bluuebunny/crossref_metadata_2025 huggingface dataset:
Analyses of the co‐localization of cellubrevin and the GLUT4 glucose transporter in rat and human insulin‐responsive tissues
Authors: Aledo J.Carlos , Hajduch Eric , Darakhshan Froogh , Hundal Harinder S
Published: July 2002

We have investigated the subcellular distribution and association of cellubrevin, a low molecular weight protein implicated in the process of membrane fusion, with intracellular membranes containing the insulin‐sensitive GLUT4 glucose transporter from rat adipocytes, rat skeletal muscle and human skeletal muscle. SDS‐PAGE and immunoblot analyses of subcellular fractions of adipocytes and skeletal muscle indicated a positive correlation between the distribution of GLUT4 and cellubrevin in intracellular membrane fractions tested from all tissues. The identity of the polypeptide reacting with antiserum against cellubrevin was further confirmed on the basis of its susceptibility to proteolysis by tetanus toxin. Immunoisolation of GLUT4‐containing vesicles from a microsomal fraction enriched with GLUT4 and cellubrevin revealed that cellubrevin could be coprecipitated with GLUT4 vesicles from adipocytes. In contrast, intracellular GLUT4 vesicles isolated from both rat and human skeletal muscle were devoid of any detectable immunoreactivity towards cellubrevin. The observation that cellubrevin does not colocalise with intracellular GLUT4 in skeletal muscle from two different species, rat and human, would strongly suggest that it is unlikely to participate in the insulininduced delivery of GLUT4 to the cell surface in skeletal muscle.
Quinazoline derivatives suppress nitric oxide production by macrophages through inhibition of NOS II gene expression
Authors: Noriko Fujiwara , Ayako Okado , Seo Han Geuk , Junichi Fujii , Kigen Kondo , Naoyuki Taniguchi
Published: July 2002

We have found three novel quinazolidine derivatives which inhibit the formation of nitrite dose‐dependently in a murine macrophage cell line, RAW264.7. The decreased nitrite formation was due not to the inhibition of nitric oxide synthase activity but to suppression of NOS II mRNA and protein expression. In rat vascular smooth muscle cells (VSMC), however, these compounds rather enhanced NOS II mRNA. These compounds also prevented LPS‐stimulated heme oxygenase‐1 (HO‐1) and cyclooxygenase‐2 (COX‐2) gene expression in RAW264.7 cells, but again not in VSMC. The three quinazolidine derivatives specifically inhibit gene expression of NOS II, HO‐1 and COX‐2 only in macrophage cells, indicating that they are selective inhibitors of inducible gene expression in macrophages.
Steroid RU 486 inducible myogenesis by 10T1/2 fibroblastic mouse cells
Authors: Bartsch Jörg W , Jäckel Monika , Perz Angela , Jockusch Harald
Published: July 2002

For reconstruction or repair of damaged tissues, an artificially regulated switch from proliferation to differentiation would be of great advantage. To achieve conditional myogenesis, we expressed MyoD in mouse C3H 10T1/2 fibroblastic cells, using a gene regulation system based on the synthetic steroid RU 486. By stable co‐transfection of a plasmid construct with the RU 486 dependent activator and an integrating inducible MyoD construct, a cell clone, designated 10T‐RM, was obtained in which MyoD expression was stringently controlled by RU 486. 12 h after addition of 10 nM RU 486 to 10T‐RM cells, saturation levels of MyoD mRNA were observed and ≥2 days later, mRNA for embryonal myosin heavy chain (MyHCemb) was abundant and mononucleated cells fused into myotubes.
Regulation of the biosynthesis of acyl analogs of platelet‐activating factor by purinergic agonist in endothelial cells
Authors: Balestrieri Maria Luisa , Lee Ten-ching
Published: July 2002

We have previously shown that platelet‐activating factor (PAF)‐dependent transacetylase (TA) contains three catalytic activities, namely PAF: lysophospholipid TA (TAL), PAF: sphingosine TA (TAS) and PAF acetylhydrolase. It serves as a modifier of PAF actions by producing different lipid signal molecules. The TAL activity is involved in the biosynthesis of acyl analogs of PAF (acyl‐PAF, 1‐acyl‐2‐acetyl‐sn‐glycero‐3‐phosphocholine, acylacetyl‐GPC) in agonist‐stimulated endothelial cells. In the present investigation, we have studied the mechanism(s) by which the TA activity is regulated in ATP‐treated endothelial cells. We have demonstrated that ATP, and thiol‐modifying agents with ATP, specifically regulate only the TAL part of the TA activities.
Investigation of the active site of Escherichia coli Cu,Zn superoxide dismutase reveals the absence of the copper‐coordinated water molecule. Is the water molecule really necessary for the enzymatic mechanism?
Authors: Sette Marco , Bozzi Manuela , Battistoni Andrea , Fasano Mauro , Paci Maurizio , Rotilio Giuseppe
Published: July 2002

The active site of the Cu,Zn superoxide dismutase from Escherichia coli in the oxidized Cu(II) state has been studied by nuclear magnetic relaxation dispersion (NMRD), optical and nuclear magnetic resonance spectroscopy. The orientation of some metal ligands is different with respect to all the other Cu,Zn superoxide dismutases. Moreover, NMRD measurements demonstrate the lack of a copper‐coordinated water molecule. In spite of these differences the enzymatic activity is still high. Azide also binds copper with normal affinity and induces modifications in the active site comparable to those previously observed in the eukaryotic enzymes. Our results suggest that, in this enzyme, the copper‐coordinated water molecule appears not necessary for the enzymatic reaction. A role for the copper‐coordinated water molecule is discussed in the light of recent crystallographic studies.
Why Ppr1p is a weak activator of transcription
Authors: Pätzold Andreas J , Lehming Norbert
Published: July 2002

Upon uracil depletion, the transcriptional activator Ppr1p stimulates expression of the Saccharomyces cerevisiae URA3 gene only four‐fold. We performed a split‐ubiquitin screen with Tup1p as bait, and we found that the global repressor Tup1p interacts with the transcriptional activator Ppr1p both in vivo and in vitro. The interaction is biologically significant, since the deletion of the TUP1 gene as well as the removal of the Tup1p‐binding domain from Ppr1p results in an increased expression of the URA3 gene. Our results suggest that Tup1p blocks Ppr1p directly, and that Ppr1p is a weak activator of transcription because of its interaction with Tup1p. Thus we were able to demonstrate that the global repressor Tup1p can modulate transcription by interacting with an activator.
Studies on endothelin receptors in the zonae fasciculata/reticularis of the rat adrenal cortex: contrast with the zona glomerulosa
Authors: Kapas S. , Cameron L.A. , Puddefoot J.R. , Hinson J.P.
Published: July 2002

This study investigated the ET
A
and ET
B
receptor subtypes in rat adrenal cortex. The ET
A
antagonist, BQ‐123, inhibited the zona glomerulosa (zg), but not the inner zone (iz) response to ET‐1. RES‐701‐1, the ET
B
antagonist, abolished the iz response to ET‐1, but had less effect on the zg. [125I]ET‐1 binding studies revealed two receptor subtypes in both zones, with ET
A
predominating in the zg, and ETB in the iz. These data suggest that the ET
A
subtype is functionally more important in the zg while the ET
B
receptor is the major subtype in the inner zones.
The role of structural domains in RIP II toxin model membrane binding
Authors: Agapov Igor I , Tonevitsky Alexander G , Shamshiev Abdijapar T , Pohl Elena , Pohl Peter , Palmer Rex A , Kirpichnikov Michail P
Published: July 2002

The interaction of plant toxin ricin and MLI binding subunits to liposomes containing monosialoganglioside (GM1), bearing a terminal galactose residue, has been examined as a possible receptor model. For the first time we demonstrate that ricin B‐chain but not ricin provokes liposome aggregation at 10 M% GM1 concentration, whereas in the presence of either ricin A‐chain or galactose the aggregation is inhibited. The B‐subunit of plant toxin MLI from Viscum album has similar lectin specificity and activity but cannot aggregate GM1 liposomes. The ability of the B‐chain to aggregate liposomes adds a new crucial step in the toxin transmembrane penetration mechanism. We demonstrate here possible ricin B‐chain interactions with membranes proceeding via two sites, namely (a) a galactose‐binding domain and (b) a hydrophobic interchain domain. In close contact with two phospholipid bilayers, ricin B‐chain may determine the geometry of the fusion site. These events can provoke A‐chain translocation which follows membrane fusion.
Ap3A and Ap4A are primers for oligoadenylate synthesis catalyzed by interferon‐inducible 2–5A synthetase1
Authors: Turpaev Kyril , Hartmann Rune , Kisselev Lev , Justesen Just
Published: July 2002

The biological role of Ap3A synthesized in cells by tryptophanyl‐tRNA synthetase (WRS) is unknown. Previously we have demonstrated that the cellular level of Ap3A significantly increases after interferon treatment. Here we show that the human 46 kDa 2‐5A synthetase efficiently utilizes Ap3A as a primer for oligoadenylate synthesis. The K
m for Ap3A is several‐fold lower than for Ap4A and 100‐fold lower than for ATP. This implies that Ap3A might be a natural primer for the 2′‐adenylation reaction catalysed by 2‐5A synthetase. Since WRS and 2‐5A synthetase are both interferon‐inducible proteins, a new link between two interferon‐dependent enzymes is established.
Antibodies to rat soluble IL‐6 receptor stimulate B9 hybridoma cell proliferation
Authors: Thibault Vincent , Richards Carl D , Botelho Fernando , Gauldie Jack
Published: July 2002

Interleukin‐6 mediates its pleiotropic effects by interacting with its membrane bound receptor (gp80) or the soluble counterpart gp54, resulting in activation of a complex that includes the transducer protein gp130. We have generated a polyclonal antibody against the rat soluble IL‐6 receptor (anti‐rat sIL‐6R) in rabbits. By Western blot analysis we show that purified anti‐rat sIL‐6R IgG antibody reacts specifically with recombinant rat sIL‐6R generated from E. coli, baculovirus or adenovirus expression systems. Anti‐rat sIL‐6R inhibited IL‐6‐induced acute phase protein synthesis in rat (H35) but not human (HepG2) hepatoma cells, and did not affect stimulation of those cells by Oncostatin‐M. Conversely, on the mouse hybridoma B9 cell line, IgG anti‐rat sIL‐6R showed a dose‐dependent stimulation of proliferation. Fab fragments of this antibody did not stimulate, but abrogated IL‐6‐mediated hepatoma cell stimulation and B9 cell proliferation. Gel shift analysis of STAT nuclear factors showed activation of STAT DNA binding in nuclei of B9 cells treated with IgG anti‐rat sIL‐6R, whereas in H35, NIH‐3T3 and M1 cells, only IL‐6 could trigger a similar STAT activation. Our data suggest that mechanisms of IL‐6 receptor activation and signalling in mouse B9 hybridoma cells show subtle but important differences from other IL‐6‐responsive cells.
Insulin activates a PD 098059‐sensitive kinase that is involved in the regulation of p70S6K and PHAS‐I
Authors: Scott Pamela H , Lawrence John C
Published: July 2002

Incubating either Chinese hamster ovary (CHO) cells or 3T3‐L1 adipocytes with insulin increased the phosphorylation of the eIF‐4E‐binding protein, PHAS‐I. Insulin also activated p70S6K and the Erk‐1 and Erk‐2 isoforms of mitogen‐activated protein kinase (MAP kinase). However, the concentrations of the hormone needed to activate MAP kinase were 10–100 times higher than those needed to increase PHAS‐I phosphorylation and p70S6K activity. Incubating cells with the inhibitor of MAP kinase kinase (MEK) activation, PD 098059, blocked the effects of low concentrations of insulin on PHAS‐I and p70S6K. The effects of the inhibitor were overcome by increasing concentrations of insulin. The results indicate that insulin activates a PD 098059‐sensitive kinase that is involved in the regulation of both p70S6K and PHAS‐I.
Effect of triphosphate modifications in 2′‐deoxynucleoside 5′‐triphosphates on their specificity towards various DNA polymerases
Authors: Martynov Boris I , Shirokova Elena A , Jasko Maxim V , Victorova Lyubov S , Krayevsky Alexander A
Published: July 2002

Some natural and glycon‐modified dNTPs with β,γ‐pyrophosphate substitution at the triphosphate residue were synthesized and studied to evaluate the effect of these modifications on substrate properties of dNTPs in DNA synthesis catalyzed by human placental DNA polymerases α and β, avian myeloblastosis virus reverse transcriptase, and calf thymus terminal deoxynucleotidyl transferase. Reverse transcriptase proved to be the enzyme least specific to such modifications; the substrate activity of β,γ‐methylenediphosphonate substituted dTTP and 3′‐azido‐3′‐deoxy‐dTTP decreased in the following order: CF2=CHF>CBr2>CFMe≫CH2. This order is individual for each DNA polymerase. It is interesting to mention that β,γ‐CBr2 substituted dTTP is neither a substrate nor an inhibitor of DNA polymerase β. This specificity distinguishes DNA polymerase β from other DNA polymerases studied.
GroEL provides a folding pathway with lower apparent activation energy compared to spontaneous refolding of human carbonic anhydrase II
Authors: Persson Malin , Carlsson Uno , Bergenhem Nils
Published: July 2002

The kinetics of the refolding of the enzyme, human carbonic anhydrase II (HCA II), at different temperatures, together with the Escherichia coli chaperonin GroEL, has been studied. The Arrhenius plots for the spontaneous, GroEL‐assisted, and GroEL/ES‐assisted refolding of HCA II show that the apparent activation energy (E
a) is lower in the presence of the chaperonin GroEL alone than for the spontaneous reaction, whereas the apparent activation energy for the GroEL/ES‐assisted reaction is almost the same as for the spontaneous reaction (85, 46, and 72 kJ/mol, for the spontaneous, GroEL, and GroEL/ES‐assisted reactions, respectively).
Alternative activation of transcriptional initiators in Drosophila melanogaster LINE promoters
Authors: Minchiotti Gabriella , Di Nocera Pier Paolo
Published: July 2002

In the Drosophila I, F and Doc LINEs, basal transcription is ensured by the functional interaction of initiator sequences with intragenic regulatory segments (B regions) which comprise distinct functional modules. Removing the B regions, as changing their composition or location, allowed different activators to stimulate transcription from novel initiators both in Doc and F promoters. The use of distinct initiators plausibly reflects the assembly of transcriptional complexes in which TFIID assumes alternative spatial conformations. The response of I, F and Doc promoters to the same enhancer is significantly influenced by the number, position and type of core elements present.
EPR spectroscopy of Escherichia coli cytochrome bo which lacks CuB
Authors: Hunter Dominic J.B , Moody A.John , Rich Peter R , Ingledew W.John
Published: July 2002

The spectroscopic and ligand‐binding properties of a copper‐deficient cytochrome bo3
, a member of the haem–copper superfamily of terminal oxidases, are reported and contrasted with those of the native enzyme. The enzyme lacks the copper atom (CuB) which is normally an integral part of the catalytic site. The consequences of loss of the CuB are the loss of antiferromagnetic coupling to the high‐spin haem and an inability to form any of the integer‐spin derivatives of the enzyme. Low‐spin compounds of the normally high‐spin haem are still formed with appropriate ligands, although these are modified.
HIV‐1 envelope protein, gp120, has no effects on inositol phosphate production and metabolism in the Jurkat T‐cell line either in the presence or absence of receptor stimulation
Authors: Sumner Martina T , Shears Stephen B
Published: July 2002

We have used HPLC techniques to investigate the effects of gp120 upon inositol phosphate turnover in Jurkat E6‐1 CD4+ T‐cells, to pursue previous reports that this viral coat protein: (a) inhibits receptor‐activated inositol phosphate release; (b) stimulates basal inositol phosphate release; (c) inhibits inositol polyphosphate 5‐phosphatase. Treatment of cells with up to 10 μg/ml gp120 from between 10 min and 24 h was without effect upon inositol phosphate turnover in both basal cells, and in C305 and OKT3 stimulated cells. This is the first report that biologically competent gp120 does not affect any aspect of inositol phosphate turnover in either basal or receptor‐activated lymphocytes.
Identification of potential ferric binding residues in the iron‐binding protein of pathogenic Neisseria meningitidis through structure‐based multiple sequence alignments
Authors: Gorinsky Beatrice , Evans Robert W.
Published: July 2002

The ferric iron‐binding proteins of pathogenic Neisseria display structural and metal‐binding properties characteristic of the transferrin family. In the absence of structural data for the ferric iron‐binding proteins, spacial folding templates have been derived for the meningococcal protein from complete and partial structure‐based multiple sequence alignments with structurally related proteins. The templates have been used to identify a number of potential iron‐binding residues. These include four residues that are identical with the iron coordinating ligands of transferrin, but only two reside within equivalent structural elements.
Regulation of prelamin A endoprotease activity by prelamin A
Authors: Kilic Fusun , Salas-Marco Joe , Garland John , Sinensky Michael
Published: July 2002

The maturation of lamin A is completed by the endoproteolytic cleavage of its farnesylated precursor protein, prelamin A. In the absence of this cleavage, prelamin A can neither give rise to lamin A nor assemble into the nuclear lamina. We call the enzyme which catalyzes this endoproteolytic step the `prelamin A endoprotease'. In this study, we begin characterization of the regulation of prelamin A endoprotease. In particular, we address the question as to whether prelamin A endoprotease activity is constitutive in cells or responds to expression of prelamin A. To do this, we compared the activity of this novel endoprotease in cells which express prelamin A with those that do not. Our data shows that the enzymatic activity of prelamin A endoprotease is enhanced by the expression of prelamin A.
Prediction of substrate‐specific pockets in cyclosporin synthetase
Authors: Husi Holger , Schörgendorfer Kurt , Stempfer Günter , Taylor Paul , Walkinshaw Malcolm D
Published: July 2002

Amino acid sequence comparisons between domains of cyclosporin synthetase have been used to identify regions of the sequence which are responsible for the recognition and binding of the individual amino acids. Using a limited set of selection rules it was possible to identify three amino acid positions in the subdomain sequences which are responsible for amino acid specificity. Homology with the firefly luciferase protein shows that these three key residues are close to each other and line the surface of a putative specific substrate binding pocket located on the amino acyl‐adenylation subdomain. These results allow us to predict a large number of cyclosporin synthetase mutants which could be used to synthesise alternative cyclosporin‐like peptides.
Crystallization and preliminary X‐ray analysis of arrestin from bovine rod outer segment
Authors: Wilden U. , Choe H.-W. , Krafft B. , Granzin J.
Published: July 2002

We present the first X‐ray study of a member of the arrestin family, the bovine retinal arrestin. Arrestin is essential for the fine regulation and termination of the light‐induced enzyme cascade in vertebrate rod outer segments. It plays an important role in quenching phototransduction by its ability to preferentially bind to phosphorylated light‐activated rhodopsin. The crystals diffract between 3 Å and 3.5 Å (space group P21212, cell dimensions a=169.17 Å, b=185.53 Å, c=90.93 Å, T=100 K). The asymmetric unit contains four molecules with a solvent content of 68.5% by volume.
Definitive chemical evidence for the constitutive ability of Candida albicans serotype A strains to synthesize β‐1,2 linked oligomannosides containing up to 14 mannose residues
Authors: Trinel P.A. , Lepage G. , Jouault T. , Strecker G. , Poulain D.
Published: July 2002

We have previously reported the presence of phosphate bound β‐1,2 linked oligomannosides with unusually high degrees of polymerization (DP>7) in the mannan of Candida albicans strain VW32. To confirm this observation, we have prepared these oligomannosides from the mannan of C. albicans strain NIH A 207. Gel filtration chromatography and TLC analysis revealed DP up to 14. For both strains, NMR analysis confirmed the exclusive presence of β‐1,2 linkages in the pools of oligomannosides with a DP higher than 6 which presented an average DP of 10.6 (VW32) and 10.4 (NIH A 207). These results are important to consider in relation with the ability of these C. albicans derived oligomannosides to trigger TNFα synthesis according to their DP.
Positive autoregulation of ras genes expression in fibroblasts
Authors: Quincoces Ana F. , Polanco Isidoro , Thomson Timothy , León Javier
Published: July 2002

We have studied the effect of ectopic overexpression of a ras gene on the expression of the other two members of the ras gene family. We obtained NIH3T3 cell lines stably transfected with inducible H‐ras and N‐ras oncogenes. The expression of these genes is driven by a glucocorticoid‐responsive promoter and the addition of dexamethasone resulted in a dramatic induction (10–20‐fold) of H‐ or N‐ras mRNA, peaking 4 h after hormone addition. The induction of the expression of ras oncogenes resulted in a transformed phenotype. In quiescent NIH3T3 cells transfected with inducible H‐ras oncogenes, the induction of H‐Ras was followed 12 h later by a 3‐fold increase in the mRNA expression of endogenous K‐ras and N‐ras. Similarly, in NIH3T3 transfected with inducible N‐ras oncogene, the induction of N‐ras was followed by an increase in the expression of endogenous K‐ and H‐ras genes. Interestingly, the effect was not limited to the mutated N‐ras, as a similar result was obtained in cells transfected with N‐ras proto‐oncogene. The induction of ras genes expression was not linked to cell cycle progression as it was reproduced in cells arrested in S‐phase by pretreatment with hydroxyurea. These results suggest the presence of a positive cross‐regulation in the expression among the members of the Ras family. This effect could play a role in Ras‐mediated carcinogenesis.
Changes in anion permeability following hypotonic challenge in rat brain endothelial cells: different responses in primary cultures and in immortalised RBE4 cells
Authors: von Weikersthal S.F , Seetharaman S , Wright K.A , Roux F.A , Barrand M.A
Published: July 2002

Hypotonicity‐induced anion permeability changes were investigated but not detected in immortalised (RBE4) rat brain endothelial cells using iodide efflux measurements. Large, rapid increases were however observed in primary cultured cells. Both cell types were reinvestigated following culture in a common growth factor‐depleted medium. Responses were still undetectable in the immortalised RBE4 cells. Reduced responses were observed in the primary cultured cells that also showed altered morphology and decreased activity of another transporter, P‐glycoprotein. Thus both immortalisation and different culture conditions may alter functional expression in these cells of transporters involved in hypotonicity‐induced anion permeability changes.
Small ligands that neither bind to nor alter the structure of d(GA·TC)n sequences in DNA
Authors: Vaquero Alejandro , Portugal José
Published: July 2002

Three minor‐groove binding ligands have been used to study the characteristics of two d(GA·CT)
n
DNAs embedded in longer DNA fragments. The binding of mithramycin, netropsin or Thia‐Net to these sequences has been studied using DNAse I footprinting. None of these ligands appeared to bind to d(GA·CT)5 nor to d(GA·CT)22 extensively, although with mithramycin some protected bonds were detected at the very edge of these sequences. In general, these small ligands did not enhance the DNAse I cleavage patterns at the alternating d(GA·CT)
n
flanking sequences located near DNA regions where the drug was bound. The d(GA·CT)
n
sequences could act as a rigid block in which it is not easy to propagate structural changes, whereas other sequences flanking the binding sites showed cleavage enhancements.
Secretion, purification and activity of two recombinant pepper endo‐β‐1,4‐glucanases expressed in the yeast Pichia pastoris
Authors: Ferrarese L , Trainotti L , Gattolin S , Casadoro G
Published: July 2002

Two pepper endo‐β‐1,4‐glucanases, involved in fruit softening (cCel1) and leaf and flower abscission (cCel2), have been expressed in Pichia pastoris. Secretion was obtained by using either the mouse α‐factor signal (cCel1) or the native signal sequence (cCel2). Times for optimal expression of the two proteins were different and cCel2 appeared very sensitive to proteolytic degradation. A one‐step purification protocol yielded cCel2 in a pure form, while an additional chromatography step was necessary to purify cCel1. The two recombinant proteins are highly active and able to degrade carboxymethylcellulose in viscometric assays. Moreover, they have both a molecular mass (54 kDa) and an isoelectric point (7.2 for cCel2 and 8.5 for cCel1) equal to those of the native proteins, thus suggesting that post‐translational modifications have properly occurred.
Effect of pea and bovine trypsin inhibitors on wild‐type and modified trypsins
Authors: Pouvreau Laurice , Chobert Jean-Marc , Briand Loı̈c , Quillien Laurence , Tran Vinh , Guéguen Jacques , Haertlé Thomas
Published: July 2002

In order to modify the catalytic properties of trypsin, lysine‐188 (S1) of the substrate binding pocket was substituted by an aromatic amino acid residue (Phe, Tyr, Trp) or by a histidyl residue. Two other mutants were obtained by displacement or elimination of the negative charge of aspartic acid‐189 (K188D/D189K and G187W/K188F/D189Y, respectively). The high affinity inhibitors, like PSTI II and BPTI, behaved as specific substrates of the trypsin and its mutants. Their inhibiting effect toward modified trypsins was studied. The bovine inhibitor had a higher affinity for all tested enzymes than pea inhibitor. The inhibition constants differed according to the mutations on the protease.
Phospholamban domain I/cytochrome b5 transmembrane sequence chimeras do not inhibit SERCA2a
Authors: Kimura Yoshihiro , Asahi Michio , Kurzydlowski Kazimierz , Tada Michihiko , MacLennan David H
Published: July 2002

A series of chimeras between the transmembrane domains of phospholamban (PLN) and cytochrome b
5 were coexpressed with the Ca2+‐ATPase of cardiac sarcoplasmic reticulum (SERCA2a). The chimeric molecules were not inhibitory, in line with our view that inhibitory PLN/SERCA2a interactions occur in transmembrane sequences, while cytoplasmic interactions regulate the inhibitory interactions in a four‐base circuit.
Somatic and germline clone analysis in mutants of the S‐adenosylmethionine synthetase encoding gene in Drosophila melanogaster
Authors: Larsson Jan , Rasmuson-Lestander Åsa
Published: July 2002

We have analysed the phenotypic consequences of homozygous mutant clones in the S‐adenosylmethionine synthetase encoding gene in Drosophila melanogaster. The results suggest that SamS function is required for cell proliferation/growth in embryonic/early larval cells and during development of imaginal disc cells. Homozygous SamS germline clones can, however, develop and give rise to viable heterozygous offspring. This offspring expresses a Minute‐like phenotype. We suggest that this phenotype is caused by an obstruction of the polyamine biosynthesis.
Cytoskeleton‐dependent inhibition of the ADP‐ribosyl cyclase activity of CD38 in thrombin‐stimulated platelets
Authors: Torti Mauro , Tolnai Festetics Enrico , Bertoni Alessandra , Sinigaglia Fabiola , Balduini Cesare
Published: July 2002

Stimulation of human platelets with thrombin caused a 42% inhibition of the ADP‐ribosyl cyclase activity of membrane CD38. This effect was mediated by the activation of the platelet thrombin receptor rather than by proteolysis of CD38, and was not due to a different distribution of the synthesised nucleotide or to a reduced accessibility of CD38 to the substrate. The inhibitory effect of thrombin required actin polymerisation and was not observed when interaction of CD38 with the cytoskeleton was prevented by cytochalasin D. Finally, we analysed whether cADPR could play a role as a Ca2+‐mobilising agent in human platelets. Using saponin‐permeabilised cells, we found that unlike IP3, cADPR did not induce any release of Ca2+ from intracellular stores. These results indicate that the enzymatic activity of membrane CD38 can be modulated by platelet activation, and that the function of this glycoprotein is probably not related to Ca2+ mobilisation.
Involvement of endotoxins or tumor necrosis factor‐α in macrophage‐mediated oxidation of low density lipoprotein
Authors: Fujiwara Kyoko , Sato Hideyo , Bannai Shiro
Published: July 2002

Oxidation of low‐density lipoprotein (LDL) can be mediated in vitro by cultured cells, including macrophages. This cellular oxidation is dependent on the production of free thiols by the cells in the presence of transition metal ions. We now report that the production of thiols by macrophages is greatly enhanced when cells are cultured with lipopolysaccharide (LPS) or tumor necrosis factor‐α (TNF‐α). Oxidation of LDL by macrophages is markedly augmented by pre‐treatment of the cells with LPS or TNF‐α. The results suggest that activation by endotoxins or TNF‐α is a necessary step for macrophages to mediate oxidation of LDL.
Design and synthesis of double‐stranded oligonucleotides containing reactive acylphosphate internucleotide groups
Authors: Kuznetsova S.A , Kanevsky I.E , Shabarova Z.A
Published: July 2002

DNA duplex and dumbbells containing chemically active acylphosphate internucleotide groups were synthesized. To obtain these compounds the chemical ligation method was used. The acylphosphate group was inserted into a DNA duplex and dumbbells as a result of template‐directed condensation of 5′‐phosphate and especially introduced 3′‐carboxy groups of oligonucleotides. 1‐Ethyl‐3‐(3′‐dimethylaminopropyl)carbodiimide (EDC) was used as a condensing agent. Oligonucleotides containing a carboxy group were obtained by the interaction of their 3′‐phosphate with glycine methyl ester under the action of EDC, followed by ester hydrolysis. The yields of acylphosphate‐containing double‐stranded oligonucleotides achieved 15–25% depending on the structure of their precursors. It was shown that these compounds are acylating agents and are efficiently cleaved in near‐physiological conditions under the action of ethylenediamine or N‐methylimidazole. These results indicate that double‐stranded oligonucleotides carrying acylphosphate internucleotide groups could constitute new crosslinking reagents for affinity modification of DNA recognizing proteins.
Alteration of mitochondrial DNA and RNA level in human fibroblasts with impaired vitamin B12 coenzyme synthesis
Authors: Cantatore Palmiro , Petruzzella Vittoria , Nicoletti Cinzia , Papadia Francesco , Fracasso Flavio , Rustin Pierre , Gadaleta Maria Nicola
Published: July 2002

Alterations of mitochondrial (mt) nucleic acid metabolism in methylmalonic aciduria (MMA) were studied in two cell lines from skin fibroblasts of patients with mitochondrial (GM00595) or cytosolic (GM10011) defects in the biosynthesis pathways of cobalamin coenzymes. The mtDNA level increased two‐fold in GM00595 cells, which carry a mt defect in the adenosylcobalamin synthesis, whereas no appreciable change was found in GM10011 cells. The content of the two rRNAs 16S and 12S mtRNAs, normalized for the mtDNA copy number, decreased by 70% and 50% in GM00595 and GM10011, respectively. The normalized content of ND1, ND2 and CO I mRNAs decreased in GM00595, but was unchanged in GM10011. Respiratory chain complex activities measured in these two cell lines were not different from control activities. These data suggest that the maintenance of the mt function is due to doubling of mtDNA and that this compensatory response takes place only in those cells in which the greater reduction of the level of rRNA might have brought the content of these transcripts below the threshold value for optimal expression of the mt genome.
Anisoosmotic regulation of the Mi‐2 autoantigen mRNA in H4IIE rat hepatoma cells and primary hepatocytes
Authors: Hammermann Rainer , Warskulat Ulrich , Häussinger Dieter
Published: July 2002

Using the differential display polymerase chain reaction (DDRT‐PCR) a 169 bp cDNA product, which is 88.8% homologous to the human Mi‐2β autoantigen, was identified in H4IIE rat hepatoma cells. At protein level 100% homology was found. The Mi‐2 mRNA was downregulated after hypoosmotic exposure and upregulated after hyperosmotic exposure in H4IIE cells and rat hepatocytes. The human Mi‐2 is an autoantigen in dermatomyositis and is a member of the SNF/RAD 54 helicase family. Accordingly, Mi‐2 may not only be a target of osmosignalling but could also be involved in the osmosignalling pathway towards gene expression in H4IIE and liver parenchymal cells.
Probing pore topology and conformational changes of Kir2.1 potassium channels by cysteine scanning mutagenesis
Authors: Kubo Yoshihiro , Yoshimichi Murata , Heinemann Stefan H
Published: July 2002

Using cysteine (Cys) scanning mutagenesis of the inward rectifier K+ channel Kir2.1, we investigated its pore structure and putative conformational changes. In the background of the Kir2.1 mutant C149F which showed no sensitivity towards Cys‐modifying reagents, Cys residues were introduced at 10 positions in the H5 pore region. Out of six functional mutants, T141C and F147C showed clear changes in current amplitude when Cys‐modifying reagents were applied from the external side. These results suggest that the corresponding sections of the H5 pore region face to the external side which is in contrast to the results previously obtained for voltage‐gated K+ (Kv) channels. Using the mutants T141C and F147C, we investigated whether or not Kir2.1 channels show state‐dependent conformational changes of the pore structure. Substantial alterations of the holding potential or external K+ concentration, however, did not cause any significant change in the speed of channel modification upon application of Cys‐specific reagents, suggesting that Kir2.1 channels do not undergo conformational changes, in contrast to C‐type inactivating Kv channels.
A conserved aspartate is essential for FAD binding and catalysis in the d‐amino acid oxidase from Trigonopsis variabilis
Authors: Ju Sheau-Shya , Lin Long-Liu , Wang Wen-Ching , Hsu Wen-Hwei
Published: July 2002

To evaluate the possible contribution of Asp206 of Trigonopsis variabilis d‐amino acid oxidase (DAO) to its flavin adenine dinucleotide (FAD) binding and catalytic function, six mutant enzymes were constructed by site‐directed mutagenesis. Western immunoblot analysis revealed that a protein with an apparent molecular mass of about 39.2 kDa was present in the cell‐free extracts of wild‐type and mutant strains. Replacement of Asp206 with Leu, Gly, and Asn resulted in the loss of DAO activity and characteristic absorption spectrum for flavoenzyme, while the other mutant DAOs, Asp206Glu, Asp206Ser, and Asp206Ala, exhibited a similar spectral profile to that of wild‐type enzyme and retained about 6–90% of the enzyme activity. These results suggested that Asp206 of T. variabilis DAO might play an important role in the binding of FAD.
Distribution of the mammalian‐wide interspersed repeats (MIRs) in the isochores of the human genome
Authors: Matassi Giorgio , Labuda Damian , Bernardi Giorgio
Published: July 2002

The distribution of MIRs (mammalian‐wide interspersed repeats) was investigated in 164 human sequences (≥100 kb), which were assigned, according to their GC level, to isochore families L, H1, H2 and H3. MIR elements, whose total number in the genome was estimated to be about 3.3×105, were found to be unevenly distributed in human isochores. The majority of MIRs (55%) were found in the L isochore family. In contrast, MIR density was highest in H2, closely followed by H1, whereas densities in L and H3 were 2‐ and 3‐fold lower than in H2, respectively. For this reason, the assessment of MIR distribution by inter‐repeat PCR led to an overestimation of MIR numbers in H2 isochore and an underestimation in L isochores.
ACTA, a fluorescent analogue of thapsigargin, is a potent inhibitor and a conformational probe of skeletal muscle Ca2+‐ATPase
Authors: Procida Kristina , Caspersen Casper , Kromann Hasse , Brøgger Christensen Søren , Treiman Marek
Published: July 2002

Thapsigargin is a highly potent and selective inhibitor of sarco‐endoplasmic reticulum (SERCA) family of Ca2+‐ATPases and a useful tool in research concerning the function of intracellular Ca2+ stores. We describe here a novel fluorescent derivative (8‐O‐(4‐aminocinnamoyl)‐8‐O‐debutanoylthapsigargin, termed ACTA) of this compound, acting as a Ca2+‐ATPase inhibitor with a potency approaching that of thapsigargin. Binding of ACTA to the skeletal muscle sarcoplasmic reticulum vesicles results in a strong fluorescence enhancement, approximately 66% of which depends on ACTA association with Ca2+‐ATPase. This specific component of ACTA fluorescence is sensitive to the E1‐E2 conformational equilibrium of the pump. The combined properties of high potency and binding‐dependent fluorescence suggest ACTA to be a useful probe for a range of studies involving the SERCA class of ATPases.
9‐Hydroxyellipticine inhibits telomerase activity in human pancreatic cancer cells
Authors: Sato Norihiro , Mizumoto Kazuhiro , Kusumoto Masahiro , Niiyama Hideaki , Maehara Naoki , Ogawa Takahiro , Tanaka Masao
Published: July 2002

There is increasing interest in identifying potent inhibitors of telomerase because the enzyme plays a crucial role in the development of cellular immortality and carcinogenesis. We hypothesized that 9‐hydroxyellipticine (9‐HE), an antitumor alkaloid, would inhibit telomerase activity because the drug has a unique mechanism of inhibiting phosphorylation of mutant p53 protein via inhibition of protein kinases, thereby restoring wild‐type p53 function. This study was conducted to examine the effect of 9‐HE on telomerase activity in human pancreatic cancer cells with differing p53 gene status. 9‐HE treatment at relatively high concentrations resulted in rapid, complete inhibition of telomerase activity, irrespective of the p53 status. We conclude that 9‐HE may exert a strong inhibitory effect on telomerase activity possibly through inhibition of protein kinases rather than through restoration of functional wild‐type p53.
Glucose induces glucose 6‐phosphatase hydrolytic subunit gene transcription in an insulinoma cell line (INS‐1)
Authors: Schmoll Dieter , Watkins Sharlene L , Wasner Christina , Walther Reinhard , Burchell Ann
Published: July 2002

Primer extension analysis and RNase protection assays revealed the identity of glucose 6‐phosphatase gene transcripts in both the insulinoma cell line INS‐1 and hepatic cells. In transient transfection assays of INS‐1 cells, using constructs between the human glucose 6‐phosphatase gene promoter and a luciferase reporter gene, the reporter gene activity was induced by dexamethasone and dibutyryl cAMP. Furthermore, the promoter was regulated by the glucose concentration in the medium. This effect was dependent on glucose metabolism. The data indicated that glucose 6‐phosphatase gene transcription is regulated in a similar way in the insulinoma cell line and in liver.
Regulation of c‐fos gene transcription and myeloid cell differentiation by acute myeloid leukemia 1 and acute myeloid leukemia‐MTG8, a chimeric leukemogenic derivative of acute myeloid leukemia 1
Authors: Hwang Eun Sook , Hong Jeong-Ho , Bae Suk Chul , Ito Yoshiaki , Lee Seung Ki
Published: July 2002

Both acute myeloid leukemia 1 and c‐Fos are regulatory factors of hematopoietic cell differentiation. We identified that the c‐fos promoter contains an acute myeloid leukemia 1 binding site at nucleotide positions −6–+14. c‐fos promoter activity was induced by transient overexpression of acute myeloid leukemia 1 in Jurkat T‐cells, but not by that of the short form of acute myeloid leukemia 1‐MTG8, a chimeric acute myeloid leukemia 1 protein. In 32Dcl3 myeloid cells, stable overexpression of acute myeloid leukemia 1‐MTG8 blocked the c‐fos gene transcription and cell differentiation, but that of acute myeloid leukemia did not. These data suggest that acute myeloid leukemia 1 and acute myeloid leukemia 1‐MTG8 reciprocally regulate the myeloid cell differentiation, possibly by the way of regulating c‐fos gene transcription.
Isolation of cDNA clones encoding two isoforms of nucleoside diphosphate kinase from bovine retina
Authors: Karaschuk Galina , Kakuev Dmitry
Published: July 2002

The cDNA encoding bovine retinal isoforms of nucleoside diphosphate kinase (NDP‐kinase, EC 2.7.4.6) has been cloned and sequenced. Based on the partial amino acid sequence of the enzyme determined after trypsin digestion of purified NDP‐kinase, primers were synthesized and used to isolate two different cDNA clones encoding the full length of two NDP‐kinase isoforms. The nucleotide sequences of these clones contained open reading frames encoding 152‐residue polypeptides with calculated molecular masses of 17.262 and 17.299 kDa, similar to that determined for the subunits of purified enzyme (17.5 and 18.5 kDa). The deduced NDP‐kinase sequences showed high similarity with the known NDP‐kinase sequences from other sources.
A low rate of nucleotide changes in Escherichia coli K‐12 estimated from a comparison of the genome sequences between two different substrains
Authors: Itoh Takeshi , Okayama Toshitsugu , Hashimoto Hiroyuki , Takeda Jun-ichi , Davis Ronald W. , Mori Hirotada , Gojobori Takashi
Published: July 2002

Two genome sequences of Escherichia coli K‐12 substrains, one partial W3110 and one complete MG1655, have been determined by Japanese and American genome projects, respectively. In order to estimate the rate of nucleotide changes, we directly compared 2 Mb of the nucleotide sequences from these closely‐related E. coli substrains. Given that the two substrains separated about 40 years ago, the rate of nucleotide changes was estimated to be less than 10−7 per site per year. This rate was supported by a further comparison between partial genome sequences of E. coli and Shigella flexneri.
Genetic heterogeneity of the immunogenic viral capsid protein region of human parvovirus B19 isolates obtained from an outbreak in a pediatric ward
Authors: Takahashi Naoko , Takada Norio , Hashimoto Takashi , Okamoto Takashi
Published: July 2002

Whereas human parvovirus B19 commonly infects children and causes erythema infectiosum, it causes more severe diseases when it infects adults. In order to examine whether different clinical outcomes of B19 infection can be ascribed to the viral genetic heterogeneity, we have determined the nucleotide sequence of highly immunogenic portions of the B19 genome obtained from six patients with various clinical manifestations in a single outbreak. Our observations demonstrated that although the B19 sequences showed a significant heterogeneity, it was not correlated with the clinical manifestation. It was thus suggested that the host immune response to B19 infection may be a major determinant of clinical presentations associated with acute B19 infection.
Thermodynamic influences on the fidelity of iron‐sulphur cluster formation in proteins
Authors: Armstrong Fraser A , Williams Robert J.P
Published: July 2002

In an organism, two thermodynamic factors are important in ensuring that homometallic [4Fe‐4S] cubane clusters are formed in preference to clusters containing heterometals such as Zn or Cu. These are the electronic resonance stabilisation, which boosts the binding of Fe(II) within an Fe‐S cluster relative to its normally low position in the Irving‐Williams order, and attenuation of the cytoplasmic concentrations of competing metals such as Zn or Cu by specific ligands.
Evolutionary divergence in the broad complex, tramtrack and bric à brac/poxviruses and zinc finger domain from the candidate tumor suppressor gene hypermethylated in cancer
Authors: Guerardel Cateline , Deltour Sophie , Leprince Dominique
Published: July 2002

Hypermethylated in cancer, a new candidate tumor suppressor gene located in 17p13.3, encodes a protein with five Krüppel‐like C2H2 zinc finger motifs and a N‐terminal protein/protein interaction domain called broad complex, tramtrack and bric à brac/poxviruses and zinc finger domain. Hypermethylated in cancer appears unique in the broad complex, tramtrack and bric à brac/poxviruses and zinc finger family since it contains a 13 amino acid insertion located in a loop between the conserved β‐strand β5 and helix α5 which are involved in dimerization and scaffolding of the broad complex, tramtrack and bric à brac/poxviruses and zinc finger domain. Cloning and sequencing of a murine hypermethylated in cancer gene suggests that this insertion has been acquired late in the evolution since it is present in two mammalian hypermethylated in cancer genes but absent in its zebrafish and avian counterparts. This is a unique example of a high divergence of the same broad complex, tramtrack and bric à brac/poxviruses and zinc finger domain in different species.
Localization of acidic residues involved in the proton pumping activity of the bovine heart mitochondrial bc1 complex
Authors: Cocco Tiziana , Di Paola Marco , Papa Sergio , Lorusso Michele
Published: July 2002

Chemical modification of carboxyl residues in polypeptide subunits of the mitochondrial bc1 complex causes a decoupling effect, that is inhibition of the proton pumping activity, without affecting the rate of electron transfer to ferricytochrome c. The study presented here is aimed at localizing and identifying the residues whose modification results in decoupling of the complex. Glutamate‐53 in subunit IX (the DCCD‐binding protein) and aspartate‐166 in the Rieske iron‐sulfur protein are the residues modified by N,N′‐dicyclohexylcarbodiimide (DCCD) and N‐(ethoxycarbonyl)‐2‐ethoxy‐1,2‐dihydroquinoline (EEDQ), respectively. The results obtained also suggest that the carboxy‐terminal sequence of the Core protein II, which is fairly rich in acidic residues, may also play a role in the vectorial proton translocation activity of the complex.
Motifer, a search tool for finding amino acid sequence patterns from nucleotide sequence databases
Authors: Jörnvall Henrik
Published: July 2002

Motifer is a software tool able to find directly in nucleotide databases very distant homologues to an amino acid query sequence. It focuses searches on a specific amino acid pattern, scoring the matching and intervening residues as specified by the user. The program has been developed for searching databases of expressed sequence tags (ESTs), but it is also well suited to search genomic sequences. The query sequence can be a variable pattern with alternative amino acids or gaps and the sequences searched can contain introns or sequencing errors with accompanying frame shifts. Other features include options to generate a searchable output, set the maximal sequencing error frequency, limit searches to given species, or exclude already known matches. Motifer can find sequence homologues that other search algorithms would deem unrelated or would not find because of sequencing errors or a too large number of other homologues. The ability of Motifer to find relatives to a given sequence is exemplified by searches for members of the transforming growth factor‐β family and for proteins containing a WW‐domain. The functions aimed at enhancing EST searches are illustrated by the ‘in silico’ cloning of a novel cytochrome P450 enzyme.