bluuebunny/crossref_metadata_2025 huggingface dataset:
The synthesis of α2‐macroglobulin by rat mammary myoepithelial cells is regulated by synergism between glucocorticoids and cytokines
Authors: Warburton Michael J. , Gusterson Barry A. , O'Hare Michael J.
Published: July 2002

Addition of hydrocortisone to the rat mammary gland myoepithelial cell line, G4.2.3, induces the synthesis and secretion of α2‐macroglobulin. Interleukin‐1β, interleukin‐6 and transforming growth factor‐β synergise with hydrocortisone, increasing the synthesis of α2‐macroglobulin 2‐ to 4‐fold, although they have no effect in the absence of hydrocortisone. Interleukin‐6 is the most potent inducer having an optimum concentration of 1 ng/ml. Interleukin‐6, unlike interleukin‐1β or tranforming growth factor‐β, decreases the lag phase from 10 h to 4 h before α2‐macroglobulin synthesis is induced by hydrocortisone. These results suggest that the mechanism of activation of transcription of the α2‐macroglobulin gene in mammary myoepithelial cells might differ from that operating in hepatic cells.
Calorimetric characterization of the stable complex of myosin subfragment 1 with ADP and beryllium fluoride
Authors: Bobkov Andrey A. , Khvorov Nikolai V. , Golitsina Nina L. , Levitsky Dmitrii I.
Published: July 2002

The thermal unfolding of the myosin subfragment 1 (S1) in its stable complex with ADP and beryllium fluoride (S1 · ADP · BeF3
−) was studied by differential scanning calorimetry. It has been shown that the structure of the S1 molecule in the S1 · ADP · BeF3
− complex is similar to that of S1 in its complex with ADP and orthovanadate (S1 · ADP · Vi but differs radically from that of nucleotide‐free S1 and S1 in the S1 · ADP complex. It is concluded that the S1 · ADP · BeF3
− complex can be considered, like the S1 · ADP · Vi complex, a stable structural analogue of the myosin head · ADP · Pi transition state of the myosin‐catalyzed ATP hydrolysis.
Nicking of the tryptophan synthase β2‐subunit at Glu‐296 prevents the conformational change undergone on binding the α‐subunit
Authors: Linkens Hans-Josef , Djavadi-Ohaniance Lisa , Goldberg Michel E.
Published: July 2002

Using a monoclonal antibody as conformational probe it has been shown that the weakly active nicked‐β2 dimer of tryptophan synthase generated by proteolytic cleavage at Glu‐296, does not undergo on association with α subunit a conformational change known to occur in intact β2 subunit. This α induced conformational change is also prevented in intact β2 by the coenzyme pyridoxal‐5'‐phosphate when the substrate l‐serine is absent.
Effect of catalytically active recruited crystallins on lens metabolism
Authors: Reddy M.C. , Groth-Vasselli Barbara , Sharon Ashwini , Farnsworth Patricia N.
Published: July 2002

The impact on duck lens metabolism of recruited ϵ‐crystallin/LDH‐B4 and τ‐crystallin/enolase was investigated by NMR spectroscopy. A comparison of the duck lens metabolite profile with that of the calf in which recruited crystallins are absent revealed significant increases in ATP, α‐glycerophosphate (α‐GP) and pyridine dinucleotide concentrations. The alterations in the concentrations of ATP and α‐GP appear to be related to both the high concentration of NAD and the elevated reduced to oxidized NADH/NAD+ ratio.
Two Tcp‐1‐related but highly divergent gene families exist in oat encoding proteins of assumed chaperone function
Authors: Ehmann Bruno , Krenz Martina , Mummert Eckart , Schäfer Eberhard
Published: July 2002

Tcp‐1‐related sequences have been isolated from a cDNA library of etiolated 6‐day‐old oat (Avena sativa) seedlings. This attempt was made to obtain cDNAs of a recently published 60 kDa plant chaperone that re‐folds denatured phytochrome and which was biochemically characterised as a Tcp‐1‐related protein [(1993) Nature 363, 644‐647]. The translation of the putative coding sequence from one full‐length cDNA clone displays no specific homologies to amino acid sequences known from peptide sequencing of the oat 60 kDa chaperone. Antibodies raised against the 60 kDa chaperone and over‐expressed protein from one full‐length coding sequence for Tcp‐1 from oat show no cross‐reactivity, whereas a monoclonal antibody raised against mouse Tcp‐1 protein recognizes both the 60 kDa protein purified from plant extracts and over‐expressed protein from Tcp‐1‐related cDNA sequences.
ATMPKs: a gene family of plant MAP kinases in Arabidopsis thaliana
Authors: Mizoguchi Tsuyoshi , Hayashida Nobuaki , Yamaguchi-Shinozaki Kazuko , Kamada Hiroshi , Shinozaki Kazuo
Published: July 2002

We previously reported two cDNAs for MAP kinases (cATMPK1 and cATMPK2) from a dicot plant, Arabidopsis thaliana. We describe here the cloning and characterization of five additional cDNAs encoding novel MAP kinases in Arabidopsis, CATMPK3, cATMPK4, cATMPK5, cATMPK6, and cATMPK7. The amino acid residues corresponding to the sites of phosphorylation (Thr‐Glu‐Tyr) that are involved in the activation of animal MAP kinases are conserved in all the seven putative ATMPK proteins. Genes for MAP kinases in Arabidopsis constitute a family that contains more than seven members. Sequence analysis suggests that there are at least three subfamilies in the family of Arabidopsis genes for MAP kinases.
Possible role of Na+ influx in phorbol ester‐induced down‐regulation of protein kinase C in HL60 cells
Authors: Takeuchi Noriko , Hashimoto Eikichi , Nakamura Toru , Takeuchi Fumito , Sada Kiyonao , Yamamura Hirohei
Published: July 2002

Amiloride, an inhibitor of Na+/H+ exchange, inhibited down‐regulation of protein kinase C in HL60 cells induced by tumor‐promoting phorbol ester in dose‐dependent manner judging from immunoblot analysis. This inhibition was observed with regard to type I (γ), type II (β), and type III (α) isozymes of protein kinase C. On the other hand, monensin, a Na+ ionophore, accelerated the down‐regulation of protein kinase C induced by phorbol ester. When we examined 22Na+ uptake by HL60 cells, the higher uptake was observed after stimulation with phorbol ester compared to the control cells and this 22Na+ uptake was strongly inhibited by the addition of amiloride. However, monensin further stimulated the 22Na+ uptake observed in phorbol ester‐treated cells. These data suggest that the increase in intracellular Na+ concentration may be one of the triggers for the induction of down regulation of protein kinase C.
Modified nucleoside 5'‐triphosphates containing 2',3'‐fused threemembered rings as substrates for different DNA polymerases
Authors: Semizarov Dimitry G. , Victorova Lyubov S. , Krayevsky Alexander A. , Kukhanova Marina K.
Published: July 2002

5'‐Triphosphates of 1‐(2',3'‐epithio‐2',3'‐dideoxy‐β‐D‐lyxofuranosy)thymine, 1‐(2',3'‐epithio‐2',3'‐dideoxy‐β‐D‐ribofuranosyl)thymine and 2',3'‐lyxoanhydrothymidine have been shown to be termination substrates for human immunodeficiency virus (HIV) and avian myeloblastosis virus (AMV) reverse transcriptases as well as DNA polymerase I from E. coli and DNA polymerase β from rat liver. At the same time they do not terminate DNA synthesis catalysed by DNA polymerase ϵ from human placenta. Km
values of ItTTP, rtTTP and laTTP incorporation into the DNA chain during catalysis by AMV reverse transcriptase agree closely with each other being 1.5–2.5 times higher than Km
value for dTTP. Furthermore, Vmax
values for modified substrates are only 2–3 times lower than Vmax
for dTTP. The evidence favours the hypothesis of high affinity of modified nucleotides with a flattened furanosyl ring for DNA polymerase active sites.
In vitro reconstitution of the proteolytic system responsible for hydrolysis of the 13S globulin in buckwheat seeds
Authors: Dunaevsky Y.E. , Belozersky M.A. , Voskoboynikova N.E.
Published: July 2002

The reconstruction of the proteolytic system responsible for the profound hydrolysis of the main storage protein of buckwheat seeds, 13S globulin, has been carried out in vitro. It was found that subsequent action of a metalloproteinase from dry buckwheat seeds and cysteine proteinase and carboxypeptidase from growing seedlings led to a profound hydrolysis of the storage protein. The 13S globulin that was not hydrolyzed with metalloproteinase was not degraded by the proteolytic enzymes of growing seedlings. A scheme is proposed, which describes the functioning of the proteolytic system hydrolyzing the storage protein in growing buckwheat seedlings in vivo.
A benzopyrylium dye as a novel effective cationic uncoupler of oxidative phosphorylation
Authors: Pronevich L.A. , Topaly E.E. , Kirpichenok M.A. , Grandberg I.I. , Topaly V.P.
Published: July 2002

Benzopyrylium dye BDBP was found to be an effective uncoupler of oxidative phosphorylation. It stimulates state 4 respiration of mitochondria 2‐fold at about 2 μM, reveals a maximum stimulation (8‐fold) at 30 μM and inhibits the respiration at higher concentrations. BDBP is also an effective proton (hydroxyl) carrier through bilayer lipid membranes (BLM) as is seen from the electrical properties of BLM with BDBP. Picrate enhances the effects of BDBP on the mitochondrial respiration and BLM conductance. The BDBP activities are accounted for by formation of dimers and complexes with picrate.
Tuftsin‐bearing liposomes as drug vehicles in the treatment of experimental aspergillosis
Authors: Owais Mohd. , Ahmed I. , Krishnakumar B. , Jaina R.K. , Bachhawat B.K. , Gupta C.M.
Published: July 2002

Encapsulation of amphotericin B in tuftsin‐bearing liposomes greatly increased its efficacy in treatment of human aspergillosis in mice. Also, the drug efficacy was significantly increased by pretreating the animals with drug‐free tuftsin‐bearing liposomes. These results demonstrate that macrophage activation can considerably enhance the therapeutic efficacy of antifungal drugs, like amphotericin B.
Inhibition of the replication of native and 3′‐azido‐2′,3′‐dideoxy‐thymidine (AZT)‐resistant simian immunodeficiency virus (SIV) by 3‐nitrosobenzamide
Authors: Chuang Ann J. , Killam Keith F. , Chuang Ronald Y. , Rice William G. , Schaeffer Catherine A. , Mendeleyev Jerome , Kun Ernest
Published: July 2002

The 3‐nitrosobenzamide (NOBA) drug abolishes SIV replication sharply at 20 μM concentration when CEM × 174 cells are preincubated for 1 h with the drug prior to viral infection. Treatment of CEM × 174 cells with 20 μM NOBA resulted in the inhibition of the synthesis of the DNA sequence coding for the gag gene, as determined by the PCR technique. Cell viability was directly proportional to the antiviral action of NOBA. Replication of AZT‐resistant SIV 23740 in MMU 23740 cells in vitro was suppressed by NOBA in a concentration‐dependent manner without significant effects on cell viability. Reverse transcriptase activity of SIVmac239 was unaffected by NOBA up to 800 μM concentration. Preincubation of two SIV strains with NOBA completely abolished their infectivity in human PHA‐PBL cells. Replication of two strains of SIV in PHA‐PBL cells was also inhibited by NOBA.
Measurement of barnase refolding rate constants under denaturing conditions
Authors: Sanz Jesús M. , Fersht Alan R.
Published: July 2002

Calculation of equilibrium and kinetic constants between the different species that occur in the folding pathway of a protein usually depends on extrapolations from conditions where these quantities can be accurately measured. Direct measurements of the extrapolated constants would allow the checking of the validity of the extrapolations. We measure here refolding constants of barnase under unfolding conditions by displacing the folding equilibrium by complexing folded barnase with its polypeptide inhibitor barstar. Formation of the enzyme‐inhibitor complex is very fast and virtually irreversible, and so refolding rate constants can be obtained. The results acquired confirm the extrapolations carried out in previous works.
Computer analyses suggest interactions of non‐muscle filamin with lipid membranes
Authors: Tempel M. , Goldmann W.H. , Isenberg G.
Published: July 2002

It is concluded from structure predictions of the primary amino acid sequence by computer analyses that two segments of non‐muscle filamin could facilitate lipid membrane attachment or anchoring. Residues 49–71 of the amino‐terminal may attach to phospholipid membranes, and residues 131–155 may anchor in the hydrophobic region of lipid membranes.
Motility and chemotaxis in Bacillus sphaericus dependence upon stage of growth
Authors: Andreev J. , Dibrov P.A. , Braun S.
Published: July 2002

Chemotaxis and motility of B. sphaericus 2362 were monitored as the function of a batch culture age. It was found that both functions changed independently during growth of the culture. Motility was low until the late logarithmic stage ensued, whereafter it increased sharply. The ability of cells to respond to chemoeffectors peaked at the mid‐logarithmic phase. A major methyl‐accepting chemotaxis protein (P53, M
r = 53 kDa) was identified. The extent of label incorporation in this protein from l‐[methyl‐3H]methionine was maximal in mid‐ and late logarithmic phases of the growth. Cells in stationary cultures incorporated very low amounts of the label. At any stage, the labeling was maximal in starved cells; it was almost abolished in cells pre‐incubated with amino acids. Although extents of P53 labeling in mid‐ and late logarithmic cells were similar, late logarithmic cells demonstrated a considerably impaired chemotaxis. Supermotile sporulating cells were practically insensitive to environmental stimuli. The difference in development of sensory and locomotive functions may be interpreted as an adaptive response. A well developed sensory apparatus would allow vegetative cells to adapt efficiently to fluctuating attractant gradients. Insensitive sporulating cells would tend to disperse randomly from the nutrient‐exhausted area. Thus, spore formation would occur in larger volume of the habitat, increasing the chance of the microbial population to survive.
Oxidase of the white rot fungus Panus tigrinus 8/18
Authors: Pozdnyakova Natalia , Leontievsky Alexey , Golovleva Ludmila
Published: July 2002

Extracellular oxidase of the white rot fungus Panus tigrinus earlier reported as laccase)contains copper but has no absorption spectrum typical of ‘blue’ oxidases. Thioglycolate and sodium azide inhibit the activity of this enzyme at concentrations 2.5–3 orders lower than those needed for fungal laccases. The oxidase of P. tigrinus oxidizes syringaldazine, coniferyl alcohol, ABTS, syringic acid, diaminobenzidine, guaiacol, catechol and vanillylacetone with different efficiencies. Oxygen consumption and no hydrogen peroxide formation were detected during substrate oxidation by P. tigrinus oxidase. It is proposed that P. tigrinus oxidase is a new ligninolytic enzyme.
Evidence for N‐glycosylation and ubiquitination of the prolactin receptor expressed in a baculovirus‐insect cell system
Authors: Cahoreau Claire , Garnier Laurence , Djiane Jean , Devauchelle Gérard , Cerutti Martine
Published: July 2002

The molecular mass of the rabbit prolactin receptor (rbPRLR) deduced from cDNA cloning is 66 kDa. However, the molecular mass of the full‐length receptor expressed in the insect Sf9 cells was found to be 94 kDa. In order to explain this discrepancy, we analyzed the possible post‐translational modifications of the PRLR. Sf9 cells were infected with recombinant baculoviruses in the presence of tunicamycin, an inhibitor of N‐glycosylation. Results showed that an additional ≈ 9 kDa of the extracellular domain could be attributed to the N‐glycosylation and another additional ≈ 20 kDa covalent modification occurred in the cytoplasmic part of the receptor. Western blot analysis, using anti‐ubiquitin antibodies, revealed that the rbPRLR was ubiquitinated in its cytoplasmic domain.
Mobile segments in rabbit skeletal muscle F‐actin detected by 1H nuclear magnetic resonance spectroscopy
Authors: Ślósarek Genowefa , Heintz Daniela , Kalbitzer Hans Robert
Published: July 2002

Polymerization of actin by increasing the ionic strength leads to a quenching of almost all 1H NMR signals. Surprisingly, distinct signals with relatively small line widths can still be observed in actin filaments (F‐actin) indicating the existence of mobile, NMR visible residues in the macromolecular structure. The intensity of the F‐actin spectrum is much reduced if one replaces Mg2+ with Ca2+, and a moderate reduction of the signal intensity can also be obtained by increasing the ionic strength. These results can be explained in a two‐state model of the actin protomers with a M‐ (mobile) state and a I‐ (immobile) state in equilibrium. In the M‐state a number of residues in the actin protomer are mobile and give rise to observable NMR signals. This equilibrium is shifted towards the I‐state specifically by replacing Mg2+ with Ca2+‐ions and unspecifically by addition of monovalent ions such as K+. The binding of phalloidin to its high‐affinity site in the filaments does not influence the equilibrium between M‐ and I‐state. Phalloidin itself is completely immobilized in F‐actin, its exchange with the solvent being slow on the NMR time scale.
Kinetics of phosphorylation of the SH2‐containing domain of phospholipase Cγ1 by the epidermal growth factor receptor
Authors: Posner Israel , Levitzki Alexander
Published: July 2002

The kinetics of the EGF receptor (EGFR) autophosphorylation and of the phosphorylation by EGFR of a fusion protein (Fp(SH2)) derived from PLC‐γ1, with two SH2 domains were studied employing purfied EGFR or membrane‐bound preparations of native and truncated EGFR. With varied ATP concentrations both reactions yielded Michaelis—Menten kinetics. KATP, for autophosphorylation was 0.35μM and for Fp(SH2) phosphorylation 1.35 μM. With Fp(SH2) as variable, the velocity curves for substrate phosphorylation by the various EGFR preparations were sigmoidal, reached peaks at 0.45μM Fp(SH2) and were followed by drops to zero velocities at about 1.0μM Fp(SH2). We conclude that (a) our data support the concept that receptor autophosphorylation is a prerequisite for the interactions between EGFR and the substrate's SH2‐domains and their eventual phosphorylation by the receptor, and (b) the interactions between EGFR and the physiological substrate seem to involve mechanisms which allow the substrate to act as an on‐off switch in the subsequent substrate phosphorylation reaction.
Isolation, biochemical characterization and N‐terminal sequence of rolipram‐sensitive cAMP phosphodiesterase from human mononuclear leukocytes
Authors: Truong V.H. , Müller T.
Published: July 2002

A cyclic AMP specific phosphodiesterase (type IV) was purified 450,000‐fold from human peripheral blood mononuclear cells through a sequence of chromatographic steps involving anion exchange, affinity chromatography on a matrix coupled to a derivative of the type IV inhibitor rolipram, and gel filtration. The enzyme showed apparent molecular masses of 70 kDa on gel filtration and 35 kDa on denaturing or native PAGE, indicating a possible dimerization or cleavage under certain conditions. The isoelectric point was 4.6. Kinetic parameters were K
m = 2.2μM, K
i = 1.2 μM (rolipram) and νmax = 80 μmol/min per mg protein. The most probable N‐terminal sequence was determined as SLTNTNIPRF, 80% identical to part of the deduced amino acid sequence from cDNA sequences of PDE IVA and PDE IVD.
Ras antagonizes cAMP stimulated glucagon gene transcription in pancreatic islet cell lines
Authors: Gherzi Roberto , Briata Paola , Fehmann Hans-Christoph , Göke Burkhard
Published: July 2002

Ras, a GTP‐binding protein, converts membrane tyrosine kinase signalling to changes in gene expression patterns. Utilising a rat glucagon promoter‐CAT construct (p[−1.1]GLU‐CAT) we demonstrate in transient transfection experiments that the oncogenic Ras inhibits cAMP‐dependent activation of p[−1.1]GLU‐CAT in both glucagonoma InR1‐G9 and insulinoma β‐TC1 cells. Conversely, the expression of a dominant negative mutant of Ras enhances the cAMP‐induced activation of p[−1.1]GLU‐CAT transcription in these cells. Our data suggests a functional interference of Ras with the cAMP‐dependent transcription of the glucagon gene.
Identification of two ets related genes in a marine worm, the polychaete annelid Nereis diversicolor
Authors: Lelièvre-Chotteau Anne , Laudet Vincent , Flourens Anne , Bègue Agnès , Leprince Dominique , Fontaine François
Published: July 2002

The ETS family includes a growing number of transcription factors with a highly conserved DNA‐binding domain, the ETS domain. We have used PCR amplification with degenerated oligonucleotides to isolate two putative ETS DNA‐binding coding domains in a primitive form of cœlomate, the polychaete annelid Nereis diversicolor. These sequences are highly related to the ETS and ERG groups of the ets gene family. For the erg sequence an adjacent region encoding for 91 amino acids has been characterized after library screening, and we show an expression in cells isolated from the cœlomic cavity of the animal. A phylogenic analysis confirms that the ets‐1/ets‐2 and the erg/fli dichotomy arose specifically in the vertebrate lineage.
Differential effects of guanine nucleotides on kainic acid binding and on adenylate cyclase activity in chick optic tectum
Authors: Paz Murilo Monteiro , Ramos Milagros , Ramírez Galo , Souza Diogo
Published: July 2002

In G protein‐coupled receptors, neurotransmitter‐induced binding of GTP to G proteins triggers the activation of effector systems while simultaneously decreasing the affinity of the transmitter for its specific binding site within the receptor—G protein complex. In the present study we show that, in the chick optic tectum, guanine nucleotides inhibit the binding of the glutamate analog, kainate, and activate adenylate cyclase by different mechanisms and acting on different sites. GMP‐PNP, a non‐hydrolyzable analog of GTP, binds tightly to G proteins so that the binding is stable even after exhaustive washing. By use of this property, we have prepared membrane samples in which G protein GTP‐binding sites are pre‐saturated with GMP‐PNP. Experiments carried out with these membranes show that GMP‐PNP, GDP‐S and GMP inhibit the binding of [3H]kainate by interacting with site(s) unrelated to G proteins, whereas GMP‐PNP activates adenylate cyclase activity by binding to G proteins.
Is Zn2+ transported by the mitochondrial calcium uniporter?
Authors: Saris Nils-Erik L. , Niva Kaija
Published: July 2002

Zinc ions were found to inhibit Ca2+ uptake by rat liver mitochondria driven by succinate respiration but not that by a valinomycin‐induced membrane potential. Zn2+ at 1 μM or higher concentrations induced a lowering of the membrane potential under the former but not the latter conditions. It is concluded that it is the lowered membrane potential in the presence of Zn2+ that reduces the rate of respiration‐driven Ca2+. Ruthenium red was found to inhibit the uptake of Zn2+ but had no influence on its action upon the membrane potential. Zn2+ did not affect the Ruthenium red‐insensitive Ca2+ efflux. Ca2+ stimulated the uptake of Zn2+. It is concluded that Zn2+ may be transported by the mitochondrial calcium uniporter but that it may have access to sites required for inhibition of respiration by other routes.
Insulin inhibits glucocorticoid‐induced stimulation of liver 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase gene transcription
Authors: Lemaigre Frédéric P. , Lause Pascale , Rousseau Guy G.
Published: July 2002

6‐Phosphofructo‐2‐kinase (PFK‐2)/fructose‐2,6‐bisphosphatase (FBPase‐2) catalyzes the synthesis and degradation of fructose 2,6‐bisphosphate, a potent stimulator of glycolysis. Transcription of the mRNA encoding rat liver PFK‐2/FBPase‐2 is stimulated by insulin and by glucocorticoids in rat hepatoma cells. We show here that insulin can also prevent and reverse this glucocorticoid effect. The inhibitory effect of insulin is independent of extracellular glucose and does not require ongoing protein synthesis. We conclude that insulin exerts opposite effects on PFK‐2/FBPase‐2 gene transcription depending on the hormonal context.
Chemical synthesis and biological activity of a novel antibacterial peptide deduced from a pig myeloid cDNA
Authors: Storici Paola , Scocchi Marco , Tossi Alessandro , Gennaro Renato , Zanetti Margherita
Published: July 2002

Several myeloid precursors of antibacterial peptides have recently been shown to share homologous pre‐ and pro‐regions. Taking advantage of this homology, a novel cDNA was cloned from pig bone marrow RNA. This encodes a 166‐residue polypeptide with highly conserved pre‐ (29 residues) and pro‐ (101 residues) sequences, followed by a unique, 36‐residue C‐terminal sequence. Structure analyses of this C‐terminal region have identified a highly cationic sequence predicted to adopt an amphipathic α‐helical conformation. A peptide corresponding to this sequence was chemically synthesized and shown to arrest the growth of both Gram‐positive and Gram‐negative bacteria. At least for Escherichia coli, the activity of this peptide appears to be mediated by its ability to permeabilize the bacterial membranes.
Isoforms of cellular fibronectin and tenascin in amniotic fluid
Authors: Linnala Auli , von Koskull Harriet , Virtanen Ismo
Published: July 2002

Amniotic fluid (AF) obtained from second trimester pregnancies presented extradomain (ED) A, B and an oncofetal (onc‐f) domain containing isoforms of cellular fibronectin (cFn) in Western blotting of gelatin‐bound polypeptides and directly of AF. Western blotting after sequential immunoprecipitation suggested at least three Fn molecules: one containing EDA and the onc‐f domain and another minor component distinctly containing all the domains, and a third one only containing EDA. The immunoblotting results for EDA‐cFn and onc‐f‐cFn were closely similar to that for total Fn, whereas in plasma samples of normal and pregnant women only traces of EDA‐cFn and onc‐f‐cFn, but no EDB‐cFn, were found. Western blotting of AF also indicated the presence of three isoforms of tenascin (Tn), M
r 190,000 and 280,000 polypeptides earlier found in many cells, and a M
r 200,000 polypeptide, novel for AF and not present in plasma. The results suggest a novel extracellular matrix polypeptide composition for AF.
Sequence similarity of mammalian epoxide hydrolases to the bacterial haloalkane dehalogenase and other related proteins
Authors: Arand Michael , Grant David F. , Beetham Jeffrey K. , Friedberg Thomas , Oesch Franz , Hammock Bruce D.
Published: July 2002

Direct comparison of the amino acid sequences of microsomal and soluble epoxide hydrolase superficially indicates that these enzymes are unrelated. Both proteins, however, share significant sequence similarity to a bacterial haloalkane dehalogenase that has earlier been shown to belong to the α/β hydrolase fold family of enzymes. The catalytic mechanism for the dehalogenase has been elucidated in detail [Verschueren et al. (1993) Nature 363, 693‐698] and proceeds via an ester intermediate where the substrate is covalently bound to the enzyme. From these observations we conclude (i) that microsomal and soluble epoxide hydrolase are distantly related enzymes that have evolved from a common ancestral protein together with the haloalkane dehalogenase and a variety of other proteins specified in the present paper, (ii) that these enzymes most likely belong to the α/β hydrolase fold family of enzymes and (iii) that the enzymatic epoxide hydrolysis proceeds via a hydroxy ester intermediate, in contrast to the presently favoured base‐catalyzed direct attack of the epoxide by an activated water.
Interaction of protein ligands with receptor fragments
Authors: Fulachier Marie-Hélène , Mourier Gilles , Cotton Joël , Servent Denis , Ménez André
Published: July 2002

Using a solid‐phase assay, we found that 3H‐labelled αCobtx from Naja naja siamensis, a long‐chain curaremimetic toxin, and 3H‐labelled toxin a from Naja nigricollis, a short‐chain toxin both bind specifically but with substantially different affinities (K
d = 4·10−7 M and 50·10−6M) to fragment 185–199 (Tα185–199) of the α‐subunit of the acetylcholine receptor (AcChoR) from Torpedo marmorata. Then we show that monoderivatizations of residues common to both long‐chain and short‐chain toxins (Tyr‐25, Lys‐27, Trp‐29, and Lys‐53) or to long‐chain toxins only (Cys‐30 and Cys‐34) do not affect the binding of the toxins to Tα185‐199, suggesting that none of these invariant residues is implicated in the recognition of this AcChoR region. αCobtx and toxin α bind to the fragment 128‐142 (Tα128‐142) with more similar affinities (K
d = 3·10−7 M and 1.4·10−6 M) and their binding is dramatically affected by the single abolition of the positive charge of Lys‐53, an invariant residue that contributes to AcChoR recognition. Therefore, the data indicate that Lys‐53 more specifically recognizes the 128–142 region of AcChoR. Other monoderivatizations have no effect on toxin binding. The approach described in this paper may be of great help to identify toxin residues that establish direct contact with receptor fragments.
Molecular cloning of the S‐adenosylmethionine synthetase gene in Drosophila melanogaster
Authors: Larsson Jan , Rasmuson-Lestander Åsa
Published: July 2002

We have isolated and sequenced cDNA clones encoding the Drosophila melanogaster
S‐adenosylmethionine synthetase. The deduced amino acid sequence contains 405 amino acid residues and shows high homology to rat, yeast, Arabidopsis and Escherichia coli counterparts. The gene is transcribed throughout Drosophila development but its main activity is seen in adult males and females. The highest transcription activity is seen in female ovaries. The transcript has an unusually long 5′‐untranslated region, which might be of importance for translational regulation.
Biochemical complementation studies in vitro of gyrase subunits from different species
Authors: Simon Hannelore , Roth Martin , Zimmer Christoph
Published: July 2002

To investigate the functional equivalence of DNA gyrase subunits from different bacterial sources hybrid enzymes were formed using purified A and B subunits from three species of Streptomycetes, E. coli and B. subtilis. The activity of gyrase hybrids composed of heterologous gyr A and gyr B proteins and of the gyrases containing homologous subunits was characterized by binding studies and a cleavage assay with two different DNA fragments. Likewise the enzyme activity was monitored by the super‐coiling and relaxation assay with pBR322 DNA. We found that cleavage reactions are largely determined by the source of the gyr B subunits whereas DNA supercoiling and relaxation reactions of pBR322 catalyzed by DNA gyrase are limited to a combination of homologous A and B subunits or of heterologous A and B subunits from the taxonomically related bacteria Streptomycetes.
Expression of recombinant pro‐neuropeptide Y, proopiomelanocortin, and proenkephalin: relative processing by ‘prohormone thiol protease’ (PTP)
Authors: Schiller Martin R , Kohn Andrea B , Mende-Mueller Liane M , Miller Kurt , Hook Vivian Y.H
Published: July 2002

The preference of the ‘prohormone thiol protease’ (PTP), a candidate prohormone processing enzyme, for different peptide precursors was assessed in vitro with recombinant prohormones near estimated in vivo levels. Pro‐neuropeptide Y (pro‐NPY), prooplomelanocortin (POMC), and proenkephalin (PE) were expressed at high levels in E. coli. Purification of prohormones utilized a combination of DEAE‐Sepharose, Mono Q, and preparative electrophoresis. PTP cleaved PE most readily, and also cleaved pro‐NPY. The processing of POMC by PTP was minimal. These results demonstrate PTP's preference for certain prohormone substrates.
Temporal regulation of gene expression in adipocyte differentiation
Authors: Martino Jeffrey J. , Chen Suzie
Published: July 2002

Cells usually become ‘committed’ to differentiate long before any actual morphological change is apparent. In one model commitment is a decision which corresponds to the expression of a control gene, while differentiation is the ultimate consequence of that decision. We have been studying adipocyte commitment and differentiation at the molecular level. Earlier we showed that the introduction of a specific DNA sequence into ‘uncommitted’ cells renders those cells committed to differentiate into adipocytes. Here we report temporal regulation of the expression of this DNA sequence; furthermore, we show that this RNA is in the non‐polyA+ fraction of total cellular RNA. These data suggest that coordinate regulation of this and other genes is important in promoting differentiation.
Genetic analysis of the cytochrome P‐450IIC18 (CYP2C18) gene and a novel member of the CYP2C subfamily
Authors: Tsuneoka Yutaka , Matsuo Yoshinori , Okuyama Eisaku , Watanabe Yasuhiro , Ichikawa Yoshiyuki
Published: July 2002

The CYP2C18 gene was investigated in order to characterize its molecular basis in the CYP2C subfamily. A mutation of the CYP2C18 gene was identified at the 5′‐flanking region of the gene, which could be detected by digestion with DdeI. The allele frequency of the mutant CYP2C18 gene was 21.4%. Genotypes of the polymorphic DdeI site of the CYP2C18 gene were found to be completely consistent with that of the polymorphic CYP2C19 gene (the m1 mutant). The CYP2C18 and CYP2C19 genes were suggested to be linked and located close together on the chromosome. Clones containing the 5′‐flanking region of a member of the CYP2C subfamily were obtained from the PCR products from human genomic DNA. The nucleotide sequence of the clone proved to be 90.5% identical to the corresponding region of the CYP2C18 gene. This is very likely to be a novel member of the CYP2C subfamily.
Glucose‐induced inactivation of isocitrate lyase in Saccharomyces cerevisiae is mediated by the cAMP‐dependent protein kinase catalytic subunits Tpk 1 and Tpk2
Authors: Ordiz Isabel , Herrero Pilar , Rodicio Rosaura , Moreno Fernando
Published: July 2002

Glucose‐induced inactivation of isocitrate lyase (Icl) has been related to protein phosphorylation. Moreover, since rapid reversible inactivation preceded irreversible inactivation of the enzyme, phosphorylation was proposed as the triggering reaction that makes the enzyme accessible to the proteolytic machinery. The protein kinase involved in the process is unknown at the moment. In this work we demonstrate that Tpk1 and Tpk2, the catalytic subunits of cAMP‐dependent protein kinase, are involved in the signalling of short‐term and long‐term inactivation processes of Icl. We also demonstrate that threonine 53 is involved in a regulatory mechanism necessary for short‐term reversible inactivation of Icl, probably mediated through its phosphorylation. Other, as yet unidentified, residues are likely to be the target of distinct protein kinases mediating the irreversible long‐term inactivation of Icl.
Alcoholytic deblocking of N‐terminally acetylated peptides and proteins for sequence analysis
Authors: Bergman Tomas , Gheorghe Madalina T. , Hjelmqvist Lars , Jörnvall Hans
Published: July 2002

N‐terminal acetylation of polypeptides is a common feature preventing direct Edman degradation. We describe a method for the removal of the acetyl group, with only a low extent of internal peptide bond cleavage, also in large proteins, by treatment at room temperature with trifluoroacetic acid and methanol. The alcohol is essential for selective deacetylation, and it is proposed that the deblocking mechanism consists of an acid‐catalyzed nucleophilic substitution involving methanol. The extent of deacetylation is limited, but the initial yield in the sequence analysis can be up to 10%. Deblocking of samples spotted or blotted onto sequencer filters is equally possible as the use of isolated samples from column separations. Deblocking on sequencer filters is also possible directly after negative results on initial sequencer attempts with samples proving to be blocked.
Analysis of SRF, SAP‐1 and ELK‐1 transcripts and proteins in human cell lines
Authors: Magnaghi-Jaulin Laura , Masutani Hiroshi , Lipinski Marc , Harel-Bellan Annick
Published: July 2002

We have analysed the expression of the genes encoding transcription factors involved in c‐fos transcriptional regulation, i.e. the serum response factor (SRF) and the ETS‐related proteins ELK‐1 and SAP‐1, in a variety of human cell lines. RNA was determined by Northern blot analysis, and proteins were detected on Western blots: the two analyses gave essentially identical results. SRF was expressed at similar levels in all cell lines tested. In contrast, SAP‐1 and ELK‐1 expression varied from one cell line to another. Interestingly, in any given cell line, high levels of one protein were accompanied by low levels of the other. Similar results were obtained by electromobility shift assays (EMSA), using antibodies directed against the proteins. Thus, our data raise the possibility of a coordinated regulation of the expression of these two Ets genes, at both RNA and protein levels.
Tissue‐specific alternative splicing of mouse brain type ryanodine receptor/calcium release channel mRNA
Authors: Miyatake Ryosuke , Furukawa Aizo , Matsushita Masayuki , Iwahashi Kazuhiko , Nakamura Kazuhiko , Ichikawa Yoshiyuki , Suwaki Hiroshi
Published: July 2002

We detected alternative splicing of the mouse brain type ryanodine receptor (RyR3) mRNA. The splicing variant was located in the transmembrane segment. The non‐splicing type (RyR3‐II) included a stretch of 341 bp, and that of the 13th codon was stop codon TAA. Reverse transcription‐polymerase chain reaction (RT‐PCR) analysis shows that RyR3‐II mRNA was expressed in various peripheral tissues and brain at all developmental stages. However, interestingly, the splicing type (RyR3‐I) mRNA was detected only in the cerebrum. These findings suggest that the splicing variants RyR3‐I and RyR3‐II may generate functional differences of RyR3 in a tissue‐specific manner.
Association in a crustacean sensory organ of two usually mutually exclusive mechanosensory cells
Authors: Crouau Yves
Published: July 2002

Summary— The external mechanosensory cells of Arthropoda are classified into two groups which are structurally and functionally different. One of the two types of cells is characteristic for exteroceptive sensory organs of terrestrial Arthropoda (mechanosensitive cell with a tubular body), while the other type is typical of Crustacea (chordotonal‐type mechanosensitive cell). However, we show that, in an exteroceptive sensory organ of a terrestrial Crustacea (Isopoda), both types of cells coexist. Only one sensory cell seems to be functional. These observations are discussed with respect to hypotheses concerning the functioning of the two types of sensory cells, the physical environment and the evolutionary position of Isopoda.
Peroxide accumulation without major mitochondrial alteration in replicative senescence
Authors: Pfeuty A. , Gueride M.
Published: July 2002

The aim of this work was to determine how ageing changes were related to each other by studying the establishment of a senescent state in cell culture, rabbit articular chondrocytes. A striking increase of the amount of peroxides appeared at 2/3 of the time of cell growth, which is responsible for an oxidative stress, as shown by the appearance of oxidized proteins, the overexpression of HSP27 gene and the accumulation of HSP27 protein. While no change of the mitochondrial membrane potential was observed all along the cell culture, p21, a protein overproduced in senescent cells, appeared with the peak of peroxides and accumulated.
Splicing before import – An intein in a mitochondrially targeted preprotein folds and is catalytically active in the cytoplasm in vivo
Authors: Williams Lisa R. , Ellis Steven R. , Hopper Anita K. , Davis Elaine O. , Martin Nancy C.
Published: July 2002

Nuclear‐encoded mitochondrial proteins are cytoplasmically synthesized and imported into the organelle. The intein‐containing RecA protein of Mycobacterium tuberculosis, with or without the CoxIVp mitochondrial targeting signal (MTS), was used to determine where a protein targeted to mitochondria folds and becomes catalytically active. Analysis of fractions from Saccharomyces cerevisiae cells expressing RecA without the MTS revealed that RecA and intein proteins remained cytoplasmic. With the MTS, most of RecA was directed to mitochondria, while most of the intein remained in the cytoplasm. The intein therefore folds into a catalytically active state in the cytoplasm prior to RecA import into mitochondria.
Differential immediate‐early gene responses to shear stress and intraluminal pressure in intact human conduit vessels
Authors: Gan Li-ming , Doroudi Roya , Hägg Ulrika , Johansson Ann-Marie , Selin-Sjögren Lena , Jern Sverker
Published: July 2002

We have previously shown distinct effects of shear stress and pressure on transcription of several potent vascular mediators. In the present study, we tested the hypothesis that c‐jun and c‐fos are regulated differentially by shear and pressure. Intact human umbilical veins were perfused with various combinations of shear and pressure during 1.5, 3 and 6 h. Protein and gene expressions were assessed by immunofluorescence and real‐time reverse transcription PCR, respectively. Shear stress and pressure exert differential temporal effects on c‐jun and c‐fos gene and protein expression, and these immediate‐early gene responses appear to be cell‐type specific for endothelial and smooth muscle cells.
The role of chromophore in the lipid–protein interactions in bacteriorhodopsin–phosphatidylcholine vesicles
Authors: Bryl Krzysztof , Yoshihara Kazuo
Published: July 2002

By fluorescence and phase properties of a 1‐acyl‐2‐[8‐(2‐anthroyl)‐octanoyl]‐sn‐glycero‐3‐phosphocholine probe, the influence of the chromophore on the phase transition of bacteriorhodopsin–lipid vesicles was investigated. It was observed that removal of the chromophore led to the down‐shifting of the phase transition temperatures. The temperatures corresponding to the beginning and ending of the gel–liquid phase transition were also influenced. This demonstrated that the liquid phase is reached more easily when the chromophore is bleached. The results indicate that removal of the chromophore alters the protein–lipid interactions. It is suggested that this alteration might be related to the change in the lipid molecular packing.
Inactivation of a MAPK‐like protein kinase and activation of a MBP kinase in germinating barley embryos
Authors: Testerink Christa , Vennik Marco , Kijne Jan W. , Wang Mei , Heimovaara-Dijkstra Sjoukje
Published: July 2002

We provide evidence for involvement of two different 45 kDa protein kinases in rehydration and germination of barley embryos. In dry embryos, a myelin basic protein (MBP) phosphorylating kinase was detected, which could be immunoprecipitated with an anti‐MAPK (mitogen‐activated protein kinase) antibody. Rehydration of the embryo induced a decrease in activity of this 45 kDa MAPK‐like protein kinase. In addition, activity of a MBP kinase of the same molecular weight was subsequently found to be induced. This second MBP kinase activity could not be immunoprecipitated with the anti‐MAPK antibody and was induced only in germinating embryos, not in dormant embryos.
On a revised mechanism of side product formation in the lignin peroxidase catalyzed oxidation of veratryl alcohol
Authors: ten Have Rimko , Franssen Maurice C.R.
Published: July 2002

The O2‐dependent formation of side products during the oxidation of veratryl alcohol (VA) by lignin peroxidase has previously been proposed to start with the attack of H2O on the VA radical cation (VA
+). This initial reaction is unlikely since it would also lead to side product formation in the absence of O2, which is not the case. In the current mechanism VA
+ reacts first with O2, whereafter H2O attacks. Furthermore, this paper describes an alternative explanation for the inhibitory effect of Mn2+ on VA side product formation. It is proposed that Mn2+ reduces reactive intermediates back to VA.
The fas locus of the phytopathogen Rhodococcus fascians affects mitosis of tobacco BY‐2 cells
Authors: Temmerman Wim , Ritsema Tita , Simón-Mateo Carmen , Van Montagu Marc , Mironov Vladimir , Inzé Dirk , Goethals Koen , Holsters Marcelle
Published: July 2002

The effect of Rhodococcus fascians, the causal agent of leafy gall disease, on the mitotic behavior of synchronized tobacco Bright Yellow‐2 (BY‐2) cells was investigated. Incubation of aphidicolin‐synchronized BY‐2 cells with R. fascians cells specifically resulted in a broader mitotic index peak, an effect that was linked to an intact and expressed fas virulence locus. The obtained results pointed towards an effect of R. fascians on the prophase of mitosis. The relevance of these results to the virulence of the bacterium is discussed.
Isolation of a putative receptor from Zea mays microsomal membranes that interacts with the G‐protein, GPα1
Authors: Wise Alan , Thomas Paul G. , White Ian R. , Millner Paul A.
Published: July 2002

The C‐terminal region of a heterotrimeric G‐protein α‐subunit is known to be one of the principal determinants governing its interaction with its cognate receptor. Use of an oligopeptide corresponding to the fifteen C‐terminal residues of the Arabidopsis Gα‐subunit (GPα1), as an affinity ligand, led to the resolution of a tightly binding 37 kDa membrane polypeptide from detergent solubilised Zea microsomal fraction membranes. An identical polypeptide bound tightly to an affinity matrix containing recombinant GPα1 protein as ligand: binding and release of this 37 kDa protein was dependent on the activation state of GPα1 which was regulated by inclusion or omission of the G‐protein activator AlF−
4. Finally, the isolated 37 kDa protein was labelled with the lectin concanavalin A, indicating it to be glycosylated. These data are consistent with the identity of the 37 kDa membrane polypeptide as a receptor that interacts with the Zea homologue of GPα1.
Charge compensated binding of divalent metals to bacterioferritin: H+ release associated with cobalt(II) and zinc(II) binding at dinuclear metal sites
Authors: E. Le Brun Nick , M. Keech Angus , R. Mauk Marcia , Grant Mauk A. , C. Andrews Simon , J. Thomson Andrew , R. Moore Geoffrey
Published: July 2002

Divalent metal ion binding to the bacterial iron‐storage protein, bacterioferritin (BFR), which contains a dinuclear metal binding site within each of its 24 subunits, was investigated by potentiometric and spectrophotometric methods. Cobalt(II) and zinc(II) were found to bind at both high‐ and low‐affinity sites. Cobalt(II) binding at the high‐affinity site was observed at a level of two per subunit with the release of ∼ 1.6 protons per metal ion, thus confirming the dinuclear metal centre as the high‐affinity site. Zinc(II) binding at the dinuclear centre (high‐affinity site) resulted in the release of ∼ 2 protons per metal ion, but exhibited a binding stoichiometry which indicated that not all dinuclear centres were capable of binding two zinc(II) ions. Competition data showed that binding affinities for the dinuclear centre were in the order zinc(II) > cobalt(II), and also confirmed the unexpected stoichiometry of zinc(II) binding. This work emphasises the importance ∼ of charge neutrality at the dinuclear centre.
ATF site of human RB gene promoter is a responsive element of myogenic differentiation
Authors: Okuyama Yusuke , Sowa Yoshihiro , Fujita Tsuyoshi , Mizuno Takakazu , Nomura Hitoshi , Nikaido Toshio , Endo Takeshi , Sakai Toshiyuki
Published: July 2002

RB mRNA increases during terminal differentiation of C2 myoblasts. We demonstrate that RB promoter activity increases about 4‐fold during differentiation. The increase of RB promoter activity was reduced when a point mutation was designed in the ATF site. In a gel shift assay of the ATF site, two specific bands were observed. One of them, with the lower mobility, disappeared during differentiation. This band reacted with an antibody against ATF‐1. We cotransfected an RB promoter‐luciferase plasmid with the TREB36/ATF‐1 plasmid. ATF‐1 suppressed the activity of the wild‐type RB promoter but not of that with a point mutation at the ATF site. These results suggest that the ATF site of the RB promoter is a responsive element during myogenic differentiation of C2 cells. We hypothesize that RB promoter activity is stimulated partially due to the dissociation of ATF‐1, which suppresses the promoter activity through the ATF site in C2 myoblasts.