bluuebunny/crossref_metadata_2025 huggingface dataset:
Respiration driven Cl− uptake by submitochondrial particles
Authors: Comerford John G. , Dawson Alan P. , Selwyn Michael J.
Published: July 2002

Both Mg2+ and oligomycin are required for the establishment of a membrane potential and the uptake of Cl− in submitochondrial particles prepared from rat liver. The effect of oligomycin is considered to be due to blocking of H+ conduction through exposed F0 channels of the ATPase complex whereas Mg2+ may more directly affect the anion‐conducting channel.
Renaturation of guanidine‐unfolded tryptophan synthase by multi‐mixing stopped‐flow dilution in D2O
Authors: Blond-Elguindi Sylvie , Friguet Bertrand , Goldberg Michel E.
Published: July 2002

Guanidine hydrochloride (GdnHCl) at high concentrations, e.g. 4 to 8 M, has been used extensively to promote reversible denaturation of several proteins. The refolding is induced by removal of the denaturing agent by diluting the denatured protein at least 50–100‐fold in a ‘renaturation buffer’. Fast kinetic studies, using a stopped‐flow apparatus to achieve such dilutions, are difficult for two reasons: firstly, injecting widely different volumes of the two reagents does not afford a proper mixing. Secondly, the density differences existing between the concentrated GdnHCl solution and the renaturation buffer often causes important mixing and redistribution artefacts particularly in vertical stopped‐flows. Here, it is shown that these difficulties can be overcome by using a multi‐mixing stopped‐flow to achieve 2 successive 7‐fold dilutions and by using heavy water (D2O) to adjust the density of the renaturation buffer. This enabled us to study the appearance of a short‐lived intermediate during the folding of the β2‐subunit of Escherichia coli tryptophan synthase.
Tuftsin stimulates growth of HL60 cells
Authors: Bump Nancy J. , Najjar Victor A.
Published: July 2002

Many functions of monocyte/macrophage and granulocyte are activated by tuftsin; principally phagocytosis, motility, immunogenic stimulation, antibacterial and antineoplastic activities. Here it is shown that tuftsin stimulates HL60 growth to twice the control rate. The uptake of [3H]uridine and [3H]leucine in a pulse of 30 min was also double that of the control. The uptake of thymidine was not stimulated.
Exploration of δ‐subunit interactions in beef heart mitochondrial F1 ‐ATPase by monoclonal antibodies
Authors: Lunardi Joël , Dupuis Alain , Frobert Yveline , Grassi Jacques , Vignais Pierre V.
Published: July 2002

Three monoclonal antibodies (mAbs) recognizing distinct epitopes on the δ‐subunit of beef heart mitochondrial F1‐ATPase were studied for their reactivity towards the δ‐subunit both in isolated F1 and in the F0‐F1 complex of submitochondrial particles. Two of the antibodies termed mAb δ 195 and mAb δ239 had free access to δ in F1 and the F0‐F1 complex. Partial hindrance was observed for the third antibody mAb δ22. By a double antibinding assay, it was found that the binding sites for mAb δ195 and mAb δ239 were close to each other and possibly overlapping. Mapping studies conducted with the isolated δ‐subunit showed that mAb δ195 and mAb δ239 interacted with the N‐terminal portion of δ extending from Ala‐1 to Met‐16, whereas mAb δ22 interacted with the fragment spanning Ser‐17‐Glu‐68. It was concluded that the Ala‐1‐Met‐16 segment of the δ‐subunit in F1 and the F0‐F1 complex is freely accessible from the outside, whereas the Ser‐17‐Glu‐68 segment of δ is partially hidden, possibly as a result of interactions with other subunits.
The tetanus toxin light chain inhibits exocytosis
Authors: Ahnert-Hilger Gudrun , Weller Ulrich , Dauzenroth Maria-Elisabeth , Habermann Ernst , Gratzl Manfred
Published: July 2002

The intracellular action on exocytosis of various forms of tetanus toxin was studied using adrenal medullary chromaffin cells, the membrane barrier of which has been removed by permeabilization with streptolysin O. Such cells still release catecholamines on stimulation with calcium. The two‐chain form of tetanus toxin (67 nmol/l) strongly inhibited exocytosis, but only if dithiothreitol was present as a reducing agent. Purified light chain completely prevented [3H]noradrenaline release with a half‐maximal effect at about 5 nmol/1. Heavy chain (up to 11 nmol/l) and unprocessed single‐chain toxin (up to 133 nmol/l) were without effect. It is concluded that the original single‐chain form of tetanus toxin has to be processed by proteolysis and reduction to yield a light chain which inhibits transmitter release.
Short model peptides having a high α‐helical tendency: Design and solution properties
Authors: Krstenansky John L. , Owen Thomas J. , Hagaman Karen A. , McLean Larry R.
Published: July 2002

Secondary structure is not typically observed for small peptides in solution. Several of the properties of α‐helical peptides are known which lead to the stabilization of the structure. The utilization of all the known factors important for α‐helical stabilization in the design of model α‐helical peptides (MAP) is reported. The peptides are based on the repeating eleven amino acid sequence, Glu‐Leu‐Leu‐Glu‐Lys‐Leu‐Leu‐Glu‐Lys‐Leu‐Lys (MAP1–11). The CD spectra of these peptides give evidence for more α‐helical content than has been reported for any short peptide (< 18 amino acids) to date. This α‐helical tendency does not require the presence of lipid or reduced temperature. For instance, Suc‐[Trp9]MAP9‐3″ amide (5), a seventeen amino acid peptide has 100% and 80% α‐helical contents at 1.7 × 10−4 M and 1.7 × 10−5 M, respectively. Suc‐[Trp9]MAP2‐11 amide (3), merely ten amino acids in length, is 51% α‐helical at 1.7 × 10−4 M in 0.1 M phosphate buffer at room temperature. In the presence of lipid or trifluoroethanol, the α‐helical content of these peptides is increased. This series of peptides demonstrates the complimentarity of various secondary structure design principles and the extent to which structure can be induced in small linear peptides.
Species‐specific variation in signal peptide design Implications for protein secretion in foreign hosts
Authors: von Heijne Gunnar , Abrahmsèn Lars
Published: July 2002

Secretory signal peptides from individual prokaryotic and eukaryotic species have been analyzed, and the lengths and amino acid compositions of the positively charged amino‐terminal region, the central hydrophobic region, and the carboxy‐terminal cleavage‐region have been compared. We find distinct differences between species in all three regions. Implications for protein secretion in foreign hosts are discussed.
Correlation between the OH bond dissociation energies of ellipticine hydroxylated derivatives and their cytotoxic properties
Authors: Rousseau-Richard C. , Auclair C. , Richard C. , Martin R.
Published: July 2002

The O−H bond dissociation energies (BDEs) of ellipticine‐derived hydroxylated compounds have been evaluated by a kinetic method and are shown to correlate with the cytotoxic activity of these derivatives.
New chloride‐activated aminopeptidase from human erythrocytes
Authors: Abramić Marija , Vitale Ljubinka
Published: July 2002

The presence of 0.2 M Cl− (Br−, F−) caused a several‐fold increase in the isolated aminopeptidase activity.
Targeting of a foreign protein into the thylakoid lumen of pea chloroplasts
Authors: Meadows Julie W. , Shackleton Jamie B. , Hulford Andrew , Robinson Colin
Published: July 2002

A chimaeric gene was constructed which encodes the pre‐sequence of the 33 kDa protein of the oxygen‐evolving complex, a thylakoid lumen protein, linked to dihydrofolate reductase, a cytoplasmic protein. The encoded fusion protein is transported into the thylakoid lumen of isolated pea chloroplasts, with concomitant removal of the pre‐sequence.
Chloride binding to photosystem II in the dark is in slow exchange
Authors: Shachar-Hill Yair , Beck Warren F. , Brudvig Gary W.
Published: July 2002

We have studied the 35Cl− NMR line broadening in the presence of photosystem II (PS II) membranes from spinach in the dark. In the presence of NH3 (which other work has shown to competitively inhibit chloride binding to PS II) we observed no decrease in 35Cl− linewidths. We conclude that binding of Cl− to the O2 evolving center in PS II in the dark (previously demonstrated by EPR) is in slow exchange on the NMR timescale. We assign the observed line broadening to interaction with non‐specific binding sites and with free paramagnetics.
Expression of the cDNA encoding lipocortin‐like 39 kDa protein of guinea pig neutrophils in yeast
Authors: Sato Eisuke F. , Tanaka Yoshikazu , Edashige Keisuke , Kobuchi Hirotsugu , Morishita Seiji , Sugino Yasuo M. , Inoue Masayasu , Utsumi Kozo
Published: July 2002

The cDNA encoding lipocortin‐like 39 kDa protein in guinea pig neutrophils was cloned into a yeast expression vector and the constructed plasmid was introduced into a yeast. The gene was expressed in an eukaryotic cell, yeast Saccharomyces
cerevisiae and the recombinant protein was purified and characterized. The purified protein was identical with the native one with respect to the antigenicity and several biochemical properties, such as inhibitory action against phospholipase A2, Ca2+‐dependent binding to acidic‐phospholipids and F‐actin and availability as a substrate for tyrosine kinase (EGF receptor/kinase) and protein kinase C.
A new proalbumin variant: Albumin Jaffna (−1 Arg→Leu)
Authors: Galliano Monica , Minchiotti Lorenzo , Stoppini Monica , Tàrnoky Andràs L.
Published: July 2002

Albumin Jaffna is an electrophoretically slowly moving genetic variant of human serum albumin found in two members of a Tamil family from Jaffna (Northern Sri Lanka), both heterozygous for the abnormal protein. Sequential analysis of albumin Jaffna, purified from serum by ion exchange chromatography on DEAE Sephadex and Mono Q columns, revealed that this variant is a new abnormal proalbumin, arising from a −1 Arg→Leu substitution, which prevents the proteolytic removal of the N‐terminal hexapeptide and allows the mutated proalbumin to enter the circulation. The presence of two additional positive charges is in keeping with the decreased electrophoretic mobility of albumin Jaffna, as well as with its isoelectric point of 5.01, determined by chromatofocusing on a Mono P column. The variant is selectively cleaved by trypsin in vitro, leaving leucine −1 as N‐terminal residue.
Evidence for the involvement of carboxyl groups in passive calcium uptake by liver plasma membrane vesicles and in agonist‐induced calcium uptake by hepatocytes
Authors: Karjalainen Ari , Bygrave Fyfe L.
Published: July 2002

The hydrophobic reagents DCCD and EEDQ, each of which reacts with protein carboxyl groups, were found to inhibit both passive Ca2+ uptake by plasma membrane vesicles isolated from rat liver and agonist‐induced Ca2+ uptake by hepatocytes. The data raise the possibility that the Ca2+ inflow pathway(s) in liver has a specific requirement for a reactive carboxyl group or groups.
Appearance of a late stage during mammalian DNA replication when cells resume formation of 10 kb DNA replication intermediates
Authors: Lönn Ulf , Lönn Sigrid , Nylen Urban , Winblad Gerard
Published: July 2002

After the joining of human large DNA replication intermediates and before the appearance of mature chromatin DNA, there exists a distinct stage — ‘the post‐elongation stage’. This stage reappears during recovery of DNA synthesis simultaneously with the reappearance of a large DNA replication intermediate, 10 kb DNA.
Thyroid hormone stimulates expression of 6‐phosphofructo‐2‐kinase in rat liver
Authors: Wall Stephen R. , van den Hove Marie-France , Crepin Karine M. , Hue Louis , Rousseau Guy G.
Published: July 2002

The activity of liver 6‐phosphofructo‐2‐kinase (PFK‐2), the enzyme that catalyses the synthesis of fructose 2,6‐bisphosphate, was markedly decreased in hypothyroid rats and partially restored after 3 days of treatment with triiodothyronine. The changes in PFK‐2 activity were accompanied by parallel changes in enzyme content measured by immunotitration and in PFK‐2 mRNA determined by dot blot and Northern blot hybridization with cDNA probes. It is concluded that thyroid hormone stimulates liver PFK‐2 gene expression by a pre‐translational mechanism.
Insertion of short hepatitis virus A amino acid sequences into poliovirus antigenic determinants results in viable progeny
Authors: Sverdlov E.D. , Tsarev S.A. , Markova S.V. , Rostapshov V.M. , Azhikina T.L. , Chernov I.P. , Gorbalenya A.E. , Kolesnikova M.S. , Romanova L.I. , Teterina N.L. , Tolskaya E.A. , Agol V.I.
Published: July 2002

In an infectious poliovirus cDNA construct, the determinant encoding antigenic epitope N‐Agl (in a loop located between two β‐strands in polypeptide VP1) was altered by site‐directed mutagenesis, to be partially similar with the determinants for presumptive epitopes in polypeptides VP1 or VP3 of hepatitis A virus (HAV). The modified constructs proved to be infectious. However, another construct, in which the same locus encoded a ‘nonsense’ and a relatively hydrophobic amino acid sequence, exhibited no infectivity. These data showed the feasibility of the insertion of foreign sequences in a specific antigenically active locus of the poliovirus icosahedron, and suggest some limitations with respect to the sequences to be ‘transplanted’.
Human Na+, K+ ‐ATPase genes
Authors: Ushkaryov Yu.A. , Monastyrskaya G.S. , Broude N.E. , Nikiforova N.N. , Bessarab D.A. , Orlova M.Yu. , Petrukhin K.E. , Modyanov N.N. , Sverdlov E.D.
Published: July 2002

The existence of a chromosome gene family containing at least one gene and one pseudogene was shown for the Na+,K+‐ATPase β‐subunit. A partial structure of the βI‐gene was determined, the coding part of which was completely homologous to cDNA of the Na+ K+‐ATPase βI‐subunit from HeLa cells [8]. The region encoding the putative protein transmembrane domain was shown to be bordered by two introns. The structure of a pseudogene (βψ) was determined. This pseudogene is processed and contains multiple stop codons. Its homology to the βI‐subunit cDNA from HeLa cells is about 88%.
A 43 kDa form of the GTP‐binding protein Gi3 in human erythrocytes
Authors: Carty Donna J. , Iyengar Ravi
Published: July 2002

Purified preparations of human erythrocyte G‐proteins contain a 43 kDa pertussis toxin substrate which appears to be the α‐subunit of a heterotrimeric GTP‐binding protein. The 43 kDa protein is recognized by antisera that are sequence‐specific for peptides encoding a sequence common to all 39–53 kDa G‐protein α‐subunits. Gαo‐specific antiserum did not recognize 43 or 40–41 kDa α‐subunits. AS/6, which recognizes the αi proteins, recognized 43 kDa as well as 40–41 kDa proteins. Of the three antisera specific for individual members of the αi family, only the Gi3‐specific antiserum recognized the 43 kDa erythrocyte G‐protein. However, 40–41 kDa forms of all three αis are present. These observations indicate that human erythrocytes contain a novel 43 kDa form of Gi3.
Could domain movements be involved in the mechanism of trypsin‐like serine proteases?
Authors: Dufton M.J.
Published: July 2002

It is hypothesised that the characteristic twin domain structure of serine proteases permits important allosteric responses in the molecule when peptide and protein substrates bind. Such movement would be ideal for stressing the scissile bond in the substrate, thereby making the task of hydrolysis substantially easier. The control of the domain movement can be closely associated with substrate binding, via the N‐ and C‐terminal regions of the enzyme. The hypothesis also suggests that certain inhibitory peptides exert their effect by binding without inducing the domain movement.
Modelling of poliovirus
Authors: Crabbe M.James C. , Evans David J. , Almond Jeffrey W.
Published: July 2002

We have used laboratory‐based molecular modelling to identify structural features of antigen chimaeras of poliovirus expressing epitopes from human immunodeficiency virus (HIV‐1) that may affect virus viability. Chimaeras were constructed by replacement of antigenic site 1 of VP1 by sequences corresponding to epitopes from HIV‐1. Loop volume, estimated by approximating the loop to an ellipsoid was significantly (P < 0.001) lower in viable (2062.1 Å3 ± 400.2) than in non‐viable (3617 Å3 ± 650.7) constructs. Our results suggest that viable virus will only be formed when antigen chimearas modified at antigenic site of VP1 have a loop occupying a similar volume in space to that occupied by the antigenic site 1 loop. In addition, the modified loop must fit with the peptide bond angles and distances at the top of the β‐barrel of VP1.
Anion binding to the chloride pump, halorhodopsin, and its implications for the transport mechanism
Authors: Lanyi Janos K. , Duschi Albert , Váro György , Zimányi László
Published: July 2002

The light‐driven chloride pump, halorhodopsin, binds and transports chloride across the membrane, and to a lesser extent nitrate. Binding and transport kinetics, and resonance Raman spectra of the retinal Schiff base, with these anions suggest the existence of two mutually exclusive binding sites. One of these may be the uptake site, and the other the release site during the transport. Plausible locations can be suggested for these sites, because halorhodopsin is a small protein with few buried positively charged residues, and the primary structure of a second pigment with similar function has recently become available for comparison.
Reduction of adenylyl cyclase activity by cholera toxin in myeloid cells
Authors: Hermouet S. , Milligan G. , Lanotte M.
Published: July 2002

In IPC‐81 cells, the adenylyl‐cyclase activation by cholera toxin produces an elevation of cAMP that causes a rapid cytolysis. A resistant clone with deficient cholera toxin‐induced cyclase activity (yet sensitive to cAMP) showed a rapid decrease in the amount of membrane‐bound Gsα (42–47 kDa) detectable soon after ADP‐ribosylation of these proteins; pertussis toxin‐sensitive G proteins (41 kDa) were not affected. Resistant cells showed a rapid decrease of Gsα that is consistent with the finding that cAMP did not accumulate in these cells. Cholera toxin treatment of resistant cells had long‐lasting effects (several weeks) on the level of Gsα in the cell membrane. The duration of Gsα decrease does not correspond to the probable life of catalytically active cholera toxin in the cells, and suggests a regulated process more complex than a proteolytic degradation targeted on ADP‐ribosylated molecules.
Investigation of the substrate specificity of thylakoid protein kinase using synthetic peptides
Authors: White I.R. , O'Donnell P.J. , Keen J.N. , Findlay J.B.C. , Millner P.A.
Published: July 2002

Synthetic peptide analogues of the N‐terminal region of the light harvesting chlorophyll a/b binding polypeptide of photosystem II (LHC II) were used to probe the effect of charged groups on the protein kinase activity of pea (Pisum sativum) thylakoid membranes. The effectiveness of the synthetic peptides as substrates for protein kinase activity or as inhibitors of LHC II phosphorylation was correlated with their net positive charge, which ranged between +2 and +5. The effects of the synthetic peptides on phosphorylation of other, non‐LHC II, thyakoid polypeptides are also discussed.
Identification of G protein‐coupled receptors by RNase H‐mediated hybrid depletion using Xenopus laevis oocytes as expression system
Authors: Meyerhof Wolfgang , Richter Dietmar
Published: July 2002

A method has been developed for rapidly identifying putative G protein‐coupled receptors isolated initially as small cDNA fragments, following reverse transcription and polymerase chain reaction (PCR) amplification of mRNA. The method is based upon the use of synthetic oligonucleotides deduced from the sequence of the amplified receptor fragments, to direct a RNase H‐mediated specific degradation of hybrids formed between the oligonucleotides and the corresponding receptor‐encoding mRNA. Loss of an agonist‐dependent receptor response in the Xenopus laevis oocyte expression system identifies the amplified receptor fragment. Taking in vitro synthesised serotonin HT2‐receptor (SR)‐encoding mRNA as a model, it was shown that following incubation with RNase H and SR antisense oligonucleotides, injection of this message no longer caused the acquisition of agonist‐dependent membrane currents in voltage‐clamped oocytes. In contrast, when corresponding sense oligonucleotides were used, the serotonin‐evoked membrane responses in oocytes were acquired as normal. The method should allow the identification of receptors which can functionally be expressed and measured in Xenopus oocytes.
Characterization of human deoxycytidine kinase correlation with cDNA sequences
Authors: Eriksson Staffan , Cederlund Ella , Bergman Tomas , Jörnvall Hans , Bohman Christina
Published: July 2002

Existing data on the structure of human deoxycytidine kinase (dCK) diverge. A monomeric 60 kDa form has been isolated and the cloning of a cDNA coding for 626 amino acids corresponding to a 71 kDa protein has been reported. However, pure dCK isolated from leukemic spleen is a dimer of 30 kDa subunits. Amino acid sequences of peptides from digests of this protein are now presented. None of the peptide structures obtained correspond to the cDNA for the 71 kDa protein, but to a cDNA for a 30.5 kDa dCK recently cloned. Furthermore, homology of the peptide sequences of dCK to parts of thymidine kinases and protein‐tyrosine kinases are detected.
Calcium is sufficient but not necessary for activation of sheep platelet cytosolic phospholipase A2
Authors: Zupan Lori A. , Kruszka Kelly K. , Gross Richard W.
Published: July 2002

In this study we demonstrate that: (1) although the major phospholipase A2 present in sheep platelets is activated by calcium ions, it can effectively catalyze hydrolysis of the sn‐2 ester linkage in phospholipids in the absence of calcium: (2) expression of calcium‐independent phospholipase A2 activity can be induced by NACl utilizing purified (but not crude) cytosolic enzyme; and (3) calcium‐independent phospholipase A2 activity is regulated by a reconstitutable cytosolic protein. Collectively, these results underscore the fundamental catalytic differences between extracellular and intracellular calcium‐dependent phospholipases A2 and demonstrate that calcium is sufficient, but not necessary, for the activation of this class of intracellular phospholipases A2
Inhibition of trypsin by the β‐amyloid protein precursor
Authors: Delvaux A. , Van der Elst L. , Octave J.-N.
Published: July 2002

Soluble β‐amyloid protein precursors (β‐APPs) were studied in human brain and cerebrospinal fluid (CSF) after partial purification by ion exchange chromatography. Proteins were analysed in immunoblotting experiments using a monoclonal antibody directed against the N‐terminal segment or the β‐APP 770, and by reverse enzymography. In the human brain and CSF, a protein which comigrates with the β‐APP 770 expressed by transfected CHO cells was able to inhibit trypsin.
The kinetics of a two‐state transition of myosin subfragment 1 A temperature‐jump relaxation study
Authors: Lin Shwu-Hwa , Cheung Herbert C.
Published: July 2002

Temperature‐jump measurements were carried out on myosin subragment 1 (S1) labeled at Cys‐707 with 5‐(iodoacetamido)fluorescein (S1‐AF). The relaxation was monitored by following the increase in the fluorescence intensity of the attached probe after a jump of 5.8°C. A single relaxation process was observed over a range of final temperatures, and the relaxation time decreased from 16.69 ms at 15°C to 3.91 ms at 27°C. The relaxation results are interpreted in terms of a two‐state transition: (S1‐AF)L (S1‐AF)11, and the observed single relaxation time (τ) equals 1/(k
+ + k
−). The individual first‐order rate constants, k
+ and k
−, were calculated from τ and the equilibrium constant previously determined. The activation energy was 21.9 kcal/mol for the forward reaction and 9.3 kcal/mol for the reverse reaction, corresponding to an enthalpy value of 12.6 kcal/mol for the two‐state transition. The results provide, for the first time, direct kinetic evidence of a two‐state transition of S1 in the absence of bound nucleotide, and support a two‐state model of unliganded myosin subfragment 1.
Reduced puromycin sensitivity of translocated polysomes after the addition of elongation factor 2 and non‐hydrolysable GTP analogues
Authors: Nilsson Lars , Nygård Odd
Published: July 2002

Treatment of reticulocyte polysomes with elongation factor cEF‐2 and GTP led to an increased sensitivity or peptidyl‐tRNA for puromycin as a result of the translocation from the ribosomal A‐site to the P‐site, upon addition of an excess of the non‐hydrolysable GTP analogue, GuoPP(CH2]P, the puromycin sensitivity decreased rapidly, The decrease in sensitivity required high concentrations of cEF‐2 with half maximal effect at an cEF‐2 concentration of around 1 μM. The data suggest either that peptidyl‐tRNA had re‐translocated back to the A‐site due to the higher affinity of cEF‐2 for the pre‐translocation than for the post‐translocation ribosome, or that the cEF‐2‐GuoPP[CH2]P complex blocks the peptidyl‐transferase activity.
Thermal injury and ozone stress affect soybean lipoxygenases expression
Authors: Maccarrone Mauro , Veldink Gerrit A. , Vliegenthart Johannes F.G.
Published: July 2002

The effects of thermal injury (cold and heat shock) and ozone treatment on lipoxygenases 1 (LOX‐1) and 2 (LOX‐2) of soybean seedlings have been investigated. Cold stress led to a decrease of the specific activities of both isoenzymes, attributable at least in part to a down‐regulation of gene expression at the translational level. Both heat shock and ozone treatment enhanced lipoxygenases‐specific activities, acting at the level of transcription of the genes. It is proposed that LOX‐1 and LOX‐2 are involved in the thermotolerance of soybeans and in the precocious aging induced by ozone.
Pulsed electric current enhances the phorbol ester induced oxidative burst in human neutrophils
Authors: Bobanović Fedja , Simc̆ic̆ Sas̆a , Kotnik Vladimir , Vodovnik Lojze
Published: July 2002

Oxidative burst (OB) response in human neutrophils, measured with chemiluminescence (CL), has been used to determine whether pulsed electric current (PEC) might induce a functional response in these electrically nonexcitable cells, and also whether it might modify cellular response to tumor‐promoting phorbol ester (PMA). Five minutes of PEC treatment caused no significant changes in neutrophil CL levels in HBSS (1.2 mM Ca2+ concentration) as well as in HBSS‐EGTA, where the extracellular Ca2+ concentration was reduced to less than 30 nM. The CL level of PMA‐activated neutrophils in HBSS was 52% higher than in HBSS‐EGTA. In HBSS the CL level, after the combined PMA and PEC treatment, was 53% higher than in PMA‐alone‐treated neutrophils. Activation of the OB in HBSS‐EGTA with PMA and PEC was 13% higher than in solely PMA treated neutrophils. The results suggest that in neutrophil OB response, the PEC effect is closely related with cellular calcium mobilization, since depletion of extracellular Ca2+ decreased the PEC effect.
The KH domain occurs in a diverse set of RNA‐binding proteins that include the antiterminator NusA and is probably involved in binding to nucleic acid
Authors: Gibson Toby J. , Thompson Julie D. , Heringa Jaap
Published: July 2002

New findings are presented for the ~ 50 residue KH motif, a domain recently discovered in RNA‐binding proteins. The conserved sequence is ~ 10 residues larger than previously reported. Profile searches have revealed new members of this family, including two, E. coli NusA and human GAP‐associated p62 phosphoprotein, for which RNA‐binding data exists. A nus A homolog was detected in the RNA polymerase gene complex of six archaebacterial species and may encode an antiterminator. All KH‐containing proteins are linked with RNA and the KH motif most probably functions as a nucleic acid binding domain.
The conformation of tRNA genes
Authors: McLaren A. , Gonos E.S. , Carr T. , Goddard J.P.
Published: July 2002

It has been suggested that eukaryotic tRNA genes might adopt a higher order stem and loop structure to facilitate transcription by interaction of their variably spaced intragenic promoter blocks. Using sodium bisulphite, which reacts specifically with cytosine residues in single‐stranded nucleic acids, no deamination of C in the TTCGAA sequence of the 3' ICR of a tRNALeu gene could be detected under conditions which caused 60% deamination of cytosine residues within the loop region of a synthetic cruciform cloned in the same negatively supercoiled plasmid vector. We conclude that, under these conditions, such structures occur in tRNA genes very rarely, if at all.
A Glu329→ Gln variant of the α‐subunit of the rat kidney Na+K+‐ATPase can sustain active transport of Na+ and K+ and Na+K+‐activated ATP hydrolysis with normal turnover number
Authors: Vilsen Bente
Published: July 2002

An allelic variant of the ouabain‐insensitive rat kidney Na+, K+‐ATPase α1‐isoform was identified by chance in a cDNA library. The variant differed from the wild‐type rat kidney Na+, K+‐ATPase by a single G‐to‐C base substitution in the cDNA, which on amino acid level gave rise to a glutamine in place of the glutamate residue Glu329 previously suggested as a likely donator of oxygen ligands for Na+ and K+ binding. The variant cDNA was transfected into COS‐1 cells and the transfectants expanded with success into stable cell lines that were able to grow in the presence of a concentration of ouabain highly cytotoxic to the parental cells containing only the endogenous COS‐1 cell Na+K+‐ATPase. Under these conditions, the viability of the cells depended on the cation transport mediated by the ouabain‐insensitive Glu329→ Gln variant, whose cDNA was shown by polymerase chain reaction amplification to be stably integrated into the COS‐1 cell genome. The maximum specific ATP hydrolysis activity of isolated plasma membranes of the Glu329→ Gln variant did not differ significantly from that of plasma membranes containing the wild type. A method was established for measurement of the phosphorylation capacity of the expressed Glu329→ Gln variant and wild‐type enzyme, and it was thereby demonstrated that the variant had a turnover number similar if not identical to that of the wild‐type.
Degradation of human apolipoprotein B‐100 by apolipoprotein(a)
Authors: Chulkova T.M. , Tertov V.V.
Published: July 2002

Human plasma low density lipoproteine (LDL) contain a very high molecular weight protein termed apoB‐100 (M
r = 550,000). In many samples of LDL, minor components designated as apoB‐74 (M
r = 407,000) and apoB‐26 (M
r = 145,000) are present. It has been shown that they can arise as a result of proteolytic degradation of apoB‐100. Our earlier studies demonstrated that the active forms of lipoprotein(a) (LP(a)) and apolipoprotein(a) (apo(a)) possess proteolytic activity. In the present study we investigated the possibility of apoB‐100 degradation in the presence of activated apo(a). LDL were incubated with the purified apo(a) and analyzed by SDS‐polyacrylamide gel electrophoresis. It was found that apoB is cleaved by apo(a) with formation of proteolytic fragments including B‐74 and B‐26. The physiological significance of apoB degradation under the action of apo(a) is considered.
Observation of inter‐subunit nuclear Overhauser effects in a dimeric protein
Authors: Burgering M.J.M. , Boelens R. , Caffrey M. , Breg J.N. , Kaptein R.
Published: July 2002

For the structure determination of symmetric protein dimers it is necessary to distinguish between intra‐ and inter‐subunit NOEs. A method is presented to measure selectively the inter‐subunit NOEs using uniform 15N and 13C isotope labelling. This is accomplished by doubly filtered 2D NOE experiments on mixtures of native protein with isotope‐labeled protein. The method has been applied to the Arc represser and allows the characterization of virtually all proton‐proton NOEs in terms of their intra‐ or inter‐subunit nature.
Cis‐acting elements in the promoter region of the human aldolase C gene
Authors: Buono Pasqualina , de Conciliis Lisa , Olivetta Eleonora , Izzo Paola , Salvatore Francesco
Published: July 2002

We investigated the cis‐acting sequences involved in the expression of the human aldolase C gene by transient transfections into human neuroblastoma cells (SKNBE). We demonstrate that 420 bp of the 5'‐flanking DNA direct at high efficiency the transcription of the CAT reporter gene. A deletion between − 420 bp and − 164 bp causes a 60% decrease of CAT activity. Gel shift and DNase I footprinting analyses revealed four protected elements: A, B, C and D. Competition analyses indicate that Spl or factors sharing a similar sequence specificity bind to elements A and B, but not to elements C and D. Sequence analysis shows a half palindromic ERE motif (GGTCA), in elements B and D. Region D binds a transactivating factor which appears also essential to stabilize the initiation complex.
Sec‐independent protein insertion into the inner E. coli membrane A phenomenon in search of an explanation
Authors: von Heijne Gunnar
Published: July 2002

Translocation of proteins through the inner membrane of E. coli is normally catalyzed by the so‐called sec‐machinery. Yet, many integral inner membrane proteins appear not to require a fully functional sec‐machinery for proper insertion, in spite of the fact that sometimes quite sizable domains have to be translocated to the periplasmic side. This review will focus on recent studies of sec‐independent translocation events in an attempt to pin‐point the main differences between sec‐dependent and sec‐independent translocation.
Apolipoprotein oxidation in the absence of lipid peroxidation enhances LDL uptake by macrophages
Authors: Hunt James V. , Bailey James R. , Schultz Donna L. , McKay Alan G. , Mitchinson Malcolm J.
Published: July 2002

A characteristic of the antioxidant, probucol, is its inability to inhibit apolipoprotein B fragmentation in low density lipoprotein (LDL), despite a pronounced ability to inhibit lipid oxidation on relatively lengthy exposure to Cu(II). Here we show that a short exposure of LDL to hydrogen peroxide and Cu(II) leads to 125I‐labelled apolipoprotein B fragmentation, the production of malondialdehyde and hydroperoxides and leads to increased uptake by macrophages on subsequent culture. However, pre‐loading LDL with probucol protects LDL from lipid oxidation but not protein fragmentation or macrophage uptake. The use of probucol to conduct studies on apolipoprotein B oxidation without extensive lipid oxidation may prove useful when studying LDL apolipoprotein damage on exposure to an aqueous free radical insult.
On the mobility behavior of a curved DNA fragment located in circular permutations
Authors: Nair T.Murlidharan , Madhusudan K. , Nagaraja V. , Kulkarni B.D. , Majumdar H.K. , Singh Rajan
Published: July 2002

Experimental and theoretical investigations on the mobility behavior of a set of permuted fragments with a K‐DNA insert is reported. The fragments with the permuted flanking sequences have the K‐DNA insert located differentially with respect to the fragment ends. The fragment wherein the insert is located in the center showed maximum retardation as compared to fragments where the insert was at the end. The experimental analysis is also in accord with the theoretical investigation.
Inhibition of fatty acid desaturation in sycamore cells deprived of iron
Authors: Pascal Nadine , Dome Albert-Jean
Published: July 2002

The growth of isolated sycamore cells in a medium devoid of iron induced a marked reduction of the unsaturation level of fatty acids: the proportion of linolenic acid (C18:3) in polar lipids (phospholipids and galactolipids) decreased whereas a parallel increase in the proportions of oleic (C18:1) and linoleic (C18:2) acid was observed. In our experimental conditions, no direct effect of iron deprivation on fatty acid and glycerolipid biosynthesis could be observed. When sycamore cells were incubated in the presence of [14C]acetate, the level of unsaturation in fatty acids was very strongly reduced: no polyunsaturated fatty acids were synthesized in iron‐deprived cells: only [14C]palmitic and [14C]oleic acids accumulated in glycerolipids. In contrast, sycamore cells grown in an iron‐containing medium in the presence of [14C]acetate were able to synthesize glycerolipids containing 14C‐labelled C18:2 and C18:3. We concluded that, in sycamore cells, iron is essential for C18:1 → C18:2 → C18:3 desaturations. In contrast, C18:0 → C18:1 desaturation is much less sensitive to iron deprivation under our experimental conditions.
Structural differences in chlorosomes from Chloroflexus aurantiacus grown under different conditions support the BChl c‐binding function of the 5.7 kDa polypeptide
Authors: Lehmann Rainer P. , Brunisholz René A. , Zuber Herbert
Published: July 2002

Structurally different chlorosomes were isolated from the green photosynthetic bacterium Chloroflexus aurantiacus grown under different conditions. They were analysed with respect to variable pigment‐protein stoichiometries in view of the presumed BChI c‐binding function of the 5.7 kDa chlorosome polypeptide. Under high‐light conditions on substrate‐limited growth medium the pigment‐protein ratio of isolated chlorosomes was several times lower than under low‐light conditions on complex medium. Proteolytic degradation of the 5.7 kDa polypeptide in high‐light chlorosomes led to a 60% decrease of the absorbance at 740 nm. The CD spectrum of high‐light chlorosomes exhibited a sixfold lower relative intensity at 740 nm (ΔA/A
740) than low‐light chlorosomes, but it showed a fivefold increase in intensity upon degradation of the 5.7 kDa polypeptide compared to a twofold increase in low‐light chlorosomes. It seems probable that BChl c in the chlorosomes is present as oligomers bound to the 5.7 kDa polypeptide. Our data suggest further that compared to low‐light chlorosomes smaller oligomers or single BChl c molecules are bound to the 5.7 kDa polypeptide in high‐light chlorosomes resulting in lower rotational strength.
Activated Gsα but not Giα prevents the thermal inactivation of adenylyl cyclase in plasma membranes derived from S49 lymphoma cells
Authors: Kvapil Petr , Novotny Jiri , Ransnäs Lennart A.
Published: July 2002

The thermal inactivation of adenylyl cyclase was studied in plasma membranes isolated from wild‐type and the mutant cell strain cyc− of S49 lymphoma. The half‐life of adenylyl cyclase activity at 30°C was decreased from 14.2 min to 3.4 min by the presence of detergents. ATP as well as forskolin prevented the adenylyl cyclase inactivation in a dose‐response manner independent of the utilized type of cell membranes. Activation of G‐proteins by GTPγS or by AlF−
4 in wild‐type membranes but not in cyc− membranes partially prevented adenylyl cyclase inactivation. Adenylyl cyclase activity in cyc− membranes was preserved in the presence of GTPγS or AlF4 from the observed detergent‐induced inactivation by complementation of these membranes with an extract from wild‐type membranes. ADP‐ribosylation of Giα in a cyc− membranes did not influence the kinetics of the inactivation process of adenylyl cyclase, whereas ADP‐ribosylated Gsα, protein protected adenylyl cyclase more effectively than non‐ribosylated Gsα in wild‐type plasma membranes when GTP was used as an activator.
A stress‐associated citrus protein is a distinct plant phospholipid hydroperoxide glutathione peroxidase
Authors: Beeor-Tzahar Talia , Ben-Hayyim Gozal , Holland Doron , Faltin Zehava , Eshdat Yuval
Published: July 2002

A protein whose level is markedly increased upon exposure of cultured citrus cells and whole plants to NaCl, was shown to specifically catalyze the reduction of phosphatidylcholine hydroperoxide in the presence of glutathione. This enzymatic activity was shown to be independent of a similar activity exhibited by glutathione S‐transferase in plants. This finding corroborates the significant homology (52%) accounted between the deduced amino acid sequence of the gene encoding for this protein and that of mammalian phospholipid hydroperoxide glutathione peroxidases. While the mammalian enzyme is known and well investigated, this study establishes the presence of this key protein also in plants.
Role of base‐backbone and base‐base interactions in alternating DNA conformations
Authors: Suzuki Masashi , Yagi Naoto , Finch John T.
Published: July 2002

Sequence‐specific conformational differences between dinucleotide steps are characterised using published crystal coordinates with special attention to steric hindrance of the methyl group of a T base to the neighbouring base, and, more importantly, to the sugar‐phosphate backbone. The TT step is inflexible and B‐like, as it has two methyl groups which interlock with each other and with the sugar‐phosphate backbones. AT slides, or overtwists, so that the methyl groups move away from the backbones, both lead the step towards the A‐conformation. TA is most flexible as it does not have such restriction. These characteristics are observed with other pyrimidine‐pyrimidine, pyrimidine‐purine, purine‐pyrimidine steps, respectively, but to less extent, depending on the number of non‐A: T basepairs in the steps.
Exciton delocalization in the antenna of purple bacteria: Exciton spectrum calculations using X‐ray data and experimental site inhomogeneity
Authors: Dracheva Tatiana V. , Novoderezhkin Vladimir I. , Razjivin Andrei P.
Published: July 2002

Electron absorption and circular dichroism spectra of the peripheral light‐harvesting complex (LH2) of photosynthetic purple bacteria were calculated taking into account the real‐life spatial arrangement and experimental inhomogeneous broadening of bacteriochlorophyll molecules. It was shown that strong excitonic interactions between 18 bacteriochlorophyll molecules (BChl850) within the circular aggregate of the LH2 complex result in an exciton delocalization over all these pigment molecules. The site inhomogeneity (spectral disorder) practically has no influence on exciton delocalization. The splitting between two lowest exciton levels corresponds to experimentally revealed splitting by hole‐burning studies of the LH2 complex.
Regulation of transcription in mammalian cells by yeast Leu3p and externally supplied inducer
Authors: Guo Hui , Kohlhaw Gunter B.
Published: July 2002

The Leu3 protein of yeast is a dual‐function regulator, stimulating transcription when the inducer α‐isopropylmalate (α‐IPM) is present and suppressing transcription when the inducer is absent. Here we show that Leu3p retains both its positive and negative regulatory properties when expressed in mammalian cells or when added to a mammalian nuclear extract. α‐IPM stimulates reporter gene expression 15–20‐fold, both in vivo and in vitro. The concentration of α‐IPM required for half‐maximal stimulation in vitro is 2.5 × 10−4 M. No yeast‐specific factors other than Leu3p itself are required for up‐ or down‐regulation. Since α‐IPM is not metabolized in mammalian cells, the Leu3p‐α‐IPM system might be useful in gene therapy and other studies as a highly specific, externally controlled on/off switch of gene expression.
Fungal ferritins: The ferritin from mycelia of Absidia spinosa is a bacterioferritin
Authors: Carrano Carl J. , Böhnke Rudi , Matzanke Berthold F.
Published: July 2002

Two distinct ferritin like iron containing proteins have been identified and isolated from the fungus Absidia spinosa; one from the spores and another from the mycelia. The mycelial protein has been purified and consists of two subunits of approx. 20 kDa. The N‐terminal sequences of both subunits have been determined. The holoprotein as isolated contains approx. 750 iron atoms/molecule and exhibits a heme‐like UV‐Vis spectrum. Based on the heme spectrum and the high degree of sequence homology found, it has been established that the mycelial protein is a bacterioferritin. This is the first example demonstrating the presence of a bacterioferritin in a eukaryotic organism.
Seasonal concordance of recurrence in mood disorder patients
Authors: Barbini B , Di Molfetta D , Gasperini M , Manfredonia MG , Smeraldi E
Published: July 2002

The aim of the present study is to evaluate the seasonal pattern of recurrences and the concordance among first episode (index episode) versus the subsequent recurrences in a sample of 210 patients affected by mood disorders, referred to the Mood Disorder Unit of San Raffaele Hospital. The most depressive recurrences are in spring for unipolar subjects and in fall for bipolars. Manic episodes are more frequent in summer. Patients presented a high concordance rate between the first and the second episode, female patients were more concordant than male subjects, and patients with low cycle of illness (one episode every six or more years) were the most concordant ones.
Involvement of histone H4 gene transcription factor 1 in downregulation of vimentin gene expression during skeletal muscle differentiation
Authors: Kryszke Marie-Hélène , Moura-Neto Vivaldo , Lilienbaum Alain , Paulin Denise , Auclair Christian
Published: July 2002

Upon in vitro myogenesis, the intermediate filament protein vimentin is replaced by desmin, the switch in gene expression occurring essentially at the transcriptional level. Trying to elucidate the molecular mechanisms of this genetic control, we show here that the vimentin promoter is specifically recognized and activated by a protein most probably identical to H4TF‐1, and that this factor is present in proliferating myoblasts but disappears upon fusion of these cells into multinucleated myotubes. Our results suggest that H4TF‐1 is a differentiation stage‐specific factor involved in the downregulation of vimentin gene expression during myogenesis.
Crystal structure of the bacterial cell division regulator MinD
Authors: Cordell Suzanne C , Löwe Jan
Published: July 2002

In bacterial cell division MinD plays a pivotal role, selecting the mid‐cell over other sites. With MinC, MinD forms a non‐specific inhibitor of division, that interacts with FtsZ. Specificity is provided by MinD's interaction with MinE at the mid‐cell. We have solved the crystal structure of MinD‐1 from Archaeoglobus fulgidus to 2.6 Å by multiple anomalous dispersion. MinD is a classic nucleotide binding protein, related to nitrogenase iron proteins, which have a fold of a seven‐stranded parallel β‐sheet, surrounded by α‐helices. Although MinD, unlike the proteins it interacts with and those it is structurally related to, is a monomer, not a dimer.
Functional identification of sterol‐4α‐methyl oxidase cDNAs from Arabidopsis thaliana by complementation of a yeast erg25 mutant lacking sterol‐4α‐methyl oxidation1
Authors: Darnet Sylvain , Bard Martin , Rahier Alain
Published: July 2002

Specific primers derived from both genomic sequence data and EST cDNA sequences were used to polymerase chain reaction amplify two full‐length cDNA sequences (AtSMO1 and AtSMO2), 801 and 783 bp, respectively, from an Arabidopsis thaliana cDNA library. The predicted proteins show 32 and 29% identity to the ERG25 gene from Saccharomyces cerevisiae which encodes the sterol‐4α‐methyl oxidase (SMO), a membrane‐bound non‐heme di‐iron oxygenase involved in lipid metabolism. Heterologous expression of AtSMO1 and AtSMO2 in a yeast erg25 ergosterol auxotroph, lacking SMO activity, restored growth and endogenous ergosterol synthesis. These results represent the first functional identification of SMO genes from plants.
Expression of stress proteins heme oxygenase‐1 and ‐2 in acute pancreatitis and pancreatic islet βTC3 and acinar AR42J cells
Authors: Sato Hideyo , Siow Richard C.M. , Bartlett Simon , Taketani Shigeru , Ishii Tetsuro , Bannai Shiro , Mann Giovanni E.
Published: July 2002

© 1997 Federation of European Biochemical Societies.