bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphorylation by Ca2+/calmodulin‐dependent protein kinase II and protein kinase C of sepiapterin reductase, the terminal enzyme in the biosynthetic pathway of tetrahydrobiopterin
Authors: Katoh Setsuko , Sueoka Terumi , Yamamoto Yoshimi , Takahashi Susumu Y.
Published: July 2002

Sepiapterin reductase, the terminal enzyme in the biosynthetic pathway of tetrahydrobiopterin, was stoichiometrically phosphorylated by Ca2+/calmodulin‐dependent protein kinase II and protein kinase C (Ca2+/phospholipid‐dependent protein kinase) in vitro. Maximal incorporation of phosphate into the enzyme subunit by these was 3.05 ± 0.05 (n = 4) and 0.74 ± 0.03 (n = 5) 32P mol per mol enzyme subunit, respectively. The enzyme was not phosphorylated by cyclic nucleotide‐dependent protein kinase of either the cAMP‐dependent or cGMP‐dependent type in this study. Dihydropteridine reductase, another enzyme working in direct supply of tetrahydrobiopterin, was also a good substrate for Ca2+/calmodulin‐dependent protein kinase II. Phosphorylation of sepiapterin reductase by these protein kinases modified the kinetic properties of the enzyme. It is likely that these multifunctional Ca2+‐activated protein kinases may play a role in the regulation of the physiological function of the BH4‐generating enzymes in vivo, as was previously found in the case of BH4‐requiring enzymes.
Glycolysis is operative in amphibian oocytes
Authors: Guixé Victoria , Preller Ana , Kessi Eduardo , Hofer H.Werner , Ureta Tito
Published: July 2002

It is generally accepted that in frog full‐grown oocytes glycolysis is absent and that carbon metabolic flux is largely directed to glycogen synthesis. Use of an anion exchange pellicular resin for analytical resolution of intermediates in perchloric acid extracts of oocytes has allowed us to observe the formation of labelled lactate after microinjection of [U‐14C]glucose. Further, formation of [32P]ATP was observed after microinjection of 32P‐labelled glucose‐6‐P, fructose‐6‐P or fructose‐1,6‐bis‐P, either in the presence or absence of 0.1 mM cyanide. The presence of phosphofructokinase activity, previously thought to be absent in oocytes, is also reported. These findings indicate that glycolysis to lactate is operative in frog oocytes.
Dual pertussis toxin‐sensitive pathway of zymosan‐induced activation in guinea pig macrophages
Authors: Hazeki Kaoru , Tamoto Koichi , Ui Michio , Mori Yoki
Published: July 2002

Complement receptor type 3 (CR3)‐mediated cellular responses in guinea pig macrophages were investigated by using zymosan and serum‐opsonized zymosan (SOZ) as the multivalent ligand for CR3. The ingestion of zymosan and SOZ was accompanied by O2
− generation and arachidonate release. These responses were suppressed by prior exposure of macrophages to pertussis toxin (PT). Opsonization of zymosan gave rise to more than 6‐fold activation of the ingestion, whereas the magnitude of either arachinonate release or O2
− generation was unchanged. The Fab' fragment of anti‐Z‐1, a monoclonal antibody specific for the α chain of guinea pig CR3, inhibited the ingestion of zymosan by 60% without affecting zymosan‐induced arachidonate release and O2
− generation. These data suggested that there might be at least two functionally distinct binding sites for zymosan. O2
− generation and arachidonate release might be regulated through one site and phagocytosis another. Both sites should be coupled to PT‐sensitive GTP binding protein.
Ionic channel activity induced by fusion of Rhodospirillum rubrum chromatophores with a planar bilayer lipid membrane
Authors: Antonenko Yuri N. , Rokitskaya Tatiana I. , Kotova Elena A. , Taisova Aleksandra S.
Published: July 2002

The present work concerns mechanisms of ionic conductivity of photosynthetic membranes. It is shown that reconstitution of vesicles of photosynthetic membranes (chromatophores) of purple bacteria Rhodospirillum rubrum into a planar bilayer lipid membrane leads to fluctuations of current showing the existence of a channel with a predominant conductance of ~ 230 pS in the presence of 100 mM KCl. Measurements under the conditions of KCl gradient prove that this channel is cation selective (P
k/P
cl = 7.2). Voltage inactivation of the channel is demonstrated which is prevented by treatment with trypsin.
Localization and expression of the closely linked cyanelle genes for RNase P RNA and two transfer RNAs
Authors: Baum Michael , Schön Astrid
Published: July 2002

The genomic region encoding the RNA subunit of the cyanelle RNase P has been characterized, rnpB, which has no homologue in chloroplasts, is flanked by two tRNA genes on the complementary DNA strand. Transcriptional control elements of all three genes have been experimentally determined, Comparison of the sequence region with the corresponding loci of chloroplast genomes from vascular plants suggeststhat major inversion may have led to a possible loss or severe truncation of the RNase P RNA coding region during the course of plastid evolution.
cDNA cloning and characterization of A3i, an alternatively spliced rat A3 adenosine receptor variant
Authors: Sajjadi Fereydoun G. , Boyle David L. , Domingo Ron C. , Firestein Gary S.
Published: July 2002

A cDNA encoding a variant form of the A3 adenosine (Ado) receptor was isolated from rat by reverse transcription of brain mRNA followed by PCR. The full‐length receptor (A3i) cDNA encodes 337 amino acids and shares complete sequence identity with the rat A3 Ado receptor, except for the presence of a seventeen amino acid insert located in the second intracellular domain. In contrast to the rat A3 receptor, stable expression of A3i in CHO cells resulted in poor coupling to Gl proteins Analysis of receptor transcripts by RT‐PCR suggests that the A3 Ado receptor mRNAs are products of alternative splicing. Sequence analysis of A3 genomic DNA Identified a 1.7 kb intron that is likely alternatively spliced to produce the A3 and A3i receptors.
Interaction of a human blood group Sd(a−) Tamm‐Horsfall glycoprotein with applied lectins
Authors: Wu June H. , Watkins Winifred M. , Chen Chie-Pein , Song Shuh-Chyung , Wu Albert M.
Published: July 2002

Unlike the human blood group Sd(a+) Tamm‐Horsfall glycoprotein (THGP), the Sd(a−) one lacks terminal GalNAcβ1 → residues at the nonreducing ends. The binding properties of this glycoprotein and its asialo product with lectins were characterized by quantitative precipitin (QPA) and precipitin inhibition assays. Among 20 lectins tested by QPA, both native and asialo Sd(a−) THGP reacted best with Abrus precatorius and Ricinus communis and completely precipitated the lectin added. They also precipitated well Wistaria floribunda (WFA), Glycine max (SBA), Bauhinia purpurea alba, abrin‐a and ricin, all of which recognize the Galβ1 → 4GlcNacβ1 → sequence, although at different strength. The lectin‐glycan interactions were inhibited by Galβ1 → 4GlcNAc and Galβ1 → 4Glc. When the precipitability of Sd(a−) THGP was compared with that of the Sd(a+) phenotype, the native Sd(a−) THGP exhibited a 40% lesser affinity for WFA, SBA, WGA and mistletoe lectin‐I (ML‐I). Mapping the precipitation and inhibition profiles of the present study and the results of THGP Sd(a+), it is concluded that Sd(a−) THGP showed a strongly diminished affinity for GalNAcβ1 → active lectins (SBA and WFA) than the Sd(a+) phenotype.
Nonenzymatically evoked and cytochrome P450‐dependent lipid peroxidation inhibits synthesis of phosphatidylethanolamine via the ethanolamine base exchange reaction in rat liver microsomes
Authors: Jasińska Renata , Rakowska Mariola , Lenart Jacek , Komańska Izabela , Pikuła Sławomir
Published: July 2002

In the present study the relationship between lipid peroxidation, changes in the redox state of membrane and phosphatidylethanolamine (PE) synthesis via base exchange reaction in rat liver microsomes was investigated. It was found that PE synthesis is enhanced in the presence of antioxidants, butylated hydroxytoluene (BHT), or unsaturated free fatty acids. Prooxidants, tert‐butyl hydroxyperoxide (BHP), ferrous ions combined with ascorbate or NADPH (via cytochrome P450‐dependent proteins), increased the amount of lipid peroxidation products in the membrane, and in consequence inhibited the reaction. The effect of BHP was fully reversed by reduced glutathione and dithiothreitol (DTT), whereas the effect of other compounds could be reversed only by BHT. In contrast, a reversal of the inhibitory effect of cadmium ions on base exchange activity was observed in the presence of DTT, but not BHT. Therefore, both the ‐SH/‐S‐S‐ ratio in the membrane, affected by BHP and cadmium ions, and the lipid hydroxyperoxides (rather than aldehydes), generated by ferrous ions and ascorbate or NADPH, are equally responsible for the inactivation of the ethanolamine base exchange enzyme in rat liver microsomes. This may suggest that the synthesis of PE via the base exchange reaction may be considered an element of the superfine cellular machinery involved in the repair of damage to unsaturated fatty acid chains of phospholipids caused by reactive oxygen species under oxidative stress.
Detection of living cells that express AP1 using a fluorolabeled DNA probe
Authors: Shimokawa Noriaki , Miura Mitsuhiko
Published: July 2002

Activator protein 1 (AP1) is a complex of Fos and Jun, and it regulates the transcription of genes possessing the AP1‐binding sequence. The purpose of this study was to detect living cells that express AP1 after stimulation with a tumor promoter. The Fos and Jun components of AP1 were induced rapidly and transiently in PC12 cells following the addition of phorbol ester (phorbol 12‐myristate 13‐acetate, PMA). The DNA fragment containing the AP1‐binding sequence was combined with ethidium bromide, which was used as a fluorescent probe. The probe was transfected into the cells using cationic liposomes. Fluorescence in the transfected cells was observed using a fluorescence microscope. The nuclei of transfected cells emitted strong fluorescence in the presence of PMA, whereas weak fluorescence was retained in the cytoplasm in its absence. The former phenomenon is evidence that AP1 combined with the fluorescent probe was transported into the nuclei. This study suggests that such a fluorolabeling method is feasible to detect living AP1‐expressed neurons.
NMR investigations of the structural properties of the nodulation protein, NodF, from Rhizobium leguminosarum and its homology with Escherichia coli acyl carrier protein
Authors: Ghose Ranajeet , Geiger Otto , Prestegard James H.
Published: July 2002

Heteronuclear NMR methods have been used to elucidate the secondary structure and the general tertiary fold of the protein NodF from Rhizobium leguminosarum. A similarity to acyl carrier proteins of the fatty acid synthase system had been suggested by the presence of a phosphopantetheine prosthetic group and a short stretch of sequence homology near the prosthetic group attachment site. NMR results suggest that the structural homology extends well beyond this region. Both proteins have three well‐formed helices which fold in a parallel‐antiparallel fashion and a prosthetic group attachment site near the beginning of the second helix.
Asp‐285 of the metal‐tetracycline/H+ antiporter of Escherichia coli is essential for substrate binding
Authors: Kimura Tomomi , Yamaguchi Akihito
Published: July 2002

The transposon Tn10‐encoded metal‐tetracycline/H+ antiporter (TetA(B)) was preferentially photolabeled when [3H]tetracycline was irradiated in the presence of energized inverted membrane vesicles containing the TetA protein. The degree of labeling depended on the duration of irradiation and the energization of the membrane. Photolabeling was not observed in vesicles containing the Asp‐285 → Asn mutant TetA protein, indicating that Asp‐285 participates in the substrate binding or the step(s) prior to substrate binding
NO2‐induced expression of specific protein kinase C isoforms and generation of phosphatidylcholine‐derived diacylglycerol in cultured pulmonary artery endothelial cells
Authors: Li Yong D. , Patel Jawaharlal M. , Block Edward R.
Published: July 2002

The present study examines whether nitrogen dioxide (NO2)‐induced activation of protein kinase C (PKC) is associated with increased expression of specific PKC isoforms and/or with enhanced generation of phosphatidylcholine(PC)‐derived diacylglycerol (DAG) in pulmonary artery endothelial cells (PAEC). Western blot analysis revealed that exposure to 5 ppm NO2 resulted in increased expression of PKC α and ε isoforms in both cytosol and membrane fractions in a time‐dependent fashion compared with controls. A time‐dependent elevated expression of PKC isoform β was observed in the cytosol fraction only of NO2‐exposed cells. PKC isoform λ was not detectable in either the cytosolic or membrane fractions from control or NO2‐exposed cells. Scatchard analysis of [3H]phorbol 12,13‐dibutyrate (PDBu) binding showed that exposure to NO2 for 24 h increased the maximal number of binding sites (B
max) from 15.2±2.3 pmollmg (control) to 42.3±5.3 pmol/mg (p < 0.01, n = 4) (NO2‐exposed). Exposure to NO2 significantly increased PC specific‐phospholipase C and phospholipase D activities in the plasma membrane of PAEC (p < 0.05 and p < 0.001, respectively). When [3H]myristic acid‐labeled cells were exposed to NO2, significantly increased radioactivity was associated with cellular DAG. These results show for the first time that exposure of PAEC to NO2 results in elevated expression of specific PKC isoforms and in enhanced generation of cellular DAG, and the latter appears to arise largely from the hydrolysis of plasma membrane PC.
DIFFERENTIAL NEURAL CREST CELL ATTACHMENT AND MIGRATION ON AVIAN LAMININ ISOFORMS
Authors: Perris Roberto , Brandenberger Ralph , Chiquet Matthias
Published: July 2002

A number of laminin isoforms have recently been identified and proposed to exert different functions during embryonic development. In the present study, we describe the purification and partial characterization of several isoforms isolated from chick heart and gizzard, and provide data on the molecular mechanisms underlying the interaction of avian neural crest cells with these molecules in vitro. Laminins extracted from heart and gizzard tissues were separated by gel filtration and purified to homogeneity by sequential lectin and immunoaffinity chromatography by utilizing monoclonal antibodies directed against the avian α2, β2 and γ1 laminin chains. The sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS—PAGE) banding pattern of the polypeptide complexes obtained and immunoblotting with polyclonal antisera allowed the identification of Laminin‐2 (α2β1γ1), Laminin‐4 (α2β2γ1), and laminins comprising the β1, β2 and γ1 chains associated with a shorter α chain which, in SDS—PAGE, co‐migrate with the β/γ complex in the 200 kDa region. These latter laminins, which are here arbitrarily denoted Laminin‐αχ (heart tissue) and Laminin‐G (gizzard tissue), are somewhat distinct in their apparent molecular weight, are differentially associated with nidogen, and appear as “T”‐shaped particles similar to Laminin‐6 and Laminin‐7 when analyzed by transmission electron microscopy following rotary shadowing. In contrast, the avian Laminin‐2 and Laminin‐4 isoforms exhibit the characteristic cruciform shape described previously for their mammalian counterparts. Isolated neural crest cells differentially attached and migrated on these laminin isoforms, showing a clear preference for Laminin‐G. Similarly to the EHS Laminin‐1, neural crest cells recognized all avian isoforms through their α1β1 integrin, shown previously to be the primary laminin‐binding receptor on these cells. Neural crest cell interaction with the avian laminins was dependent upon maintenance of the secondary and tertiary structure of the molecules, as shown by the marked reduction in cell attachment and migration upon disruption of the α‐helical coiled‐coil structure of their constituent chains. The results demonstrate that different laminin isoforms may be differentially involved in the regulation of neural crest cell migration and suggest that this regulation operates through interaction of the cells with a structurally conserved cell binding site recognized by the α1β1 integrin.
Sleep, morning alertness and quality of life in subjects treated with zopiclone and in good sleepers. Study comparing 167 patients and 381 good sleepers
Authors: Léger D , Janus C , Pellois A , Quera-Salva MA , Dreyfus JP
Published: July 2002

From 20,000 households regularly surveyed by a national survey institute (Sofres), two groups were selected and questioned (using a 23-item questionnaire) about sleep vigilance and quality of life. The first group was composed of insomniacs using zopiclone for the last 12 months and no other CNS treatments (167 subjects). The second group was composed of 381 “good sleepers” selected as having no or occasionally one sleep disturbance in the last 12 months. No difference was found between the two groups in average total sleep time. Sleep disturbances were statistically equal in the two groups except for difficulties in initiating sleep which is more present for zopiclone users (13% vs 3%). Vigilance was mostly comparable in both groups.Five aspects of quality of life explored by the questionnaires (the professional, relational and sentimental, domestic, leisure and safety aspects) appeared to be comparable in the two groups.
GroES co‐chaperonin small‐angle X‐ray scattering study shows ring orifice increase in solution
Authors: Timchenko A.A. , Melnik B.S. , Kihara H. , Kimura K. , Semisotnov G.V.
Published: July 2002

GroES consists of seven identical 10 kDa subunits and is involved in assisting protein folding as the partner of another oligomeric protein, the GroEL chaperonin. Here we studied the GroES structure in solution using small‐angle X‐ray scattering (SAXS). The SAXS pattern, calculated for the GroES crystal structure, was found to be different from the experimental one measured in solution. The synchronic shift in the radial direction and some turning of the protein subunits eliminate the difference and result in the increase of the hole diameter in the GroES ring‐like structure from 8 Å in the crystal to 21 Å in solution.
Effect of DNA topology on plasmid DNA repair in vivo
Authors: Park Jik-Young , Ahn Byungchan
Published: July 2002

Escherichia coli nucleotide excision repair (NER) is responsible for removing bulky DNA adducts by dual incisions of the UvrABC endonuclease. Although the activity of the UvrAB complex which can induce DNA conformational change is employed in NER, the involvement of DNA topology and DNA topoisomerases remains unclear. We examined the effect of topoisomerase inhibitions on a NER in vivo system. The repair analysis of intracellular plasmid revealed that the DNA damage on positive supercoils generated by gyrase inhibition remained unrepaired, whereas the DNA damage was repaired in topoisomerase I mutants. These results suggest that DNA topology affects the NER process and the removal of positive supercoils by gyrase is vital for the efficiency of the E. coli NER system.
Conservation of the 3′‐untranslated region of the Rab1a gene in amniote vertebrates: exceptional structure in marsupials and possible role for posttranscriptional regulation
Authors: Wedemeyer Niels , Schmitt-John Thomas , Evers Dirk , Thiel Cora , Eberhard Daniel , Jockusch Harald
Published: July 2002

The YPT1/RAB1 protein, a key regulator of the intracellular vesicle transport in eukaryotes, is highly conserved in function and amino acid sequence. Here we report that the most highly conserved nucleotide sequence of the Rab1a gene of amniote vertebrates corresponds to the 3′‐untranslated region (3′‐UTR) of the mRNA. Sequences of 27 species ranging from mammals to sauropsida are >91% identical in this region. Secondary structure prediction procedures applied to the 3′‐UTR sequences between positions 750 and 984 and 1428 (mouse cDNA: Y00094), respectively, of the RAB1a mRNAs revealed families of alternative structures around nucleotide position 800 as recurrent features. The two hairpin loops are also predicted for marsupials, despite of their exceptional extension of the A‐rich sequence in between. Yet, sequence conservation is much higher than required to conserve secondary structure. Implications for posttranscriptional regulation and protein binding are discussed.
Functional specificity conferred by the unique plasticity of fully α‐helical Ras and Rho GAPs
Authors: Souchet Michel , Poupon Anne , Callebaut Isabelle , Léger Isabelle , Mornon Jean-Paul , Bril Antoine , Calmels Thierry P.G.
Published: July 2002

Structural comparisons of the two GTPase activating proteins (GAPs) p120 and p50 in complex with Ras and Rho, respectively, allowed us to decipher the functional role of specific structural features, such as helix α8c of p120 and helix A1 of p50, necessary for small GTPase recognition. We identified important residues that may be critical for stabilization of the GAP/GTPase binary complexes. Detection of topohydrophobic positions (positions which are most often occupied by hydrophobic amino acids within a family of protein domains) conserved between the two GAP families led to the characterization of a common flexible four‐helix bundle. Altogether, these data are consistent with a rearrangement of several helices around a common core, which strongly supports the assumption that p50 and p120 GAPs derive from a unique fold. Considered as a whole, the remarkable plasticity of GAPs appears to be a means used by nature to accurately confer functional specificity.
Molecular dynamics simulation of four‐α‐helix bundles that bind the anesthetic halothane
Authors: Davies Lowri A. , Zhong Qingfeng , Klein Michael L. , Scharf Daphna
Published: July 2002

The mutation of a single leucine residue (L38) to methionine (M) is known experimentally to significantly increase the affinity of the synthetic four‐α‐helix bundle (Aα2)2 for the anesthetic halothane. We present a molecular dynamics study of the mutant (Aα2–L38M)2 peptide, which consists of a dimer of 62‐residue U‐shaped di‐α‐helical monomers assembled in an anti topology. A comparison between the simulation results and those obtained for the native (Aα2)2 peptide indicates that the overall secondary structure of the bundle is not affected by the mutation, but that the side chains within the monomers are better packed in the mutant structure. Unlike the native peptide, binding of a single halothane molecule to the hydrophobic core of (Aα2–L38M)2 deforms the helical nature of one monomer in a region close to the mutation site. Increased exposure of the cysteine side chain to the hydrophobic core in the mutant structure leads to the enhancement of the attractive interaction between halothane and this specific residue. Since the mutated residues are located outside the hydrophobic core the observed increased affinity for halothane appears to be an indirect effect of the mutation.
Cholestan‐3β,5α,6β‐triol, but not 7‐ketocholesterol, suppresses taurocholate‐induced mucin secretion by cultured dog gallbladder epithelial cells
Authors: Yoshida Tadashi , Klinkspoor J.Henriëtte , Kuver Rahul , Wrenn Steven P. , Kaler Eric W. , Lee Sum P.
Published: July 2002

In order to investigate oxysterol‐mediated effects on the biliary system, we studied the effects of cholestan‐3β,5α,6β‐triol (TriolC) and 7‐ketocholesterol (7KC) on gallbladder epithelial cells. We compared their cell proliferation effects in cultured dog gallbladder epithelial cells (DGBE) to their effects in cultured human pulmonary artery endothelial cells (HPAE). Oxysterols inhibited cell proliferation in a dose‐dependent fashion. Oxysterols inhibited cell growth to 50% of control at a higher dose for DGBE cells than for HPAE cells. TriolC was more cytotoxic than 7KC. We also investigated the effect of oxysterols on bile salt‐induced mucin secretion by DGBE cells. TriolC suppressed mucin secretion by DGBE cells, whereas 7KC did not. These findings support the hypothesis that biliary oxysterols affect gallbladder mucosal function.
Selective phosphotyrosine phosphatase inhibition and increased ceramide formation is associated with B‐cell death by apoptosis
Authors: Dawson Glyn , Kilkus John , Schieven Gary L.
Published: July 2002

Bis(maltolato)oxovanadium(IV) (BMOV), a protein phosphotyrosine phosphatase inhibitor, selectively induced apoptosis (as quantitated by TUNEL staining) in a B‐cell line (Ramos) but not in a T‐cell line (Jurkat). The pattern of BMOV‐induced protein tyrosine phosphorylation was different in B‐cells versus T‐cells. Further, BMOV induced a 2‐fold increase in ceramide levels in B‐cells but not in T‐cells and this resembled the ceramide increase following activation of the B‐cell antigen receptor. A 2‐fold increase in the ratio of ceramide to sphingomyelin in B‐cells treated with BMOV suggested that sphingomyelinase activation was the result of the sustained tyrosine phosphorylation of specific proteins and activated the cell death pathway.
Interaction of different oligomeric states of hexameric DNA‐helicase RepA with single‐stranded DNA studied by analytical ultracentrifugation
Authors: Xu Hai , Frank Joachim , Holzwarth Josef F. , Saenger Wolfram , Behlke Joachim
Published: July 2002

Analytical ultracentrifugation was used to determine the molecular mass, M, of hexameric DNA‐helicase RepA at pH 5.8 and 7.6. At pH 7.6, a molecular mass of 179.5±2.6 kDa was found, consistent with the known hexameric state of RepA, (RepA)6. At pH 5.8, (RepA)6 associates to form a dimer with a molecular mass of 366.2±4.1 kDa. Analytical ultracentrifugation was also applied to characterize the interaction of single‐stranded DNA (ssDNA) with the two different oligomeric states of (RepA)6 at pH 5.8 and 7.6. The dissociation constants, K
d, for the equilibrium binding of (dA)30 to the (RepA)6 dimer at pH 5.8 and to (RepA)6 at pH 7.6 were determined at 10°C in the presence of 0.5 mM ATPγS, 10 mM MgCl2 and 60 mM NaCl as K
d5.8=0.94±0.13 μM at pH 5.8 and K
d7.6=25.4±6.4 μM at pH 7.6. The stoichiometries, n, for the two complexes (dA)30/(RepA)6 dimer and (dA)30/(RepA)6 at pH 5.8 and 7.6 were calculated from the corresponding binding curves. At pH 5.8 one (dA)30 molecule was bound per (RepA)6 dimer, while at pH 7.6 one (dA)30 molecule was bound to one (RepA)6. Binding curves were compatible with a single ssDNA binding site present on the (RepA)6 dimer and on (RepA)6, respectively, with no indication of cooperativity. (RepA)6 tends to form larger aggregates under acidic conditions (pH<6.0) which are optimal for ssDNA binding. In contrast, at pH 5.8 in the presence of 60 mM NaCl, only the (RepA)6 dimer was observed both in the absence and presence of (dA)30.
Observation of RecA protein monomer by small angle X‐ray scattering with synchrotron radiation
Authors: Kato Ryuichi , Kataoka Mikio , Mikawa Tsutomu , Masui Ryoji , Nakagawa Noriko , Kamikubo Hironari , Kuramitsu Seiki
Published: July 2002

RecA protein is capable of forming homo‐oligomers in solution. The oligomeric and monomeric states of Thermus thermophilus RecA protein were studied by small angle X‐ray scattering, a direct method used to measure the overall dimensions of a macromolecule. In the presence of 3 M urea or 0.2 M lithium perchlorate, RecA dissociates from higher oligomeric states to form a hexamer with a radius of gyration (R
g) of 52 Å. The value of R
g decreased to 36 Å at a higher lithium perchlorate concentration (1.0 M). The zero angle intensity, I(0), was consistent with the identification of the former state as a hexamer and the latter as a monomer.
Increased synthesis and decreased stability of mitochondrial translation products in yeast as a result of loss of mitochondrial (NAD+)‐dependent isocitrate dehydrogenase
Authors: de Jong Liesbeth , Elzinga Sandra D.J , McCammon Mark T , Grivell Leslie A , van der Spek Hans
Published: July 2002

We have previously demonstrated that the yeast Krebs cycle enzyme NAD+‐dependent isocitrate dehydrogenase (Idh) binds specifically and with high affinity to the 5′‐untranslated leader sequences of mitochondrial mRNAs in vitro and have proposed a role for the enzyme in the regulation of mitochondrial translation [Elzinga, S.D.J. et al. (2000) Curr. Genet., in press]. Although our studies initially failed to reveal any consistent correlation between idh disruption and mitochondrial translational activity, it is now apparent that compensatory extragenic suppressor mutations readily accumulate in idh disruption strains thereby masking mutant behaviour. Now, pulse‐chase protein labelling of isolated mitochondria from an Idh disruption mutant lacking suppressor mutations reveals a strong (2–3‐fold) increase in the synthesis of mitochondrial translation products. Strikingly, the newly synthesised proteins are more short‐lived than in mitochondria from wild‐type cells, their degradation occurring with a 2–3‐fold reduced half‐life. Enhanced degradation of translation products is also a feature of yeast mutants in which tethering/docking of mitochondrial mRNAs is disturbed. We therefore suggest that binding of Idh to mitochondrial mRNAs may suppress inappropriate translation of mitochondrial mRNAs.
The N‐terminal domain of the Caulobacter crescentus CgtA protein does not function as a guanine nucleotide exchange factor
Authors: Lin Bin , Maddock Janine R
Published: July 2002

The Caulobacter crescentus GTP binding protein CgtA is a member of the Obg/GTP1 subfamily of monomeric GTP binding proteins. In vitro, CgtA displays moderate affinity for both GDP and GTP, and rapid exchange rate constants for either nucleotide. One possible explanation for the observed rapid guanine nucleotide exchange factor (GEF) rates is that CgtA is a bimodal protein with a C‐terminal GTP binding domain and an N‐terminal GEF domain. In this study we demonstrate that although the N‐terminus of CgtA is required for function in vivo, this domain plays no significant role in the guanine nucleotide binding, exchange or GTPase activity.
A tetanus toxin sensitive protein other than VAMP 2 is required for exocytosis in the pancreatic acinar cell
Authors: Padfield Philip J
Published: July 2002

The neurotoxin sensitivity of regulated exocytosis in the pancreatic acinar cell was investigated using streptolysin‐O permeabilized pancreatic acini. Treatment of permeabilized acini with botulinum toxin B (BoNT/B) or botulinum toxin D (BoNT/D) had no detectable effect on Ca2+‐dependent amylase secretion but did result in the complete cleavage of VAMP 2. In comparison, tetanus toxin (TeTx) treatment both significantly inhibited Ca2+‐dependent amylase secretion and cleaved VAMP 2. These results indicate that regulated exocytosis in the pancreatic acinar cell requires a tetanus toxin sensitive protein(s) other than VAMP 2.
Phosphoinositide 3‐kinase inhibition reverses platelet aggregation triggered by the combination of the neutrophil proteinases elastase and cathepsin G without impairing αIIbβ3 integrin activation
Authors: Trumel Cathy , Si-Tahar Mustapha , Balloy Viviane , Chignard Michel , Chap Hugues , Payrastre Bernard , Plantavid Monique , Pidard Dominique
Published: July 2002

Neutrophil elastase (NE) upregulates the fibrinogen binding activity of the platelet integrin αIIbβ3 through proteolysis of the αIIb subunit. This cleavage allows a strong potentiation of platelet aggregation induced by low concentrations of cathepsin G (CG), another neutrophil serine proteinase. During this activation process, we observed a strong fibrinogen binding and aggregation‐dependent phosphatidylinositol 3,4‐bis‐phosphate (PtdIns(3,4)P2) accumulation. PtdIns(3,4)P2 has been suggested to play a role in the stabilization of platelet aggregation, possibly through the control of a maintained αIIbβ3 integrin activation. Here we show that inhibition of phosphoinositide 3‐kinase (PI 3‐K) by very low concentrations of wortmannin or LY294002 transformed the irreversible platelet aggregation induced by a combination of NE and low concentrations of CG into a reversible aggregation. However, although inhibition of PI 3‐K was very efficient in inducing platelet disaggregation, it did not modify the level of αIIbβ3 activation as assessed by binding of an activation‐dependent antibody. These results indicate that PI 3‐K activity can control the irreversibility of platelet aggregation even under conditions where αIIbβ3 integrin remains activated.
p1,p4‐Diadenosine 5′‐tetraphosphate induces the uptake of arginine and citrulline by a pore on the plasma membrane of bovine aortic endothelial cells
Authors: Cooper Heidi M. , Rosenberg Christina R. , Hilderman Richard H.
Published: July 2002

We have previously demonstrated that p1,p4‐diadenosine 5′‐tetraphosphate (Ap4A) induces the release of nitric oxide (NO) and modulates the uptake of extracellular L‐arginine (L‐Arg) and L‐citrulline (L‐Cit) by bovine aortic endothelial cells (BAEC) [Hilderman, R.H. and Christensen, E.F. (1998) FEBS Lett. 427, 320–324 and Hilderman, R.H., Casey, T.E. and Pojoga, L.H. (2000) Arch. Biochem. Biophys. 375, 124–130]. In this communication we report that extracellular Ap4A enhances the uptake of L‐Arg and L‐Cit through a pore on the plasma membrane of BAEC that is selective for these two amino acids. We also demonstrate that Ap2A, which induces NO release, enhances L‐Arg uptake while Ap5A, a vasoconstrictor, does not enhance the uptake of L‐Arg. The potential physiological significance of the uptake of these two amino acids in relation to NO synthesis is discussed.
Down‐regulation of peroxisome proliferator‐activated receptor‐γ gene expression by sphingomyelins
Authors: Al-Makdissy Nehmann , Bianchi Arnaud , Younsi Mohamed , Picard Evelyne , Valet Philippe , Martinet Nadine , Dauça Michel , Donner Mireille
Published: July 2002

We recently demonstrated that the sphingomyelin (SM) content of adipocyte membranes was negatively correlated with the expression of peroxisome proliferator‐activated receptor‐γ (PPARγ) in the subcutaneous adipose tissue of obese women with variable degrees of insulin resistance. We have now investigated whether SM really does have an impact on the expression of PPARγ in 3T3‐F442A adipocytes. Adding SM to the culture medium for 24 h caused a significant increase in SM content of adipocyte membranes and an acyl chain length‐dependent decrease in the levels of PPARγ mRNA and protein. The longer the acyl chain of the fatty acid of SM, the greater was the decrease in PPARγ. These data suggest that the nature of the fatty acid is important in the regulation of PPARγ by the SM pathway.
A small upstream open reading frame causes inhibition of human major vault protein expression from a ubiquitous mRNA splice variant
Authors: Holzmann Klaus , Ambrosch Ingo , Elbling Leonilla , Micksche Michael , Berger Walter
Published: July 2002

Overexpression of the major vault protein (MVP) has been linked to a multidrug resistance (MDR) phenotype. We describe a ubiquitously expressed MVP mRNA splice variant (long (L)‐MVP) differing from the regular isoform (short (S)‐MVP) within the 5′‐leader. Only L‐MVP mRNA contains a small upstream open reading frame which was proven to inhibit in vitro and in vivo MVP expression in cis. L‐MVP represented an almost constant portion of total MVP mRNA in diverse normal tissues, but was more variable in malignant cell types. MDR sublines with altered MVP expression displayed changed S‐MVP/L‐MVP ratios as compared to their drug‐sensitive counterparts. Our results suggest alternative splicing as one mechanism for regulation of MVP expression.
Pulmonary surfactant protein SP‐B is significantly more immunoreactive in anionic than in zwitterionic bilayers
Authors: Marı́a Oviedo Jose , Casals Cristina , Pérez-Gil Jesús
Published: July 2002

Binding of polyclonal and monoclonal antibodies, quantitated by enzyme‐linked immunosorbent assay, to porcine SP‐B reconstituted in different phospholipid bilayers has been used to assess differences in protein structure due to lipid–protein interactions. SP‐B bound significantly more antibodies when it was reconstituted in bilayers made of anionic phospholipids (phosphatidic acid, cardiolipin, phosphatidylglycerol, phosphatidylinositol or phosphatidylserine) than in zwitterionic bilayers (phosphatidylcholine, phosphatidylcholine/cholesterol, or phosphatidylethanolamine) or in fatty acid micelles (made of salts of palmitic or stearic acids). These differences in immunoreactivity can be important in the development of quantitation methods for SP‐B in clinical samples based on immunological techniques.
Proper receptor signalling in a mutant catfish gonadotropin‐releasing hormone receptor lacking the highly conserved Asp90 residue
Authors: Blomenröhr Marion , Kühne Ronald , Hund Eveline , Leurs Rob , Bogerd Jan , ter Laak Ton
Published: July 2002

The negatively charged side chain of an Asp residue in transmembrane domain 2 is likely to play an important role in receptor signalling since it is highly conserved in the whole family of G protein‐coupled receptors, except in mammalian gonadotropin‐releasing hormone (GnRH) receptors. In this paper we show that the conserved Asp90 of the catfish GnRH receptor can be substituted by a neutral Asn90 without abolishing receptor signalling if another negatively charged Glu93 is introduced in a proximal region of the receptor interior, thereby mimicking the Glu90–Lys121 salt bridge of mammalian GnRH receptors.
Distribution of GyrA intein in non‐tuberculous mycobacteria and genomic heterogeneity of Mycobacterium gastri
Authors: Isabelle Saves , Mamadou Daffé , Jean-Michel Masson
Published: July 2002

To gain further insights into the understanding of the intein invasion process in mycobacteria, intein sequences in the gyrA gene of 42 mycobacterial strains were searched and a new gyrA intein was found in Mycobacterium gastri (Mga). This 1260 bp intein, named MgaGyrA, inserted at the GyrA‐a site, is highly homologous to the members of the Mycobacterium leprae GyrA allelic family. As the recA intein, MgaGyrA was detected in only one out of six Mga strains examined, while the pps1 intein was a constant character of Mga. This data supports the genomic heterogeneity of Mga towards intein invasion, a finding that may have phylogenetic implications.
Is nebulin truly a component of the thin filament?
Authors: Cuneo Paola , Trombetta Giorgio , Adami Raffaella , Grazi Enrico
Published: July 2002

Thin filaments were prepared from rabbit and beef skeletal muscle with three different procedures, both at high and low ionic strength. Nebulin was always found to be associated with the myosin fraction and was always absent from the thin filament fraction.
Lysine‐87 is a functionally important residue in human prothymosin α
Authors: Rubtsov Yuri , Vartapetian Andrey
Published: July 2002

Human prothymosin a mutants were generated with the aid of random mutagenesis and screened for their ability to inhibit yeast Saccharomyces cerevisiae cell growth. Conversion of Lys‐87 to Glu resulted in an inactivated prothymosin α mutant, which lost the ability of the wild‐type protein to block yeast cell growth. We propose that prothymosin α may possess a bipartite rather than monopartite nuclear localization signal, which includes Lys‐87, and that the above mutation destroys one part of the nuclear localization signal, thus preventing efficient nuclear uptake of prothymosin α.
The algorithm of estimation of the Km values for primers in DNA synthesis catalyzed by human DNA polymerase α
Authors: Kolocheva Tat'yana I. , Maksakova Galiya A. , Zakharova Ol'ga D. , Nevinsky Georgy A.
Published: July 2002

DNA synthesis with various deoxyribo homo‐ and heterooligoprimers in the presence of complementary templates was investigated. The lengthening of d(pN)
n
primers (n =1–10) by a unit resulted in an increase of the primer affinity and a maximal rate of polymerization. The coefficient of the affinity enhancement of primers due to formation of one hydrogen bond between primer and template was found to be 1.35. The dependence of the primer affinity and polymerization rate on template‐primer structure in solution was analyzed and the objective laws of the changes of the KM and V
max values were revealed.
Vacuolating toxin from Helicobacter pylori activates cellular signaling and pepsinogen secretion in human gastric adenocarcinoma cells
Authors: Chan Err-Cheng , Chen Kuei-Tian , Lin Yuh-Ling
Published: July 2002

We investigated cellular signaling and pepsinogen secretion in the human gastric adenocarcinoma cell line AGS which was pretreated with the purified vacuolating cytotoxin from Helicobacter pylori. Results indicated that vacuolating toxin increased the levels of inositol phosphates, cytosolic free calcium concentration, adenosine 3′,5′‐cyclic monophosphate, and phosphorylation of 31 kDa and 22 kDa proteins in the host cells. Moreover, pepsinogen secretion from AGS cells was stimulated with increasing concentrations of cytotoxin. We conclude that besides the H. pylori cytotoxin‐induced cellular vacuoles, cytotoxin‐stimulated signaling mediators and pepsinogen release are important factors involved in the etiology of chronic active gastritis and peptic ulcer disease.
Phosphoenzyme formation by purified, reconstituted copper ATPase of Enterococcus hirae
Authors: Wyler-Duda Petra , Solioz Marc
Published: July 2002

The Enterococcus hirae CopB ATPase serves in the secretion of excess copper from cells and belongs to the recently discovered, new class of heavy metal transport ATPases. We here report the affinity purification of CopB to near homogeneity and its reconstitution into phospholipid vesicles. In these proteoliposomes, the ATPase formed an acylphosphate reaction intermediate with the γ‐phosphate of ATP. ATPase activity and phosphoenzyme formation were inhibited by vanadate with an I
50 of 0.1 mM. Our results suggest that heavy metal and non‐heavy metal ATPases operate by the same underlying mechanism.
Expression of a transactivation‐deficient form of thyroid transcription factor I decreases the activity of co‐transfected thyroglobulin and thyroperoxidase promoters
Authors: Christophe-Hobertus Christiane , van Renterghem Pierre , Pichon Bruno , Christophe Daniel
Published: July 2002

Thyroid transcription factor I (TTF‐1) plays a critical role in thyroid organogenesis and in the control of expression of several thyroid‐specific genes, like those coding for thyroglobulin and thyroperoxidase. We have expressed the isolated DNA‐binding homeodomain of TTF‐1 in cultured thyroid cells by transient transfection. A specific reduction in the activity of cotransfected thyroglobulin and thyroperoxidase promoters was observed in the presence of the isolated TTF‐1 homeodomain, as compared to their activity measured in the presence of a mutated homeodomain unable to bind DNA. The activity of the SV40 early promoter, used as a control, was only marginally affected in these experiments. The transactivation‐deficient form of TTF‐1 described here may thus be used for investigating other cellular processes that are dependent on TTF‐1 transcriptional activity.
Very fast electron transfer from cytochrome to the bacterial photosynthetic reaction center at low temperature
Authors: Ortega José M , Dohse Barbara , Oesterhelt Dieter , Mathis Paul
Published: July 2002

Electron transfer from the proximal heme c‐559 to the primary donor P has been studied in reaction centers of the photosynthetic bacterium Rhodopseudomonas viridis in which the tyrosine residue L162 was replaced by threonine. In the wild type, when the two high‐potential hemes of the tetraheme cytochrome are reduced before flash excitation, a rapid electron transfer (t
1/2=190 ns) observed at ambient temperature disappears below 190 K. In the mutant, the reaction is partly maintained down to 8 K, leading to irreversible charge separation. The reaction rate is nearly temperature‐independent between 294 K and 8 K (t
1/2≈450 ns). The different behavior of wild type and mutant reaction centers is attributed to differences in a network of water molecules, the freezing of which may block structural reorganizations associated with cytochrome oxidation, in the wild type but not in the mutant.
Functional expression, activation and desensitization of opioid receptor‐like receptor ORL1 in neuroblastoma×glioma NG108‐15 hybrid cells
Authors: Ma Lan , Cheng Zhi-Jie , Fan Guo-Huang , Cai Ying-Chun , Jiang Li-Zhen , Pei Gang
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Amounts of proteins altered by mutations in the dnaA gene of Escherichia coli
Authors: Ohba Akiko , Mizushima Tohru , Katayama Tsutomu , Sekimizu Kazuhisa
Published: July 2002

© 1997 Federation of European Biochemical Societies.
The basal subcellular distribution of β‐adrenergic receptor kinase is independent of G‐protein βγ subunits
Authors: Murga Cristina , Esteban Nuria , Ruiz-Gómez Ana , Mayor Federico
Published: July 2002

β‐adrenergic receptor kinase (βARK‐1 or GRK2) is a key regulatory protein involved in the regulation of G‐protein‐coupled receptors which associates with microsomal and plasma membranes. βγ subunits of G‐proteins have been suggested to mediate agonist‐dependent membrane translocation of βARK, but their possible role in maintaining the complex subcellular distribution of the kinase is not known. In this study we show that lovastatin‐mediated inhibition of Gγ subunits isoprenylation in HEK‐293 cells stably transfected with βARK1 leads to a significant release of Gβ subunits to the cytosol without causing changes in total particulate βARK or in the association of this kinase to plasma or microsomal membrane fractions. In addition, transient overexpression of mutant forms of Gγ unable to become isoprenylated resulted in a marked sequestration of Gβ to the soluble compartment, but caused no rearrangement in the distribution of cotransfected βARK. These results indicate that anchoring of βARK to cellular membranes under basal conditions is independent of the availability of heterotrimeric G‐protein subunits.
Stimulatory effects of parathyroid hormone and 1,25‐dihydroxyvitamin D3 on insulin‐like growth factor‐binding protein‐5 mRNA expression in osteoblastic UMR‐106 cells: the difference between transient and continuous treatments
Authors: Nasu Masamichi , Sugimoto Toshitsugu , Chihara Kazuo
Published: July 2002

The transient treatment with parathyroid hormone (PTH) for 12 h, followed by its removal for 36 h, stimulated insulin‐like growth factor‐binding protein (IGFBP)‐5 mRNA expression more strongly than the continuous treatment for 48 h in osteoblastic UMR‐106 cells. The transient but not continuous treatment with A23187 also stimulated it. In contrast, 1,25‐dihydroxyvitamin D3 stimulated it, irrespective of the treatment design. IGFBP‐5 stimulated type‐1 procollagen mRNA expression. The present study first indicated that the transient treatment with PTH more effectively stimulated IGFBP‐5 mRNA expression than its continuous treatment partly via an increase in intracellular calcium and suggested that IGFBP‐5 might be involved in the anabolic action of PTH in bone.
Liposome‐mediated peptide loading of MHC‐DR molecules in vivo
Authors: 't Hart Bert A. , Elferink Diënne G. , Drijfhout Jan Wouter , Storm Gert , van Blooijs Louis , Bontrop Ronald E. , de Vries René R.P.
Published: July 2002

Amino acid residues 3–15 of mycobacterial HSP60 define a dominant T‐cell epitope for HLA‐DR3+ve humans and Mamu‐DR3+ve rhesus monkeys. Our results show that Mamu‐DR3 molecules on PBMC can be efficiently loaded in vivo with the above‐mentioned peptides when they are intravenously injected encapsulated in liposomes, but not in the free form. Mamu‐DR3 loading is abolished by encapsulation of a nonstimulatory peptide. These results have implications for the delivery of therapeutic peptides in vivo.