bluuebunny/crossref_metadata_2025 huggingface dataset:
Variability in the nucleic acid binding site size and the amount of single‐stranded DNA‐binding protein in Escherichia coli
Authors: Bobst E.V. , Bobst A.M. , Perrino F.W. , Meyer R.R. , Rein D.C.
Published: July 2002

The Escherichia coli single‐stranded DNA binding protein (SSB), essential for DNA replication, recombination and repair, can undergo a thermally induced irreversible conformational change which does not eliminate its biological activity, but changes the number of nucleotides it covers (binding site size) when binding to a single‐stranded nucleic acid lattice. The binding site size of native and conformationally changed SSB was also found to be a function of the molecular mass of the polynucleotide, an observation which is unusual for single‐stranded DNA binding proteins and will greatly affect the affinity relationship of this protein for nucleic acids. A radioimmunoassay used to quantitate in SSB level in cells revealed the number of SSB tetramers to be larger than initial estimates by a factor of as much as six. All these data suggest that the biological role of SSB and its mechanism of action is by far more complex than originally assumed.
Antigens reactive with prestalk/prespore specific monoclonal antibodies in Dictyostelium discoideum
Authors: Noce Toshiaki , Takeuchi Ikuo
Published: July 2002

Dictyostelium discoideum
Monoclonal antibody
Immunoblotting
Prestalk
Prespore
Cell differentiation
ADP stimulates IP3 formation in human platelets
Authors: Daniel James L. , Dangelmaier Carol A. , Selak Mary , Smith J.Bryan
Published: July 2002

Aspirinated human platelets labeled with 32PO4 showed a 1.7‐fold increase in [32P]IP3 when stimulated with ADP. ADP‐stimulated mobilization of internal Ca2+ and phosphorylation of myosin were enhanced in the presence of extracellular Ca2+ but the increase in IP3 was not significantly affected by external Ca2+. The Ca2+ ionophore, ionomycin, elevated internal Ca2+ and induced myosin phosphorylation without a detectable change in IP3. These results indicate that the mechanism of ADP stimulation of human platelets is similar to that of other platelet agonists and supports the theory that IP3 functions to liberate internal Ca2+.
Effect of electrostatic repulsion on the morphology and thermotropic transitions of anionic phospholipids
Authors: Epand Richard M. , Hui Sek-Wen
Published: July 2002

Samples of dimyristoylphosphatidylglycerol and dimyristoylphosphatidylserine which exhibit highly cooperative phase transitions in suspension with 0.5 M NaCl, 10 mM sodium phosphate, pH 7.0, demonstrate a marked broadening of the phase transition when suspended in distilled water at pH 7. The cooperativity of the thermal transition of dimyristoylphosphatidic acid at pH 7.0, in contrast, was little affected by the presence or absence of 0.5 M NaCl. The most dramatic changes in phase transition properties were observed with phosphatidylglycerol. The morphology of phosphatidylglycerol was also altered by NaCl. At high salt concentrations, typical multilamellar structures were observed by freeze‐fracture electron microscopy. However, in distilled water the large multilamellar structures are disrupted, with opened shells and smaller particles of 20–30 nm diameter being observed, in addition to a few larger vesicles. Structures of differing morphology could be partially separated from aqueous ‘solutions’ of phosphatidylglycerol by ultracentrifugation. The supernate contained only small particles. The results are discussed in terms of the large effective head group volume resulting from strong electrostatic repulsion at low ionic strengths. This allows the anionic lipids, particularly phosphatidylglycerol, to form hydrophilic surfaces with high curvature so as to seal the bilayer edges of discs or shells. Thus, certain anionic lipids in distilled water destabilize bilayers by a mechanism which is opposite to that found with lipids which destabilize bilayers by forming the hexagonal phase.
Kinship of cephalopod photoreceptor G‐protein with vertebrate transducin
Authors: Tsuda Motoyuki , Tsuda Tomoko , Terayama Yoshio , Fukada Yoshitaka , Akino Toyoaki , Yamanaka Gregory , Stryer Lubert , Katada Toshiaki , Ui Michio , Ebrey Thomas
Published: July 2002

G‐protein
Transducin
(cephalopod)
Photoeceptor
Evidence for a second phosphorylation site on eIF‐2α from rabbit reticulocytes
Authors: Kudlicki Wieslaw , Wettenhall Richard E.H. , Kemp Bruce E. , Szyszka Ryszard , Kramer Gisela , Hardesty Boyd
Published: July 2002

Ser 51 in the NH2‐terminal sequence of the α‐subunit of eukaryotic peptide initiation factor 2 (eIF‐2) has been identified as a second phosphorylation site for the heme‐controlled eIF‐2α kinase from rabbit reticulocytes. Increased phosphorylation of this serine relative to the previously described phosphorylation site (Ser 48) is observed when the kinase reaction is carried out in the presence of the α‐subunit of spectrin. A synthetic peptide corresponding to eIF‐2α(41–54) is phosphorylated only in Ser 51 by the eIF‐2α kinase.
Electrogenic photocycle of the 13‐cis retinal‐containing bacteriorhodopsin with an M intermediate involved
Authors: Drachev L.A. , Kaulen A.D. , Skulachev V.P. , Zorina V.V.
Published: July 2002

It has been found that bacteriorhodopsin containing 13‐cis‐retinal as the chromophore (13‐cis‐bR) is competent in a one‐photon photocycle which involves an M‐type intermediate. Such an effect can be shown in 13‐cis‐Br obtained by means of 13‐cis‐retinal treatment of white bacterioopsin membrane sheets, as well as in dark‐adapted purple sheets, solubilized Br and Br proteoliposomes. The 13‐cis‐Br photocycle with the M intermediate is found to be proton‐motive, the electrogenic phases resembling those of the all‐trans‐Br photocycle. All of these effects are observed only when the Ph is above some critical value, namely Ph 8.5–9.5 for the Br sheets, 7.6 for proteoliposomes and 7.0 for the solubilized Br.
FITC‐labeled lipopolysaccharide: use as a probe for liposomal membrane incorporation studies
Authors: Trubetskoy V.S. , Koshkina N.V. , Omel'yanenko V.G. , L'vov V.L. , Dmitriev B.A. , Petrov A.B. , Torchilin V.P.
Published: July 2002

FITC‐labeled LPS from Neisseria meningitidis can be used as a probe to follow the process of LPS incorporation into liposomal membrane and to study its interaction with a bilayer. The incorporation of FITC‐LPS into the bilayer was proved by physicochemical methods as well as by liposomal LPS toxirity decrease in actinomycin D‐sensitized mice. Fluorescence intensity increase was observed upon the insertion of FITC‐LPS into the membrane of dehydration/rehydration vesicles and vesicles obtained by co‐sonication of lipid suspension and FITC‐LPS. Following FITC‐LPS fluorescence polarization it was shown that the substance seems to be clusterized m the liposomal membrane starting from FITC‐LPS/lipid molar ratio 1:800.
K‐252a inhibits the response of tomato cells to fungal elicitors in vivo and their microsomal protein kinase in vitro
Authors: Grosskopf Debora G. , Felix Georg , Boller Thomas
Published: July 2002

Two characteristic responses of plant cells to fungal elicitors, induction of phenylalanine ammonia‐lyase activity and of ethylene biosynthesis, were studied in suspension‐cultured tomato cells. Induction of both responses was completely blocked by 500 nM K‐252a, a known inhibitor of mammalian protein kinases. About 100 nM K‐252a caused half‐maximal inhibition. In vitro, K‐252a inhibited protein kinase activity in microsomal preparations from tomato cells. Inhibition was competitive with respect to ATP and had a K
1 of about 15 nM. Thus, protein kinases sensitive to K‐252a occur in plants and might be important for the plant's response to fungal elicitors.
Physico‐chemical properties of actin cleaved with bacterial protease from E. coli A2 strain
Authors: Khaitlina S.Yu. , Collins J.H. , Kuznetsova I.M. , Pershina V.P. , Synakevich I.G. , Turoverov K.K. , Usmanova A.M.
Published: July 2002

The 36 kDa fragment of actin molecule obtained with the protease from E. coli A2 strain [(1988) FEBS Lett. 228, 172] was shown to begin with Val‐43 and retain the COOH‐terminal amino acid residues of the parent molecule. The E. coli protease split actin preserves the NH2‐terminal part of the polypeptide chain as well as the native conformation of actin molecule. However, the E. coli protease split actin failed to polymerize in 0.1 M KCl, suggesting that integrity of actin molecule between Gly‐42 and Val‐43 is crucial for actin polymerization.
Consensus sequence for processing of peptide precursors at monobasic sites
Authors: Devi Lakshmi
Published: July 2002

Many regulatory peptide precursors undergo post‐translational processing at mono‐ and/or dibasic residues. Comparison of amino acids around the monobasic cleavage sites suggests that these cleavages follow certain sequence motifs and can be described as the rules that govern monobasic cleavages: (i) a basic amino acid it present at either 3, 5, or 7 amino acids N‐terminal to the cleavage site, (ii) hydrophobic aliphatic amino acids (leucine, isoleucine, valine, or methionine) are never present in the position C‐terminal to the monobasic amino acid at the cleavage site, (iii) a cysteine is never present in the vicinity of the cleavage site, and (iv) an aromatic amino acid is never present at the position N‐terminal to the monobasic amino acid at the cleavage site. In addition to these rules, the monobasic cleavages follow certain tendencies: (i) the amino acid at the cleavage site tends to be predominantly arginine, (ii) the amino acid at the position C‐terminal to the cleavage site tends to be serine, alanine or glycine in more than 60% of the cases, (iii) the amino acid at either 3, 5, or 7 position N‐terminal to the cleavage site tends to be arginine, (iv) aromatic amino acids are rare at the position C‐terminal to the monobasic amino acid at the cleavage site, and (v) aliphatic amino acids tend to be in the two positions N‐terminal to and the two positions C‐terminal to the cleavage site, except as noted above. When compared with a large number of sequence containing single basic amino acids, these rules and tendencies are capable of not only correctly predicting the processing sites, but also are capable of excluding most of the single basic sequences that are known to be uncleaved. Many or these rules can also be applied to correctly predict the dibasic and multibasic cleavage sites suggesting that the rules and tendencies could govern endoproteolytic processing at the monobasic, dibasic and multibasic sites.
Monitoring a mammalian nuclear membrane phase transition by intrinsic ultraweak light emission
Authors: Scott R.Q. , Roschger P. , Devaraj B. , Inaba H.
Published: July 2002

The first thermodynamic measurements of the intensity of light emission associated with native lipid peroxidation in a biological membrane are described. Kinetics of the radical chain reaction are shown to be sensitive to membrane structural phase and lipid dynamics. This is demonstrated by a novel measurement of a phase transition in the membrane of the intact mammalian nucleus. The apparent activation energies of lipid peroxidation in this system are also obtained for the first time. We suggest that this measurement may be more generally applicable as a method for monitoring membrane phase transitions.
PDGF suppresses the activation of group II phospholipase A2gene expression by interleukin I and forskolin in mesangial cells
Authors: Mühl Heiko , Geiger Thomas , Pignat Werner , Märki Fritz , van den Bosch Henk , Vosbeck Klaus , Pfeilschifter Josef
Published: July 2002

Treatment of rat mesangial cells with interleukin 1β (IL‐1β) and forskolin greatly enhanced the expression of group II phospholipase A2(PLA2) mRNA, with subsequent increased synthesis and secretion of PLA2as detected by PLA2activity measurements and immunoprecipitation of culture media of [35S]methionine‐labelled mesangial cells. PDGF—BB dose‐dependently suppressed the IL‐1β‐ and forskolin‐induced elevation of PLA2mRNA, as well as PLA2synthesis and secretion. In contrast, PDGF—AA had no inhibitory effect. The tyrosine kinase inhibitor genistein dose‐dependently antagonized the inhibitory effect of PDGF‐BB on IL‐1β‐stimulated PLA2secretion, thus suggesting that tyrosine phosphorylation may be required for PDGF‐BB inhibition of PLA2gene expression in mesangial cells.
Measurements of cytoplasmic free Ca2+ concentration in human pancreatic islets and insulinoma cells
Authors: Kindmark Hendrik , Köhler Martin , Nilsson Thomas , Arkhammar Per , Wiechel Karl-Ludvig , Rorsman Patrik , Efendić Suad , Berggren Per-Olaf
Published: July 2002

In human pancreatic islets an increase in the glucose concentration from 3 to 20 mM raised the free cytoplasmic Ca2+ concentration ([Ca2+]i), an effect being reversible upon withdrawal of the sugar. Depolarization with a high concentration of K+ or the sulphonylurea tolbutamide also raised [Ca2+]i. Addition of extracellular ATP produced a transient rapid rise in [Ca2+]i. Oscillations in [Ca2+]i were observed in the presence of 10 mM glucose. Insulinoma cells responded to glucose and tolbutamide with increases in [Ca2+]i, whereas the sulphonamide diazoxide caused a decrease in [Ca2+]i. These findings confirm previous results obtained in rodent β‐cells.
Fluorescence study on cardiac glycoside binding to the Na,K‐pump Ouabain binding is associated with movement of electrical charge
Authors: Stürmer W. , Apell H.-J.
Published: July 2002

Recently we have presented evidence that the fluorescence probe RH 421 can be used to detect binding and release of ions at the extracellular face of the pump since these processes are associated with translocation of electrical charge [1]. Applying this method to experiments with cardiac glycosides we found that: (1) ouabain induced fluorescence changes of the electrochromic dye, RH 421, were caused by the changes of charges bound to the enzyme: (2) independent of the sodium concentration, the final fluorescence amplitude indicated that approximately 2 Na+ ions were bound to the pump: (3) the sodium release to the extracellular side involved two distinct electrogenic steps: (4) the kinetics of inhibition depended on the Na+ concentration. Experiments with hydrophobic ions indicated that the kinetics of ouabain binding to the Na‐ATPase is voltage dependent and (5) the applied techniques is a convenient tool to characterized binding of cardiac glycosides to the Na,K‐pump.
The secondary structure of influenza A M2 transmembrane domain A circular dichroism study
Authors: Duff Kevin C. , Kelly Sharon M , Price Nicholas C. , Bradshaw Jeremy P.
Published: July 2002

Using circular dichroism, this study investigated the secondary structure of the influenza A M2 transmembrane domain. When reconstituted into 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine liposomes, the M2 transmembrane domain was found to adopt a predominantly α‐helical secondary structure which was unaffected by both temperature and the addition of 1‐aminoadamantane hydrochloride. Reconstitution into 1,2‐dioleoyl‐sn‐glycero‐3‐phosphoglycerol liposomes resulted in a marked decrease in helical content.
Adhesive interactions of neutrophils and leukotriene synthesis
Authors: Sud'ina Galina F. , Galkina Svetlana I. , Barsky Oleg A. , Margolis Leonid B.
Published: July 2002

Cell‐substrate and cell‐cell adhesion of neutrophils has been found to slow down the calcium ionophore A23187‐induced synthesis of 5‐lipoxygenase (5‐LO) metabolites of arachidonic add. Addition of the exogenous substrate, arachidonic acid (AA), together with A23187, resulted in the enhanced production of leukotriene B4 (LTB4) and 5‐hydroxyeicosatetraenoic acid (5‐HETE) by adherent neutrophils in comparison with those by the cells in suspension. We observed also the enhanced production of 5‐LO metabolites in attached cells when we stimulated the cells by the combined action of phorbol 12‐myristate 13‐acetate (PMA) and A23187. Thus, the adhesion to solid substrate and to other cells, an important regulatory factor for the activity of many cells, is a powerful regulator of leukotriene production by neutrophils.
Regulation of Cu,Zn‐ and Mn‐superoxide dismutase transcription in Saccharomyces cerevisiae
Authors: Galiazzo Francesca , Labbe-Bois Rosine
Published: July 2002

The regulation of Cu,Zn‐ and Mn‐superoxide dismutases (SOD) was investigated by Northern blotting and gene fusions of SOD1 and S0D2 promoters with the β‐galactosidase reporter gene. Cu,ZnSOD expression was increased 3‐fold under glucose derepressing conditions, and decreased 4‐ to 6‐fold by oxygen or heme deficiency. MnSOD expression was increased 5‐fold by glucose derepression, and decreased 8‐ to 10‐fold by anaerobiosis and 4‐ to 5‐fold by heme deficiency. Induction by paraquat was modest, about 50% for SOD1 and 100% for SOD2; it was apparently independent of the respiratory chain function.
Transcription of rat mitochondrial NADH‐dehydrogenase subunits presence of antisense and precursor RNA species
Authors: Tullo Apollonia , Tanzariello Filomena , D'Erchia Anna Maria , Nardelli Maria , Papeo Pasquale Antonio , Sbisà Elisabetta , Saccone Cecilia
Published: July 2002

We have characterized the transcriptional pattern of the rat mitochondrial ND6‐containing region in vivo. We have identified a stable polyadenylated RNA species complementary for the full length of the ND6 mRNA. The analysis of the ND5 region has revealed the presence of an antisense RNA only at its 3′ end. The presence of these stable antisense species complementary to structural genes is intriguing and suggests a possible regulatory function. The quantitative analyses have demonstrated that the H transcripts, both codogenic and non‐codogenic, are more stable than the L transcripts. We have defined the 5′ end of the ND6 mRNA at the level of the ATG downstream of the tRNAGlu. The mapping of the ND1 5′ end has demonstrated that GTG is the first codon of the mRNA. Our findings suggest that the post‐transcriptional mechanisms involved in the expression of the mt genome are much more numerous and complex than those already described in the literature.
The α‐isoform of glycogen synthase kinase‐3 from rabbit skeletal muscle is inactivated by p70 S6 kinase or MAP kinase‐activated protein kinase‐1 in vitro
Authors: Sutherland Calum , Cohen Philip
Published: July 2002

The α‐isoform of glycogen synthase kinase‐3 (GSK3α) was inactivated by 80% towards a synthetic peptide substrate upon incubation with Mg‐ATP and either MAP kinase‐activated protein (MAPKAP) kinase‐1 or p70 S6 kinase. Inactivation by either kinase resulted from the phosphorylation of Ser‐21 and was reversed by treatment with protein phosphatase 2A1. Phosphorylation also decreased GSK3α activity towards glycogen synthase, inhibitor‐2 and c‐jun. The specificity of GSK3a was similar to GSK3β, but with the synthetic peptide substrate heparin stimulated the dephosphorylated form of GSK3α (6‐fold) more than GSK3β(1.8‐fold). After phosphorylation, both isoforms were stimulated 15–20‐fold by heparin.
Regulation of triacylglycerol synthesis in permeabilized rat hepatocytes
Authors: Stals Hedwig K. , Top W. , Declercq Peter E.
Published: July 2002

Isolated hepatocytes from fed and starved rats, permeabilized with Staphylococcus aureus α‐toxin, were incubated with increasing concentrations of radiolabelled fatty acids, in the presence of a saturating concentration of 3‐GP. Incorporation of label into LPA, PA and DAG was lower in cells from starved rats than in cells from fed rats, apparently reflecting the lower activity of GPAT after starvation. This enzyme approached saturation at high fatty acid levels and determined the overall flux through the esterification pathway. TAG synthesis, however, was the same in both nutritional states and could not be saturated with fatty acid under the given conditions. Taken together with the observed accumulation of DAG, these data suggest that the rate of TAG synthesis is controlled by the fatty acid supply and, more particularly, by the affinity of DGAT for acyl‐CoA.
Mechanical unfolding of a2‐macroglobulin molecules with atomic force microscope
Authors: Mitsui Keita , Hara Masahiko , Ikai Atsushi
Published: July 2002

α
2‐Macroglobutin was derivatized with a sulfhydryl cross‐linker and sandwiched between a mica substrate and a solicon nitride tip, both coated with gold, of an atomic force microscope and force curve measurement was carried out. An extensive downward deflection of the cantilever was observed in the retracting realm of the curve, when and only when the substrate was covered with the derivatized protein. The result was interpreted in terms of the mechanical stretching and unfolding of a single or a few protein molecules.
Evolution of legumin genes: loss of an ancestral intron at the beginning of angiosperm diversification
Authors: Häger K.-P. , Müller B. , Wind C. , Erbach S. , Fischer H.
Published: July 2002

The polymerase chain reaction was used to survey gymnosperm legumin genes. Characterization of 46 cloned amplificates, differing in sequence and size (1.2–1.6 kb), revealed the ubiquitous occurrence of legumin genes and their organization in small subfamilies in the 22 species investigated. The 3′ portions of the genes, coding for the legumin β‐polypeptides, show a highly conserved intron/exon structure divergent from those of angiosperms: an additional intron (intron IV) uniformly interrupts the region coding for the C‐terminal part of the β‐polypeptides. Phylogenetic analysis of the respective coding sequences as well as the organization of the Magnolia B14 legumin gene also investigated here both indicate that intron IV is ancestral and was lost during early angiosperm evolution. Taking into account the intronlexon structures from all legumin genes known, our results suggest that legumin genes evolved by subsequent loss of introns, providing also further evidence for a common origin of legumins and vicilins.
Molecular characterisation of meaB, a novel gene affecting nitrogen metabolite repression in Aspergillus nidulans
Authors: Polley Spencer D. , Caddick Mark X.
Published: July 2002

Mutations within the meaB gene elicit the inappropriate expression of several activities subject to nitrogen metabolite repression in Aspergillus nidulans and also have some unrelated phenotypic effects. We have cloned meaB and isolated a full length cDNA clone. Northern analysis has shown that meaB expression is not subject to nitrogen metabolite repression. meaB encodes a novel protein of 418 amino acids and contains a significantly high number of S/TPXX motifs, a motif common in transcriptional regulatory proteins. We have sequenced three mutations within meaB, two of which have an identical phenotype to that produced by gene disruption.
Time, voltage and ionic concentration dependence of rectification of h‐erg expressed in Xenopus oocytes
Authors: Wang Shimin , Morales Michael J. , Liu Shuguang , Strauss Harold C. , Rasmusson Randall L.
Published: July 2002

The rapid delayed rectifier, IKr is believed to have h‐erg (uman ther‐à‐go‐go elated ene) as its molecular basis. A recent study has shown that rectification of h‐erg involves a rapid inactivation process that involves rapid closure of the external mouth of the pore or C‐type inactivation. We measured the instantaneous current to voltage relationship for h‐erg channels using the saponin permeabilized variation of the cut‐open oocyte clamp technique. In contrast to C‐type inactivation in other voltage‐gated K+ channels, the rate of inactivation was strongly voltage dependent at depolarized potentials. This voltage dependence could be modulated independently of activation by increasing [K+]o from 2 to 98 mM. These results suggest that inactivation of h‐erg has its own intrinsic voltage sensor.
Closed loops of nearly standard size: common basic element of protein structure
Authors: Berezovsky Igor N , Grosberg Alexander Y , Trifonov Edward N
Published: July 2002

By screening the crystal protein structure database for close Cα–Cα contacts, a size distribution of the closed loops is generated. The distribution reveals a maximum at 27±5 residues, the same for eukaryotic and prokaryotic proteins. This is apparently a consequence of polymer statistic properties of protein chain trajectory. That is, closure into the loops depends on the flexibility (persistence length) of the chain. The observed preferential loop size is consistent with the theoretical optimal loop closure size. The mapping of the detected unit‐size loops on the sequences of major typical folds reveals an almost regular compact consecutive arrangement of the loops. Thus, a novel basic element of protein architecture is discovered; structurally diverse closed loops of the particular size.
Detection of partially phosphorylated forms of ERK by monoclonal antibodies reveals spatial regulation of ERK activity by phosphatases
Authors: Yao Zhong , Dolginov Yakov , Hanoch Tamar , Yung Yuval , Ridner Gabriela , Lando Zeev , Zharhary Dorit , Seger Rony
Published: July 2002

When cells are stimulated by mitogens, extracellular signal‐regulated kinase (ERK) is activated by phosphorylation of its regulatory threonine (Thr) and tyrosine (Tyr) residues. The inactivation of ERK may occur by phosphatase‐mediated removal of the phosphates from these Tyr, Thr or both residues together. In this study, antibodies that selectively recognize all combinations of phosphorylation of the regulatory Thr and Tyr residues of ERK were developed, and used to study the inactivation of ERK upon mitogenic stimulation. We found that inactivation of ERK in the early stages of mitogenic stimulation involves separate Thr and Tyr phosphatases which operate differently in the nucleus and in the cytoplasm. Thus, ERK is differentially regulated in various subcellular compartments to secure proper length and strength of activation, which eventually determine the physiological outcome of many external signals.
Antibacterial activities of temporin A analogs
Authors: Wade David , Silberring Jerzy , Soliymani Rabah , Heikkinen Sami , Kilpeläinen Ilkka , Lankinen Hilkka , Kuusela Pentti
Published: July 2002

Temporin A (TA) is a small, basic, highly hydrophobic, antimicrobial peptide amide (FLPLIGRVLSGIL‐NH2) found in the skin of the European red frog, Rana temporaria. It has variable antibiotic activities against a broad spectrum of microorganisms, including clinically important methicillin‐sensitive and ‐resistant Staphylococcus aureus as well as vancomycin‐resistant Enterococcus faecium strains. In this investigation the antimicrobial activity and structural characteristics of TA synthetic analogs were studied. For antibacterial activity against S. aureus and enterococcal strains, the hydrophobicity of the N‐terminal amino acid of TA was found to be important as well as a positive charge at amino acid position 7, and bulky hydrophobic side chains at positions 5 and 12. Replacing isoleucine with leucine at amino acid positions 5 and 12 resulted in the greatest enhancement of antibacterial activity. In addition, there was little difference between the activities of TA and its all‐D enantiomer, indicating that the peptide probably exerts its effect on bacteria via non‐chiral interactions with membrane lipids.
Erv1p from Saccharomyces cerevisiae is a FAD‐linked sulfhydryl oxidase
Authors: Lee Jeung-Eun , Hofhaus Götz , Lisowsky Thomas
Published: July 2002

The yeast ERV1 gene encodes a small polypeptide of 189 amino acids that is essential for mitochondrial function and for the viability of the cell. In this study we report the enzymatic activity of this protein as a flavin‐linked sulfhydryl oxidase catalyzing the formation of disulfide bridges. Deletion of the amino‐terminal part of Erv1p shows that the enzyme activity is located in the 15 kDa carboxy‐terminal domain of the protein. This fragment of Erv1p still binds FAD and catalyzes the formation of disulfide bonds but is no longer able to form dimers like the complete protein. The carboxy‐terminal fragment contains a conserved CXXC motif that is present in all homologous proteins from yeast to human. Thus Erv1p represents the first FAD‐linked sulfhydryl oxidase from yeast and the first of these enzymes that is involved in mitochondrial biogenesis.
Substrate binding and enzyme function investigated by infrared spectroscopy
Authors: Barth Andreas , Zscherp Christian
Published: July 2002

Protein conformational changes triggered by molecule binding are increasingly investigated by infrared spectroscopy often using caged compounds. Several examples of molecule‐protein recognition studies are given, which focus on nucleotide binding to proteins. The investigation of enzyme mechanisms is illustrated in detail using the Ca2+‐ATPase of the sarcoplasmic reticulum membrane as an example. It is shown that infrared spectroscopy provides valuable information on general aspects of enzyme function as well as on molecular details of molecule–protein interactions and the mechanism of catalysis.
Characterization of two distinct DP‐related genes from Arabidopsis thaliana1
Authors: Magyar Zoltán , Atanassova Ana , De Veylder Lieven , Rombauts Stéphane , Inzé Dirk
Published: July 2002

E2F/DP complexes play a pivotal role in the regulation of the G1/S transition in animals. Recently, plant E2F homologs have been cloned, but DP‐related sequences have not been identified so far. Here we report that Arabidopsis thaliana contains at least two different DP‐related genes, AtDPa and AtDPb. They exhibit an overall domain organization similar to that of their animal counterparts, although phylogenetic analysis demonstrated that they form a separate subgroup. AtDPs efficiently heterodimerize in vitro with the Arabidopsis E2F‐related proteins, AtE2Fa and AtE2Fb through their dimerization domains. AtDPa and AtE2Fa are predominantly produced in actively dividing cells with highest transcript levels in early S phase cells.
Functional characterization of sphingolipid C4‐hydroxylase genes from Arabidopsis thaliana
Authors: Sperling Petra , Ternes Philipp , Moll Hermann , Franke Stephan , Zähringer Ulrich , Heinz Ernst
Published: July 2002

In the genome of Arabidopsis thaliana, two genes were identified encoding isoenzymes for C4‐hydroxylation of long chain bases (LCB) in plant sphingolipids. Both predicted proteins consist of 258 amino acid residues (77% identity) which show sequence similarity to di‐iron‐binding enzymes, such as Sur2p and Erg3p from yeast, involved in oxygen‐dependent lipid modifications. Heterologous expression of these genes in a yeast sur2Δ‐null mutant lacking C4‐LCB hydroxylation resulted in the formation of D‐ribo‐C18‐ and ‐C20‐phytosphinganine. The identity and stereochemical configuration of the isolated trihydroxybases was confirmed by electrospray ionization‐mass spectroscopy, gas–liquid chromatography–mass spectrometry and 1H‐nuclear magnetic resonance spectroscopy. These results represent the first functional identification of SUR2 genes from plants as well as from any organism other than yeast.
Suppression of inducible cyclooxygenase and nitric oxide synthase through activation of peroxisome proliferator‐activated receptor‐γ by flavonoids in mouse macrophages
Authors: Liang Yu-Chih , Tsai Shu-Huei , Tsai De-Cheng , Lin-Shiau Shoei-Yn , Lin Jen-Kun
Published: July 2002

Peroxisome proliferator‐activated receptor (PPAR)γ transcription factor has been implicated in anti‐inflammatory response. Of the compounds tested, apigenin, chrysin, and kaempferol significantly stimulated PPARγ transcriptional activity in a transient reporter assay. In addition, these three flavonoids strongly enhanced the inhibition of inducible cyclooxygenase and inducible nitric oxide synthase promoter activities in lipopolysaccharide‐activated macrophages which contain the PPARγ expression plasmids. However, these three flavonoids exhibited weak PPARγ agonist activities in an in vitro competitive binding assay. Limited protease digestion of PPARγ suggested these three flavonoids produced a conformational change in PPARγ and the conformation differs in the receptor bound to BRL49653 versus these three flavonoids. These results suggested that these three flavonoids might act as allosteric effectors and were able to bind to PPARγ and activate it, but its binding site might be different from the natural ligand BRL49653.
Subunit topology of the Rhodococcus proteasome
Authors: Zühl Frank , Tamura Tomohiro , Dolenc Iztok , Cejka Zdenka , Nagy István , De Mot René , Baumeister Wolfgang
Published: July 2002

The 20S proteasome, isolated from the nocardioform actinomycete Rhodococcus erythropolis strain NI86/21, is built from two α‐type and two β‐type subunits. In order to probe the subunit topology, we have set up an expression system which allows coexpression of the genes encoding the α‐ and β‐subunits in all possible combinations. The four respective constructs obtained yielded fully assembled and proteolytically active proteasomes. Biochemical, kinetic and electron microscopy analysis allow us to rule out several of the models which were originally envisaged for the subunit topology of the Rhodococcus proteasome. The experiments further indicate that the assembly pathways of the Rhodococcus and of the Thermoplasma proteasome differ in some important details.
Fructan synthesis in transgenic tobacco and chicory plants expressing barley sucrose:fructan 6‐fructosyltransferase
Authors: Sprenger Norbert , Schellenbaum Luisa , van Dun Kees , Boller Thomas , Wiemken Andres
Published: July 2002

We have recently cloned a cDNA encoding sucrose:fructan 6‐fructosyltransferase (6‐SFT), a key enzyme of fructan synthesis forming the β‐2,6 linkages typical of the grass fructans, graminans and phleins [Sprenger et al. (1995) Proc. Natl. Acad. Sci. USA 92, 11652–11656]. Here we report functional expression of 6‐SFT from barley in transgenic tobacco and chicory. Transformants of tobacco, a plant naturally unable to form fructans, synthesized the trisaccharide kestose and a series of unbranched fructans of the phlein type (β‐2,6 linkages). Transformants of chicory, a plant naturally producing only unbranched fructans of the inulin type (β‐2,1 linkages), synthesized in addition branched fructans of the graminan type, particularly the tetrasaccharide bifurcose which is also a main fructan in barley leaves.
Symmetric GroEL‐GroES complexes can contain substrate simultaneously in both GroEL rings
Authors: Llorca Oscar , Marco Sergio , Carrascosa José L. , Valpuesta José M.
Published: July 2002

© 1997 Federation of European Biochemical Societies.
CKβ8, a novel CC chemokine that predominantly acts on monocytes
Authors: Forssmann Ulf , Delgado Maria Belen , Uguccioni Mariagrazia , Loetscher Pius , Garotta Gianni , Baggiolini Marco
Published: July 2002

We have studied the biological properties of a new human CC chemokine, CKβ8, consisting of 99 amino acids including six cysteines. CKβ8 mRNA transcripts were induced in monocytes by IL‐1β and, to a lesser extent, by IFNγ, and were detected in RNA extracted from normal human liver and gastrointestinal tract. CKβ8 is chemotactic for monocytes, but is inactive on IL‐2 conditioned T lymphocytes, eosinophils and neutrophils. Desensitization experiments indicate that CKβ8 and MIP‐1β completely share receptors on monocytes and that the CKβ8 receptor, which appears to differ from the known ones, is also recognized by MCP‐1, MCP‐2, MCP‐3, MCP‐4, MIP‐1α and RANTES.
Cyclic AMP signalling and cellular proliferation: regulation of CREB and CREM
Authors: Della Fazia Maria Agnese , Servillo Giuseppe , Sassone-Corsi Paolo
Published: July 2002

In eukaryotes, transcriptional regulation upon stimulation of the adenylyl cyclase signalling pathway is mediated by a family of cAMP‐responsive nuclear factors. This family consists of a large number of members which may act as activators or repressors. These factors contain the basic domain/leucine zipper motifs and bind as dimers to cAMP‐response elements (CRE). The function of CRE‐binding proteins (CREB) is modulated by phosphorylation by the cAMP‐dependent protein kinase. The ICER (inducible cAMP early repressor) protein is the only inducible member of this family and is a product of the CREM gene. The induction of this powerful repressor is likely to be important for the transient nature of cAMP‐induced gene expression. CREB proteins have been found to play an important role in the physiology of neuroendocrine functions. In addition, recent results indicate that CREB and CREM could be involved in the proliferation of hepatocytes which follows partial hepatectomy.
Repression of apoC‐III gene expression by TNFα involves C/EBPδ/NF‐IL6β via an IL‐1 independent pathway
Authors: Lacorte Jean-Marc , Beigneux Anne , Parant Monique , Chambaz Jean
Published: July 2002

Apolipoproteins A‐II and C‐III, which participate in the control of cholesterolemia and triglyceridemia, are negative acute phase proteins. Treatment of HepG2 cells with TNFα showed that apoA‐II and apoC‐III mRNA levels were decreased. Using transient transfection, we found that apoC‐III gene expression is controlled at the transcriptional level. By competition and supershift experiments, we demonstrate that TNFα‐induced complexes were related to C/EBPδ/NF‐IL6β and p50 and that overexpression of C/EBPδ was able to reproduce the inhibitory effect of TNFα on the apoC‐III promoter. RT‐PCR failed to detect the IL‐1 transcript in TNFα‐treated HepG2 cells, suggesting that activation of C/EBPδ by TNFα is not related to the IL‐1‐signalling pathway.
Regulatory phosphorylation of plant phosphoenolpyruvate carboxylase: role of a conserved basic residue upstream of the phosphorylation site
Authors: Ueno Yoshihisa , Hata Shingo , Izui Katsura
Published: July 2002

In order to mimic regulatory phosphorylation of the Ser‐15 of maize C4‐form phosphoenolpyruvate carboxylase (PEPC), we replaced Ser‐15 and Lys‐12 with Asp (S15D) and Asn (K12N), respectively, by site‐directed mutagenesis. Although both mutant enzymes were catalytically as active as the wild‐type PEPC, they showed much less sensitivity to malate, an allosteric inhibitor, similarly to the phosphorylated wild‐type PEPC. A maize protein kinase of 30 kDa which is known to be specific to PEPC (PEPC‐PK), phosphorylated K12N as well as the wild‐type PEPC but not S15D. The phosphorylation of K12N further diminished the sensitivity to malate. Thus, a positive charge of the conserved Lys‐12 is not required for the recognition by PEPC‐PK but contributes to the intrinsic sensitivity to malate inhibition.
Involvement of integrins and the cytoskeleton in modulation of skeletal muscle glycogen synthesis by changes in cell volume
Authors: Low Sylvia Y , Rennie Michael J , Taylor Peter M
Published: July 2002

Muscle glycogen synthesis is modulated by physiologically relevant changes in cell volume. We have investigated the possible involvement of integrin‐extracellular matrix interactions in this process using primary cultures of rat skeletal muscle subject to hypo‐ or hyper‐osmotic exposure with integrin binding peptide GRGDTP to disrupt integrin actions and the inactive analogue GRGESP as control. Osmotically induced increases (77%) and decreases (34%) in glycogen synthesis (d‐[14C]‐glucose incorporation into glycogen) were prevented by GRGDTP (but not GRGESP) without affecting glucose transport. Cytoskeletal disruption with cytochalasin D or colchicine had similar effects to GRGDTP. Osmotically induced modulation of muscle glycogen synthesis involves integrin‐extracellular matrix interactions and cytoskeletal elements, possibly as components of a cell‐volume `sensing' mechanism.
FT‐IR study of the Ca2+‐binding to bovine α‐lactalbumin
Authors: Mizuguchi Mineyuki , Nara Masayuki , Kawano Keiichi , Nitta Katsutoshi
Published: July 2002

Fourier‐transform infrared spectroscopy (FT‐IR) was applied to examine relationships between the type of coordination and the COO− antisymmetric and symmetric stretches of the COO− groups in the Ca2+‐binding site of bovine α‐lactalbumin. The peaks at 1593, 1578, 1425, and 1403 cm−1 were assigned to the COO− groups of Asp‐82, 87, and 88 coordinating to Ca2+ in the pseudo bridging mode, according to the results of X‐ray crystallography. The bands due to the COO− groups were quite similar to each other between α‐lactalbumin and EDTA which is the model compound for the pseudo bridging state.
Arabidopsis thaliana class IV chitinase is early induced during the interaction with Xanthomonas campestris
Authors: de A. Gerhardt Liliane B , Sachetto-Martins Gilberto , Contarini Maria G , Sandroni Mariana , de P. Ferreira Rodrigo , de Lima Viviane M , Cordeiro Maria C , de Oliveira Dulce E , Margis-Pinheiro Márcia
Published: July 2002

Endochitinases are widely distributed among higher plants, including a number of important crop species. They are generally considered to be involved in plant defence against potential pathogens. We have cloned a class IV chitinase gene (AtchitIV) from Arabidopsis thaliana. Southern blot analysis allowed the detection of two cross‐hybridising genes in the A. thaliana genome. AtchitIV transcripts are detected in seedpods, but not in roots, inflorescence stems, leaves and flowers of healthy plants. The transcripts accumulated very rapidly in leaves after inoculation with Xanthomonas campestris. Maximum mRNA accumulation was reached one hour after infection and decreased to very low levels 72 hours after induction. This result suggests an involvement of AtchitIV in the initial events of the hypersensitive reaction. Nevertheless, A. thaliana plants transformed with the gus gene under the control of a class IV chitinase bean promoter, showed GUS activity in seed embryos. These data, together with the constitutive expression of the endogenous gene in the seedpods, points to additional physiological roles for this protein.
Prostaglandin E2biphasic control of lymphocyte proliferation: inhibition by picomolar concentrations
Authors: Sergeeva Marina G , Gonchar Maria V , Mevkh Alevtina T , Varfolomeyev Sergey D
Published: July 2002

Prostaglandins (PGs) have an important physiological role in the modulation of various cell immune functions. The main sources of PGs during immune responses are monocyte cells. We report here the ability of non‐stimulated macrophages to synthesize prostanoids and show that peritoneal mouse macrophages synthesize PGE2, PGF2aand thromboxane B2, spleen macrophages produce PGE2and PGF2a, and in a fresh medium this synthesis reaches a constant basal level in a few hours. We studied the kinetics of Con A‐induced proliferation of murine splenocytes under the influence of a wide range of PGE2concentrations (10−14–10−7M). The suppressive effect of PGE2decreased when its concentration was lowered and disappeared at 10−9M PGE2(this concentration corresponded to the basal level of non‐stimulated macrophage synthesis of PGE2). Further lowering of the concentration became essential for the proliferation process once again, and at picomolar concentrations PGE2caused a suppressive effect comparable with that for 10−8M PGE2. We also found that PGE2significantly inhibited cell proliferation when it was added 1 h before the addition of mitogen, as compared with simultaneous mitogen addition. The effect was obtained for both low (10−12M) and high (10−8M) PGE2concentrations. This phenomenon of PGE2biphasic control of lymphocyte proliferation may play an important role in cellular homeostasis, in particular in immune cell function regulation.
Molecular assembling of DNA with amphipathic peptides
Authors: Dufourcq J , Neri W , Henry-Toulmé N
Published: July 2002

The self‐assembling of double‐stranded DNA with short synthetic peptides has been analysed using the fluorescent properties of the intercalating dye, ethidium bromide. Two membrane‐active peptides with appropriate sequences of lysine and leucine amino acids and a short polylysine have been probed. The results revealed that the secondary structure of the peptide decisively aimed the peptide‐DNA complex formation: only the longest peptide, which is the only one to exhibit an α‐helical structure in solution, could achieve DNA compacting before charge neutralisation. The obtained complex retained a significant membrane activity as demonstrated by calcein leakage experiments. This shows that short synthetic peptides of elementary sequence can combine both membrane activity and DNA‐condensing properties. The potential of these constructs as DNA carriers will be discussed.
Restoration of phytanic acid oxidation in Refsum disease fibroblasts from patients with mutations in the phytanoyl‐CoA hydroxylase gene
Authors: Chahal Amarjit , Khan Mushfiquddin , Pai Shashidhar G , Barbosa Ernest , Singh Inderjit
Published: July 2002

Refsum disease (RD) is biochemically characterized by the excessive accumulation of phytanic acid in tissues and body fluids due to deficiency of phytanoyl‐CoA hydroxylase (PAHX). In this study, we screened three RD patients and identified a novel deletion (88 amino acids), and a missense mutation (Arg275Trp) in the previously reported PAHX cDNA (Jansen et al., 1997; Mihalik et al., 1997). Moreover, transfection of skin fibroblasts from two RD patients with wild‐type PAHX gene restored the activity for α‐oxidation of phytanic acid. Southern analysis on a somatic cell hybrid panel detected the PAHX gene on chromosome 10, corroborating radiation hybrid and homozygosity mapping data (Mihalik et al., 1997; Nadal et al., 1995).
T‐cadherin and signal‐transducing molecules co‐localize in caveolin‐rich membrane domains of vascular smooth muscle cells
Authors: Philippova M.P , Bochkov V.N , Stambolsky D.V , Tkachuk V.A , Resink T.J
Published: July 2002

Cadherins are a family of cellular adhesion proteins mediating homotypic cell‐cell binding. In contrast to classical cadherins, T‐cadherin does not possess the transmembrane and cytosolic domains known to be essential for tight mechanical coupling of cells, and is instead attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. This study explores the hypothesis that T‐cadherin might function as a signal‐transducing protein. Membranes from human and rat vascular smooth muscle cells were fractionated using Triton X‐100 solubilization and density gradient centrifugation techniques. We demonstrate that T‐cadherin is enriched in a minor detergent‐insoluble low‐density membrane domain and co‐distributes with caveolin, a marker of caveolae. This domain was enriched in other GPI‐anchored proteins (CD‐59, uPA receptor) and signal‐transducing molecules (Gαs protein and Src‐family kinases), but completely excluded cell‐cell and cell‐matrix adhesion molecules (N‐cadherin and β1‐integrin). Coupling of T‐cadherin with signalling molecules within caveolae might enable cellular signal transduction.
Skeletal muscle UCP2 and UCP3 gene expression in a rat cancer cachexia model
Authors: Sanchı́s Daniel , Busquets Sı́lvia , Alvarez Belén , Ricquier Daniel , López-Soriano Francisco J , Argilés Josep M
Published: July 2002

Rats bearing the Yoshida AH‐130 ascites hepatoma showed an increased expression of both uncoupling protein‐2 (UCP2) (194%) and UCP3 (189%) mRNA levels in skeletal muscle 7 days after tumour inoculation. Interestingly, an even greater increase was observed in mRNA for both UCP2 (278%) and UCP3 (797%) in the pair‐fed animals, suggesting that the increase in gene expression was the result of the anorexia associated with tumour burden. The results constitute the first report of UCP2 and UCP3 gene expression during cancer cachexia and agree to their possible role in the increase of energy expenditure associated with tumour growth.
Cytochrome c release from brain mitochondria is independent of the mitochondrial permeability transition
Authors: Andreyev Alexander Y. , Fahy Brenda , Fiskum Gary
Published: July 2002

Ca2+ uptake by brain mitochondria induces the release of up to 40% of total cytochrome c in a cyclosporin A‐insensitive manner. In the presence of ATP and Mg2+, this process is not accompanied by mitochondrial swelling. There is a moderate decrease in membrane potential under these conditions, but it is completely reversible upon removal of accumulated Ca2+ by addition of EGTA+A23187 but not by EGTA alone. These observations provide evidence that cytochrome c release from brain mitochondria does not require the membrane permeability transition. However, brain mitochondria can undergo the permeability transition in the absence of ATP and Mg2+, which results in cyclosporin A‐sensitive large amplitude swelling, loss of Ca2+ uptake capacity and release of matrix solutes.