bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of the gene encoding α1‐acid glycoprotein in rabbit liver under acute‐phase conditions involves induction and activation of β and δ CCAAT‐enhancer‐binding proteins
Authors: RAY Bimal K. , RAY Alpana
Published: March 2005

Transcription of the gene encoding α1‐acid glycoprotein is highly induced during acute inflammation which has been previously shown to be mediated by some inducible members of the CCAAT‐enhancer‐binding (C/EBP) transcription‐factor family. In this study, we demonstrate that the involved inducible C/EBP isoforms are C/EBP‐β and C/EBP‐δ, and together they control the high‐level induction of the α1‐acid glycoprotein gene in response to inflammatory signals. We observed that dephosphorylation severely inhibits the DNA‐binding ability of C/EBP‐δ and its transactivating potential increases in the presence of cellular phosphatase inhibitors, such as okadaic acid and sodium orthovanadate. These results suggest that C/EBP‐δ is regulated by phosphorylation. Transient transfections using expression vectors of C/EBP‐α, C/EBP‐β and C/EBP‐δ have shown that while individually all three isoforms can transactivate the α1‐acid glycoprotein–chloramphenicol‐acetyltransferase gene transcription, co‐expression of C/EBP‐α and C/EBP‐β isoforms results in lower levels of reporter gene expression than the levels predicted from their additive transactivation level. In vitro DNA‐binding studies have shown that C/EBP‐α and C/EBP‐β isoforms both interact and form complexes with the α1‐acid glycoprotein gene C/EBP‐binding element under normal non‐induced conditions during which α1‐acid glycoprotein is expressed at a very low level. Higher than additive levels of reporter gene expression are observed when combinations of C/EBP‐δ and C/EBP‐β or C/EBP‐δ and C/EBP‐α are used. Together, these data demonstrate that C/EBP‐β and C/EBP‐δ are the major proteins responsible for the acute‐phase induction of α1‐acid‐glycoprotein gene expression and they require phosphorylation for transactivation potential.
NMR studies of recombinant Coprinus peroxidase and three site‐directed mutants
Authors: VEITCH Nigel C. , TAMS Jeppe W. , VIND Jesper , DALBØGE Henrik , WELINDER Karen G.
Published: March 2005

Proton nuclear magnetic resonance spectroscopy has been used to characterise and compare wild‐type fungal and recombinant Coprinus cinereus peroxidase (CIP) and three mutants in which Gly156 and/or Asn157 was replaced by Phe. Analysis of one‐ and two‐dimensional NMR spectra of recombinant CIP was undertaken for comparison with the fungal enzyme and in order to establish a meaningful basis for solution studies of CIP mutants. Proton resonance assignments of haem and haem‐linked residues obtained for the cyanide‐ligated form of recombinant CIP revealed a high degree of spectral similarity with those of lignin and manganese‐dependent peroxidases and extend previously reported NMR data for fungal CIP.

The three mutants examined by NMR spectroscopy comprised site‐specific substitutions made to a region of the structure believed to form part of the peroxidase haem group access channel for substrate and ligand molecules. Proton resonances of the aromatic side‐chains of Phe156 and Phe157 were found to have similar spectral characteristics to those of two phenylalanine residues known to be involved in the binding of aromatic donor molecules to the plant peroxidase, horseradish peroxidase isoenzyme C. The results are discussed in the context of complementary reactivity studies on the mutants in order to develop a more detailed understanding of aromatic donor molecule binding to fungal and plant peroxidases.
Structural changes of high‐density‐lipoprotein apolipoproteins following incubation with human polymorphonuclear cells
Authors: COGNY Anne , PAUL J. Louis , ATGER Véronique , SONI Théophile , MOATTI Nicole
Published: March 2005

Based on the analogy in mechanisms and events between the pathogenesis of atherosclerosis and the inflammatory reaction, we investigated the impact of human polymorphonuclear leukocyte (PMN) degranulation and oxidative process on high‐density‐lipoprotein (HDL) structure.

HDL were incubated (37°C) with PMN at a physiological ratio (370 nmol cholesterol‐HDL/ml with 2X106 PMN/ml) for 15, 30 and 60 min with or without stimulating agent. PMN activation was assessed by measurement of superoxide anion generation and elastase production, which both reached peak concentration at 15 min.

HDL apolipoproteins (apo) analysed by immunoblotting after SDS/PAGE and electrofocusing evidenced the following modifications: (a) a slow hydrolysis of apo AII and apo Cs; (b) a rapid hydrolysis of apo E; (c) a change in apo AI isoform distribution with an increase in the most acidic isoform (AI‐2) at the expense of a less acidic form (AI‐1); (d) a shift of the major apo AII isoform into two more basic forms.

In contrast, no quantifiable lipid modification nor lipid oxidation, assessed by thiobarbituric‐acid‐reactive substances (TBARS) were noted. Despite a lack of variation of TBARS, a decrease in HDL vitamin E content by 80% was observed. Since this decrease was prevented by addition of superoxide dismutase in the medium, we concluded the occurence of an oxidative process affecting HDL.

Experiments with proteolytic inhibitors showed that elastase caused the proteolytic cleavage of apolipoprotein E, AII and Cs. In contrast, apo AI modification might involve both oxidative and proteolytic processes.
Metabolism of malate in bovine adrenocortical mitochondria studied by 13C‐NMR spectroscopy
Authors: PERRIN Anne , GOUT Elisabeth , CHAMBAZ Edmond M. , DEFAYE Geneviève
Published: March 2005

13C‐NMR spectroscopy was used to study the metabolism of [13C]malate in bovine coupled adrenocortical mitochondria. The most apparent difference between the mitochondria from steroidogenic tissues and mitochondria from other tissues is the presence, in addition to the normal respiratory chain, of a second electron‐transport system responsible for steroid hydroxylation.

[13C]malate was synthesized from [13C]succinate by isolated adrenocortical mitochondria. The basic functional suspension consisted of oxygenated mitochondria to which were added ADP, inorganic phosphate (Pi) and [13C]malate, both in the absence or presence of the steroid substrate, deoxycorticosterone. These mitochondria synthesized [13C]citrate and [13C]pyruvate from [13C]malate. The 13C labeling of these two metabolites demonstrated an important role of the malic enzyme and the kinetics depended on the presence of the steroid substrate; the citric acid cycle was stopped during the hydroxylation pathway. The addition of cyanide, a strong inhibitor of the respiratory chain, confirmed an increased malic enzyme activity when hydroxylation occurred, since pyruvate was trapped by formation of a cyanohydrin.

The relative enzymic activities of malic enzyme and isocitrate dehydrogenase were compared, both in the absence or presence of the steroid substrate, by supplementing the basic suspension with unlabeled exogenous metabolites, such as pyruvate or oxaloacetate.
Expression of the major surface glycoprotein of Leishmania, gp63, in wild‐type and sinefungin‐resistant promastigotes
Authors: SOTERIADOU Ketty P. , TZINIA Athina K. , MAMALAKI Avgi , PHELOUZAT Marie‐Anne , LAWRENCE Francoise , ROBERT‐GERO Malka
Published: March 2005

In this study, we have surveyed gp63 expression in sinefungin‐(SF)‐resistant and wild‐type Leishmania promastigotes. Documentation of gp63 expression in Leishmania promastigotes was carried out by Western blotting, purification of the protein and assessment of gp63 protease activity. We demonstrated a 3–4‐fold and 1.5–2‐fold increase of gp63 protein in SF‐resistant Leishmania donovani and Leishmania tropica promastigotes compared to wild‐type, respectively. Northern blot analysis showed that the increase in the amount of gp63 protein in SF‐resistant compared to wild‐type parasites was concomitant with an increase in gp63 mRNA. No extrachromosomal DNA was identified by alkaline lysis of isolated DNA samples and Southern blot analysis. Treatment of SF‐resistant and wild‐type L. donovani promastigotes with cycloheximide resulted in an increase of the steady state levels of gp63 mRNA in the SF‐resistant parasites to approximately fivefold that of the wild type. After treating parasites with actinomycin D, estimated gp63 mRNA t1/2 in the wild type was 40 min and increased to 83 min in SF‐resistant promastigotes. Therefore, the overexpression of gp63 may be mediated, at least in part, by post‐transcriptional stabilization of a gp63 transcript by a protein factor. Down regulation of the latter factor may account for the observed increase in gp63 expression in SF‐resistant promastigotes. Attempts to correlate gp63 expression with promastigote virulence suggested that the observed increase in gp63 expression did not result in a significant change in the virulence of SF‐resistant compared to wild‐type L. donovani promastigotes.
Expression of phytochrome apoprotein from Avena sativa in Escherichia coli and formation of photoactive chromoproteins by assembly with phycocyanobilin
Authors: HILL Christiane , GÄRTNER Wolfgang , TOWNER Paul , BRASLAVSKY Silvia E. , SCHAFFNER Kurt
Published: March 2005

Phytochrome DNAs from oat (Avena sativa L.) encoding the full‐length 124‐kDa polypeptide, a 118‐kDa fragment lacking the first 65 amino acids, and two N‐terminal fragments of 65 kDa and 45 kDa were subcloned and expressed in Escherichia coli. Reducing the temperature to 25°C during cell growth and the coexpression of chaperones improved the folding into a functional conformation for most of the polypeptides, and in one case the yield of polypeptides was also enhanced. A maximum yield of reconstitutable apoprotein was obtained by expressing the 65‐kDa fragment consisting of 595 amino acids. The apoproteins could be assembled in the dark with phycocyanobilin into photoreversible chromoproteins. The yield of photoreversible pigment could be further increased by far‐red/red irradiation cycles, indicating that the presence of the chromophore promotes the correct folding of the binding site. The chromoproteins with an intact N‐terminal domain exhibit Pr and Pfr absorption bands, which are blue‐shifted relative to the corresponding bands of native phytochrome due to the particular phycocyanobilin structure. The 118‐kDa fragment, only lacking the 6‐kDa N‐terminus, exhibits a strong Pr band, but only a weak Pfr absorbance. This indicates an essential role of the front 6‐kDa region of the protein in the formation of the far‐red absorbing chromophore‐protein complex. Otherwise, the C‐terminal region seems to be less important for photoreversibility as indicated by the function of the shorter fragments.
Desulfoviridin, a multimeric‐dissimilatory sulfite reductase from Desulfovibrio vulgaris (Hildenborough) Purification, characterization, kinetics and EPR studies
Authors: WOLFE Bonnie M. , LUI Siu Man , COWAN James A.
Published: March 2005

Conditions for the rigorous purification of desulfoviridin, the dissimilatory sulfite reductase from the sulfate‐reducing bacterium Desulfovibrio vulgaris (Hildenborough) have been established. A final purification by fast protein liquid chromatography yields at least three distinct bands that each exhibit the characteristic absorption spectrum of desulfoviridin. Two of these have been extensively characterized by amino acid analysis, isoelectric focusing, polyacrylamide gel electrophoresis, and formulation of the prosthetic centers. Each contains two pairs of [Fe4S4] and siroheme units. These results stand in marked contrast to recent work claiming significant demetallation of siroheme, excess iron content, and the presence of Fe6S6 clusters. These proposals are critically assessed in light of our results and other published work. Steady‐state kinetic parameters have been determined: kcat(SO32‐) = 0.31 mol SO32‐· s−1· mol heme−1, Km= 0.06 mM; kcat(NO2−) = 0.038 mol NO2−· s−1· mol heme−1, Km= 0.028 mM; kcat(NH2OH) = 29 mol NH2OH · s−1· mol heme−1, Km= 48 mM. A detailed comparison is made with the Escherichia coli and spinach‐assimilatory sulfite reductase enzymes and spinach nitrite reductase. Highly purified samples of dissimilatory sulfite reductase display an electron paramagnetic resonance spectrum characteristic of rhombic high spin ferric heme centers, while the fully reduced enzyme shows EPR features typical of [Fe4S4] clusters. The magnetic properties of the prosthetic centers are further characterized by variable temperature experiments and spin quantitation.
Purification and characterisation of a second cathepsin L proteinase secreted by the parasitic trematode Fasciola hepatica
Authors: DOWD Andrew J. , SMITH Angela M. , McGONIGLE Sharon , DALTON John P.
Published: March 2005

A 29.5‐kDa cysteine proteinase was purified from medium in which mature Fasciola hepatica parasites were maintained. The N‐terminal sequence (14 residues) of the purified protein is similar to known cathepsin L proteinases, including a 27‐kDa cathepsin L proteinase, also secreted by this parasite, which had been isolated previously in our laboratory [Smith, A. M., Dowd, A. J., Mc Gonigle, S., Keegan, P. S., Brennan, G., Trudgett, A. & Dalton, J. P. (1993) Mol. Biochem. Parasitol. 62, 1–8]. The N‐terminal sequences of the 29.5‐kDa and 27‐kDa cathepsin L proteinases differ only in residue number seven (arginine and proline, respectively). Immunoblot studies, using antiserum that reacts with both cathepsin L proteinases, rule out the possibility of both enzymes arising from a higher molecular sized parent molecule. The reaction kinetics of the two F. hepatica cathepsin L proteinases on a variety of peptide substrates revealed that the two enzymes differ in their substrate specificity. Five peptide substrates that are cleaved with high affinity by the 29.5‐kDa cathepsin L isolated in this study are not cleaved by the previously purified 27‐kDa cathepsin L. The protein‐modifying reagent, tetranitromethane, affected the 29.5‐kDa cathepsin L proteinase only, causing inactivation of the enzyme and changing its migration in polyacrylamide gel electrophoresis. Our studies suggest that the two F. hepatica cysteine proteinases represent two distinct subclasses within the cathepsin L class.
Probing hydrogen‐bonding interactions of bovine heart galectin‐1 and methyl β‐lactoside by use of engineered ligands
Authors: SOLÍS Dolores , JIMÉNEZ‐BARBERO Jesús , MARTÍN‐LOMAS Manuel , DÍAZ‐MAURIÑO Teresa
Published: March 2005

The binding of different synthetic monodeoxy, O‐methyl and fluorodeoxy derivatives of methyl β‐lactoside to galectin‐1 from bovine heart has been studied to probe the role of hydrogen bonding in the recognition and binding. The energetic contributions of the hydroxyl groups of methyl β‐lactoside directly involved in the interaction have been estimated and the nature of the protein residues involved has been predicted on the basis of the free energy data. Interpretations of the results have been sustained by molecular modeling of the three‐dimensional structure of the sugars in solution. One side of the disaccharide molecule is not involved (HO‐6 and HO‐2′) or only marginally involved (HO‐3′) in hydrogen bonding. Moreover, O‐methylation at these positions causes an enhancement of the binding, suggesting favourable interactions of the methyl groups which may come into contact with hydrophobic residues at the periphery of the combining site. Hydrogen‐bonding interactions are almost exclusively restricted to the other side of the molecule: the C‐4′ and C‐6′ hydroxyl groups act as donors of the strongest hydrogen bonds to charged groups of the lectin, while the C‐3 hydroxyl group participates in a strong hydrogen bond with a neutral group. The results also suggest that the N‐acetyl NH group in N‐acetyllactosamine, as well as the hydroxyl group at position C‐2 in methyl β‐lactoside, are involved in a polar interaction with neutral groups of the combining site. This hydrogen‐bonding pattern contrasts markedly with that previously reported for the two galactose‐specific Ricinus communis lectins. The recognition of different epitopes of the same basic structure underlies the differences in the oligosaccharide‐binding specificities of galectin‐1 and the R. communis lectins.
Modification at C2 of myo‐inositol 1,4,5‐trisphosphate produces inositol trisphosphates and tetrakisphosphates with potent biological activities
Authors: WILCOX Robert A. , SAFRANY Stephen T. , LAMPE Dethard , MILLS Stephen J. , NAHORSKI Stefan R. , POTTER Barry V. L.
Published: March 2005

Novel 2‐position‐modified d‐myo‐inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3] analogues, dl‐2‐deoxy‐2‐fluoro‐myo‐inositol 1,4,5‐trisphosphate [dl‐2F‐Ins(1,4,5)P3], dl‐myo‐inositol 1,2,4,5‐tetrakisphosphate [dl‐Ins(1,2,4,5)P4], dl‐scyllo‐inositol 1,2,4‐trisphosphate [dl‐sc‐Ins(1,2,4)P3], scyllo‐inositol 1,2,4,5‐tetrakisphosphate [sc‐Ins(1,2,4,5)P4] and scyllo‐inositol 1,2,4,5‐tetrakisphos‐phorothioate [sc‐Ins(1,2,4,5)PS4] were investigated for their ability to bind to the Ins(1,4,5)P3 receptor, mobilise intracellular Ca2+ stores and interact with metabolic enzymes. With the exception of sc‐Ins(1,2,4,5)PS4, all the Ins(1,4,5)P3 analogues potently displaced [3H]Ins(1,4,5)P3 from its receptor in bovine adrenal cortex and were apparently potent full agonists at the Ca2+ mobilising Ins(1,4,5)P3 receptor of SH‐SY5Y cells, giving respective IC50 and EC50 values of: sc‐Ins(1,2,4,5)P4 (IC50 14 nM, EC50 77 nM), dl‐2F‐Ins(1,4,5)P3 (IC50 25 nM, EC50 105 nM), dl‐Ins(1,2,4,5)P4 (IC50 26 nM, EC50 163 nM), dl‐sc‐Ins(1,2,4)P3 (IC50 52 nM, EC50 171 nM), compared to Ins(1,4,5)P3 (IC50 4 nM, EC50 52 nM). sc‐Ins(1,2,4,5)P4 was equipotent to Ins(1,4,5)P3 for Ca2+ release making it the most potent inositol tetrakisphosphate and indeed Ins(1,4,5)P3 analogue yet characterised. In contrast, although sc‐Ins(1,2,4,5)PS4 (IC50 425 nM, EC50 1603 nM) was a significantly weaker ligand and agonist than Ins(1,4,5)P3, it was a partial agonist of high intrinsic activity with maximally effective concentrations releasing only about 80% of Ins(1,4,5)P3‐sensitive Ca2+ stores of SH‐SY5Y cells. Ins(1,4,5)P3 and sc‐Ins(1,2,4,5)P4 were readily metabolised by Ins(1,4,5)P3 3‐kinase and 5‐phosphatase activities, dl‐2F‐Ins(1,4,5)P3 and dl‐sc‐Ins(1,2,4)P3 were resistant to 5‐phosphatase, while sc‐Ins(1,2,4,5)PS4 and dl‐Ins(1,2,4,5)P4 were resistant to both 3‐kinase and 5‐phosphatase activity and were potent inhibitors of the 5‐phosphatase enzyme (Ki= 300 nM and 2.9 μM, respectively). These results demonstrate that modification of the 2‐position of Ins(1,4,5)P3, even with an anionic group, does not critically affect Ins(1,4,5)P3 receptor binding interaction or Ca2+ release, suggesting that the 2‐OH of Ins(1,4,5)P3 fails to interact significantly with the binding site of its receptor. However, modification remote from the crucial vicinal 4,5‐bisphosphate can affect analogue efficacy in Ca2+ release.
Rat platelets contain glycosylated and non‐glycosylated forms of platelet factor 4
Authors: RAVANAT Catherine , GACHET Christian , HERBERT Jean‐Marc , SCHUHLER Simone , GUILLEMOT Jean‐Claude , UZABIAGA Françoise , PICARD Claudine , FERRARA Pascual , FREUND Monique , CAZENAVE Jean‐Pierre
Published: March 2005

Platelet factor 4 is a heparin‐binding protein released from the α granules of activated platelets. This study describes the purification and identification of two forms of rat platelet factor 4, the previously characterized non‐glycosylated form of 7 kDa and an additional glycosylated form of molecular mass 9 kDa. The two proteins both neutralized the antithrombin‐III‐dependent inhibitory activity of heparin. Although their amino acid composition was found to be the same, in the N‐terminal sequence of the 9‐kDa protein, the second threonine residue could not be detected and a difference of 976Da was determined by mass spectrometry. After digestion with O‐glycanase and sialidase, the two proteins showed the same molecular mass. Overall consideration of these data led to identification of the higher‐molecular‐mass protein as a glycosylated form of rat platelet factor 4 with O‐glycosylation at the second N‐terminal amino acid, while the structure of the oligosaccharide core was established by mass spectrometry and sugar differentiation with lectins. The two forms of platelet factor 4 are both present in platelets and secreted after platelet activation.
Isolation, structural determination, and calcium‐binding properties of the major glycoprotein present in Bufo japonicus japonicus egg jelly
Authors: SHIMODA Yasushi , KITAJIMA Ken , INOUE Sadako , INOUE Yasuo
Published: March 2005

Although the previous studies showed that the jelly coat is essential in anuran fertilization under natural conditions, identification and structural studies of the macromolecules that play functional roles have remained to be elucidated. In the present study we isolated acidic glycoproteins (JGP) from the solubilized egg jelly of Bufo japonicus japonicus, and showed that they were the major non‐dialyzable macromolecular components of the jelly coat. JGP was a typical mucin‐type glycoprotein, and it showed high degree of polydispersity in molecular masses ranging over 100–4000 kDa, but both amino acid and carbohydrate compositions were practically identical among fractions, suggesting that JGP was composed of a repeating glycoprotein unit. Four types of short O‐glycan chains were isolated from JGP by reductive β‐elimination and their structures were determined as: Galβ1→3[NeuAcα2→6]GalNAcol (=N‐acetylgalactosaminitol), Fucα1→2Galβ1→3 [NeuAcα2→6]GalNAcol, Fucα1→2Galβ1→3[GlcNAcβ1→6]GalNAcol, and Fucα1→2Galβ1→3‐GalNAcol. These carbohydrate units (about 80% of the mass of JGP) were linked to nearly all the serine and threonine residues which accounted for 55% of total amino acid residues. The Ca2+‐binding property of JGP was studied by equilibrium dialysis. The high Ca2+‐binding capacity of JGP was abolished by its desialylation of JGP and was highly dependent on the JGP concentration. When the low JGP concentrations as in the hydrated Bufo jelly were used, a 50% increment of both n (the number of binding sites) and Kd (the dissociation constant of JGP‐Ca2+) values was observed. This property of JGP is suited to retaining Ca2+ and keeping its concentration at that just necessary for fertilizing sperm.
The effect of denervation on myosin isoform synthesis in rabbit slow‐type and fast‐type muscles during terminal differentiation
Authors: d'ALBIS Anne , GOUBEL Francis , COUTEAUX René , JANMOT Chantal , MIRA Jean‐Claude
Published: March 2005

The soleus and gastrocnemius medialis of eight‐day‐old rabbits were denervated and the effects were examined after fifty‐two days by biochemical, cytochemical and mechanical methods. The contralateral soleus exhibited the properties of slow‐type muscle, namely a predominance of slow‐type myosin isoforms and slow‐type oxidative fibers, slow twitch and low maximal velocity for shortening. The contralateral gastrocnemius exhibited the properties of fast‐type muscle, namely a predominance of fast‐type myosin isoforms and fast‐type non‐oxidative fibers, fast twitch and high maximal velocity of shortening. Denervation of muscles caused the differentiation of the two muscles towards slow‐type muscles. Both denervated soleus and gastrocnemius muscles exhibited a predominance of slow‐type myosins (either the normal type, made up of slow heavy and light chains, or the hybrid type, made up of slow heavy and regulatory light chains and fast essential light chains), a predominance of slow‐type fibers, and slow mechanical properties. Thus, innervation in rabbit appears to be a determining factor for differentiation into fast‐type muscle, but it is not necessary for differentiation into slow‐type muscle. This conclusion contradicts the findings of previous studies in rat and thus raises new questions concerning the role of nerves in controlling the expression of myosin isoforms and the differentiation of muscle fibers.
Biosynthesis and half‐life of the interleukin‐6 receptor and its signal transducer gp130
Authors: GERHARTZ Claudia , DITTRICH Elke , STOYAN Tanja , ROSE‐JOHN Stefan , YASUKAWA Kiyoshi , HEINRICH Peter C. , GRAEVE Lutz
Published: March 2005

Interleukin‐6 (IL‐6) exerts its action via a receptor complex composed of a ligand‐binding sub‐unit (gp80) and a signal transducer (gp130) which both belong to the hematopoietic receptor super‐family. Very little is known about the biosynthesis and the biological half‐lives of proteins of this superfamily. Therefore, we studied the biosynthesis and maturation of the interleukin‐6 receptor and its signaling subunit gp130 by pulse chase experiments in stably transfected Madin‐Darby canine kidney cells. We found that both proteins are synthesized as precursors with apparent molecular masses of 67 kDa and 130 kDa, respectively. These receptor forms are processed within 45–60 min into mature proteins of 82 kDa and 150 kDa containing complex‐type oligosaccharides. The signal transducer gp130 shows a similar maturation in human hepatoma cells HepG2. The IL‐6 receptor appears at the cell surface 45 min after completion of its synthesis in the endoplasmic reticulum. In both cell types studied, gp80 and gp130 are rapidly turned over with half‐lives of 2–3 h. These half‐lives were unaffected by the presence of the ligand IL‐6.
NMR spectroscopy of exchangeable protons of glucoamylase and of complexes with inhibitors in the 9–15‐ppm range
Authors: FIRSOV Leonid M. , NEUSTROEV Kirill N. , ALESHIN Alexander E. , METZLER Carol M. , METZLER David E. , SCOTT Robert D. , STOFFER Bjarne , CHRISTENSEN Trine , SVENSSON Birte
Published: March 2005

1H‐NMR spectra have been recorded for glucoamylases I and II from Aspergillus awamori var. X100 and from A. niger in the 9–15‐ppm reion. At least 17 distinct peaks, many of them arising from single protons, are observed. These are designated A–Q, A being the furthest downfield. At least 9 of these are lost rapidly by exchange when the enzyme is placed in D2O. Peaks A, B, E and H undergo distinct shifts with pH change in the pH region 3–7. Several others undergo smaller shifts. Small differences are also seen between the enzymes from the two different sources. Binding of the pseudotetrasaccharide inhibitor acarbose leads to a 0.50‐ppm downfield shift of peak B, other smaller changes, and retention of two additional protons in D2O. δ‐d‐Gluconolactone induces shifts in peaks E, H, and L. The slow substrate maltitol causes peak A to broaden and shift, peaks J and K to shift and a new or greatly shifted resonance to appear at 15.4 ppm. It disappears as the maltitol is hydrolyzed.

Treatment with iodoacetamide or diethyl pyrocarbonate leads to disappearance of peak D at 12.3 ppm. When this peak was irradiated strong nuclear Overhauser effects (NOE) were observed at 8.01 ppm and 7.22 ppm, positions expected for the Cε1 and Cδ2 protons of an uncharged imidazole ring. We identify D as arising from the Nε2 proton of His254 which is uncharged except at the lowest pH values. Other NOE and two‐dimensional NOE spectra have provided additional information.

Three mutant forms of the A. niger enzyme, in which tryptophan residues have been replaced by phenylalanine, have been examined. Because of shifts induced by changes in ring current and other environmental effects it is hard to make a direct identification of the resonances from the replaced indole NH protons. However, on the basis of a distinct NOE between peaks E and H we have identified these resonances as arising from the indole NH protons of Trp52 and Trp120. Other possible assignments are considered.

The NMR spectra of the glucoamylases I, which have a starch binding domain of about 104 residues at the carboxyl terminus, show four sharp resonances in the 9.7–10.6‐ppm range that are not present in the glucoamylases II, which lack this domain. These resonances no doubt represent the four indole NH ring protons from Trp543, Trp562, Trp590 and Trp615. Three of these are very sharp suggesting a high mobility of this domain.
The thermostability of natural variants of bacterial plasminogen‐activator staphylokinase
Authors: GASE Ariane , BIRCH‐HIRSCHFELD Eckhard , GÜHRS Karl‐Heinz , HARTMANN Manfred , VETTERMAN Stefan , DAMASCHUN Gregor , DAMASCHUN Hilde , GAST Klaus , MISSELWITZ Rolf , ZIRWER Dietrich , COLLEN Désiré , SCHLOTT Bernhard
Published: March 2005

Three natural variants (wild‐type staphylokinase, [R36G, R43H]staphylokinase, and [G34S, R36G, R43H]staphylokinase) of the bacterial plasminogen‐activator staphylokinase, a 136‐amino‐acid protein secreted by certain Staphylococcus aureus strains, have been characterized. These variants differ at amino acid positions 34, 36 and 43 only, and have a very similar plasminogen‐activating capacity and conformation in solution, as revealed by fluorescence spectroscopy, dynamic light scattering and circular dichroism. However, the thermostability of these variants is significantly different.

At 70°C and 0.5 mg protein/ml, irreversible inactivation occurred with apparent half‐life (t1/2) values 0.54 ± 0.13, 0.81 ± 0.20 and 3.7 ± 0.7 h (mean ± SEM) for wild‐type staphylokinase, [R36G, R43H]staphylokinase, and [G34S, R36G, R43H]staphylokinase, respectively, with corresponding values at 0.08 mg/ml of 5.3 ± 1.4 h and 11 ± 2.0 h for wild‐type staphylokinase and [R36G, R43H]staphylokinase, respectively. Dynamic light‐scattering measurements indicated that inactivation was associated with protein aggregation, which precluded accurate determination of transition temperatures and enthalpies of unfolding. 0.08–0.34 mg/ml [G34S, R36G, R43H]staphylokinase, however, did not aggregate at 70°C but underwent unfolding as revealed by a 20% increase in the Stokes’ radius and a 30% decrease in circular dichroism. The unfolding was reversible upon cooling and was associated with full recovery of functional activity.

Thus, these natural variants of staphylokinase have a different sensitivity to thermal inactivation, that is mediated by reversible unfolding of the protein and concentration‐dependent irreversible aggregation. [G34S, R36G, R43H]staphylokinase, the most resistant natural variant, has a stability approaching the minimal requirements for pasteurization, which would facilitate its development for clinical use.
The interaction of DNA with the DNA‐binding domain encoded by the Drosophila gene fork head
Authors: KAUFMANN Eckhard , HOCH Michael , JÄCKLE Herbert
Published: March 2005

The Drosophila gene fork head (fkh) encodes a nuclear protein which shares sequence similarity with the rat hepatocyte‐enriched transcription factor family HNF3α–γ. The sequence similarity is restricted to the region that has been defined as the DNA‐binding domain of these proteins, termed the fork head domain. In this study, we investigate the structural properties of the fork head domain of the prototype of this protein family encoded by fkh and its interaction with DNA. The core sequence required for DNA binding of the fork head domain consists of 114 amino acids and represents a stable and highly compact monomer of globular structure with an α‐helix content of 37%. The fork head domain binds specifically to the DNA target sequence of HNF3α–γ and to an enhancer element that is derived from a regulatory sequence of an in vivo Drosophila target gene. The specific interaction between the DNA‐binding domain of the fkh‐encoded protein and its target DNA is mediated by two contact regions which are separated from each other by one turn of the DNA. Our data are consistent with a structural model which derived rom X‐ray diffraction analysis of the DNA‐binding domain of HNF3γ. Differences concerning the DNA contact sites between the DNA‐binding domain of the fkh‐encoded protein and the HNF3 protein family are discussed.
Molecular cloning of low‐Ca2+‐sensitive‐type non‐muscle α‐actinin
Authors: IMAMURA Michihiro , SAKURAI Takeshi , OGAWA Yoshikatsu , ISHIKAWA Takeshi , GOTO Katsutoshi , MASAKI Tomoh
Published: March 2005

We previously reported the purification and characterization of a novel non‐muscle α‐actinin from chicken lung [Imamura, M. & Masaki, T. (1992) J. Biol. Chem. 267, 25927–25933]. The Ca2+ sensitivity of the lung α‐actinin for the interaction with polymerized actin (F‐actin) was much lower than those of the other reported non‐muscle α‐actinins. Here, we isolated a cDNA clone encoding the novel α‐actinin by screening a chicken lung γgt11 cDNA library with antibody specific for the low‐Ca2+‐sensitive α‐actinin. The deduced amino acid sequence of the lung α‐actinin showed 76%, 82% and 83% identity to those of chicken skeletal‐muscle, smooth‐muscle and fibroblast‐type α‐actinin, respectively. Marked difference in the structure between the lung‐type and the other α‐actinins was found in the extreme NH2‐terminal and in the COOH‐terminal half; in the third and fourth regions of four spectrin‐like repeats, and in two Ca2+‐binding EF‐hand consensus regions. The NH2‐terminal‐side EF‐hand contained a notable defect in one of the five oxygen‐containing amino acid side chains involved in chelating Ca2+, suggesting that the lower Ca2+ sensitivity of the lung α‐actinin is ascribable to this defect. Northern blot analysis showed that the expression pattern of lung‐type α‐actinin mRNA in various non‐muscle tissues differed from that of the other known non‐muscle‐type (fibroblast‐type) α‐actinin. The present results clearly demonstrate the existence of two structurally and functionally different types of non‐muscle α‐actinin; high‐Ca2+‐sensitive‐type (NM1) and low‐Ca2+‐sensitive‐type (NM2) α‐actinin.
Induction of metallothionein and stomatin by interleukin‐6 and glucocorticoids in a human amniotic cell line
Authors: SNYERS Luc , CONTENT Jean
Published: March 2005

Interleukin 6 (IL‐6) is an important mediator of various kinds of inflammatory and immune responses. The human amniotic cell line UAC has an increased number of IL‐6 receptors after treatment by glucocorticoids. To find a possible activity of IL‐6 on these cells, a cDNA library of IL‐6‐ and dexamethasone‐treated cells was screened with cDNA probes from both induced and non‐induced cells.

Two cDNAs showed a differential hybridization signal. The first one corresponds to metallothionein, a group of small cysteine‐rich proteins thought to participate in the metabolism and storage of zinc and to protect cells against oxidative damage.

A second cDNA corresponds to the recently cloned cDNA of band 7 integral membrane protein also called stomatin. In hereditary stomatocytosis, absence of this protein in erythrocyte membranes is associated with high Na+ and low K+ intracellular concentrations [Stewart, G. W., Hepworth‐Jones, B. E., Keen, J. N., Dash, B. C. J., Argent, A. C. & Casimir, C. M. (1992) Blood 79, 1593–1601].

In UAC cells both metallothionein and stomatin are induced by dexamethasone and IL‐6 in a more than additive manner. Western blot analysis shows that stomatin protein is induced in a similar way as its mRNA.

IL‐6 and dexamethasone induce a state of resistance against hydrogen peroxide toxicity in UAC cells. Metallothionein induction might be partly responsible for this cytoprotection against oxidative stress.
Mechanism of resistance to the antibiotic trichothecin in the producing fungi
Authors: IGLESIAS Maite , BALLESTA Juan P. G.
Published: March 2005

Trichothecium roseum, an imperfecti fungus producer of the translation inhibitor trichothecin, is constitutively resistant to its product. Fusarium oxysporum, a fungi not described as a toxin producer, is sensitive to trichothecin but becomes resistant when grown in the presence of the drug. In both cases, the resistance occurs at the level of the ribosomes. In cell‐free polypeptide polymerization systems, trichothecin resistance is associated with the presence of 60S subunits from the resistant organisms. Resistant ribosomes can be prepared in vitro by incubating sensitive ribosomes, from either non‐induced F. oxysporum or Saccharomyces cerevisiae, with cell extracts from the resistant cells in the presence of S‐adenosylmethionine. An in‐vitro specific differential methylation is detected in the sensitive ribosomes but not in resistant particles using radioactive S‐adenosylmethionine.

The results indicate for the first time the existence in eukaryotic organims of an antibiotic‐resistance mechanism involving a ribosomal methylation similar to that described previously in prokaryotic systems.
Structure requirements for disulfide bridge sulfitolysis of oxidized Escherichia coli thioredoxin studied by fluorescence spectroscopy
Authors: HÄBERLEIN Ingo
Published: March 2005

Sulfitolysis of wild‐type and four mutated Escherichia coli thioredoxins ([D26A]thioredoxin, [P34H]thioredoxin, [K36E]thioredoxin and endo‐Arg33a‐thioredoxin) has been investigated at millimolar concentrations of sulfite in the absence of protein‐denaturing agents by fluorescence spectroscopy. Sulfitolysis of the single disulfide bridge of these proteins is associated with an increase in fluorescence emissions at 345 nm. Evaluation of the fluorescence emission spectra revealed that sulfitolysis of thioredoxins is a homogenous process. The reactivities of the thioredoxins are determined by negatively charged (Asp26) or positively charged (Lys36) amino acid residues near the active site disulfide bridge.
Role of a distal promoter element in the S‐phase control of the human H1.2 histone gene transcription
Authors: EILERS Andreas , BOUTERFA Hakim , TRIEBE Suzane , DOENECKE Detlef
Published: March 2005

The expression of one of the human main type H1 histone genes (termed H1.2) appears to be regulated by several trans‐acting factors. Upstream of consensus regulatory regions, such as the TATA‐, CCAAT‐ and H1‐box (AAACACA) sequences, a crucial control site is located between nucleotide positions −536 and −412 (relative to the ATG initiation site). Removal of this promoter portion causes in chloramphenicol acetyl transferase reporter gene constructs a loss of the S‐phase control function of the H1.2 promoter in HeLa cells. Electrophoretic mobility‐shift assay and DNase I footprinting analysis suggest that the H1‐box variant AAACAGA is a potential control element within the distal promoter region.
Expression of novel 400‐kDa laminin chains by mouse and bovine endothelial cells
Authors: SOROKIN Lydia , GIRG Winfred , GÖPFERT Thea , HALLMANN Rupert , DEUTZMANN Rainer
Published: March 2005

Laminin expression was studied in endothelial cells derived from different mouse tissues and primary cultures of bovine aortic endothelium (BAEC). Immunoprecipitation with polyclonal antilaminin‐1 revealed two 200‐kDa chains, a novel 400‐kDa chain and nidogen in all cells studied. Two‐dimensional electrophoresis of laminin complexes immunoprecipitated from endothelial‐cell‐conditioned medium demonstrated that the 400‐kDa chain was disulphide‐linked to the laminin β1 and γ1 chains. In addition, rotary shadowing illustrated the secretion of molecules from the endothelial cells which were morphologically similar to laminin‐1. Immunoblotting and Northern blot analysis confirmed the presence of β1 and γ1 polypeptides and the corresponding LamB1 and LamC1 mRNAs in all cells investigated. However, both polyclonal anti‐laminin‐1 and α1‐specific monoclonal antibodies failed to react with the 400‐kDa polypeptide in immunoblots and immunoprecipitations. Similarly, the expression of the gene coding for α1, LamA1, was not detected in the majority of cells by Northern blot analysis using three different cDNAs. Only in two cases, BAEC and a Simian‐virus‐40‐transformed mouse endothelial cell line (SVEC), a 10‐kb mRNA was detected by Northern analysis using a cDNA specific for the 3′ coding region (LAC) of LamA1 mRNA. However, cDNAs specific for the central and 5′ coding region of LamA1 mRNA did not show any reaction. Hybridization of LAC to BAEC and SVEC mRNA is consistant with the laminin‐like nature of the 400‐kDa chain expressed by these cells. The results demonstrate the existence of at least one and possibly two novel 400‐kDa laminin chains which complex with β1 and γ1 and occur in mouse and bovine endothelial cell basement membranes.
Molecular cloning, sequence analysis and differential expression of an intron‐containing gene encoding tomato histone H1
Authors: JAYAWARDENE Nishan , RIGGS C. Daniel
Published: March 2005

A tomato genomic clone has been identified which encodes histone H1. The deduced polypeptide is 287 amino acids in length, and exhibits the tripartite organization typical of histones H1. The central globular domain is highly similar to those regions from other H1 molecules, and the carboxyl‐terminal domain contains a repeating hexapeptide motif, variants of which are conserved among H1 molecules. RNA gel blotting revealed that histone H1 mRNA is expressed at higher levels in organs which contain meristematic tissue and/or which have a high proportion of actively cycling cells. DNA gel blotting and dot‐blot hybridization studies revealed that histone H1 in tomato is encoded by a small gene family. By employing the polymerase chain reaction on genomic DNA and on cDNA, it was determined that the gene is interrupted by an intron. The location and approximate length of the intron are conserved in both the tomato and Arabidopsis genes, with the intron separating the ‘nose’ region (encoded by exon 1) from the central globular domain (exon 2). The promoter region was found to contain several conserved sequence motifs which likely participate in the regulation of the gene.
Expression of mRNA encoding neurotrophins and neurotrophin receptors in rat thymus, spleen tissue and immunocompetent cells
Authors: LAURENZI Maria Assunta , BARBANY Gisela , TIMMUSK Tõnis , LINDGREN Jan Åke , PERSSON Håkan
Published: March 2005

The expression of neurotrophin and neurotrophin receptor mRNAs was examined using RNase protection assays and Northern‐blot analysis in rat thymus, spleen tissue and immunocompetent mononuclear cells purified from these two organs. Nerve growth factor, brain‐derived neurotrophic factor, neurotrophin‐3 and neurotrophin‐4 mRNAs were all expressed in thymus and spleen tissue although at different levels, while immunocompetent cells expressed neurotrophin‐3 and neurotrophin‐4 mRNAs. Thymus and spleen tissue expressed mRNAs encoding the low‐affinity nervegrowth‐factor receptor, the non‐neuronal TrkA I receptor, the truncated (kinase deficient) and full‐length TrkB, and the TrkC receptor. Low‐affinity nerve‐growth‐factor receptor and non‐neuronal TrkA I mRNAs were detected in both thymus and spleen immunocompetent cells. In addition, thymus cells expressed neuronal TrkA II mRNA and spleen cells expressed truncated TrkB mRNA. The expression of TrkA I and TrkA II mRNAs was enhanced in both thymus and spleen cells after cell culture. Enhanced levels of neurotrophin‐4 mRNA were observed in spleen immunocompetent cells after adrenalectomy. Moreover, the expression of neurotrophin‐4 mRNA was up‐regulated after stimulation of immune cells with the mitogens concanavalin A or lipopolysaccharide or with the inflammatory mediator leukotriene B4. This suggests that neurotrophin‐4 could be secreted by immunocompetent cells and may be involved in inflammatory processes.
Calcium and 2‐oxoglutarate‐mediated control of aspartate formation by rat heart mitochondria
Authors: SCADUTO Russell C.
Published: March 2005

Studies of the influence of calcium on the metabolism of cardiac mitochondria have indicated that calcium activates key enzymes involved in the citric acid cycle. Calcium‐mediated activation of one of these enzymes, 2‐oxoglutarate dehydrogenase, has been shown to cause a marked decrease in the steady‐state concentration of 2‐oxoglutarate in both heart and liver mitochondria. In liver, 2‐oxoglutarate is a potent inhibitor of oxalacetate transamination to aspartate and activation of this enzyme by calcium‐mobilizing hormones leads to a stimulation of aspartate formation and gluconeogenesis. Since mitochondrial aspartate formation is a key step in the malate/aspartate shuttle, we investigated the control of aspartate formation by cardiac mitochondria. In mitochondria incubated with glutamate and malate, activation of 2‐oxoglutarate dehydrogenase by calcium led to an inhibition of aspartate formation. However, calcium caused a stimulation of aspartate production when incubations were supplemented with pyruvate as an additional substrate. Estimates of the mitochondrial redox potential (NADH/NAD+) indicated that both calcium and pyruvate increased the redox potential. The observed influence of calcium on aspartate formation was found to be due to a balance between its inhibitory effect, caused by an increased redox potential, and its stimulatory effect, caused by a decreased 2‐oxoglutarate concentration. Under conditions in which the redox component was held constant, a kinetic analysis indicated that the apparentKifor 2‐oxoglutarate inhibition of aspartate formation is 0.2 mM. The data suggest that activation of cardiac 2‐oxoglutarate dehydrogenase by calcium could lead to stimulation of the mitochondrial oxidation of cytosolic NADH via the malate/aspartate cycle.
Cross‐linking of nucleic acids to proteins
Authors: WOJTECH Eva , BRIMACOMBE Richard , HÄCKEL Andreas , PROCHNOW Detlef , FASOLD Hugo
Published: March 2005

Poly(adenylic acid) was modified by methylchlorotetrolic ester in a reproducible and defined content of the derivatized bases. The nucleic acid derivative is protein reactive and was coupled to 70S ribosomes from Escherichia coli, in order to identify proteins along the mRNA pathway. The binding of the label becomes specific under the direction of tRNALys and is then almost exclusively located on the small subunit. The proteins S1, S12, S18 and S21 were labeled, as shown by an antibody assay. The yield of the affinity label was 5.4%, as calculated from the labeled nucleic acid. This compares favourably with the yields from photolabile compounds.
Sequence determination of three variable surface glycoproteins from Trypanosoma congolense
Authors: RAUSCH Sebastian , SHAYAN Parviz , SALNIKOFF Johann , REINWALD Erwin
Published: March 2005

The full‐length cDNA sequences of three variable surface glycoproteins from bloodstream forms of Trypanosoma congolense have been determined. They encode preproteins of 429, 449, and 428 amino acids. These proteins contain the typical N‐terminal leader sequences of secreted eukaryotic proteins, and display hydrophobic amino acids at their C‐termini characteristic of variable surface glycoproteins; these leader sequences serve as transient membrane anchors after protein synthesis. By performing sequence comparisons of all currently known variable surface glycoproteins from T. congolense, several conserved elements could be identified. These elements included positional conservation of most of the cysteine residues, conservation of the flanking sequences surrounding these cysteine residues, clustering of proline residues near the C‐termini, and a hydrophobic heptad motif near the end of the N‐terminal domains. The N‐terminal domains seem to be closely related to the B domains of Trypanosoma brucei variable surface glycoproteins, whereas the C domains have up to now only been identified in T. congolense variable surface glycoproteins. The data suggest that T. congolense variable surface glycoproteins, despite low sequence similarities, could have conserved tertiary structures.