bluuebunny/crossref_metadata_2025 huggingface dataset:
Conformations of the alternating (C‐T)n sequence under neutral and low pH
Authors: Jaishree T.N. , Wang Andrew H.-J.
Published: July 2002

The structures of the (C‐T)n sequence at two different pHs have been analyzed by 500 MHz 2D‐NMR using a modified DNA decamer d(CT[m5C]TCU[m5C]UCT) as a model system. The chemical modifications serve to perturb the monotonous C‐T repeat, and consequently to yield a better chemical shift dispersion. The results reinforce our earlier suggestion that there are three major pH‐depedent confonnational species: two antiparallel‐stranded (APS) duplexes at pH 7 and pH 3, and a different structure near pH 5. Structural refinement of the decamer duplexes at pH = 7.5 and pH = 2.9 using 2D‐NOE data suggests that the C : T or C+ : T base pairs are continuously stacked. Exchangeable proton NMR spectra at pH 7.5 and pH 2.9 are consistent with C : T or C+ : T base pairing schemes in which a water molecule bridges the two bases.
Characterization by 1H NMR of a C32S,C35S double mutant of Escherichia coli thioredoxin confirms its resemblance to the reduced wild‐type protein
Authors: Jane Dyson H. , Jeng Mei-Fen , Model Peter , Holmgren Arne
Published: July 2002

A mutant of Escherichia coli thioredoxin containing serine residues in place of the two activesite cysteines, termed C32S,C35S, previously shown to be partially able to substitute for reduced thioredoxin in certain phage systems, has been characterized by 1H NMR spectroscopy at pH values between 5.5 and 10. The 1H NMR spectrum of the mutant at pH 5.5 is very similar to that of the wild‐type protein in either the reduced or oxidized state. Chemical shift changes in the vicinity of the active site serines indicate that the nearby hydrophobic pocket is somewhat changed, probably as a result of the replacement of the cysteine thiols with the smaller, more hydrophilic hydroxyl side chains and a change in the preferred χ1 angles of the side chains. Although the pattern of amide protons persistent in 2H2O differs only slightly between the two forms of the wild‐type protein, the pattern observed for the C32S,C35S mutant shows characteristic features that correspond closely with those of the reduced wild‐type protein rather than with the oxidized form. The pH dependence of the mutant protein shows a single group titrating close to the active site with a pK
a, of 8.3, which we assign to the buried carboxyl group of Asp 26 by analogy with the behavior of wild‐type thioredoxin. The pK
a, is significantly higher for the mutant protein, consistent with an increase in the hydrophobicity of the pocket where the carboxyl is buried, probably due to repacking caused by the removal of the cysteine thiols and the placement of the serine hydroxyls in positions where they interact better with solvent. The results demonstrate that the solution behavior of the mutant protein is similar in many ways to that of reduced wild‐type thioredoxin, explaining its partial activity in the two essential roles of reduced thioredoxin as a subunit of phage T7 DNA polymerase and in the assembly of filamentous phage.
Detection of specific glucose‐3‐phosphatase activity in rat liver
Authors: Canales José , Buitrago Francisco , Faraldo Angeles , Cameselle José Carlos
Published: July 2002

Sugar‐3‐phosphates are related to aspects of diabetes which depend on protein glycosylation events. Sorbitol‐3‐phosphate and fructose‐3‐phosphate occur in normal and diabetic individuals, and glucose‐3‐phosphate is a potential intermediate in their biosynthesis. Almost nothing is known about enzyme pathways for their metabolic turnover. We have found that part of the phosphohydrolytic activity on glucose‐3‐phosphate in rat liver supernatants corresponds to a specific, Mg2+‐dependent, glucose‐3‐phosphatase much less or not active on other phosphate esters, including glucose‐1‐phosphate, glucose‐6‐phosphate, fructose‐1‐phosphate, fructose‐6‐phosphate and p‐nitrophenyl‐phosphate. This finding opens a route to a better understanding of the metabolism and role of sugar‐3‐phosphates.
Phospholipase A2 enhances [3H]AMPA binding to a putative homomeric GluR‐B receptor in the rat spinal cord
Authors: Cruickshank Alison M. , Henley Jeremy M.
Published: July 2002

[3H]AMPA and [3H]kanine binding to rat spinal cord was localised most densely in the substantia gelatinosa of the dorsal horn. Phospholipase A2 elicited a dose‐dependent increase in specific [3H]AMPA binding but not in [3H]kainate binding. The enhancement of [3H]AMPA binding was blocked by bromophenacyi bromide and at 0'C and was not mimicked by arachidonic acid. Since GluR‐B is the only AMPA receptor subunit detectable in the rat spinal cord, and recombinant homomeric GluR‐B assemblies do not bind [3H]kainate, these results suggest phospholipase A2 may modulate [3H]AMPA binding to putative homomeric GluR‐B receptors.
Formation and properties of S‐protein complex with S‐peptide‐containing fusion protein
Authors: Karpeisky M.Ya. , Senchenko V.N. , Dianova M.V. , Kanevsky V.Yu.
Published: July 2002

A fusion protein (FP) comprised of the RNase A S‐peptide and human epidermal growth factor was shown to form a stable noncovalent catalytically active complex with the RNase A S‐protein at a stoichiometric ratio 1:1 with K
diss = 5.0 × 10−7M. The S‐protein complex with FP exhibits the pyrimidine specificity toward substrates in both reactions catalyzed by RNase S, transesterification and hydrolysis. The fusion protein can be determined specifically and quantitatively in the presence of S‐protein by RNase activity assays. The possibility of effective purification of S‐peptide‐containing proteins by affinity chromatography on an S‐protein‐Sepharose column has been demonstrated.
Induction of apoptotic cell death by pancreatitis‐associated ascitic fluid in Madin‐Darby canine kidney cells
Authors: Nishikawa Junsuke , Takeyama Yoshifumi , Ueda Takashi , Hori Yuichi , Ueno Nozomi , Yamamoto Masahiro , Saitoh Yoichi
Published: July 2002

We investigated the cytotoxicity on Madin‐Darby canine kidney (MDCK) cells of pancreatitis‐associated ascitic fluid (PAAF) collected from rats with experimental necrotizing pancreatitis. PAAF reduced viability of MDCK cells in a time‐ and dose‐dependent manner. We detected DNA fragmentation on the PAAF‐treated MDCK cells, indicating that the cytocidal action of PAAF is via apoptosis. From the results obtained, we conclude that PAAF contains factor(s) inducing apoptosis on MDCK cells, and we assume that apoptotic cell death is involved in the mechanism of organ failure in acute pancreatitis.
Differential up‐regulation by tRNAs of ribosome‐inactivating proteins
Authors: Brigotti Maurizio , Carnicelli Domenica , Alvergna Paola , Pallanca Alessandra , Sperti Simonetta , Montanaro Lucio
Published: July 2002

Some plant ribosome‐inactivating proteins (RIPs) with RNA‐N‐glycosidase activity on 28S RNA require, for the inactivation of ribosomes, the presence of macromolecular cofactors present in post‐ribosomal supernatants. In the case of gelonin one of the cofactors is tRNATrp lacking one or two nucleotides at the 3′‐CCA end [Brigotti, M., Carnicelli, D., Alvergna, P., Pallanca, A., Lorenzetti, R., Denaro, M., Sperti, S. and Montanaro, L. (1995) Biochem. J. 310, 249–253]. In the present study it is shown that tRNAs are involved in the up‐regulation of all the cofactor‐requiring RIPs up to now identified (agrostin, barley RIP, PAP and tritin, besides gelonin). Polyacrylamide gel electrophoresis shows that tRNA fractions with different mobilities stimulate different RIPs. With the identification of agrostin, the cofactor‐requiring RIPs (italics) add to five out of a total of thirteen investigated: barley RIP, bryodin‐R, gelonin, lychnin, momordin, momorcochin‐S, PAP, saporin‐6, tritin [Carnicelli, D., Brigotti, M., Montanaro, L. and Sperti, S. (1992) Biochem. Biophys. Res. Commun. 182, 579–582], agrostin, luffin, trichokirin and trichosanthin (present study).
DNA methylation in the promoter of ribosomal RNA genes in human cells as determined by genomic sequencing
Authors: Kochanek Stefan , Hosokawa Keiichi , Schiedner Gudrun , Renz Doris , Doerfler Walter
Published: July 2002

Many RNA polymerase II‐ or III‐transcribed genes are inactive when their promoter is methylated at critical CpG dinucleotides. We have applied the genomic sequencing method and a direct DNA blotting technique to analyze the extent of DNA methylation in the 5′‐CpG‐3′ rich promoter region of the RNA polymerase I‐transcribed ribosomal RNA genes (rDNA) in DNA from primary human cells, primary human tumor cells and human cell lines. In none of the analyzed primary human cells and primary human tumor cells was the DNA in the rDNA promoter region found to be detectably methylated. In contrast, in some of the cell lines this promoter is methylated in all 5′‐CpG‐3′ dinucleotides in the majority of the approximately 200 ribosomal RNA gene copies. In actively growing cells, rDNA gene activity is a prerequisite for cell viability. The high levels of DNA methylation in the promoter region of rDNA in the human cell lines raise questions on the role of promoter methylation in these RNA polymerase I‐transcribed genes. It is, however, conceivable that a subset of the about 200 rDNA copies per haploid genome have escaped methylation and account for the rRNA synthesis in these cell lines. Alternatively, complete 5′‐CpG‐3′ promoter methylation may be compatible with promoter activity as demonstrated for certain viral genomes.
Cross‐linking of SsoII restriction endonuclease to cognate and non‐cognate DNAs
Authors: Sheflyan G.Ya. , Kubareva E.A. , Kuznetsova S.A. , Karyagina A.S. , Nikolskaya I.I. , Gromova E.S. , Shabarova Z.A.
Published: July 2002

Specific and non‐specific interactions SsoII restriction endonuclease (R·SsoII) were probed by the method of covalent attachment to modified DNA containing an active monosubstituted pyrophosphate internucleotide bond instead of a phosphodiester one. R·SsoII with six N‐terminal His residues was shown to be cross‐linked to duplexes with this type of modification, either containing or not the recognition sequence. Competition experiments with covalent attachment of R·SsoII to activated DNAs demonstrated the similar affinity of the enzyme to cognate and non‐cognate DNAs in the absence of cofactor, Mg2+ ions.
Preferential recognition of the very low‐density lipoprotein receptor ligand binding site by antibodies from phage display libraries
Authors: Pfistermueller Doris M. , Blaas Dieter , Hodits Regina A.
Published: July 2002

Screening of a phage library displaying single chain fragments of the variable regions of human immunoglobulins (scFv) for binding to the ovarian chicken very low‐density lipoprotein/vitellogenin receptor (OVR) led to the isolation of several antibody fragments with high affinity. As for the natural ligands of OVR, receptor binding of all antibody fragments is strictly Ca2+‐dependent and is prevented by receptor‐associated protein (RAP). Moreover, attachment of human rhinovirus serotype 2 (HRV2) to this receptor is inhibited by all scFvs. In contrast to conventional immunization, the in vitro selection method thus exclusively led to antibodies that attach to or close to the ligand binding site and thereby block the receptor‐ligand interaction.
Modified nucleotides as substrates and inhibitors of adenylate kinase from different sources
Authors: Skoblov Juri S , Frank-Kamenetskaya Maria D , Chernov Dmitry N , Krayevsky Alexander A
Published: July 2002

The substrate and inhibitory properties of modified nucleotides with respect to adenylate kinase from rabbit muscles, human placenta and Escherichia coli were studied. A number of 5′‐hydrogenphosphonates and 5′‐fluorophosphates of modified nucleotides were shown to inhibit the phosphorylation reaction catalyzed by these enzymes. A clear difference between phosphonates of 3′‐deoxyribonucleotides and the corresponding ribo‐ and 2′,3′‐dideoxyribonucleotides was found. 3′‐Azido‐2′,3′‐dideoxythymidine and its phosphorus derivatives did not inhibit the adenylate kinase reaction.
Pain peptides. Solution structure of orphanin FQ2
Authors: Amodeo Pietro , López Méndez Blanca , Guerrini Remo , Salvadori Severo , Temussi Piero A , Tancredi Teodorico
Published: July 2002

Orphanin FQ2 (OFQ2) is a novel heptadecapeptide generated from prepronociceptin (PPNOC), the same precursor of nociceptin/orphanin FQ and nocistatin. OFQ2 is a potent analgesic when administered both supraspinally and spinally. In order to clarify the structural relationship with all peptides generated from PPNOC, we have undertaken the conformational study of OFQ2 in water and in structure‐promoting solvent media. Nuclear magnetic resonance data and theoretical calculations are consistent with a well defined helical structure from Met5 to Ser16. The uniform distribution of hydrophobic residues along the helix suggests that OFQ2 may interact with the transmembrane helices of a receptor akin to those of nociceptin and opioids.
The structure of denatured bovine pancreatic trypsin inhibitor (BPTI)
Authors: Chang Jui-Yoa , Ballatore Annie
Published: July 2002

In the presence of denaturant and thiol initiator, the native bovine pancreatic trypsin inhibitor (BPTI) denatures by shuffling its native disulfide bonds and converts to a mixture of scrambled isomers. The extent of denaturation is evaluated by the relative yields of the scrambled and native species of BPTI. BPTI is an exceedingly stable molecule and can be effectively denatured only by guanidine thiocyanate (GdmSCN) at concentrations higher than 3–4 M. The denatured BPTI consists of at least eight fractions of scrambled isomers. Their composition varies under increasing concentrations of GdmSCN. In the presence of 6 M GdmSCN, the most predominant fraction of scrambled BPTI accounts for 56% of the total structure of denatured BPTI. Structural analysis reveals that this predominant fraction contains the bead‐form isomer of scrambled BPTI, bridged by three pairs of neighboring cysteines, Cys5‐Cys14, Cys30‐Cys38 and Cys51‐Cys55. The extreme conformational stability of BPTI has important implications in its distinctive folding pathway.
An analysis of the lamellar structure of sea urchin egg cortical granules using X‐ray scattering
Authors: Whalley T. , Grossmann J.G. , Wess T.J.
Published: July 2002

Cortical granules (CGs) are secretory vesicles associated with egg and oocyte plasma membranes that undergo exocytosis at fertilisation. In the sea urchin Strongylocentrotus purpuratus, the internal organisation of these CGs exhibits a lamellar‐type morphology. The different lamellar layers correspond to proteoglycans, structural proteins and enzymes required for fertilisation envelope assembly and modification of the post‐fertilisation egg surface. We have studied the lamellar structure of CGs using X‐ray scattering and reveal the contrast density variation of the lamellae in the native state. The structure of functionally competent CGs in situ differs significantly from that determined by electron microscopic studies. We observed a strong periodicity of the lamellar structure of 280 Å as opposed to the 590 Å repeat observed previously. Fusion of the CGs produced a loss of the lamellar repeat and the development of a broad peak corresponding to a 20 Å periodicity that may be indicative of the molecular packing in the resulting hydrated gel structure.
An endogenous redox molecule, thioredoxin, regulates transactivation of epidermal growth factor receptor and activation of NF‐κB by lysophosphatidic acid
Authors: Hirota Kiichi , Murata Miyahiko , Itoh Tatsuya , Yodoi Junji , Fukuda Kazuhiko
Published: July 2002

Lysophosphatidic acid (LPA) is the smallest and simplest of all the glycerophospholipids that activates a specific GTP‐binding protein coupled receptor to evoke multiple cellular responses. In this paper, we have demonstrated that LPA stimulates nuclear factor (NF)‐κB‐dependent gene induction in a neuronal cell line, NG108‐15 and that this is under redox regulation by an endogenous molecule, thioredoxin. We also have shown that redox‐sensitive transactivation of epidermal growth factor receptor by LPA confers NF‐κB activation and small GTPase proteins are involved in this pathway.
Overexpression of endonuclease III protects Escherichia coli mutants defective in alkylation repair against lethal effects of methylmethanesulphonate
Authors: Eide Lars , Fosberg Ellen , Hoff Elsebeth , Seeberg Erling
Published: July 2002

Endonuclease III of Escherichia coli is normally involved in the repair of oxidative DNA damage. Here, we have investigated a possible role of EndoIII in the repair of alkylation damage because of its structural similarity to the alkylation repair enzyme 3‐methyladenine DNA glycosylase II. It was found that overproduction of EndoIII partially relieved the alkylation sensitivity of alkA mutant cells. Site‐directed mutagenesis to make the active site of EndoIII more similar to AlkA (K120W) had an adverse effect on the complementation and the mutant protein apparently inhibited repair by competing for the substrate without base release. These results suggest that EndoIII might replace AlkA in some aspect of alkylation repair, although high expression levels are needed to produce this effect.
Pigment conformation and pigment–protein interactions in the reconstituted Lhcb4 antenna protein
Authors: Pascal Andy , Gastaldelli Mirko , Ceoldo Stefania , Bassi Roberto , Robert Bruno
Published: July 2002

Resonance Raman spectra of the native Lhcb4 antenna protein are compared with those of a recombinant protein prepared by in vitro refolding of its polypeptide, over‐expressed in Escherichia coli, with added pigments [Giuffra et al. (1996) Eur. J. Biochem. 238, 112–120]. The results indicate that the native pigment conformation is reproduced almost perfectly in the reconstituted protein, with only small differences which are attributed to a slight shift in the Soret absorption peak of two or more chlorophylls. This procedure therefore represents a model system for the investigation of site‐directed mutant LHC proteins, which are otherwise very difficult to obtain.
The speckling domain of the Wilms tumor suppressor WT1 overlaps with the transcriptional repression domain
Authors: Herzer Ute , Lutz Bertram , Hartmann Kristina , Englert Christoph
Published: July 2002

The Wilms tumor suppressor gene WT1 encodes a zinc finger protein, expressed as different splicing variants, that has all the hallmarks of a transcription factor. The −KTS form of WT1 displays a homogeneous localization within the nucleus and has been shown to activate or repress the activity of various target genes. In contrast, the WT1(+KTS) variant demonstrates a speckled pattern of expression within the nucleus. This and its association with factors of the splicing machinery has led to the hypothesis that WT1(+KTS) might play a role in post‐transcriptional processes. By the generation of a series of deletion constructs and subsequent immunofluorescence analysis, we have identified and characterized the domain which is responsible for the localization of WT1 variants in nuclear speckles. The speckling domain comprises amino acids 76–120 within the N‐terminus of WT1 and is sufficient to target other proteins into distinct nuclear domains. Interestingly the WT1 speckling domain does not overlap with the domain required for interaction with the splicing factor U2AF65 but overlaps with the transcriptional repression domain. Thus our data challenge the view that association of WT1 with spliceosomes is responsible for the speckling phenotype.
Lysophosphatidic acid‐independent platelet activation by low‐density lipoprotein
Authors: Korporaal Suzanne J.A , Relou Ingrid A.M , van Rijn Herman J.M , Akkerman Jan-Willem N
Published: July 2002

Mildly oxidized low‐density lipoprotein activates platelets through lysophosphatidic acid (LPA). Hence, the platelet‐activating properties attributed to native low‐density lipoprotein (nLDL) might be caused by LPA contamination. We show that nLDL enhances thrombin receptor‐activating peptide (TRAP)‐induced fibrinogen binding to αIIbβ3. The LPA receptor blocker N‐palmitoyl‐L‐serine‐phosphoric acid did not affect nLDL‐enhanced fibrinogen binding induced by TRAP, but reduced TRAP‐induced binding. cAMP and inhibitors of protein kinase C and Ca2+ rises completely blocked ligand binding by TRAP and nLDL/TRAP. Inhibitors of p38MAPK and ADP secretion interfered only partially. Blockade of Rho‐kinase increased ligand binding 2–3‐fold. We conclude that nLDL enhances TRAP‐induced fibrinogen binding independent of LPA.
Inhibition of mitochondrial protein synthesis promotes autonomous regulation of mtDNA expression and generation of a new mitochondrial RNA species
Authors: Selwood Simon P , McGregor Alistair , Lightowlers Robert N , Chrzanowska-Lightowlers Zofia M.A
Published: July 2002

Mammalian mitochondria are known to proliferate in response to several stimuli. Proliferation requires an increase in expression of genes encoding proteins involved in mitochondrial biogenesis, as well as in the replication and expression of mitochondrial DNA (mtDNA). In contrast, we report that inhibiting mitochondrial protein synthesis causes a modulation in mtDNA gene expression without the concomitant increase in proliferative markers. Further, inhibition results in the production of a previously unidentified light‐strand mitochondrial RNA that spans the entire displacement loop, the function of which is currently unknown.
Functions of the conserved anionic amino acids and those interacting with the substrate phosphate group of phosphoglucose isomerase
Authors: Meng Menghsiao , Lin Hua-Yang , Hsieh Chia-Jung , Chen Yen-Ting
Published: July 2002

Phosphoglucose isomerase catalyzes the isomerization between glucose 6‐phosphate and fructose 6‐phosphate in cytoplasm, and functions as autocrine motility factor and neuroleukin outside the cells. A phosphoglucose isomerase from Bacillus stearothermophilus (pgiA) was subjected to mutagenesis study to address the catalytic function of the conserved anionic residues and those probably interacting with the phosphate group of substrates. The results suggest that Glu290 works concertedly with His311 as a general acid–base pair to initiate the isomerization step, and Glu150 assists the base function of His311. The conserved loop structure consisting of Gly205–Gly206–Arg207 plays a critical role for the recognition of substrates.
Isotope effects in the study of enzymatic phosphoryl transfer reactions
Authors: Hengge Alvan C
Published: July 2002

Protein‐tyrosine phosphatases and serine/threonine protein phosphatases utilize very different catalytic machinery to catalyze phosphoryl transfer reactions. Tyrosine is a better leaving group than serine or threonine, having a pK
a more than three units lower. Has the difference in the catalytic machinery used by these enzyme families evolved as a result of the difference in the lability of their substrates? Are the transition states for phosphoryl transfer similar for the two classes of enzymes? This review summarizes what has been learned from kinetic isotope effects about the nature of enzymatic phosphoryl transfer, and how the enzymatic mechanisms compare to uncatalyzed phosphoryl transfer reactions.
Investigating the conformational coupling between the transmembrane and cytoplasmic domains of a single‐spanning membrane protein
Authors: Mousson Florence , Beswick Veronica , Coı̈c Yves-Marie , Huynh-Dinh Tam , Sanson Alain , Neumann Jean-Michel
Published: July 2002

PMP1 is a 38‐residue single‐spanning membrane protein whose C‐terminal cytoplasmic domain, Y25–F38, is highly positively charged. The conformational coupling between the transmembrane span and the cytoplasmic domain of PMP1 was investigated from 1H‐nuclear magnetic resonance data of two synthetic fragments: F9–F38, i.e. 80% of the whole sequence, and Y25–F38, the isolated cytoplasmic domain. Highly disordered in aqueous solution, the Y25–F38 peptide adopts a well‐defined conformation in the presence of dodecylphosphocholine micelles. Compared with the long PMP1 fragment, this structure exhibits both native and non‐native elements. Our results make it possible to assess the influence of a hydrophobic anchor on the intrinsic conformational propensity of a cytoplasmic domain.
The heat shock factor and mitochondrial Hsp70 are necessary for survival of heat shock in Saccharomyces cerevisiae
Authors: Nwaka Solomon , Mechler Bernd , von Ahsen Oliver , Holzer Helmut
Published: July 2002

A heat shock recovery assay on solid medium (Nwaka et al. (1995) J. Biol. Chem. 270, 10193–10198) as well as the classical cell counting method were used to investigate the function of some heat shock proteins in thermotolerance. We show that expression of intact heat shock factor protein (HSF), which regulates the stress induced expression of heat shock proteins (HSPs), is necessary for recovery from heat shock. A HSF1 mutant (hsf1‐m3) which does not induce the expression of some heat shock proteins at heat stress (37–40°C) is defective in recovery after heat shock at 50–52 C compared to a corresponding wild‐type strain in both stationary and exponentially growing cells. Using two temperature sensitive mutants of the mitochondrial Hsp70 (ssc1–2 and ssc1–3) encoded by the SSC1 gene, we show that the ssc1–3 mutant, which has a mutation in the ATPase domain, is defective in recovery after heat shock in contrast to the ssc1–2 mutant, which has a mutation in the peptide binding domain. Different binding capacities for unfolded proteins are shown to be the molecular reason for the observed phenotypes. The thermotolerance defect of the hsf1‐m3 and ssc1–3 mutants is demonstrated for both glucose and glycerol media.
Augmentation of ouabain sensitivity of rat liver Na/K‐ATPase by in vivo adenovirus‐mediated expression of the Na/K‐ATPase α2 subunit
Authors: Askari Frederick K , Hitomi Yoshinori , Xie Zijian
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Modulation of cell surface lectin receptors on K562 human erythroleukemia cells induced by transfection with annexin IV cDNA
Authors: Satoh Ayano , Takayama Eiji , Kojima Kyoko , Ogawa Haruko , Katsura Yoshimoto , Kina Tatsuo , Irimura Tatsuro , Matsumoto Isamu
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Caged cysteine and thiophosphoryl peptides
Authors: Pan Peng , Bayley Hagan
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Cytotoxic protein from human platelets
Authors: Golubeva T.S. , Paromov V.M. , Tuguz A.R. , Chertov O.Yu. , Kiselevsky M.V.
Published: July 2002

The 14 kDa protein was purified from human platelets. It displays high cytotoxic activity to the human ACL cells at 10 M concentration (21.8±7.1%). Its N‐terminal sequence is YAPQXQFGP–, being highly homologous to region 241–249 residues of the human Cls complement component.
Influence of antibody binding on oxygen binding behavior of Panulirus interruptus hemocyanin
Authors: Perton Frank G , Beintema Jaap J , Decker Heinz
Published: July 2002

Oxygen binding behavior of monomeric subunit a and the hexameric form of this subunit of hemocyanin of Panulirus interruptus is influenced by the binding of various monoclonal antibodies. These antibodies react with other surface parts of the subunit than its second domain in which the oxygen binding site is located. The influence of three monoclonal antibodies and their antigen binding fragments (Fab) has been investigated. Two antibodies increase the oxygen affinity of monomeric hemocyanin from that observed in its low affinity T‐state, while the third has little influence on this property. Fab fragments abolish almost completely the cooperativity of oxygen binding by the hexameric hemocyanin molecule. The two antibodies which increase the oxygen affinity of the monomeric molecule stabilize high‐affinity states of the hexameric molecule, while the third stabilizes the low‐affinity state.
pH dependence of bovine mast cell tryptase catalytic activity and of its inhibition by 4′,6‐diamidino‐2‐phenylindole
Authors: Fiorucci Laura , Erba Fulvio , Bolognesi Martino , Massimiliano Coletta  , Ascoli Franca
Published: July 2002

Tryptases are oligomeric enzymes localized in the secretory granules of mast cells. Their role(s) in vivo has yet to be clarified and the lack of powerful and specific inhibitors has hampered the comprehension of the biological functions of these enzymes. In this paper, we identify 4′,6‐diamidino‐2‐phenylindole as a potent inhibitor for bovine tryptase. This inhibitory effect and the enzyme catalyzed hydrolysis of the synthetic substrate Boc‐Phe‐Ser‐Arg‐methyl‐coumarin were investigated in the pH range of 6.0–9.0. On the basis of the pK shifts occurring upon formation of the inhibitor(substrate)/enzyme complexes, some aminoacidic groups are proposed to play a role in such interactions.
Hydrogenosomes in the anaerobic fungus Neocallimastix frontalis have a double membrane but lack an associated organelle genome
Authors: van der Giezen Mark , Sjollema Klaas A , Artz Rebekka R.E , Alkema Wynand , Prins Rudolf A
Published: July 2002

The presence of hydrogenosomes in phylogenetically distinct anaerobic eukaryotes implies that they have been acquired independently, and previously reported differences in ultrastructure among taxa have suggested that some hydrogenosomes have different origins. Of particular interest are reports that Neocallimastix frontalis hydrogenosomes resemble microbodies in possessing a single membrane, in contrast to those in ciliates and trichomonads which have two and thus resemble mitochondria. In this investigation we have clearly demonstrated that N. frontalis hydrogenosomes possess two, rather than one, closely apposed membranes and in some preparations cristae‐like structures were observed. These observations have led us to reject the microbody hypothesis and provide some indirect support for a possible mitochondrion origin as proposed for other hydrogenosomes. N. frontalis hydrogenosomes were shown to lack an associated genome as previously demonstrated for trichomonad hydrogenosomes. This might be explained by assuming that a mitochondrial genome encoding proteins for aerobic function is no longer necessary for either organelle.
The leech excitatory peptide, a member of the GGNG peptide family: isolation and comparison with the earthworm GGNG peptides
Authors: Minakata Hiroyuki , Fujita Tsuyoshi , Kawano Tsuyoshi , Nagahama Tomoaki , Oumi Tomoyuki , Ukena Kazuyoshi , Matsushima Osamu , Muneoka Yojiro , Nomoto Kyosuke
Published: July 2002

A member of the GGNG peptide family was isolated from Hirudo nipponia (leech). GGNG peptides had only been isolated previously from earthworms. The C‐terminus structure of the leech peptide, LEP (leech excitatory peptide), was –Gly–Gly–Asn–amide, while that of the earthworm peptides, EEP (earthworm excitatory peptide), was –Gly–Gly–Asn–Gly. LEP exerted 1000‐fold more potent activities on leech gut than did EEP‐2. On the other hand, EEP‐2 was 1000‐fold more potent than LEP on the crop‐gizzard of the earthworm. Analog peptides of LEP and EEP‐2 were synthesized, and the myoactive potency of each analog on the leech and earthworm tissues was compared.
Spectral studies on the calcium‐binding properties of Mts1 protein and its interaction with target protein
Authors: Dukhanina E.A , Dukhanin A.S , Lomonosov M.Y , Lukanidin E.M , Georgiev G.P
Published: July 2002

Two calcium‐binding sites of the Mts1 protein, a member of S‐100 protein family, were distinguished with the Fluo‐3 fluorescent technique. The geometric mean of the apparent dissociation constant () for these two sites is 2.6 μM; the Hill coefficient (n
H) is 0.98. In the presence of a novel target protein p37, isolated from the mouse adenocarcinoma cell line CSML‐100, Mts1 binds Ca2+ ions with higher affinity and with strong positive cooperativity (=0.2 μM, n
H=1.91). Interaction of Mts1 with p37 is confirmed by the fluorescent probe 2‐p‐toluidinylnaphthalene‐6‐sulfonate (TNS). Reaction with TNS shows that p37 interacts with the hydrophobic site of Mts1 which is exposed due to the binding of Ca2+ ions.
Centrifugation does not alter spatial distribution of ‘BEP4’ mRNA in paracentrotus lividus EGG
Authors: Costa Caterina , Rinaldi Anna Maria , Romancino Daniele P , Cavalcante Carmela , Vizzini Aiti , Di Carlo Marta
Published: July 2002

Paracentrotus lividus unfertilized eggs were centrifuged in a sucrose gradient, so to split each into two parts: a nucleated light fragment and an anucleated heavy fragment. Northern blot analyses utilizing a bep4 probe as animal marker and H2A histone gene and 12S‐mit RNA as controls indicate that the eggs are elongated along the animal‐vegetal axis during centrifugation and thereafter split into an animal and a vegetal half. Treatment of the eggs with colchicine before centrifugation abolishes the animal localization of bep4 mRNA.
Apoptosis induced by HIV‐gp120 in a Th1 clone involves the generation of reactive oxygen intermediates downstream CD95 triggering
Authors: Radrizzani Marina , Accornero Paola , Delia Domenico , Kurrle Roland , Colombo Mario P.
Published: July 2002

HIV‐gp120 sensitizes Th1 clones from seronegative donors to apoptosis, which occurs through two distinct events: expression of CD95L followed by its interaction with CD95 to trigger cell death. gp120‐apoptosis of the Th1 clone 103 was inhibited by Cyclosporin A, the PTK inhibitors Genistein and PNU152518, as well as the anti‐oxidants Ascorbic Acid and Glutathione. Cyclosporin A interfered with CD95L expression, Ascorbic Acid and Glutathione inhibited cell death triggered by CD95/CD95L interaction; Genistein and PNU152518 acted on both steps. The occurrence of oxidative stress during CD95‐dependent apoptosis was supported by the direct evidence of ROI production.
Differential action of AGCF2 upon cell type‐dependent expression of human angiotensinogen gene
Authors: Yanai Kazuyuki , Matsuyama Shigeki , Murakami Kazuo , Fukamizu Akiyoshi
Published: July 2002

To investigate the regulatory mechanisms of human angiotensinogen (ANG) gene expression in the brain, we analyzed the 1.3‐kb promoter by transfection studies and gel shift assays. The region from −106 to +44 was sufficient for promoter activity in glioblastoma cells, and multiple nuclear factors including AGCF2 (human
N

ore promoter binding
actor
) bound within this 150‐bp region. The mutations within AGCF2‐binding elements decreased the transcriptional activity in glioblastoma cells but rather increased it in hepatoma cells. These results indicate that AGCF2 has a differential function between these cells and contributes to the glia‐dependent angiotensinogen promoter activity.
Isolation of mRNAs induced by a hazardous chemical in white‐rot fungus, Coriolus versicolor, by differential display
Authors: Iimura Yosuke , Tatsumi Kenji
Published: July 2002

White‐rot fungus Coriolus versicolor, a ligninolytic basidiomycete, has been studied because of its ability to degrade hazardous chemicals. In this study, we searched for genes that are induced by a hazardous chemical using the mRNA differential‐display technique and C. versicolor IFO30340 that has been exposed to pentachlorophenol (PCP). Five cDNA fragments were cloned and the DNA sequences of two fragments were analyzed in further detail. The clones corresponded to novel genes that have not previously been identified in C. versicolor. One of the cDNAs exhibited strong sequence homology to the gene for an enolase and the other exhibited homology to a heat shock protein. The expression of the two genes was up‐regulated in PCP‐treated C. versicolor.
Burdock, a novel retrotransposon in Drosophila melanogaster, integrates into the coding region of the cut locus
Authors: Ponomarenko N.A. , Bannikov V.M. , Anashchenko V.A. , Tchurikov N.A.
Published: July 2002

The burdock element is known to be the 2.6‐kb insertion into the same region of the cut locus in 12 independently obtained ct‐lethal mutants. Here we have determined the complete sequences of this insertion and of the hot spot region. It was found that the burdock is a short retrotransposon with long terminal repeats and a single open reading frame (ORF). The polypeptide encoded by the burdock ORF contains two adjacent regions homologous to the gag and pol polyproteins of the gypsy mobile element. The burdock insertion interrupts the short ORF of the cut locus. The target site sequence of the burdock insertions is similar to the Drosophila topoisomerase II cleavage site.
Small‐angle X‐ray scattering study on CEL‐III, a hemolytic lectin from Holothuroidea Cucumaria echinata, and its oligomer induced by the binding of specific carbohydrate
Authors: Fujisawa Tetsuro , Kuwahara Hiromiki , Hiromasa Yasuaki , Niidome Takuro , Aoyagi Haruhiko , Hatakeyama Tomomitsu
Published: July 2002

Hemolytic lectin CEL‐III from a marine invertebrate Cucumaria echinata forms an oligomer upon binding of specific carbohydrate such as lactose at high pH values and in the presence of high concentrations of salt. In this study, using small‐angle X‐ray scattering, we characterized CEL‐III and its oligomer induced by the binding of lactose. The molecular mass of the oligomer was determined as 1019 kDa from its forward scattering value, compared with 47 490 Da for the monomer. This oligomer size is much larger than that estimated using SDS–polyacrylamide gel electrophoresis (SDS‐PAGE, 270 kDa). The monomer has a 24.6 Å radius of gyration and can be approximated by a rod which has a 20 Å radius and a height of 75 Å, while the oligomer has a 101.4Å radius of gyration. Together with the comparison of the radii of gyration and the forward scattering of the cross‐section of the monomer and oligomer, it is suggested that in aqueous solution the oligomer comprises three or four molecules of a smaller unit which was observed by SDS‐PAGE (270 kDa), held by a relatively weak interaction. The scattering profile also suggests that the oligomer has a hole in its central axis which might be associated with the formation of ion‐permeable pores in the erythrocyte membrane by CEL‐III during the hemolytic process.
Permeation and gating of α1 glycine‐gated channels expressed at low and high density in Xenopus oocyte
Authors: Maammar Mohammed , Rodeau Jean-Luc , Taleb Omar
Published: July 2002

When a high density of α1‐subunit glycine receptor (GlyR) is expressed in Xenopus oocytes, two populations of channels can be distinguished according to their apparent affinity for glycine which differs 5‐ to 6‐fold. To compare the open pore diameter of these channels, the relative permeability of formate with respect to chloride (P
formate/P
Cl) was determined in bionic conditions. For the low‐affinity GlyR P
formate/P
Cl was comparable to that reported for glycine‐gated channels in cultured spinal cord and hippocampal neurons. In contrast, the high‐affinity GlyR had a 56% larger P
formate/P
Cl. In addition, the open probability of the channels was differentially sensitive to voltage. These results show that the high expression of α1 GlyR resulted in two populations of GlyR which differed not only in the affinity to agonists but also in permeation and gating mechanisms.
Crystal structure of cytochrome P‐450cam complexed with the (1S)‐camphor enantiomer
Authors: Schlichting Ilme , Jung Christiane , Schulze Heike
Published: July 2002

The crystal structure of cytochrome P‐450cam complexed with the enantiomer (1S)‐camphor has been solved to 1.8 Å resolution and compared with the structure of the (1R)‐camphor P‐450cam complex. The overall protein structure is the same for both enantiomer complexes. However, the orientation of the substrates in the heme pocket differs. In contrast to (1R)‐camphor, the (1S)‐enantiomer binds in at least two orientations. The major binding mode of (1S)‐camphor resembles the one of the (1R)‐enantiomer in that there is a hydrogen bond between Tyr‐96 and the quinone group of camphor, and the 10‐methyl group points towards the I‐helix. The binding differs in that C‐5 is not at a position suitable for hydroxylation. In the other orientation (1S)‐camphor is not hydrogen bonded, but C‐5 is located suitably for hydroxylation.
Resistance to tumor necrosis factor (TNF) cytotoxicity by autocrine TNF production is independent of intracellular signaling pathways
Authors: Decoster Els , Vandevoorde Veronique , Beyaert Rudi , Haegeman Guy , Fiers Walter
Published: July 2002

We previously showed that autocrine tumor necrosis factor (TNF) production in the TNF‐sensitive L929sA fibrosarcoma cell line induced TNF resistance, which is correlated with downmodulation of both TNF receptors on the cell surface. We now analyzed whether autocrine TNF production also interfered with intracellular TNF signaling pathways. The L929sA‐CAT‐R55i cell line, in which cell death can be induced by controlled cytoplasmic expression of a trimeric fusion protein between chloramphenicol acetyltransferase and the intracellular domain of TNF‐R55 (CAT‐R55i), was supertransfected with the murine TNF gene. Expression of the latter conferred resistance to cell death induced by exogenous TNF, while cytotoxicity induced by CAT‐R55i was not impaired. This demonstrates that autocrine TNF did not induce intracellular mechanisms that block TNF signaling leading to cell death. Thus the induction of TNF resistance via autocrine TNF production in L929sA cells is solely due to downmodulation of TNF receptors on the cell surface.
Sequence analysis of a 24‐kb contiguous genomic region at the Arabidopsis thaliana PFL locus on chromosome 11
Authors: Terryn Nancy , Neyt Pia , De Clercq Rebecca , De Keyser Annick , Van Den Daele Hilde , Ardiles Wilson , Déhais Patrice , Rouzé Pierre , Gielen Jan , Villarroel Raimundo , Van Montagu Marc
Published: July 2002

As part of the European Union program of European Scientist Sequencing Arabidopsis (ESSA), the DNA sequence of a 24.053‐bp insert of cosmid clone CC17J13 was determined. The cosmid is located on chromosome 1 at the PFL locus (position 30 cM). Analysis of the sequence and comparison to public databases predicts seven genes in this area, thus approximately one gene every 3.3 kb. Three cDNAs corresponding to genes in this region were also sequenced. The homologies and/or possible functions of the (putative) genes are discussed. Proteins encoded by genes in this region include a polyadenylate‐binding protein (PAB‐3) and a GTP‐binding protein (Rab7) as well as a novel protein, possibly involved in double‐stranded RNA unwinding and apoptosis. Intriguingly, the gene encoding the PAB‐3 protein, which is very specifically expressed, is flanked by putative matrix attachment regions.
Synthesis of a new zinc finger peptide; comparison of its `code' deduced and `CASTing' derived binding sites
Authors: Corbi Nicoletta , Perez Marie , Maione Rossella , Passananti Claudio
Published: July 2002

Using two synthetic oligonucleotides, we have constructed a new gene containing three zinc finger motifs of the Cys2‐His2 type. We named this artificial gene `Mago'. The Mago nucleotide triplets encoding the amino acid positions, described to be crucial for DNA binding specificity, have been chosen on the basis of the proposed recognition `code' that relates the zinc finger's primary structure to the DNA binding target. Here we demonstrate that Mago protein specifically binds the `code' DNA target, with a dissociation constant (K
d) comparable to the K
d of the well known Zif268 protein with its binding site. Moreover, we show that the deduced Mago `code' and the `experimental' selected DNA binding sites are almost identical, differing only in two nucleotides at the side positions.
Modulation of fructose‐2,6‐bisphosphate metabolism by components of the extracellular matrix in cultured cells. Interaction with epidermal growth factor
Authors: Baulida Josep , Onetti Rafael , Bassols Anna
Published: July 2002

The use of NIH3T3 fibroblasts overexpressing different mutations of the EGF receptor shows that regulation of fructose‐2,6‐bisphosphate (Fru‐2,6‐P2) metabolism by EGF is mediated by the kinase activity of the EGF receptor and suggests a PLCγ1‐mediated mechanism. The effect of several extracellular matrix components on glucose metabolism was assessed by incubating A431 cells and NIH3T3 fibroblasts with heparin, laminin, fibronectin, collagen and PG‐I and PG‐II proteoglycans and measuring the levels of Fru‐2,6‐P2. Laminin increased the levels of Fru‐2,6‐P2 and heparin decreased the levels of the metabolite, whereas the other molecules did not have any effect. No effect of laminin or heparin in glucose uptake by the cell was observed. Laminin was able to modulate the effects of EGF on Fru‐2,6‐P2 concentration, suggesting cross‐talk between these agents.
Regulation of mammalian pyruvate dehydrogenase kinase
Authors: Popov Kirill M
Published: July 2002

It is generally believed that mammalian pyruvate dehydrogenase kinase is a heterodimer consisting of catalytic and regulatory subunits. However, the contribution of the two subunits to the kinase‐mediated signal transduction has remained undefined. In the present study recombinant components of mammalian pyruvate dehydrogenase complex were employed in order to characterize the role of the kinase catalytic subunit in the regulation of pyruvate dehydrogenase reaction. The results provide the first evidence strongly suggesting that the catalytic subunit of pyruvate dehydrogenase kinase is competent to respond to known effectors of kinase activity as well as to interact with the E2‐core without assistance of a regulatory subunit.
Jacalin interacts with Asn‐linked glycopeptides containing multi‐antennary oligosaccharide structure with terminal α‐linked galactose
Authors: Do Su-Il , Lee Ki-Young
Published: July 2002

The carbohydrate binding properties of jacalin lectin were examined using RAF9 cell‐derived d‐[6‐3H]glucosamine‐radiolabeled total glycopeptides containing N‐linked and O‐linked oligosaccharides. The binding of N‐linked glycopeptides to jacalin was abolished by treatment of α‐galactosidase whereas O‐linked glycopeptides were still bound lectin after this treatment. The removal of O‐linked oligosaccharides by mild alkaline/borohydride treatment completely eliminated the lectin binding of α‐galactosidase treated glycopeptides. These results demonstrate that jacalin interacts with cellular glycopeptides containing N‐linked oligosaccharides with terminal α‐galactose residues as well as glycopeptides containing O‐linked oligosaccharides.
Antibodies against EMC virus RNA‐VPg recognize Tyr‐(5′P→O)‐pU and immunostain infected cells
Authors: Bordunova Olga A , Turina Oksana V , Nadezhdina Elena S , Shatskaya Galina S , Veiko Vladimir P , Drygin Yuri F
Published: July 2002

Covalent complexes of nucleic acids and proteins are widespread among viruses. Covalent complexes of RNA and proteins are proposed to exist in eukaryotic cells. The goal of this work was to obtain specific antibodies to the covalent linkage unit (CLU) between virus RNA and protein to search cellular RNA‐protein complexes. Antibodies were generated by direct immunization of a rabbit with the BSA‐coupled EMC virus RNA‐VPg complex. By a dot‐blot immunoassay and immunofluorescent microscopy it was found that the antibodies specifically recognize both EMC virus RNA‐VPg and synthetic CLU‐containing compounds. Thus, a fraction of the antibodies was directed to CLU.
The transcription factor GHF‐1, but not the splice variant GHF‐2, cooperates with thyroid hormone and retinoic acid receptors to stimulate rat growth hormone gene expression
Authors: Sánchez-Pacheco Aurora , Peña Pilar , Palomino Teresa , Güell Ana , Castrillo José Luis , Aranda Ana
Published: July 2002

The rat growth hormone (GH) promoter was significantly activated in non‐pituitary cells by the expression of unliganded trioodothyronine (T3) and retinoic acid (RA) receptors. Furthermore, a strong ligand‐dependent activation was found in the presence of the pituitary‐specific transcription factor GHF‐1. When compared with GHF‐1, the splice variant GHF‐2 showed a decreased ability to bind the cognate site in the GH promoter. As a consequence, expression of GHF‐2 had little stimulatory effect on the GH promoter and did not show cooperation with T3 or RA receptors even in the presence of ligands. Furthermore, over‐expression of GHF‐2 inhibited the response to T3 and RA in pituitary cells. These results show that alternative splicing of the GHF‐1 gene gives rise to two isoforms that differ in their transactivating properties and in their ability to synergize with the nuclear thyroid hormone and retinoic acid receptors on GH gene expression.