bluuebunny/crossref_metadata_2025 huggingface dataset:
Dissection of the enzymatic and immunologic functions of macrophage migration inhibitory factor
Authors: Kleemann Robert , Rorsman Hans , Rosengren Evald , Mischke Ralf , Mai Nguyen Tuyet , Bernhagen Jürgen
Published: March 2003

Macrophage migration inhibitory factor (MIF) is a cytokine with broad regulatory functions in innate immunity. MIF belongs to the few cytokines displaying catalytic activities, i.e. MIF has a Pro2‐dependent tautomerase and a Cys‐Ala‐Leu‐Cys (CALC) cysteine‐based thiol‐protein oxidoreductase activity. Previous studies have addressed the roles of the catalytic site residues and the C‐terminus. The two activities have not been directly compared. Here we report on the N‐terminal mutational analysis and minimization of MIF and on a dissection of the two catalytic activities by comparing mutants P2AMIF, Δ4MIF, Δ5MIF, Δ6MIF, Δ7MIF, Δ8MIF, and Δ10MIF with the cysteine mutants of MIF. As N‐terminal deletion was predicted to interfere with protein structure due to disruption of the central β sheet, it was surprising that deletion of up to six N‐terminal residues resulted in normally expressed proteins with wild‐type conformation. Strikingly, such mutants exhibited full MIF‐specific immunologic activity. While mutation of Pro2 eliminated tautomerase activity, the CALC cysteine residues had no influence on this activity. However, mutant C81SMIF, which otherwise has full biologic activity, only had 32% tautomerase activity. Deletion of four N‐terminal residues did not interfere with insulin reduction by MIF. By contrast, reduction of 2‐hydroxyethyldisulfide (HED) was markedly affected by N‐terminal manipulation, with P2AMIF and Δ2MIF exhibiting 40% activity, and Δ4MIF completely failing to reduce HED. This study constitutes the first comparison of the two catalytic activities of MIF and should assist in understanding the molecular links between the catalytic and immunologic activities of this cytokine and in providing guidelines for N‐terminal protein minimization.
The rat ortholog of the presumptive flounder antifreeze enhancer‐binding protein is a helicase domain‐containing protein
Authors: Miao Ming , Chan Shing‐Leng , Fletcher Garth L. , Hew Choy L.
Published: March 2003

The expression of winter flounder liver‐type antifreeze protein (wflAFP) genes is tissue‐specific and under seasonal and hormonal regulation. The only intron of the major wflAFP gene was demonstrated to be a liver‐specific enhancer in both mammalian cell lines and flounder hepatocytes. Element B, the core enhancer sequence, was shown to interact specifically with a liver‐enriched transcription factor, CCAAT/enhancer‐binding protein α (C/EBPα), as well as a presumptive antifreeze enhancer‐binding protein (AEP). In this study, the identity of the rat AEP ortholog was revealed via its DNA–protein interaction with element B. It is a helicase‐domain‐containing protein, 988 amino acids in length, and is homologous to mouse Smubp‐2, hamster Rip‐1 and human Smubp‐2. The specific binding between element B and AEP was confirmed by South‐Western analysis and gel retardation assays. Residues in element B important to this interaction were identified by methylation interference assays. Mutation on one of the residues disrupted the binding between element B and AEP and its enhancer activity was significantly reduced, suggesting that AEP is essential for the transactivation of the wflAFP gene intron. The rat AEP is ubiquitously expressed in various tissues, and the flounder homolog is present as shown by genomic Southern analysis. The potential role of AEP in regulating the flounder AFP gene expression is discussed.
Construction and functional evaluation of a single‐chain antibody fragment that neutralizes toxin AahI from the venom of the scorpion Androctonus australis hector
Authors: Devaux Christiane , Moreau Emmanuel , Goyffon Max , Rochat Hervé , Billiald Philippe
Published: March 2003

9C2 is a murine monoclonal IgG that participates in the neutralization of Androctonus australis hector scorpion venom. It recognizes AahI and AahIII, two of the three main neurotoxins responsible for almost all the toxicity of the venom when injected into mammals. Using PCR we cloned the antibody variable region coding genes from 9C2 hybridoma cells and constructed a gene encoding a single‐chain antibody variable fragment molecule (scFv). This scFv was produced in the periplasm of Escherichia coli in a soluble and functional form and purified in a single step using protein L−agarose beads yielding 1–2 mg·L−1 of bacterial culture. scFv9C2 was predominantly monomeric but also tended to form dimeric and oligomeric structures, all capable of binding toxin AahI. The affinity of scFv and the parental mAb for toxin AahI and homologous toxin AahIII was of the same magnitude, in the nanomolar range. Similarly, purified forms of scFv9C2 completely inhibited the binding of toxin AahI to rat brain synaptosomes. Finally, scFv9C2 was efficient in protecting mice against the toxic effects of AahI after injection of the toxin and scFv to mice by the intracerebroventricular route in a molar ratio as low as 0.36 : 1. Thus, we produced a recombinant scFv that reproduces the recognition properties of the parent antibody and neutralizes the scorpion neurotoxin AahI, thereby opening new prospects for the treatment of envenomation.
Ligand binding and physico‐chemical properties of ASP2, a recombinant odorant‐binding protein from honeybee (Apis mellifera L.)
Authors: Briand Loïc , Nespoulous Claude , Huet Jean‐Claude , Takahashi Masayuki , Pernollet Jean‐Claude
Published: March 2003

In insects, the transport of airborne, hydrophobic odorants and pheromones through the sensillum lymph is generally thought to be accomplished by odorant‐binding proteins (OBPs). We report the structural and functional properties of a honeybee OBP called ASP2, heterologously expressed by the yeast Pichia pastoris. ASP2 disulfide bonds were assigned after classic trypsinolysis followed by ion‐spray mass spectrometry combined with microsequencing. The pairing [Cys(I)‐Cys(III), Cys(II)‐Cys(V), Cys(IV)‐Cys(VI)] was found to be identical to that of Bombyx mori OBP, suggesting that this pattern occurs commonly throughout the highly divergent insect OBPs. CD measurements revealed that ASP2 is mainly constituted of α helices, like other insect OBPs, but different from lipocalin‐like vertebrate OBPs. Gel filtration analysis showed that ASP2 is homodimeric at neutral pH, but monomerizes upon acidification or addition of a chaotropic agent. A general volatile‐odorant binding assay allowed us to examine the uptake of some odorants and pheromones by ASP2. Recombinant ASP2 bound all tested molecules, except β‐ionone, which could not interact with it at all. The affinity constants of ASP2 for these ligands, determined at neutral pH by isothermal titration calorimetry, are in the micromolar range, as observed for vertebrate OBP. These results suggest that odorants occupy three binding sites per dimer, probably one in the core of each monomer and another whose location and biological role are questionable. At acidic pH, no binding was observed, in correlation with monomerization and a local conformational change supported by CD experiments.
Role of the electrostatic loop of Cu,Zn superoxide dismutase in the copper uptake process
Authors: Ciriolo Maria R. , Battistoni Andrea , Falconi Mattia , Filomeni Giuseppe , Rotilio Giuseppe
Published: March 2003

Cu,Zn superoxide dismutases are characterized by the presence of four highly conserved charged residues (Lys120, Glu/Asp130, Glu131 and Lys134), which are placed at the edge of the active site channel and have been shown to be individually involved in the electrostatic attraction of the substrate toward the catalytically active copper ion. By genetic engineering we mutated these four residues into neutrally charged ones (Leu120, Gln130, Gln131, Thr134). The effects of these mutations on the rate of superoxide dismutation were not dramatic. In fact, at two different pH and ionic strength values, the mutant enzyme had a catalytic constant even higher with respect to the wild‐type protein, showing that electrostatic interaction at these surface sites is not essential for high catalytic efficiency of the enzyme. The mutant and the wild‐type enzyme showed the same degree of inhibition by CN−, and both were not affected by I−, showing that mutations did not alter the sensitivity of the enzyme to anions. On the other hand, reconstitution of active enzyme from either the wild‐type or mutant copper‐free enzymes with a copper(I)‐glutathione [Cu(I)–GSH] complex showed that metal uptake by the mutant was much slower than by the wild‐type enzyme. The demonstration that the ‘electrostatic loop’ is apparently conserved to assure optimal copper uptake by the enzyme, rather than fast dismutation, may provide further support to the idea that Cu,Zn superoxide dismutase is a bifunctional protein, acting in cellular defense against oxidative stress both as a copper buffer and as a superoxide radical scavenger.
Dopamine is a key factor for the induction of egg diapause of the silkworm, Bombyx mori
Authors: Noguchi Hirofumi , Hayakawa Yoichi
Published: March 2003

The silkworm, Bombyx mori, enters diapause in the early embryonic stage. Embryonic diapause is induced by incubating eggs of the maternal generation at high temperature (diapause type), whereas incubation at low temperature results in non‐diapausing progeny (non‐diapause type). Measurement of catecholamine concentrations in haemolymph and brain–subesophageal ganglia (Br‐SGs) showed that only dopamine concentrations in both tissues are consistently higher in diapause‐type than non‐diapause‐type larvae and pupae. In particular, the difference in dopamine concentrations in both tissues increases around pupal ecdysis. During the early pupal stage, Dopa decarboxylase activities and mRNA concentrations in Br‐SGs were also much higher in diapause‐type than non‐diapause‐type insects. Elevation of dopamine levels induced by feeding Dopa to penultimate‐instar and last‐instar larvae, and by injecting Dopa or dopamine into pupae 2 days after pupation made the non‐diapause‐destined insects lay diapause‐destined eggs at 59% and ≈ 70% frequencies, respectively. Furthermore, injection of Dopa or dopamine elevated mRNA levels of the diapause hormone in the Br‐SGs of non‐diapause‐type pupae 1 day after injection. Incubation of Br‐SGs isolated from non‐diapause‐type day‐2 pupae with Dopa or dopamine also stimulated the expression of diapause hormone mRNA. These data indicate that environmental stimuli during embryonic development increase dopamine levels in both hemolymph and Br‐SGs from the larval stage to early pupal stage, which results in laying of diapause‐destined eggs by female adults through enhanced expression of the diapause hormone gene.
Critical role of glutamic acid 202 in the enzymatic activity of stromelysin‐1 (MMP‐3)
Authors: Arza Begoña , De Maeyer Marc , Félez Jordi , Collen Désiré , Lijnen H. Roger
Published: March 2003

To test the hypothesis that Glu202, adjacent to the His201 residue that participates in the coordination of Zn2+ in matrix metalloproteinase‐3 (MMP‐3 or stromelysin‐1), plays a role in its enzymatic activity it was substituted with Ala, Lys or Asp by site‐specific mutagenesis. Wild‐type proMMP‐3, proMMP‐3(E202A), proMMP‐3(E202K) and proMMP‐3(E202D) were expressed in Escherichia coli and purified to apparent homogeneity. Whereas 33‐kDa wild‐type proMMP‐3 (consisting of the propeptide and catalytic domains) was quantitatively converted to 24‐kDa active MMP‐3 by treatment with p‐aminophenyl‐mercuric acetate (APMA), proMMP‐3(E202A) and proMMP‐3 (E202K) were fully resistant to APMA and proMMP‐3 (E202D) was quantitatively converted into a 14‐kDa species. In contrast, treatment with plasmin quantitatively converted the wild‐type and the three mutant proMMP‐3 moieties into the corresponding 24‐kDa MMP‐3 moieties. Biospecific interaction analysis revealed comparable affinity for binding to plasminogen of wild‐type and mutant proMMP‐3 (Ka of 2.6–6.3 × 106 m−1) or MMP‐3 (Ka of 33–58 × 106 m−1) moieties. The affinity for binding to single‐chain urokinase‐type plasminogen activator (scu‐PA) was also similar for wild‐type and mutant proMMP‐3 (Ka of 5.0–6.9 × 106 m−1) or MMP‐3 (Ka of 37–72 × 106 m−1) moieties. However, MMP‐3(E202A) and MMP‐3(E202K) did not hydrolyze plasminogen whereas MMP‐3(E202D) showed an activity of 20–30% of wild‐type MMP‐3. All three mutants were inactive towards scu‐PA under conditions where this was quantitatively cleaved by wild‐type MMP‐3. Furthermore, MMP‐3(E202A) and MMP‐3(E202K) were inactive toward a fluorogenic substrate and MMP‐3 (E202D) displayed about 15% of the activity of wild‐type MMP‐3. Taken together, these data suggest that Glu202 plays a crucial role in the enzymatic activity of MMP‐3.
Identification and molecular cloning of germinal vesicle lamin B3 in goldfish (Carassius auratus) oocytes
Authors: Yamaguchi Akihiko , Yamashita Masakane , Yoshikuni Michiyasu , Nagahama Yoshitaka
Published: March 2003

A bulk isolation method was developed to collect a large number of germinal vesicles (GV) from postvitellogenic oocytes of goldfish (Carassius auratus). Using this method, we obtained GV lamina which are resistant to high salt and nonionic detergent. 2D PAGE revealed that the goldfish GV lamina contained several spots with similar molecular masses (67 kDa) and slightly different neutral isoelectrofocusing values (pI 5.8–6.2). After trypsin digestion and extraction of a major spot (pI 6.1), the peptide was subjected to RP‐HPLC and sequenced. A homology search identified this spot as a nuclear lamin. A cDNA encoding goldfish GV lamin was isolated by RT‐PCR using degenerate primers designed from the GV lamin tryptic peptide sequence. The goldfish GV lamin cDNA encodes a predicted molecular mass of 67 455 Da with a pI of 5.84. Phylogenetic analysis indicates that the amino‐acid sequence is most similar to Xenopus oocyte‐specific GV lamin B3, but differs from somatic lamins (A, B1 or B2). In contrast to somatic lamins, neither goldfish nor Xenopus GV lamin contain conserved phosphorylation sites for nuclear transport, except the nuclear localization sequence. Therefore, we conclude that the goldfish oocyte GV is mainly comprised of GV‐type lamin (the homolog of Xenopus lamin B3).
Identification of myogenesis‐dependent transcriptional enhancers in promoter region of mouse γ‐sarcoglycan gene
Authors: Wakabayashi‐Takai Eriko , Noguchi Satoru , Ozawa Eijiro
Published: March 2003

Four sarcoglycan subunit proteins, α‐, β‐, γ‐ and δ‐sarcoglycans, form a complex on the skeletal muscle cell surface membrane and a gene defect in any one of them causes the loss or marked decrease of whole sarcoglycan complex, resulting in an autosomal recessive muscular dystrophy, sarcoglycanopathy. To characterize the regulation of sarcoglycan transcription during myocyte differentiation, we isolated the promoter regions for all sarcoglycan transcripts and measured the level of transcriptional activity of these promoter regions in the C2C12 skeletal muscle cell line. The promoters of γ‐sarcoglycan and one of two promoters of α‐sarcoglycan exhibited marked transcriptional activation following differentiation to myotubes. Then, we characterized the 1.5‐kb region of the γ‐sarcoglycan promoter by generating reporter‐constructs having various deletions and measuring their transcriptional activities. In this promoter, we identified a basal promoter region and two enhancer regions dependent on differentiation. We also showed that A/T‐rich and E box elements in the upstream enhancer region are essential for the activation of γ‐sarcoglycan transcription following myotube formation. Furthermore, from the identification of binding proteins to these elements together with the cotransfection experiments with the γ‐sarcoglycan promoter reporter construct and cDNAs encoding these binding factors to 10T1/2 fibroblast cell line, it was suggested that MyoD directs the transcription of γ‐sarcoglycan gene as one of the trans activators.
Purification and structure elucidation of the N‐acetylbacillosamine‐containing polysaccharide from Bacillus licheniformis ATCC 9945
Authors: Schäffer Christina , Scherf Tali , Christian Rudolf , Kosma Paul , Zayni Sonja , Messner Paul , Sharon Nathan
Published: March 2003

The exopolysaccharide of Bacillus licheniformis ATCC 9945 (formerly B. subtilis ATCC 9945) contains among other glycoses 4‐acetamido‐2‐amino‐2,4,6‐trideoxy‐d‐glucose, termed N‐acetylbacillosamine (Bac2N4NAc). A similar diamino glycose, 2‐acetamido‐4‐amino‐2,4,6‐trideoxy‐d‐glucose, was found in a surface layer (S‐layer) glycoprotein preparation of Clostridium symbiosum HB25. Electron microscopic studies, however, showed that B. licheniformis ATCC 9945 is not covered with an S‐layer lattice, indicating that the N‐acetylbacillosamine present in that organism might be a constituent of a cell wall‐associated polymer. For elucidation of the structure of the N‐acetylbacillosamine‐containing polysaccharide, it was purified from a trichloroacetic acid extract of B. licheniformis ATCC 9945 cells. Using different hydrolysis protocols and a hydrolysate of the S‐layer glycoprotein preparation from C. symbiosum HB25 as reference, the purified polysaccharide was found to contain 2,4‐diamino‐2,4,6‐trideoxy‐glucose, 2‐acetamido‐2‐deoxy‐glucose, 2‐acetamido‐2‐deoxy‐galactose and galactose in a molar ratio of 1 : 1 : 1 : 2. One‐ and two‐dimensional NMR spectroscopy, including 800 MHz proton magnetic resonance measurements, in combination with chemical modification and degradation experiments, revealed that the polysaccharide consists of identical pyruvylated pentasaccharide repeating units with the structure: {→3)‐[(S)Py‐(3,4)‐β‐d‐Galp‐(1→6)]‐α‐d‐GlcpNAc‐(1→3)‐β‐d‐Bacp2N4NAc‐(1→3)‐[(S)Py‐(3,4)‐β‐d‐Galp‐(1→6)]‐β‐d‐GalpNAc‐(1→}n
Phosphoinositide fatty acids regulate phosphatidylinositol 5‐kinase, phospholipase C and protein kinase C activities
Authors: Carricaburu Valérie , Fournier Bernard
Published: March 2003

PtdIns(4,5)P2 generally results from phosphorylation of PtdIns(4)P by the phosphatidylinositol 5‐kinase (PtdIns5‐K). Its hydrolysis by phospholipase C (PLC) yields inositol 1,4,5‐trisphosphate and diacylglycerol, which stimulates protein kinase C (PKC). We show that epithelial cells of the cockroach rectum contain three different inositol lipids: PtdIns(4,5)P2, PtdIns(4)P, and PtdIns. They are composed of six major fatty acids: palmitic (16:0) stearic (18:0), oleic (18:1n−9), linoleic (18:2n−6), linolenic (18:3n−3), and arachidonic (20:4n−6) acids. The fatty acid preference of each of the above enzymes was evaluated by incorporating different fatty acids in pairs into membrane lipids. Incorporation of 16:0 plus 18:1n−9 provoked an increase in PtdIns(4,5)P2‐PLC activity and a decrease in PtdIns5‐K activity. In contrast, incorporation of 16:0 plus 18:3n−3 led to a potentiation of PtdIns5‐K activity and a decrease in PtdIns(4,5)P2‐PLC activity. Furthermore, PLC and PtdIns5‐K acted preferentially on substrates containing 18:3n−3, and 18:3n−3‐containing diacylglycerol specifically potentiated PKC activity. Thus, we propose that the fatty acids that make up the phosphoinositides function as intracellular modulators of the activity of certain enzymes.
Sugar‐based tertiary amino gemini surfactants with a vesicle‐to‐micelle transition in the endosomal pH range mediate efficient transfection in vitro
Authors: Fielden Matthew L. , Perrin Christele , Kremer Andreas , Bergsma Mark , Stuart Marc C. , Camilleri Patrick , Engberts Jan B. F. N.
Published: March 2003

Novel reduced sugar gemini amphiphiles linked through their tertiary amino head groups via alkyl spacers of 4 or 6 carbons, and with varying (unsaturated) alkyl tail lengths of 12–18, have been synthesized and tested for transfection in vitro in an adherent Chinese hamster ovary cell line (CHO‐K1). Transfection efficiencies peaked at 2.7 times that of the commercial standard Lipofectamine Plus/2000 for pure solutions of the compound bearing unsaturated (oleyl) alkyl tails. For those compounds bearing saturated alkyl tails, transfection efficiency peaked at a tail length of 16, at a level similar to Lipofectamine Plus/2000. All of the amphiphiles formed bilayer vesicles at physiological pH. Some of the amino groups at the surface were protonated, and vesicles therefore bore a positive charge. Increased protonation with reduced pH resulted in greatly increased monomer solubility and a morphology change from vesicle to micelle at characteristic pH values, dependent on the tail length. For the compounds promoting high transfection efficiency, this characteristic pH was within the range found in the endosomal compartment (7.4–4.0). Formation of mixed micelles between gemini surfactant and membrane phospholipids at reduced pH may therefore provide a method of endosome rupture and subsequent escape of entrapped DNA, thus discarding the need for extra fusogenic or endosomolytic agents. The positive charge on the vesicles at physiological pH drives the colloidal association with DNA. Small angle X‐ray scattering measurements indicate that lamellar aggregates are formed, which have a d spacing of 48–54 Å. Preliminary differential scanning calorimetric measurements suggest that reduction of pH causes a disordering of the hydrocarbon region of the DNA–surfactant complex.
Multiple signaling cascades are differentially involved in gene induction by double stranded RNA in interferon‐α‐primed cells
Authors: Harcourt Jennifer L. , Offermann Margaret K.
Published: March 2003

Priming with interfon (IFN)α enhanced the ability of the synthetic double‐stranded RNA polyriboinosinic acid: polyribocytidilic acid (pI:C), but not interleukin‐1β, to activate both p38 mitogen‐activated kinase (MAPK) and extracellular signal‐regulated kinase (ERK) signaling cascades. Activation by pI:C in IFNα‐primed cells was delayed compared to activation with interleukin‐1β, and this delay was followed by high, sustained activation of p38 MAPK and a modest elevation of ERK activation. Pharmacologic inhibition of either the ERK or the p38 MAPK pathway, using U0126 and SB203580, respectively, reduced interleukin‐6 protein induction by at least 70%, and combined inhibition of both pathways fully blocked interleukin‐6 protein expression and reduced interleukin‐6 mRNA induction by more than 80%. In contrast, induction of double‐stranded RNA‐activated protein kinase (PKR) mRNA and protein by IFNα and/or pI:C was minimally affected by either inhibitor. Induction of interferon‐regulatory factor‐1 (IRF‐1) by pI:C in IFNα primed cells was profoundly inhibited by U0126 but not by SB203580. Thus, IFNα priming enhances activation of p38 MAPK and ERK pathways by pI:C but not by interleukin‐1β, thereby enhancing the expression of some, but not all, genes that are induced by pI:C.
Reduction of ubiquinone by lipoamide dehydrogenase
Authors: Xia Ling , Björnstedt Mikael , Nordman Tomas , Eriksson Lennart C. , Olsson Jerker M.
Published: March 2003

Lipoamide dehydrogenase belongs to a family of pyridine nucleotide disulfide oxidoreductases and is ubiquitous in aerobic organisms. This enzyme also reduces ubiquinone (the only endogenously synthesized lipid‐soluble antioxidant) to ubiquinol, the form in which it functions as an antioxidant. The reduction of ubiquinone was linear with time and exhibited turnover numbers of 5 and 1.2 min−1 in the presence and absence of zinc, respectively. The reaction was stimulated by zinc and cadmium but not by the other divalent ions tested. The zinc/cadmium‐dependent stimulation of the reaction increased rapidly and linearly up to a concentration of 0.1 mm and was even further increased at 0.5 mm. At pH 6, the activity was three times higher than at physiological pH. Alteration of the NADPH : NADP+ ratio revealed that the reaction is inhibited by higher concentrations of the oxidized cofactors. FAD reduced ubiquinone in a dose‐dependent manner at a considerably lower rate, suggesting that the reduction of ubiquinone by lipoamide dehydrogenase involves the FAD moiety of the enzyme.
The role of the Cdk5–p35 kinase in neuronal development
Authors: Paglini Gabriela , Cáceres Alfredo
Published: March 2003

Cyclin‐dependent kinase 5 (Cdk5) plays a key role in proper development of the nervous system. To be activated, Cdk5 associates with regulatory subunits not related to cyclins, such as p35 (the regulatory subunit of Cdk5). In this article, we review some of the experimental evidence supporting a central role for the Cdk5/p35 kinase in neuronal migration and process formation.
Development and characterization of an animal model of carnitine deficiency
Authors: Spaniol Markus , Brooks Hilary , Auer Lucas , Zimmermann Arthur , Solioz Marc , Stieger Bruno , Krähenbühl Stephan
Published: March 2003

Mammals cover their carnitine needs by diet and biosynthesis. The last step of carnitine biosynthesis is the conversion of butyrobetaine to carnitine by butyrobetaine hydroxylase. We investigated the effect of N‐trimethyl‐hydrazine‐3‐propionate (THP), a butyrobetaine analogue, on butyrobetaine hydroxylase kinetics, and carnitine biosynthesis and body homeostasis in rats fed a casein‐based or a vegetarian diet. The Km of butyrobetaine hydroxylase purified from rat liver was 41 ± 9 µmol·L−1 for butyrobetaine and 37 ± 5 µmol·L−1 for THP, and THP was a competitive inhibitor of butyrobetaine hydroxylase (Ki 16 ± 2 µmol·L−1). In rats fed a vegetarian diet, renal excretion of total carnitine was increased by THP (20 mg·100 g−1·day−1 for three weeks), averaging 96 ± 36 and 5.3 ± 1.2 µmol·day−1 in THP‐treated and control rats, respectively. After three weeks of treatment, the total carnitine plasma concentration (8.8 ± 2.1 versus 52.8 ± 11.4 µmol·L−1) and tissue levels were decreased in THP‐treated rats (liver 0.19 ± 0.03 versus 0.59 ± 0.08 and muscle 0.24 ± 0.04 versus 1.07 ± 0.13 µmol·g−1). Carnitine biosynthesis was blocked in THP‐treated rats (−0.22 ± 0.13 versus 0.57 ± 0.21 µmol·100 g−1·day−1). Similar results were obtained in rats treated with the casein‐based diet. THP inhibited carnitine transport by rat renal brush‐border membrane vesicles competitively (Ki 41 ± 3 µmol·L−1). Palmitate metabolism in vivo was impaired in THP‐treated rats and the livers showed mixed steatosis. Steady‐state mRNA levels of the carnitine transporter rat OCTN2 were increased in THP‐treated rats in skeletal muscle and small intestine. In conclusion, THP inhibits butyrobetaine hydroxylase competitively, blocks carnitine biosynthesis in vivo and interacts competitively with renal carnitine reabsorption. THP‐treated rats develop systemic carnitine deficiency over three weeks and can therefore serve as an animal model for human carnitine deficiency.
Amine oxidase‐like activity of polyphenols
Authors: Akagawa Mitsugu , Suyama Kyozo
Published: March 2003

Polyphenols in several oxidation systems gained amine oxidase‐like activity, probably due to the formation of the corresponding quinones. In the presence of Cu(II), o‐ and p‐phenolic compounds exhibited amine oxidase‐like activity, whereas only the o‐phenolic compounds showed the activity in the presence of 1,1‐diphenyl‐2‐picrylhydrazyl radical. The activity was determined by measuring the conversion of benzylamine to benzaldehyde by HPLC. Moreover, gallic acid, chlorogenic acid, and caffeic acid, which are plant polyphenols, converted the lysine residue of bovine serum albumin to α‐amino‐adipic semialdehyde residue, indicating lysyl oxidase‐like activity. We also characterized the activity of pyrocatechol, hydroquinone, and pyrogallol in the presence of Cu(II). The oxidative deamination was accelerated at a higher pH, and required O2 and transition metal ions. Furthermore, EDTA markedly inhibited the reaction but not β‐aminopropionitrile, which is a specific inhibitor of lysyl oxidase. Catalase significantly inhibited the oxidation, implying the participation of hydroxyl radical in the reaction, but superoxide dismutase stimulated the oxidation, probably due to its radical formation activity. We discussed the mechanism of the oxidative deamination by polyphenols and the possible significance of the activity for biological systems.
Assembly and clustering of acetylcholine receptors containing GFP‐tagged ε or γ subunits
Authors: Gensler Sven , Sander Andreas , Korngreen Alon , Traina Giovanna , Giese Günther , Witzemann Veit
Published: March 2003

Acetylcholine receptor (AChR) γ and ε subunits were tagged by green fluorescent protein (GFP) to analyse assembly and targeting in live muscle fibers at the neuromuscular junction. N‐ or C‐terminal fusion polypeptides showed no fluorescence upon transfection of HEK cells. When GFP was inserted into the cytoplasmic loop connecting putative transmembrane regions M3 and M4, the γ/GFP and ε/GFP subunits were fluorescent and formed together with the α, β, and δ subunits GFP‐tagged AChR complexes that were integrated into the plasma membrane. As the AChR were also clustered by rapsyn, the results indicate that the cytoplasmatic domains of the γ and ε subunits may not be required for assembly and rapsyn‐dependent clustering. The γ/GFP and ε/GFP subunit‐containing receptors were expressed in X. laevis oocytes and have affinities for acetylcholine similar to that of the wild‐type receptors. Direct gene transfer into single muscle fibers reveals that γ/GFP or ε/GFP polypeptides are expressed at the site of injection and are transported within the endoplasmatic reticulum. When reaching subsynaptic regions, both γ/GFP or ε/GFP subunits compete with endogenous ε subunits to assemble GFP‐tagged receptors, which are selectively targeted to the postsynaptic membrane.
Oxidized adrenodoxin acts as a competitive inhibitor of cytochrome P450scc in mitochondria from the human placenta
Authors: Tuckey Robert C. , McKinley Allan J. , Headlam Madeleine J.
Published: March 2003

The conversion of cholesterol to pregnenolone by cytochrome P450scc is the rate‐determining step in placental progesterone synthesis. The limiting component for placental cytochrome P450scc activity is the concentration of adrenodoxin reductase in the mitochondria, where it permits cytochrome P450scc to work at only 16% of maximum velocity. Adrenodoxin reductase serves to reduce adrenodoxin as part of the electron transfer from NADPH to cytochrome P450scc. We therefore measured the proportion of adrenodoxin in the reduced form in intact mitochondria from the human placenta during active pregnenolone synthesis, using EPR. We found that the adrenodoxin pool was only 30% reduced, indicating that the adrenodoxin reductase concentration was insufficient to maintain the adrenodoxin in the fully reduced state. As both oxidized and reduced adrenodoxin can bind to cytochrome P450scc we tested the ability of oxidized adrenodoxin to act as a competitive inhibitor of pregnenolone synthesis. This was done in a fully reconstituted system comprising 0.3% Tween 20 and purified proteins, and in a partially reconstituted system comprising submitochondrial particles, purified adrenodoxin and adrenodoxin reductase. We found that oxidized adrenodoxin is an effective competitive inhibitor of placental cytochrome P450scc with a Ki value half that of the Km for reduced adrenodoxin. We conclude that the limiting concentration of adrenodoxin reductase present in placental mitochondria has a two‐fold effect on cytochrome P450scc activity. It limits the amount of reduced adrenodoxin that is available to donate electrons to cytochrome P450scc and the oxidized adrenodoxin that remains, competitively inhibits the cytochrome.
Molecular cloning of a mammalian nuclear phosphoprotein NUCKS, which serves as a substrate for Cdk1 in vivo
Authors: Østvold Anne C. , Norum Jens H. , Mathiesen Siri , Wanvik Birgit , Sefland Iren , Grundt Kirsten
Published: March 2003

We have isolated and characterized a cDNA encoding a mammalian nuclear phosphoprotein NUCKS, previously designated P1. Molecular analyses of several overlapping and full‐length cDNAs from HeLa cells and rat brain revealed a protein with an apparent molecular mass of 27 kDa in both species. The deduced amino‐acid sequences are highly conserved between human and rodents, but show no homology with primary structures in protein databases or with translated sequences of cDNAs in cDNA databanks. Although the protein has some features in common with the high mobility group proteins HMGI/Y, attempts to find a putative protein family by database query using both sequence alignment methods and amino‐acid composition have failed. Northern blot analyses revealed that human and rat tissues contain three NUCKS transcripts varying in size from 1.5 to 6.5 kb. All human and rat tissues express the gene, but the level of transcripts varies among different tissues. Circular dichroism analysis and secondary structure predictions based on the amino‐acid sequence indicate a low level of α helical content and substantial amounts of β turn structures. The protein is phosphorylated in all phases of the cell cycle and exhibits mitosis‐specific phosphorylation of threonine residues. Phosphopeptide mapping and back‐phosphorylation experiments employing NUCKS from HeLa interphase and metaphase cells show that the protein is phosphorylated by Cdk1 during mitosis of the cell cycle.
P‐Glycoprotein conformational changes detected by antibody competition
Authors: Nagy Henrietta , Goda Katalin , Arceci Robert , Cianfriglia Maurizio , Mechetner Eugene , Szabó Gábor
Published: March 2003

Conformational changes accompanying P‐glycoprotein (Pgp) mediated drug transport are reflected by changes in the avidity of certain monoclonal antibodies (mAbs). More of the UIC2 mAb binds to Pgp‐expressing cells in the presence of substrates or modulators [Mechetner, E.B., Schott, B., Morse, S.B., Stein, W., Druley, T., Dvis, K.A., Tsuruo, T. & Roninson, I.B. (1997) Proc. Natl Acad. Sci. USA94, 12908–12913], while the binding of other mAbs (e.g. MM12.10, MRK16, 4E3) is not conformation sensitive. Pre‐staining of Pgp+ cells with UIC2 decreased the subsequent binding of MM12.10 mAb by about 30–40%, suggesting that there are Pgp molecules available for both UIC2 and MM12.10, and others accessible only for MM12.10. In the presence of certain substrates/modulators such as vinblastin, cyclosporin A or valinomycin, the MM12.10 reactivity was completely abolished by preincubation with UIC2. However, verapamil, Tween‐80 and nifedipine did not influence the ratio of bound mAbs significantly. This is the first assay to our knowledge, sharply distinguishing two classes of modulators. The conformational changes accompanying the mAb competition phenomenon appear to be closely related, though not identical to those accompanying the UIC2‐shift, as suggested by the simultaneous assessment of the two phenomena.
Central carbon metabolism of Saccharomyces cerevisiae explored by biosynthetic fractional 13C labeling of common amino acids
Authors: Maaheimo Hannu , Fiaux Jocelyne , Çakar Z. Petek , Bailey James E. , Sauer Uwe , Szyperski Thomas
Published: March 2003

Aerobic and anaerobic central metabolism of Saccharomyces cerevisiae cells was explored in batch cultures on a minimal medium containing glucose as the sole carbon source, using biosynthetic fractional 13C labeling of proteinogenic amino acids. This allowed, firstly, unravelling of the network of active central pathways in cytosol and mitochondria, secondly, determination of flux ratios characterizing glycolysis, pentose phosphate cycle, tricarboxylic acid cycle and C1‐metabolism, and thirdly, assessment of intercompartmental transport fluxes of pyruvate, acetyl‐CoA, oxaloacetate and glycine. The data also revealed that alanine aminotransferase is located in the mitochondria, and that amino acids are synthesized according to documented pathways. In both the aerobic and the anaerobic regime: (a) the mitochondrial glycine cleavage pathway is active, and efflux of glycine into the cytosol is observed; (b) the pentose phosphate pathways serve for biosynthesis only, i.e. phosphoenolpyruvate is entirely generated via glycolysis; (c) the majority of the cytosolic oxaloacetate is synthesized via anaplerotic carboxylation of pyruvate; (d) the malic enzyme plays a key role for mitochondrial pyruvate metabolism; (e) the transfer of oxaloacetate from the cytosol to the mitochondria is largely unidirectional, and the activity of the malate–aspartate shuttle and the succinate‐fumarate carrier is low; (e) a large fraction of the mitochondrial pyruvate is imported from the cytosol; and (f) the glyoxylate cycle is inactive. In the aerobic regime, 75% of mitochondrial oxaloacetate arises from anaplerotic carboxylation of pyruvate, while in the anaerobic regime, the tricarboxylic acid cycle is operating in a branched fashion to fulfill biosynthetic demands only. The present study shows that fractional 13C labeling of amino acids represents a powerful approach to study compartmented eukaryotic systems.
Molecular characterization of an anti‐epilepsy peptide from the scorpion Buthus martensi Karsch
Authors: Wang Chun‐Guang , He Xiao‐Lin , Shao Feng , Liu Wei , Ling Min‐Hua , Wang Da‐Cheng , Chi Cheng‐Wu
Published: March 2003

For a long time Asian scorpion Buthus martensi Karsch (BmK) has been used in Chinese traditional medicine to cure many diseases of nervous system. Here we report the purification and characterization of a pharmacologically active neurotoxin from the scorpion BmK. This toxin had little toxicity in mice and insects but was found to have an anti‐epilepsy effect in rats, and is thus named as BmK anti‐epilepsy peptide (BmK AEP). Its amino‐acid sequence was determined by lysylendopeptidase digestion, Edman degradation and mass spectrographic analysis. Based on the determined sequence, the gene coding for this peptide was also cloned and sequenced by the 3′ and 5′ RACE methods. It encodes a precursor of 85 amino‐acid residues including a signal peptide of 21 residues, a mature peptide of 61 residues and three additional residues Gly‐Lys‐Lys at the C‐terminus. The additional Gly sometimes followed by one or two basic residues is prerequisite for the amidation of its C‐terminus. C‐terminal amidation was also verified by the molecular‐mass determination of BmK AEP. This anti‐epilepsy peptide toxin shares homology with other depressant insect toxins. The remarkable difference between them was mainly focused at residues 6, 7 and 39; these residues might relate to the unique action of BmK AEP.
The oxidative mechanism of heparin interferes with radical production by glucose and reduces the degree of glycooxidative modifications on human serum albumin
Authors: Finotti Paola , Pagetta Andrea , Ashton Tony
Published: March 2003

Among substances which may prove useful in preventing or reducing the progression of glycooxidative modifications of proteins, heparin plays a unique role. To elucidate the mechanism whereby heparin may favourably influence the protein structure during glycation, human serum albumin (HSA) was glycated with both 25 and 50 mm glucose in the absence and presence of 12 µg·mL−1 low‐molecular‐mass heparin. Glycation caused: (a) modifications of fluorescence emission and excitation spectra consistent with the covalent attachment of glucose to protein; (b) a significant increase in the esterase activity of HSA on p‐nitrophenyl acetate; (c) a reduced susceptibility to tryptic digestion and (d) enhanced formation of high‐molecular mass aggregates of HSA. These alterations were accompanied by oxidative reactions, as the EPR spectra showed a clear‐cut radical signal, dependent on glucose concentration, further confirmed by measurement of the carbonyl content of HSA, as an indirect proof of oxidative damage. In the presence of heparin all the above alterations, especially at 25 mm glucose, turned out to be antagonized. The effects of heparin were dependent on its specific binding to HSA, which triggered an oxidative mechanism strikingly different from that caused by glucose. In the presence of heparin, only the radical species catalyzed by heparin was detected across all samples of glycated HSA, irrespective of glucose concentration. In addition, at 25 mm glucose, enhancement of the oxidative capacity of heparin was also observed. The results demonstrate that the oxidative mechanism sustained by heparin mediates biological effects that may be beneficial in reducing the extent of glycooxidative damage on HSA.
Purification and cloning of chloroplast 6‐phosphogluconate dehydrogenase from spinach
Authors: Krepinsky Karsten , Plaumann Marlies , Martin William , Schnarrenberger Claus
Published: March 2003

Previous attempts to purify chloroplast 6‐phosphogluconate dehydrogenase (cp6PGDH), a key enzyme of the oxidative pentose phosphate pathway, have been unsuccessful due to rapid activity loss. An efficient purification protocol was developed and the enzyme from spinach leaves was purified 1000‐fold to apparent homogeneity with a specific activity of 60 U·mg−1. The enzyme is a homodimer with subunits of 50 kDa. Antibodies raised against the purified cp6PGDH detected a 53‐kDa protein from a crude extract, indicating alterations during purification. Purified cp6PGDH was microsequenced and the corresponding spinach cDNA was cloned using PCR techniques and degenerate primers. The cDNA for cytosolic 6PGDH from spinach was cloned for comparison. Phylogenetic analysis in the context of available homologues from eukaryotes and eubacteria revealed that animal and fungal cytosolic 6PGDH sequences are more similar to their homologues from γ‐proteobacteria, whereas plant 6PGDH is more similar to its cyanobacterial homologues. The ancestral gene for higher plant 6PGDH was acquired from the antecedent of plastids through endosymbiosis and gene transfer to the nucleus. A subsequent gene duplication gave rise to higher plant cytosolic 6PGDH, which assumed the function of its pre‐existing cytosolic homologue through endosymbiotic gene replacement. The protein phylogeny of both 6PGDH and of the first enzyme of the oxidative pentose phosphate pathway, glucose‐6‐phosphate dehydrogenase, indicate a surprisingly close relationship between the plant and Trypanosoma brucei lineages, suggesting that T. brucei (a relative of Euglena gracilis) may be secondarily nonphotosynthetic.
Nuclear import and DNA‐binding activity of RFX1
Authors: Katan‐Khaykovich Yael , Shaul Yosef
Published: March 2003

RFX1 binds and regulates the enhancers of a number of viruses and cellular genes. RFX1 belongs to the evolutionarily conserved RFX protein family that shares a DNA‐binding domain and a conserved C‐terminal region. In RFX1 this conserved region mediates dimerization, and is followed by a unique C‐terminal tail, containing a highly acidic stretch. In HL‐60 cells nuclear translocation of RFX1 is regulated by protein kinase C with unknown mechanisms. By confocal fluorescence microscopy, we have identified a nonclassical nuclear localization signal (NLS) at the extreme C‐terminus. The adjacent ‘acidic region’, which showed no independent NLS activity, potentiated the function of the NLS. Subcellular fractionation showed that the tight association of RFX1 with the nucleus is mediated by its DNA‐binding domain and enhanced by the dimerization domain. In contrast, the acidic region inhibited nuclear association, by down‐regulating the DNA‐binding activity of RFX1. These data suggest an autoinhibitory interaction, which may regulate the function of RFX1 at the level of DNA binding. The C‐terminal tail thus constitutes a composite localization domain, which on the one hand mediates nuclear import of RFX1, and on the other hand inhibits its association with the nucleus and binding to DNA. The participation of the acidic region in both activities suggests a mechanism by which the nuclear import and DNA‐binding activity of RFX1 may be coordinately regulated by phosphorylation by kinases such as PKC.
p300/CBP/p53 interaction and regulation of the p53 response
Authors: Grossman Steven R.
Published: March 2003

Substantial evidence points to a critical role for the p300/CREB binding protein (CBP) coactivators in p53 responses to DNA damage. p300/CBP and the associated protein P/CAF bind to and acetylate p53 during the DNA damage response, and are needed for full p53 transactivation as well as downstream p53 effects of growth arrest and/or apoptosis. Beyond this simplistic model, p300/CBP appear to be complex integrators of signals that regulate p53, and biochemically, the multipartite p53/p300/CBP interaction is equally complex. Through physical interaction with p53, p300/CBP can both positively and negatively regulate p53 transactivation, as well as p53 protein turnover depending on cellular context and environmental stimuli, such as DNA damage.
Human neutrophils employ the myeloperoxidase/hydrogen peroxide/chloride system to oxidatively damage apolipoprotein A‐I
Authors: Bergt Constanze , Marsche Gunther , Panzenboeck Ute , Heinecke Jay W. , Malle Ernst , Sattler Wolfgang
Published: March 2003

The structural integrity of apolipoprotein A‐I (apo A‐I) is critical to the physiological function of high‐density lipoprotein (HDL). Oxidized lipoproteins are thought to be of central importance in atherogenesis, and oxidation products characteristic of myeloperoxidase, a heme protein secreted by activated phagocytes, have been detected in human atherosclerotic tissue. At plasma concentrations of halide ion, hypochlorous acid is a major product of the myeloperoxidase‐hydrogen peroxide‐chloride system. We therefore investigated the effects of activated human neutrophils, a potent source of myeloperoxidase and hydrogen peroxide, on the protein and lipid components of HDL. Both free and HDL‐associated apo A‐I exposed to activated human neutrophils underwent extensive degradation as monitored by RP‐HPLC and Western blotting with a polyclonal antibody to apo A‐I. Replacement of the neutrophils with reagent HOCl resulted in comparable damage (at molar oxidant : HDL subclass 3 ratio = 100) as observed in the presence of activated phagocytes. Apo A‐I degradation by activated neutrophils was partially inhibited by the HOCl scavenger methionine, by the heme inhibitor azide, by chloride‐free conditions, by the peroxide scavenger catalase, and by a combination of superoxide dismutase (SOD)/catalase, implicating HOCl in the cell‐mediated reaction. The addition of a protease inhibitor (3,4‐dichloroisocoumarin) further reduced the extent of apo A‐I damage. In contrast to the protein moiety, there was little evidence for oxidation of unsaturated fatty acids or cholesterol in HDL3 exposed to activated neutrophils, suggesting that HOCl was selectively damaging apo A‐I. Our observations indicate that HOCl generated by myeloperoxidase represents one pathway for protein degradation in HDL3 exposed to activated phagocytes.
Monitoring of secondary and tertiary structure changes in the gastric H+/K+‐ATPase by infrared spectroscopy
Authors: Scheirlinckx Frantz , Buchet René , Ruysschaert Jean‐Marie , Goormaghtigh Erik
Published: March 2003

Conformational changes occurring in the catalytic cycle of the H+/K+‐ATPase were monitored by Fourier transform infrared spectroscopy (FTIR). Caged compounds were used to release ATP, in the presence of Ca2+, to induce the transition between the E1 and E1‐P conformation of the H+/K+‐ATPase. In addition to bands associated with the photolysis of the caged compounds, some peaks of the difference infrared spectra were associated with changes in secondary structure and modifications of the ionization in the side chains of amino‐acid residues (Glu or Asp). These changes were specific to the reaction between the ligand and the enzyme. We estimated that 39 amino acids changed their secondary structure during the reaction and four amino‐acid residues were deprotonated. Similar spectral changes appeared when ADP was released from its precursor. The release of Pi from the same caged molecule did not induce similar changes. Changes in tertiary structure occurring during the binding of adenosine and phosphorylation of the enzyme were demonstrated by recording hydrogen/deuterium exchange kinetics by attenuated total reflectance FTIR spectroscopy (ATR‐FTIR). At least 129 amide protons were involved in a tertiary structure change induced by ATP. This suggested that secondary structure change transduced a much larger tertiary structure modification.