bluuebunny/crossref_metadata_2025 huggingface dataset:
DNA triple‐helix formation on nucleosome core particles
Authors: Brown Philip M. , Fox Keith R.
Published: March 2003

We have used DNase I footprinting to examine the formation of intermolecular triplexes on DNA fragments which have been complexed with nucleosome core particles. We have prepared five DNA fragments, based on the 160‐bp tyrT sequence, which contain different length oligopurine tracts (up to 25 bp) at two different positions along the fragment, and have examined their availability for triple‐helix formation after reconstituting onto nucleosome core particles. These results are compared with the formation of shorter triplexes in the same regions. In general we find that increasing the length of the complex does not facilitate nucleosomal triplex formation and that the most important factor affecting triplex formation is the position of the target site within the nucleosome‐bound fragment. In some instances we find that longer oligonucleotides inhibit triplex formation. Although successful triplex formation was achieved on the longest nucleosome‐bound oligopurine tracts, this was accompanied by changes in cleavage pattern that suggest oligonucleotide‐induced changes in nucleosome structure.
Human mitochondrial thioredoxin reductase
Authors: Miranda‐Vizuete Antonio , Damdimopoulos Anastasios E. , Pedrajas José Rafael , Gustafsson Jan‐Åke , Spyrou Giannis
Published: March 2003

We have isolated a 1918‐bp cDNA from a human adrenal cDNA library which encodes a novel thioredoxin reductase (TrxR2) of 521 amino acid residues with a calculated molecular mass of 56.2 kDa. It is highly homologous to the previously described cytosolic enzyme (TrxR1), including the conserved active site CVNVGC and the FAD‐binding and NADPH‐binding domains. However, human TrxR2 differs from human TrxR1 by the presence of a 33‐amino acid extension at the N‐terminus which has properties characteristic of a mitochondrial translocation signal. Northern‐blot analysis identified one mRNA species of 2.2 kb with highest expression in prostate, testis and liver. We expressed human TrxR2 as a fusion protein with green fluorescent protein and showed that in vivo it is localized in mitochondria. Removal of the mitochondrial targeting sequence abolishes the mitochondrial translocation. Finally, we determined the genomic organization of the human TrxR2 gene, which consists of 18 exons spanning about 67 kb, and its chromosomal localization at position 22q11.2.
Bioenergetic and structural consequences of allotopic expression of subunit 8 of yeast mitochondrial ATP synthase
Authors: Roucou Xavier , Artika I. Made , Devenish Rodney J. , Nagley Phillip
Published: March 2003

Subunit 8 (Y8), a mitochondrially encoded subunit of the F0 sector of the F1F0‐ATP synthase is essential for oxidative phosphorylation. We have previously introduced the technique of allotopic expression to study the structure/function of Y8, whereby an artificial Y8 gene is expressed in the nucleus of cells lacking a functional mitochondrial Y8, thus generating assembly of a functional F1F0‐ATPase complex. In this paper we show that when a gene encoding an essentially unmodified version of Y8 is allotopically expressed, ATP synthesis and hydrolysis rates, as well as efficiency of oxidative phosphorylation, were similar to those of the parental wild‐type strain in which Y8 is naturally expressed in mitochondria. We then tested the requirement for the hydrophobicity of the central domain (residues 14–32), which possibly represents a transmembrane stem, by introducing adjacent negative charges at different positions of Y8. One of the variants thus generated, which carries the double substitution Leu23→Asp, Leu24→Asp, when expressed in a strain lacking endogenous Y8, gave rise to cells which grew very slowly by oxidative phosphorylation.  Measurement of bioenergetic parameters showed two major defects in these cells relative to control cells allotopically expressing unmodified Y8. First, the activity of the F1F0‐ATP synthase was significantly decreased. ATP synthesis and state 3 of respiration were reduced by ≈ 30–40%. ATP hydrolysis was reduced by ≈ 30% and was almost insensitive to the F0 inhibitor oligomycin. Second, the physical coupling between the two sectors of the enzyme, as well as the stability of the F1 sector itself, were affected as shown by decreased recovery of F0 sector [8, 9, b, oligomycin sensitivity‐conferring protein (OSCP), d, h and f] and F1 sector (α, γ, δ) subunits in immunoprecipitates of ATP synthase. This study indicates that Y8 not only performs an important role in the structure of the mitochondrial complex but also in its activity. We conclude that the hydrophobic character of amino acids 23 and 24 in the middle of the putative transmembrane stem of Y8 is essential for coupling proton transport through F0 to ATP synthesis on F1.
Elucidation of the structure of an alanine‐lacking core tetrasaccharide trisphosphate from the lipopolysaccharide of Pseudomonas aeruginosa mutant H4
Authors: Sánchez Carballo Patricia M. , Rietschel Ernst Th. , Kosma Paul , Zähringer Ulrich
Published: March 2003

Lipopolysaccharide (LPS) of Pseudomonas aeruginosa rough mutant H4 was isolated by hot water/phenol extraction followed by a modified phenol/chloroform/petroleum ether procedure. Upon SDS/PAGE, the LPS showed a strong major band corresponding to the expected rough‐type LPS. Additional faint high molecular‐mass bands revealed that the O‐chain was present, indicating that the H4 mutant is genetically unstable. Mild acid hydrolysis of the LPS removed lipid A and released a phosphorylated core oligosaccharide that was purified by gel‐permeation chromatography and high‐performance anion‐exchange liquid chromatography. The oligosaccharide contained two residues of lglycerodmanno‐heptose (Hep) and one residue each of 3‐deoxy‐dmanno‐oct‐2‐ulosonic acid (Kdo) and GalNAc. Upon matrix‐assisted laser desorption/ionization mass spectroscopy in the negative ion mode, the main fraction expressed a peak for the molecular ion [M‐H]− at m/z 1106.41, which was compatible with a carbamoylated, trisphosphorylated tetrasaccharide. The structure was further investigated using one‐ and two‐dimensional homonuclear and heteronuclear correlated NMR spectroscopy at pD 3 and, after borohydride reduction, at pD 9. The NMR data of the two phosphorylated tetrasaccharides recorded at different pD allowed determination of the positions of the three phosphate (P) groups and the carbamoyl group (Cm) thus establishing the following structure of the core oligosaccharide:

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Two unusual structural features in the core oligosaccharide of P. aeruginosa were identified for the first time, i.e. the replacement of an amide‐linked alanyl group in GalN with an acetyl group and the phosphorylation at position 6 of HepII.
Biogenesis of α6β4 integrin in a human colonic adenocarcinoma cell line
Authors: Rigot Véronique , André Frédéric , Lehmann Maxime , Lissitzky Jean‐Claude , Marvaldi Jacques , Luis José
Published: March 2003

The heterodimer α6β4 is a major integrin and the main laminin receptor in epithelia. The α6 integrin subunit is proteolytically cleaved, probably by furin, and glycosylated during its biosynthesis. In the present work, we have investigated the kinetics of the assembly process of α6β4 heterodimers in the colonic adenocarcinoma cell line HT29‐D4. We demonstrate that the association of α6 and β4 precursors occurs within the ER, while the endoproteolytic cleavage of pro‐α6 occurs later, probably in the trans‐Golgi network. When pro‐α6 was blocked within the ER by treatment with brefeldin A, its maturation processing was completely prevented without any consequence on its association with β4 subunit. Low temperature (20 °C) also blocked pro‐α6 maturation, like brefeldin A, but in addition impaired the integrin assembly. Calnexin, an ER resident protein chaperone, was found to be associated with both the α6 and β4 subunit precursors. Our data suggest that calnexin might be responsible for the prolonged retention of pro‐α6 within the ER compartment and for the defect of integrin subunit association observed at low temperature.
The occurrence of two types of collagen proα‐chain in the abalone Haliotis discus muscle
Authors: Yoneda Chie , Hirayama Yasushi , Nakaya Misako , Matsubara Youco , Irie Shinkichi , Hatae Keiko , Watabe Shugo
Published: March 2003

Acid‐soluble collagens were prepared from connective tissues in the abalone Haliotis discus foot and adductor muscles with limited proteolysis using pepsin. Collagen preparation solubilized with 1% pepsin contained two types of α‐chains which were different in their N‐terminal amino acid sequences. Accordingly, two types of full‐length cDNAs coding for collagen proα‐chains were isolated from the foot muscle of the same animal and these proteins were named Hdcols (Haliotis discus collagens) 1α and 2α. The two N‐terminal amino acid sequences of the abalone pepsin‐solubilized collagen preparation corresponded to either of the two sequences deduced from the cDNA clones. In addition, several tryptic peptides prepared from the pepsin‐solubilized collagen and fractionated by HPLC showed N‐terminal amino acid sequences identical to those deduced from the two cDNA clones. Hdcols 1α and 2α consisted of 1378 and 1439 amino acids, respectively, showing the primary structure typical to those of fibril‐forming collagens. The N‐terminal propeptides of the two collagen proα‐chains contained cysteine‐rich globular domains. It is of note that Hdcol 1α completely lacked a short Gly‐X‐Y triplet repeat sequence in its propeptide. An unusual structure such as this has never before been reported for any fibril‐forming collagen. The main triple‐helical domains for both chains consisted of 1014 amino acids, where a supposed glycine residue in the triplet at the 598th position from the N‐terminus was replaced by alanine in Hdcol 1α and by serine in Hdcol 2α. Both proα‐chains of abalone collagens contained six cysteine residues in the carboxyl‐terminal propeptide, lacking two cysteine residues usually found in vertebrate collagens. Northern blot analysis demonstrated that the mRNA levels of Hdcols 1α and 2α in various tissues including muscles were similar to each other.
Iron dependence of tryptophan hydroxylase activity in RBL2H3 cells and its manipulation by chelators
Authors: Hasegawa Hiroyuki , Oguro Kazuya , Naito Yasuhisa , Ichiyama Arata
Published: March 2003

Tryptophan hydroxylase requires Fe2+ for in vitro enzyme activity. In this study, the intracellular activity of tryptophan hydroxylase was assessed by applying 3‐hydroxybenzylhydrazine (NSD‐1015), an inhibitor of aromatic l‐amino acid decarboxylase, to monolayer cultures of RBL2H3 cells, a serotonin producing mast cell line. The effect of manipulating intracellular ‘free’ iron levels on enzyme activity was analyzed by administration of iron chelators. Desferrioxamine (DFO) suppressed the intracellular enzyme activity. Salicylaldehyde isonicotinoyl hydrazone (SIH) also suppressed enzyme activity, but stimulated it when administered in the Fe‐bound form. Hemin also stimulated enzyme activity, which progressively increased over several hours to more than sixfold the initial level. DFO and SIH inhibited the hemin stimulatory effect when administered simultaneously with hemin. Both suppression and stimulation with these chelators took place without a significant decrease or increase in the amount of enzyme. These results indicate that there was an inadequate supply of Fe2+ in the cells to support full activity of tryptophan hydroxylase.
Characterization of catalytic centre mutants of macrophage migration inhibitory factor (MIF) and comparison to Cys81Ser MIF
Authors: Kleemann Robert , Kapurniotu Aphrodite , Mischke Ralf , Held Jürgen , Bernhagen Jürgen
Published: March 2003

Macrophage migration inhibitory factor (MIF) displays both cytokine and enzyme activities, but its molecular mode of action is still unclear. MIF contains three cysteine residues and we showed recently that the conserved Cys57‐Ala‐Leu‐Cys60 (CALC) motif is critical for the oxidoreductase and macrophage‐activating activities of MIF. Here we probed further the role of this catalytic centre by expression, purification, and characterization of the cysteine→serine mutants Cys60Ser, Cys57Ser/Cys60Ser, and Cys81Ser of human MIF and of mutants Ala58Gly/Leu59Pro and Ala58Gly/Leu59His, containing a thioredoxin (Trx)‐like and protein disulphide isomerase (PDI)‐like dipeptide, respectively. The catalytic centre mutants formed inclusion bodies and the resultant mutant proteins Cys57Ser/Cys60Ser, Ala58Gly/Leu59Pro, and Als58Gly/Leu59His were only soluble in organic solvent or 6 m GdmHCl when reconstituted at concentrations above 1 µg·mL−1. This made it necessary to devise new purification methods. By contrast, mutant Cys81Ser was soluble. Effects of pH, solvent, and ionic strength conditions on the conformation of the mutants were analysed by far‐UV CD spectropolarimetry and mutant stability was examined by denaturant‐induced unfolding. The mutants, except for mutant Cys81Ser, showed a close conformational similarity to wild‐type (wt) MIF, and stabilization of the mutants was due mainly to acid pH conditions. Intramolecular disulphide bond formation at the CALC region was confirmed by near‐UV CD of mutant Cys60Ser. Mutant Cys81Ser was not involved in disulphide bond formation, yet had decreased stability. Analysis in the oxidoreductase and a MIF‐specific cytokine assay revealed that only substitution of the active site residues led to inactivation of MIF. Mutant Cys60Ser had no enzyme and markedly reduced cytokine activity, whereas mutant Cys81Ser was active in both tests. The Trx‐like variant showed significant enzyme activity but was less active than wtMIF; PDI‐like MIF was enzymatically inactive. However, both variants had full cytokine activity. Together with the low but nonzero cytokine activity of mutant Cys60Ser, this indicated that the cytokine activity of MIF may not be tightly regulated by redox effects or that a distinguishable receptor mechanism exists. This study provides evidence for a role of the CALC motif in the oxidoreductase and cytokine activities of MIF, and suggests that Cys81 could mediate conformational effects. Availability and characterization of the mutants should greatly aid in the further elucidation of the mechanism of action of the unusual cytokine MIF.
Co‐operative binding of Ca2+ ions to the regulatory binding sites of gelsolin
Authors: Gremm Dagmar , Wegner Albrecht
Published: March 2003

The rate of association of actin with gelsolin was measured at various Ca2+ and ATP concentrations. The fraction of Ca2+‐activated gelsolin was determined by quantitative evaluation of the association rates thereby assuming that Ca2+‐binding gelsolin associates with actin and Ca2+‐free gelsolin does not. A plot of the fraction of Ca2+‐activated gelsolin vs. the free Ca2+ concentration revealed a sigmoidal shape suggesting that co‐operative binding of Ca2+ ions is required for activation of gelsolin. A good fit of the experimental data by calculated binding curves was obtained if two Ca2+ ions were assumed to bind to actin in a highly co‐operative manner. ATP decreased the rate of association of gelsolin with actin and bound to gelsolin at a low affinity (Kd = 32 µm for Ca2+‐free and Kd = 400 µm for Ca2+‐activated gelsolin). In contrast, a 1 : 1 gelsolin–actin complex was found to be activated for association with actin by a single Ca2+ ion in a non‐co‐operative manner.
The isocitrate dehydrogenase kinase/phosphatase from Escherichia coli is highly sensitive to in‐vitro oxidative conditions
Authors: Oudot Christelle , Jaquinod Michel , Cortay Jean‐Claude , Cozzone Alain J. , Jault Jean‐Michel
Published: March 2003

Isocitrate dehydrogenase kinase/phosphatase (IDHK/P) is a homodimeric enzyme which controls the oxidative metabolism of Escherichia coli, and exibits a high intrinsic ATPase activity. When subjected to electrophoresis under nonreducing conditions, the purified enzyme migrates partially as a dimer. The proportion of the dimer over the monomer is greatly increased by treatment with cupric 1,10 phenanthrolinate or 5,5′‐dithio‐bis(2‐nitrobenzoic acid), and fully reversed by dithiothreitol, indicating that covalent dimerization is produced by a disulfide bond. To identify the residue(s) involved in this intermolecular disulfide‐bond, each of the eight cysteines of the enzyme was individually mutated into a serine. It was found that, under nonreducing conditions, the electrophoretic patterns of all corresponding mutants are identical to that of the wild‐type, except for the Cys67→Ser which migrates exclusively as a monomer and for the Cys108→Ser which migrates preferentially as a dimer. Furthermore, in contrast to the wild‐type enzyme and all the other mutants, the Cys67→Ser mutant still migrates as a monomer after treatment with cupric 1,10 phenanthrolinate. This result indicates that the intermolecular disulfide bond involves only Cys67 in each IDHK/P wild‐type monomer. This was further supported by mass spectrum analysis of the tryptic peptides derived from either the cupric 1,10 phenanthrolinate‐treated wild‐type enzyme or the native Cys108→Ser mutant, which show that they both contain a Cys67–Cys67 disulfide bond. Moreover, both the cupric 1,10 phenanthrolinate‐treated wild‐type enzyme and the native Cys108→Ser mutant contain another disulfide bond between Cys356 and Cys480. Previous results have shown that this additional Cys356–Cys480 disulfide bond is intramolecular [Oudot, C., Jault, J.‐M., Jaquinod, M., Negre, D., Prost, J.‐F., Cozzone, A.J. & Cortay, J.‐C. (1998) Eur. J. Biochem. 258, 579–585].
Mechanism of inhibition of aldehyde dehydrogenase by citral, a retinoid antagonist
Authors: Kikonyogo Alexandra , Abriola Darryl P. , Dryjanski Marek , Pietruszko Regina
Published: March 2003

Low concentrations of citral (3,7‐dimethyl‐2,6‐octadienal), an inhibitor of retinoic acid biosynthesis, inhibited E1, E2 and E3 isozymes of human aldehyde dehydrogenase (EC1.2.1.3). The inhibition was reversible on dilution and upon long incubation in the presence of NAD+; it occurred with simultaneous formation of NADH and of geranic acid. Thus, citral is an inhibitor and also a substrate. Km values for citral were 4 µm for E1, 1 µm for E2 and 0.1 µm for E3; Vmax values were highest for E1 (73 nmol·min−1·mg−1), intermediate for E2 (17 nmol·min−1·mg−1) and lowest (0.07 nmol·min−1·mg−1) for the E3 isozyme. Citral is a 1 : 2 mixture of isomers: cis isomer neral and trans isomer, geranial; the latter structurally resembles physiologically important retinoids. Both were utilized by all three isozymes; a preference for the trans isomer, geranial, was observed by HPLC and by enzyme kinetics. With the E1 isozyme, both geranial and neral, and with the E2 isozyme, only neral obeyed Michaelis–Menten kinetics. With the E2 isozyme and geranial sigmoidal saturation curves were observed with S0.5 of ≈ 50 nm; the n‐values of 2–2.5 indicated positive cooperativity. Geranial was a better substrate and a better inhibitor than neral. The low Vmax, which appeared to be controlled by either the slow formation, or decomposition via the hydride transfer, of the thiohemiacetal reaction intermediate, makes citral an excellent inhibitor whose selectivity is enhanced by low Km values. The Vmax for citral with the E1 isozyme was higher than those of the E2 and E3 isozymes which explains its fast recovery following inhibition by citral and suggests that E1 may be the enzyme involved in vivo citral metabolism.
The human calcium‐independent phospholipase A2 gene
Authors: Larsson Forsell Pontus K. A. , Kennedy Brian P. , Claesson Hans‐Erik
Published: March 2003

Recently, we reported the human 88‐kDa calcium‐independent phospholipase A2 (iPLA2) cDNA sequence, as well as extensive alternative splicing of the iPLA2 mRNA. In this report we identified the gene coding for iPLA2, which was localized on chromosome 22q13.1. The gene consists of at least 17 exons spanning > 69 kb. Based on the iPLA2 gene organization the splice variants can be explained. The putative promotor for the iPLA2 gene lacks a TATA‐box and contains a CpG island as well as several potential Sp‐1‐binding sites. Furthermore, the 5′‐flanking region also contains one medium reiteration frequency repeat (MER53) and an Alu repetitive sequence. Northern blot analysis of iPLA2 mRNA in various human tissues demonstrated tissue‐specific expression of four distinct iPLA2 transcripts. The native human 3.2‐kb iPLA2 transcript was predominantly expressed in heart, brain, skeletal muscle, prostate, testis, thyroid and spinal cord, and to a lesser extent in peripheral blood leucocytes, stomach, trachea and bone marrow. Studies on the subcellular localization of the native iPLA2 protein were performed in COS‐7 cells overexpressing this enzyme. The cytosolic fraction of untransfected and cells overexpressing iPLA2 contained equal amounts of calcium‐independent PLA2 activity. However, the membrane fraction displayed a 5.5‐fold increased activity in iPLA2 overexpressing cells. This increased calcium‐independent PLA2 activity correlated with the presence of iPLA2 immunoreactive protein in the membrane fraction, indicating that this form of iPLA2 protein was membrane associated. Studies of iPLA2 in rat vascular smooth muscle cells verified the membrane association of this form of iPLA2. The major difference between this form of iPLA2 enzyme and the soluble forms of iPLA2 studied previously is the presence of 54 additional amino acid residues derived from exon 9. We suggest that the addition of these 54 amino acids leads to a membrane‐associated protein. In summary, these results demonstrate that alternative splicing of the human iPLA2 transcript generates multiple iPLA2 isoforms with distinct tissue distribution and cellular localization.
Denervation of rabbit gastrocnemius and soleus muscles
Authors: Nozais Muriel , Merkulova Tatyana , Keller Angélica , Janmot Chantal , Lompré Anne‐Marie , D’Albis Anne , Lucas Marguerite
Published: March 2003

We report here, for the first time, the expression of the muscle‐specific isoform of the glycolytic enzyme, enolase (EC 4.2.1.11) (β enolase), in rabbit skeletal muscles. We have analysed the fast‐twitch gastrocnemius and the slow‐twitch soleus muscles during normal postnatal development and following denervation. We show that, in rabbit, as already described in rodents, β enolase gene expression behaves as a good marker of the fast‐twitch fibers. In soleus muscle, the β enolase transcript level is 10–20% of that found in gastrocnemius. Denervation, performed at 8 postnatal days, induces an important drop of β enolase transcript levels in both developing soleus and gastrocnemius muscles, with a 80% decrease observed 1 week after denervation in the operated muscles, as compared to the corresponding contralateral muscles. Thereafter, the β enolase transcript level continues to decrease in the fast‐twitch muscle, with the β enolase subunit being detectable only in the atrophic fast‐twitch fibers. In contrast, the β transcript level tends to increase in the denervated slow‐twitch muscle, reaching about 50% of that in contralateral soleus, at 7 weeks after surgery. The level of β enolase transcripts still expressed after denervation seems to stabilize at the same low level in both types of inactive muscles. This suggests that the small fraction of β enolase expression which is not controlled by the nerve, or by the contractile activity imposed by it, is independent of the muscle phenotype.
Tropomodulin isolated from rabbit skeletal muscle inhibits filament formation of actin in the presence of tropomyosin and troponin
Authors: Kimura Sumiko , Ichikawa Akira , Ishizuka Junko , Ohkouchi Shiho , Kake Takei , Maruyama Koscak
Published: March 2003

Tropomodulin is a tropomyosin‐binding protein, originally isolated from human erythrocytes. Tropomodulin is currently regarded as the sole actin pointed‐end capping protein [Weber, A., Pennise, C.R., Babcock, G.G. & Fowler, V.M. (1994) J. Cell Biol.127, 1627–1635]. This work first describes a procedure for the purification of tropomodulin from rabbit skeletal muscle. Tropomodulin almost completely inhibited filament formation of actin in the presence of tropomyosin and troponin. For the maximal inhibition of actin polymerization, ≈ 0.10, 0.12 and 0.003 mol of tropomyosin, troponin and tropomodulin per mol of actin were required, respectively. Fluorescence‐intensity measurements, electron‐microscopy and sedimentation experiments revealed that only very short fragments and amorphous aggregates, but not filaments, were formed when actin was copolymerized with tropomyosin, troponin and tropomodulin by the addition of 50 mm KCl at pH 8.0. The effects of tropomyosin, troponin and tropomodulin were more remarkable on Ca‐actin than on Mg‐actin. It appears that tropomodulin caps both the pointed and barbed ends of tropomyosin‐ and troponin‐bound actin filaments.
A cytosolic domain of the erythropoietin receptor contributes to endoplasmic reticulum‐associated degradation
Authors: Supino‐Rosin Lia , Yoshimura Akihiko , Altaratz Hanna , Neumann Drorit
Published: March 2003

The erythropoietin receptor (EPO‐R) is the cellular target for erythropoietin (EPO), the primary hormone that mediates the proliferation of immature erythroblasts and their differentiation into mature erythrocytes. Unusual features of the EPO‐R are its short half‐life (t1\2 1–2 h), its degradation via multiple pathways and the fact that less than 1% of total cellular EPO‐R molecules are found on the cell surface. The contribution of EPO‐R structural determinants to the regulation of its intracellular metabolism is still unclear. The epidermal growth factor receptor (EGF‐R), unlike the EPO‐R, is efficiently transported to the cell surface and displays a much longer metabolic half‐life. To determine which EPO‐R cytosolic domains are involved in intracellular degradation, we studied chimeric receptor molecules constructed of EGF‐R extracellular and transmembrane parts, linked to the full length or truncated cytosolic part of the EPO‐R. The chimeras were expressed in transiently transfected COS 7 cells and stably expressed in Ba/F3 cells. Our experiments indicate that the cytosolic part of the EPO‐R contains determinants that mark it for rapid degradation, in association with the endoplasmic reticulum (ER). This degradation was insensitive to brefeldin A and was inhibited by specific proteasomal inhibitors. A truncated EGF‐R/EPO‐R chimera containing only 50 amino acids of the EPO‐R membrane‐proximal cytosolic part was also rapidly degraded suggesting that these 50 amino acids are involved in receptor degradation.
Plasminogen activator inhibitor type‐2 (PAI‐2) gene transcription requires a novel NF‐κB‐like transcriptional regulatory motif
Authors: Mahony Donna , Kalionis Bill , Antalis Toni M.
Published: March 2003

Induction of human plasminogen activator inhibitor type‐2 (PAI‐2) gene transcription is the response of macrophages to inflammatory stimuli, such as the pleiotropic cytokine, tumour necrosis factor‐α (TNFα). Here we have examined whether PAI‐2 gene transcription in response to TNFα may be mediated through a regulatory pathway involving the transcription factor, NF‐κB. We have tested the function of two potential NF‐κB‐like sites present in the PAI‐2 proximal promoter for responsiveness to TNFα using chloramphenicol acetyl transferase reporter gene deletion and mutation analyses. While no evidence was found for TNFα regulation of the PAI‐2 gene through either of these two sites, one of the NF‐κB‐like motifs, transcriptional regulatory motif (TRM), present at position −400 was found to be essential for constitutive PAI‐2 transcription, as mutation of this motif abolished basal PAI‐2 promoter activity in both monocyte‐like U937 cells and HT1080 fibrosarcoma cells. Competition electrophoretic mobility shift assays identified four TRM‐binding proteins present in U937, HT1080 and HeLa cell extracts, which bound to this motif but were not components of the NF‐κB regulatory complex. Expression screening of a HeLa cell cDNA library using the −400 TRM as a probe identified two cDNAs encoding partial peptides which specifically bound the TRM motif. DNA sequence analysis revealed that one cDNA was novel, and the second cDNA encoded exon 5 of the nephroblastoma overexpressed (novH) proto‐oncogene, suggesting a new role for this peptide in gene regulation. Taken together, these findings identify a new regulatory element required for constitutive PAI‐2 transcription, and identify potential DNA‐binding proteins associated with this element that may play a role in PAI‐2 gene regulation.
Developmental expression of the protein kinase C substrate/binding protein (clone 72/SSeCKS) in rat testis
Authors: Erlichman Jack , Gutierrez‐Juarez Roger , Zucker Shoshanna , Mei Xiaohua , Orr George A.
Published: March 2003

The coordinated interaction of kinases, phosphatases and other regulatory molecules with scaffolding proteins is emerging as a major theme in intracellular signaling networks. In this report we show that a cDNA isolated from a rat testis expression library by interactive cloning using the regulatory subunit (R) of a type‐II protein kinase A (PKA) is identical with a previously characterized protein kinase C (PKC)‐binding protein termed either clone 72 [Chapline, C., Mousseau, B., Ramsay, K., Duddy, S., Li, Y., Kiley, S.C. & Jaken, S. (1996)J. Biol. Chem.271, 6417–6422] or SSeCKS [Lin, X., Tombler, E., B., Nelson, P.J., Ross, M. & Gelman, I.H. (1996)J. Biol. Chem.271, 28430–28438]. Deletion mutagenesis demonstrated that amino acids 1495–1524 of clone 72/SSeCKS had the ability to interact with RII. Antibodies prepared against the recombinant protein recognized a 280/290‐kDa doublet and a 240‐kDa protein on Western blots of rat testis cytosolic and Triton X‐100 extracts. Expression of clone 72/SSeCKS mRNA and protein levels was developmentally regulated in rat testis. Northern‐blot analysis showed a dramatic increase in clone 72/SSeCKS‐hybridizing mRNA starting 30 days after birth. Immunohistochemical examination showed high expression levels in elongating spermatids. Clone 72/SSeCKS was not detected in mature sperm. These studies suggest a role for clone 72/SSeCKS, a PKA/PKC scaffolding protein, during the process of spermiogenesis.
High prevalence of 2‐mono‐ and 2,6‐di‐substituted Manol‐terminating sequences among O‐glycans released from brain glycopeptides by reductive alkaline hydrolysis
Authors: Chai Wengang , Yuen Chun‐Ting , Kogelberg Heide , Carruthers Robert A. , Margolis Richard U. , Feizi Ten , Lawson Alexander M.
Published: March 2003

Di‐ to heptasaccharides isolated from total nondialyzable brain glycopeptides after release by alkaline borohydride treatment have been subjected to mass spectrometric and nuclear magnetic resonance spectroscopic analyses supplemented by TLC‐MS analyses of derived neoglycolipids. A family of Manol‐terminating oligosaccharides has been revealed which includes novel sequences with a 2,6‐disubstituted Manol:

In contrast to the Manol‐terminating HNK‐1 antigen‐positive chains described previously that occur as a minor population [Yuen, C.‐T., Chai, W., Loveless, R.W., Lawson, A.M., Margolis, R.U. & Feizi, T. (1997) J. Biol. Chem.272, 8924–8931], the above oligosaccharides are abundant. The ratio of these compounds to the classical N‐acetylgalactosaminitol‐terminating oligosaccharides is about 1 : 3. Thus, there appears to be in higher eukaryotes a major alternative pathway related to the yeast‐type protein O‐mannosylation, the enzymatic basis and functional importance of which now require investigation.
Solution structures of a duplex containing an adenine opposite a gap (absence of one nucleotide)
Authors: Roll Claus , Ketterlé Christophe , Fazakerley G. Victor , Boulard Yves
Published: March 2003

We investigated the behaviour of a 15mer DNA duplex, [5′d(CAGAGTCACTGGCTC)3′]. [5′d(GAGCCAG)3′ + 5′d(GACTCTG)3′] which contained an adenine opposite the gap. Analysis of the NMR data showed the existence of one major species, which was in equilibrium with two minor species. Their relative concentrations varied as a function of pH with a pKa of ≈ 4.5. For the major species, the duplex was globally in B conformation with the central adenine stacked in the helix. The two G·C base pairs adjacent to the central adenine were well formed and a gap was present in front of this adenine. For the minor species, major structural perturbations occurred in the centre of the duplex. At neutral pH, the central adenine was involved in a G·A mismatch with G23 adjacent to the gap. Cytosine C7 was then extrahelical and no gap was observed. Under these conditions, the major neutral species corresponded to 70% of the total and the minor species to 30%. At acidic pH, the central adenine of the minor species was protonated and was involved in a G(syn)·A+(anti) mismatch. The difference is that C9 is now extrahelical and G22 is implicated in the mispair. Three‐dimensional models were built to initiate molecular dynamic simulations, which were in good agreement with the NMR data. Their structural stability in terms of hydrogen bonding and their flexibility are discussed and the biological significance for the interaction with DNA polymerase is evoked.
Mechanism of porcine pancreatic α‐amylase
Authors: Koukiekolo Roger , Le Berre‐Anton Véronique , Desseaux Véronique , Moreau Yann , Rougé Pierre , Marchis‐Mouren Guy , Santimone Marius
Published: March 2003

The effects of Phaseolus vulgaris inhibitor (α‐AI) on the amylose and maltopentaose hydrolysis catalysed by porcine pancreatic α‐amylase (PPA) were investigated. Based on a statistical analysis of the kinetic data and using the general velocity equation, which is valid at equilibrium for all types of inhibition in a single‐substrate reaction, it was concluded that the inhibitory mode is of the mixed noncompetitive type involving two molecules of inhibitor. In line with this conclusion, the Lineweaver–Burk primary plots intersect in the second quadrant and the secondary plots of the slopes and the intercepts versus the inhibitor concentrations are parabolic curves, whether the substrate used was amylose or maltopentaose. A specific inhibition model of the mixed noncompetitive type applies here. This model differs from those previously proposed for acarbose [Al Kazaz, M., Desseaux, V., Marchis‐Mouren, G., Payan, F., Forest, E. & Santimone, M. (1996) Eur. J. Biochem. 241, 787–796 and Al Kazaz, M., Desseaux, V., Marchis‐Mouren, G., Prodanov, E. & Santimone, M. (1998) Eur. J. Biochem.252, 100–107]. In particular, with α‐AI, the inhibition takes place only when PPA and α‐AI are preincubated together before the substrate is added. This shows that the inhibitory PPA–αAI complex is formed during the preincubation period. Secondly, other inhibitory complexes are formed, in which two molecules of inhibitor are bound to either the free enzyme or the enzyme–substrate complex. The catalytic efficiency was determined both with and without inhibitor. Using the same molar concentration of inhibitor, α‐AI was found to be a much stronger inhibitor than acarbose. However, when the inhibitor amount is expressed on a weight basis (mg·L−1), the opposite conclusion is drawn. In addition, limited proteolysis was performed on PPA alone and on the α‐AI–PPA complex. The results show that, in the complex, PPA is more sensitive to subtilisin attack, and shorter fragments are obtained. These data reflect the conformational changes undergone by PPA as the result of the protein inhibitor binding, which differ from those previously observed with acarbose.
Type I ‘antifreeze’ proteins
Authors: Harding Margaret M. , Ward Leanne G. , Haymet A. D. J.
Published: March 2003

The type I ‘antifreeze’ proteins, found in the body fluids of fish inhabiting polar oceans, are alanine‐rich α‐helical proteins that are able to inhibit the growth of ice. Within this class there are two distinct subclasses of proteins: those related to the winter flounder sequence HPLC6 and which contain 11‐residue repeat units commencing with threonine; and those from the sculpins that are unique in the N‐terminal region that contains established helix breakers and lacks the 11‐residue repeat structure present in the rest of the protein. Although 14 type I proteins have been isolated, almost all research has focused on HPLC6, the 37‐residue protein from the winter flounder Pseudopleuronectes americanus. This protein modifies both the rate and shape (or ‘habit’) of ice crystal growth, displays hysteresis and accumulates specifically at the {2 0 2¯ 1} ice plane. Until very recently, all models to explain the mechanism for this specific interaction have relied on the interaction of the four threonine hydroxyls, which are spaced equally apart on one face of the helix, with the ice lattice. In contrast, proteins belonging to the sculpin family accumulate specifically at the {2 1¯ 1¯ 0} plane. The molecular origin of this difference in specificity between the flounder and sculpin proteins is not understood. This review will summarize the structure–activity and molecular modelling and dynamics studies on HPLC6, with an emphasis on recent studies in which the threonine residues have been mutated. These studies have identified important hydrophobic contributions to the ice growth inhibition mechanism. Some 50 mutants of HPLC6 have been reported and the data is consistent with the following requirements for ice growth inhibition: (a) a minimum length of approx. 25 residues; (b) an alanine‐rich sequence in order to induce a highly helical conformation; (c) a hydrophobic face; (d) a number of charged/polar residues which are involved in solubility and/or interaction with the ice surface. The emerging picture, that requires further dynamics studies including accurate modelling of the ice/water interface, suggests that a hydrophobic interaction between the surface of the protein and ice is the key to explaining accumulation at specific ice planes, and thus the molecular level mechanism for ice growth inhibition.
Human formyl peptide receptor function
Authors: Lala Amitabha , Gwinn Maureen , De Nardin Ernesto
Published: March 2003

In this study, we investigated the role of charged residues in ligand binding interactions of f‐Met‐Leu‐Phe receptors (FPR). Charged residues of FPR, both conserved and nonconserved, which are located close to the membrane interface were mutated to alanine to determine their role in ligand binding. The mutated residues belonged to specific domains of FPR which have previously been implicated in FPR ligand binding interactions. We demonstrate that nonconserved charged residues such as Arg84, Lys85, Arg205 and Asp284 and conserved charge residue Arg163 seem to play a role in ligand binding. However, alteration of nonconserved charged residue Asp106 did not have any effect. In conclusion, specific charged residues of FPR, both conserved nonconserved, may contribute to FPR function either directly or indirectly.
Remodeling of the AB site of rat parvalbumin and oncomodulin into a canonical EF‐hand
Authors: Cox Jos A. , Durussel Isabelle , Scott David J. , Berchtold Martin W.
Published: March 2003

Parvalbumin (PV) and the homologous protein oncomodulin (OM) contain three EF‐hand motifs, but the first site (AB) cannot bind Ca2+. Here we aimed to recreate the putative ancestral proteins [D19‐28E]PV and [D19‐28E]OM by replacing the 10‐residue‐long nonfunctional loop in the AB site by a 12‐residue canonical loop. To create an optical conformational probe we also expressed the homologs with a F102W replacement. Unexpectedly, in none of the proteins did the mutation reactivate the AB site. The AB‐remodeled parvalbumins bind two Ca2+ ions with strong positive cooperativity (nH = 2) and moderate affinity ([Ca2+]0.5 = 2 µm), compared with [Ca2+]0.5 = 37 nm and nH = 1 for the wild‐type protein. Increasing Mg2+ concentrations changed nH from 2 to 0.65, but without modification of the [Ca2+]0.5‐value. CD revealed that the Ca2+ and Mg2+ forms of the remodeled parvalbumins lost one‐third of their α helix content compared with the Ca2+ form of wild‐type parvalbumin. However, the microenvironment of single Trp residues in the hydrophobic cores, monitored using intrinsic fluorescence and difference optical density, is the same. The metal‐free remodeled parvalbumins possess unfolded conformations. The AB‐remodeled oncomodulins also bind two Ca2+ with [Ca2+]0.5 = 43 µm and nH = 1.45. Mg2+ does not affect Ca2+ binding. Again the Ca2+ forms display two‐thirds of the α‐helical content in the wild‐type, while their core is still strongly hydrophobic as monitored by Trp and Tyr fluorescence. The metal‐free oncomodulins are partially unfolded and seem not to possess a hydrophobic core. Our data indicate that AB‐remodeled parvalbumin has the potential to regulate cell functions, whereas it is unlikely that [D19‐28E]OM can play a regulatory role in vivo. The predicted evolution of the AB site from a canonical to an abortive EF‐hand may have been dictated by the need for stronger interaction with Mg2+ and Ca2+, and a high conformational stability of the metal‐free forms.
Fluorescence and FTIR study of the pressure‐induced denaturation of bovine pancreas trypsin
Authors: Ruan Kangcheng , Lange Reinhard , Meersman Filip , Heremans Karel , Balny Claude
Published: March 2003

The pressure denaturation of trypsin from bovine pancreas was investigated by fluorescence spectroscopy in the pressure range 0.1–700 MPa and by FTIR spectroscopy up to 1000 MPa. The tryptophan fluorescence measurements indicated that at pH 3.0 and 0 °C the pressure denaturation of trypsin is reversible but with a large hysteresis in the renaturation profile. The standard volume changes upon denaturation and renaturation are −78 mL·mol−1 and +73 mL·mol−1, respectively. However, the free energy calculated from the data in the compression and decompression directions are quite different in absolute values with + 36.6 kJ·mol−1 for the denaturation and −5 kJ·mol−1 for the renaturation. For the pressure denaturation at pH 7.3 the tryptophan fluorescence measurement and enzymatic activity assays indicated that the pressure denaturation of trypsin is irreversible. Interestingly, the study on 8‐anilinonaphthalene‐1‐sulfonate (ANS) binding to trypsin under pressure leads to the opposite conclusion that the denaturation is reversible. FTIR spectroscopy was used to follow the changes in secondary structure. The pressure stability data found by fluorescence measurements are confirmed but the denaturation was irreversible at low and high pH in the FTIR investigation. These findings confirm that the trypsin molecule has two domains: one is related to the enzyme active site and the tryptophan residues; the other is related to the ANS binding. This is in agreement with the study on urea unfolding of trypsin and the knowledge of the molecular structure of trypsin.
Location of the catalytic nucleophile of phospholipase D of Streptomyces antibioticus in the C‐terminal half domain
Authors: Iwasaki Yugo , Horiike Satoko , Matsushima Kayoko , Yamane Tsuneo
Published: March 2003

Phospholipase D (PLD) of Streptomyces antibioticus was labelled with fluorescent‐labelled substrate, 1‐hexanoyl‐2‐{6‐[(7‐nitro‐2‐1,3‐benzoxadiazol‐4‐yl)‐amino]hexanoyl}‐sn‐glycero‐3‐phosphocholine, when it was incubated with the substrate and the reaction followed by SDS/PAGE. Mutant enzymes lacking the catalytic activity were not labelled under the same conditions, indicating that labelling of the PLD occurred as the result of its catalytic action. This confirmed that the labelled protein was the phosphatidyl PLD intermediate. PLDs contain two copies of the highly conserved catalytic HxKxxxxD (HKD) motif. Therefore, two protein fragments were separately prepared with recombinant strains of Escherichia coli. One of the fragments was the N‐terminal half of the intact PLD containing one HKD motif, and the other was the C‐terminal half with the other motif. An active enzyme was reconstructed from these two fragments, and therefore designated fragmentary PLD (fPLD). When fPLD was subjected to the labelling experiment, only the C‐terminal half was labelled. Therefore, it was concluded that the catalytic nucleophile that bound directly to the phosphatidyl group of the substrate was located on the C‐terminal half of PLD, and that the N‐terminal half did not contain such a nucleophile.
Functional complementation analysis of yeastbc1mutants
Authors: van Wilpe Sandra , Boumans Hans , Lobo‐Hajdu Gisele , Grivell Leslie A. , Berden Jan A.
Published: March 2003

Previous complementation studies with yeastbc1mutants, defective in subunit VII or VIII, using heterologous and hybrid subunits, suggested that the requirement for import into mitochondria might significantly restrict the scope of this test for compatible proteins. Prediction algorithms indicate that the N‐terminal domain of subunit VII contains all known characteristics of a mitochondrial targeting signal, whereas in subunit VIII such a signal is absent from the N‐terminal domain, but possibly present in an internal region of the protein. Despite the fact that the characteristics of a mitochondrial import signal are found in the N‐terminus of all known subunit‐VII orthologues,in vitroimport experiments show that the protein of human origin is not imported into yeast mitochondria.In vitroimport can be restored, however, by replacement of the N‐terminal part of the human protein by the N‐terminus of theSaccharomyces cerevisiaeorthologue, indicating a requirement for species‐specific elements.

 Similar experiments were performed with subunit VIII and orthologues thereof, including a hybrid protein in which the N‐terminus of the bovine heart orthologue was replaced by that ofS. cerevisiae. The ability of yeast mitochondria to import this hybrid protein, in contrast with the bovine subunit‐VIII orthologue itself, indicates that for subunit VIII also the N‐terminus, in contradiction of theoretical predictions, contributes to the targeting signal, most likely via species‐specific elements.

 Our findings expose the limitations of the currently available criteria for prediction of the presence and location of a mitochondrial targeting sequence and highlight the necessity of performing separate import studies for interpreting complementation studies as long as the species‐specific characteristics of the import signals have not been identified.
Expression, purification and copper‐binding studies of the first metal‐binding domain of Menkes protein
Authors: Jensen Pia Y. , Bonander Nicklas , Horn Nina , Tümer Zeynep , Farver Ole
Published: March 2003

The cDNA, coding for the first metal‐binding domain (MBD1) of Menkes protein, was cloned into the T7‐system based vector, pCA. The T7 lysozyme‐encoding plasmid, pLysS, is shown to be crucial for expression, suggesting that the protein is toxic to the cells. Adding copper to the growth medium did not affect the plasmid stability. MBD1 is purified in two steps with a typical yield of 12 mg·L−1.

 Menkes protein, a P‐type ATPase, contains a sequence GMXCXSC that is repeated six times, at the N‐terminus. The paired cysteine residues are involved in metal binding. MBD1 has only two cysteine residues, which can exist as free thiol groups (reduced), as a disulphide bond (oxidized) or bound to a metal ion [e.g. Cu(I)‐MBD1]. These three MBD1 forms have been investigated using CD. No major spectral change was seen between the different MBD1 forms, indicating that the folding is not changed upon metal binding.

 A copper‐bound MBD1 was also studied by EPR, and the lack of an EPR signal suggests that the oxidation state of copper bound to MBD1 is Cu(I). Cu(I) binding studies were performed by equilibrium dialysis and revealed a stoichiometry of 1 : 1 and an apparent Kd = 46 µm. Oxidized MBD1, however, is not able to bind copper. Different copper complexes were investigated for their ability to reconstitute apo–MBD1. Given the same total copper concentration CuCl43– was superior to Cu(I)–thiourea (structural analogue of metallothionein) and Cu(I)–glutathione (used at fivefold higher copper concentration) although the latter two were able to partially reconstitute apo–MBD1. Cu(II) was not able to reconstitute apo–MBD1, presumably due to Cu(II)‐induced oxidation of the thiol groups. Based on our results, glutathione and/or metallothionein are likely candidates for the in vivo incorporation of copper to Menkes protein.