bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of −1 ribosomal frameshifting directed by Cocksfoot mottle sobemovirus genome
Authors: Lucchesi Jimmy , Mäkeläinen Katri , Merits Andres , Tamm Tiina , Mäkinen Kristiina
Published: March 2003

The polyprotein of Cocksfoot mottle virus (CfMV) is encoded by two overlapping open reading frames (ORFs). The putative replicase of CfMV is produced as a part of the polyprotein from ORF2b by the −1 ribosomal frameshifting mechanism. The signals leading to −1 ribosomal frameshifting directed by CfMV RNA are the slippery heptamer UUUAAAC and a stem–loop structure starting seven nucleotides downstream from the heptamer. We studied the effect of different parts of the CfMV genome on the −1 ribosomal frameshifting efficiency using a wheat germ extract transcription/translation system. A point mutation in the slippery heptamer and a mutation deleting the stem–loop structure prevented frameshifting. Seventy nucleotides of CfMV sequence, including the slippery sequence and the stem–loop structure, was found to act as a minimal region for frameshifting. Interestingly, a termination codon introduced into the −1‐frame 27 nucleotides downstream of the stem–loop structure increased frameshift efficiency threefold, while a similarly located termination codon in the 0‐frame had no effect. Even fourfold to fivefold efficiencies were observed when the polyprotein encoding ORFs were fused together, which led simultaneously to the formation of a termination codon downstream of the frameshift signal. Possible reasons underlying these observations are discussed.
Inhibition by unsaturated fatty acids of type II secretory phospholipase A2 synthesis in guinea‐pig alveolar macrophages
Authors: Alaoui El Azher Mounia , Havet Nathalie , Singer Monique , Dumarey Claude , Touqui Lhousseine
Published: March 2003

The effect of arachidonic acid (C20:4) on the production of secretory type II phospholipase A2 (sPLA2‐II) by guinea‐pig alveolar macrophages was investigated. We show that incubation of these cells with 1–30 µm of arachidonic acid inhibits the synthesis of sPLA2‐II in a concentration‐dependent manner with an IC50 of ≈ 7.5 µm. The inhibition by low concentrations (5 µm) of arachidonic acid was partially reduced by pretreatment of alveolar macrophages with cyclooxygenase or cytochrome P450 inhibitors (aspirin and 1‐aminobenzotriazole, respectively), but not by lipoxygenase inhibitor, BW A4C. However, these inhibitors failed to interfere with the effect of high concentrations (30 µm) of arachidonic acid, suggesting that the latter may act on the expression of sPLA2‐II, at least in part, independently of eicosanoid generation. Indeed, a similar inhibitory effect on sPLA2‐II activity and mRNA expression was observed with other unsaturated fatty acids such as eicosapentaenoic (C20:5) and oleic (C18:1) acids, but not with the saturated fatty acid, palmitic acid (C16:0). In addition, arachidonic acid partially reduced the secretion of tumor necrosis factor α, an important intermediate in the induction of sPLA2‐II synthesis by guinea‐pig alveolar macrophages. However, addition of recombinant tumor necrosis factor α failed to reverse the inhibitory effect of arachidonic acid on sPLA2‐II expression, suggesting that this process occurs downstream of tumor necrosis factor α secretion. We conclude that the expression of sPLA2‐II in alveolar macrophages is down‐regulated at the transcriptional level by arachidonic acid either directly or via its cyclooxygenase and cytochrome P450‐derived metabolites.
Liposome‐mediated cytosolic delivery of macromolecules and its possible use in vaccine development
Authors: Owais M. , Gupta C. M.
Published: March 2003

In the majority of bacterial and viral infections the generation of cytotoxic T cells is of particular interest because such pathogens are able to escape the host defence mechanisms by surviving intracellularly within the phagocytic cells. To generate a CD8+ T lymphocyte response against exogenous antigens, the prerequisite is their delivery into the cytosol followed by processing and presentation along with class I major histocompatibility complex (MHC‐I) molecules. In the present study we describe the method of liposome‐based delivery of antigens and other macromolecules into the cytosol of target cells. To develop safe and effective methods for generating CD8+ T lymphocytes, we exploited the fusogenic character of lipids derived from lower organisms, that is baker's yeast (Saccharomyces cerevisiae). The degree of fusion with model membrane systems using yeast lipid liposomes varied from 40–70%, as opposed to 1–8% observed with egg PtdCho liposomes, depending on the assay system used. The fusion of yeast lipid liposomes with macrophages resulted in effective delivery of the entrapped solutes into the cytoplasmic compartment. This was further supported by the inhibition of cellular protein synthesis in J774 A1 cells by ricin A, encapsulated in the yeast lipid liposomes. Interestingly, the model antigen ovalbumin, when entrapped in the yeast lipid liposomes, successfully elicited antigen reactive CD8+ T cell responses. It may be concluded that the liposomes made of lipids derived from S. cerevisiae can spontaneously fuse with macrophages, delivering a significant portion of their contents into the cytoplasmic compartment of the cells.
Cadmium blocks hypoxia‐inducible factor (HIF)‐1‐mediated response to hypoxia by stimulating the proteasome‐dependent degradation of HIF‐1α
Authors: Chun Yang‐Sook , Choi Eunjoo , Kim Gi‐Tae , Choi Hong , Kim Chan‐Hyung , Lee Min‐Jae , Kim Myung‐Suk , Park Jong‐Wan
Published: March 2003

Cadmium is a substantial industrial and environmental pollutant which seriously impairs erythropoiesis. Cd has been demonstrated to aggravate anemia by suppressing erythropoietin gene expression in anemic patients. As hypoxic induction of erythropoietin mRNA depends on a transcription factor, hypoxia‐inducible factor 1 (HIF‐1), we hypothesized that Cd suppresses the hypoxic activation of HIF‐1. In hypoxic Hep3B cells, all mRNAs of various genes, which are known to be upregulated by HIF‐1 activation under hypoxia, were suppressed by Cd in a dose‐dependent manner. Cd inhibited the hypoxia‐induced activity of luciferase in 293 cells which was transfected with a reporter plasmid carrying a hypoxia response element. By electrophoretic mobility gel shift assay, Cd inhibited the DNA‐binding activity of HIF‐1 in hypoxic Hep3B cells. Cd reduced the amount of HIF‐1α protein in hypoxia, whereas it didn’t affect HIF‐1 α mRNA levels. Moreover, Cd inhibited HIF‐1α accumulation induced by cobalt and desferrioxamine. Antioxidants and a proteasome inhibitor prevented the HIF‐1α degradation caused by Cd. The possibility that oxidative stress mediates this action of Cd was examined. Cd didn’t affect protein oxidation and reduced glutathione levels in hypoxic cells. These results indicate that Cd triggers a redox/proteasome‐dependent degradation of HIF‐1α protein, reducing HIF‐1 activity and in turn suppressing the hypoxic induction of hypoxia‐inducible genes.
Hydrogen ion gradients across the mitochondrial, endosomal and plasma membranes in bloodstream forms of Trypanosoma brucei
Authors: Nolan Derek P. , Voorheis H. Paul
Published: March 2003

Conditions for the use of both [14C]methylamine and 5,5‐dimethyl[14C]oxa‐azolidine‐2,4‐dione (DMO) to measure the H+ concentration of intracellular compartments of monomorphic long thin bloodstream forms of Trypanosoma brucei were established. Neither probe was actively transported or bound to internal components of the cell and both probes equilibrated passively with a t1/2 close to 8 min. DMO was excluded from cells, while methylamine was accumulated but not metabolized. Solution of the three‐compartment problem revealed that, when cells were respiring aerobically on glucose at an external pH of 7.5, the cytoplasmic pH was in the range 6.99–7.03, the pH of the mitochondrial matrix was 7.71–7.73, and the algebraic average pH of the various endosomal compartments was 5.19–5.50. Similar values were found when cells were respiring aerobically on glycerol. However, bloodstream forms of T. brucei could not maintain a constant internal H+ concentration outside the external pH range 7.0–7.5, and no evidence for the presence of an H+/Na+ exchanger was found. Full motility and levels of pyruvate production were maintained as the external pH was raised as high as 9.5, suggesting that these cells tolerate significant internal alkalinisation. However, both motility and pyruvate production were severely inhibited under acidic conditions, and the cells deteriorated rapidly below an external pH of 6.5. Physiologically, the plasma membrane of T. brucei had low permeability to H+ and the internal pH was unaffected by changes in Δψp, which is dominated by the potassium diffusion potential. However, in the presence of FCCP, the internal pH fell rapidly about 0.5 pH unit and came into equilibrium with Δψp. Oligomycin abolished the mitochondrial pH gradient (ΔpHm) selectively, whereas chloroquine abolished only the endosomal pH gradient (ΔpHe). The pH gradient across the plasma membrane (ΔpHp) alone could be abolished by careful osmotic swelling of cells. The plasma membrane had an inwardly directed proton‐motive force (ΔPp) of −52 mV and an inwardly directed sodium‐motive force (ΔNp) of −149 mV, whereas the mitochondrial inner membrane had only an inwardly directed ΔPm of −195 mV. The pH gradient across the endosomal membranes was not accompanied by an electrical gradient. Consequently, endosomal membranes had an algebraically average outwardly directed ΔPl within the range + 89 to + 110 mV, depending on the measurement method.
Homodimeric reverse transcriptases from Rous sarcoma virus mutated within the polymerase or RNase H active site of one subunit are active
Authors: Werner Susanne , Wöhrl Birgitta M.
Published: March 2003

Heterodimeric reverse transcriptase (RT) αβ from Rous sarcoma virus (RSV) possesses an asymmetric subunit organization with the polymerase and RNase H active sites localized in the α subunit. To determine whether homodimeric RSV RTs α (63 kDa) or β (95 kDa) assume α subunit organization similar to that of the heterodimer, an essential aspartic acid residue was mutated in the active site of either the polymerase (Asp181 > Asn) or the RNase H (Asp505 > Asn). Homodimeric α or β RT consisting of one wild‐type and one mutated subunit exhibit polymerase or RNase H activity, respectively, whereas the corresponding doubly mutated enzymes are inactive, indicating that the catalytic sites of the polymerase and RNase H domains are formed by only one subunit of the homodimer.
Cloning, expression and characterization of a Family 48 exocellulase, Cel48A, from Thermobifida fusca
Authors: Irwin Diana C. , Zhang Sheng , Wilson David B.
Published: March 2003

The gene for a 104‐kDa exocellulase, Cel48A, formerly E6, was cloned from Thermobifida fusca into Escherichia coli and Streptomyces lividans. The DNA sequence revealed a type II cellulose‐binding domain at the N‐terminus, followed by a FNIII‐like domain and ending with a glycosyl hydrolase Family 48 catalytic domain. The enzyme and catalytic domain alone were each expressed in and purified from S. lividans and had very low catalytic activity on swollen cellulose, carboxymethyl cellulose, bacterial microcrystalline cellulose and filter paper. However, in synergistic assays on filter paper, the addition of Cel48A to a balanced mixture of T. fusca endocellulase and exocellulase increased the specific activity from 7.9 to 11.7 µmol cellobiose·min−1·mL−1, more than 15‐fold higher than any single enzyme alone. Cel48A retained > 50% of its maximum activity from pH 5 to 9 and from 40 to 60 °C. Using swissmodel, the amino‐acid sequence of the Cel48Acd was modeled to the known structure of Clostridium cellulolyticum CelF. Family 48 enzymes are remarkably homologous at 35% identity for all their catalytic domains and some of the properties of the 10 members are discussed.
Nicotinamide adenine dinucleotide species in the horseradish peroxidase–oxidase oscillator
Authors: Kirkor Ewa S. , Scheeline Alexander
Published: March 2003

NADH chemistry ancillary to the oscillatory peroxidase–oxidase (PO) reaction has been reexamined. Previously, (NAD)2 has been thought of as a terminal, inert product of the PO reaction. We now show that (NAD)2 is a central reactant in this system. Although we found traces of the dimer after several hours of the PO reaction, no accumulation of the dimer occurred, regardless of the reaction time or the number of oscillations. (NAD)2 can convert horseradish peroxidase (HRP) compound I (CpI) to compound II (CpII) with apparent rate constant (2.7 ± 0.2) × 105 m−1·s−1 and CpII to HRP at 1 × 105 m−1·s−1. Moreover, a reduction of HRP compound III (CpIII) to CpI by (NAD)2 occurs with a rate constant faster than 5 × 106 m−1·s−1. The (NAD)2 reduction of CpIII provides an alternative to the reduction by NAD radical suggested by Yokota and Yamazaki. HRP catalyzes oxidation of α‐NADH, not only the β anomer as previously assumed. Rate constants of α‐ and β‐NADH reactions with CpI are (7.4 ± 0.4) × 105 m−1·s−1, and (1.7 ± 0.2) × 105 m−1·s−1, and with CpII are estimated as 5 × 104 m−1·s−1, and 4 × 104 m−1·s−1.

Apparent rate constants of reduction of methylene blue (MB) to leuco‐methylene blue (MBH) are 3.8 × 104 m−1·s−1 for NADH and 6.4 × 104 m−1·s−1 for NAD dimer, (NAD)2, while reoxidation of MBH proceeds at (2.1 ± 0.2) × 103 m−1·s−1 All the rates were measured in 0.1 m acetate buffer, pH 5.1.
Kinetics of the coupled reaction catalysed by a fusion protein of yeast mitochondrial malate dehydrogenase and citrate synthase
Authors: Pettersson Henrik , Olsson Peter , Bülow Leif , Pettersson Gösta
Published: March 2003

The mechanistic implications of the kinetic behaviour of a fusion protein of mitochondrial malate dehydrogenase and citrate synthase have been reanalysed in view of predictions based on experimentally determined kinetic parameter values for the dehydrogenase and synthase activities of the protein. The results show that the time‐course of citrate formation from malate in the coupled reaction catalysed by the fusion protein can be most satisfactorily accounted for in terms of a free‐diffusion mechanism when consideration is taken to the inhibitory effects of NADH and oxaloacetate on the malate dehydrogenase activity. The effect of aspartate aminotransferase on the coupled reaction is likewise fully consistent with that expected for a free‐diffusion mechanism. It is concluded that no tenable kinetic evidence is available to support the proposal that the fusion protein catalyses citrate formation from malate by a mechanism involving channelling of the intermediate oxaloacetate.
Novel polysialogangliosides of skate brain
Authors: Nakamura Kazuo , Kojima Hisako , Suzuki Minoru , Suzuki Akemi , Tamai Yoichi
Published: March 2003

The gangliosides in the brain of a cartilaginous fish, skate (Bathyraja smirnovi), have been isolated and characterized by means of methylation analysis, antibody binding, enzymatic hydrolysis and MALDI‐TOF MS. In addition to gangliosides with known structures (GM2, fucosyl‐GM1, GD3, GD2, GT3 and GT2), five polysialogangliosides were isolated and characterized as having the following structures. (1) IV3NeuAc, III6NeuAc, II3NeuAc‐Gg4Cer; (2) IV3NeuAc2, III6NeuAc, II3NeuAc‐Gg4Cer; (3) IV3NeuAc, III6NeuAc, II3NeuAc2‐Gg4Cer; (4) IV3NeuAc, III6NeuAc, II3NeuAc3‐Gg4Cer; and (5) IV3NeuAc2, III6NeuAc, II3NeuAc3‐Gg4Cer. These structures are ‘hybrid‐type’ which comprise combinations of α‐series and either a, b or c‐series structures. Three gangliosides (2), (4) and (5), were novel. The main features of the ganglioside composition of skate brain were an abundance of gangliotriaosyl species, a lack of gangliotetraosyl species (except fucosyl‐GM1), and an abundance of hybrid‐types. These characteristics closely resemble those in shark brain which we reported previously [Nakamura, K., Tamai, Y. & Kasama, T. (1997) Neurochem. Int.30, 593–604]. Two of the hybrid‐type gangliosides (1) and (4), were examined for their neuritogenic activity toward cultured neuronal cells (Neuro‐2A), and were found to have more potent activity than nonhybrid‐type gangliosides such as GM1.
Biochemical characterization of human cathepsin X revealed that the enzyme is an exopeptidase, acting as carboxymonopeptidase or carboxydipeptidase
Authors: Klemenčič Ivica , Carmona Adriana K. , Cezari Maria Helena S. , Juliano Maria A. , Juliano Luiz , Gunčar Gregor , Turk Dušan , Križaj Igor , Turk Vito , Turk Boris
Published: March 2003

Cathepsin X, purified to homogeneity from human liver, is a single chain glycoprotein with a molecular mass of ≈ 33 kDa and pI 5.1–5.3. Cathepsin X was inhibited by stefin A, cystatin C and chicken cystatin (Ki = 1.7–15.0 nm), but poorly or not at all by stefin B (Ki > 250 nm) and l‐kininogen, respectively. The enzyme was also inhibited by two specific synthetic cathepsin B inhibitors, CA‐074 and GFG‐semicarbazone. Cathepsin X was similar to cathepsin B and found to be a carboxypeptidase with preference for a positively charged Arg in P1 position. Contrary to the preference of cathepsin B, cathepsin X normally acts as a carboxymonopeptidase. However, the preference for Arg in the P1 position is so strong that cathepsin X cleaves substrates with Arg in antepenultimate position, acting also as a carboxydipeptidase. A large hydrophobic residue such as Trp is preferred in the P1′ position, although the enzyme cleaved all P1′ residues investigated (Trp, Phe, Ala, Arg, Pro). Cathepsin X also cleaved substrates with amide‐blocked C‐terminal carboxyl group with rates similar to those of the unblocked substrates. In contrast, no endopeptidase activity of cathepsin X could be detected on a series of o‐aminobenzoic acid‐peptidyl‐N‐[2,‐dinitrophenyl]ethylenediamine substrates. Furthermore, the standard cysteine protease methylcoumarine amide substrates (kcat/Km≈ 5.0 × 103 m−1·s−1) were degraded ≈ 25‐fold less efficiently than the carboxypeptidase substrates (kcat/Km ≈ 120.0 × 103 m−1·s−1).
Glutathione, oxidative stress and neurodegeneration
Authors: Schulz Jörg B. , Lindenau Jörg , Seyfried Jan , Dichgans Johannes
Published: March 2003

There is significant evidence that the pathogenesis of several neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, Friedreich's ataxia and amyotrophic lateral sclerosis, may involve the generation of reactive oxygen species and mitochondrial dysfunction. Here, we review the evidence for a disturbance of glutathione homeostasis that may either lead to or result from oxidative stress in neurodegenerative disorders. Glutathione is an important intracellular antioxidant that protects against a variety of different antioxidant species. An important role for glutathione was proposed for the pathogenesis of Parkinson's disease, because a decrease in total glutathione concentrations in the substantia nigra has been observed in preclinical stages, at a time at which other biochemical changes are not yet detectable. Because glutathione does not cross the blood–brain barrier other treatment options to increase brain concentrations of glutathione including glutathione analogs, mimetics or precursors are discussed.
Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity
Authors: O'Brien John , Wilson Ian , Orton Terry , Pognan François
Published: March 2003

We show here the identity of Alamar Blue as resazurin. The ‘resazurin reduction test’ has been used for about 50 years to monitor bacterial and yeast contamination of milk, and also for assessing semen quality. Resazurin (blue and nonfluorescent) is reduced to resorufin (pink and highly fluorescent) which is further reduced to hydroresorufin (uncoloured and nonfluorescent). It is still not known how this reduction occurs, intracellularly via enzyme activity or in the medium as a chemical reaction, although the reduced fluorescent form of Alamar Blue was found in the cytoplasm and of living cells nucleus of dead cells. Recently, the dye has gained popularity as a very simple and versatile way of measuring cell proliferation and cytotoxicity. This dye presents numerous advantages over other cytotoxicity or proliferation tests but we observed several drawbacks to the routine use of Alamar Blue. Tests with several toxicants in different cell lines and rat primary hepatocytes have shown accumulation of the fluorescent product of Alamar Blue in the medium which could lead to an overestimation of cell population. Also, the extensive reduction of Alamar Blue by metabolically active cells led to a final nonfluorescent product, and hence an underestimation of cellular activity.
Unique poly(glycerophosphate) lipoteichoic acid and the glycolipids of a Streptococcus sp. closely related to Streptococcus pneumoniae
Authors: Roethlisberger Peter , Iida‐Tanaka Naoko , Hollemeyer Klaus , Heinzle Elmar , Ishizuka Ineo , Fischer Werner
Published: March 2003

The Streptococcus sp. studied here is closely related to Streptococcus pneumoniae with 98.6% 16S rRNA similarity and 65% DNA/DNA homology. We isolated the lipoteichoic acid and the membrane glycolipids whose structures were established using conventional procedures and NMR spectroscopy. The lipoteichoic acid contains a linear 1,3‐linked poly(glycerophosphate) chain which is partly substituted with d‐alanine ester and is phosphodiester‐linked to O6 of β‐d‐Galf(1→3)acyl2Gro. This lipoteichoic acid is the first example in which a monohexosylglycerol serves as the glycolipid anchor; and with an average chain length of 10 glycerophosphate residues it is the shortest known to date. MS analysis, applied for the first time to a native acylated lipoteichoic acid, revealed a continuous increase in chain length from seven to 17 glycerophosphate residues with a maximum at 10, and allowed identification of the fatty acid combinations. Membrane glycolipids consisted of β‐d‐Galf(1→3)acyl2Gro (9%), α‐d‐Glcp(1→3)acyl2Gro (22%), α‐d‐Galp(1→2)‐α‐d‐Glcp(1→3)acyl2Gro (64%) and α‐d‐Galp(1→2)‐(6‐O‐acyl)‐α‐d‐Glcp(1→3)acyl2Gro (5%). It is noteworthy that in lipoteichoic acid biosynthesis, Galfacyl2Gro, a less abundant membrane glycolipid, is selected as the lipid anchor. Despite the genetic relatedness to Streptococcus pneumoniae, the lipoteichoic acid structure is quite different to the complex structure of pneumococcal lipoteichoic acid [T. Behr et al. (1992) Eur. J. Biochem.207, 1063–1075], thus providing an example that minor differences in DNA sequence exert major changes in macromolecular structure.
Implications of the generation of reactive oxygen species by photoactivated calcein for mitochondrial studies
Authors: Beghetto Claudia , Renken Christian , Eriksson Ove , Jori Giulio , Bernardi Paolo , Ricchelli Fernanda
Published: March 2003

Calcein is a fluorescent probe that is widely used in studies of cell viability and mitochondrial function by microscopy fluorescence imaging. It was found to have a strong photosensitizing action that prevalently involves the generation of reactive oxygen species (ROS). The photooxidation properties of calcein in solution were studied in the presence of histidine and tryptophan as oxidizable substrates. The photodegradation of histidine was mainly mediated by singlet oxygen (1O2), as shown by the inhibitory effect of sodium azide, a specific 1O2 scavenger. On the other hand, mixed photosensitization mechanisms were present when tryptophan was used as the target of the calcein‐stimulated photoprocess. In addition to 1O2, hydroxyl radicals and hydrogen peroxide were involved as reactive species, as shown by using mannitol and catalase as scavengers.

The calcein‐photosensitized alterations of mitochondria as a potential source of artifacts in confocal microscopy studies of cells were considered. Irradiation of isolated mitochondria with visible light (500–600 nm) in the presence of calcein induced opening of the permeability transition (PT) pore. The extent of the mitochondrial membrane photodamage, however, was modulated by the nature of the calcein environment. Thus, pore opening was triggered at short irradiation times and low dye concentrations when calcein was dissolved in the bulk medium. On the contrary, calcein concentrated in the matrix space was rather inefficient as photosensitizer even at concentrations 10 times higher than those present in the external medium.
Changes induced by oxygen in rat liver proteins identified by high‐resolution two‐dimensional gel electrophoresis
Authors: Miralles Cristina , Agustí Alvar G. N. , Aubry Christiane , Sanchez Jean‐Charles , Walzer Claude , Hochstrasser Denis , Busquets Xavier
Published: March 2003

Molecular oxygen (O2) regulates the expression of a variety of genes. Several of the proteins that respond to changes in oxygen concentration have been identified in a variety of cell lines. We extend these previous studies by analyzing the effect of oxygen on the entire protein expression profile of an intact organ using high‐resolution two‐dimensional gel electrophoresis. To this end, we used an isolated, in vitro perfused organ preparation to produce two groups of rat livers perfused with high (95% O2, 5% CO2) or low (95% N2, 5% CO2) oxygen concentrations. Using two‐dimensional gel electrophoresis we compared the protein expression profiles of both groups of livers. Computer analysis of the files obtained after laser densitometry of the two‐dimensional gels revealed two spots that were strongly up‐regulated in high PO2 perfused livers compared with low PO2 perfused livers. These spots were analyzed by peptide mass fingerprinting analysis. These spots were identified as arginase 1 (liver‐type arginase; EC 3.5.3.1) and mitochondrial enoyl‐CoA hydratase 1 (EC 4.2.1.17). The possible role of these proteins in its new context of oxygen availability is discussed.
Monitoring of the internalization of neuropeptide Y on neuroblastoma cell line SK‐N‐MC
Authors: Fabry Marlies , Langer Michael , Rothen‐Rutishauser Barbara , Wunderli‐Allenspach Heidi , Höcker Hartwig , Beck‐Sickinger Annette G.
Published: March 2003

Neuropeptide Y (NPY) is an important neuromodulator in the central and peripheral nervous system. The peptide acts through different NPY receptor subtypes (Y1–Y5, y6) that belong to the family of G protein‐coupled receptors. In general, cellular responses to prolonged exposure to agonists of G protein‐coupled receptors are attenuated, often through internalization of the receptors and their bound ligands. In this study, a fluorescent labeled NPY derivative was synthesized and characterized to investigate the internalization of NPY in the human neuroblastoma cell line SK‐N‐MC. Internalization was proven by binding experiments and subsequent acidic washing as well as by direct visualization by means of confocal laser scanning microscopy. Approximately 20–30% of the fluorescent labeled NPY and a tritium‐marked NPY were resistant to acid removal of cell surface‐bound ligands indicating internalization. Extracellular fluorescent labeled NPY was found to be distributed heterogeneously in a clustered pattern, which suggests that the ligand‐receptor complex is collected in pits and caveolae followed by endocytosis.
Amine group of guanine enhances the binding of norfloxacin antibiotics to DNA
Authors: Lee Eun‐Jeong , Yeo Jeong‐Ah , Cho Chang‐Beom , Lee Gil‐Jun , Han Sung Wook , Kim Seog K.
Published: March 2003

The binding mode of norfloxacin, a quinolone antibacterial agent, in the synthetic polynucleotides poly[d(G–C)2], poly[d(I–C)2] and poly[d(A–T)2] was studied using polarized light spectroscopy, fluorescence spectroscopy and melting profiles. The absorption, circular and linear dichroism properties of norfloxacin are essentially the same for all the complexes, and the angle of electric transition dipole moment I and II of norfloxacin relative to the DNA helix axis is measured as 68–75° for all complexes. These similarities indicate that the binding mode of norfloxacin is similar for all the polynucleotides. The decrease in the linear dichroism (LD) magnitude at 260 nm upon binding norfloxacin, which is strongest for the norfloxacin‐poly[d(G–C)2] complex, and the identical melting temperature of poly[d(A–T)2] and poly[d(I–C)2] in the presence and absence of norfloxacin rule out the possibility of classic intercalation and minor groove binding. However, the characteristics of the fluorescence emission spectra of norfloxacin bound to poly[d(A–T)2] and to poly[d(I–C)2] are similar but are different to that of norfloxacin bound to poly[d(G–C)2]. As the amine group of the guanine base protrudes to the minor groove, this result strongly suggests that norfloxacin binds in the minor groove of B‐form DNA in a nonclassic manner.
The archaeal elongation factor 1α bound to GTP forms a ternary complex with eubacterial and eukaryal aminoacyl‐tRNA
Authors: Raimo Gennaro , Masullo Mariorosario , Lombardo Barbara , Bocchini Vincenzo
Published: March 2003

The archaeal Sulfolobus solfataricus elongation factor 1α (SsEF‐1α) bound to GTP or to its analogue guanyl‐5′‐yl imido diphosphate [Gpp(NH)p] formed a ternary complex with either Escherichia coli Val‐tRNAVal or Saccharomyces cerevisiae Phe‐tRNAPhe as demonstrated by gel‐shift and gel‐filtration experiments. Evidence of such an interaction also came from the observation that SsEF‐1αz.rad;Gpp(NH)p was able to display a protective effect against either the spontaneous deacylation or the digestion of aminoacyl‐tRNA by RNase A. Protection against the deacylation of aminoacyl‐tRNA allowed evaluatation of the affinity of SsEF‐1αz.rad;Gpp(NH)p for both aminoacyl‐tRNAs used. The K′d values of the ternary complex containing S. cerevisiae Phe‐tRNAPhe or E. coli Val‐tRNAVal were 0.3 µm and 4.4 µm, respectively. In both cases, the affinity of SsEF‐1αz.rad;Gpp(NH)p for aminoacyl‐tRNA was three orders of magnitude lower than that of the homologous eubacterial ternary complexes, but comparable with the affinity shown by the ternary complex involving eukaryal EF‐1α[Negrutskii, B.S. & El’skaya, A.V. (1998) Prog. Nucleic Acids Res.60, 47–77]. As already observed with eukaryal EF‐1α, SsEF‐1α in its GDP‐bound form was also able to protect the ester bond of aminoacyl‐tRNA, even though with a 10‐fold lower efficiency compared with SsEF‐1αz.rad;Gpp(NH)p. The overall results indicated that the archaeal elongation factor 1α shares several properties with eukaryal EF‐1α but not with eubacterial EF‐Tu.
Dual metabolic pathway of 25‐hydroxyvitamin D3 catalyzed by human CYP24
Authors: Sakaki Toshiyuki , Sawada Natsumi , Komai Koichiro , Shiozawa Shunichi , Yamada Sachiko , Yamamoto Keiko , Ohyama Yoshihiko , Inouye Kuniyo
Published: March 2003

Human 25‐hydroxyvitamin D3 (25(OH)D3) 24‐hydroxylase (CYP24) cDNA was expressed in Escherichia coli, and its enzymatic and spectral properties were revealed. The reconstituted system containing the membrane fraction prepared from recombinant E. coli cells, adrenodoxin and adrenodoxin reductase was examined for the metabolism of 25(OH)D3, 1α,25(OH)2D3 and their related compounds. Human CYP24 demonstrated a remarkable metabolism consisting of both C‐23 and C‐24 hydroxylation pathways towards both 25(OH)D3 and 1α,25(OH)2D3, whereas rat CYP24 showed almost no C‐23 hydroxylation pathway [Sakaki, T. Sawada, N. Nonaka, Y. Ohyama, Y. & Inouye, K. (1999) Eur. J. Biochem. 262, 43–48]. HPLC analysis and mass spectrometric analysis revealed that human CYP24 catalyzed all the steps of the C‐23 hydroxylation pathway from 25(OH)D3 via 23S,25(OH)2D3, 23S,25,26(OH)3D3 and 25(OH)D3‐26,23‐lactol to 25(OH)D3‐26,23‐lactone in addition to the C‐24 hydroxylation pathway from 25(OH)D3 via 24R,25(OH)2D3, 24‐oxo‐25(OH)D3, 24‐oxo‐23S,25(OH)2D3 to 24,25,26,27‐tetranor‐23(OH)D3. On 1α,25(OH)2D3 metabolism, similar results were observed. These results strongly suggest that the single enzyme human CYP24 is greatly responsible for the metabolism of both 25(OH)D3 and 1α,25(OH)2D3. We also succeeded in the coexpression of CYP24, adrenodoxin and NADPH‐adrenodoxin reductase in E. coli. Addition of 25(OH)D3 to the recombinant E. coli cell culture yielded most of the metabolites in both the C‐23 and C‐24 hydroxylation pathways. Thus, the E. coli expression system for human CYP24 appears quite useful in predicting the metabolism of vitamin D analogs used as drugs.
Potency and selectivity of inhibition of cathepsin K, L and S by their respective propeptides
Authors: Guay Jocelyne , Falgueyret Jean‐Pierre , Ducret Axel , Percival M. David , Mancini Joseph A.
Published: March 2003

The prodomains of several cysteine proteases of the papain family have been shown to be potent inhibitors of their parent enzymes. An increased interest in cysteine proteases inhibitors has been generated with potential therapeutic targets such as cathepsin K for osteoporosis and cathepsin S for immune modulation. The propeptides of cathepsin S, L and K were expressed as glutathione S‐transferase‐fusion proteins in Escherichia coli. The proteins were purified on glutathione affinity columns and the glutathione S‐transferase was removed by thrombin cleavage. All three propeptides were tested for inhibitor potency and found to be selective within the cathepsin L subfamily (cathepsins K, L and S) compared with cathepsin B or papain. Inhibition of cathepsin K by either procathepsin K, L or S was time‐dependent and occurred by an apparent one‐step mechanism. The cathepsin K propeptide had a Ki of 3.6–6.3 nm for each of the three cathepsins K, L and S. The cathepsin L propeptide was at least a 240‐fold selective inhibitor of cathepsin K (Ki = 0.27 nm) and cathepsin L (Ki = 0.12 nm) compared with cathepsin S (Ki = 65 nm). Interestingly, the cathepsin S propeptide was more selective for inhibition of cathepsin L (Ki = 0.46 nm) than cathepsin S (Ki = 7.6 nm) itself or cathepsin K (Ki = 7.0 nm). This is in sharp contrast to previously published data demonstrating that the cathepsin S propeptide is equipotent for inhibition of human cathepsin S and rat and paramecium cathepsin L [Maubach, G., Schilling, K., Rommerskirch, W., Wenz, I., Schultz, J.E., Weber, E. & Wiederanders, B. (1997), Eur J. Biochem. 250, 745–750]. These results demonstrate that limited selectivity of inhibition can be measured for the procathepsins K, L and S vs. the parent enzymes, but selective inhibition vs. cathepsin B and papain was obtained.
Molecular cloning, functional complementation in Saccharomyces cerevisiae and enzymatic properties of phosphatidylinositol synthase from the protozoan parasite Toxoplasma gondii
Authors: Séron Karin , Dzierszinski Florence , Tomavo Stanislas
Published: March 2003

The obligate intracellular parasite Toxoplasma gondii, the causative agent of toxoplasmosis, switches between the rapidly dividing tachyzoite and the slowly replicating bradyzoite in intermediate hosts such as humans and domestic animals. We have recently identified a bradyzoite cDNA encoding a putative phosphatidylinositol (PtdIns) synthase using a subtractive library [Yahiaoui, B., Dzierszinski, F., Bernigaud, A., Slomianny, C., Camus, D., and Tomavo, S. (1999) Mol. Biochem. Parasitol. 99, 223–235]. Here, we report the cloning of another cDNA encoding PtdIns synthase that is exclusively expressed in the tachyzoite stage. The two transcripts are encoded by two different genes, which are stage‐specifically regulated. The deduced amino‐acid sequence (258 amino acids with a calculated total molecular mass of 27.8 kDa) of the tachyzoite‐specific cDNA shares a significant degree of identity (between 26.5 and 30.1%) to the PtdIns synthases from human, rat, Arabidopsis thaliana and yeast. Interestingly, the putative protein encompasses an N‐terminal extension that is approximately 40 amino‐acids longer than that of PtdIns synthases from other organisms. Functional complementation realized by tetrad analysis of segregants of a Saccharomyces cerevisiae PtdIns synthase‐deficient mutant (PIS1/pis1::kanMX4) showed that only the T. gondii putative PtdIns synthase truncated at its N‐terminal extension is able to restore the viability of the cells. We demonstrate that this protein expressed in yeast transformants is functionally active in the membrane preparation and requires manganese and magnesium ions for activity. To our knowledge, this is the first report on the molecular cloning and functional analysis of a gene encoding a PtdIns synthase in protozoan parasites.
Comparative functional characterization in vitro of heptosyltransferase I (WaaC) and II (WaaF) from Escherichia coli
Authors: Gronow Sabine , Brabetz Werner , Brade Helmut
Published: March 2003

Heptosyltransferase II, encoded by the waaF gene of Escherichia coli, is a glycosyltransferase involved in the synthesis of the inner core region of lipopolysaccharide. The gene was subcloned from plasmid pWSB33 [Brabetz, W., Müller‐Loennies, S., Holst, O. & Brade, H. (1997) Eur. J. Biochem. 247, 716–724] into a shuttle vector for the expression in the gram‐positive host Corynebacterium glutamicum. The in vitro activity of the enzyme was investigated in comparison to that of heptosyltransferase I (WaaC) using as a source for the sugar nucleotide donor, ADP‐lglycerodmanno‐heptose, a low molecular mass filtrate from a ΔwaaCF E. coli strain. Synthetic lipid A analogues varying in the acylation or phosphorylation pattern or both were tested as acceptors for the subsequent transfer of 3‐deoxy‐dmanno‐oct‐2‐ulosonic acid (Kdo) and heptose by successive action of Kdo transferase (WaaA), heptosyltransferase I (WaaC) and heptosyltransferase II (WaaF). The reaction products were characterized after separation by TLC and blotting with monoclonal antibodies specific for the acceptor, the intermediates and the final products.
Targeted mutations of transforming growth factor‐β genes reveal important roles in mouse development and adult homeostasis
Authors: Dünker Nicole , Krieglstein Kerstin
Published: March 2003

Transforming growth factors‐beta (TGF‐β) are multifunctional molecules with profound biological effects in many developmental processes including regulation of cell proliferation, differentiation, cell adhesion, skeletal development, haematopoiesis, inflammatory responses, and wound healing. To learn about the role of TGF‐βin vivo, phenotypes of targeted mutations of molecules within the TGF‐β signalling pathway, TGF‐β1, ‐β2, ‐β3, TGF‐β receptor (TβR‐II) and the signalling molecules SMAD2, SMAD3 and SMAD4, are discussed in this review. The three individual TGF‐β mutants show distinct and only partially overlapping phenotypes. In mice, targeted disruption of the TGF‐β1 gene results in diffuse and lethal inflammation about 3 weeks after birth, suggesting a prominent role of TGF‐β in the regulation of immune cell proliferation and extravasation into tissues. However, just half of the TGF‐β1 (–/–) conceptuses actually reach partuition due to defective haematopoiesis and endothelial differentiation. Targeted disruption of both TGF‐β2 and TGF‐β3 genes results in perinatal lethality. TGF‐β2 null mice exhibit a broad range of developmental defects, including cardiac, lung, craniofacial, limb, eye, ear and urogenital defects, whereas TGF‐β3 gene ablation results exclusively in defective palatogenesis and delayed pulmonary development. The TβR‐II null phenotype closely resembles that of TGF‐β1 (–/–) conceptuses, which die in utero by E10.5. Loss of SMAD2 or SMAD4 results in related phenotypes: the mutants fail to form an organized egg cylinder, lack mesoderm required for gastrulation and die prior to E8.5. Together, gene ablation within the TGF‐β signalling pathway supports the notion of a prominent role of TGF‐β during development.
Expression and characterization of truncated forms of humanized L243 IgG1
Authors: Lund John , Takahashi Noriko , Popplewell Andrew , Goodall Margaret , Pound John D. , Tyler Ruth , King David J. , Jefferis Royston
Published: March 2003

The properties of IgG and its subcomponents are being exploited to generate new therapeutics with selected biological activities. In this study, a series of truncated, humanized IgG1 antibodies was expressed in Chinese hamster ovary cells, to evaluate the contribution of structural components to glycosylation and function. The series includes L243 IgG1 (α‐MHC Class II) lacking a CH3 domain pair (ΔCH3‐IgG1), single‐chain Fv fusion proteins with Fc or a hinge‐CH2 domain, Fc with/out a hinge, and a single CH2 domain.

Glycosylation of IgG Fc is important for recognition by effector ligands such as Fcγ receptors. HPLC analysis of released and pyridylaminated oligosaccharides indicates that intact IgG1 and scFvFc antibodies are galactosylated and sialylated to levels similar to those observed previously for normal human IgG1. The truncated forms express increased levels of digalactosylated (30–83%) or sialylated (9–21%) oligosaccharide chains with the highest levels observed for the single CH2 domain. These data show which architectural components influence IgG glycosylation processing and that the (CH3)2 pair is particularly influential.

When MHC Class II bearing (JY) cells were sensitized with L243 ΔCH3‐IgG1, scFvFc, or scFvhCH2 they elicited superoxide production, from U937 cells, at levels of 35–45% relative to that obtained for intact L243 IgG1 (100%). Mild reduction and alkylation of the hinge disulphide bonds of scFvhCH2 greatly decreased its capacity to trigger superoxide production. Thus, the L243 scFvhCH2 homo‐dimer constitutes the minimal truncated form that binds the MHC Class II antigen and triggers superoxide production through FcγRI.
Leakage and aggregation of phospholipid vesicles induced by the BH3‐only Bcl‐2 family member, BID
Authors: Zhai Dayong , Miao Qi , Xin Xiaofeng , Yang Fuyu
Published: March 2003

BID is a BH3 domain‐only member of the Bcl‐2 family that acts as an apoptotic agonist in programmed cell death. After cleavage by caspase‐8, the N‐terminal of BID (N‐BID) stays in the cytosol while the C‐terminal of BID (C‐BID) translocates to mitochondria, leading to cytochrome c release in vivo and in vitro. We have previously reported that BID or truncated BID (tBID) can induce the release of entrapped trypsin and cytochrome c from large unilamellar vesicles (LUVs). Further studies have been performed and are presented here; the results demonstrate that C‐BID, like BID and tBID, induces vesicle leakage, whereas N‐BID or the BID mutants BID (D59A) and BID (G94E) fail to have any significant effects. The affinity of the above‐mentioned proteins for soybean phospholipid LUVs (SLUVs) decreased in an order similar to their leakage‐inducing capability: tBID > BID > BID (D59A), while N‐BID and BID (G94E) were unable to bind to the vesicles at all. BID‐induced leakage was dependent on the lipid composition of vesicles. Acidic phospholipid (e.g. phosphatidic acid or phosphatidylglycerol) was necessary for BID‐induced leakage while the presence of phosphatidylethanolamine or cholesterol reduced the leakage. It was also found C‐BID is better able to penetrate the soybean phospholipid monolayer than BID or tBID. A further finding was that tBID, but not full‐length BID, could stimulate the aggregation of SLUVs. Finally, Bcl‐xL, an apoptotic antagonist in programmed cell death, can prevent the aggregation of LUVs induced by tBID, but not the release of entrapped trypsin. It is postulated that two separate domains of tBID are responsible for inducing leakage and aggregation of phospholipid vesicles.
Soluble gp130 is the natural inhibitor of soluble interleukin‐6 receptor transsignaling responses
Authors: Jostock Thomas , Müllberg Jürgen , Özbek Suat , Atreya Raja , Blinn Guido , Voltz Nicole , Fischer Martina , Neurath Markus F. , Rose‐John Stefan
Published: March 2003

Signal transduction in response to interleukin‐6 (IL‐6) requires binding of the cytokine to its receptor (IL‐6R) and subsequent homodimerization of the signal transducer gp130. The complex of IL‐6 and soluble IL‐6R (sIL‐6R) triggers dimerization of gp130 and induces responses on cells that do not express membrane bound IL‐6R. Naturally occurring soluble gp130 (sgp130) can be found in a ternary complex with IL‐6 and sIL‐6R. We created recombinant sgp130 proteins that showed binding to IL‐6 in complex with sIL‐6R and inhibited IL‐6/sIL‐6R induced proliferation of BAF/3 cells expressing gp130. Surprisingly, sgp130 proteins did not affect IL‐6 stimulated proliferation of BAF/3 cells expressing gp130 and membrane bound IL‐6R, indicating that sgp130 did not interfere with IL‐6 bound to IL‐6R on the cell surface. Additionally, sgp130 partially inhibited proliferation induced by leukemia inhibitory factor (LIF) and oncostatin M (OSM) albeit at higher concentrations. Recombinant sgp130 protein could be used to block the anti‐apoptotic effect of sIL‐6R on lamina propria cells from Crohn disease patients. We conclude that sgp130 is the natural inhibitor of IL‐6 responses dependent on sIL‐6R. Furthermore, recombinant sgp130 is expected to be a valuable therapeutic tool to specifically block disease states in which sIL‐6R transsignaling responses exist, e.g. in morbus Crohn disease.
Construction, separation and properties of hybrid hexamers of glutamate dehydrogenase in which five of the six subunits are contributed by the catalytically inert D165S
Authors: Hayden Bronagh M. , Engel Paul C.
Published: March 2003

In vitro subunit hybridization was used to explore the basis of putative allosteric behaviour in clostridial glutamate dehydrogenase. C320S and D165S mutant enzymes were chosen to construct the hybrid proteins. The C320S mutant protein is fully active and shows normal allosteric properties but lacks the reactive cysteine. D165S is capable of binding both glutamate and NAD+ but is catalytically inactive. The mutant proteins were denatured separately in 4 m urea, mixed in a 5 : 1 (D165S/C320S) ratio and diluted into a refolding mixture composed of 2 mm NAD+, 1 m fluoride and artificial chaperones (4 mm polyoxyethylene 10 lauryl ether and 1.6 mmβ‐cyclodextrin). Under these conditions ≈ 50% refolding was achieved for both mutant proteins separately. The renatured mixture was concentrated and separated from denatured proteins and the components of the refolding mixture by ultrafiltration and ion‐exchange chromatography. Ellman’s reagent, 5,5′‐dithiobis (2‐nitrobenzoic acid) (DTNB), which binds close to the NAD+ binding site, thus abolishing coenzyme binding in the wild‐type enzyme, also reacts with D165S but has no effect on C320S. Modification by DTNB was coupled with dye‐ligand affinity chromatography on a Procion Red HE‐3B column in order to separate the hybrid mixture into fractions of defined composition. An optimized procedure based on salt gradient elution was developed. DTNB‐modified 5 : 1 hybrids, with only one subunit capable of binding coenzyme, showed classical Michaelis–Menten kinetics when the NAD+ concentration was varied, whereas removal of the thionitrobenzoate moieties that blocked the other five coenzyme binding sites in the hexamer reinstated nonlinear behaviour, suggesting that ‘nonlinear’ behaviour of the native enzyme and the hybrid with six coenzyme binding sites depends on binding to multiple sites. When assayed at high pH with increasing glutamate concentration, the sample with only one active subunit showed reduced sigmoidicity in the dependence of reaction rate on glutamate concentration (h = 3.0) compared with native C320S with six active subunits (h = 5.2) suggesting that the interaction between the subunits was reduced but not abolished completely. Catalytically silent subunits can thus still contribute to cooperativity.