bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation, characterization and immunolocalization of phosphorylcholine‐substituted glycolipids in developmental stages of Caenorhabditis elegans
Authors: Gerdt Stefan , Dennis Roger D. , Borgonie Gaetan , Schnabel Ralf , Geyer Rudolf
Published: March 2003

Caenorhabditis elegans displays three neutral glycosphingolipids with structural homology to glycosphingolipids from the porcine nematode parasite, Ascaris suum. The present findings extend the degree of structural conservation between the two nematode species to glycosphingolipids with a phosphodiester substitution. Using a combination of hydrofluoric acid pretreatment, immunochemical characterization and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry, three zwitterionic, phosphorylcholine‐substituted glycosphingolipids could be identified in the neutral glycolipid fraction of C. elegans. The components were isolated as their zwitterionic, phosphorylcholine‐substituted, pyridylaminated oligosaccharides by HPLC. Structural analysis was performed using hydrofluoric acid treatment, partial acid hydrolysis, methylation analysis, gas chromatography–mass spectrometry, cleavage with exoglycosidases and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. Their chemical structures are proposed as: component Nz1, GalNAc(β1–4)[phosphorylcholine]GlcNAc(β1–3)Man(β1–4)Glc‐ceramide; component Nz2, Gal(α1–3)GalNAc(β1–4)[phosphorylcholine]‐GlcNAc(β1–3)Man(β1–4)Glc‐ceramide; and component Nz3, Gal(β1–3)‐ Gal(α1–3)GalNAc(β1–4)[phosphorylcholine]GlcNAc(β1–3)Man(β1–4)Glc–ceramide. The oligosaccharide core is characteristic of the biosynthetic arthro‐carbohydrate series of protostomial glycosphingolipids. The ceramide moiety was specified by a d17 : 1 sphingoid‐base with iso‐branching and anteiso‐branching, and 2‐hydroxy, saturated fatty acids as represented by docosanoic and tetracosanoic acids. Analysis of the spatial and temporal expression of the phosphorylcholine epitope, during embryonic and postembryonic development, showed it to be localized predominantly in seam cells and basement membranes, respectively. In early embryonic ontogenesis the phosphorylcholine epitope was only lipid bound, while in late embryonic and postembryonic development this epitope was both lipid bound and protein bound.
The CCKB/gastrin receptor is coupled to the regulation of enzyme secretion, protein synthesis and p70 S6 kinase activity in acinar cells from ElasCCKB transgenic mice
Authors: Desbois Christine , Le Huërou‐Luron Isabelle , Dufresne Marlène , Estival Agnès , Clerc Pascal , Romé Véronique , Clemente François , Guilloteau Paul , Fourmy Daniel
Published: March 2003

In order to determine which physiological functions can be regulated by the pancreatic CCKB/gastrin receptor, studies were carried out on pancreatic acini from mice expressing transgenic CCKB/gastrin receptors in the exocrine pancreas (ElasCCKB mice). Acini were stimulated by sulfated gastrin in the presence of SR 27897 (1.8 µm), blocking endogenous CCKA receptors. After 30 min incubation with gastrin, the secretion of chymotrypsinogen and amylase showed superimposable monophasic dose–response curves. Enzyme secretion was detectable and maximal at 100 pm and 1 nm of gastrin, respectively. No increase in chymotrypsinogen and amylase mRNAs was detected for doses of gastrin which specifically occupy the CCKB/gastrin receptor. In contrast, gastrin stimulated total protein synthesis in isolated acini from ElasCCKB mice. [35S]Methionine incorporation into total proteins was increased dose‐dependently to a maximum for 30 pm gastrin and inhibited with higher doses (> 300 pm). Gastrin stimulated p70 S6 kinase activity for concentrations ranging from 10 pm to 1 nm. Gastrin‐stimulated p70 S6 kinase activity and protein synthesis were blocked by rapamycin and wortmannin. Therefore, in ElasCCKB mice acinar cells, the CCKB/gastrin receptor mediates enzyme release and protein synthesis. However, a more efficient coupling of the CCKB/gastrin receptor to protein synthesis than to enzyme secretion was demonstrated. CCKB/gastrin receptor‐stimulated protein synthesis likely results from an enhancement of mRNA translation and involves phosphatidyl inositol 3‐kinase and p70 S6 kinase.
Circular dichroism and fluorescence spectroscopic properties of the major core protein of feline immunodeficiency virus and its tryptophan mutants
Authors: Yélamos Belén , Núñez Elena , Gómez‐Gutiérrez Julián , Datta Manisha , Pacheco Beatriz , Peterson Darrell L. , Gavilanes Francisco
Published: March 2003

The gene coding for the major capsid protein of feline immunodeficiency virus (FIV) has been cloned into the expression vector pQE60, which allows protein purification by affinity chromatography on a nitrilotriacetic acid/Ni/agarose column. The gene was expressed in Escherichia coli and the resultant soluble protein (FIV‐rp24) purified to electrophoretic homogeneity. The amino‐acid composition of the recombinant protein is almost identical to that predicted from the DNA sequence. This protein has two tryptophan residues at positions 40 and 126 that have been replaced by phenylalanine by site‐directed mutagenesis to obtain two single mutants and a double mutant. Circular dichroism and fluorescence spectroscopy were employed to study the structural features of FIV‐rp24 protein and its tryptophan mutants. The analysis of the CD spectra indicated that α‐helix is the major secondary structural element (48–52%) and that the overall three‐dimensional structure is not modified by the mutations. The fluorescence emission spectra showed that both tryptophan residues occupy a highly hydrophobic environment. Moreover, the different tyrosine fluorescence intensities of wild‐type and mutant proteins are indicative of the existence of resonance energy transfer processes to nearby tryptophan. The individual contributions of each tryptophan residue to the spectroscopic properties of the wild‐type protein were obtained from the spectra of all these proteins. Thermal denaturation studies indicate that the two tryptophan residues do not contribute equally to the stabilization of the three‐dimensional structure.
Important role of membrane‐associated CD14 in the induction of IFN‐β and subsequent nitric oxide production by murine macrophages in response to bacterial lipopolysaccharide
Authors: Saito Shinji , Matsuura Motohiro , Tominaga Kaoru , Kirikae Teruo , Nakano Masayasu
Published: March 2003

The surface antigen CD14 is known to play a central role in the recognition of lipopolysaccharide by macrophages. We characterized a mutant cell line, J7.DEF.3, derived from a murine macrophage‐like cell line, J774.1, to be defective in the ability to express the membrane‐associated form of CD14 (mCD14) but not in the ability to release the soluble form of CD14 (sCD14), and used these parent and mutant cells to investigate the role of CD14 in lipopolysaccharide signaling. In response to lipopolysaccharide stimulation, mutant cells produced slightly less tumor necrosis factor than parent cells, and produced much less (negligible level) nitric oxide than parent cells. Production of both tumor necrosis factor and nitric oxide by parent cells upon lipopolysaccharide stimulation was suppressed by anti‐CD14 serum. Expression of interferon‐β mRNA by stimulation with lipopolysaccharide, detected in parent cells, was barely detectable in mutant cells and in enzymatically mCD14‐eliminated parent cells. Lipopolysaccharide‐induced nitric oxide production in parent cells was suppressed by anti‐(murine interferon‐β), and its production in the mutant cells appeared and increased dose dependently on exogenously supplied murine interferon‐β in the presence of lipopolysaccharide. These results provide new insight into the lipopolysaccharide signaling pathway, indicating that the lipopolysaccharide signal for interferon‐β production is transduced through a mCD14‐dependent pathway and that the endogenously generated interferon‐β is an essential cofactor leading to nitric oxide production. Nuclear translocation of a transcription factor, nuclear factor κB, was observed in both parent and mutant cells following stimulation with a low dose of lipopolysaccharide, and mitogen‐activated protein kinases were also activated in both types of cell, although a higher dose of lipopolysaccharide was required by the mutant cells than by the parent cells. These results indicate that these signaling factors may participate in the mCD14‐independent lipopolysaccharide signaling pathway rather than in the mCD14‐dependent interferon‐β‐producing pathway.
Nitrilase ofRhodococcus rhodochrousJ1
Authors: Nagasawa Toru , Wieser Marco , Nakamura Tetsuji , Iwahara Hitomi , Yoshida Toyokazu , Gekko Kunihiko
Published: March 2003

Nitrilase‐containing resting cells ofRhodococcus rhodochrousJ1 converted acrylonitrile and benzonitrile to the corresponding acids, but the purified nitrilase hydrolyzed only benzonitrile, and not acrylonitrile. The activity of the purified enzyme towards acrylonitrile was recovered by preincubation with 10 mmbenzonitrile, but not by preincubation with aliphatic nitriles such as acrylonitrile. It was shown by light‐scattering experiments, that preincubation with benzonitrile led to the assembly of the inactive, purified and homodimeric 80‐kDa enzyme to its active 410‐kDa aggregate, which was proposed to be a decamer. Furthermore, the association concomitant with the activation was reached after dialysis of the enzyme against various salts and organic solvents, with the highest recovery reached at 10% saturated ammonium sulfate and 50% (v/v) glycerol, and by preincubation at increased temperatures or enzyme concentrations.
The multiple nicotinamide nucleotide‐binding subunits of bovine heart mitochondrial NADH:ubiquinone oxidoreductase (complex I)
Authors: Yamaguchi Mutsuo , Belogrudov Grigory I. , Matsuno‐Yagi Akemi , Hatefi Youssef
Published: March 2003

Direct photoaffinity labeling of purified bovine heart NADH:ubiquinone oxidoreductase (complex I) with 32P‐labeled NAD(H), NADP(H) and ADP has shown that five polypeptides become labeled, with molecular masses of 51, 42, 39, 30, and 18–20 kDa. The 51 and the 30‐kDa polypeptides were labeled with either [32P]NAD(H), [32P]NADP(H) or [β‐32P]ADP. The 42‐kDa polypeptide was labeled with [32P]NAD(H) and to a small extent with [β‐32P]ADP. It was not labeled with [32P]NADP(H). The 39‐kDa polypeptide was labeled with [32P]NADPH and to a small extent with [β‐32P]ADP. Our previous studies had shown that this subunit also binds NADP, but not NAD(H) [Yamaguchi, M., Belogrudov, G.I. & Hatefi, Y. (1998) J. Biol. Chem.273, 8094–8098]. The 18–20‐kDa polypeptide was labeled only with [32P]NADPH. Among these polypeptides, the 51‐kDa subunit is known to contain FMN and a [4Fe–4S] cluster, and is the NAD(P)H‐binding subunit of the primary dehydrogenase domain of complex I. The possible roles of the other nucleotide‐binding subunits of complex I have been discussed.
Regiospecific glycosidase‐assisted synthesis of lacto‐N‐biose I (Galβ1–3GlcNAc) and 3′‐sialyl‐lacto‐N‐biose I (NeuAcα2–3Galβ1–3GlcNAc)
Authors: Vetere Amedeo , Miletich Marinella , Bosco Marco , Paoletti Sergio
Published: March 2003

The all‐transglycolytic synthesis of lacto‐N‐biose I (Galβ1–3GlcNAc) and 3′‐sialyl‐lacto‐N‐biose I (NeuAcα2–3Galβ1–3GlcNAc) was performed. The disaccharide lacto‐N‐biose I was obtained by use of p‐nitrophenyl β‐d‐galactopyranoside as the donor, 2‐acetamido‐2‐deoxy‐d‐glucopyranose as the acceptor and Xanthomonas manihotisβ‐d‐galactosidase as the catalyst. The reaction was shown to be regiospecific, with a high molar yield (about 55%) with respect to the donor. Lacto‐N‐biose I obtained by this method was used as the acceptor for a subsequent enzymatic reaction catalyzed by Trypanosoma cruzi trans‐sialidase in which 2′‐(4‐methylumbellyferyl)‐α‐d‐N‐acetylneuraminic was used as the donor of the N‐acetylneuraminil moiety. The reaction generated the product, 3′‐sialyl‐lacto‐N‐biose I, regiospecifically and with a molar yield of about 35%.
A novel α‐amylase gene is transiently upregulated during low temperature exposure in apple fruit
Authors: Wegrzyn Teresa , Reilly Katrina , Cipriani Guido , Murphy Peter , Newcomb Richard , Gardner Richard , MacRae Elspeth
Published: March 2003

An α‐amylase gene product was isolated from apple fruit by reverse‐transcriptase PCR using redundant primers, followed by 5′ and 3′ RACE. The gene is a member of a small gene family. It encodes a putative 46.9 kDa protein that is most similar to an α‐amylase gene from potato (GenBank accession M79328). In apple fruit this new gene was expressed at low levels, as detected by reverse‐transcriptase PCR, in a number of plant tissues and during fruit development. Highest levels of mRNA for this transcript were observed 3 to 9 days after placing apple fruit at 0.5 °C. Phylogenetic analysis of amino acid sequence places the potato and apple proteins as a distinct and separate new subgroup within the plant α‐amylases, which appears to have diverged prior to the split between monocotyledonous and dicotyledonous plants. These two divergent α‐amylases lack the standard signal peptide structures found in all other plant α‐amylases, and have sequence differences within the B‐domain and C‐domain. However, comparisons with structures of known starch hydrolases suggest that these differences are unlikely to affect the enzymatic α‐1,4‐amylase function of the protein. This is the first report of upregulation of a dicotyledonous α‐amylase in response to low temperature, and confirms the presence of a new family of α‐amylases in plants.
Resonance Raman study of multihemic c‐type cytochromes from Desulfuromonas acetoxidans
Authors: Chottard Geneviève , Kazanskaya Irina , Bruschi Mireille
Published: March 2003

Two multihemic cytochromes c from the sulfur reducing bacteria Desulfuromonas acetoxidans have been studied by optical and resonance Raman spectroscopy: cytochrome c551.5, a trihemic cytochrome and cytochrome c Mr 50 000, a recently isolated high molecular mass cytochrome. The redox and Raman characteristics of cytochrome c551.5 are compared to those of the tetrahemic cytochromes c3 from Desulfovibrio. While the redox behavior, followed by spectroelectrochemistry, is similar to that of cytochrome c3, showing the same conformational change after reduction of the highest potential heme, the Raman data show a contribution from a His− form of the axial ligands and lead to the assignment of a band at 218 cm−1 to the Fe(III)–(His)2 stretching vibration. The Raman data on cytochrome c Mr 50 000 are in favor of an entirely low spin species with two different sets of axial ligands. A partially reduced state is easily accessible by ascorbate addition.
Complementation of NADPH oxidase in p67‐phox‐deficient CGD patients
Authors: Vergnaud Sabrina , Paclet Marie‐Hélène , El Benna Jamel , Pocidalo Marie‐Anne , Morel Françoise
Published: March 2003

Chronic granulomatous disease (CGD) is due to a functional defect of the O2− generating NADPH oxidase of phagocytes. Epstein–Barr‐virus‐immortalized B lymphocytes express all the constituents of oxidase with activity 100 times less than that of neutrophils. As in neutrophils, oxidase activity of Epstein–Barr‐virus‐immortalized B lymphocytes was shown to be defective in the different forms of CGD; these cells were used as a model for the complementation studies of two p67‐phox‐deficient CGD patients. Reconstitution of oxidase activity was performed in vitro by using a heterologous cell‐free assay consisting of membrane‐suspended or solubilized and purified cytochrome b558 that was associated with cytosol or with the isolated cytosolic‐activating factors (p67‐phox, p47‐phox, p40‐phox) from healthy or CGD patients. In p67‐phox‐deficient CGD patients, two cytosolic factors are deficient or missing: p67‐phox and p40‐phox. Not more than 20% of oxidase activity was recovered by complementing the cytosol of p67‐phox‐deficient patients with recombinant p67‐phox. On the contrary, a complete restoration of oxidase activity was observed when, instead of cytosol, the cytosolic factors were added in the cell‐free assay after isolation in combination with cytochrome b558 purified from neutrophil membrane. Moreover, the simultaneous addition of recombinant p67‐phox and recombinant p40‐phox reversed the previous complementation in a p40‐phox dose‐dependent process.

These results suggest that in the reconstitution of oxidase activity, p67‐phox is the limiting factor; the efficiency of complementation depends on the membrane tissue and the cytosolic environment. In vitro, the transition from the resting to the activated state of oxidase, which results from assembling, requires the dissociation of p40‐phox from p67‐phox for efficient oxidase activity. In the process, p40‐phox could function as a negative regulatory factor and stabilize the resting state.
Characterization of hydroxylaminobenzene mutase from pNBZ139 cloned from Pseudomonas pseudoalcaligenes JS45
Authors: He Zhongqi , Nadeau Lloyd J. , Spain Jim C.
Published: March 2003

Hydroxylaminobenzene mutase is the enzyme that converts intermediates formed during initial steps in the degradation of nitrobenzene to a novel ring‐fission lower pathway in Pseudomonas pseudoalcaligenes JS45. The mutase catalyzes a rearrangement of hydroxylaminobenzene to 2‐aminophenol. The mechanism of the reactions and the properties of the enzymes are unknown. In crude extracts, the hydroxylaminobenzene mutase was stable at SDS concentrations as high as 2%. A procedure including Hitrap‐SP, Hitrap‐Q and Cu(II)‐chelating chromatography was used to partially purify the enzyme from an Escherichia coli clone. The partially purified enzyme was eluted in the void volume of a Superose‐12 gel‐filtration column even in the presence of 0.05% SDS in 25 mm Tris/HCl buffer, which indicated that it was highly associated. When the enzymatic conversion of hydroxylaminobenzene to 2‐aminophenol was carried out in 18O‐labeled water, the product did not contain 18O, as determined by GC‐MS. The results indicate that the reaction proceeded by intramolecular transfer of the hydroxy group from the nitrogen to the C‐2 position of the ring. The mechanism is clearly different from the intermolecular transfer of the hydroxy group in the non‐enzymatic Bamberger rearrangement of hydroxylaminobenzene to 4‐aminophenol and in the enzymatic hydroxymutation of chorismate to isochorismate.
The wide binding properties of a wheat nonspecific lipid transfer protein
Authors: Tassin‐Moindrot Séverine , Caille Anita , Douliez Jean‐Paul , Marion Didier , Vovelle Françoise
Published: March 2003

The 3D solution structure of wheat nonspecific lipid transfer protein (ns‐LTP) complexed with prostaglandin B2, a lipid with both vinyl and hydroxylated groups, has been determined by 1H 2D NMR. The global fold of the protein is close to the previously published structures of wheat, maize, barley and rice ns‐LTPs. The ligand is almost completely embedded in the hydrophobic core of the protein. Structure comparisons of free and bound wheat ns‐LTP reveal that the binding of prostaglandin B2 hardly affects the global fold of the protein. The structural data on this unusual complex are discussed and compared with other known ns‐LTP lipid‐complexes.
Favourable side‐chain orientation of cleavage site dibasic residues of prohormone in proteolytic processing by prohormone convertase 1/3
Authors: Brakch Noureddine , Rholam Mohamed , Simonetti Mario , Cohen Paul
Published: March 2003

Previous studies using selectively modified pro‐ocytocin/neurophysin substrate analogues and the purified metalloprotease, pro‐ocytocin/neurophysin convertase (magnolysin; EC 3.4 24.62), have shown that dibasic cleavage site processing is associated with a prohormone sequence organized in a β‐turn structure. We have used various peptide analogues of the pro‐ocytocin‐neurophysin processing domain, and recombinant prohormone convertase 1/3, to test the validity of this property towards this member of the family of prohormone convertases (PCs). The enzymatic cleavage analysis and kinetics showed that: (a) with methyl amide (N‐Met) modification, a secondary structure β‐turn breaker, the enzyme substrate interaction was abolished; (b) cleavage was favoured when the dibasic substrate side‐chains were oriented in opposite directions; (c) the amino acid present at the P′1 position is important in the enzyme–substrate interaction; (d) the flexibility of the peptide substrate is necessary for the interaction; (e) Addition of dimethylsulfoxide to the cleavage assay favoured the cleavage of the pro‐ocytocin/neurophysin large substrate over that of the smaller one pGlu‐Arg‐Thr‐Lys‐Arg‐methyl coumarin amide. These data allowed us to conclude that proteolytic processing of pro‐ocytocin‐related peptide substrates by PC1/3 as well as by the metalloenzyme, magnolysin, involves selective recognition of precise cleavage site local secondary structure by the processing enzyme. It is hypothesized that this may represent a general property of peptide precursor proteolytic processing systems.
Degradation of transcription factor IRF‐1 by the ubiquitin–proteasome pathway
Authors: Nakagawa Koji , Yokosawa Hideyoshi
Published: March 2003

Interferon regulatory factor‐1(IRF‐1) is a transcriptional activator of interferon genes and interferon‐inducible genes. It has been shown that IRF‐1 functions not only as a regulator of the interferon‐responsive system but also as a regulator of cell growth and apoptosis. In addition, it is known that IRF‐1 is a short‐lived protein, but the mechanism that regulates its stability has not yet been clarified. Here, we show that IRF‐1 is degraded via the ubiquitin–proteasome pathway. IRF‐1 protein degradation in HeLa and NIH3T3 cells was inhibited by treatment with proteasome‐specific inhibitors. Overexpression of IRF‐1 protein and ubiquitin in COS7 cells revealed specific multiubiquitination of IRF‐1. Although the full‐length IRF‐1 was unstable, IRF‐1 mutants with C‐terminal truncations larger than 39 amino acids were found to be almost stable, suggesting that the 39‐residue C‐terminal region controls the stability of IRF‐1. Further analysis of the stability of a green fluorescent protein‐fusion protein containing the 39‐residue C‐terminal region of IRF‐1 showed that this C‐terminal region confers instability on green fluorescent protein, a normally stable protein, suggesting that this region functions as a protein‐degradation signal. Taking the results together, it can be concluded that the 39‐residue C‐terminal region is necessary and sufficient to control the stability of the IRF‐1 protein.
The role of amino‐acid residues in the hydrophobic patch surrounding the haem group of cytochrome f in the interaction with plastocyanin
Authors: Gong Xiao‐Song , Wen Jiang Qi , Gray John C.
Published: March 2003

Soluble turnip cytochrome f has been purified from the periplasmic fraction of Escherichia coli expressing a truncated petA gene encoding the precursor protein lacking the C‐terminal 33 amino‐acid residues. The protein is identical [as judged by 1H‐NMR spectroscopy, midpoint redox potential (+ 365 mV) and electron transfer reactions with plastocyanin] to cytochrome f purified from turnip leaves. Several residues in the hydrophobic patch surrounding the haem group have been changed by site‐directed mutagenesis, and the proteins purified from E. coli. The Y1F and Q7N mutants showed only minor changes in the plastocyanin‐binding constant Ka and the second‐order rate constant for electron transfer to plastocyanin, whereas the Y160S mutant showed a 30% decrease in the overall rate of electron transfer caused in part by a 60% decrease in binding constant and partially compensated by an increased driving force due to a 27‐mV decrease in redox potential. In contrast, the F4Y mutant showed increased rates of electron transfer which may be ascribed to an increased binding constant and a 14‐mV decrease in midpoint redox potential. This indicates that subtle changes in the hydrophobic patch can influence rates of electron transfer to plastocyanin by changing the binding constants and altering the midpoint redox potential of the cytochrome haem group.
Intracellular trafficking and release of intact edible mushroom lectin from HT29 human colon cancer cells
Authors: Yu Lu‐Gang , Fernig David G. , Rhodes Jonathan M.
Published: March 2003

Our previous studies have shown that the Galβ1–3GalNAcα‐ (Thomsen–Friedenreich antigen)‐binding lectin from the common edible mushroom Agaricus bisporus (ABL) reversibly inhibits cell proliferation, and this effect is a consequence of inhibition of nuclear localization sequence‐dependent nuclear protein import after ABL internalization [Yu, L.G., Fernig, D.G., White, M.R.H., Spiller, D.G., Appleton, P., Evans, R.C., Grierson, I., Smith, J.A., Davies, H., Gerasimenko, O.V., Petersen, O.H., Milton, J.D. & Rhodes, J.M. (1999) J. Biol. Chem.274, 4890–4899]. Here, we have investigated further the intracellular trafficking and fate of ABL after internalization in HT29 human colon cancer cells. Internalization of 125I‐ABL occurred within 30 min of the lectin being bound to the cell surface. Subcellular fractionation after pulse labelling of the cells with 125I‐ABL for 2 h at 4 °C followed by culture of the cells at 37 °C demonstrated a steady increase in radioactivity in a crude nuclear extract. The radioactivity in this extract reached a maximum after 10 h and declined after 20 h. Release of ABL from the cell, after pulse labelling, was assessed using both fluorescein isothiocyanate‐labelled ABL and 125I‐ABL and was slow, with a t1/2 of 48 h. Most of the 125I‐ABL both inside cells and in the medium remained intact, as determined by trichloroacetic acid precipitation and SDS/PAGE, and after 48 h only 22 ± 2% of ABL in the medium and 14 ± 2% inside the cells was degraded. This study suggests that the reversibility of the antiproliferative effect of ABL is associated with its release from cells after internalization. The internalization and subsequent slow release, with little degradation of ABL, reflects the tendency of lectins to resist biodegradation and implies that other endogenous or exogenous lectins may be processed in this way by intestinal epithelial cells.
Compact residual structure in lentil lectin at pH 2
Authors: Marcos María J. , Villar Enrique , Gavilanes Francisco , Zhadan Galina G. , Shnyrov Valery L.
Published: March 2003

Lentil lectin obtained from Lens culinaris collected in the La Armuña area (Salamanca, Spain) was examined by high‐sensitivity differential scanning calorimetry, fluorimetry and measurements of circular dichroism at pH 2.0 and 7.4. At pH 2.0 the lentil lectin is not in the native state; however, at this pH it does show signs of a residual structure that breaks down upon heating. The lentil lectin at pH 2 shares some similarities with what has become known as the molten globule state. The thermal denaturation of intact (pH 7.4) and partially unfolded (pH 2.0) lentil lectin was irreversible and strongly dependent upon the scan rate, suggesting that its denaturation is under kinetic control. The process of lentil lectin denaturation is interpreted in terms of the simple kinetic model, Nk → D, where k is a first‐order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denatured state.
Complementation of deletion mutants in the genes encoding the F1‐ATPase by expression of the corresponding bovine subunits in yeast S. cerevisiae
Authors: Lai‐Zhang Jie , Mueller David M.
Published: March 2003

The F1F0 ATP synthase is composed of the F1‐ATPase which is bound to F0, in the inner membrane of the mitochondrion. Assembly and function of the enzyme is a complicated task requiring the interactions of many proteins for the folding, import, assembly, and function of the enzyme. The F1‐ATPase is a multimeric enzyme composed of five subunits in the stoichiometry of α3β3γδε. This study demonstrates that four of the five bovine subunits of the F1‐ATPase can be imported and function in an otherwise yeast enzyme effectively complementing mutations in the genes encoding the corresponding yeast ATPase subunits. In order to demonstrate this, the coding regions of each of the five genes were separately deleted in yeast providing five null mutant strains. All of the strains displayed negative or a slow growth phenotype on medium containing glycerol as the carbon source and strains with a null mutation in the gene encoding the γ‐, δ‐ or ε‐gene became completely, or at a high frequency, cytoplasmically petite. The subunits of bovine F1 were expressed individually in the yeast strains with the corresponding null mutations and targeted to the mitochondrion using a yeast mitochondrial leader peptide. Expression of the bovine α‐, β‐, γ‐, and ε‐, but not the δ‐, subunit complemented the corresponding null mutations in yeast correcting the corresponding negative phenotypes. These results indicate that yeast is able to import, assemble subunits of bovine F1‐ATPase in mitochondria and form a functional chimeric yeast/bovine enzyme complex.
Biophysical studies of the development of amyloid fibrils from a peptide fragment of cold shock protein B
Authors: Wilkins Deborah K. , Dobson Christopher M. , Groß Michael
Published: March 2003

The peptide CspB‐1, which represents residues 1–22 of the cold shock protein CspB from Bacillus subtilis, has been shown to form amyloid fibrils when solutions containing this peptide in aqueous (50%) acetonitrile are diluted in water [M. Großet al. (1999) Protein Science8, 1350–1357] We established conditions in which reproducible kinetic steps associated with the formation of these fibrils can be observed. Studies combining these conditions with a range of biophysical methods reveal that a variety of distinct events occurs during the process that results in amyloid fibrils. A CD spectrum indicative of β structure is observed within 1 min of the solvent shift, and its intensity increases on a longer timescale in at least two kinetic phases. The characteristic wavelength shift of the amyloid‐binding dye Congo Red is established within 30 min of the initiation of the aggregation process and corresponds to one of the phases observed by CD and to changes in the Fourier transform‐infrared spectrum indicative of β structure. Short fibrillar structures begin to be visible under the electron microscope after these events, and longer, well‐defined amyloid fibrils are established on a timescale of hours. NMR spectroscopy shows that there are no significant changes in the concentration of monomeric species in solution during the events leading to fibril formation, but that soluble aggregates too large to be visible in NMR spectra are present throughout the process. A model for amyloid formation by this peptide is presented which is consistent with these kinetic data and with published work on a variety of disease‐related systems. These findings support the concept that the ability to form amyloid fibrils is a generic property of polypeptide chains, and that the mechanism of their formation is similar for different peptides and proteins.
Human oxytocin receptors in cholesterol‐rich vs. cholesterol‐poor microdomains of the plasma membrane
Authors: Gimpl Gerald , Fahrenholz Falk
Published: March 2003

We analyzed the properties of a G protein‐coupled receptor localized in cholesterol‐poor vs. cholesterol‐rich microdomains of the plasma membrane. For this purpose, the human oxytocin receptor, which is very sensitive against alterations of the membrane cholesterol level, was stably expressed in HEK293 cells. To calculate the total number of receptors independent of ligand binding studies, the oxytocin receptor was tagged with an enhanced green fluorescent protein (EGFP) which did not change the functional properties of the receptor. Only 1% of the oxytocin receptors were present in cholesterol‐rich detergent‐insoluble domains. In contrast, employing a detergent‐free fractionation scheme that preserves the functional activity of the receptor, we detected 10–15% of the receptors in cholesterol‐rich low‐density membranes and therein the high‐affinity state receptors were twofold enriched. In cholesterol‐poor vs. cholesterol‐rich domains, high‐affinity oxytocin receptors behaved similar with respect to their agonist binding kinetics and GTP sensitivity. However, high‐affinity oxytocin receptors localized in cholesterol‐rich low‐density membranes showed a markedly enhanced (t ½≈ threefold) stability at 37 °C as compared with the oxytocin receptors localized in the cholesterol‐poor high‐density membranes. Addition of cholesterol to the high‐density membranes fully protected the oxytocin receptors against loss of function. The importance of cholesterol to stabilize the oxytocin receptor was supported in experiments with solubilized receptors. Cholesterol markedly delayed the inactivation of oxytocin receptors solubilized with Chapso. In conclusion, the data of this report suggest that functional properties of heptahelical receptor proteins could differ in dependence of their localization in different membrane microdomains.
Correct folding of a ribozyme induced by nonspecific macromolecules
Authors: Nashimoto Masayuki
Published: March 2003

The 50‐nucleotide hammerhead ribozyme HH‐S was tested for self‐cleavage. The self‐cleavage was very inefficient, and only 13% of HH‐S was transformed to its cleavage products. Surprisingly, the percentage of cleavage of HH‐S was increased to 30% when 1 µg of tRNA was added to the reaction mixture (6 µL). Other macromolecules such as DNAs and proteins were examined to see if they also augmented cleavage of HH‐S , and it was found that most of the macromolecules tested, except nucleotide monomers, did indeed enhance HH‐S cleavage. The self‐cleaving reaction was almost saturated in 30 min, and only 13% of HH‐S was cleaved at 37 °C for a 70‐min reaction, indicating that 87% of HH‐S was in kinetically trapped inactive conformations. Time courses for the reaction of the HH‐S self‐cleavage were also measured in the presence of tRNA, an oligodeoxyribonucleotide, or BSA. Cleavage of HH‐S, which had already reached a plateau of 13% cleaved, increased gradually after the addition of the effector molecules. The first‐order rate constant for the self‐cleavage reaction in the absence of an effector was comparable to that in the presence of BSA, indicating that the effector molecules do not affect the chemical step of self‐cleavage. These results demonstrate that a variety of nonspecific macromolecules can induce conformational change of the hammerhead even in such a low concentration as 0.003% (w/v). This conformational change may occur by macromolecular collisions, or nonspecific weak interactions between HH‐S and effectors. Alternatively, a molecular crowding effect may cause the conformational change.
Interferon‐α synergistically enhances induction of interleukin‐6 by double stranded RNA in HeLa cells
Authors: Harcourt Jennifer L. , Offermann Margaret K.
Published: March 2003

Double stranded RNA (dsRNA), an intermediate that is common during viral infection, directly induces much higher levels of expression of interleukin‐6 (IL‐6) mRNA than does the cytokine IL‐1β. Interferon α (IFNα) by itself does not induce expression of IL‐6; nonetheless, IFNα pretreatment dramatically enhances IL‐6 induction by dsRNA but not by IL‐1β. Mutation of either the activating transcription factor/cyclic AMP response element binding protein (ATF/CREB) or the NF‐IL‐6 binding element within the IL‐6 promoter eliminates most responsiveness of CAT reporter constructs to either dsRNA or to IL‐1β. IFNα pretreatment partially restores responsiveness to dsRNA but not to IL‐1β when either the ATF/CREB site or the NF‐IL‐6 site is mutated, but at least one of these sites must be intact for responsiveness to be restored. Mutation of the κB binding site in the IL‐6 promoter eliminates responsiveness to either IL‐1β or to dsRNA, and pretreatment with IFNα does not restore any responsiveness. Incubation with dsRNA leads to a decrease in protein translation, especially in cells that have been pretreated with IFNα. Nonetheless, IFNα pretreatment followed by dsRNA leads to very high IL‐6 protein levels. These studies demonstrate that major differences exist in the induction of IL‐6 at both the mRNA and protein levels by dsRNA compared to cytokines and that IFNα pretreatment selectively enhances IL‐6 induction by dsRNA but not by IL‐1β. The high levels of IL‐6 expression that result when cells encounter class I IFN prior to dsRNA suggest a mechanism for a heightened host response to viral infection with heightened production of this pleotropic cytokine.
Biogenesis of the yeast frataxin homolog Yfh1p
Authors: Geissler Andreas , Krimmer Thomas , Schönfisch Birgit , Meijer Michiel , Rassow Joachim
Published: March 2003

Tim44 is an essential component of the mitochondrial inner membrane protein import machinery. In this study we asked if Tim44 is of relevance in intramitochondrial protein folding. We investigated the role of Tim44 in the biogenesis of the authentic mitochondrial protein Yfh1p, the yeast homolog of mammalian frataxin, which was recently implicated in Friedreich ataxia. After inactivation of Tim44, binding of mitochondrial heat shock protein (mtHsp)70 to translocating Yfh1p and subsequent folding to the native state was nearly completely blocked. Residual amounts of imported Yfh1p showed an increased tendency to aggregate. To further characterize the functions of Tim44 in the matrix, we imported dihydrofolate reductase (DHFR) as a model protein. Depletion of Tim44 allowed import of DHFR, although folding of the newly imported DHFR was delayed. Moreover, the depletion of Tim44 caused a strongly reduced binding of mtHsp70 and Mge1 to the translocating polypeptide. Subsequent dissociation of mtHsp70 from imported DHFR was delayed, indicating that mtHsp70–substrate complexes formed independently of Tim44 differ from the complexes that form under the control of Tim44. We conclude that Tim44 not only plays a role in protein translocation but also in the pathways of mitochondrial protein folding.
Activation of phosphatidylinositol 3‐kinase by a complex of p59fyn and the receptor tyrosine kinase Xmrk is involved in malignant transformation of pigment cells
Authors: Wellbrock Claudia , Schartl Manfred
Published: March 2003

Malignant melanoma in the fish Xiphophorus is induced by overexpression of the Xmrk‐oncogene, encoding a subclass I receptor tyrosine kinase. The mutationally activated Xmrk protein triggers constitutive mitogenic signalling in fish melanoma cells. In recent studies we showed that in melanoma cells phosphatidylinositol (PtdIns) 3‐kinase, as well as p59fyn, has elevated levels of kinase activity. Both bind directly to different phosphotyrosine residues in the Xmrk receptor C‐terminus through their SH2 domains. To analyse the mechanism of regulation of these Xmrk‐associated kinases in melanoma we characterized the protein–protein interactions between PtdIns 3‐kinase, p59fyn and the Xmrk receptor in detail. A ternary complex in which the p85 subunit of PtdIns 3‐kinase is associated with p59fyn as well as with Xmrk was identified. Contrary to complexes described for other receptors, the adaptor protein p120Cbl was not involved in these interactions. Thus, we describe here a new mechanism of activation of PtdIns 3‐kinase by a receptor of the epidermal growth factor receptor family in which p59fyn acts as an adaptor as well as an activator of PtdIns 3‐kinase. Activation of PtdIns 3‐kinase activity by fyn was also found in vivo. The fact that this was only detectable in highly transformed Xmrk overexpressing melanomas but not in benign lesions points to the essential role of the Xmrk receptor in this mechanism of regulation.
Folding of a β‐hairpin peptide derived from the N‐terminus of ubiquitin
Authors: Jourdan Muriel , Griffiths‐Jones Samuel R. , Searle Mark S.
Published: March 2003

The role of the non‐native β‐turn sequence (NPDG) in nucleating the folding of a β‐hairpin peptide derived from the N‐terminus of ubiquitin, has been examined by NMR and CD spectroscopy. The NPDG sequence, while representing a common two‐residue type I turn sequence in proteins, folds to give a G1‐bulged type I turn in the context of a β‐hairpin peptide, to the exclusion of other possible conformations. The turn conformation results in misalignment of the two β strands and a β hairpin with non‐native side chain interactions. A truncated 12‐residue analogue of the hairpin, in which the majority of residues in the N‐terminal β strand have been deleted, shows some weak propensity to fold into a G‐bulged type I turn conformation in the absence of interstrand stabilizing interactions. The NPDG turn sequence pays some of the entropic cost in initiating folding allowing interstrand interactions, which in this case arise from the non‐native pairing of residue side chains, to stabilize a significant population of the folded state. Examination of the relative abundance of the Pro–Asp type I turn, with G in the +B1 position, vs. the type I G‐bulged turn PXG, in a database of high resolution structures, reveals 48 instances of PXG bulged turns for which X = Asp is by far the most common residue with 20 occurrences. Strikingly, there are no examples of a type I PD turn with G at the +B1 position, in good agreement with our experimental observations that the PDG G‐bulged turn is populated preferentially in solution.
Transcriptional control of adrenomedullin induction by phorbol ester in human monocytic leukemia cells
Authors: Nakayama Masaharu , Takahashi Kazuhiro , Kitamuro Tomomi , Murakami Osamu , Shirato Kunio , Shibahara Shigeki
Published: March 2003

Adrenomedullin is a potent vasodilator peptide that was originally identified from human pheochromocytoma. In this study, we investigated the induction of adrenomedullin gene expression in THP‐1 acute monocytic leukemia cells during differentiation into macrophage‐like cells by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), and identified a cis‐regulatory region of the human adrenomedullin gene responsible for TPA‐induced adrenomedullin expression. Upon treatment with TPA (100 ng·mL−1) for 24 h, immunoreactive adrenomedullin concentrations in the culture medium and adrenomedullin mRNA levels were increased more than 10‐fold, concomitant with the differentiation of THP‐1 cells into macrophage‐like cells. Actinomycin D abolished the TPA‐induced adrenomedullin expression, indicating that the induction of ADM gene expression by TPA was regulated at the transcriptional level. Transient transfection assay revealed that a cis‐acting region (positions −70 to −30) of human adrenomedullin gene was necessary for TPA‐induced reporter gene expression. This region contains multiple copies of activator protein 2 (AP‐2) binding sites, which are bound by purified AP‐2 protein, as judged by electrophoretic mobility shift assay. The binding activity to this region was undetectable in nuclear extracts prepared from untreated THP‐1 cells, but was increased in extracts prepared from TPA‐treated cells. The protein binding was abolished by unlabeled oligonucleotides containing the AP‐2 consensus sequence. These results indicate that the region (−70 to −30) of the human ADM gene containing multiple AP‐2 binding sites is responsible for TPA‐induced adrenomedullin expression in THP‐1 cells.
Phosphoinositides determine specificity of the guanine‐nucleotide exchange activity of cytohesin‐1 for ADP‐ribosylation factors derived from a mammalian expression system
Authors: Knorr Thomas , Nagel Wolfgang , Kolanus Waldemar
Published: March 2003

ADP‐ribosylation factors (ARFs) are small Ras‐like GTPases which play important roles in intracellular vesicle transport and in the remodeling of the actin cytoskeleton. Guanine nucleotide exchange factors (GEFs) for ARFs have recently been identified. One of them, cytohesin‐1, a 47‐kDa cytoplasmic protein acts as an inside‐out signaling molecule and regulates binding of the β2 integrin leukocyte function antigen 1 (LFA‐1) to its ligand intercellular adhesion molecule 1 (ICAM‐1). In this study, we address the regulation of the GEF activity of cytohesin‐1 by phosphoinositides, using mammalian expression of functional ARF–Ig chimeras. The fusion proteins, which can be quantitatively immunoprecipitated on protein A–Sepharose, target to the expected intracellular compartments, and they are readily induced to bind GTP in vitro. We show that both ARF1–Ig and ARF6–Ig chimeras are activated in vitro by cytohesin‐1. However, GEF activity towards ARF6 is strongly suppressed by phosphatidylinositol‐(3,4,5)‐trisphosphate (PtdInsP3). In contrast, cytohesin‐1‐dependent GTP binding of ARF1 is significantly enhanced by PtdInsP3. We conclude that the membrane phospholipid PtdInsP3 determines the specificity of the GEF activity of cytohesin‐1.
Guanidine hydrochloride induced reversible dissociation and denaturation of duck δ2‐crystallin
Authors: Lee Hwei‐Jen , Chang Gu‐Gang
Published: March 2003

The tetrameric δ2‐crystallin from duck lens exhibits a reversible dissociation‐denaturation process in solutions containing guanidine hydrochloride (GdnHCl). Sigmoidal or biphasic curves for the dissociation/denaturation processes, obtained using different methods of structural analysis, as a function of GdnHCl concentration were not coincidental with each other. δ2‐crystallin in 0.91 m GdnHCl existed primarily as a monomer, which had no endogenous argininosuccinate lyase activity. After dilution of the GdnHCl‐treated protein, the monomers reassociated into tetramers with concomitant recovery of enzyme activity. The sigmoidal recovery of enzyme activity demonstrates a cooperative hysteretic reactivation process. When the concentration of GdnHCl was higher than 1.2 m, various partially unfolded soluble forms of δ2‐crystallin were produced from the dissociated monomers as shown by size‐exclusion chromatography. The formation of a partially unfolded intermediate during the dissociation–denaturation process is proposed.
HMG1 protein stimulates DNA end joining by promoting association of DNA molecules via their ends
Authors: Štros Michal , Cherny Dmitry , Jovin Thomas M.
Published: March 2003

High mobility group (HMG) 1 protein is a highly abundant and an evolutionarily conserved chromosomal protein with two homologous DNA‐binding domains (HMG boxes), A and B, attached by a short basic region to an acidic C‐terminal tail. The protein has been implicated in a number of fundamental biological processes including DNA replication, transcription, recombination and repair. We demonstrate that HMG1 is able to enhance cohesive‐end and blunt‐end DNA ligation by T4 DNA ligase via its B domain. The C‐terminal flanking sequence of the B domain (seven basic residues out of ≈ 18) and a number of conserved amino‐acid residues within the HMG box (mainly basic or hydrophobic) are required for efficient stimulation of ligation. Pull‐down assays, electron and scanning force microscopy revealed that HMG1 can associate two DNA molecules via their ends even in the absence of complementary overhangs. We propose that HMG1 protein may be involved in the rejoining of DNA breaks by different DNA ligases due to its ability to bring DNA duplexes and their termini into a close proximity while leaving the ends accessible for ligation.