bluuebunny/crossref_metadata_2025 huggingface dataset:
Pine stilbene synthase cDNA, a tool for probing environmental stress
Authors: Schwekendiek Axel , Pfeffer Gerda , Kindl Helmut
Published: July 2002

Stilbene synthase cDNAs were isolated from a pine (Pinus sylvestris) cDNA library, Poly(A)'RNA required for the preparation was obtained from young seedlings challenged with Botrytis cinerea. A full‐length cDNA encoding pinosylvin‐forming stilbene synthase was sequenced, and the deduced amino acid sequence was compared with sequences of resveratrol‐forming stilbene synthases. The cDNA coding for the key enzyme of pinosylvin formation is a valuable tool for detecting early effects of environmental stress in pine needles.
Southern molecular hybridization experiments with parallel complementary DNA probes
Authors: Tchurikov Nickolai A. , Shchyolkina Anna K. , Borissova Olga F. , Chernov Boris K.
Published: July 2002

We have detected the specific binding in Southern blot hybridization experiments of both complementary antiparallel and parallel 40 bp synthetic DNA probes, corresponding to a cloned Drosophila DNA fragment. The highly cooperative annealing and melting were observed in solution with these probes, which are complementary in the same direction and possess 17 GC pairs. The binding of ethidium bromide is indicative of formation of a perfect parallel DNA duplex. The specific binding was also detected in both genomic and in plaque hybridization experiments.
Multidomain enzymes involved in peptide synthesis
Authors: Marahiel Mohamed A.
Published: July 2002

Biosynthesis of peptides in non‐ribosomal systems is catalyzed by multifunctional enzymes that employ the thio‐template mechanism. Recent studies on the analysis of the primary structure of several peptide synthetases have revealed that they are organized in highly conserved and repeated functional domains. The aligned domains provide the template for peptide synthesis, and their order determines the sequence of the peptide product.
Divergent effects of fluoroaluminates on the peptide chain elongation factors EF‐Tu and EF‐G as members of the GTPase superfamily
Authors: Mesters Jeroen R. , de Graaf J.Martien , Kraal Barend
Published: July 2002

Fluoroaluminates are thought to mimic the γ‐phosphate of GTP and thus, together with GDP, perturb the functioning of heterotrimeric GTP‐binding G‐proteins. Here we show they do inhibit the ribosome‐stimulated GTPase activity of EF‐G from Escherichia coli via the formation of a stable complex with EF‐G·GDP and ribosomes. In contrast, no perturbed interactions were observed in a similar ribosomal complex with EF‐Tu. Interestingly, in the absence of ribosomes both EF‐Tu and EF‐G remain totally unaffected by fluoroaluminates. For members of the GTPase superfamily such differential effects have not been described before.
Up‐regulation of system A activity in the regenerating rat liver
Authors: Martinez-Mas Joan-Vicenç , Ruiz-Montasell Bonaventura , Felipe Antonio , Casado Javier , Pastor-Anglada Marçal
Published: July 2002

System A activity for neutral amino acid transport, measured as the MeAIB‐sensitive Na+‐dependent l‐alanine uptake, is induced 6 h after partial hepatectomy in plasma membrane vesicles from rat livers. Other Na+‐dependent transporters, like system ASC (MeAIB‐insensitive Na+‐dependent l‐alanine transport) and the nucleoside carrier show similar inductions. Up‐regulation of system A is not explained by changes in the dissipation rate of the Na+ transmembrane gradient, as deduced from uptake measurements performed in the presence of monensin. To determine whether induced system A shared any similarity with the activity found in hepatoma cell lines, we analyzed the N‐ethylmaleimide (NEM) sensitivity of system A in both regenerating and control rat liver plasma membrane vesicles. NEM treatment was equally effective in inhibiting system A in both experimental groups. Thus, during the prereplicative phase of liver growth, a transport activity similar to basal system A is up‐regulated in liver parenchymal cells, by a stable mechanism that does not involve changes in the Na+ transmembrane gradient.
Sequence homology between bacteriorhodopsin and G‐protein coupled receptors: exon shuffling or evolution by duplication?
Authors: Taylor E.Will , Agarwal Atul
Published: July 2002

Bacteriorhodopsin (BR) is a membrane protein of known structure, widely used for the homology modeling of G‐protein‐coupled receptors (GPCR). The observation of apparently transposed sequence similarities between some of the helical domains of BR and GPCR has led to the suggestion that exon shuffling may have occurred in the later evolution of GPCR, which would necessitate a different folding pattern for the seven transmembrane helices of GPCR. An alternate hypothesis is that duplication occurred in the evolution of an ancestral gene, such that helices 5–7 originated as duplicates of helices 1–3, leading to intragenic as well as intergenic similarities between helices 1–3 and 5–7 of BR and various GPCR. Analyses of GPCR and BR sequences suggest that such a duplication may have occurred; symmetry within the BR structure is also consistent with homology between these two regions. The hypothesis of evolution by duplication is consistent with the conventional, unshuffled homology model, which is also supported by the obvious conservation of the retinal binding Lys moiety on helix 7 in both BR and the mammalian opsins.
cDNA cloning and expression of an Arabidopsis GTP‐binding protein of the ARF family
Authors: Regad Farid , Bardet Claude , Tremousaygue Dominique , Moisan Annick , Lescure Bernard , Axelos Michèle
Published: July 2002

A cDNA clone encoding a small GTP‐binding protein, the ADP‐ribosylation factor (ARF) was isolated from a cDNA library of Arabidopsis thaliana cultured cells. The predicted amino acid sequence was highly homologous to the known yeast, bovine and human ARF sequences. Southern analysis of Arabidopsis genomic DNA suggested the existence of at least two copies of ARF genes. The level of ARF mRNA was found to be nearly constant during all cell growth stages in suspension cultures.
A protein produced by a monocytic human cell line can induce apoptosis on tumor cells
Authors: Ghibelli L. , Coppola S. , Nosseri C. , Bergamini A. , Beninati S.
Published: July 2002

A serum‐free medium conditioned by U937, a human cell line of monocyte/macrophage origin, was found capable of inducing apoptosis on exponentially growing U937 cells themselves (autocrine suicide). The apoptosis‐inducing agent is a macromolecule and possibly a protein (SKT factor), with a relative molecular mass in the range of 18–25 kDa. All human tumor cell lines examined have been induced to apoptosis with high efficiency, whereas non transformed human lymphocytes and monocytes are insensitive to the apoptosis‐inducing activity; moreover, partially differentiated U937 are not killed but induced to full maturation. These observations suggest that the SKT factor could possibly be a cytokine with a specific cytotoxic tropism, that resembles in many respects the cytokine tumor necrosis factor (TNF), even though no TNF is detectable in the conditioned medium.
ADP binding and ATP synthesis by reconstituted H+‐ATPase from chloroplasts
Authors: Creczynski-Pasa Tania Beatriz , Gräber Peter
Published: July 2002

The H+‐ATPase from chloroplasts, CF0F1, was isolated, purified and reconstituted into asolectin liposomes. The enzyme was brought either into the oxidized state or into the reduced state, and the rate of ATP synthesis was measured after energisation of the proteoliposomes with an acid—base transition ΔpH (pHin = 5.0, pHout = 8.5) and a K+/valinomycin diffusion potential, Δφ (K+
in = 0.6 mM, K+
out = 60 mM). A rate of 250 s−1 was observed with the reduced enzyme (85 s−1 in the absence of Δφ). A rate of 50 s−1 was observed with the oxidized enzyme under the same conditions (15 s−1 in the absence of Δφ). The reconstituted enzyme contained 2 ATPbound per CF0F1 and 1 ADPbound per CF0F1. Upon energisation the enzyme was activated and 0.9 ADP per CF0F1, was released. Binding of ADP to the active reduced enzyme was observed under different conditions. In the absence of phosphate the rate constant for ADP binding was 105 M−1·s−1 under energized and de‐energized conditions. In the presence of phosphate the rate of ADP binding drastically increased under energized conditions, and strongly decreased under de‐energized conditions.
Evidence for the ligand‐induced conversion from a dimer to a tetramer of the granulocyte colony‐stimulating factor receptor
Authors: Hiraoka Osamu , Anaguchi Hiroyuki , Ota Yoshimi
Published: July 2002

An extracellular portion of granulocyte colony‐stimulating factor (G‐CSF) receptor, which contains an immunoglobulin‐like (Ig) domain and cytokine receptor homologous (CRH) region, was secreted into the medium using Trichoplusia ni‐Autographa californica nuclear polyhedrosis virus system. The gene product was purified to homogeneity mainly as a dimer (85 kDa) using G‐CSF affinity column chromatography and gel filtration HPLC, although the product existed as a monomer (45 kDa) in the medium. Scatchard analyses suggested that only the dimer had high affinity ligand binding (K
d = about 100 pM), which is comparable with the K
d value of the cell surface receptor. The binding of G‐CSF to Ig‐CRH induced its tetramerization (200–250 kDa). The molecular composition of the tetrameric complex showed a stoichiometry of four ligands bound to four Ig‐CRH. These results suggested that the oligomeric mechanism of the G‐CSF receptor differs from that reported for growth hormone (GH) receptor, although CD spectrum spectroscopy suggested that the Ig‐CRH has a GH receptor‐like structure.
The binding of natural variants of human factor IX to endothelial cells
Authors: Mayhew Mark , Handford Penny , Brownlee George G.
Published: July 2002

The Gladomain of human factor IX contains a specific element required for the binding of factor IX to an endothelial cell surface protein. We have investigated the dependence of this interaction on the structural integrity of the adjacent hydrophobic stack and epidermal growth factor‐like domains. The ability of purified natural variants of human factor IX to compete with wild‐type factor IX binding to the endothelial cell surface was used to obtain apparent Ki
values of the variants. Our data suggest that the functional integrity of the Gla domain, enabling factor IX to specifically interact with an endothelial cell surface protein, depends on the structural and functional integrity of both the hydrophobic stack domain and the first epidermal growth factor‐like domain.
Lysophosphatidic acid‐induced activation of protein Ser/Thr kinases in cultured rat 3Y1 fibroblasts Possible involvement in rhop21‐mediated signalling
Authors: Kumagai Naokazu , Morii Narito , Ishizaki Toshimasa , Watanabe Naoki , Fujisawa Kazuko , Saito Yuji , Narumiya Shuh
Published: July 2002

Renaturation kinase assay was used to detect protein kinases activated by lysophosphatidic acid (LPA) in cultured rat 3Y1 fibroblasts. LPA activated several Ser/Thr protein kinases with apparent molecular weights of 145K, 85K, 64–65K (a doublet), and 60K (each named p145, p85, p64165 and p60, respectively) in addition to p43 mitogen activated protein (MAP)‐kinase. Experiments using pertussis toxin and botulinum C3 exoenzyme showed that p145, p85, and p64165 kinases were activated by a pertussis toxin‐insensitive rho
p21‐dependent pathway and that the activation of MAP‐kinase was mediated by both the pertussis toxin‐sensitive rho
p21‐independent and the pertussis toxin‐insensitive rho
p21‐dependent pathways.
Role of multiple cellular proteases in the execution of programmed cell death
Authors: Kumar Sharad , Harvey Natasha L
Published: July 2002

A family of mammalian homologues of the Caenorhabditis elegans cell death protein Ced‐3 has been recently discovered. These mammalian proteins encode novel cysteine proteases with homology to the interleukin‐1β converting enzyme (ICE). Although several studies support a role for one or more of these proteases in mediating apoptosis, their mechanism of action is far from understood. The presence of multiple mammalian ICE‐like proteases, with apparently similar apoptotic function indicates that, despite its conservation during evolution, the cell death pathway is much more complex in mammals than in the worm. In addition to ICE‐like proteases, several other proteases of different cleavage specificities have been implicated in apoptosis. There is now a growing body of evidence suggesting that apoptosis involves the activation of a cascade of proteases. This article summarises the presently available evidence and discusses how multiple proteases might be required in the effector phase of cell death.
(RP)‐cAMPS inhibits the cAMP‐dependent protein kinase by blocking the cAMP‐induced conformational transition
Authors: Dostmann Wolfgang R.G.
Published: July 2002

(RP)‐cAMPS is known to inhibit competitively the cAMP‐induced activation of cAMP‐dependent protein kinase (PKA). The molecular nature of this inhibition, however, is unknown. By monitoring the intrinsic tryptophan fluorescence of recombinant type I regulatory subunit of PKA under unfolding conditions, a free energy value (ΔGDH2O) of 8.23 ± 0.22 was calculated. The cAMP‐free form of the regulatory subunit was less stable withΔGDH2O= 6.04 ± 0.05 kcal/mol. Native stability was recovered by treatment of the cAMP‐free protein with either cAMP or (SP)‐cAMPS but not with (RP)‐cAMPS. Thus, (RP)‐cAMPS binding to the regulatory subunit keeps the protein in a locked conformation, unable to release the catalytic subunit. This finding was further supported by demonstrating that holoenzyme formation was greatly accelerated only when bound cAMP was replaced with (RP)‐cAMPS but not with cAMP or (SP)‐cAMPS.
Suppression of Bcl‐2 gene expression by sphingosine in the apoptosis of human leukemic HL‐60 cells during phorbol ester‐induced terminal differentiation
Authors: Sakakura Chouhei , Sweeney Elizabeth A. , Shirahama Tsutomu , Hakomori Sen-itiroh , lgarashi Yasuyuki
Published: July 2002

Our recent studies have shown that intracellular levels of sphingosine, an endogenous PKC inhibitor, increase during apoptosis resulting from phorbol ester (PMA)‐induced terminal differentiation of human myeloid leukemic HL‐60 cells, and have suggested that sphingosine may function as an endogenous mediator of apoptosis in these cells [Ohta, et al. (1995) Cancer Res. 55, 691–697]. We report here that apoptosis induced by PMA, sphingosine, and N,N‐dimethylsphingosine (DMS) was accompanied by a concomitant decrease of bcl‐2 expression in both RNA and protein levels in HL‐60 cells, while expression of bcl‐XL and bax mRNA did not change, and neither sphingosine nor DMS induced differentiation of HL‐60 cells. In contrast, in apoptotic cells induced by pharmaceutical PKC inhibitors H7 or staurosporine, expression of bcl‐2 did not change nor did the intracellular sphingosine concentration. These results suggest that sphingosine may function as an endogenous mediator of apoptotic signaling in PMA‐induced terminal differentiation of HL‐60 cells through bcl‐2 down‐regulation, probably independent from PKC inhibition.
Aspirin‐DNA interaction studied by FTIR and laser Raman difference spectroscopy
Authors: Neault J.F , Naoui M , Manfait M , Tajmir-Riahi H.A
Published: July 2002

The interaction of calf‐thymus DNA with aspirin is investigated in aqueous solution at pH 7‐6 with drug/DNA(phosphate) molar ratios of r = 1/40, 1/20, 1/15/ 1/2, 1 and 2. Fourier transform infrared (FTIR) and laser Raman difference spectroscopy are used to determine drug binding sites, sequence preference and DNA secondary structure, as well as the structural variations of aspirin‐DNA complexes in aqueous solution. Spectroscopic evidence showed that at low aspirin concentration (r = 1/40), drug‐DNA interaction is mainly through the backbone PO2 groups and the A‐T base pairs. Such interaction largely perturbs the phosphate vibration at 1222 cm−1 and the A‐T bands at 1663 and 1609 cm−1 with no major helix destabilization. At higher drug concentration (r > 1/20), the participation of the G‐C bases in drug‐DNA complexation was evident by strong perturbations of the guanine and cytosine vibrations at 1717 and 1494 cm−1, with a partial helix destabilization. A major alteration of the B‐DNA structure towards A‐DNA occurs on drug complexation. The aspirin interaction was through anion CO and COOCH3 donor atoms with those of the backbone PO2 group and DNA bases donor sites (directly or indirectly via H2O molecules).
An active‐site cysteine of sorghum leaf NADP‐malate dehydrogenase studied by site‐directed mutagenesis
Authors: Lemaire Martine , Issakidis Emmanuelle , Ruelland Eric , Decottignies Paulette , Miginiac-Maslow Myroslawa
Published: July 2002

The chlorplast NADP‐malate dehydrogenase is activated through the reduction of two different disulfides per subunit. The activated enzyme, as well as a permanently active mutant where all four regulatory cysteines were replaced are still sensitive to thiol reagents. This observation suggested the presence of an additional important cysteine at the active site. In an attempt to identify that cysteine, site‐directed mutagenesis was performed on the cDNA encoding sorghum leaf NADP‐malate dehydrogenase. The replacement of Cys‐175 by an alanine yielded an enzyme whose sensitivity to thiol reagents was markedly decreased whereas its catalytic activity was enhanced. This finding suggests that Cys‐175 has no catalytic function but is located close to the active site.
The micelle to vesicle transition of lipids and detergents in the presence of a membrane protein: towards a rationale for 2D crystallization
Authors: Dolder Max , Engel Andreas , Zulauf Martin
Published: July 2002

The assembly of two‐dimensional membrane protein crystals in the presence of lipids was analyzed with quasielastic light scattering and electron microscopy. Mixtures of detergent‐solubilized lipids and/or proteins were submitted to slow or rapid dilution while measuring the hydrodynamic radii of the aggregates. Lipids alone exhibited λ‐shaped dilution curves with intermediate rod‐shaped particles that converted into small vesicles. Depending on the protein‐protein and protein‐lipid interactions, detergent‐solubilized protein‐lipid mixtures showed a sharp transition from micelles to large, densely packed proteoliposomes. Electron microscopy revealed that formation of crystals occurred shortly after this phase transition.
Crystal structures of human procathepsin B at 3.2 and 3.3 Å resolution reveal an interaction motif between a papain‐like cysteine protease and its propeptide
Authors: Turk Dušan , Podobnik Marjeta , Kuhelj Robert , Dolinar Marko , Turk Vito
Published: July 2002

A wild‐type human procathepsin B was expressed, crystallized in two crystal forms and its crystal structure determined at 3.2 and 3.3 Å resolution. The structure reveals that the propeptide folds on the cathepsin B surface, shielding the enzyme active site from exposure to solvent. The structure of the enzymatically active domains is virtually identical to that of the native enzyme [Musil et al. (1991) EMBO J. 10, 2321–2330]: the main difference is that the occluding loop residues are lifted above the body of the mature enzyme, supporting the propeptide structure.
Antioxidant activities of carotenes and xanthophylls
Authors: Miller Nicholas J. , Sampson Julia , Candeias Luis P. , Bramley Peter M. , Rice-Evans Catherine A.
Published: July 2002

The purpose of this study was to assess the relative antioxidant activities of a range of carotenes and xanthophylls through the extent of their abilities to scavenge the ABTS·+ radical cation. The results show that the relative abilities of the carotenoids to scavenge the ABTS·+ radical cation are influenced by the presence of functional groups with increasing polarities, such as carbonyl and hydroxyl groups, in the terminal rings, as well as by the number of conjugated double bonds.
Characterisation of the heptameric pore‐forming complex of the Aeromonas toxin aerolysin using MALDI‐TOF mass spectrometry
Authors: Moniatte M. , van der Goot F.G. , Buckley J.T. , Pattus F. , van Dorsselaer A.
Published: July 2002

Aerolysin, a virulence factor secreted by Aeromonas hydrophila, is representative of a group of β‐sheet toxins that must form stable homooligomers in order to be able to insert into biological membranes and generate channels. Electron microscopy and image analysis of two‐dimensional membrane crystals had previously revealed a structure with 7‐fold symmetry et al. (1992) EMBO J. 11, 2457–2463]. However, this unusual et al. (1992) EMBO J. 11, 2457–2463]. However, this unusual molecularity of the channel remained to be confirmed by an independent method since low‐resolution electron crystallography had led to artefactual data for other pore‐forming toxins. In this study, matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) was used to measure the mass of the aerolysin oligomer preparation. A mass of 333 850 Da was measured, fitting very well with a heptameric complex (expected mass: 332 300 Da). These results confirm the earlier evidence that the aerolysin oligomer is a heptamer and also show that MALDI‐TOF mass spectrometry could be a valuable tool to study non‐covalent association of proteins.
NMR evidence for helix geometry modifications by a G‐U wobble base pair in the acceptor arm ofE. colitRNAAla
Authors: Limmera Stefan , Reifa Bernd , otta Günther , Arnold Lubos , Sprinzl Mathias
Published: July 2002

A ribooligonucleotide duplex representing the acceptor stem ofE. coliRNAAlawith a G3‐U70 wobble base pair, which is the main identity element for the recognition by the alanine‐tRNA synthetase, has been characterized by 2D‐NMR, as having two sequence variants with a regular Watson‐Crick G3‐C70 and an I3‐U70 wobble pair, respectively. As compared to a regular A‐RNA, the G‐U base pair gives rise to variations of the local helix geometry which are reflected in distinct local chemical shift changes. Structural differences between the duplex possessing an 13‐U70 base pair and the wild‐type G3‐U70 sequence have also been found. The nucleotides in the ubiquitous single‐stranded NCCA terminus display a surprisingly high degree of stacking order, especially between A73, C74, and C75.
The role of the C‐terminal lysine in the hinge bending mechanism of yeast phosphoglycerate kinase
Authors: Adams Benjamin , Fowler Richard , Hudson Martin , Pain Roger H.
Published: July 2002

Treatment of yeast phosphoglycerate kinase (PGK) with trypsin results in a fourfold increase in the V
max of this enzyme, without affecting the Km. This activation is shown to be due to the removal of the C‐terminal lysine residue. The C‐terminal sequence folds back over the N‐terminal domain and contacts the extreme N‐terminal sequence which folds onto the C‐terminal domain, thus making many of the inter‐domain contacts in this two domain protein. Previous studies have shown that this C‐terminal region is important in mediating the conformational changes required during catalysis by yeast PGK. Observation of the three‐dimensional structure of this enzyme suggests that removal of the C‐terminal lysine residue will strengthen the interaction between K5 and E413. This indicates that this salt bridge stabilises the enzyme in the higher activity form, while the presence of K415 reduces the strength of that interaction.
Identification of the prolyl isomerase domain of Escherichia coli trigger factor
Authors: Hesterkamp Thomas , Bukau Bernd
Published: July 2002

E. coli trigger factor is a protein of 48 kDa which was recently identified as a ribosome‐bound peptidyl‐prolyl‐cis/trans‐isomerase (PPIase) capable of catalysing protein folding in vitro. We found trigger factor in association with nascent polypeptide chains, suggesting a function in the co‐translational folding of proteins. Sequence comparisons revealed a homology of a segment of trigger factor with PPlases of the FK506 binding protein (FKBP) family. Here, we report on the purification of trigger factor and a domain assignment of its polypeptide chain by microsequencing and mass spectroscopy of proteolytic fragments. Two proteases generated fragments of 12–13 kDa molecular weight that encompass the predicted FKBP domain and possess PPIase activity in vitro. Sequence alignment of the known trigger factor proteins demonstrates a high degree of conservation within this central functional domain of the protein.
Calculation of Cys 30 ΔpKa's and oxidising power for DsbA mutants
Authors: Warwicker J. , Gane P.J.
Published: July 2002

DsbA possesses a redox active disulphide, with the equilibrium strongly shifted towards the reduced form as compared to its structural homologue, thioredoxin. It is widely believed that the two amino acids that separate the active site cysteines play a crucial role in determining oxidising power within the thioredoxin family. Data concerning redox and pKa properties for DsbA mutants in this region are available. Electrostatics calculations show reasonable agreement with the experimental data, and support the suggestion that amino acids outside of the CXXC active site sequence are as important in determining oxidising power within the thioredoxin family as are those within it.
Site‐directed mutants designed to test back‐door hypotheses of acetylcholinesterase function
Authors: Faerman Carlos , Ripoll Daniel , Bon Suzanne , Le Feuvre Yves , Morel Nathalie , Massoulié Jean , Sussman Joel L. , Silman Israel
Published: July 2002

The location of the active site of the rapid enzyme, acetylcholinesterase, near the bottom of a deep and narrow gorge indicates that alternative routes may exist for traffic of substrate, products or solute into and out of the gorge. Molecular dynamics suggest the existence of a shutter‐like back door near Trp84, a key residue in the binding site for acetylcholine, in the Torpedo californica enzyme. The homology of the Ω loop, bearing Trp84, with the lid which sequesters the substrate in neutral lipases displaying structural homology with acetylcholinesterase, suggests a flap‐like back door. Both possibilities were examined by site‐directed mutagenesis. The shutter‐like back door was tested by generating a salt bridge which might impede opening of the shutter. The flap‐like back door was tested by de novo insertion of a disulfide bridge which tethered the Ω loop to the body of the enzyme. Neither type of mutation produced significant changes in catalytic activity, thus failing to provide experimental support for either back door model. Molecular dynamics revealed, however, substantial mobility of the Ω loop in the immediate vicinity of Trp84, even when the loop was tethered, supporting the possibility that access to the active site, involving limited movement of a segment of the loop, is indeed possible.
Increased levels of 4‐hydroxynonenal in human monocytes fed with malarial pigment hemozoin A possible clue for hemozoin toxicity
Authors: Schwarzer Evelin , Müller Oliver , Arese Paolo , Siems Werner G. , Grune Tilman
Published: July 2002

In human monocytes, lipoperoxides were increased 3‐fold at 2 h, 6‐fold at 5 h and 7.5‐fold at 12 h after hemozoin phagocytosis. 4‐Hydroxynonenal (HNE) was also increased, reaching 40 nmol/1010 cells at 2 h (approximate intracellular concentration [AIE] 8 μM), 230 nmol/1010 cells at 5 h (AIE 46 μM) and 79 nmol/1010 cells (AIE 16 μM) at 12 h. A moderate increase in HNE, approx. 20 nmol/1010 cells (AIE 4 μM) was also observed after phagocytosis of anti‐D IgG‐opsonized erythrocytes. HNE in unfed controls was approx. 5 nmol/1010 cells (AIE 1 μM) during the whole incubation period. An increased amount of protein kinase C (PKC)/HNE adduct was demonstrated in hemozoin‐fed monocytes. Purified PKC was profoundly inhibited at HNE > 10 μM. The impairment of PKC previously observed in hemozoin‐fed monocytes can thus be explained by direct interaction with increased HNE levels.
Inhibition of MAP kinase kinase (MEK) blocks endothelial PGI2 release but has no effect on von Willebrand factor secretion or E‐selectin expression
Authors: Wheeler-Jones Caroline P.D. , May Michael J. , Houliston Rebecca A. , Pearson Jeremy D.
Published: July 2002

We have examined the potential role of MAP kinase in the regulation of endothelial cell PGI2 synthesis, vWF secretion and E‐selectin expression using the specific MEK inhibitor PD98059. PD98059 dose‐dependently attenuated the tyrosine phosphorylation and activation of p42mapk in response to thrombin or inflammatory cytokines. Inhibition of thrombin‐induced p42mapk activation was paralleled by an inhibitory effect of PD98059 on thrombin‐driven PGI2 generation but not on vWF secretion or IL‐1α/TNFα‐induced E‐selectin expression. These results provide evidence for a key role for p42mapk in the acute regulation of PGI2 synthesis in human endothelial cells and suggest that activation of the MAP kinase cascade is not obligatory for cytokine‐stimulated E‐selectin expression.
Insulin‐stimulated phosphorylation of initiation factor 4E is mediated by the MAP kinase pathway
Authors: Flynn Andrea , Proud Christopher G.
Published: July 2002

The cap‐binding initiation factor 4E (eIF4E) is regulated by phosphorylation and by the inhibitory binding protein 4E‐BP1. Here we show that insulin‐induced phosphorylation of eIF4E is not significantly affected by rapamycin, but is sensitive to wortmannin, which inhibits phosphatidylinositol 3′‐kinase and blocks the activation of MAP kinase. Since PD098059, an inhibitor of MAP kinase activation, also blocks insulin‐induced phosphorylation of eIF4E, the MAP kinase pathway seems to mediate this effect. Phosphorylated eIF4E can still bind to 4E‐BP1. These data illustrate that (i) distinct signalling pathways mediate the phosphorylation of eIF4E and 4E‐BP1 and (ii) phosphorylation of eIF4E, unlike that of 4EBPI, does not lead directly to the release of 4E‐BP1.
The protein fold of the hyaluronate‐binding proteoglycan tandem repeat domain of link protein, aggrecan and CD44 is similar to that of the C‐type lectin superfamily
Authors: Brissett Nigel C. , Perkins Stephen J.
Published: July 2002

Link protein and aggrecan of the extracellular matrix each contain two proteoglycan tandem repeat (PTR) domains that interact with hyaluronate. Consensus secondary structure predictions for 59 PTR sequences and 129 C‐type lectin sequences give similar patterns of two a‐helices and up to seven β‐strands. Protein fold recognition analyses show that the 59 PTR sequences are highly compatible with the C‐type lectin crystal structure. The predicted fold consists of a conserved motif formed from an antiparallel β‐sheet flanked by two α‐helices, the motif being attached to two distinct types of sheet region in the two superfamilies. Arg9 or Lys11 on an exposed loop and up to three other Arg residues in the β‐sheet region are conserved and may form part of a hyaluronate binding site.
In vivo gene electroinjection and expression in rat liver
Authors: Heller Richard , Jaroszeski Mark , Atkin Andrew , Moradpour Darius , Gilbert Richard , Wands Jack , Nicolau Claude
Published: July 2002

In vivo targeted gene transfer by non‐viral vectors is subjected to anatomical constraints depending on the route of administration. Transfection efficiency and gene expression in vivo using non‐viral vectors is also relatively low. We report that in vivo electropermeabilization of the liver tissue of rats in the presence of genes encoding luciferase or β‐galactosidase resulted in the strong expression of these genetic markers in rat liver cells. About 30–40% of the rat liver cells electroporated expressed the β‐galactosidase genetic marker 48 h after electroporation. The marker expression was also detected at least 21 days after transfection at about 5% of the level 48 h after electroporation. The results indicate that gene transfer by electroporation in vivo may avoid anatomical constraints and low transfection efficiency.
Thimerosal modulates the agonist‐specific cytosolic Ca2+ oscillatory patterns in single pancreatic acinar cells of mouse
Authors: Wu Jie , Takeo Teruko , Kamimura Noritaka , Wada Junro , Sechiko Suga , Hoshina Yuko , Wakui Makoto
Published: July 2002

Modulation of the agonist‐specific cytosolic Ca2+ oscillatory pattern by thimerosal has been investigated in single pancreatic acinar cells using patch‐clamp perforated whole‐cell recording to measure the calcium‐dependent chloride current (I
C1(Ca2+)). 1 μM thimerosal, which fails to evoke Ca2+ oscillation f alone, clearly changed the pattern of Ca2+ oscillation from pulsatile spikes (evoked by low concentrations of activators) to sinusoidal or transient oscillations. The mimetic action of thimerosal was independent of extracellular Ca2+, was blocked by extracellular application of dithiothreitol or 10 mM caffeine, as well as by internal perfusion with heparin; but was unaffected by ruthenium red. We conclude that thimerosal modulates the agonist‐specific cytosolic Ca2+ oscillatory patterns mediated by sensitizing the InsP3‐induced Ca2+ release.
Gluconeogenesis from ascorbic acid: ascorbate recycling in isolated murine hepatocytes
Authors: Braun László , Puskás Ferenc , Csala Miklós , Gyórffy Erika , Garzó Tamás , Mandl József , Bánhegyi Gábor
Published: July 2002

Ascorbic acid synthesis and breakdown were investigated in isolated hepatocytes prepared from fasted mice. Stimulation of gluconeogenesis by alanine or xylitol led to ascorbate synthesis. On the other hand, ascorbate or dehydroascorbate addition resulted in concentration‐dependent glucose production and elevation of the pentose phosphate pathway intermediate xylulose 5‐phosphate. Stimulation of ascorbate oxidation and/or the inhibition of dehydroascorbate reduction increased glucose formation. Inhibition of the pentose phosphate pathway decreased glucose production from dehydroascorbate with increased accumulation of xylulose 5‐phosphate. These results suggest that ascorbate can be recycled by a novel way involving intermediates of the pentose phosphate pathway, gluconeogenesis and hexuronic acid pathway.
Probing the higher order structure of RNA with peroxonitrous acid
Authors: Götte Matthias , Marquet Roland , Isel Catherine , Anderson Vernon E. , Keith Gérard , Gross Hans J. , Ehresmann Chantal , Ehresmann Bernard , Heumann Hermann
Published: July 2002

Potassium peroxinitrite (ONOOK) and [Fe(EDTA)]2− were used to analyze the influence of chemically entirely different hydroxyl radical sources on tRNA cleavage profiles. [Fe(EDTA)]2− gives rise to hydroxyl radicals via a Fenton‐like reaction during the oxidation of chelated Fe2+, while ONOOK generates hydroxyl radicals via its conjugate acid (ONOOK) when adding a stable alkaline solution of ONOOK in samples buffered at neutral pH. [Fe(EDTA)]2− is known to induce oxidative strand scission at sugar moieties thought to be solvent accessible, while those residues located in the ‘inside’ of structured RNAs are protected. Although ONOOH is neutral and significantly smaller than the metal complex, both reagents generate the same protection pattern on tRNAs, suggesting that access of the commonly formed hydroxyl radical, rather than access of its source, is the determining factor when probing the higher order structure of RNA. Strong difference in reactivity is only seen at the modified 2‐thiouridine S34 of tRNALys3 which shows hyperreactivity towards ONOOK treatment. This particular reaction may require interaction between the peroxonitrite anion and the thiocarbonyl group of the base, since hyperreactivity is not observed when probing the dethiolated tRNALys3.
Sequential activation of three distinct ICE‐like activities in Fas‐ligated Jurkat cells
Authors: Greidinger Eric L. , Miller Douglas K. , Yamin Ting-Ting , Casciola-Rosen Livia , Rosen Antony
Published: July 2002

ICE family proteases have been implicated as important effectors of the apoptotic pathway, perhaps acting hierarchically in a protease cascade. Using cleavage of endogenous protease substrates as probes, three distinct tiers of ICE‐like activity were observed after Fas ligation in Jurkat cells. The earliest cleavage detected (30 min) was of fodrin, and produced a 150 kDa fragment. The second phase of cleavage (50 min) involved PARP, U1‐70kDa and DNA‐PKcs, all substrates of the CPP32‐like proteases. Lamin B cleavage was observed during the third cleavage phase (90 min). Distinct inhibition profiles obtained using a panel of peptide‐based inhibitors of ICE‐like proteases clearly distinguished the three different cleavage phases. These studies provide evidence for a sequence of ICE‐like proteolytic activity during apoptosis. The early fodrin cleavage, producing a 150 kDa fragment, identifies an ICE‐like activity proximal to CPP32 in Fas‐induced Jurkat cell apoptosis.
Widespread expression of human cysteine string proteins
Authors: Coppola Thierry , Gundersen Cameron
Published: July 2002

We present the nucleotide and deduced amino acid sequences of a human cysteine string protein (csp) and a unique truncated csp variant that derives from the retention of an exonic sequence that introduces a premature, in‐frame stop codon. Low stringency Southern analysis is compatible with the presence of a single human csp gene. Northern analysis reveals that human csp mRNA has both a more heterogeneous size distribution and a more widespread tissue distribution than previously reported for other caps. Exemplifying this is the fact that csp immunoreactivity is detected in Triton X‐114 extracts of human blood, an observation which may facilitate blood‐based diagnostic assays of csp status in man.
Modulation of tumor necrosis factor‐α release by anisoosmolarity and betaine in rat liver macrophages (Kupffer cells)
Authors: Zhang Fan , Warskulat Ulrich , Häussinger Dieter
Published: July 2002

Hypoosmotic exposure (205 mosmol/l) of rat liver macrophages together with lipopolysaccharide (LPS) inhibited the LPS‐induced tumor necrosis factor‐α (TNF‐α) release by about 60% and markedly diminished the LPS‐induced increase of TNF‐α mRNA levels. Hyperosmotic exposure (405 mosmol/l) had no effect on total TNF‐α release, however, both TNF‐α accumulation in the medium and the LPS‐induced increase of TNF‐α mRNA levels were significantly delayed under these conditions. This delay was abolished upon addition of betaine, which acts as an osmolyte in Kupffer cells. When LPS was added to Kupffer cells that had been preexposed to hyperosmotic medium for 24 h, the LPS‐induced TNF‐α release was inhibited by 90% when compared to normoosmotic conditions. Likewise, the LPS‐induced increase in TNF‐α mRNA levels was largely abolished. Inhibition of TNF‐α release and of the increase in the TNF‐α mRNA level in response to hyperosmolarity/LPS, however, was largely overcome when indomethacin or betaine was present during the hyperosmotic preincubation period. Because betaine has recently been shown to inhibit the hyperosmolarity‐induced induction of cyclooxygenase‐2 and stimulation of prostaglandin production, these findings suggest that the effect of betaine in restoring the LPS‐induced TNF‐α response in hyperosmotically exposed Kupffer cells is mediated by an inhibition of prostaglandin synthesis. The findings point to a regulatory role of cell volume and betaine for TNF‐α production by liver macrophages, suggesting a new role of osmolytes in modulating immune function.
Mitochondrial ATP synthase F1‐β‐subunit is a calcium‐binding protein
Authors: Hubbard Michael J. , McHugh Nicola J.
Published: July 2002

Mitochondrial ATP synthase is responsive to changes in cytosolic calcium concentration, but the regulatory mechanisms are unclear. Here we identified a major 52 kDa calcium‐binding protein in rat enamel cells as the mitochondrial ATP synthase F1‐β‐subunit. The F1‐β‐subunit behaved as a low affinity and moderate capacity calcium‐binding protein during comparative 45Ca overlay analyses. Equivalent behavior was shown by the F1‐β‐subunit from rat liver mitochondria, but not by the homologous F1‐α‐subunit, supporting the specificity of calcium binding. Evidence that the catalytic F1‐β‐subunit binds calcium specifically introduces new mechanistic possibilities for regulating ATP synthase, and thereby coordinating ATP production with demand for ATP‐fuelled calcium pump activity.
Dissociation of affinity and efficacy in KOR‐3 chimeras
Authors: Ying-Xing Pan  , Jin Xu  , Ryan-Moro Jennifer , Mathis John , Hom Judy S.H , Jianfeng Mei  , Pasternak Gavril W
Published: July 2002

KOR‐3 chimeras were constructed in which the first coding exon of KOR‐3 was exchanged for the corresponding first coding exon of either MOR‐1 (MOR‐1/KOR‐3) or DOR‐1 (DOR‐1/KOR‐3). All three clones were expressed in CHO cells and characterized with regards to their binding profiles for orphanin FQ/nociceptin (OFQ/N) and a variety of opioids as well as their functional activities in cyclase studies. 125I[Tyr14]OFQ/N labels both KOR‐3 (K
D 37 pM) and the MOR‐1/KOR‐3 chimera (K
D 39 pM) equally well. Although its affinity for the DOR‐1/KOR‐3 chimera is quite good (K
D 135 pM), it is slightly lower than the other two. Competition studies confirm the high affinity of OFQ/N for all three clones. However, several competitors clearly distinguish the chimeras from KOR‐3. OFQ/N(1‐11) competes KOR‐3 (K
i 55 nM) over 6‐fold more potently than either of the chimeras (K
i values > 350 nM). Conversely, the modest affinity of naloxone benzoylhydrazone for KOR‐3 (310 nM) is greatly increased in both the MOR‐1/KOR‐3 (K
i 69 nM) and DOR‐1/KOR‐3 (K

i
) chimeras. The remainder of the opioids tested have no appreciable affinity against any of the clones. Functionally, OFQ/N inhibits forskolin‐stimulated cAMP accumulation in both the KOR‐3 and the MOR‐1/KOR‐3 chimera by almost 40%, with IC50 values in the low nanomolar range. Little activity is seen against the DOR‐1/KOR‐3 chimera. Naloxone benzoylhydrazone inhibits cAMP accumulation in the KOR‐3 and the DOR‐1/KOR‐3 chimera. Although naloxone benzoylhydrazone has higher affinity for the MOR‐1/KOR‐3 chimera in binding studies than KOR‐3 itself, it is inactive in cyclase studies using the MOR‐1/KOR‐3 chimera, implying that the replacement of the first coding exon increases affinity while decreasing intrinsic activity.
Induction of γ‐glutamylcysteine synthetase by cigarette smoke is associated with AP‐1 in human alveolar epithelial cells
Authors: Rahman Irfan , Smith Christopher A.D. , Lawson Mark F. , Harrison David J. , MacNee William
Published: July 2002

Increased levels of glutathione (GSH) occur in the epithelial lining fluid (ELF) of chronic cigarette smokers. Therefore we investigated the effect of cigarette smoke condensate solution (CSC) on GSH synthesis and the regulation of γ‐glutamylcysteine synthetase (γGCS) in human type II alveolar epithelial cells (A549). CSC exposure increased GSH levels, γGCS activity and γGCS heavy subunit (HS) mRNA, as well as increasing DNA binding of the activator protein‐1 (AP‐1) and the human antioxidant response element (hARE). Transfection of deletion constructs of the γGCS‐HS promoter in a chloramphenicol acetyl transferase (CAT) reporter system revealed that an hARE, present within promoter, is not required for the CSC mediated induction. We conclude that CSC induction of γGCS‐HS expression is associated with AP‐1/AP‐1‐like responsive elements.
In vivo modulated N‐acyl side chain of N‐acetylneuraminic acid modulates the cell contact‐dependent inhibition of growth
Authors: Wieser J.Raimund , Heisner Anja , Stehling Peer , Oesch Franz , Reutter Werner
Published: July 2002

Sialytation of plasma membrane glycoproteins is thought to be involved in the regulation of differentiation and in the process of tumorigenesis. Here we show that sialylation also affects cell‐cell contact‐dependent growth regulation. When cultured in the presence of non‐physiological synthetic sialic acid precursors, human diploid fibroblasts no longer exhibited density‐dependent inhibition of growth. Concomitantly, increased sialylation of contactinhibin, a glycoprotein involved in density‐dependent inhibition of growth, was observed. These results indicate that sialidase‐resistant sialic acid modifications lead to dysregulated growth control. The modifications have been induced by N‐propanoyl and other N‐acyl derivatives of d‐mannosamine.
Galectin‐3 stimulates uptake of extracellular Ca2+ in human Jurkat T‐cells
Authors: Dong Sucai , Hughes R.Colin
Published: July 2002

Galectin‐3, a mammalian galactoside‐binding protein, is not expressed in the Jurkat T‐lymphoblastoid cell line. However, Jurkat cells express surface glycoprotein receptors for galectin‐3, one of which is shown to be the glycosylated heavy chain of CD98 (4F2 antigen), a T‐cell activation marker. Addition of galectin‐3 to Jurkat cells triggers a sustained influx of extracellular Ca2+ in a concentration dependent manner. The induced increase in cytosolic [Ca2+]
i
is blocked by sugar hapten inhibitors of galectin‐3. The galectin‐3‐induced effect is insensitive to voltage‐gated Ca2+ channel antagonists such as prenylamine, nifedipine and diltiazem and to pertussis toxin but is inhibited by cholera toxin. The results suggest that galectin‐3 released by accessory cells such as macrophages may bind in vivo to T‐cell activation antigens and also participate in Ca2+ signalling.
Yeasties and beasties: 7 years of genome sequencing
Authors: Thomas Karen
Published: July 2002

The Saccharomyces cerevisiae genome sequencing project was the first of many projects aimed at sequencing the entire genomes of model organisms. Since its initiation in 1989, there have been numerous debates about the validity of genome sequencing, especially with reference to the model organisms. Seven years on, I hope to satisfy some of the critics by demonstrating that, as a consequence of the mass of data now becoming available from such projects, and the beginning of the major collaborative effort to sequence the human genome, we are now entering an exciting and dynamic time for those involved not only in genome sequencing, but also in all areas of the biological sciences.
Recurrent squamous cell carcinoma of the cervix with pulmonary lymphangitic metastasis
Authors: Sawin S.W. , Aikins J.K. , Van Hoeuen K.H. , Prioleau Y. , Morgan M.A. , Mikuta J.J.
Published: July 2002

A case report and review of the world literature are presented to examine all the reported cases of cervical carcinoma manifesting as pulmonary lymphangitic carcinomatosis in order to better understand this rare condition. The clinical and pathologic features of this disease process are reviewed, as are potential treatment options. We present the first reported case of an immunocompromised patient with cervical carcinoma and pulmonary lymphangitic metastasis with a prospective diagnosis made by transbronchial biopsy. Given that this condition carries a uniformly fatal prognosis, unwanted therapy may result from a missed diagnosis. A prospective pathologic diagnosis by transbronchial biopsy may guide appropriate therapy in these patients.