bluuebunny/crossref_metadata_2025 huggingface dataset:
Biochemical and structural characterization of recombinant copper‐metallothionein from Saccharomyces cerevisiae
Authors: Sayers Zehra , Brouillon Patricia , Svergun Dimitri I. , Zielenkiewicz Piotr , Koch Michel H. J.
Published: March 2003

Methods were developed for large‐scale purification of recombinant Cu‐metallothionein (Cu‐MT) for structural investigations and the determination of Cu‐binding stoichiometry. Cu‐MT of Saccharomyces cerevisiae overexpressed in Escherichia coli was purified using a procedure based on ion exchange and gel filtration chromatography followed by reversed‐phase HPLC. The purified protein was fully characterized by electrophoresis, amino acid analysis, atomic absorption spectroscopy and elemental analysis, and was shown to contain 10 ± 2 Cu(I) per molecule of protein. Small angle X‐ray scattering measurements yielded a radius of gyration of 1.2 nm for the recombinant protein, indicating a more extended structure in solution than that derived from the recent NMR data [Peterson, C.W., Narula, S.S. & Armitage, I.A. (1996) FEBS Lett.379, 85–93].
Effect of high hydrostatic pressures on 20S proteasome activity
Authors: Gardrat Florence , Fraigneau Bettina , Montel Valérie , Raymond Jacques , Azanza Jean‐Louis
Published: March 2003

The 20S proteasome is the catalytic core of the ubiquitin proteolytic pathway, which is implicated in many cellular processes. The cylindrical structure of this complex consists of four stacked rings of seven subunits each. The central cavity is formed by two β catalytic subunit rings in which protein substrates are progressively degraded. The 20S proteasome is isolated in a latent form which can be activated in vitro by various chemical and physical treatments. In this study, the effects of high hydrostatic pressures on 20S proteasome enzymatic activity were investigated. When proteasomes were subjected to increasing hydrostatic pressures, a progressive loss of peptidase activities was observed between 75 and 150 MPa. The inactivation also occurred when proteasomes were pressurized in the presence of synthetic peptide substrates; this may be the result of the dissociation of the 20S particle into its subunits under pressure, as was shown by PAGE. Pressurized proteasomes also lost their caseinolytic activity. In contrast, in the presence of casein, the pressure‐induced inactivation and the dissociation of the 20S particles were prevented. In addition, in comparison to that observed at atmospheric pressure, their caseinolytic activity was increased under pressure. Following depressurization, the caseinolytic activity returned to basal levels but was further enhanced following an additional pressurization treatment. Thus, the structure of the 20S particle exhibits a certain degree of plasticity. This pressure‐induced activation of the 20S proteasome is discussed in relation to its hollow structure, its currently accepted proteolytic mechanism and the general effect of high pressures on the biochemical reactions and structures of biopolymers.
Analysis of functional regions of YPM, a superantigen derived from gram‐negative bacteria
Authors: Ito Yasuhiko , Seprényi György , Abe Jun , Kohsaka Takao
Published: March 2003

The bacterial superantigens, staphylococcal enterotoxins and streptococcal pyrogenic exotoxins, are grouped in a family by the conservation of amino acid sequence and polypeptide folding patterns. In the case of Yersinia pseudotuberculosis‐derived mitogen (YPM), however, there is no noticeable homology with this family, although many of the in vitro functional features conform to the criteria for a superantigen. To study the mode of action of YPM at the molecular level, we first generated a number of YPM point mutants with reduced T‐cell proliferative activity using random mutagenesis and localized the amino acid positions involved in either major histocompatibility complex class II or T‐cell receptor Vβ‐interaction. Plotting the elucidated positions on the hydrophilicity profile suggested that they reside mostly on the outer portion of the molecule. We also report that the two cysteines positioned almost at opposing ends of the YPM molecule are connected by an S–S bond the destruction of which causes fatal damage. Finally, we obtained evidence that YPM partially competes with staphylococcal enterotoxin E for human leukocyte antigen‐DR binding. This raises the question of whether these different types of superantigens have acquired the same function by genetic convergence or originated from a common ancestral gene.
Ionophoretic properties and mitochondrial effects of cereulide
Authors: Mikkola Raimo , Saris Nils‐Erik L. , Grigoriev Pavel A. , Andersson Maria A. , Salkinoja‐Salonen Mirja S.
Published: March 2003

The emetic toxin of Bacillus cereus, found to cause immobilization of spermatozoa and swelling of their mitochondria, was purified and its structure found to be identical to the earlier known toxin cereulide. It increased the conductance in black‐lipid membranes in KCl solutions in an ionophore‐like manner. It formed adducts with K+, Na+, and NH4+ but the conductance was highly selective for K+ in relation to Na+ and H+ (three orders of magnitude). The increase in the kinetics of conductance indicated a stoichiometric ratio between the cereulide and K+. Its ionophoretic properties are thus similar to those of valinomycin. In addition, its effects on rat liver mitochondria were similar: it stimulated swelling and respiration in respiring mitochondria in the presence but not in the absence of K+, it reduced the transmembrane potential under these conditions. In nonrespiring mitochondria, swelling was seen in KNO3‐ but not in NaNO3‐containing media, less in acetate. In NaNO3 media addition of the cereulide caused a transient diffusion potential which was reduced by adding K+. It is concluded that the toxic effects of cereulide are due to it being a K+ ionophore.
The concentration of adrenodoxin reductase limits cytochrome P450scc activity in the human placenta
Authors: Tuckey Robert C. , Sadleir Jade
Published: March 2003

We have previously reported that cytochrome P450scc activity in the human placenta is limited by the supply of electrons to the P450scc [Tuckey, R. C., Woods, S. T. & Tajbakhsh, M. (1997) Eur. J. Biochem.244, 835–839]. The aim of the present study was to determine whether it is adrenodoxin reductase, adrenodoxin or both which limits cytochrome P450scc activity and hence progesterone synthesis in the placenta. We found that the concentrations of adrenodoxin reductase and adrenodoxin in placental mitochondria were both considerably lower than the concentrations of these proteins in the bovine adrenal cortex. When P450scc activity assays were carried out at high mitochondrial protein concentrations, we found that the addition of exogenous adrenodoxin reductase to sonicated mitochondria rescued pregnenolone synthesis to a level above that for intact mitochondria, showing that adrenodoxin is near‐saturating in vivo. In contrast, pregnenolone synthesis by sonicated mitochondria was almost zero even after the addition of human adrenodoxin. This shows that the concentration of endogenous adrenodoxin reductase was insufficient to support appreciable rates of pregnenolone synthesis, even when concentrated mitochondrial samples were used. Comparative studies with human and bovine adrenodoxin reductase have revealed that a twofold higher concentration of human adrenodoxin reductase is required for maximal P450scc activity in the presence of saturating human adrenodoxin. Thus, not only is the adrenodoxin concentration low in placental mitochondria, but the amount required for maximal P450scc activity is higher than that for the bovine reductase. Overall, the data indicate that the adrenodoxin reductase concentration limits the activity of P450scc in placental mitochondria and hence determines the rate of progesterone synthesis.
α Subunit compositions of Kv1.1‐containing K+channel subtypes fractionated from rat brain using dendrotoxins
Authors: Wang Fan C. , Parcej David N. , Dolly J. Oliver
Published: March 2003

K+channels from the Kv1 subfamily contain four α‐subunits and the combinations (from Kv1.1–1.6) determine susceptibility to dendrotoxin (DTX) homologues. The subunit composition of certain subtypes in rat brain was investigated using DTXkwhich only interacts with Kv1.1‐containing channels and αDTX (and its closely related homologue DTXi) that binds preferentially to Kv1.2‐possessing homo‐ or hetero‐oligomers. Covalent attachment of [125I]DTXkbound to channels in synaptic membranes unveiled subunits ofMr = 78 000 and 96 000. Immunoprecipitation of these solubilized and dissociated cross‐linked proteins with IgG specific for each of the α‐subunits identified Kv1.1, 1.2 and 1.4; this led to assemblies of Kv1.1/1.2 and 1.1/1.4 being established. Kv1.2‐enriched channels, purified from rat brain by chromatography on immobilized DTXi, contained Kv1.1, 1.2 and 1.6 confirming one of the above‐noted pairs and indicating an additional Kv1.1‐containing oligomer (Kv1.1/1.2/1.6); the notable lack of Kv1.4 excludes a Kv1.1/1.2/1.4 combination. On the other hand, channels with Kv1.1 as a constituent, isolated using DTXk, possessed Kv1.4 in addition to those found in the DTXi‐purified oligomers; this provides convergent support for the occurrence of the three combinations established above but adds a possible fourth (Kv1.1/1.4/1.6), though this was not confirmed. Moreover, sequential purification on DTXiand DTXkresins yielded channels containing only Kv1.1/1.2 but with an apparent predominance of Kv1.1, reaffirming the latter multimer.
Rhodamine phalloidin F‐actin
Authors: Adami Raffaella , Choquet Daniel , Grazi Enrico
Published: March 2003

The mechanic and elastic properties of rhodamine phalloidin F‐actin were investigated as a function of the ionic strength and in the absence of Mg2+. By increasing ionic strength from 3 to 19 mm, critical concentration decreased from 146 to 36 nm and the yield strength increased from 5.6 pN to 28.6 pN. At the ionic strength of 12–13 mm, the elastic modulus by stretching increased by 330–430 kP·nm−1 up to the break point, where it was 38–44.2 MP. The work required to break the filament, 403–439 kJ·m−1 provides an estimate of the free energy of annealing of rhodamine phalloidin F‐actin, the annealing constant being 2.8 × 1074 m−1.
Detection and characterization of a mannan‐binding lectin from the mosquito, Anopheles stephensi (Liston)
Authors: Chen Changlin , Billingsley Peter F.
Published: March 2003

Two lectins from the serum of the mosquito, Anopheles stephensi (Liston), with distinct characteristics, were detected by agglutination of various animal erythrocytes. The lectins were developmental stage‐specific and/or sex‐related. One adult female‐specific lectin was identified as mannan‐specific, and named mosquito mannan‐binding lectin (MBL). MBL cross‐reacted immunologically with antibodies against a previously characterized cockroach lectin, Blaberus discoidalis lectin (BDL1), and its activity was almost completely blocked by the antibodies. Mosquito MBL agglutinated erythrocytes from human, sheep, goat and rabbit, but not chicken or mouse, and agglutination was inhibited by mannan and nitrophenol‐modified sugar derivatives, but not by simple sugars. Using affinity chromatography with immobilized mannan on Sepharose 6B, the mosquito MBL was partially purified. Purified mosquito MBL shared biochemical properties with BDL1, containing two subunits of molecular mass of 28 and 30 kDa under reducing conditions in SDS/PAGE. Its activity is dependent on Ca2+, and it is stable at pH 7–9 and at temperatures less than 30 °C.
Structural and kinetic analysis of drug resistant mutants of HIV‐1 protease
Authors: Mahalingam Bhuvaneshwari , Louis John M. , Reed Charles C. , Adomat Jill M. , Krouse Jennifer , Wang Yuan‐Fang , Harrison Robert W. , Weber Irene T.
Published: March 2003

Mutants of HIV‐1 protease that are commonly selected on exposure to different drugs, V82S, G48V, N88D and L90M, showed reduced catalytic activity compared to the wild‐type protease on cleavage site peptides, CA‐p2, p6pol‐PR and PR‐RT, critical for viral maturation. Mutant V82S is the least active (2–20% of wild‐type protease), mutants N88D, R8Q, and L90M exhibit activities ranging from 20 to 40% and G48V from 50 to 80% of the wild‐type activity. In contrast, D30N is variable in its activity on different substrates (10–110% of wild‐type), with the PR‐RT site being the most affected. Mutants K45I and M46L, usually selected in combination with other mutations, showed activities that are similar to (60–110%) or greater than (110–530%) wild‐type, respectively. No direct relationship was observed between catalytic activity, inhibition, and structural stability. The mutants D30N and V82S were similar to wild‐type protease in their stability toward urea denaturation, while R8Q, G48V, and L90M showed 1.5 to 2.7‐fold decreased stability, and N88D and K45I showed 1.6 to 1.7‐fold increased stability. The crystal structures of R8Q, K45I and L90M mutants complexed with a CA‐p2 analog inhibitor were determined at 2.0, 1.55 and 1.88 Å resolution, respectively, and compared to the wild‐type structure. The intersubunit hydrophobic contacts observed in the crystal structures are in good agreement with the relative structural stability of the mutant proteases. All these results suggest that viral resistance does not arise by a single mechanism.
Molecular cloning of a human UDP‐galactose:GlcNAcβ1,3GalNAc β1,3 galactosyltransferase gene encoding an O‐linked core3‐elongation enzyme
Authors: Zhou Dapeng , Berger Eric G. , Hennet Thierry
Published: March 2003

Using the full‐length amino‐acid sequences of the human β1,3 galactosyltransferase (β3GalT)‐I, ‐II and III enzymes as query, we have identified an additional member of the β3GalT gene family within a sequenced region of the human chromosome 21 as found in GenBank. The novel human β3GalT‐V gene included an open reading frame of 933 bp encoding a protein of 310 amino acids with a short N‐terminal cytoplasmic tail, a single predicted transmembrane domain and a large lumenal catalytic domain. The human β3GalT‐V protein showed 34%, 27%, 31% and 23% sequence identity with the human β3GalT‐I, ‐II, ‐III and ‐IV enzymes, respectively. The expression of β3GalT‐V as a recombinant protein in Sf9 insect cells confirmed the galactosyltransferase activity catalyzed by this enzyme. Similarly to β3GalT‐I, ‐II and ‐III, the β3GalT‐V enzyme used β‐linked GlcNAc as an acceptor, but unlike the former enzymes β3GalT‐V exhibited a marked preference for the O‐linked core3 GlcNAcβ1,3GalNAc substrate. The β3GalT‐V gene was mainly expressed in human small intestine and to a lesser extent in pancreas and testis. Although β3GalT‐V transcripts were not detected in normal colon tissue, based on Northern analysis, β3GalT‐V mRNA was found in the adenocarcinoma cell line Colo 205.
Cyclic AMP‐dependent synthesis and release of adrenomedullin and proadrenomedullin N‐terminal 20 peptide in cultured bovine adrenal chromaffin cells
Authors: Kobayashi Hideyuki , Yamamoto Ryuichi , Kitamura Kazuo , Niina Hiromi , Masumoto Keizo , Minami Shin‐ichi , Yanagita Toshihiko , Izumi Futoshi , Aunis Dominique , Eto Tanenao , Wada Akihiko
Published: March 2003

Adrenomedullin and proadrenomedullin N‐terminal 20 peptide are peptides with multiple physiological functions and are most abundant in adrenal medulla. We studied whether the cAMP‐dependent pathway is involved in the regulation of synthesis and release of adrenomedullin and proadrenomedullin N‐terminal 20 peptide in cultured bovine adrenal chromaffin cells. Exposure of the cells to dibutyryl cAMP (dbcAMP) increased a progressive accumulation of immunoreactive‐adrenomedullin and immunoreactive‐proadrenomedullin N‐terminal 20 peptide in the extracellular medium, while reciprocally decreasing their cellular content in a time‐dependent manner. The decrease of levels of both peptides in the cells was much greater in extent than the increase of the peptides in the medium. H89, an inhibitor of cAMP‐dependent protein kinase attenuated these changes, induced by dbcAMP. The resulting changes by dbcAMP and H89 were similar to those of chromogranin B, a marker peptide of chromaffin granule. Northern blot analysis showed that the mRNA encoding these peptides, detected as a band of 1.6 kb, was decreased by the treatment with dbcAMP. The effect of dbcAMP on mRNA was attenuated by H89, and was reversible as the decreased mRNA level caused by dbcAMP could be returned to control levels by culturing cells after removal of dbcAMP. These results suggest that the cAMP‐dependent protein kinase pathway stimulates the release of adrenomedullin and proadrenomedullin N‐terminal 20 peptide, whereas it lowers synthesis of these peptides via the reduction of their transcript level.
Kinetic mechanisms of cholesterol oxidase fromStreptomyces hygroscopicusandBrevibacterium sterolicum
Authors: Pollegioni Loredano , Wels Gaby , Pilone Mirella S. , Ghisla Sandro
Published: March 2003

The kinetic properties of two cholesterol oxidases, one fromBrevibacterium sterolicum(BCO) the other fromStreptomyces hygroscopicus(SCO) were investigated. BCO works via a ping‐pong mechanism, whereas the catalytic pathway of SCO is sequential. The turnover numbers at infinite cholesterol and oxygen concentrations are 202 s−1and 105 s−1for SCO and BCO, respectively. The rates of flavin reduction extrapolated to saturating substrate concentration, under anaerobic conditions, are 235 s−1for BCO and 232 s−1for SCO (in the presence of 1% Thesit and 10% 2‐propanol). With reduced SCO the rate of Δ5‐6→Δ4‐5 isomerization of the intermediate 5‐cholesten‐3‐one to final product is slow (0.3 s−1). With oxidized SCO and BCO the rate of isomerization is much faster (≈ 300 s−1), thus it is not rate‐limiting for catalysis. The kinetic behaviour of both reduced COs towards oxygen is unusual in that they exhibit apparent saturation with increasing oxygen concentrations (extrapolated rates ≈ 250 s−1and 1.3 s−1, for BCO and SCO, respectively): too slow to account for catalysis. For BCO the kinetic data are compatible with a step preceding the reaction with oxygen, involving interconversion of reactive and nonreactive forms of the enzyme. We suggest that the presence of micelles in the reaction medium, due to the necessary presence of detergents to solubilize the substrate, influence the availability or reactivity of oxygen towards the enzyme. The rate of re‐oxidation of SCO in the presence of product is also too slow to account for catalysis, probably due to the impossibility of producing quantitatively the reduced enzyme–product complexes.
Inhibition of tyrosine phosphatases antagonizes CD95‐mediated apoptosis
Authors: Hehner Steffen P. , Hofmann Thomas G. , Ratter Frank , Dröge Wulf , Schmitz M. Lienhard
Published: March 2003

Ligation of the CD95 receptor resulted in a transient increase of cellular tyrosine phosphorylation. The inhibition of protein tyrosine phosphatases by pervanadate, a potent activator of B cells and T cells through the induction of tyrosine phosphorylation and downstream signaling events in the activation cascade, antagonized CD95‐triggered apoptosis. Pervanadate exerted its inhibitory effect only during the early phase of apoptosis prior to the CD95‐induced decrease of the mitochondrial transmembrane potential. Inhibition of tyrosine phosphatases delayed the cleavage and activation of caspase‐8 and caspase‐3 and antagonized the tyrosine dephosphorylation of the CD95 receptor‐associated phosphoproteins p61 and p89/92. In contrast, ligation of the tumor necrosis factor (TNF) receptor resulted in a continuous tyrosine dephosphorylation of cellular proteins. Pervanadate‐induced tyrosine phosphorylation increased the TNF‐α‐induced cytotoxicity and NF‐κB activation, suggesting that it stimulates early signaling events prior to the separation of the two signaling pathways.
Characterization of the Asp94 and Glu242 mutants in myeloperoxidase, the residues linking the heme group via ester bonds
Authors: Kooter Ingeborg M. , Moguilevsky Nicole , Bollen Alex , Sijtsema Nanna M. , Otto Cees , Dekker Henk L. , Wever Ron
Published: March 2003

The heme group of all mammalian peroxidases is covalently linked to the protein matrix via two esterbonds, as we have recently shown by Fourier transform infrared (FTIR) difference spectroscopy [Kooter, I.M., Pierik, A.J., Merkx, M., Averill, B.A., Moguilevsky, N., Bollen, A. & Wever, R. (1997) J. Am. Chem. Soc.119, 11542–11543]. We have examined the effects of mutation of Asp94 and Glu242, responsible for those ester bonds in myeloperoxidase, on the spectroscopic properties and catalytic activity of this enzyme. Mutation of Asp94 in myeloperoxidase results in two species. The first species has spectroscopic characteristics similar to that of wild‐type myeloperoxidase. The second species has spectroscopic characteristics similar to that of Met243→Gln mutant, and it is therefore concluded that, besides loss of the ester bond involving Asp94, this species also has lost the sulfonium ion linkage that is also characteristic of myeloperoxidase. The Asp94→Asn mutant still has about 30% residual peroxidase activity while for the Asp94→Val mutant only a few percentage activity is left. When Glu242 is mutated the sulfonium ion linkage is not affected, but this residue together with its neighbouring residue Met243, according to resonance Raman spectra, is responsible for the low symmetry of the heme group. Mutation of either of these residues results in loss of the bowed distortion from the planar conformation, and in a heme group with higher symmetry. For the Glu242→Gln mutant 8% residual peroxidase activity is found.
Xenopus laevis occludin
Authors: Cordenonsi Michelangelo , Turco Franca , D’atri Fabio , Hammar Eva , Martinucci Giambruno , Meggio Flavio , Citi Sandra
Published: March 2003

Occludin is a protein component of the membrane domain of tight junctions, and has been shown to be phosphorylated in vivo in cultured cells and Xenopus laevis embryos. However, nothing is known about the identity of specific occludin kinase(s) and occludin phosphorylation site(s). Furthermore, nothing is known about the interaction of occludin with cingulin, a cytoplasmic plaque component of tight junctions. Here we report the isolation and sequencing of a complete X. laevis occludin cDNA, and experiments aimed at mapping X. laevis occludin in vitro phosphorylation site(s) and characterizing occludin interaction with cingulin. The sequence of Xenopus occludin is homologous to that of occludins from other species, with identities ranging from 41% to 58%. Bacterially expressed domain E of Xenopus occludin (amino acids 247–493) was a good substrate for protein kinase CK2 (stoichiometry 10.8%, Km 8.4 μm) but not for CK1 kinase, protein kinase A, cdc2 kinase, MAP kinase or syk kinase. Residues Thr375 and Ser379 were identified as potential CK2 phosphorylation sites in this region based on sequence analysis. Mutation of Ser379 to aspartic acid or alanine reduced phosphorylation by CK2 by ≈ 50%, and double mutation of Ser379 into aspartic acid and Thr375 into aspartic acid essentially abolished phosphorylation. Glutathione S‐transferase (GST) pull‐down experiments using extracts of Xenopus A6 epithelial cells showed that constructs of GST fused to wild‐type and mutant forms of the C‐terminal region of X. laevis occludin associate with several polypeptides, and immunoblot analysis showed that one of these polypeptides is cingulin. GST pull‐down experiments using in vitro translated, full‐length Xenopus cingulin indicated that cingulin interacts directly with the C‐terminal region of occludin.
Comparison of expression patterns and cell adhesion properties of the mouse biliary glycoproteins Bgp1 and Bgp2
Authors: Robitaille Julie , Izzi Luisa , Daniels Eugene , Zelus Bruce , Holmes Kathryn V. , Beauchemin Nicole
Published: March 2003

Biliary glycoproteins are members of the carcinoembryonic antigen (CEA) family and behave as cell adhesion molecules. The mouse genome contains two very similar Bgp genes, Bgp1 and Bgp2, whereas the human and rat genomes contain only one BGP gene. A Bgp2 isoform was previously identified as an alternative receptor for the mouse coronavirus mouse hepatitis virus. This isoform consists of two extracellular immunoglobulin domains, a transmembrane domain and a cytoplasmic tail of five amino acids. In this report, we have examined whether the Bgp2 gene can express other isoforms in different mouse tissues. We found only one other isoform, which has a long cytoplasmic tail of 73 amino acids. The long cytodomain of the Bgp2 protein is highly similar to that of the Bgp1/4L isoform. The Bgp2 protein is expressed in low amounts in kidney and in a rectal carcinoma cell line. Antibodies specific to Bgp2 detected a 42‐kDa protein, which is expressed at the cell surface of these samples. Bgp2 was found by immunocytochemistry in smooth muscle layers of the kidney, the uterus, in gut mononuclear cells and in the crypt epithelia of intestinal tissues. Transfection studies showed that, in contrast with Bgp1, the Bgp2 glycoprotein was not directly involved in intercellular adhesion. However, this protein is found in the proliferative compartment of the intestinal crypts and in cells involved in immune recognition. This suggests that the Bgp2 protein represents a distinctive member of the CEA family; its unusual expression patterns in mouse tissues and the unique functions it may be fulfilling may provide novel clues about the multiple functions mediated by a common BGP protein in humans and rats.
Structural bistability of repetitive DNA elements featuring CA/TG dinucleotide steps and mode of evolution of satellite DNA
Authors: Kato Mikio
Published: March 2003

Satellite DNA sequences are known to be important components required for the construction of centromeres and are common to all higher eukaryotes. Nevertheless, their nucleotide sequences vary significantly, even in evolutionarily related species. In order to elucidate how the nucleotide sequences define the conformational character of centromeric satellite DNA, an evolutionary path toward repetitive units has been hypothesized. In that context, the DNA conformation of fish satellite DNA was evaluated in two ways: the organization of subrepeats and sequence characteristics were compared, and the differences in stacking energies between A‐helix and B‐helix and the sequence‐dependent bendability of the helices were evaluated. Our findings suggest that the monomeric units making up currently observed repetitive sequences have evolved through stepwise amplification of shorter, ancestral sequences by increasing the length of the units. In addition, we suggest that potentially key sequences required for DNA amplification comprise highly flexible structures. Thus flexibility of the DNA structure may be a primary prerequisite for DNA amplification.
Purification of the human apical conjugate export pump MRP2
Authors: Hagmann Wolfgang , Nies Anne T. , König Jörg , Frey Manfred , Zentgraf Hanswalter , Keppler Dietrich
Published: March 2003

The multidrug resistance protein MRP2 (ABCC2) acts as an ATP‐dependent conjugate export pump in apical membranes of polarized cells and confers multidrug resistance. Purified MRP2 is essential for the detailed functional characterization of this member of the family of ATP‐binding cassette (ABC) transporter proteins. In human embryonic kidney cells (HEK293), we have permanently expressed MRP2 containing an additional C‐terminal (His)6‐tag. Immunoblot and immunofluorescence analyses detected the MRP2–(His)6 overexpressing clones. Isolated membrane vesicles from the MRP2–(His)6‐expressing cells were active in ATP‐dependent transport of the glutathione S‐conjugate leukotriene C4 and were photoaffinity‐labelled with 8‐azido‐[α‐32P]ATP. MRP2–(His)6 was solubilized from membranes of MRP2–(His)6‐cells and purified to homogeneity in a three‐step procedure using immobilized metal affinity chromatography, desalting, and immunoaffinity chromatography. The identity of the pure MRP2–(His)6 was verified by MS analysis of tryptic peptides. The purified MRP2–(His)6 glycoprotein was reconstituted into proteoliposomes and showed functional activity as ATPase in a protein‐dependent manner with a Km for ATP of 2.1 mm and a Vmax of 25 nmol ADP·mg MRP2−1·min−1. This ATPase activity was substrate‐stimulated by oxidized and reduced glutathione and by S‐decyl‐glutathione. Future studies using pure MRP2 reconstituted in proteoliposomes should allow further insight into the molecular parameters contributing to MRP2 transport function and to define its intracellular partners for transport and multidrug resistance.
Denaturation of apolipoprotein A‐I and the monomer form of apolipoprotein A‐IMilano
Authors: Suurkuusk Malin , Hallén Dan
Published: March 2003

In this study the thermal and denaturant induced unfolding of apolipoprotein A‐I (apo A‐I) and the monomer form of apolipoprotein A‐IMilano (apo A‐IM) was followed. Dimer apo A‐IM was reduced with dithiothreitol, which was present in the protein solutions in all experiments. Thermal denaturation is followed by differential scanning calorimetry (DSC) and far‐UV and near‐UV CD. Both apo A‐I and monomer apo A‐IM have a broad asymmetric DSC peak that could be deconvoluted into three non two‐state transitions, apo A‐I being more stable than the monomer apo A‐IM. Estimation of melting of tertiary structure by near‐UV CD is lower than that for secondary structure determined from far‐UV. This together with the non two‐state unfolding of the proteins observed with DSC is indicative of unfolding via a molten globular‐like state.

 Apo A‐I and monomer apo A‐IM are equally susceptible to guanidinum chloride, half‐unfolded at 1.2 m denaturant. The presence of 0.5 and 1.0 m denaturant, lower and equalize the denaturation temperatures of the proteins, respectively.
Pearl millet cysteine protease inhibitor
Authors: Joshi Bimba N. , Sainani Mohini N. , Bastawade Kulbhushan B. , Deshpande Vasanti V. , Gupta Vidya S. , Ranjekar Prabhakar K.
Published: March 2003

Recently, pearl millet cysteine protease inhibitor (CPI) was, for the first time, shown to possess anti‐fungal activity in addition to its anti‐feedent (protease inhibitory) activity [Joshi, B.N. et al. (1998) Biochem. Biophys. Res. Commun. 246, 382–387]. Characterization of CPI revealed that it has a reversible mode of action for protease inhibition. The CD spectrum exhibited a 35% α helix and 65% random coil structure. The intrinsic fluorescence spectrum was typical of a protein devoid of tryptophan residues. Demetallation of Zn2+ resulted in a substantial change in the secondary and tertiary structure of CPI accompanied by the complete loss of anti‐fungal and inhibitory activity indicating that Zn2+ plays an important role in maintaining both structural integrity and biological function. The differential response of anti‐fungal and inhibitory activities to specific modifiers showed that there are two different reactive sites associated with anti‐fungal and anti‐feedent activity in CPI located on a single protein as revealed from its N‐terminal sequence data (AGVCYGVLGNNLP). Modification of cysteine, glutamic/aspartic acid or argnine resulted in abolition of the anti‐fungal activity of CPI, whereas modification of arginine led to an enhancement of the inhibitory activity in solution. Modification of histidine resulted in a twofold increase in the protease inhibitory activity without affecting the anti‐fungal activity, whereas modification of serine led to selective inhibition of the protease inhibitory activity. The differential nature of the two activities was further supported by differences in the temperature stabilities of the anti‐fungal (60 °C) and inhibitory (40 °C) activities. Binding of papain to CPI did not abolish the anti‐fungal activity of CPI, supporting the presence of two active sites on CPI. The differential behavior of CPI towards anti‐fungal and anti‐feedent activity cannot be attributed to changes in conformation, as assessed by their CD and fluorescence spectra. The interaction of CPI modified for arginine or histidine with papain resulted in an enhancement of CPI activity accompanied by a slight decrease in fluorescence intensity of 15–20% at 343 nm. In contrast, modification of serine resulted in inhibition of CPI activity with a concomitant increase of 20% in the fluorescence intensity when complexed by the enzyme. This implies the involvement of enzyme‐based tryptophan in the formation of a biologically active enzyme–inhibitor complex. The presence of anti‐fungal and anti‐feedent activity on a single protein, as evidenced in pearl millet CPI, opens up a new possibility of raising a transgenic plant resistant to pathogens, as well as pests, by transfer of a single CPI gene.
The four murine peroxisomal ABC‐transporter genes differ in constitutive, inducible and developmental expression
Authors: Berger Johannes , Albet Sabrina , Bentejac Marc , Netik Angela , Holzinger Andreas , Roscher Adelbert A. , Bugaut Maurice , Forss‐Petter Sonja
Published: March 2003

Four ATP‐binding cassette (ABC) half‐transporters have been identified in mammalian peroxisomes: adrenoleukodystrophy protein (ALDP), adrenoleukodystrophy‐related protein (ALDRP), 70‐kDa peroxisomal membrane protein (PMP70) and PMP70‐related protein (P70R). Inherited defects in ALDP cause the neurodegenerative disorder X‐linked adrenoleukodystrophy (X‐ALD). By comparative Northern blot analyses we found each of the four murine peroxisomal ABC transporter mRNA species at maximum abundance only in a few tissues, which differed for each family member. The four genes were also regulated differentially during mouse brain development: ALDP mRNA was most abundant in embryonic brain and gradually decreased during maturation; ALDRP and P70R mRNA accumulated in the early postnatal period; and the amount of PMP70 transcript increased slightly during the second and third postnatal week. The different expression patterns could explain why β‐oxidation is defective in X‐ALD, although ALDRP and PMP70 can replace ALDP functionally in fibroblasts. Dietary fenofibrate had no effect on the ALD and P70R genes, but strongly increased expression of the ALDR and PMP70 genes in mouse liver. However, in P‐glycoprotein Mdr1a‐deficient mice fenofibrate treatment increased ALDR gene expression also in the brain, suggesting that the multidrug‐transporter P‐glycoprotein restricts entry of fenofibrate to the brain at the blood–brain barrier. Analysis of the promoter sequences revealed a cryptic nuclear hormone receptor response element of the DR+4 type in the ALDR promoter and a novel 18‐bp sequence motif present only in the 5′ flanking DNA of the ALDR and PMP70 genes. The mouse ALDR gene uses a single transcription start site but alternative polyadenylation sites. These data are of importance for the use of ALDP‐deficient mice as a model in pharmacological gene therapy studies.
Cloning and characterization of CcEcR
Authors: Verras Meletis , Mavroidis Manolis , Kokolakis Giorgos , Gourzi Polyxeni , Zacharopoulou Antigone , Mintzas Anastassios C.
Published: March 2003

In order to understand the role that 20‐hydroxyecdysone plays during development of the Mediterranean fruit fly Ceratitis capitata (medfly), a major agricultural pest, we have cloned a Ceratitis ecdysone receptor (CcEcR) and studied its expression and its binding properties to an ecdysone response element. Using the conserved DNA binding region of the Drosophila melanogaster ecdysone receptor (DmEcR) B1 cDNA as a probe, we isolated a medfly cDNA clone containing the coding region, a part of the 5′‐untranslated region and the complete 3′‐untranslated region of a CcEcR. The deduced CcEcR polypeptide contained all five domains typical of a nuclear receptor. Alignment comparisons and phylogenetic analyses indicated that CcEcR most closely resembled the B1 isoform of DmEcR and Lucilia cuprina EcR homolog (LcEcR) relative to all other known ecdysone receptors. In situ hybridization analysis showed that the CcEcR gene is mapped in the region 53B of the 4R chromosome arm, while Northern hybridization analysis showed that CcEcR transcripts have a size of approximately 8 kb. Significant levels of CcEcR transcripts were detected in eggs, middle and late embryos, late third instar larvae and middle prepupae. The levels of the CcEcR transcripts during the other larval stages as well as during pupal and adult stages were much lower, while during the early stages of embryogenesis were very low. Electrophoretic mobility shift assays indicated that CcEcR binds specifically to the Drosophila hsp27 ecdysone response element as a heterodimer with Drosophila USP, the product of the ultraspiracle gene. Our structural and biochemical data suggest that CcEcR is the functional homolog of the B1 isoform of DmEcR.
Cross‐talk mechanisms in the development of insulin resistance of skeletal muscle cells
Authors: Storz Peter , Döppler Heike , Wernig Anton , Pfizenmaier Klaus , Müller Gertraud
Published: March 2003

Insulin resistance in skeletal muscle is one of the earliest symptoms associated with non‐insulin‐dependent diabetes mellitus (NIDDM). Tumour necrosis factor (TNF) and nonesterified fatty acids have been proposed to be crucial factors in the development of the insulin‐resistant state. We here show that, although TNF downregulated insulin‐induced insulin receptor (IR) and IR substrate (IRS)‐1 phosphorylation as well as phosphoinositide 3‐kinase (PI3‐kinase) activity in pmi28 myotubes, this was, unlike in adipocytes, not sufficient to affect insulin‐induced glucose transport. Rather, TNF increased membrane expression of GLUT1 and glucose transport in these muscle cells. In contrast, the nonesterified fatty acid palmitate inhibited insulin‐induced signalling cascades not only at the level of IR and IRS‐1 phosphorylation, but also at the level protein kinase B (PKB/Akt), which is thought to be directly involved in the insulin‐induced translocation of GLUT4, and inhibited insulin‐induced glucose uptake. Palmitate also abrogated TNF‐dependent enhancement of basal glucose uptake, suggesting that palmitate has the capacity to render muscle cells resistant not only to insulin but also to TNF with respect to glucose transport by GLUT4 and GLUT1, respectively. Our data illustrate the complexity of the mechanisms governing insulin resistance of skeletal muscle, questioning the role of TNF as a direct inhibitor of glucose homoeostasis in this tissue and shedding new light on an as yet unrecognized multifunctional role for the predominant nonesterified fatty acid palmitate in this process.
Autoantibodies interacting with purified native thyrotropin receptor
Authors: Atger Michel , Misrahi Micheline , Young Jacques , Jolivet André , Orgiazzi Jacques , Schaison Gilbert , Milgrom Edwin
Published: March 2003

Native thyrotropin receptor (TSHR) was purified by immunoaffinity chromatography from membrane extracts of stably transfected L cells. An ELISA test was devised to study anti‐TSHR autoantibodies directly. Comparison of native TSHR with bacterially expressed, denatured TSHR showed that the latter was not recognized by the autoantibodies, suggesting that they bind to conformational epitopes only present on the native receptor. The use of deglycosylated TSHR and of purified receptor ectodomain (α‐subunit) showed that the autoantibodies recognized only the protein backbone moiety of the receptor and that their epitopes were localized entirely in its ectodomain.

 Autoantibodies were detected in 45 of 48 subjects with untreated Graves’ disease and in 26 of 47 healthy volunteers. The affinity for the receptor was similar in the two groups (Kd = 0.25–1 × 10−10 m) and the autoantibodies belonged to the IgG class in all cases. Although the concentration of autoantibodies was higher in Graves’ disease patients (3.50 ± 0.36 mg·L−1) than in control subjects (1.76 ± 0.21) (mean ± SEM), there was an overlap between the groups.

 Receptor‐stimulating autoantibodies (TSAb) were studied by measuring cAMP synthesis in stably transfected HEK 293 cells. Their characteristics (recognition of α‐subunit, of deglycosylated TSHR, nonrecognition of bacterially expressed denatured receptor) were similar to those of the antibodies detected by the ELISA test. TSAb were only found in individuals with Graves’ disease.

 The ELISA test measures total anti‐TSHR antibodies, whereas the test using adenylate cyclase stimulation measures antibodies that recognize specific epitopes involved in receptor activation. Our observations thus disprove the hypothesis according to which Graves’ disease is related to the appearance of anti‐TSHR antibodies not present in normal subjects. Actually, anti‐TSHR antibodies exist in many euthyroid subjects, in some cases even at concentrations higher than those found in patients with Graves’ disease. What distinguishes the latter from normal subjects is the existence of subpopulation(s) of antibodies directed against specific epitope(s) of the receptor involved in its activation.
High‐affinity binding of fatty acyl‐CoAs and peroxisome proliferator‐CoA esters to glutathione S‐transferases
Authors: Silva Cecilia , Loyola Gloria , Valenzuela Rodrigo , García‐Huidobro Tea , Monasterio Octavio , Bronfman Miguel
Published: March 2003

Acyl‐CoAs are present at high concentrations within the cell, yet are strongly buffered by specific binding proteins in order to maintain a low intracellular unbound acyl‐CoA concentration, compatible with their metabolic role, their importance in cell signaling, and as protection from their detergent properties. This intracellular regulation may be disrupted by nonmetabolizables acyl‐CoA esters of xenobiotics, such as peroxisome proliferators, which are formed at relatively high concentration within the liver cell. The low molecular mass acyl‐CoA binding protein (ACBP) and fatty acyl‐CoA binding protein (FABP) have been proposed as the buffering system for fatty acyl‐CoAs. Whether these proteins also bind xenobiotic‐CoA is not known. Here we have identified new liver cytosolic fatty acyl‐CoA and xenobiotic‐CoA binding sites as glutathione S‐transferase (GST), using fluorescent polarization and a acyl‐etheno‐CoA derivative of the peroxisome proliferator nafenopin as ligand. Rat liver GST and human liver recombinant GSTA1‐1, GSTP1‐1 and GSTM1‐1 were used. Only class alpha rat liver GST and human GSTA1‐1 bind xenobiotic‐CoAs and fatty acyl‐CoAs, with Kd values ranging from 200 nm to 5 µm. One mol of acyl‐CoA is bound per mol of dimeric enzyme, and no metabolization or hydrolysis was observed. Binding results in strong inhibition of rat liver GST and human recombinant GSTA1‐1 (IC50 at the nanomolar level for palmitoyl‐CoA) but not GSTP1‐1 and GSTM1‐1. Acyl‐CoAs do not interact with the GSTA1‐1 substrate binding site, but probably with a different domain. Results suggest that under increased acyl‐CoA concentration, as occurs after exposure to peroxisome proliferators, acyl‐CoA binding to the abundant class alpha GSTs may result in strong inhibition of xenobiotic detoxification. Analysis of the binding properties of GSTs and other acyl‐CoA binding proteins suggest that under increased acyl‐CoA concentration GSTs would be responsible for xenobiotic‐CoA binding whereas ACBP would preferentially bind fatty acyl‐CoAs.
Neu5Acα3Gal is part of the Helicobacter pylori binding epitope in polyglycosylceramides of human erythrocytes
Authors: Johansson Lena , Johansson Petra , Miller‐Podraza Halina
Published: March 2003

The sialic acid dependent binding by the human pathogen Helicobacter pylori to polyglycosylceramides of human erythrocytes was investigated. Polyglycosylceramides, complex glycosphingolipids with a branched N‐acetyllactosamine core, were isolated from human erythrocytes, blood group O, and subfractionated after peracetylation by anion‐exchange chromatography. Three subfractions were deacetylated, analysed by matrix‐assisted laser desorption ionization‐time of flight MS and 2D 1H NMR spectroscopy. The observed mass ranges were m/z = 3093–7622, 3968–7255 and 3459–7987 in the mass spectra of the first, second and third fractions, respectively. The observed ions agreed with the general formula Hex(x+2)HexNAcxFucyNeu5AczCer. Two‐dimensional 1H total correlation spectra of the mixtures showed that the first fraction contained 3‐linked sialic acid and the second and third fractions contained both 3‐linked and 6‐linked sialic acid. Thin‐layer chromatogram binding assays using the lectins from Maackia amurensis, specific for Neu5Acα3Galβ4GlcNAc, and Sambucus nigra, specific for Neu5Acα6Gal/GalNAc, were used to confirm this distribution. H. pylori recognized all three fractions in the binding assay, indicating that the 3‐linked, rather than 6‐linked, sialic acid is essential for binding.
Protection against thermal denaturation by trehalose on the plasma membrane H+‐ATPase from yeast
Authors: Felix Carla Fagundas , Moreira Clarisse Cortes , Oliveira Mylene Santos , Sola‐Penna Mauro , Meyer‐Fernandes José R. , Scofano Helena M. , Ferreira‐Pereira Antônio
Published: March 2003

Yeast cells have had to develop mechanisms in order to protect themselves from chemical and physical agents of the environment to which they are exposed. One of these physical agents is thermal variation. Some yeast cells are known to accumulate high concentrations of trehalose when submitted to heat shock. In this work, we have studied the effect of trehalose on the protection against thermal inactivation of purified plasma membrane H+‐ATPase from Schizosaccharomyces pombe, in the solubilized and in the reconstituted state. We observed that after 1 min of incubation at 51 °C in the presence of 1 m trehalose, about 50% of soluble enzyme remains active. In the same conditions, but in the absence of trehalose, the activity was completely abolished. The t0.5 for the enzyme inactivation increased from 10 to 50 s after reconstitution into asolectin liposomes. Curiously, in the presence of 1 m trehalose, the t0.5 for inactivation of the reconstituted enzyme was further increased to higher than 300 s, regardless of whether trehalose was added inside or outside the liposome. Additionally, the concentration that confers 50% for the protection by trehalose (K0.5) decreased from 0.5 m, in the solubilized state, to 0.04 m in the reconstituted state, suggesting a synergetic effect between sugar and lipids. Gel electrophoresis revealed that the pattern of H+‐ATPase cleavage by trypsin changed when 1 m trehalose was present in the buffer. It is suggested that both in a soluble and in a phospholipid environment, accumulation of trehalose leads to a more heat‐stable conformation of the enzyme, probably an E2‐like form.
Anti‐proliferative effect of two novel palmitoyl‐carnitine analogs, selective inhibitors of protein kinase C conventional isoenzymes
Authors: Garcia‐Huidobro Tea , Valenzuela Enrique , Leisewitz Andrea V. , Valderrama Jaime , Bronfman Miguel
Published: March 2003

Previous studies have shown that palmitoyl‐carnitine is an anti‐proliferative agent and a protein kinase C inhibitor. Two new palmitoyl‐carnitine analogs were synthesized by replacing the ester bond with a metabolically more stable ether bond. An LD50 value in the nm range was found in anti‐proliferative assays using HL‐60 cells and was dependent on the alkyl‐chain length. The inhibitory action of these water‐soluble compounds on protein kinase C in vitro was greatly increased with respect to palmitoyl‐carnitine and was dependent on the length of the alkyl chain. Its effect was mediated by an increase in the enzyme’s requirement for phosphatidylserine. Inhibition of the in situ phosphorylation of a physiological platelet protein kinase C substrate and of phorbol ester‐induced differentiation of HL‐60 cells was also observed. Finally, to test for isoenzyme selectivity, several human recombinant protein kinase C isoforms were used. Only the Ca2+‐dependent classic protein kinase Cs (α, βΙ, βΙΙ and γ) were inhibited by these compounds, yet the activities of casein kinase I, Ca2+/calmodulin‐dependent kinase and cAMP‐dependent protein kinase were unaffected. Thus, these novel inhibitors appear to be both protein kinase C and isozyme selective. They may be useful in assessing the individual roles of protein kinase C isoforms in cell proliferation and tumor development and may be rational candidates for anti‐neoplasic drug design.