bluuebunny/crossref_metadata_2025 huggingface dataset:
Close vicinity of Lhc b1 and Lhc b4 in Photosystem II
Authors: Miao Jihong , Irrgang Klaus‐Dieter , Salnikow Johann , Franke Peter , Vater Joachim
Published: March 2003

The nearest neighbourhood of pigment‐protein complexes within Photosystem II (PSII) membrane fragments has been studied by means of chemical cross‐linking with o‐phthalaldehyde (OPA) in conjunction with protein‐chemical techniques. By means of OPA‐induced cross‐linking a major conjugate of about 60 kDa has been identified. This conjugate was shown to consist of two pigment‐protein complexes of light‐harvesting complex II (LHC II), Lhc b1 (CP27) and Lhc b4 (CP29) by means of SDS/PAGE in combination with an immunological analysis using mAbs directed against Lhc b4 and by matrix‐assisted‐laser‐desorption−ionization mass spectrometry (MALDI‐MS) and sequence analysis of peptides derived from a proteolytic digest of the conjugate. Domains of Lhc b1 and Lhc b4 have been localized to a distance of not more than 5 Å within LHC II. Our results are discussed in the light of recent models on the topography of the two subunits within the antenna system of Photosystem II.
Self‐association of the C‐terminal domain of the hepatitis‐C virus core protein
Authors: Yan Bo‐Shiun , Tam Ming H , Syu Wan‐Jr
Published: March 2003

The N‐terminal region of the hepatitis‐C virus (HCV) core protein is rich in basic residues, while the C‐terminal end of the protein comprises of a stretch of hydrophobic amino acids. Between these two extremes is an amphipathic region with two predicted α‐helical segments. This region embodies Leu or hydrophobic residues in positions of heptad repeats and is possibly capable of self‐association. To investigate this possibility, the core sequence was divided into two fragments and expressed separately as recombinant proteins. Recombinant proteins with the N‐terminal fragment remained as monomers even at high concentrations in SDS/PAGE. Recombinant protein with the C‐terminal fragment appeared largely monomeric on denaturing gels but some oligomers were also detected. Furthermore, proline mutations in either one of the predicted α helices adversely affected the observed oligomerization. The self‐association capacity of the core protein C‐terminal region was further supported by results from a yeast two‐hybrid system. To affirm our conclusion, a peptide covering the heptad repeats and the predicted α helices was synthesized. Data from mass spectrometry and gel‐filtration chromatography concluded that this peptide readily self‐associated into the homodimer. Therefore, our results suggest that the oligomerization motifs of the HCV core protein may not be limited to the previously suggested N‐terminal region.
An SP1‐like 5′‐GACCACGCC‐3′ sequence is critical for activity of the inflammatory phospholipase A2 promoter and binds several non‐zinc finger proteins
Authors: Paradon Michel , Salvat Colette , Fan Qishi , Bereziat Gilbert , Olivier Jean Luc
Published: March 2003

We have previously shown that the promoter of the type IIa secreted phospholipase A2 gene contains a strong positive regulatory proximal element [−125 to −85] element B. Mutation of this element abolishes the activation of the phospholipase A2 promoter by C/EBPβ in HepG2 cells. Liver nuclear proteins form three major and two minor complexes with this element. The [−107 to −99] 5′‐GACCACGCC‐3′ sequence is critical for the formation of these complexes and the activity of the promoter. Although the sequence of element B is highly similar to those of Sp1 binding sites, it does not bind Sp1 or other zinc‐finger proteins. Each major complex contains a single protein, the molecular masses of these proteins being 100, 90 and 75 kDa. These proteins have the same nucleotide requirements for binding, with the cytosines at positions −102, −100, −99 and the adenosine at −103 being the most important nucleotides. The activity of the phospholipase A2 promoter in HeLa cells was lower than in HepG2 cells, and was correlated with the absence of complex 3 in HeLa cell nuclear extracts. Our results suggest different roles for the proteins bound to the 5′‐GACCACGCC‐3′ sequence. In particular, the 75‐kDa protein which forms the third complex is critical for the activity of the promoter of the secretory phospholipase A2 gene.
Protein engineering of the restriction endonuclease EcoRV
Authors: Schöttler Sylvia , Wenz Christian , Lanio Thomas , Jeltsch Albert , Pingoud Alfred
Published: March 2003

We generated variants of the restriction endonuclease EcoRV that discriminate between recognition sites with different flanking sequences. This was achieved by designing new contacts to the bases in the major groove of the DNA preceding and following the EcoRV recognition site. We selected Ala181 as the starting point for the extension of the site specificity of EcoRV because, according to the structure of the specific EcoRV  DNA complex, this residue is involved in a water mediated contact with the bases flanking the recognition sequence on the 5′ side. A substitution of this alanine residue by other amino acid residues changes the protein‐DNA interface in this region and potentially creates new contacts, such that EcoRV variants could have an extended specificity, i.e. a greater selectivity for EcoRV sites within a particular sequence context. EcoRV variants with naturally occuring amino acid residues at position 181 were produced and their selectivity analyzed with oligodeoxynucleotide and plasmid substrates that differ only in the base pairs immediately flanking the EcoRV site. Some variants, having amino acid residues with long or bulky side chains at position 181 showed altered preferences for the base pairs flanking the recognition sequence with oligodeoxynucleotide substrates without loosing their catalytic efficiency. One variant, A181K, is able to discriminate between purine and pyrimidine bases on the 5′ side of the recognition sequence, probably by means of a new hydrogen bond to the N7 of the purine base. Another variant, A181E, strongly prefers a thymine base on the 5′ side of the recognition sequence, presumably due to a hydrogen bond formed between the protonated glutamic acid residue and the O4 of thymine.
Side‐chain selective and covalent labelling of proteins by organometallic complexes of heavy transition metals
Authors: Salmain Michèle , Gorfti Abdellaziz , Jaouen Gerard
Published: March 2003

Organo‐rhenium and organo‐tungsten N‐succinimidyl and N‐sulfosuccinimidyl esters react at amino groups of proteins to form stable amide bonds between the protein and the heavy transition metal complexes. We show here that factors such as pH of the reaction medium and nature of the reagent (coordinating metal, surrounding ligands, type of ester) had a marked influence on the final number of coupled organometallic groups per protein molecule, defined as the coupling ratio. By operating with stoichiometric quantities of reagent with respect to the number of amino groups, up to 30 organometallic groups were introduced into the model protein, bovine serum albumin (BSA). BSA conjugates were characterized by gel electrophoresis in non‐denaturating conditions. Hen egg‐white lysozyme organo‐tungsten conjugates were prepared in the same manner and the peptides resulting from their tryptic hydrolysis were analyzed by reverse‐phase HPLC. A tentative assignment of the preferential amino sites of conjugation is suggested from the latter results.
Mechanisms of control of heart glycolysis
Authors: Depré Christophe , Rider Mark H. , Hue Louis
Published: March 2003

This review focuses on the mechanisms of control of heart glycolysis under conditions of normal and reduced oxygen supply. The kinetic properties and the biochemical characteristics of control steps (glucose transporters, hexokinase, glycogen phosphorylase and phosphofructokinases) in the heart are reviewed in the light of recent findings and are considered together to explain the control of glycolysis by substrate supply and availability, energy demand, oxygen deprivation and hormones. The role of fructose 2,6‐bisphosphate in the control of glycolysis is analysed in detail. This regulator participates in the stimulation of heart glycolysis in response to glucose, workload, insulin and adrenaline, and it decreases the glycolytic flux when alternative fuels are oxidized. Fructose 2,6‐bisphosphate integrates information from various metabolic and signalling pathways and acts as a glycolytic signal. Moreover, a hierarchy in the control of glycolysis occurs and is evidenced in the presence of adrenaline or cyclic AMP, which relieve the inhibition of glycolysis by alternative fuels and stimulate fatty acid oxidation. Insulin and glucose also stimulate glycolysis, but inhibit fatty acid oxidation. The mechanisms of control underlying this fuel selection are discussed. Finally, the study of the metabolic adaptation of glucose metabolism to oxygen deprivation revealed the implication of nitric oxide and cyclic GMP in the control of heart glucose metabolism.
X‐ray crystal structure of the Desulfovibrio vulgaris (Hildenborough) apoflavodoxin‐riboflavin complex
Authors: Walsh Martin A. , McCarthy Andrew , O'Farrell Paul A. , McArdle Patrick , Cunningham P. Desmond , Mayhew Stephen G. , Higgins Timothy M.
Published: March 2003

The apoprotein of flavodoxin from Desulfovibrio vulgaris forms a complex with riboflavin. The ability to bind riboflavin distinguishes this flavodoxin from other short‐chain flavodoxins which require the phosphate of FMN for flavin binding. The redox potential of the semiquinone/hydroquinone couple of the bound riboflavin is 180 mV less negative than the corresponding complex with FMN. To elucidate the binding of riboflavin, the complex has been crystallized and the crystal structure solved by molecular replacement using native flavodoxin as a search model to a resolution of 0.183 nm. Compared to the FMN complex, the hydrogen‐bonding network at the isoalloxazine sub‐site of the riboflavin complex is severely disrupted by movement of the loop residues Ser58−Ile64 (60‐loop) which contact the isoalloxazine by over 0.35 nm, and by a small displacement of the isoalloxazine moiety. The 60‐loop movement away from the flavin increases the solvent exposure of the flavin‐binding site. The conformation of the site at which 5′‐phosphate of FMN normally binds is similar in the two complexes, but in the riboflavin complex a sulphate or phosphate ion from the crystallization buffer occupies the space. This causes small structural perturbations in the phosphate‐binding site. The flexibility of the 60‐loop in D. vulgaris flavodoxin appears to be a contributing factor to the binding of riboflavin by the apoprotein, and a feature that distinguishes the protein from other ’short chain' flavodoxins. In the absence of the terminal phosphate group, free movement at the 5′‐OH group of the ribityl chain can occur. Thus, the 5′‐phosphate of FMN secures the cofactor at the binding site and positions it optimally. The structural changes which occur in the 60‐loop in the riboflavin complex probably account for most of the positive shift that is observed in the midpoint potential of the semiquinone/hydroquinone couple of the riboflavin complex compared to that of the FMN complex.
A peptide isolated from a random phage peptide library is a structural mimic to the P1, P4‐diadenosine 5′‐tetraphosphate binding site on its receptor
Authors: Hilderman Richard H. , Liu Guang , Zimmerman James K.
Published: March 2003

We have previously demonstrated that a monoclonal antibody (mAb TL4), which inhibits P1, P4‐diadenosine 5′‐tetraphosphate (Ap4A) binding to its receptor, selected a consensus RGS tripeptide from a random phage hexapeptide library. RGS interfered with Ap4A binding to its membrane receptor [Liu, G., Bryant, R. T. & Hilderman, R. H. (1996) Biochemistry 35, 197−201]. However the mechanism by which RGS interfered with Ap4A binding to its receptor was not determined. In this communication, we demonstrate that RGS interacts with Ap4A to prevent [3H]Ap4A binding to the Ap4A membrane receptor. To further characterize the mechanism by which RGS inhibits Ap4A binding to its receptor, we used mAb TL4 to screen a 15‐residue random peptide phage library and DNAs from 24 clones were sequenced. 20 clones contain a RGSSS sequence while 17 of these clones contained an identical 15‐amino‐acid insert (clone A). Gel‐filtration studies of previously equilibrated clone A phage and [3H]Ap4A support the idea that [3H]Ap4A interacts specifically with clone A phage while RGS effectively competes for [3H]Ap4A interaction on clone A phage. These data are consistent with RGS mimicking a sequence on the receptor essential for Ap4A binding.
Structure/function correlation of spermine‐analogue‐induced modulation of peptidyltransferase activity
Authors: Karahalios Panagiotis , Mamos Petros , Karigiannis George , Kalpaxis Dimitrios L.
Published: March 2003

In a cell‐free system derived from Escherichia coli,various analogues of spermine were used to study their effect on the binding of AcPhe‐tRNA to poly (U)‐programmed ribosomes and on the puromycin reaction carried out at 6 mM Mg2+ (Ac, acetyl). In the absence of factors washable from ribosomes (FWR fraction), mono‐acylated or di‐acylated analogues of spermine stimulate the binding of AcPhe‐tRNA to a lesser degree than spermine, in the order : N1‐acetylspermine > N1,N12‐diacetylspermine ≅N1,N12‐dipivaloylspermine. Also, the above analogues do not show any sparing effect on Mg2+ requirements for AcPhe‐tRNA binding to ribosomes, in contrast to spermine. The presence of FWR fraction during the binding or acetylation of the secondary amines of spermine moderates or abolishes the stimulatory effect. In addition, all analogues tested enhance the stability of the ternary complex AcPhe‐tRNA‐poly(U)‐ribosome and the extent of AcPhe‐puromycin synthesis, particularly in the absence of the FWR fraction. At the kinetic phase of AcPhe‐puromycin synthesis, the analogues display both stimulatory and inhibitory effects, depending on the absence (partial noncompetitive inhibition) or the presence of the FWR fraction (nonessential activation in concert with partial noncompetitive inhibition). Detailed kinetic analysis shows that the analogues tested can mimic the behaviour of spermine, however, the potency to affect the peptidyltransferase activity depends on their degree of acylation, acyl‐substituent size, charge distribution and on their chain flexibility.
Solution structure of an artificial Fe8S8 ferredoxin : the D13C variant of Bacillus schlegelii Fe7S8 ferredoxin
Authors: Aono Shigetoshi , Bentrop Detlef , Bertini Ivano , Cosenza Grazia , Luchinat Claudio
Published: March 2003

The solution structure of the D13C variant of the thermostable Fe7S8 ferredoxin from Bacillus schlegelii has been determined by 1H‐NMR spectroscopy in its oxidized form. In a variable‐temperature NMR study the D13C variant was as thermostable (up to 90 +C) as the wild‐type protein (WT). Seventy‐five out of 77 amino acid residues and 81 % of all theoretically expected proton resonances in the D13C Fe8S8 protein have been assigned. Its structure was determined through torsion angle dynamics calculations with the program DYANA, using 935 meaningful NOEs (from a total of 1251), hydrogen bond constraints, and NMR‐derived dihedral angle constraints for the cluster‐ligating cysteines. Afterwards, restrained energy minimization and restrained molecular dynamics were applied to each conformer of the family. The final family of 20 structures has RMSD values from the mean structure of 0.055 nm for the backbone atoms and of 0.099 nm for all heavy atoms. The overall folding of the WT is maintained in the mutant, except for the immediate vicinity of the new cysteine, which becomes much more similar to native Fe8S8 proteins. The two residues at positions 11 and 12, which constitute an insertion with respect to all known Fe8S8 proteins, assume a conformation that does not prevent the preceding and following residues from folding like in native Fe8S8 proteins. Clear evidence for the existence of two conformations involving almost half of the amino acid residues was found. The two conformations are structurally indistinguishable. Temperature‐dependent NMR experiments show that one of them is thermodynamically more stable than the other.
The secondary structure and architecture of human elastin
Authors: Debelle Laurent , Alix Alain J. P. , Wei Shao M. , Jacob Marie‐Paule , Huvenne Jean‐Pierre , Berjot Maurice , Legrand Pierre
Published: March 2003

The presented work constitutes the first structural characterization of both insoluble human elastin and its solubilized form, κ‐elastin. Structural data were reached following the use of Fourier transform infrared, near infrared Fourier transform Raman and circular dichroism optical spectroscopic methods and their quantitative analysis permitted us to estimate ≈10 %α‐helices, ≈35 %β‐strands and ≈55 % undefined conformations in the global secondary structure of insoluble human elastin in the solid state. Following the use of the LINK method, the probable local distribution of the secondary‐structure elements along the sequence was determined and compared to that obtained for bovine elastin, the historical standard of elastin. This comparison led us to propose a globular architecture for the human elastomer and permitted us to delineate some elements of its structure‐elasticity relationship.
Amino‐acid‐sequence determination and biological activity of tessulin, a naturally occurring trypsin‐chymotrypsin inhibitor isolated from the leech Theromyzon tessulatum
Authors: Chopin Vincent , Stefano George B. , Salzet Michel
Published: March 2003

We purified a new trypsin−chymotrypsin inhibitor, designated tessulin, from the rhynchobdellid leech Theromyzon tessulatum. This 9‐kDa peptide was purified to apparent homogeneity by gel‐permeation and anion‐exchange chromatographies followed by reverse‐phase HPLC. The structure of tessulin was determined by reduction, S‐β‐pyridylethylation, trypsin digestion, automated Edman degradation and matrix‐assisted laser desorption mass spectrometry (m/z 8985 Da). The 81‐amino‐acid peptide possesses 16 cysteines and exhibits a 16 % sequence similarity with antistasin‐type inhibitors. Tessulin inhibits trypsin (Ki 1 pM) and chymotrypsin (Ki 150 pM) and exhibits no activity with thrombin, factor Xa, cathepsin G and elastase. This is the first trypsin−chymotrypsin inhibitor isolated from leeches that does not inhibit elastase or cathepsin G, except for cytin and therin. Furthermore, tessulin, in conjunction with other serine‐protease inhibitors isolated from Theromyzon (therin, theromin), significantly diminishes the level of human granulocyte and monocyte activation induced by lipopolysaccharides (10 μg). The combined level of inhibition is higher than that of aprotinin, another serine‐protease inhibitor used biomedically. Thus, tessulin may be clinically significant in reducing inflammatory events.
Two γ‐interferon‐activation sites (GAS) on the promoter of the human intercellular adhesion molecule (ICAM‐1) gene are required for induction of transcription by IFN‐γ
Authors: Tessitore Alessandra , Pastore Laura , Rispoli Alessandra , Cilenti Lucia , Toniato Elena , Flati Vincenzo , Farina Antonietta R. , Frati Luigi , Gulino Alberto , Martinotti Stefano
Published: March 2003

We describe the molecular features of the interferon (IFN)‐γ‐mediated transcription of the human intercellular adhesion molecule (ICAM‐1) gene. We identified putative IFN‐γ‐activated sites (GAS) distributed throughout a large segment of the ICAM‐1 promoter (4.0 kb region). Using computer‐assisted search, these sequences were similar to potential IFN‐γ responsive elements that have a core sequence 5′‐TTNCNNNAA‐3′. In this report we show that in the ICAM‐1 promoter a GAS site is located at −115 from the translation initiation site, and binds with strong affinity to IFN‐γ‐activated Signal Transducers and Activators of Transcription (STAT1) homodimers. The same sequence is responsible for the IFN‐γ‐mediated transcription of the ICAM‐1 gene. Moreover, we present evidence that a more distal GAS element that maps at −2787 from the translation initiation site, binds IFN‐γ‐activated STAT1 dimers with lower affinity. Multimeric copies of such GAS sequence inserted into a tkCAT minimal promoter can drive transcription, demonstrating that the −2787 bp GAS element has an independent functional activity upon binding of IFN‐γ‐activated STAT1 proteins as documented by in vitro binding assays. Furthermore, using recombinant ICAM‐CAT mutants, we show that, in vivo, the −2787 GAS, but not a mutagenized −2787 GAS site, when coupled to the more proximal −115 GAS element, has an additive effect in enhancing the IFN‐γ‐mediated transcription of ICAM‐1 promoter. Nevertheless, using a recombinant construct bearing the wild type −2787 GAS element and a mutagenized −115 GAS element, we could not detect any transcription after transfection of U937 recipient cells, suggesting that the −2787 bp GAS element is not sufficient as such for gene activation, but can cooperate with its cognate proximal sequence to give full function to the ICAM‐1 promoter during the IFN‐γ response. Taken together these data provide evidence that two GAS sites are required for the full potential activity in the mechanism of ICAM‐1 gene activation by IFN‐γ.
Modification of the unfolding region in bovine pancreatic ribonuclease and its influence on the thermal stability and proteolytic fragmentation
Authors: Arnold Ulrich , Schierhorn Angelika , Ulbrich‐Hofmann Renate
Published: March 2003

Ribonuclease (RNase) A and the more stable glycosylated RNase B differ by a carbohydrate moiety (GlcNAc2Man5–9) attached to Asn34. As previously shown, the first proteolytic cleavage sites to appear on thermal denaturation of both enzymes are in the structural region around Asn34. To discriminate the contribution of the modifying moiety to the stabilization toward thermal unfolding, on the one hand, and proteolytic fragmentation, on the other hand, the carbohydrate chain of RNase B was shortened by treatment with glycosidases to obtain GlcNAc‐RNase and (GlcNAc)2Man3‐RNase and extended by binding to concanavalin A or concanavalin A‐agarose. The results show a saltatory increase of the thermal unfolding constants and transition temperatures of GlcNAc‐RNase in comparison to RNase A, whereas the extension of the modification at Asn34 in the other RNase species does not further increase thermal stability. Therefore, the stability difference between RNase A and RNase B derivatives is attributed to the first carbohydrate unit. In contrast, the rate of proteolysis decreases gradually with increasing volume of the modifying moiety. As concluded from the analysis of the primary cleavage fragments, the main degradation pathway is shifted from the Asn34‐Leu35 to the Thr45‐Phe46 peptide bond due to increasing shielding effects.
The gelatin‐binding site of the second type‐II domain of gelatinase  A/MMP‐2
Authors: Tordai Hedvig , Patthy László
Published: March 2003

We have shown previously that all three fibronectin type‐II modules of gelatinase A contribute to its gelatin affinity. In the present work the second type‐II module was subjected to site‐directed mutagenesis in order to localize its gelatin‐binding site. The functional integrity of mutant proteins was assessed by their affinity for gelatin using gelatin–Sepharose affinity chromatography. The structural integrity of the mutant proteins, i.e. their resistance to thermal and chaotropic agent‐induced denaturation, was characterized by CD spectroscopy. Our studies show that, in the case of mutants R19L, R38L, K50G, K50R and R19L/R38L, the mutations had no significant effect on the structure and gelatin affinity of the type‐II module, excluding the direct involvement of these residues in ligand binding. In the case of mutants Y25A, Y46A, D49A and Y52A, the mutations yielded proteins that were devoid of gelatin affinity. Structural characterization of these proteins, however, indicated that they had also lost their ability to fold into the native structure characteristic of the wild‐type domain. In the case of mutant Y37A, the structure and stability of the mutant protein is similar to the wild‐type module. However, its gelatin affinity was severely impaired compared with the wild‐type protein. The fact that the Y37A mutation impairs ligand binding without detectable distortion of the module's architecture suggests that Y37 is directly involved in ligand binding. Homology modeling based on the three‐dimensional structure of the second type‐II module of PDC‐109 places Y37 on the right‐hand rim of a hydrophobic pocket that includes residues F20, W39, Y46, Y52 and F54, and thus provides proof for the involvement of this pocket in ligand binding.
Identification of the reactive cysteine residue (Cys227) in human carbonyl reductase
Authors: Tinguely Josiane N. , Wermuth Bendicht
Published: March 2003

Carbonyl reductase is highly susceptible to inactivation by organomercurials suggesting the presence of a reactive cysteine residue in, or close to, the active site. This residue is also close to a site which binds glutathione. Structurally, carbonyl reductase belongs to the short‐chain dehydrogenase/reductase family and contains five cysteine residues, none of which is conserved within the family. In order to identify the reactive residue and investigate its possible role in glutathione binding, alanine was substituted for each cysteine residue of human carbonyl reductase by site‐directed mutagenesis. The mutant enzymes were expressed in Escherichia coli and purified to homogeneity. Four of the five mutants (C26A, C122A C150A and C226A) exhibited wild‐type‐like enzyme activity, although Km values of C226A for three structurally different substrates were increased threefold to 10‐fold. The fifth mutant, C227A, showed a 10–15‐fold decrease in kcat and a threefold to 40‐fold increase in Km, resulting in a 30–500‐fold drop in kcat/Km. NaCl (300 m
m) increased the activity of C227A 16‐fold, whereas the activity of the wild‐type enzyme was only doubled. Substitution of serine rather than alanine for Cys227 similarly affected the kinetic constants with the exception that NaCl did not activate the enzyme. Both C227A and C227S mutants were insensitive to inactivation by 4‐hydroxymercuribenzoate. Unlike the parent carbonyl compounds, the glutathione adducts of menadione and prostaglandin A1 were better substrates for the C227A and C227S mutants than the wild‐type enzyme. Conversely, the binding of free glutathione to both mutants was reduced. Our findings indicate that Cys227 is the reactive residue and suggest that it is involved in the binding of both substrate and glutathione.
Na,K‐ATPase mRNAβ1 expression in rat myocardium – effect of thyroid status
Authors: Shao Yvonne , Pressley Thomas A. , Ismail‐Beigi Faramarz
Published: March 2003

The abundance of Na,K‐ATPase and its α and β subunit mRNAs is upregulated in cardiac and other target tissue by thyroid hormone (T3). Multiple Na,K‐ATPase mRNAβ1 species encoding an identical β1 polypeptide are expressed in the heart. The different mRNAβ1 species result from utilization of two transcription start‐sites in the first exon and multiple (five) poly(A) signals in the terminal exon of the β1 gene. In the present study we identify the mRNAβ1 species that are expressed in rat ventricular myocardium under basal conditions, and determine whether they are differentially regulated by T3. mRNAβ1 species were identified by 3′‐RACE followed by DNA sequencing, and by Northern blotting using probes derived from different regions of rat cDNAβ1. Five mRNAβ1 species are expressed in rat heart: mRNAβ1 species that are initiated at the first transcription start‐site and end at the first, second and fifth poly(A) sites (resulting in mRNAs of 1630, 1810, and 2780 nucleotides), and mRNAβ1 species initiated at the second transcription start‐site and ending at the second and fifth poly(A) sites (resulting in mRNAs of 1500 and 2490 nucleotides); in order of increasing length, the five mRNAs constitute 0.04, 0.15, 0.38, 0.11 and 0.32 of total mRNAβ1 content. In hypothyroid rats (induced by addition of propyl‐thiouracil to the drinking water for 3 weeks), total mRNAβ1 content decreased to 0.18 euthyroid levels, which was associated with a disproportionate 7.5‐fold decrease in the abundance of the longest transcript (P < 0.05); transcripts initiating at the first transcription start‐site and ending at the second poly(A) signal in hypothyroid hearts were 0.26 euthyroid levels (P < 0.05). Hyperthyroidism induced by injection of normal rats with three doses of 100 µg T3/100 g body weight every 48 h resulted in an overall ≈ 2‐fold increase in mRNAβ1 content with no change in the fractional contribution of any of the mRNAβ1 species. The results indicate a complex heterogeneity in the expression of mRNAβ1 in myocardium.
Role of autocrine stimulation on the effects of cyclic AMP on protein  and lipid phosphorylation in collagen‐activated and thrombin‐activated platelets
Authors: Ryningen Anita , Jensen Baard Olav , Holmsen Holm
Published: March 2003

We compared several responses in thrombin‐stimulated and collagen (type I)‐stimulated platelets with and without forskolin and inhibitors of autocrine stimulation (IAS: an ADP‐removing system of creatine phosphate/creatine phosphokinase, Arg‐Gly‐Asp‐Ser peptide to prevent fibrinogen/fibronectin binding to GPIIb/IIIa, SQ 29.548 as a thromboxane A2 receptor antagonist, cyproheptadine as a serotonin receptor antagonist, BN 52021 as a platelet‐activating factor receptor antagonist). The pattern of tyrosine‐phosphorylated proteins, the phosphorylation of lipids in the polyphosphoinositide cycle and phosphorylation of pleckstrin (P47) were studied as markers for signal‐transducing responses, exposure of CD62 (P‐selectin) and CD63 (Glycoprotein 53), as well as secretion of ADP + ATP and β‐N‐acetyl‐glycosaminidase were studied as final activation responses. Clear differences between thrombin‐stimulated and collagen‐stimulated platelets were observed. First, practically all protein‐tyrosine phosphorylation induced by thrombin was inhibited by IAS, while a partial inhibition was observed for collagen; the phosphorylation due to collagen alone was apparently stimulated by elevation of cAMP. Secondly, the other responses to thrombin were inhibited by increased levels of cAMP, independent of autocrine stimulation. In contrast, only the autocrine part of the collagen‐induced responses was inhibited by elevation of cAMP. Thus, the inhibition by elevated cAMP seen in collagen‐stimulated platelets seems to be due to removal of the G‐protein‐mediated activation from secreted autocrine stimulators either by IAS or forskolin. The remaining activity is a pure collagen effect which is not affected by elevated levels of cAMP.
Crystal structure of a hybrid between ribonuclease A and bovine seminal ribonuclease – the basic surface, at 2.0 Å resolution
Authors: Vatzaki Efstratia H. , Allen Simon C. , Leonidas Demetres D. , Trautwein‐Fritz Katrin , Stackhouse Joseph , Benner Steven A. , Acharya K. Ravi
Published: March 2003

A variant of bovine pancreatic ribonuclease A has been prepared with seven amino acid substitutions (Q55K, N62K, A64T, Y76K, S80R, E111G, N113K). These substitutions recreate in RNase A the basic surface found in bovine seminal RNase, a homologue of pancreatic RNase that diverged some 35 million years ago. Substitution of a portion of this basic surface (positions 55, 62, 64, 111 and 113) enhances the immunosuppressive activity of the RNase variant, activity found in native seminal RNase, while substitution of another portion (positions 76 and 80) attenuates the activity. Further, introduction of Gly at position 111 has been shown to increase the catalytic activity of RNase against double‐stranded RNA. The variant and the wild‐type (recombinant) protein were crystallized and their structures determined to a resolution of 2.0 Å. Each of the mutated amino acids is seen in the electron density map. The main change observed in the mutant structure compared with the wild‐type is the region encompassing residues 16–22, where the structure is more disordered. This loop is the region where the polypeptide chain of RNase A is cleaved by subtilisin to form RNase S, and undergoes conformational change to allow residues 1–20 of the RNase to swap between subunits in the covalent seminal RNase dimer.
The covalent attachment of polyamines to proteins in plant mitochondria
Authors: Votyakova Tatyana V. , Wallace Heather M. , Dunbar Bryan , Wilson S. Brian
Published: March 2003

Plant mitochondria from both potato and mung bean incorporated radioactivity into acid insoluble material when incubated with labelled polyamines (spermine, spermidine and putrescine). Extensive washing of mitochondrial precipitates with trichloroacetic acid and the excess of cold polyamine failed to remove bound radioactivity. Addition of nonradioactive polyamine stopped further incorporation of radioactivity but did not release radioactivity already bound. The radioactivity is incorporated into the membrane fraction. The labelling process has all the features of an enzymatic reaction: it is long lasting with distinctive kinetics peculiar to each polyamine, it is temperature dependent and is affected by N‐ethylmaleimide. The latter inhibits the incorporation of putrescine but stimulates the incorporation of spermine and spermidine. Treatment of prelabelled mitochondria with pepsin releases bound radioactivity thus indicating protein to be the ligand for the attachment of polyamines. HPLC of mitochondrial hydrolysates revealed that the radioactivity bound to mitochondria is polyamines; traces of acetyl polyamines were also found in some samples. On autoradiograms of SDS/PAGE gels several radioactive bands of proteins were detected. Protein sequencing of labelled spots from a 2D gel gave a sequence which was 60% identical to catalase. We suggest that the attachment of polyamines to mitochondrial proteins occurs cotranslationally possibly via transglutaminases.
Iron regulatory protein as an endogenous sensor of iron in rat intestinal mucosa
Authors: Schümann Klaus , Moret Rémy , Künzle Heinz , Kühn Lukas C.
Published: March 2003

Duodenal enterocytes adjust intestinal iron absorption to the body’s state of iron repletion. Here we tested how iron supply from the blood modulates the RNA‐binding activity of iron regulatory proteins (IRP‐1 and IRP‐2) in immature duodenal rat enterocytes, and whether the modulation is compatible with the hypothesis that IRPs, in turn, may regulate the expression of iron transport proteins in maturating enterocytes during migration to the villus tips. Tissue uptake of parenterally applied 59Fe along the duodenal crypt–villus axis was compared to local IRP‐1 and IRP‐2 activity and to duodenal 59Fe transport capacity 12 h, 48 h, and 72 h after intravenous iron administration to iron‐deficient rats. IRP‐1 and IRP‐2 activity was significantly increased in iron‐deficiency. 59Fe administrated from the blood side was almost exclusively taken up by crypt enterocytes. Accordingly, the activity of IRP‐1 decreased at this site 12 h after parenteral iron administration, but remained high at the villus tips. After 48 h the bulk of 59Fe containing enterocytes had migrated to the villus tips. Correspondingly, IRP‐1 activity was decreased at duodenal villus tips after 48 h. IRP‐2 activity also tended to decrease, though the change was statistically not significant. IRP‐2 activity remained significantly higher at duodenal villus tips than in crypts, even after 72 h. Intestinal iron absorption capacity decreased with the same delay as IRP‐1 activity after intravenous iron administration. In the ileum 59Fe uptake from the blood and IRP activity showed no significant difference between crypt and villus region. Luminal administration of iron decreased duodenal IRP‐1 and IRP‐2 activity at tips and crypts within 2 h. Thus, recently absorbed iron becomes available to cytosolic IRP during its passage through the enterocyte. Our results are compatible with a role of IRPs in gearing the expression of intestinal iron transporters in the duodenal brushborder to the body’s state of iron repletion.
Chemical shift perturbation studies of the interactions of the second RNA‐binding domain of the Drosophila sex‐lethal protein with the transformer pre‐mRNA polyuridine tract and 3′ splice‐site sequences
Authors: Chi Seung‐Wook , Muto Yutaka , Inoue Makoto , Kim Insil , Sakamoto Hiroshi , Shimura Yoshiro , Yokoyama Shigeyuki , Choi Byong‐Seok , Kim Hyoungman
Published: March 2003

The interactions of the second RNA‐binding domain of the Drosophila melanogaster Sex‐lethal protein (Sxl RBD2) with the oligoribonucleotides, GUUUUUUUU (GU8) and CUAGUG, representing the sequences surrounding an alternative 3′‐splicing site of the transformer pre‐mRNA (GU8CUAGUG), were studied using heteronuclear two‐dimensional NMR techniques. The 1H and 15N chemical shifts of the backbone amide resonances upon titration of Sxl RBD2 with each of these RNAs were recorded. It was found that Sxl RBD2 can bind not only to the polyuridine tract, GU8, but also to the downstream 3′ splice‐site sequence, CUAGUG, with similar affinities. In contrast, a nonspecific sequence, C8, did not bind to Sxl RBD2. This result is consistent with previous in vitro RNA‐selection and UV‐cross‐linking results which indicated that the Sex‐lethal protein binds to the uridine stretch and the AG dinucleotide in the consensus sequence, AUnNnAGU. In both cases, the chemical‐shift perturbations were significant for almost the same amino acid residues, including the two central β‐strands formed by the RNP2‐motif and RNP1‐motif with the two highly conserved aromatic residues (Y214 and F256) in the middle. As the first RNA‐binding domain of Sex‐lethal (Sxl RBD1) has a characteristic aliphatic residue at one of the two corresponding positions (I128 and F170), Y214 of Sxl RBD2 was replaced by Ile using site‐directed mutagenesis. On the one hand, the 1H and 15N chemical‐shift perturbations indicated that GU8 binds to the same interface of mutant Sxl RBD2 as of wild‐type Sxl RBD2, although its binding affinity was decreased significantly. On the other hand, the specific binding of Sxl RBD2 to CUAGUG was abolished almost completely by the Y→I mutation. Taken together, the present results indicate that the interface residues that bind with GU8 and CUAGUG are much the same, but the role of the Y214 residue is clearly different between these two target sequences.
Distribution of microsomal glutathione transferase 1 in mammalian tissues
Authors: Estonius Mats , Forsberg Lena , Danielsson Olle , Weinander Rolf , Kelner Michael J. , Morgenstern Ralf
Published: March 2003

An extensive Northern blot analysis of microsomal glutathione transferase 1 in human and rat tissues was performed. When normalized against the glyceraldehyde‐3‐phosphate dehydrogenase or actin expression it was evident that the predominant expression occurs in liver and pancreas. An ontogenetic, as well as a functional, basis for the high levels in these two organs is possible. The relative expression levels in man ranged from: liver and pancreas (100%), to kidney, prostate, colon (30–40%), heart, brain, lung, testis, ovary, small intestine (10–20%), placenta, skeletal muscle, spleen, thymus and peripheral blood leucocytes (1–10%). Liver‐enriched expression was detected in human fetal tissues with lung and kidney displaying lower levels (10–20%). No transcripts could be detected in fetal brain or heart. When comparing the expression levels between rat and man it is apparent that human extrahepatic mRNA levels are much higher relative to liver. Rat microsomal glutathione transferase mRNA expression ranges from 0.2 to 10% that of liver, with adrenal, uterus, ovary and stomach displaying the highest levels of the organs tested. Based on these observations, and the fact that the enzyme is encoded by a highly conserved single‐copy gene, it is suggested that microsomal glutathione transferase 1 performs essential functions vital to most mammalian cell types. We suggest that protection against oxidative stress constitutes one such function. Human expressed sequence tag (EST) characterization yielded four alternate mRNA transcripts with different 5′‐ends (four alternate noncoding exons 1). The predominant exon (based on the observed EST frequency) revealed a tissue distribution similar to that obtained using the reading frame as probe. Thus, it appears that one exon preferentially gives rise to mature mRNA in the human tissues examined. This exon is different from the one reported in the original cDNA characterized.
Transcriptional regulation of fatty acid synthase gene by insulin/glucose, polyunsaturated fatty acid and leptin in hepatocytes and adipocytes in normal and genetically obese rats
Authors: Fukuda Hitomi , Iritani Nobuko , Sugimoto Tomomi , Ikeda Hitoshi
Published: March 2003

Transcriptional regulation of the fatty acid synthase (FAS) gene by insulin/glucose, polyunsaturated fatty acids and leptin was investigated in hepatocytes and adipocytes of Wistar fatty rats and their lean littermates. The sequence spanning nucleotides –57 to –35 of FAS gene, which is responsive to insulin/glucose stimulation [Fukuda, H., Iritani, N. & Noguchi, T. (1997) FEBS Lett.406, 243–248], was linked to a reporter gene containing a heterologous promoter and transfected into rat hepatocytes or adipocytes. The activity of the reporter, chloramphenicol acetyltransferase, in the presence of glucose alone was similar in the primary cultured cells from the lean and obese rats. In the presence of insulin/glucose, however, chloramphenicol acetyltransferase activity was markedly increased in hepatocytes of lean rats, but was not significantly increased in those of obese rats. The stimulation by insulin/glucose was reduced in arachidonic acid‐treated cells of lean rats. Similarly, the stimulation by insulin/glucose was reduced in leptin‐treated cells and in cells from lean rats containing an expression vector encoding leptin. However, neither polyunsaturated fatty acids nor leptin‐treated cells from obese rats responded to insulin‐stimulation. The same effects were observed at endogenous FAS mRNA and enzyme levels. Similar results were seen in adipocytes, although the stimulation and suppression were much smaller than in hepatocytes. The insulin‐binding capacities of the receptors of liver and adipose tissue were reduced in the presence of leptin or polyunsaturated fatty acids. Leptin and polyunsaturated fatty acids appeared to suppress the insulin stimulation of FAS transcription by reducing the insulin‐binding capacities of receptors. Leptin converged on the insulin/glucose response element of FAS gene and suppressed the transcription.
Expression of δ, κ and µ human opioid receptors in Escherichia coli and reconstitution of the high‐affinity state for agonist with heterotrimeric G proteins
Authors: Stanasila Laura , Massotte Dominique , Kieffer Brigitte L. , Pattus Franc
Published: March 2003

Human opioid receptors of the δ, µ and κ subtypes were successfully expressed in Escherichia coli as fusions to the C‐terminus of the periplasmic maltose‐binding protein, MBP. Expression levels of correctly folded receptor molecules were comparable for the three subtypes and reached an average of 30 receptors·cell−1 or 0.5 pmol·mg−1 membrane protein. Binding of [3H]diprenorphine to intact cells or membrane preparations was saturatable, with a dissociation constant, KD, of 2.5 nm, 0.66 nm and 0.75 nm for human δ, µ and κ opioid receptors (hDOR, hMOR and hKOR, respectively). Recombinant receptors of the three subtypes retained selectivity and nanomolar affinity for their specific antagonists. Agonist affinities were decreased by one to three orders of magnitude as compared to values measured for receptors expressed in mammalian cells. The effect of sodium on agonist binding to E. coli‐expressed receptors was investigated. Receptor high‐affinity state for agonists was reconstituted in the presence of heterotrimeric G proteins. We also report affinity values of endomorphins 1 and 2 for µ opioid receptors expressed both in E. coli and in COS cells. Our results confirm that opioid receptors can be expressed in a functional form in bacteria and point out the advantages of E. coli as an expression system for pharmacological studies.
Functional differentiation in trematode hemoglobin isoforms
Authors: Rashid Aftab K. , Weber Roy E.
Published: March 2003

The Hbs and the major electrophoretic Hb components (isoHbs) were isolated from three species of the trematodes, Explanatum explanatum (Ee), Gastrothylax crumenifer (Gc) and Paramphistomum epiclitum (Pe), that parasitise the common Indian water buffalo Bubalus bubalis.

 The Hbs are monomeric and resemble the so‐called nonfunctional mutant hemoglobins that have Tyr at B10 or E7 positions (replacing Leu and the His residues, respectively). However, they are capable of binding with O2 and CO.

 O2 equilibrium studies of trematode Hb isoforms reveal extremely high O2 affinities, with half‐saturation O2 tension (P50) values up to 800 times lower than those of human hemoglobins. This correlates with Tyr residues at B10 and at the distal position (E7) that decrease the O2 dissociation rate by contributing hydrogen bonds (H‐bonds) to the bound O2. These substitutions also increase the O2 association rates either due to orientation of E7‐Tyr towards the solvent and/or by sterically hindering the entry of water molecules into the heme pocket. The latter may account for the low rate of autoxidation of trematode Hbs.

 The Hbs and their isoforms from different species exhibited pronounced variation in O2 affinity, which may relate to subtle differences in the structure of the heme pocket. The O2 affinities of the composite (unfractionated) Hbs were intermediate to those of the individual Hb isoform.

 The P50 values of Hbs here obtained by direct O2 equilibrium measurements differed from those calculated from kinetic data already published [Kiger, L., Rashid, A. K., Griffon, N., Haque, M., Moens, L.,Gibson, Q. H., Poyart, C., & Marden, M. C. (1998). Biophys. J. 75, 990–998.] Intermediate state(s) due to slow reorientation of E7‐Tyr may account for this difference.

 Some Hb isoforms showed slight (either normal or reverse) Bohr effects. The hyperbolic O2 equilibrium curve, Hill coefficient (n) values near unity accord with a monomeric nature of trematode Hbs.

 In marked contrast to vertebrate Hbs, CO does not seem to compete effectively with O2 in trematode Hbs, as evident from partition coefficient values (M) below 1.
D1‐D2 protein degradation in the chloroplast
Authors: Jansen Marcel A. K. , Mattoo Autar K. , Edelman Marvin
Published: March 2003

The D1 and D2 proteins of the photosystem II (PSII) reaction center are stable in the dark, while rapid degradation occurs in the light. Thus far, a quantitative correlation between degradation and photon fluences has not been determined. In Spirodela oligorrhiza, D1‐D2 degradation increases with photon flux. We find that kinetics for D2 degradation mirror those for D1, except that the actual half‐life times of the D2 protein are about three times larger than those of the D1. The degradation ratio, D2/D1, is fluence independent, supporting the proposal [Jansen, M.A.K., Greenberg, B.M., Edelman, M., Mattoo, A.K. & Gaba, V. (1996), Photochem. Photobiol.63, 814–817] that degradation of the two proteins is coupled. It is commonly conceived that D1 degradation is predominantly associated with photon fluences that are supersaturating for photosynthesis. We now show that a fluence as low as 5 µmol·m−2·s−1 elicited a reaction constituting > 25% of the total degradation response, while > 90% of the degradation potential was attained at intensities below saturation for photosynthesis (≈750 µmol·m−2·s−1). Thus, in intact plants, D1 degradation is overwhelmingly associated with fluences limiting for photosynthesis. D1 degradation increases with photon flux in a complex, multiphasic manner. Four phases were uncovered over the fluence range from 0–1600 µmol·m−2·s−1. The multiphasic saturation kinetics underscore that the D1 and D2 degradation response is complex, and emanates from more than one parameter. The physiological processes associated with each phase remain to be determined.