bluuebunny/crossref_metadata_2025 huggingface dataset:
Blockage of a pump‐related calcium‐efflux pathway in light sarcoplasmic reticulum vesicles by Mops
Authors: STEPHAN Sibylle , MIGALA Andrea , HASSELBACH Wilhelm
Published: March 2005

Mops, used as a proton buffer, specifically enhances the accumulation of calcium or strontium by light sarcoplasmic reticulum vesicles driven by ATP or dinitrophenylphosphate as energy‐yielding substrates when calcium‐precipitating agents are absent. The enhancement of ion uptake by Mops is much greater for strontium than for calcium and is further increased when potassium is replaced by sodium as the dominant monovalent cation. Mops affects neither the activity of the calcium‐ or strontium‐activated transport enzyme nor the active accumulation of calcium in the presence of oxalate, i.e. when the pump runs unidirectionally forward. Passive calcium and strontium efflux rates of approximately 40–50 nmol · mg−1· min−1 are considerably reduced when histidine/glycerophosphate or Tris/maleate are exchanged for Mops. The observed passive efflux rates and their modulation by Mops are too small, in relation to the rate of ion influx, to account for either the relatively small calcium and strontium load in the absence of precipitating agents or for its modulation by Mops.

The results imply that the pump itself mediates ion efflux dependent on pump activity and the different degree of saturation of lumenal ion‐binding sites by calcium and strontium, as well as their susceptibility to Mops.
Interferons and bone
Authors: BERESFORD Jon N. , TAYLOR Guy T. , TRIFFITT James T.
Published: March 2005

Recombinant human interferon‐α2C and recombinant human interferon‐γ(5–1000 U/ml) inhibit the proliferation of normal human bone‐derived cells and a human osteosarcoma cell line. In the bone‐derived cells the inhibitory effect of interferon‐γ was significantly greater than that of interferon‐α, whereas in the osteosarcoma cell line the inhibitory effects of both interferons were quantitatively similar. Interferon‐α did not affect the alkaline phosphatase activity of either type of cells. In contrast, interferon‐γ affected the activity of the enzyme in both cell types: in the bone‐derived cells the effect of interferon‐γ was stimulatory whereas in the osteosarcoma cells the effect was inhibitory. In both cell types interferon‐γ selectively inhibited the incorporation of radiolabelled proline into type I collagen. In the osteosarcoma cells, the effects of both interferons on collagen synthesis were quantitatively similar. In the bone‐derived cells, however, interferon‐α decreased proline incorporation into collagen and non‐collagen proteins to a similar extent and thus did not affect collagen synthesis when expressed as a percentage of total protein synthesis. Two‐dimensional polyacrylamide gel electrophoresis of the radiolabelled proteins of the cell layer synthesised by both cell types in the presence of either interferon demonstrated that this treatment enhanced or induced the synthesis of a total of 21 individual proteins (19 in bone cells, 14 in osteosarcoma), ranging in apparent molecular mass over 14–87 kDa. The set of proteins induced was different in all four combinations of cells and interferon. A tentative identification of several of the proteins was possible based upon estimation of molecular mass, preferential induction by interferon‐α or interferon‐γ and differential induction in normal and transformed bone‐derived cells. The results of this study demonstrate that interferons have complex effects upon the proliferative and biosynthetic activities of human bone‐derived cells and demonstrate significant differences between the responses of normal cells and transformed bone‐derived cell line. Further investigations will be required in order to determine whether or not these differences are unique to the osteosarcoma cell line or are a characteristic of the effects of interferons on bone‐derived cells in general.
Structure of the snake short‐chain neurotoxin, erabutoxin c, precursor gene
Authors: FUSE Naoyuki , TSUCHIYA Takahide , NONOMURA Yoshiaki , MENEZ Andre , TAMIYA Toru
Published: March 2005

A 2.4‐kb genomic clone, which encodes the precursor of a snake neurotoxin, erabutoxin c, was isolated from the liver of the sea snake, Laticauda semifasciata. The erabutoxin c gene is about 1.2 kb long and consists of three exons and two introns. The first intron is found at the position corresponding to the signal peptide between amino acid residues 18(Leu) and 19(Gly). The second intron is located at the position corresponding to the central loop of the mature toxin, between amino acid residues 33(Arg) and 34(Gly). A TATA box consensus sequence, characteristic of promoter regions, is identified 29–33 nucleotides upstream from the transcription‐initiation site identified by nuclease S1 analysis. The erabutoxin c cDNA nucleotide sequence, deduced from the present work, is compared with the cDNA sequences of erabutoxins a and b reported previously. Replacements are found at three positions, two of which correspond to the amino acid replacements found among the toxins, while the other is silent.
Translational control by the Epstein‐Barr virus small RNA EBER‐1
Authors: CLARKE Paul A. , SHARP Nigel A. , CLEMENS Michael J.
Published: March 2005

A role for the Epstein‐Barr virus small RNA species EBER‐1 in the regulation of protein synthesis has been investigated in the reticulocyte‐lysate cell‐free translation system. Recombinant EBER‐1 was synthesized by in vitro transcription of a plasmid containing the viral gene and purified by CF11‐cellulose chromatography and ribonuclease III treatment. When added to the reticulocyte lysate at 10–20 μg/ml or more, EBER‐1 prevents the inhibition of protein synthesis caused by low concentrations of synthetic double‐stranded RNA, poly(I) · poly(C). This effect is eliminated by treatment of the recombinant EBER‐1 with ribonuclease T1. Disruption of the secondary structure of EBER‐1 by substitution of inosine for guanosine in the in‐vitro‐syntheized RNA impairs the ability of EBER‐1 to prevent the poly(I) · poly(C)‐mediated inhibition of protein synthesis. These results suggest that high concentrations of EBER‐1 regulate protein synthesis by blocking the activation of the double‐stranded RNA‐dependent eukaryotic initiation factor 2α (eIF‐2α) protein kinase DAI (p68), and that this property is dependent on the secondary structure of the small RNA molecule.
Partial release of AcPhe‐Phe‐tRNA from ribosomes during poly(U)‐dependent poly(Phe) synthesis and the effects of chloramphenicol
Authors: RHEINBERGER Hans‐Jörg , NIERHAUS Knud H.
Published: March 2005

Poly(U)‐programmed 70S ribosomes can be shown to be 80% to 100% active in binding the peptidyl‐tRNA analogue AcPhe‐tRNA to their A or P sites, respectively. Despite this fact, only a fraction of such ribosomes primed with AcPhe‐tRNA participate in poly(U)‐directed poly(Phe) synthesis (up to 65%) at 14 mM Mg2+ and 160 mM NH+4. Here it is demonstrated that the apparently ‘inactive’ ribosomes (≧35%) are able to participate in peptide‐bond formation, but lose their nascent peptidyl‐tRNA at the stage of Ac(Phe)n‐tRNA, with n≥2. The relative loss of early peptidyl‐tRNAs is largely independent of the degree of initial saturation with AcPhe‐tRNA and is observed in a poly(A) system as well. This observation resolves a current controversy concerning the active fraction of ribosomes. The loss of Ac(Phe)n‐tRNA is reduced but still significant if more physiological conditions for Ac(Phe)n synthesis are applied (3 mM Mg2+, 150 mM NH+4, 2 mM spermidine, 0.05mM spermine).

Chloramphenicol (0.1 mM) blocks the puromycin reaction with AcPhe‐tRNA as expected but, surprisingly, does not affect the puromycin reaction with Ac(Phe)2‐tRNA nor peptide bond formation between AcPhe‐tRNA and Phe‐tRNA. The drug facilitates the release of Ac(Phe)2–4‐tRNA from ribosomes at 14 mM Mg2+ while it hardly affects the overall synthesis of poly(Phe) or poly(Lys).
Regulation of Ki‐ras expression in Reuber H35 cells
Authors: CHAN Sai‐On , WONG Susanna Siu‐Chun , YEUNG Desmond Chak‐Yew
Published: March 2005

Glucagon at a low concentration has a stimulatory effect on Ki‐ras expression, whereas, at high concentrations, the hormone suppresses the level of the Ki‐ras transcripts. Incubation of the hepatoma cells with 10 μM dibutyryl cyclic AMP results in suppression of Ki‐ras expression but the phorbol ester, 21‐O‐tetradecanoylphorbol 13‐acetate (TPA) causes an increase. Down regulation of protein kinase C by prolonged exposure of hepatoma cells to TPA causes a dramatic decrease in the glucagon‐stimulated effect on Ki‐ras expression. The presence of diacylglycerol for 2 h in the culture medium results in a significant increase in Ki‐ras expression, while treatment of the cells with 1‐(5‐isoquinolinylsulphonyl)‐2‐methylpiperazine, a potent inhibitor of protein kinase C, leads to a dramatic reduction. The calcium ionophore, A23187 is able to stimulate Ki‐ras expression, whereas, addition of verapamil or EGTA results in its suppression.

The present findings suggest that the inductive effect of glucagon on Ki‐ras expression at low concentrations is via the activation of protein kinase C which causes phosphorylation of some regulatory proteins that may eventually affect the level of Ki‐ras mRNA. The suppressive effect of glucagon at higher concentrations is via an increase in cAMP through activation of adenylate cyclase.
A study on the regulation of N‐glycoloylneuraminic acid biosynthesis and utilization in rat and mouse liver
Authors: LEPERS Anne , SHAW Lee , SCHNECKENBURGER Petra , Cacan René , VERBERT André , SCHAUER Roland
Published: March 2005

The relative contribution of N‐glycoloyl‐β‐d‐neuraminic acid (Neu5Gc) to total sialic acids expressed in mouse and rat liver glycoconjugates was found to be 95% and 11%, respectively. This considerable difference in sialic acid composition made these two tissues suitable modles for a comparative investigation into the regulation of Neu5Gc biosynthesis and utilization.

An examination of the CMP‐glycoside specificity of Golgi‐associated sialyltransferases using CMP‐N‐acetyl‐β‐d‐neuraminic acid (CMP‐Neu5Ac) and CMP‐Neu5Gc revealed no significant tissue‐dependent differences. The Golgi membrane CMP–sialic acid transport system from rat liver did, however, exhibit a slightly higher internalisation rate for CMP‐Neu5Ac, though no preferential affinity for this sugar nucleotide over CMP‐Neu5Gc was observed.

In experiments, where Golgi membrane preparations were incubated with an equimolar mixture of labelled CMP‐Neu5Ac and CMP‐Neu5Gc, no significant tissue‐dependent differences in [14C]sialic acid composition were observed, either in the luminal soluble sialic acid fraction or in the precipitable sialic acid fraction, results which are consistent with the above observations. From this experiment, evidence was also obtained for the presence of a Golgi‐lumen‐associated CMP–sialic acid hydrolase which exhibited no apparent specificity for either CMP‐Neu5Ac or CMP‐Neu5Gc.

The specific activity of the CMP‐Neu5Ac hydroxylase, the enzyme responsible for the biosynthesis of Neu5Gc, was found to be 28‐fold greater in high‐speed supernatants of mouse liver than of rat liver. No hydroxylase activity was detected in the Golgi membrane preparations. It is therefore proposed that the cytoplasmic ratio of CMP‐Neu5Ac and CMP‐Neu5Gc produced by the hydroxylase, remains largely unmodified after CMP‐glycoside uptake into the Golgi apparatus and transfer on to growing glycoconjugate glycan chains. The close relationship between the total sialic acid composition and the sialic acid pattern in the CMP‐glycoside pools of the tissues lends considerable weight to this hypothesis.
Molecular cloning of cDNAs for two subunits of rat multicatalytic proteinase
Authors: SORIMACHI Hiroyuki , TSUKAHARA Toshifumi , KAWASAKI Hiroshi , ISHIURA Shoichi , EMORI Yasufumi , SUGITA Hideo , SUZUKI Koichi
Published: March 2005

cDNA clones for two subunits (designated subunits K and L) of rat liver multicatalytic proteinase (MCP) were isolated using oligonucleotide probes synthesized according to their partial amino acid sequences. The encoded polypeptides of subunits K and L consisted of 255 and 261 amino acid residues with calculated molecular mass of 28.3 kDa and 29.5 kDa, respectively. Northern blot analysis revealed that subunits K and L were expressed in all tissues examined and their expression patterns were almost identical. The deduced amino acid sequences showed no similarities to known protein sequences other than the recently reported sequences of rat and Drosophila MCP subunits. Sequence comparison of MCP subunits of rat and Drosophila revealed that the N‐terminal two‐thirds of the sequence (especially the N‐terminal ∼ 20 residues) is conserved, but the C‐terminal third of the sequence shows no similarity, suggesting functional and structural roles for both regions. Implications for the structural and functional aspects of MCP subunits are discussed based on the sequence similarity.
The collagen‐binding site of type‐II units of bovine seminal fluid protein PDC‐109 and fibronectin
Authors: BÁNYAI László , TREXLER Mária , KONCZ Sándor , GYENES Marianne , SIPOS György , PATTHY László
Published: March 2005

A single type‐II domain has been isolated by limited proteolysis of the collagen‐binding bovine seminal fluid protein, PDC‐109. The 45‐residue fragment corresponding to the second type‐II domain of the parent molecule was found to have retained affinity for immobilized collagen, indicating that this minidomain carries critical regions of the collagen‐binding site. Studies on various fragments of fibronectin have also implicated the two type‐II units of this molecule in collagen‐binding. In the present work we have found that type‐II domains of human fibronectin, expressed in Escherichia coli as β‐galactosidase fusion proteins, bind specifically to immobilized collagen.
Modification of ATP regulatory function in sacroplasmic reticulum Ca2+‐ATPase by hydrophobic molecules
Authors: WOLOSKER Herman , PETRETSKI Jorge H. , de MEIS Leopoldo
Published: March 2005

The effects of the three hydrophobic molecules triphenylphosphine, trifluoperazine and 3‐nitrophenol on Ca2+ uptake and ATPase activity in sarcoplasmic reticulum vesicles was investigated. When ATP was the substrate, triphenylphosphine (3 μM) increased the amount of Ca2+ accumulated by the vesicles. At high concentrations triphenylphosphine inhibited Ca2+ uptake. This effect varied depending on the ATP concentration and the type of nucleotide used. With ITP there was only inhibition and no activation of Ca2+ uptake by triphenylphosphine. On the other hand, trifluoperazine inhibited Ca2+ accumulation regardless of whether ATP or ITP was used as substrate. When 5 mM oxalate was included in the medium in order to avoid binding of Ca2+ to the low‐affinity Ca2+‐binding sites of the enzyme, both stimulation by triphenylphosphine and inhibition by trifluoperazine were reduced. In leaky vesicles at low Ca2+ concentrations, triphenylphosphine and 3‐nitrophenol were competitive inhibitors of ATPase activity at the regulatory site of the enzyme (0.1–1 mM ATP). A striking difference was observed when both the high‐ and low‐affinity Ca2+‐binding sites were saturated. In this condition, triphenylphosphine and 3‐nitrophenol promoted a 3–4 fold increase in the apparent affinity for ATP at its regulatory site.
Analysis of wheat‐germ RNA polymerase II by trypsin cleavage
Authors: TEISSERE Marcel , SERGI Isabelle , JOB Claudette , JOB Dominique
Published: March 2005

When wheat‐germ RNA polymerase II is subjected to mild proteolytic attack in the presence of trypsin, the resulting form of the enzyme migrates as a single species on electrophoresis in native polyacrylamide gels, with an apparent Mr significantly smaller than that of the native enzyme. Analysis by denaturing gel electrophoresis of the truncated eukaryotic polymerase revealed that the two largest subunits of the native enzyme, i.e. the 220 000‐Mr and 140000‐Mr subunits, were cleaved, giving rise to shorter polypeptide chains of Mr 172800, 155000, 143000, 133800, 125000 and 115000. The use of affinity‐purified antibodies directed against each of the two large subunits of the native enzyme allowed us to probe for possible precursor/product relationships between the 220000‐Mr and 140000‐Mr subunits of wheat‐germ RNA polymerase II and their breakdown products generated in the presence of trypsin.

None of the smaller subunits of the plant RNA polymerase II appeared to be sensitive to trypsin attack. The results indicate that the truncated RNA polymerase retained a multimeric structure, and therefore that the proteolyzed largest subunits of the enzyme remained associated with the smaller ones.

Furthermore, in transcription of a poly[d(A‐T)] template, the catalytic activity of the proteolyzed form of wheat‐germ RNA polymerase II was identical to that of the native enzyme. Therefore, the protein domains that can be deleted by the action of trypsin from the two large subunits of the plant transcriptase are not involved in DNA binding and/or nucleotide binding, and do not play an important role in template‐directed catalysis of phosphodiester bond formation.
KRDS, a new peptide derived from human lactotransferrin, inhibits platelet aggregation and release reaction
Authors: MAZOYER Elisabeth , LÉVY‐TOLEDANO Sylviane , RENDU Francine , HERMANT Laurence , LU He , FIAT Anne‐Marie , JOLLÈS Pierre , CAEN Jacques
Published: March 2005

KRDS (Lys‐Arg‐Asp‐Ser), a tetrapeptide from human lactotransferrin, was tested in vitro on human platelet function, and its effects were compared to those of RGDS, a tetrapeptide from human fibrinogen. Both peptides had a high probability of initiating a β‐turn and were highly hydrophilic. KRDS inhibited ADP‐induced platelet aggregation [median inhibitory concentration (IC50) 350 μM] and fibrinogen binding (IC50 360 μM) to a lesser extent than RGDS (IC50 75 μM and 20 μM, respectively). Different from RGDS, thrombin‐induced serotonin release was inhibited by KRDS (750 μM) on normal platelets (55 ± 10%) and type I Glanzmann's thrombasthenia platelets (43%± 1). However, KRDS had no effect on cytoplasmic Ca2+ mobilization, inositol phospholipid metabolism or protein phosphorylation (myosin light chain P20 and P43). In contrast to RGDS, KRDS does not inhibit the binding of monoclonal antibody PAC‐1 to activated platelets. KRDS and RGDS inhibited 4β‐phorbol‐12‐myristate‐13‐acetate (PMA)‐induced aggregation and fibrinogen binding, while proteins were normally phosphorylated. Thus, the tetrapeptide KRDS is (a) an inhibitor of serotonin release by a mechanism independent of protein phosphorylation and (b) an inhibitor of fibrinogen binding and, hence, aggregation by a mechanism that may not necessarily involve its direct binding to the glycoprotein IIb‐IIIa‐complex.
The structure of gramicidin A in dimethylsulfoxide/acetone
Authors: ROUX Benoit , BRÜSCHWEILER Rafael , ERNST Richard R.
Published: March 2005

It has been demonstrated by two‐dimensional NMR cross‐relaxation spectroscopy that gramicidin A exists in dimethylsulfoxide/acetone solution in random coil form. This contradicts earlier conclusions by Hawkes et al. [Hawkes, G. E., Lian, L. Y., Randall, E. W., Sales, K. D. & Curzon, E. H. (1987) Eur. J. Biochem. 166, 437–445] that were based on the interpretation of vicinal proton coupling constants.
Interaction of hemoglobin with chloride and 2,3‐bisphosphoglycerate
Authors: GIARDINA Bruno , BRIX Ole , COLOSIMO Alfredo , PETRUZZELLI Raffaele , CERRONI Loredana , CONDO Saverio G.
Published: March 2005

The equilibrium oxygen‐binding properties of hemoglobins from reindeer (Rangifer tarandus tarandus), musk ox (Ovibos muschatos) and a bat (Rousettus aegyptiacus) have been investigated with special reference to the effect of heterotrophic ligands such as chloride and 2,3‐bisphosphoglycerate [Gri(2,3)P2].

The results obtained with hemoglobins from reindeer and musk ox indicate that their low oxygen affinity and their insensitivity to Gri(2,3)P2 are not only an intrinsic property of the molecule, as proposed in the case of ruminant hemoglobins, but also the results of the interplay between chloride and Gri(2,3)P2 interactions. In other words, insensitivity of reindeer and musk ox hemoglobins to Gri(2,3)P2 is mainly due to a decreased affinity constant for this cofactor and to an increased affinity constant for chloride anions; this renders more effective the competition of chloride for the anion‐binding site.

On the other hand bat hemoglobin behaves in a completely different way and could be regarded as a type case of low‐affinity hemoglobin since its functional properties are modulated neither by chloride nor by Gri(2,3)P2.

The results are discussed in the light of the amino acid residues which are known to be involved in the binding of organic phosphates.
Possible interaction of α1‐adrenergic receptor with pertussis‐toxin‐sensitive guanine‐nucleotide‐binding regulatory proteins (G proteins) responsible for phospholipase C activation in rat liver plasma membranes
Authors: TOHKIN Masahiro , YAGAMI Tatsurou , KATADA Toshiaki , MATSUBARA Takashi
Published: March 2005

Islet‐activating protein (IAP; pertussis toxin) was employed to test the hypothesis that IAP‐sensitive GTP‐binding regulatory proteins (G proteins) are coupled with α1‐adrenergic receptor in rat liver plasma membranes. The high‐affinity state of the binding of α2‐adrenergic agonist, which is known to be coupled with IAP‐sensitive G protein, was abolished in IAP‐treated plasma membranes. IAP treatment of plasma membranes could also diminish the high‐affinity state of the α1‐adrenergic receptor for the agonist. Restoration of the high‐affinity state of the α1‐adrenergic receptor for the agonist occurred on reconstitution of the bovine brain IAP‐sensitive G proteins. The α1‐adrenergic receptor agonist stimulated inositol triphosphate (InsP3) production from [3H]inositollabeled liver plasma membranes in a concentration‐dependent manner. IAP treatment also decreased α1‐adrenergic‐agonist‐induced InsP3 production but not completely. From these results, we concluded that there is a possibility that both IAP‐sensitive and IAP‐insensitive G proteins were involved in α1‐adrenergic‐receptor‐stimulated phospholipase C activation in rat liver plasma membranes.
A d(GpG)‐platinated decanucleotide duplex is kinked
Authors: HERMAN Frédéric , KOZELKA Jiřarí , STOVEN Véronique , GUITTET Eric , GIRAULT Jean‐Pierre , HUYNH‐DINH Tam , IGOLEN Jean , LALLEMAND Jean‐Yves , CHOTTARD Jean‐Claude
Published: March 2005

A conformational study of the double‐stranded decanucleotide d(GCCG*G*ATCGC) · d(GCGATCCGGC), with the G* guanines chelating a cis‐Pt(NH3)2 moiety, has been accomplished using 1H and 31P NMR, and molecular mechanics. Correlation of the NMR data with molecular models has disclosed an equilibrium between several kinked conformations and has ruled out an unkinked structure. The deformation is localized at the CG*G*· CCG trinucleotide where the helix is kinked by approximately 60° towards the major groove and unwound by 12–19°. The models revealed an unexpected mobility of the cytosine complementary to the 5′‐G*. This cytosine can stack on either branch of the kinked complementary strand. The energy barrier between the two positions has been calculated to be ≤ 12 kJ/mol. The NMR data are in support of rapid flip‐flopping of this cytosine. An explanation for the strong downfield shift observed in the 31P resonance of the G*pG* phosphate is given.
Biosynthesis of the lantibiotic Pep5
Authors: WEIL Hans‐Peter , BECK‐SICKINGER Annette G. , METZGER Jörg , STEVANOVIC Stefan , JUNG Günther , JOSTEN Michaele , SAHL Hans‐Georg
Published: March 2005

Pep5 is a tricyclic peptide antibiotic which contains the unusual amino acids dehydrobutyrine, lanthionine and 3‐methyllanthionine. It is matured from a 60‐amino‐acid precursor peptide (pre‐Pep5) deduced from the sequence of the structural gene pep A. To study the biosynthesis of Pep5 we tried to isolate the primary translation product. We identified a peptide in crude extracts of the Pep5‐producing Staphylococcus epidermidis strain using antibodies raised against a synthetic 26‐residue peptide representing the leader peptide region of pre‐Pep5. The putative precursor was purified by reversed‐phase HPLC. The isolated peptide did not react with antibodies directed against a C‐terminal fragment of mature Pep5 containing two sulfide bridges. Neither lanthionine nor 3‐methyllanthionine was detected in amino acid analysis of the isolated precursor. Its amino acid sequence was identical with the sequence predicted from pepA, but Edman degradation stopped at the first threonine residue of the pro‐lantibiotic region indicating a posttranslational modification at this position. The molecular mass of the isolated peptide was 6575.4 ± 1.7 Da, determined by ion‐spray mass spectrometry. This is in agreement with a molecule being dehydrated at the four threonine and the two serine residues in the propeptide region; such a peptide has a calculated molecular mass of 6576.7 Da. The results strongly suggest that maturation of the lantibiotic Pep5 is initiated by selective dehydration of hydroxyamino acids in the propeptide region of the primary translation product and that thioether ring formation is not closely linked to dehydration.
ADP‐ribosylation of actin isoforms by Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin
Authors: MAUSS Stefan , CHAPONNIER Christine , JUST Ingo , AKTORIES Klaus , GABBIANI Giulio
Published: March 2005

The substrate specificities of the actin‐ADP‐ribosylating toxins, Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin were studied by using five different preparations of actin isoforms: α‐skeletal muscle actin, α‐cardiac muscle actin, gizzard γ‐smooth muscle actin, spleen β‐ and γ‐cytoplasmic actin, and aortic smooth muscle actin containing α‐ and γ‐smooth muscle actin isoforms. C. perfringens iota toxin ADP‐ribosylated all actin isoforms tested, whereas C. botulinum C2 toxin did not modify α‐skeletal muscle actin or α‐cardiac muscle actin. Spleen β/γ‐cytoplasmic actin and gizzard γ‐smooth muscle actin were substrates of C. botulinum C2 toxin. In the aortic smooth muscle actin preparation, γ‐smooth muscle actin but not α‐smooth muscle actin was ADP‐ribosylated by C. botulinum C2 toxin. The data indicate that, in contrast to C. perfringens iota toxin, C. botulinum C2 toxin ADP‐ribosylates only β/γ‐cytoplasmic and γ‐smooth muscle actin and suggest that the N‐terminal region of actin isoforms define the substrate specificity for ADP‐ribosylation by C. botulinum C2 toxin.
Herpes simplex virus type‐1 immediate‐early gene expression and shut off of host protein synthesis are inhibited in neomycin‐treated human epidermoid carcinoma 2 cells
Authors: GARCIN Dominique , MASSÉ Thierry , MADJAR Jean‐Jacques , JACQUEMONT Bernard
Published: March 2005

Infection of human epidermoid carcinoma‐2 (HEp‐2) cells by Herpes simplex virus type 1 (HSV‐1) leads to significant activation of inositol phospholipid turnover after 15 min. The effect of neomycin, an inhibitor of inositol phospholipid turnover, has been investigated for its effect on HSV‐1 multiplication in HEp‐2 cells. HSV‐1 multiplication is inhibited by neomycin. This inhibition is not due to a block of virus adsorption or penetration. Neomycin inhibits the expression of virus immediate‐early genes, as well as expression of early genes and viral DNA synthesis. In neomycin‐treated cells, the usual virion‐associated shut off of host protein synthesis does not occur. These results indicate that the inositol phospholipid pathway is involved in immediate‐early gene expression and shut off of host protein synthesis in HEp‐2 cells.
Herpes simplex virus type‐1‐induced stimulation of ribosomal protein S6 phosphorylation is inhibited in neomycin‐treated human epidermoid carcinoma 2 cells and in ras‐transformed cells
Authors: MASSÉ Thierry , GARCIN Dominique , JACQUEMONT Bernard , MADJAR Jean‐Jacques
Published: March 2005

Neomycin, an inhibitor of inositol phospholipid turnover, prevents Herpes‐simplex‐virus‐type‐1(HSV‐1)‐induced stimulation of ribosomal protein S6 phosphorylation, but does not impair the S6 phosphorylation induced by serum. Long‐term treatment with phorbol 12‐myristate 13‐acetate, which down‐regulates protein kinase C activity, does not inhibit virus‐induced S6 phosphorylation. In ras‐transformed cells, S6 phosphorylation is not stimulated after HSV‐1 infection. These results suggest that activation of the inositol phospholipid pathway is involved in the HSV‐1‐induced stimulation of S6 phosphorylation. However, protein kinase C activation does not appear to be necessary for HSV‐1‐induced S6 phosphorylation.
Respiratory burst of rabbit peritoneal neutrophils
Authors: LAPORTE François , DOUSSIERE Jacques , VIGNAIS Pierre V.
Published: March 2005

Superoxide (·O−2) production by the NADPH oxidase of a membrane fraction derived from rabbit peritoneal neutrophils activated by 4β‐phorbol1Zmyristate 13‐acetate (PMA) was studied at 25°C under different conditions, and measured by the superoxide dismutase inhibitable reduction of cytochrome c. Whereas PMA‐activated rabbit neutrophils incubated in a glucose‐supplemented medium exhibited a substantial rate of production of ·O−2, the membranes prepared by sonication of the activated neutrophils were virtually unable to generate ·O−2, in the presence of NADPH. Instead, they exhibited an NADPH‐dependent diaphorase activity, measured by the superoxide‐dismutase‐insensitive reduction of cytochrome c. Upon addition of arachidonic acid, which is known to elicit oxidase activation, the NADPH diaphorase activity of the rabbit neutrophil membranes vanished and was stoichiometrically replaced by an NADPH oxidase activity. The emerging oxidase activity was fully sensitive to iodonium biphenyl, a potent inhibitor of the respiratory burst, whereas the diaphorase activity was not affected. Addition of 0.1 % Triton X‐100 or an excess of arachidonic acid, acting as detergent, resulted in the reappearance of the diaphorase activity at the expense of the oxidase activity. These results indicate that the diaphorase‐ oxidase transition is reversible. When the rabbit neutrophil membranes were supplemented with rabbit neutrophil cytosol, guanosine 5′‐[pthio]triphosphate and Mg2+ in addition to arachidonic acid, not only the NADPH diaphorase activity disappeared, but the emerging NADPH oxidase activity was markedly enhanced (about 10 times compared to that of membranes treated with arachidonic acid alone). The diaphorase ‐ oxidase transition was accompanied by a 10‐fold increase in the K, for NADPH, suggesting a change of conformation propagated to the NADPH‐binding site during the transition. The treatment of PMA‐activated rabbit neutrophils with cross‐ linking reagents, like glutaraldehyde or 1‐(3‐dimethylaminopropyl)‐3‐ethyl carbodiimide, prevented the loss of the PMA‐elicited oxidase activity upon disruption of the cells by sonication, suggesting that the interactions between the components of the oxidase complex are stabilized by cross‐linking.
Human liver manganese superoxide dismutase
Authors: MATSUDA Yukihiko , HIGASHIYAMA Shigeki , KIJIMA Yoshiyuki , SUZUKI Keiichiro , KAWANO Kiyoshi , AKIYAMA Morio , KAWATA Sumio , TARUI Seiichiro , DEUTSCH Harold F. , TANIGUCHI Naoyuki
Published: March 2005

Manganese superoxide dismutase (Mn‐SOD) has been purified with a high yield (320 mg) from human liver (2 kg) and crystallized. Low‐angle laser light scattering of the enzyme has shown that native enzyme is a tetrametic form.

Four of the eight cysteine residues in the tetramer reacted with−5,5′dithiobis(2‐nitrobenzoic acid) or with iodoacetamide. The others were only reactive in protein heated with SDS or urea after reduction with dithiothreitol or 2‐mercaptoethanol. The reactive sulfhydryl group was found to be located at Cys196 by amino acid sequence analysis of Nbs2‐reactive peptides isolated by activated thiol‐Sepharose covalent chromatography. Incubation of Mn‐SOD in 1% SDS for 2 or 3 days at 25°C or 5 min at 100°C gave material showing two prominent components on polyacrylamide gel electrophoresis in the presence of 0.1% SDS. The major component had a molecular mass of 23 kDa; the other, 25 kDa. Reduction of the protein by dithiothreitol or 2‐mercaptoethanol heated in SDS produced only the 25‐kDa monomer species. Essentially, no thiol groups were detected in the 23‐kDa form, in which two cysteine residues appear to have been oxidized to form an intrasubunit disulfide. This indicates that Cys196 has a reactive sulfhydryl and appears to be a likely candidate for a mixed disulfide formation in vivo.
Immunological characterization of rapeseed myrosinase
Authors: LENMAN Marit , RÖDIN Joakim , JOSEFSSON Lars‐Göran , RASK Lars
Published: March 2005

A purified 75‐kDa myrosinase and a crude rapeseed myrosinase fraction were used as antigens to produce mouse anti‐myrosinase monoclonal antibodies. The 75‐kDa myrosinase was also used to produce a polyclonal rabbit antiserum. The antiserum and one monoclonal antibody reacted with three distinct rapeseed polypeptides of 75, 70 and 65 kDa (M75, M70 and M65, respectively). A second set of monoclonal antibodies reacted exclusively with the 75‐kDa form of myrosinase, and a third set showed specificity towards two components of 52 and 50 kDa (myrosinase‐binding proteins, MBP52 and MBP50, respectively). MBP52 and MBP50 lack inherent myrosinase activity, but are nevertheless capable of mediating immunoprecipitation of myrosinase due to their interaction with myrosinase. Gel chromatography and glycerol gradient centrifugation experiments resolved two myrosinase‐containing fractions. One of these had an approximate molecular mass of 140 kDa and consisted of disulfide‐linked dimers of the 75‐kDa myrosinase. The other fraction was heterogeneous in size with molecular masses ranging from 250 kDa to approximately 1 MDa. The high‐molecular‐mass fractions contained complexes consisting of disulfide‐linked 70‐kDa and 65‐kDa myrosinases and non‐covalently bound 52‐kDa and 50‐kDa myrosinase‐binding proteins.
Purification and partial characterization of pyruvate decarboxylase from Oryza sativa L.
Authors: RIVOAL Jean , RICARD Bérénice , PRADET Alain
Published: March 2005

Pyruvate decarboxylase(PyrDC) was purified from rice bran to a specific activity of 1 μkat/mg and partially characterized. The holoenzyme is a tetramer of two types of subunits with molecular masses 64 kDa and 62 kDa. Purified rice PyrDC exhibits positive cooperative kinetics with respect to pyruvate and functions with a significant lag phase. When compared to other plant PyrDC, the lag phase was shorter at low pyruvate concentrations and the S0.5 was smaller. The optimum pH (6.25) was also less acidic and the enzyme retained 30% of its maximal activity at neutral pH. In contrast to other plant PyrDC, rice PyrDC could be active at the onset of anoxia and would be activated by small changes in pyruvate concentration.
Intracellular accumulation and oligosaccharide processing of alkaline phosphatase under disassembly of the Golgi complex caused by brefeldin A
Authors: TAKAMI Noboru , ODA Kimimitsu , FUJIWARA Toshiyuki , IKEHARA Yukio
Published: March 2005

Electron microscopic observations showed that the fungal metabolite brefeldin A caused disassembly of the Golgi complex in human choriocarcinoma cells and accumulation of alkaline phosphatase (ALP) in the endoplasmic reticulum (ER) and nuclear envelope, where ALP was not apparently detectable in control cells. Pulse/chase experiments with [35S]methionine demonstrated that in the control cells, ALP synthesized as a 63‐kDa precursor form was rapidly converted to a 66‐kDa form, by processing of its N‐linked oligosaccharides from the high‐mannose type to the complex type, which was expressed on the cell surface after 30 min of chase. In contrast, in the brefeldin‐A‐treated cells the precursor was gradually converted to a 65‐kDa form, slightly smaller than the control mature form, which was not expressed on the cell surface even after a prolonged time of chase. Kinetics of the ALP processing in the brefeldin‐A‐treated cells demonstrated that the precursor was initially converted to an intermediate form, partially sensitive to endo‐β‐N‐acetylglucosaminidase H (endo H), then to an endo‐H‐resistant 65‐kDa form. In addition, this form was found to be sensitive to neuraminidase digestion, though its sialylation was not so complete as that of the control mature form. Taken together, these results suggest that under disassembly of the Golgi complex caused by brefeldin A, oligosaccharide‐processing enzymes including sialyltransferase, an enzyme in the trans Golgi cisterna(e) and/or the trans Golgi network, might be redistributed into the ER and involved in processing of the oligosaccharides of ALP accumulating there.
Purification and characterization of lipoamide dehydrogenase from Trypanosoma cruzi
Authors: LOHRER Helmut , KRAUTH‐SIEGEL R. Luise
Published: March 2005

From Trypanosoma cruzi, the causative agent of Chagas' disease, a lipoamide dehydrogenase was isolated. The enzyme, an FAD —cystine oxidoreductase, shares many physical and chemical properties with T. cruzi trypanothione reductase, the key enzyme of the parasite's thiol metabolism.

From 60 g epimastigotic T. cruzi cells, 2.7 mg lipoamide dehydrogenase was extracted. The flavoenzyme was purified 3000‐fold to homogeneity with an overall yield of 26%.

The enzyme is a dimer with a subunit Mr of 55000. With 1 mM lipoamide (Km∼ 5 mM) and 100 μM NADH (Km= 23 μM), the specific activity at pH 7.0 is 297 U/mg.

With excess NADH, the enzyme is reduced to the EH2· NADH complex and, by addition of lipoamide, it is reoxidized, indicating that it can cycle between the oxidized state E and the two‐electron‐reduced state, EH2.

As shown by N‐terminal sequencing of the enzyme, 21 out of 30 positions are identical with those of pig heart and human liver lipoamide dehydrogenase. The sequenced section comprises the GGGPGG stretch, which represents the binding site for the pyrophosphate moiety of FAD.

After reduction of Eox to the two‐electron‐reduced state, the enzyme is specifically inhibited by the nitro‐sourea drug 1,3‐bis(2‐chloroethyl)‐1‐nitrosourea (Carmustine), presumably by carbamoylation at one of the nascent active‐site thiols.

Polyclonal rabbit antibodies raised against T. cruzi lipoamide dehydrogenase and trypanothione reductase are specific for the respective enzyme, as shown by immunoblots of the pure proteins and of cell extracts.
Kinetics and thermodynamics of catalysis by the inorganic pyrophosphatase of Escherichia coli in both directions
Authors: BAYKOV Alexander A. , SHESTAKOV Alexander S. , KASHO Vladimir N. , VENER Alexander V. , IVANOV Artemiy H.
Published: March 2005

Combined evidence obtained from the measurements of pyrophosphate hydrolysis and synthesis, oxygen exchange between phosphate and water, enzyme‐bound pyrophosphate formation and Mg2+ binding enabled us to deduce the overall scheme of catalysis by Escherichia coli inorganic pyrophosphatase in the presence of Mg2+. We determined the equilibrium constants for Mg2+ binding to various enzyme species and forward and reverse rate constants for the four steps of the catalytic reaction, namely, binding/release of PPi, hydrolysis/synthesis of PPi and successive binding/release of two Pi molecules. Catalysis by the E. coli enzyme in both directions, in contrast to baker's yeast pyrophosphatase, occurs via a single pathway, which requires the binding of Mg2+ to the sites of four types. Three of them can be filled in the absence of the substrates, and the affinity of one of them to Mg2+ is increased by two orders of magnitude in the enzyme‐substrate complexes. The distribution of 18O‐labelled phosphate isotopomers during the exchange indicated that hydrolysis of pyrophosphate in the active site is appreciably reversible. The equilibrium constant for this process estimated from direct measurements is 5.0. The ratio of the maximal velocities of pyrophosphate hydrolysis and synthesis is 69. The rate of the synthesis is almost entirely determined by the rate of the release of pyrophosphate from the enzyme. In the hydrolytic reaction, enzyme‐bound pyrophosphate hydrolysis and successive release of two phosphate molecules proceed with nearly equal rate constants.
NMR studies of bipyrimidine cyclobutane photodimers
Authors: KONING Thea M. G. , van SOEST Jeroen J. G. , KAPTEIN Robert
Published: March 2005

Cyclobutane‐type photodimers of dinucleoside monophosphates dCpdT, dTpdC and dTpdT were prepared by ultraviolet irradiation in the presence of acetophenone as photosensitizer. The cytosine‐containing derivatives were found to deaminate forming uracil products. Using one‐ and two‐dimensional NMR, the photoproducts were characterized as cis‐syn and trans‐syn cyclobutane photodimers. On the basis of NOE data the structures of the cis‐syn and trans‐syn products of dUpdT were determined using distance‐geometry and restrained‐energy‐minimization methods. The cis‐syn structures showed (high‐ANTI/SYN)/high‐ANTI glycosidic linkages while the trans‐syn structures were in the SYN‐ANTI region. The backbone conformations of both structures were in fair agreement with the coupling‐constant‐data. The trans‐syn structures were found to be very rigid and similar in all three products. For the three cis‐syn structures more conformational freedom and more variation among the three structures was observed.
Activation of the double‐stranded RNA‐dependent eIF‐2α kinase by cellular RNA from 3T3‐F442A cells
Authors: LI Jinhe , PETRYSHYN Raymond A.
Published: March 2005

The interferon induced double‐stranded‐RNA‐dependent eIF‐2α kinase has an established role in mediating part of interferons anti‐viral effects. Several studies have suggested that it may have additional functions in cells not infected with virus. The mechanism of activation of the kinase and the consequences of its activity in uninfected cells remain to be determined. Our previous results have indicated that the activation (phosphorylation) of this kinase may be an important regulatory signal to the arrest of growth of mouse 3T3‐F442A fibroblasts and their subsequent differentiation to adipocytes. We have found that the phosphorylation of the kinase occurred in vivo in the absence of viral infection and in vitro without the addition of dsRNA. We demonstrate here that total cytoplasmic RNA from 3T3‐F442A cells contains a regulatory RNA(s) capable of activating dsRNA‐dependent eIF‐2α kinase. Fractionation of the cytoplasmic RNA by oligo(dT)‐cellulose indicated that the regulatory RNA eluted with the poly(A)‐rich RNA fraction. It bound tightly to the dsRNA‐dependent eIF‐2α kinase and was immune‐precipitated with its antibodies as a complex of regulatory RNA and dsRNA‐dependent eIF‐2α kinase. The regulatory RNA activity was further purified by phenol extraction of immune precipitates containing this complex. These findings indicated that the regulatory RNA forms a specific complex with the dsRNA‐dependent eIF‐2α kinase. The activity of the regulatory RNA was sensitive to the dsRNA‐specific RNase VI but not to proteinase K, DNase I or ssRNA‐specific RNase T1. The activation of the dsRNA‐dependent eIF‐2α kinase by regulatory RNA was prevented by addition of a high concentration of poly(I) · poly(C). The regulatory RNA was also shown to activate partially purified dsRNA‐dependent eIF‐2α kinase prepared from rabbit reticulocyte lysates and to inhibit protein synthesis in reticulocyte lysates. Our findings, that cellular RNAs can specifically activate the dsRNA‐dependent eIF‐2α kinase, are consistent with a physiological role for the dsRNA‐dependent eIF‐2α kinase and interferon during cell growth and differentiation. The relationship of the regulatory RNA activity to growth and differentiation of 3T3‐F442A cells is discussed.
Thymidine kinase from Tetrahymena thermophila
Authors: KINYANJUI Peter W. , PEARLMAN Ronald E.
Published: March 2005

Thymidine kinase is an enzyme involved in DNA precursor metabolism and DNA replication. The synthesis of this enzyme is highly regulated during the cell cycle and the activity of the enzyme is also regulated by feedback inhibition. Genes encoding thymidine kinase have been extremely useful as selectable markers for introducing DNA into a number of cells. In order to study cell cycle regulation of thymidine kinase, the gene which encodes this enzyme, as well as aspects of DNA replication in the ciliated protozoan Tetrahymena thermophila, we have purified thymidine kinase from Tetrahymena. Two forms of thymidine kinase with native molecular masses of 59 kDa and 80 kDa have been identified and purified 6800‐ and 4600‐fold, respectively. The 59‐kDa enzyme, a homodimer of 30‐kDa subunits, has been purified to near homogeneity and polyclonal antibodies have been raised against the 30‐kDa subunit. Serological studies indicate that the two enzymes are antigenically distinct. The antibody against the Tetrahymena protein cross‐reacts with a polypeptide in Chinese hamster ovary (CHO) cell extracts of 26 kDa which corresponds to the reported size of Chinese hamster thymidine kinase protein.
Mechanism and energetics of a citrate‐transport system of Klebsiella pneumoniae
Authors: VAN DER REST Michel E. , ABEE Tjakko , MOLENAAR Douwe , KONINGS Wil N.
Published: March 2005

The citrate‐transport determinant of plasmid pES1 from Klebsiella pneumoniae [Schwarz, E. & Oesterhelt, D. (1985) EM BO J. 4, 1599–1603] has been subcloned in Escherichia coli DH1. Uptake of citrate in E. coli membrane vesicles via this uptake system is an electrogenic process, although the pH gradient is the main driving force for citrate uptake. The rate of citrate uptake, driven by artificially imposed ion‐gradients, is high in the presence of an artificial ΔpH and low in the presence of an artificial ΔΨ. Citrate transport does not depend on the presence of Na+ or Mg2+ as has been observed for other citrate‐transport systems. Citrate has three pK values: 3.14, 4.77 and 5.40. Citrate forms a stable complex with Mg2+ with a stability constant of 3.2. Kinetic parameters and calculations of the different citrate (Cit) species at a given pH, indicate that the HCit2− is the species transported and that transport occurs in symport with three protons. This citrate‐transport system is thus a unique example of a 3H+/solute symport system.