bluuebunny/crossref_metadata_2025_split huggingface dataset:
Increased methyl esterification of altered aspartyl residues in erythrocyte membrane proteins in response to oxidative stress
Authors: Ingrosso Diego , D’Angelo Stefania , di Carlo Enza , Perna Alessandra F. , Zappia Vincenzo , Galletti Patrizia 
Published: March 2003

Protein‐l‐isoaspartate (d‐aspartate) O‐methyltransferase (PCMT; EC 2.1.1.77) catalyses the methyl esterification of the free α‐carboxyl group of abnormal l‐isoaspartyl residues, which occur spontaneously in protein and peptide substrates as a consequence of molecular ageing. The biological function of this transmethylation reaction is related to the repair or degradation of age‐damaged proteins. Methyl ester formation in erythrocyte membrane proteins has also been used as a marker reaction to tag these abnormal residues and to monitor their increase associated with erythrocyte ageing diseases, such as hereditary spherocytosis, or cell stress (thermal or osmotic) conditions.The study shows that levels of l‐isoaspartyl residues rise in membrane proteins of human erythrocytes exposed to oxidative stress, induced by t‐butyl hydroperoxide or H2O2. The increase in malondialdehyde content confirmed that the cell membrane is a primary target of oxidative alterations. A parallel rise in the methaemoglobin content indicates that proteins are heavily affected by the molecular alterations induced by oxidative treatments in erythrocytes. Antioxidants largely prevented the increase in membrane protein methylation, underscoring the specificity of the effect. Conversely, we found that PCMT activity, consistent with its repair function, remained remarkably stable under oxidative conditions, while damaged membrane protein substrates increased significantly. The latter include ankyrin, band 4.1 and 4.2, and the integral membrane protein band 3 (the anion exchanger). The main target was found to be particularly protein 4.1, a crucial element in the maintenance of membrane‐cytoskeleton network stability. We conclude that the increased formation/exposure of l‐isoaspartyl residues is one of the major structural alterations occurring in erythrocyte membrane proteins as a result of an oxidative stress event. In the light of these and previous findings, the occurrence of isoaspartyl sites in membrane proteins as a key event in erythrocyte spleen conditioning and hemocatheresis is proposed.
Structure elucidation and biological activity of an unusual adipokinetic hormone from corpora cardiaca of the butterfly, Vanessa cardui
Authors: Köllisch Gabriele V. , Lorenz Matthias W. , Kellner Roland , Verhaert Peter D. , Hoffmann Klaus H. 
Published: March 2003

A structurally unusual member of the adipokinetic hormone/red pigment‐concentrating hormone peptide family was isolated from corpora cardiaca of the painted lady butterfly, Vanessa cardui. Its primary structure was assigned by Edman degradation and nano‐electrospray‐time‐of‐flight mass spectrometry as pQLTFTSSWGGK (Vac‐AKH). Vac‐AKH represents the first 11mer and the first nonamidated peptide in this family.The peptide shows significant adipokinetic activity in adult specimens of V. cardui. Injection of 10 pmol of synthetic Vac‐AKH into 4‐day‐old decapitated males resulted in an ≈ 150% increase of hemolymph lipids after 90 min. Half maximal adipokinetic activity was achieved with about 0.1 pmol of Vac‐AKH. During a 2‐h incubation of corpora cardiaca/corpora allata complexes in medium containing 50 mm KCl, significant amounts of Vac‐AKH were released from the glands.
Substrate and solvent isotope effects on the fate of the active oxygen species in substrate‐modulated reactions of putidamonooxin
Authors: Twilfer Hans , Sandfort Georg , Bernhardt Frithjof‐Hans 
Published: March 2003

Using 4‐methoxybenzoate monooxygenase from Pseudomonas putida, the substrate deuterium isotope effect on product formation and the solvent isotope effect on the stoichiometry of oxygen uptake, NADH oxidation, product and/or H2O2 (D2O2) formation for tight couplers, partial uncouplers, and uncouplers as substrates were measured. These studies revealed for the true, intrinsic substrate deuterium isotope effect on the oxygenation reaction a k1H/k2H ratio of < 2.0, derived from the inter‐ and intramolecular substrate isotope effects. This value favours a concerted oxygenation mechanism of the substrate. Deuterium substitution in a tightly coupling substrate initiated a partial uncoupling of oxygen reduction and substrate oxygenation, with release of H2O2 corresponding to 20% of the overall oxygen uptake. This H2O2 (D2O2) formation (oxidase reaction) almost completely disappeared when the oxygenase function was increased by deuterium substitution in the solvent. The electron transfer from NADH to oxygen, however, was not affected by deuterium substitution in the substrate and/or the solvent.With 4‐trifluoromethylbenzoate as uncoupling substrate and D2O as solvent, a reduction (peroxidase reaction) of the active oxygen complex was initiated in consequence of its extended lifetime. These additional two electron‐transfer reactions to the active oxygen complex were accompanied by a decrease of both NADH oxidation and oxygen uptake rates. These findings lead to the following conclusions: (a) under tightly coupling conditions the rate‐limiting step must be the formation time and lifetime of an active transient intermediate within the ternary complex iron/peroxo/substrate, rather than an oxygenative attack on a suitable C–H bond or electron transfer from NADH to oxygen. Water is released after the monooxygenation reaction; (b) under uncoupling conditions there is competition in the detoxification of the active oxygen complex between its protonation (deuteronation), with formation of H2O2 (D2O2) and its further reduction to water. The additional two electron‐transfer reactions onto the active oxygen complex then become rate limiting for the oxygen uptake rate.
Evidence for an amphipathicity independent cellular uptake of amphipathic cell‐penetrating peptides
Authors: Scheller Anne , Wiesner Burkhard , Melzig Matthias , Bienert Michael , Oehlke Johannes 
Published: March 2003

The cellular uptake of a peptide set derived from membrane‐permeable α‐helical amphipathic peptides by stepwise alterations of structure forming propensity and charge was studied by confocal laser scanning microscopy (CLSM) combined with HPLC. For CLSM monitoring, an online protocol was employed that avoided bias of the uptake results by washout. Using this protocol, extensive fluorescence, approaching the intensity of the external peptide, was observed in the cytosol and nucleus within minutes in all cases, irrespective of the degree of amphipathicity. HPLC analyses of the cell lysates revealed the unmetabolized peptides to be the predominant source of the intracellular fluorescence. Significant amphipathicity‐dependent differences became apparent only after washing the peptide‐loaded cells, reflecting the effects of amphipathicity on resistance to wash out. Exposure of the cells to the peptides at 37 and 0 °C led to similar results, indicating the nonendocytic character of the uptake.With a view to practical applications , the results of the present study open the possibility of exploiting nonamphipathic peptides as vectors for translocating polar compounds into the cell interior, which would circumvent substantial obstacles currently connected with the use of amphipathic vector peptides, such as membrane toxicity and low solubility. Moreover, differences in the uptake of several members of the investigated peptide series into different cell types present a promising basis for the design of cell‐type specific vector peptides.
Thioredoxin reductase as a pathophysiological factor and drug target
Authors: Becker Katja , Gromer Stephan , Schirmer R. Heiner , Müller Sylke 
Published: March 2003

Human cytosolic thioredoxin reductase (TrxR), a homodimeric protein containing 1 selenocysteine and 1 FAD per subunit of 55 kDa, catalyses the NADPH‐dependent reduction of thioredoxin disulfide and of numerous other oxidized cell constituents. As a general reducing enzyme with little substrate specificity, it also contributes to redox homeostasis and is involved in prevention, intervention and repair of damage caused by H2O2‐based oxidative stress.Being a selenite‐reducing enzyme as well as a selenol‐containing enzyme, human TrxR plays a central role in selenium (patho)physiology. Both dietary selenium deficiency and selenium oversupplementation, a lifestyle phenomenon of our time, appear to interfere with the activity of TrxR. Selenocysteine 496 of human TrxR is a major target of the anti‐rheumatic gold‐containing drug auranofin, the formal Ki for the stoichiometric inhibition being 4 nm. The hypothesis that TrxR and extracellular thioredoxin play a pathophysiologic role in chronic diseases such as rheumatoid arthritis, Sjögren's syndrom, AIDS, and certain malignancies, is substantiated by biochemical, virological, and clinical evidence. Reduced thioredoxin acts as an autocrine growth factor in various tumour diseases, as a chemoattractant, and it synergises with interleukins 1 and 2. The effects of anti‐tumour drugs such as carmustine and cisplatin can be explained in part by the inhibition of TrxR. Consistently, high levels of the enzyme can support drug resistance.TrxRs from different organisms such as Escherichia coli, Mycobacterium leprae, Plasmodium falciparum, Drosophila melanogaster, and man show a surprising diversity in their chemical mechanism of thioredoxin reduction. This is the basis for attempts to develop specific TrxR inhibitors as drugs against bacterial infections like leprosy and parasitic diseases like amebiasis and malaria.
Signal transduction pathways triggered by fibroblast growth factor receptor 1 expressed in Xenopus laevis oocytes after fibroblast growth factor 1 addition
Authors: Browaeys‐Poly Edith , Cailliau Katia , Vilain Jean‐Pierre 
Published: March 2003

Xenopus oocytes expressing fibroblast growth factor receptor 1 (FGFR1) were used as a biological model system to analyse the signal transduction pathways that are triggered by fibroblast growth factor 1 (FGF1). Germinal vesicle breakdown (GVBD) and phosphorylation of extracellular signal‐regulated protein kinase 2 (ERK2) occured 15 h after FGF1 addition. These events were Ras‐dependent as they were blocked by a Ras dominant negative form. The Ras activity was promoted by three upstream effectors, growth factor‐bound protein 2 (Grb2), phosphatidylinositol 3‐kinase (PI3K) and Src cytoplasmic kinase. Ras activation was inhibited by a Grb2 dominant negative form (P49L), by PI3K inhibitors, including wortmannin, LY294002, the N‐SH2 domain of p85α PI3K and by the SH2 domain of Src. Src activation induced by FGF1 was blocked by the SH2 domain of Src and PP2, a specific inhibitor of Src. The Grb2 adaptor was recruited by the upstream Src homology 2/α‐collagen‐related (Shc) effector, as the SH2‐Shc domain prevented the GVBD and the ERK2 phosphorylation induced by FGF1. The importance of another signalling pathway involving phospholipase Cγ (PLCγ) was also investigated. The use of the PLCγ inhibitory peptide, neomycin and the calcium chelator BAPTA‐AM on oocytes expressing FGFR1 or the stimulation by PDGF‐BB of oocytes expressing PDGFR‐FGFR1 mutated on the PLCγ binding site, prevented GVBD and ERK2 phosphorylation. This study shows that the transduction cascade induced by the FGFR1–FGF1 interaction in Xenopus oocytes represents the sum of Ras‐dependent and PLCγ‐dependent pathways. It emphasizes the role played by PI3K and Src and their connections with the Ras cascade in the FGFR1 signal transduction.
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.
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.
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.
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.