p and Cox2p
Authors: Lode Anja , Kuschel Margret , Paret Claudia , Rödel Gerhard
Published: July 2002

Yeast mitochondrial Sco1p is required for the formation of a functional cytochrome c oxidase (COX). It was suggested that Sco1p aids copper delivery to the catalytic center of COX. Here we show by affinity chromatography and coimmunoprecipitation that Sco1p interacts with subunit Cox2p. In addition we provide evidence that Sco1p can form homomeric complexes. Both homomer formation and binding of Cox2p are neither dependent on the presence of copper nor affected by mutations of His‐239, Cys‐148 or Cys‐152. These amino acids, which are conserved among the members of the Sco1p family, have been suggested to act in the reduction of the cysteines in the copper binding center of Cox2p and are discussed as ligands for copper.
Mitochondrial Hsp78, a member of the Clp/Hsp100 family in Saccharomyces cerevisiae, cooperates with Hsp70 in protein refolding
Authors: Krzewska Joanna , Langer Thomas , Liberek Krzysztof
Published: July 2002

The molecular chaperone protein Hsp78, a member of the Clp/Hsp100 family localized in the mitochondria of Saccharomyces cerevisiae, is required for maintenance of mitochondrial functions under heat stress. To characterize the biochemical mechanisms of Hsp78 function, Hsp78 was purified to homogeneity and its role in the reactivation of chemically and heat‐denatured substrate protein was analyzed in vitro. Hsp78 alone was not able to mediate reactivation of firefly luciferase. Rather, efficient refolding was dependent on the simultaneous presence of Hsp78 and the mitochondrial Hsp70 machinery, composed of Ssc1p/Mdj1p/Mge1p. Bacterial DnaK/DnaJ/GrpE, which cooperates with the Hsp78 homolog, ClpB in Escherichia coli, could not substitute for the mitochondrial Hsp70 system. However, efficient Hsp78‐dependent refolding of luciferase was observed if DnaK was replaced by Ssc1p in these experiments, suggesting a specific functional interaction of both chaperone proteins. These findings establish the cooperation of Hsp78 with the Hsp70 machinery in the refolding of heat‐inactivated proteins and demonstrate a conserved mode of action of ClpB homologs.
Imperatoxin A (IpTxa) from Pandinus imperator stimulates [3H]ryanodine binding to RyR3 channels
Authors: Simeoni Ilenia , Rossi Daniela , Zhu Xinsheng , Garcı́a Jesus , Valdivia Hector H. , Sorrentino Vincenzo
Published: July 2002

The effect of imperatoxin A (IpTxa) on the ryanodine receptor type 3 (RyR3) was studied. IpTxa stimulates [3H]ryanodine binding to RyR3‐containing microsomes, but this effect requires toxin concentrations higher than those required to stimulate RyR1 channels. The effect of IpTxa on RyR3 channels was observed at calcium concentrations in the range 0.1 μM to 10 mM. By contrast, RyR2 channels were not significantly affected by IpTxa in the same calcium ranges. Single channel current measurements indicated that IpTxa induced subconductance state in RyR3 channels that was similar to those observed with RyR1 and RyR2 channels. These results indicate that IpTxa is capable of inducing similar subconductance states in all three RyR isoforms, while stimulation of [3H]ryanodine binding by this toxin results in isoform‐specific responses, with RyR1 being the most sensitive channel, RyR3 displaying an intermediate response and RyR2 the least responsive ones.
Genomic organization of the KTX2 gene, encoding a ‘short’ scorpion toxin active on K+ channels
Authors: Legros Christian , Bougis Pierre E , Martin-Eauclaire Marie-France
Published: July 2002

A single intron of 87 bp, close to the region encoding the C‐terminal part of the signal peptide, was found in the gene of the ‘short’ scorpion toxin kaliotoxin 2 of Androctonus australis acting on various types of K+ channels. Its A+T content was particularly high (up to 86%). By walking and ligation‐mediated PCR, the promoter sequences of the kaliotoxin 2 gene of Androctonus australis were studied. The transcription unit of the gene is 390 bp long. Consensus sequences were identified. The genes of ‘short’ scorpion toxins active on K+ channels are organized similarly to those of the ‘long’ scorpion toxins active on Na+ channels and not like those of structurally related insect defensins, which are intronless.
Dexamethasone differently modulates TNF‐α‐ and IL‐1β‐induced transcription of the hepatic Mn‐superoxide dismutase gene
Authors: Antras-Ferry Jocelyne , Mahéo Karine , Morel Fabrice , Guillouzo André , Cillard Pierre , Cillard Josiane
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Molecular cloning and characterization of the human p27Kip1 gene promoter1
Authors: Minami Shinji , Ohtani-Fujita Naoko , Igata Eiji , Tamaki Tetsuya , Sakai Toshiyuki
Published: July 2002

p27Kip1 is an inhibitor of multiple cyclin‐dependent kinases (cdk), and can arrest the cell‐cycle progression by inhibiting the phosphorylation of the retinoblastoma gene family products. Tumor formation in p27Kip1 knockout mice clearly shows that p27Kip1 plays an important role in inhibiting tumor formation and progression. To investigate the mechanism of transcriptional p27Kip1 gene expression, we isolated the genomic DNA fragment of the 5′ flanking region of the human p27Kip1 gene and characterized its promoter region. The human p27Kip1 promoter is TATA‐less, and the sequence is highly homologous to the murine p27Kip1 promoter sequence. In the promoter assay, deletion from −774 to −435 relative to the initiating codon resulted in a 15–20‐fold reduction of the p27Kip1 promoter activity, suggesting that the elements for basal promoter activity exist in this highly conserved 340 bp region, where putative CTF and ATF sites are conserved.
Primary structure of matrilin‐3, a new member of a family of extracellular matrix proteins related to cartilage matrix protein (matrilin‐1) and von Willebrand factor1
Authors: Wagener Raimund , Kobbe Birgit , Paulsson Mats
Published: July 2002

A mouse cDNA encoding for matrilin‐3, the third member of the novel matrilin family of extracellular matrix proteins, was cloned. The protein precursor of 481 amino acids consists of a putative signal peptide, a short positively charged sequence, a single vWFA‐like domain followed by four epidermal growth factor‐like modules and a potential coiled‐coil α‐helical oligomerization domain at the C‐terminus. It is the smallest member of the matrilin family with a predicted M
r of the mature protein of 48 902. The primary structure of a C‐terminal portion of 310 amino acids of the human matrilin‐3 was determined and showed a sequence identity to the mouse matrilin‐3 of 84.8%. Northern blot hybridization of mouse matrilin‐3 mRNA showed a 2.9 kb mRNA expressed in sternum, femur and trachea and indicates a cartilage‐specific expression.
Arachidonic acid, a principal product of Rac‐activated phospholipase A2, stimulates c‐fos serum response element via Rho‐dependent mechanism
Authors: Kim Byung-Chul , Lim Chang-Jin , Kim Jae-Hong
Published: July 2002

Previously, we have reported that phospholipase A2 (PLA2) is one of the major downstream targets by which Rac GTPase mediates the activation of c‐fos serum response element (SRE) in response to agonists such as EGF [FEBS Lett. 407 (1997) 7–12]. Thus, the potential activity of arachidonic acid (AA), a principal product of Rac‐activated PLA2, on c‐fos SRE stimulation has been suggested. Here, we provide evidence about the biological activity of AA on c‐fos SRE activation. Further, we observed that co‐transfection with expression plasmid of either RhoN19, a dominant negative RhoA mutant, or botulinum C3 transferase which inhibits Rho via ADP ribosylation, selectively repressed AA‐ or Rac‐induced SRE activation, suggesting that Rho activity is critical for the signaling cascade of `Rac‐PLA2‐AA' to c‐fos SRE. Thus, Rac signaling to the nucleus appears to be, at least partly, mediated by a Rho‐linked pathway and this Rac‐Rho signaling connection is mediated by AA. In accordance with the role of Rho as a potential mediator of AA signaling to the nucleus, AA induces a rapid translocation of RhoA.
The human α3b is a `full‐sized' laminin chain variant with a more widespread tissue expression than the truncated α3a
Authors: Doliana Roberto , Bellina Isabella , Bucciotti Francesco , Mongiat Maurizio , Perris Roberto , Colombatti Alfonso
Published: July 2002

We report the molecular cloning of the human laminin α3b chain variant and its mRNA expression pattern in adult human tissues when compared to the α3a variant. The mRNA encoding for the α3b variant is about 11 kb and the predicted translation product carries the complete set of domains typical for a `full‐sized' laminin α chain. Apart from the similar domain structure of α3b also the sequence of α3 resulted more closely related to the α5 than to the α4 chain. Quantitative analysis of the RNA expression in a broad panel of adult human tissues indicated that the α3b variant is more widely distributed than the α3a shorter variant.
Chemical oxidation and DNA damage catalysed by inorganic sunscreen ingredients
Authors: Dunford Rosemary , Salinaro Angela , Cai Lezhen , Serpone Nick , Horikoshi Satoshi , Hidaka Hisao , Knowland John
Published: July 2002

Titanium dioxide (TiO2) has been noted (US Federal Register, 43FR38206, 25 August 1978) to be a safe physical sunscreen because it reflects and scatters UVB and UVA in sunlight. However, TiO2 absorbs about 70% of incident UV, and in aqueous environments this leads to the generation of hydroxyl radicals which can initiate oxidations. Using chemical methods, we show that all sunscreen TiO2 samples tested catalyse the photo‐oxidation of a representative organic substrate (phenol). We also show that sunlight‐illuminated TiO2 catalyses DNA damage both in vitro and in human cells. These results may be relevant to the overall effects of sunscreens.
Atomic force microscopy for studying gene transfection mediated by cationic liposomes with a cationic cholesterol derivative
Authors: Kawaura Chiyo , Noguchi Ari , Furuno Tadahide , Nakanishi Mamoru
Published: July 2002

Atomic force microscopy (AFM) was used for studying gene transfection mediated by cationic liposomes which contain a cationic cholesterol derivative with a different spacer arm. Cationic liposomes were made by a mixture of one of eight cationic cholesterol derivatives and 1,2‐dioleoyl‐sn‐glycero‐3‐phosphatidyl ethanolamine (DOPE). AFM images showed that vesicles made of the liposome/DNA complex had various diameters depending on each cationic cholesterol derivative with a different spacer arm. The results showed that the diameter of the liposome/DNA complex was well related to the transfection activity of plasmid pSV2CAT DNA to a cultured cell line (NIH3T3). From the results it was found that the vesicles with moderate diameters (from 0.4 to 1.4 μm) were moste effective for gene transfection of plasmid pSV2CAT DNA into the target cell. Neither smaller vesicles (<400 nm) nor larger vesicles (>1.4 μm) were adequate for gene transfection. As the gene transfection by the cationic liposomes was mostly inhibited by wortmannin, an inhibitor of endocytosis, it is suggested that the vesicles with moderate diameters were useful for gene transfection by endocytosis.
Spermine binding to liver mitochondria deenergized by ruthenium red plus either FCCP or antimycin A
Authors: Dalla Via Lisa , Di Noto Vito , Toninello Antonio
Published: July 2002

Thermodynamic analysis of spermine binding to mitochondria treated with ruthenium red and deenergized with either FCCP or antimycin A confirms the presence of two polyamine binding sites, S1 and S2, both with monocoordination, as previously observed in energized mitochondria [Dalla Via et al., Biochim. Biophys. Acta 1284 (1996) 247–252]. Both sites undergo a marked change in binding capacity and binding affinity upon mitochondrial deenergization. This change is most likely responsible for the incomplete or delayed spermine‐mediated inhibition of the permeability transition induced in deenergized mitochondria.