bluuebunny/crossref_metadata_2025 huggingface dataset:
Kinetics of the Reaction between Human C1‐Esterase Inhibitor and C1r or C1s
Authors: NILSSON Torbjörn , WIMAN Björn
Published: July 2010

The kinetics of the reactions of C‐esterase inhibitor with Cs or Cr has been studied in purified systems. Both reactions were found to proceed in two steps: a fast reversible second‐order reaction followed by a slower irreversible first‐order transition according to the following scheme: . The rate constant k1 for the reaction between Cs and C‐esterase inhibitor was found to be 1.4 × 105 M−1 s−1 at 25°C and 4.7 × 105 M−1 s−1 at 37°C, while the corresponding values for the reaction between Cr and C‐esterase inhibitor were 2.5 × 103 M−1 s−1 and 4.4 × 104 M−1 s−1, respectively. The dissociation constant K of the initial complex was estimated as 1–2 nM for the complex with Cs and 20–50 nM for the complex with Cr. Heparin was found to accelerate the rate of the Cs/C‐esterase inhibitor reaction by a 16‐fold increase in k1 at heparin concentrations above 100 mg/l, whereas no effect was obtained on the thermodynamic equilibrium. The inhibition of purified Cs added to dilutions of plasma or serum was found to proceed as expected from their known concentrations of C‐esterase inhibitor and the rate constants determined in the purified system. Thus, the half‐life of Cs in plasma under pseudo‐first‐order kinetics would be about 1 s
Differential and Synergistic Actions of Somatomedin‐Like Growth Factors, Fibroblast Growth Factor and Epidermal Growth Factor in Rabbit Costal Chondrocytes
Authors: KATO Yukio , HIRAKI Yuji , INOUE Hiroyuki , KINOSHITA Masahiko , YUTANI Yasutaka , SUZUKI Fujio
Published: July 2010

Epidermal growth factor (EGF) stimulated DNA synthesis, RNA synthesis and cell division of rabbit costal chondrocytes in culture, but did not stimulate nor inhibit proteoglycan synthesis. Multiplication‐stimulating activity (MSA) and cartilage‐derived factor (CDF) with somatomedin‐like activity had relatively small effects on DNA synthesis and cell division of chondrocytes, but markedly enhanced the synthesis of sulfated proteoglycans. MSA and EGF had additive or synergistic effects on the growth of rabbit chondrocytes. MSA and fibroblast growth factor (FGF) also had additive or synergistic effects on the growth of rabbit chondrocytes. CDF mimicked these effects of MSA; CDF and MSA had similar stimulatory effects on DNA synthesis in rabbit chondrocytes even in the presence of EGF, FGF or both. MSA and CDF did not reduce the concentrations of EGF and FGF required for maximum stimulation of DNA synthesis. In addition, EGF and FGF had additive effects on DNA synthesis and cell division of rabbit costal chondrocytes in culture. These findings suggest that the somatomedin‐like factors, EGF and FGF have complementary effects in chondrocytes and provide further evidence that CDF produced by chondrocytes resembles somatomedin in its biological action in chondrocytes.
Free N‐Acetylneuraminic Acid in Tissues in Salla Disease and the Enzymes Involved in Its Metabolism
Authors: RENLUND Martin , CHESTER M. Alan , LUNDBLAD Arne , PARKKINEN Jaakko , KRUSIUS Tom
Published: July 2010

Salla disease is a lysosomal storage disorder of unknown etiology, characterized biochemically by increased urinary excretion of N‐acetylneuraminic acid. This compounds has now been shown to occur in abnormally large amounts in liver and cultured skin fibroblasts from these patients. Quantification of N‐acetylneuraminic acid was performed using a new gas‐chromatography/mass spectrometric single‐ion method which is sensitive and specific. No abrormalities in the activity of several enzymes involved in sialic acid metabolism (N‐acetylneuraminate:pyruvate lyase, neuraminidase, CMP‐N‐acetylneuraminate N‐acylneuraminohydrolase and CTP:N‐acylneuraminate cytidylyltransferase) were demonstrable. A possible explanation for the defect is a malfunctioning active transport of N‐acetylneuraminic acid across the lysosomal membrane.
Interaction of β1‐Anticollagenase from Human Plasma with Collagenases from Various Tissues and Competition with α2‐Macroglobulin
Authors: MACARTNEY Henry W. , TSCHESCHE Harald
Published: July 2010

β1‐Anticollagenase from human plasma forms an inactive enzyme‐inhibitor complex with active human polymorphonuclear leukocyte collagenase [Macartney, H. W. and Tschesche, H. (1983) Eur. J. Biochem. 130, 85–92]. The inhibitor was shown to contain a reactive sulfhydryl group necessary for inhibition of the collagenase. Inhibition of the enzyme by β1‐anticollagenase was shown to take place via a thiol/disulfide interchange reaction similar to the mechanism reported for the reaction between polymorphonuclear leukocyte collagenase and the polymorphonuclear collagenase inhibitor [Macartney, H. W. and Tschesche, H. (1980) FEBS Lett. 119, 327–332].

The observations made in this paper have shown that the collagenases obtained from other human tissues, such as rheumatoid synovial fluid, embryonic skin fibroblasts, diabetic skin fibroblasts and normal skin fibroblasts, all react with β1‐anticollagenase via a thiol/disulfide interchange mechanism, and are inhibited in a 1:1 stoichiometric reaction.

Reaction of the inhibitor with the collagenases can be blocked by alkylation of the inhibitor's free sulfhydryl group with iodoacetamide, or by reaction of the inhibitor with disulfide‐containing compounds such as cystine, oxidised glutathione, insulin, relaxin or trypsinogen.

The β1‐anticollagenase–collagenase complexes (latent enzyme) can all be reactivated with disulfide‐containing compounds, such as those mentioned above. An activation process has been reported for human latent polymorphonuclear leukocyte collagenase via a thiol/disulfide interchange as catalysed by the glutathione cycle in a peroxidase‐coupled reaction to glucose metabolism [Tschesche, H. and Macartney, H. W. (1981) Eur. J. Biochem. 120, 183–190]. The same physiological activation process is capable of reactivating β1‐anticollagenase–collagenase complexes in a reversible manner.

It was further shown in competition experiments with β1‐anticollagenase (and with human leukocyte collagenase inhibitor) and α2‐macroglobulin that vertebrate collagenases were preferentially inhibited by the low‐molecular‐weight inhibitors. It could also be shown that there was no transfer of the enzyme to α2‐macroglobulin from either β1‐anticollagenase–collagenase, or leukocyte inhibitor–collagenase complexes indicating a true inhibitory function for the β1‐anticollagenase and the leukocyte collagenase inhibitor.
Natural‐Abundance 13C‐NMR Spectroscopy of Two Glyco‐asparagines Derived from the Core of N‐Glycosidic Carbohydrate Chains
Authors: DIJKSTRA Bauke W. , VLIEGENTHART Johannes F. G. , STRECKER Gérard , MONTREUIL Jean
Published: July 2010

13C‐NMR spectroscopic studies were carried out on Manα(1→6)Manβ(1→4)GlcNAcβ(1→4)GlcNAcβ(1→N)‐Asn and Manα(1→6)Manβ(1→4)GlcNAcβ(1→4)[Fucα(1→6)]GlcNAcβ(1→N)Asn, which are part of the invariant core of N‐glycosidically linked carbohydrates of glycoproteins. All resonances of proton‐bearing carbon atoms were unambiguously assigned. Where possible, the assignments were confirmed by selective proton‐decoupling experiments.
Elicitor Induction of mRNA Activity
Authors: LAWTON Michael A. , DIXON Richard A. , HAHLBROCK Klaus , LAMB Chris J.
Published: July 2010

Changes in the activity levels of mRNAs encoding phenylalanine ammonia‐lyase and chalcone synthase, two characteristic enzymes of phenylpropanoid biosynthesis, in elicitor‐treated cells of dwarf French bean (Phaseolus vulgaris L.) have been investigated by immunoprecipitation of [35S]methionine‐labelled enzyme subunits synthesised in vitro in an mRNA‐dependent rabbit reticulocyte lysate translation system.

Elicitor heat‐released from cell walls of Colletotrichum lindemuthianum, the causal agent of anthracnose disease of bean, causes marked rapid increases in the polysomal activities of the mRNAs encoding the two enzymes concomitant with the phase of rapid increase in enzyme activity at the onset of phaseollin accumulation during the phytoalexin defence response.

Increased polysomal mRNA activities encoding the two enzymes can be observed 30 min after elicitor treatment. The patterns of induction of the mRNA activities are broadly similar with respect to time and elicitor concentration although small but distinct differences between the enzymes were observed in the elicitor concentration giving maximum induction. There is a close correlation between the induction of polysomal mRNA activity and the induction of enzyme synthesis in vivo by elicitor treatment with respect to both the kinetics of induction and the dependence on elicitor concentration. The data indicate that elicitor stimulation of phenylalanine ammonia‐lyase and chalcone synthase synthesis in vivo is largely a result of increased polysomal activity of the mRNAs encoding these enzymes. Similar patterns of induction of polysomal mRNA activity are observed with elicitor preparations from a variety of sources.

The marked increases in polysomal mRNA activities encoding phenylalanine ammonia‐lyase and chalcone synthase are increases as a proportion of total cellular mRNA activity, indicating that elicitor does not increase these polysomal mRNA activities by stimulation of selective recruitment from the total pool of cellular mRNA.
On the Relationship between Calcium and Phosphate Transport, Transmembrane Potential and Acetoacetate‐Induced Oxidation of Pyridine Nucleotides in Rat‐Liver Mitochondria
Authors: SILIPRANDI Dagmar , SILIPRANDI Noris , TONINELLO Antonio
Published: July 2010

Acetoacetate addition to rat liver mitochondria induces a complete oxidation of pyridine nucleotides, a collapse of membrane potential, a release of mitochondrial Ca2+ and a loss of respiratory control only in the presence of external phosphate. Acetoacetate also enhances the efflux of mitochondrial Mg2+ promoted by phosphate. All these effects are not only prevented but also reversed, except the oxidation of pyridine nucleotides, by the combined addition of Mg2+, ADP and dithioerythritol to damaged mitochondria. It is concluded that acetoacetate, through the oxidation of mitochondrial pyridine nucleotides, potentiates the action of phosphate in altering the mitochondrial permeability barrier, which is closely dependent on the maintenance of membrane thiol groups in a reduced form.
Inactivation of 3‐Oxoacyl Synthetase Activity of Pigeon Liver Fatty Acid Synthetase by S‐(4‐Bromo‐2,3‐dioxobutyl)‐coenzyme A
Authors: KATIYAR Sarvagya S. , PAN David , PORTER John W.
Published: July 2010

Pigeon liver fatty acid synthetase was inactivated by treatment with S‐(4‐bromo‐2,3‐dioxobutyl)‐CoA. This inactivation is fast, irreversible and dependent on both time and inhibitor concentration. It is also specific and selective as only the 3‐oxoacyl synthetase and acetyl‐CoA transacylase component activities, out of the seven component activities possessed by the fatty acid synthetase complex, were inhibited. The binding of bromo‐dioxobutyl‐CoA to the enzyme is covalent as evidenced by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The modified enzyme showed characteristic absorption peaks at 266 nm and 315 nm that are identical to the absorption maxima of pure inhibitor. All the available evidence suggests that the mechanism of inhibition of fatty acid synthetase by S‐(4‐bromo‐2,3‐dioxobutyl)‐CoA involved its binding to critical thiols, namely the cysteine — SH of the condensing enzyme and cysteamine — SH of the 4′‐phosphopantetheine prosthetic group. Both subsrates, acetyl‐CoA and malonyl‐CoA, also protected the enzyme against inhibition by S‐(4‐bromo‐2,3‐dioxobutyl)‐CoA. Comparison of high‐voltage electrophoretic patterns of peptic peptides obtained from [1‐14C]acetyl‐labeled native and S‐(4‐bromo‐2,3‐dioxobutyl)‐CoA‐inactivated fatty acid synthetase confirmed the involvement of both cysteine and 4′‐phosphopantetheine — SH groups in the binding with S‐(4‐bromo‐2,3‐dioxobutyl)‐CoA. Scatchard analysis of the results of binding studies has established that 4 mol of the inhibitor are bound/mol of the enzyme complex, which indicates the presence of two moles condensing activities and two moles 4′‐phosphopantetheine prosthetic groups/mole of enzyme. Our data do not support a half‐site reactivity for the fatty acid synthetase as proposed earlier [Clements et al. (1979) Biochem. Biophys. Res. Commun. 86, 278–284].
The Multifunctional Polypeptide Chains of Rabbit‐Mammary Fatty‐Acid Synthase
Authors: McCARTHY Alun D. , HARDIE D. Grahame
Published: July 2010

Several methods have been used to label active centres on the multifunctional polypeptide chains of rabbit mammary fatty acid synthase. Experiments using [14C]acetyl‐CoA or [14C]malonyl‐CoA have shown that there is a single non‐thiol site which binds either acetyl or malonyl groups, present at a stoichiometry of two per enzyme dimer, and representing an intermediate in the acyl transferase reaction. This adds further support to the view that the two subunits are identical and that each polypeptide chain contains up to seven active centres. However, two novel and independent methods for the quantification of the pantetheine thiol demonstrate that this prosthetic group can be present in sub‐stoichiometric amounts. By studying intermediates during limited elastase digestion of fatty acid synthase labelled in different active centres, we have been able to map the positions of four active centres within the polypeptide chain. The thioesterase domain is present in a terminal location on both polypeptide chains as previously reported. The acyl carrier domain (pantetheine thiol) is located in a region of molecular weight 9000 immediately adjacent to the thioesterase domain. The acyl transferase (acyl‐O‐ester site) and the 3‐oxoacylsynthase thiol are located in a region of molecular weight 110000 at the opposite end of the polypeptide chain to the thioesterase domain. The relationship between the disposition of the activities on the multifunctional polypeptide chains of yeast and mammalian fatty acid synthase is discussed.
Translational Stability of Plant Viral RNAs Microinjected into Living Cells
Authors: HUEZ Georges , CLEUTER Yvette , BRUCK Claudine , VAN VLOTEN‐DOTING Lous , GOLDBACH Rob , VERDUIN Benedictus
Published: July 2010

Three different alternative structural features have been shown to be present at the 3′ terminus of plant viral RNAs: (a) a poly(A) track, (b) a tRNA‐like structure, (c) no special structural or sequence characteristic. We have compared the translational stability after injection into frog oocytes of a representative of each type: (a) the small genomic RNA (M‐RNA) of cowpea mosaic virus (CPMV), (b) the subgenomic mRNA for coat protein (RNA 4) of brome mosaic virus (BMV), (c) the subgenomic mRNA for coat protein (RNA 4) of alfalfa mosaic virus (AIMV).

It has been shown that CPMV M‐RNA exhibits the highest translational stability. However, the stability of AIMV RNA 4 is remarkably high and moreover significantly higher than that of BMV RNA 4.

We demonstrate that, for all three viral RNA species considered, the presence of a poly(A) segment at the 3′ end of the molecules improves the translational stability. From a comparative investigation in which AIMV RNA 4 was also injected into HeLa cells, it is concluded that the stability of a given non‐adenylylated mRNA depends on the nature of the cytoplastic environment.
Unmetabolized 5,6‐Dichlororibofuranosylbenzimidazole Rather than Its Monophosphate Metabolites Is Probably the Active Transcription Inhibitor
Authors: EGYHÁZI Endre , HOLST Mikael , TAYIP Umit
Published: July 2010

The question of whether or not the monophosphate metabolites DRB2′P and DRB5′P of the transcription inhibitor 5,6‐dichloro‐1‐β‐d‐ribofuranosylbenzimidazole (DRB) constitute the active form of the inhibitor was investigated in intact salivary gland cells of Chironomus tentants. The intracellular phosphorylation of DRB to DRB2′P as well as to DRB5′P could be lowered to a considerable extent by addition of a 15‐fold excess of adenosine or 3′‐deoxyadenosine without lowering the intracellular concentration of unmetabolized [3H]DRB. Nevertheless, the inhibition of hnRNA synthesis remained essentially unaffected. The addition of excess guanosine interfered only with the formation of 3H‐labelled DRB5′P isomer while uridine was without any detectable effect on the metabolism of DRB to any of the DRB monophosphate isomers. When in another type of experiment with DRB‐inhibited cells the unmetabolized DRB was washed out, whereas the monophosphate isomers, formed before removal of DRB, remained trapped intracellularly, it was found that the inhibitory activity was abolished and the initiation of RNA synthesis was resumed. The experimental data taken together strongly indicate that the DRB metabolites DRB2′P and DRB5′P are not involved in the inhibitory activity, and that it is the unmetabolized compound that acts as active transcription inhibitor.
cDNA cloning, nucleotide sequence and expression of the gene for arylsulfatase in the sea urchin (Hemicentrotus pulcherrimus) embryo
Authors: SASAKI Hiroshi , YAMADA Kazumi , AKASAKA Koji , KAWASAKI Hiroshi , SUZUKI Koichi , SAITO Akiko , SATO Moriyuki , SHIMADA Hiraku
Published: July 2010

Arylsulfatase is known to be synthesized in large amounts at the early gastrula stage of sea urchin development. We determined the amino acid sequence of a portion of the purified sea urchin embryonic arylsulfatase, and then isolated a cDNA clone for arylsulfatase by screening a sea urchin plutei λgt10 cDNA library with an oligodeoxynucleotide probe synthesized according to the determined amino acid sequence. The longest cDNA clones were selected and the nucleotide sequence determined. The cDNA is 2422 nucleotides long and encodes 551 amino acids. The deduced amino acid sequence has no sequence similarity with any of the peptides registered in NBRF peptide databank. Northern blot analysis revealed that the arylsulfatase cDNA hybridizes to a 2.9‐kb mRNA. This mRNA exists in the unfertilized egg in small amounts, but markedly increases after the blastula stage preceding the increase of the arylsulfatase activity.
Gallidermin: a new lanthionine‐containing polypeptide antibiotic
Authors: KELLNER Roland , JUNG Günther , HÖRNER Thomas , ZÄHNER Hans , SCHNELL Norbert , ENTIAN Karl‐Dieter , GÖTZ Friedrich
Published: July 2010

Gallidermin is a new member of the class of lanthionine‐containing peptide antibiotics, which are summarized under the common name lantibiotics. The lantibiotic gallidermin is produced by Staphylococcus gallinarum (F16/P57) Tü3928, and it exhibits activities against the Propionibacteria, involved in acne disease. Gallidermin differs from the recently discovered tetracyclic 21‐residue peptide antibiotic epidermin only in a Leu/Ile exchange in position 6. The isolation procedures for gallidermin included adsorption directly from the culture broth, ion‐exchange chromatography of the amphiphilic and basic polypeptide followed by desalting, and final purification by reversed‐phase HPLC. The structural elucidation of the polypeptide containing four thioether bridges involved mainly a combination of automated gas‐phase sequencing, thermospray liquid chromatography/mass spectrometry and fast‐atom‐bombardment mass spectrometry.
Anti‐(Arg‐Gly‐Asp‐Ser) antibody and its interaction with fibronectin, fibrinogen and platelets
Authors: CIERNIEWSKI Czeslaw S. , SWIATKOWSKA Maria , PONIATOWSKI Jacek , NIEWIAROWSKA Jolanta
Published: July 2010

An antibody population recognizing the sequence Arg‐Gly‐Asp‐Ser (RGDS) in fibronectin, anti‐(RGDS)N, was isolated by immunoadsorption. Between 2.5% and 4.9% of antibodies were obtained from two different antifibronectin sera indicating that this region represents an antigenic epitope in native fibronectin. Complete inhibition of binding of 125I‐fibronectin to anti‐(RGDS)N was produced only by nonreduced and reduced fibronectin. Fibrinogen and synthetic RGDS tetrapeptide, each at concentration of 10 μM, showed only a slight inhibition of 22% and 17%, respectively. Measurements of the conformational constant, the equilibrium constant for the interconversion of the non‐native and native conformations of this epitope, showed that less than 0.0001% of the RGDS molecules adopt the native conformation in aqueous solutions. It indicates that long‐range interactions in fibronectin and fibrinogen result in different conformations of the RGDS sequence in both proteins. Anti‐(RGDS)N antibodies purified from anti‐fibronectin serum had a strong inhibitory effect on thrombinstimulated platelet aggregation. They also inhibited binding of fibronectin and fibrinogen to thrombin‐stimulated platelets, supporting the primary role of the RGDS sequence in the direct interaction of these proteins with platelet membrane receptors.
Refolding of bacteriorhodopsin
Authors: SIGRIST Hans , WENGER Roland H. , KISLIG Elisabeth , WÜTHRICH Manuel
Published: July 2010

Staphylococcus aureus protease V8 cleaves bacteriorhodopsin to two main fragments, V‐1 and V‐2. Proteolytic digestion of the purple membrane integrated protein is carried out in the presence of limited amounts of sodium dodecyl sulfate (0.5 g detergent/g bacteriorhodopsin). The fragment V‐1 includes the arylisothiocyanate binding site (Lys41). The V‐2 fragment comprises the two C‐terminal transmembrane segments of bacteriorhodopsin. Improved renaturation of bacteriorhodopsin and the ternary complex, reformed from its V8 proteolytic fragments, is attained by peptide extraction in chloroform/methanol/0.1 M ammonium acetate and subsequent incorporation into phospholipid/detergent micelles. In the presence of retinal, V8 fragments reform chromophoric ternary complexes. Light‐adapted reconstituted chromophores absorb incident light at 560 nm. Protein secondary structures are partially conserved in the course of solvent extraction and are restored in the reconstituted system. Vesicles prepared from the reconstituted complexes show light‐dependent proton translocation activity.
Regulation of cyclic GMP binding to retinal rod membranes by calcium
Authors: CARETTA Antonio , CAVAGGIONI Andrea , GRIMALDI Roberta , SORBI Robert T.
Published: July 2010

The apparently cooperative binding of 8‐(5‐thioacetamidofluorescein)‐cGMP (SAF‐cGMP) to cGMP‐binding sites of the rod outer segments is regulated by Ca2+ in the 0.1‐1 μM activity range. High Ca2+ reduces, and low Ca2+ increases the affinity of SAF‐cGMP binding. This regulation involves only intrinsic membrane components. It is proposed that an allosteric regulation of cGMP binding by Ca2+ can contribute to photoreceptor potential adaptation.
Mechanism of galanin‐inhibited insulin release
Authors: AMIRANOFF Brigitte , LORINET Anne‐Marie , LAGNY‐POURMIR Isabelle , LABURTHE Marc
Published: July 2010

In the insulin‐secreting beta cell line Rin m 5F, galanin, a newly discovered ubiquitous neuropeptide, inhibited, by 50%, the stimulation of insulin release induced by gastric inhibitory polypeptide (GIP) or forskolin, i.e. two cAMP‐generating effectors. In contrast, it failed to decrease the stimulation of insulin release elicited by either the Ca2+‐mobilizing agent, carbamoylcholine, or by dibutyryl‐cAMP. Concomitantly, galanin inhibited the GIP‐and forskolin‐stimulated cAMP production. Furthermore, adenylate cyclase in membranes from Rin m 5F cells was highly sensitive to galanin, which exerted a marked inhibitory effect on the forskolin‐stimulated enzyme activity. All these galanin effects were observed at low physiological doses, in the nanomolar range. Overnight treatment of the Rin m 5F cells with pertussis toxin completely abolished the inhibitory effect of galanin on insulin release, cAMP production and adenylate cyclase activity. Moreover, pertussis toxin specifically ADP‐ribosylated a 39‐kDa protein present in membranes from those cells. Taken together, these data show that the galanin inhibition of insulin release most likely occurs through the inhibition of adenylate cyclase, involving a petussis‐toxin‐sensitive inhibitory GTP‐binding regulatory protein.
Primary structure of the major allergen of yellow mustard (Sinapis alba L.) seed, Sin a I
Authors: MENÉNDEZ‐ARIAS Luis , MONEO Ignacio , DOMÍNGUEZ Javier , RODRÍGUEZ Rosalía
Published: July 2010

Sin a I, a 2‐S albumin from the seeds of yellow mustard, is herein described as the major allergen of these seeds. This protein is composed of two disulfide‐linked polypeptide chains of 39 and 88 amino acids, whose primary structures are reported. The Sin a I allergen is found to be related to other low‐molecular‐mass albumins, such as those isolated from rapeseed, castor bean and Brazil nut. Additional structural similarity has also been found between the glutamine‐rich large chain of Sin a I and a proline‐rich zein, a gliadin, and trypsin and α‐amylase inhibitors isolated from the seeds of several monocotyledons. Internal amino acid sequence similarity has been detected at both termini of the small and large chains of Sin a I and involves the location of proline and glycine residues at similar positions in relation to the processing cleavage sites. Prediction of secondary structure, based on the amino acid sequences of the mature chains of the mustard allergen, indicates that the precursor polypeptide is cleaved at regions showing a high α‐turn probability. This is also observed with the amino acid sequence deduced from the rapeseed napin gene nucleotide sequence.
Neomycin inhibits platelet functions and inositol phospholipid metabolism upon stimulation with thrombin, but not with ionomycin or 12‐O‐tetradecanoyl‐phorbol 13‐acetate
Authors: TYSNES Ole‐BjØrn , STEEN Vidar M. , HOLMSEN Holm
Published: July 2010

Gel‐filtered human platelets that had been pre‐labelled with [32P]Pi were stimulated with thrombin, ionomycin or the phorbol ester 12‐O‐tetradecanoyl‐phorbol 13‐acetate (TPA). The effect of the hexacationic aminoglycoside antibiotic, neomycin, on platelet physiological responses, such as aggregation and secretion, as well as changes in phosphoinositide metabolism was studied. Neomycin strongly inhibited thrombin‐induced aggregation and secretion whereas the antibiotic had no effect on ionomycin‐ or TPA‐induced platelet functions. The thrombin‐induced enhancement of inositol phospholipid metabolism was strongly inhibited by the presence of neomycin whereas the TPA‐ or ionomycin‐induced increase in inositol [32P]polyphospholipids remained unaffected. The inhibitory effect of some other aminoglycoside antibiotics was compared to that of neomycin and the data demonstrate that the inhibition of platelet secretion and phosphatidic acid production was dependent on the cationic charge of the antibiotic. It is suggested that neomycin inhibits signal transduction in platelets at a level prior to the inositol‐phospholipid‐specific phosphodiesterase.
Identification of an Endonuclease Responsible for Apoptosis in Rat Thymocytes
Authors: Shiokawa Daisuke , Ohyama Harumi , Yamada Takeshi , Takahashi Kenji , Tanuma Sei‐ichi
Published: July 2010

Analyses of cleavage ends of DNA fragments in apoptotic rat thymocytes induced by γ‐ray irradiation or by treatment with dexamethasone revealed that in both cases the fragments produced had 3′‐hydroxyl (OH) and 5′‐phosphoryl (P) ends of DNA chains. Rat thymocyte nuclei contained at least three endonuclease activities (deoxyribonucleases α, β and γ) that were able to cleave chromatin to mononucleosomal and oligonucleosomal fragments. The nuclei of apoptotic rat thymocytes induced by γ‐ray irradiation or dexamethasone retained considerable deoxyribonuclease γ activity, but not α or β deoxyribonuclease activity. During the induction of apoptosis, treatment with cycloheximide, which suppressed apoptosis, resulted in marked decreases of deoxyribonucleases α and β activities. After release of cycloheximide inhibition, DNA fragmentation associated with apoptosis occurred in the cycloheximide‐treated thymocyte nuclei, in which deoxyribonuclease γ activity was only observed. The purified deoxyribonucleases α and β were divalent cation‐independent acidic endonucleases, which were separated on a CM5PW column by HPLC. The molecular masses of deoxyribonucleases α and β were 28 kDa and 30 kDa, respectively, as determined by TSK G‐2000SW gel‐filtration HPLC, and both were 32 kDa in molecular mass as determined by SDS/PAGE. In contrast, deoxyribonuclease γ, a neutral endonuclease, required both Ca2+ and Mg2+ for full activity and was inhibited by Zn2+. The molecular mass of deoxyribonuclease γ was 31 kDa and 33 kDa when measured by gel filtration and SDS/PAGE, respectively. Under these optimal conditions, deoxyribonuclease γ was shown to produce 3′‐OH/5′‐P ends of nucleosomal DNA fragments, while deoxyribonucleases α and β both formed DNA fragments with 3′‐P/5′‐OH ends. The ends formed by cleavage with deoxyribonuclease γ were the same as those produced in apoptotic rat thymocytes. On the basis of these results, it seems likely that deoxyribonuclease γ is responsible for internucleosomal cleavage of chromatin during thymic apoptosis.
1 H‐NMR Study of a Cobalt‐Substituted Blue Copper Protein: Pseudomonas Aeruginosa Co(II)‐Azurin
Authors: Salgado Jesus , Jiménez Hermas R. , Donaire Antonio , Moratal Jose M.
Published: July 2010

Substitution of copper by cobalt in blue copper proteins gives a paramagnetic metalloderivative suitable for paramagnetic NMR studies. A thorough analysis of the 1H‐NMR spectrum of Pseudomonas aeruginosa Co(II)‐azurin is presented here. All the observable contact‐shifted signals as well as many other paramagnetic signals from protons placed up to about 1.0 nm around the metal center, including some residues belonging to functionally important parts of the protein like the hydrophobic patch and the His35 region, have been assigned. The results obtained permit the detection and study of structural variations like those originated by the His35 ionization, and allow us to draw a feasible picture of the metal coordination site. Contact‐shifted signals correspond to the same five residues which are found in the coordination sphere of the native Cu(II)‐azurin, i.e. His46, His117, Cys112, Met121 and Gly45. Among them, the histidine residues present a pattern of resonances typical for histidines coordinated to cobalt in other cobalt protein derivatives, and the cysteine signals clearly indicate a strong interaction with the paramagnetic Co(II) ion. In contrast, the Met121 signals indicate a weak but still existent contact interaction with the metal center. On the other hand, the very weak copper ligand, Gly45, appears here as clearly coordinated to cobalt. Results are consistent with a distorted tetrahedral metal site with the cobalt deviated from the N2S plane towards the Gly45 O axial position and weakly interacting with the Met121 sulfur.
Kinetics of Streptolysin O Self‐Assembly
Authors: Palmer Michael , Valeva Angela , Kehoe Michael , Bhakdi Sucharit
Published: July 2010

Streptolysin O is a member of a family of membrane‐damaging toxins that bind to cell membranes containing cholesterol and then polymerize to form large pores. We have examined the kinetics of toxin action using 125I‐labelled streptolysin O. Binding of toxin monomers to membranes displays first‐order kinetics and is reversible; the rate of desorption from red cells shows a marked dependence on temperature. To study oligomerization, toxin was bound to erythrocytes at 0°C. Oligomer formation was then triggered by a sudden temperature shift and stopped by solubilization of membranes with deoxycholate. While at moderately high streptolysin O concentrations oligomerization behaves as a reaction of second order, the kinetic pattern changes with increasing toxin concentration. We show that this can be accounted for by the assumption of a two‐step reaction mechanism: two membrane‐bound monomers first associate into a start complex, which then is rapidly extended by the sequential addition of further monomers up to the final oligomer size.
Human Pterin‐4α‐Carbinolamine Dehydratase/Dimerization Cofactor of Hepatocyte Nuclear Factor‐1α
Authors: Köster Sandra , Thony Beat , Macheroux Peter , Curtius Hans‐Christoph , Heizmann Claus W. , Pfleiderer Wolfgang , Ghisla Sandro
Published: July 2010

Pterin‐4a‐carbinolamine dehydratase/dimerization cofactor for hepatocyte nuclear factor‐1α is a protein with two different functions. We have overexpressed and purified the human wild‐type protein, and its Cys81Ser and Cys81Arg mutants. The Cys81Arg mutant has been proposed to be causative in a hyperphenylalaninaemic patient [Citron, B. A., Kaufman, S., Milstien, S., Naylor, E. W., Greene, C. L. & Davis, M. D. (1993) Am. J. Hum. Genet. 53, 768–774]. The dehydratase behaves as a tetramer on gel filtration, while cross‐linking experiments showed mono‐, di‐, tri‐, and tetrameric forms, irrespective of the presence of the single Cys81. Sulfhydryl‐modifying reagents did not affect the activity, but rather showed that Cys81 is exposed. Various pterins bind and quench the tryptophan fluorescence suggesting the presence of a specific binding site. The fluorescence is destroyed upon light irradiation. Wild‐type and the Cys81Ser protein enhance the rate of the phenylalanine hydroxylase assay ≈ 10‐fold, a value similar to that of native dehydratase from rat liver; the Cys81Arg mutant, in contrast, has significantly lower activity. This is compatible with the hypothesis that the dehydratase is a rate‐limiting factor for the in vivo phenylalanine hydroxylase reaction. The three proteins enhance the spontaneous dehydration of the synthetic substrate 6,6‐dimethyl‐7,8‐dihydropterin‐4a‐carbinolamine ≈50–70‐fold at 4°C and pH 8.5. The results are discussed in view of the recently solved three‐dimensional structure of the enzyme [Ficner, R., Sauer, U. W., Stier, G. & Suck, D. (1995) EMBO J. 14, 2032–2042].
Structure of Six 3‐Deoxy‐d‐Glycero‐d‐Galacto‐Nonulosonic Acid(Kdn)‐Containing Oligosaccharide‐Alditols Released from Oviduct Secretions of Ambystoma Maculatum
Authors: Fontaine Marie‐Dominique , Wieruszeski Jean‐Michel , Plancke Yves , Delplace Florence , Strecker Gerard
Published: July 2010

TheO‐linked acidic oligosaccharides of the jelly coat surrounding the eggs of Ambystoma maculatum were analyzed by NMR spectroscopy. The structures of the major oligosaccharides were established as follows where Kdn represents 3‐deoxy‐d‐glycero‐d‐galacto‐nonulosonic acid and GalNAc‐ol is N‐acetylgalactosaminitol:

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As shown for five other amphibian species, the structures of these carbohydrate chains appear to be species specific and can afford a basis for molecular taxonomy. These new sequences also reflect the occurrence of specific fucosyltransferase activities that are characteristic of Ambystoma maculatum.
The Species‐Specific Differences in the cAMP Regulation of the Tissue‐Type Plasminogen Activator Gene between Rat, Mouse and Human is Caused by a One‐Nucleotide Substitution in the cAMP‐Responsive Element of the Promoters
Authors: Holmberg Monica , Leonardsson Göran , Tor NY
Published: July 2010

In rat ovarian cells tissue‐type plasminogen activator (tPA) is induced by gonadotropins, by a cAMP‐dependent pathway and the induction correlates with the time of follicle rupture in vivo. However, in mice, gonadotropins induce the related but distinct protease urokinase‐type plasminogen activator (uPA). Comparison of rat, mouse and human tPA genes reveal that there is a species‐specific difference in the promoter that could explain the difference in regulation of the tPA gene between these species. At the position where the rat promoter contains a consensus cAMP‐responsive element (CRE), the mouse and human counterparts contains a CRE variant with a one‐nucleotide substitution. Transient transfection experiments of rat glial and granulosa cells demonstrated that reporter constructs driven by rat but not mouse or human tPA promoters were efficiently induced by the cAMP‐inducing agents forskolin or follicle‐stimulating hormone. Following the conversion of the mouse and human CRE‐like sequences to rat consensus CRE these promoters became cAMP responsive. In contrast the rat promoter, following conversion of the consensus CRE to the corresponding mouse and human CRE‐like sequence, lost the ability to efficiently respond to cAMP. Deoxyribonuclease I footprinting analysis and electrophoretic mobility shift assays were used to examine interactions of nuclear factors with the consensus and variant CRE. Compared to rat CRE, the mouse and human CRE‐like sequences had a drastically reduced binding affinity for a nuclear factor identified as the cAMP‐responsive element binding protein. Thus the inability of the mouse and human tPA promoters to respond efficiently to forskolin and follicle‐stimulating hormone seem to be due to the inability of these CRE‐like sequences to efficiently bind transcription factor CRE binding protein.
Epitope Mapping of Monoclonal Antibodies to Keratin 19 Using Keratin Fragments, Synthetic Peptides and Phage Peptide Libraries
Authors: Böttger Volker , Stasiak Pauline C. , Harrison Dolores L. , Mellerick Dervla M. , Lane E. Birgitte
Published: July 2010

To generate tools for monitoring processing and folding in keratin intermediate filaments, a group of monoclonal antibodies reacting with the intermediate filament protein keratin 19 were studied using different approaches to define the structure and localization of their epitopes. The binding pattern to bacterially expressed human keratin 19 fragments allowed the definition of minimal amino acid sequences required for antibody binding. The screening of overlapping 15‐residue peptides confirmed and further specified the epitope locations for a subset of the tested antibodies. In addition, the epitope of an antibody with apparent species‐restricted specificity (LE64) was revealed by isolating and characterizing a full‐length keratin 19 clone from a PtK2 cDNA library. Taken together with species cross‐reactivity of individual antibodies and sequence information obtained by probing a phage display library, specific amino acid residues could be highlighted as likely to be involved in the antibody binding.
Octanoate Affects 2,4‐Dinitrophenol Uncoupling in Intact Isolated Rat Hepatocytes
Authors: Sibille Brigitte , Keriel Christiane , Fontaine Eric , Catelloni Françoise , Rigoulet Michel , Leverve Xavier M.
Published: July 2010

When intact isolated rat hepatocytes, either incubated or perifused, were uncoupled by 2,4‐dinitrophenol, we found that the effect on glucose and lactate+pyruvate fluxes, cytosolic and mitochondrial redox states and ATP/ADP ratios were dependent on the nature of the exogenous substrate added. 2,4‐Dinitro‐phenol addition (0.25 mmol/1) to cells perifused with dihydroxyacetone (10 mmol/1) resulted in a modest and transient activation of oxygen uptake accompanied by a surprising rise in lactate/pyruvate ratio indicating an increase in the cytosolic NADH/NAD+ ratio. In addition, such uncoupling, fully abolished glucose production, enhanced lactate+pyruvate flux, and strongly decreased cytosolic and mitochondrial ATP/ADP ratios. In these steady‐state conditions, further addition of Octanoate (0.4 mmol/1) induced a large and sustained enhancement of respiration with a concomitant decrease in the lactate/pyruvate ratio, whereas glucose flux was restored to some extent and cytosolic and mitochondrial ATP/ADP ratios increased. Inhibition of the malate‐aspartate shuttle by the transaminase inhibitor aminooxyacetate (0.3 mmol/1) did not modify the effect of 2,4‐dinitrophenol with dihydroxyacetone alone whereas it decreased the maximal stimulation of oxygen uptake after octanoate addition.

In view of these results we propose the following conclusions.

The uncoupling of intact cells by 2,4‐dinitrophenol inhibits the translocation of reducing equivalents into the mitochondrial matrix probably by impairing the malate‐aspartate shuttle. This explains the increase in the cytosolic NADH/NAD4 ratio and the transient activation of respiration with dihydroxyacetone.

Fatty acid addition to cells uncoupled with 2,4‐dinitrophenol appears to restore a mitochondrial membrane potential, probably by providing the respiratory chain with reduced cofactors directly in the matrix, thus allowing the transfer of reducing equivalents across the mitochondrial membrane.

The restoration, to some extent, of a protonmotive force to uncoupled cells by fatty acid addition is also supported by an increase in ATP synthesis as evidenced by a glucose synthesis with dihydroxyacetone as gluconeogenic substrate.
Surface Mapping of the Ligand‐Filled C‐Terminal Half of the Porcine Estradiol Receptor by Restricted Proteolysis
Authors: Thole Hubert H. , Maschler Itzhak , Jungblut Peter W.
Published: July 2010

The ligand‐filled 32‐kDa fragment of the porcine estradiol receptor extending from His267 to the C‐terminal Ile595 was purified to homogeneity by adsorption to mAb 13H2. The native protein was exposed at 4 °C to a panel of proteases: thermolysin, subtilisin, pronase, elastase, ficin, bromelain, endopeptidase Lys‐C, both in the dimer and the monomer state, and chymotrypsin at pH 8.2 only. The digests were analysed by SDS/PAGE/Western blotting for Coomassie staining and immunostaining. Peptides were sequenced from blots. The majority of cleavage sites in upper domain E (8 out of 11) amassed in the Leu296–Leu310 stretch. Cleavage at Leu319 was seen with subtilisin and at Tyr328 with chymotrypsin. Susceptability to enzymic proteolysis was also pronounced in Thr465–Glu470 at the center of domain E. Three peptides, 13 kDa with thermolysin, beginning at Leu337, 6 kDa and, in low yield, 5 kDa with endopeptidase Lys‐C beginning at Asp473 resp. Cys417 were only obtained from the monomer substrate. The various digests featured either 27–23‐kDa peptides or mixtures of 17–13‐kDa and 12–7‐kDa peptides separable by SDS/PAGE. All peptides with N‐termini between Leu297 and Ser329 reacted with mAb 13H2. The digests showed high peaks of bound estradiol in the dimer position of 32‐kDa fragment controls on density gradient centrifugation at pH 7.4. However, the property of proton‐driven dissociation was only preserved in the pronase, elastase and chymotrypsin digests with peptides extending beyond the His547‐ArgLeuHis550 motif. The preservation of the estradiol‐binding niche in the tightly complexed peptides of domain E was also demonstrated by refilling after steroid removal. The sites exposed to proteolytic enzymes and the epitope for 13H2 attachment are in good agreement with surface probability plots.
The Ability of Actinic Light to Modify the Bacteriorhodopsin Photocycle
Authors: Shrager Richard I. , Hendler Richard W. , Bose Salil
Published: July 2010

The focus of this paper is on the established observation that the bacteriorhodopsin (BR) photocycle responds to the level of actinic light by altering the proportions of two forms of the M intermediate. The first form of M, called M‐fast or MF, decays to the O intermediate. In contrast, the second form of M, called M‐slow or MS, decays directly to the ground state, and its decay rate is slower than that of MF. Any proposed scheme for the BR photocycle must account for this light‐dependent phenomenon. Several papers have attempted to explain the observation on the basis of photocooperativity, or on the basis of heterogeneous populations. In this paper, we test previously proposed cooperative models with experimental data, and find those models to be inadequate. We show that two new models, one purely cooperative, the other purely heterogeneous, can both fit the data, hence such modelling will not resolve the mechanism. Taking into account the demonstration of heterogeneity, the trimer structure of BR, and certain experimental evidence in favor of cooperativity, it appears likely that both heterogeneity and cooperativity are involved in the adaptation of the BR photocycle to different levels of actinic light.
The Gene of Chicken Axonin‐1
Authors: Giger Roman J. , Vogt Lorenz , Zuellig Richard A. , Rader Christoph , Henehan‐Beatty Anne , Wolfer David P. , Sonderegger Peter
Published: July 2010

We have isolated and characterised the gene encoding the chicken axonal cell adhesion molecule axonin‐1. This gene comprises 23 exons distributed over approximately 40 kb. Each of the six immunoglobulin‐like domains and the four fibronectin‐type‐III‐like domains of axonin‐1 is encoded by two exons. The introns between two domains are exclusively phase I. Their exonintron borders correspond to the domain borders of the protein, suggesting that the gene of axonin‐1 had been generated by exon shuffling. Three transcripts with a length of 4.3 kb, 5 kb, and 8 kb are found, and we provide evidence that they result from alternative use of polyadenylation signals. In situ hybridization revealed co‐localisation of these transcripts in time and space in the developing chicken retina. Several identical transcription initiation sites were found in retina, brain, and cerebellum by RNase protection assay and anchored polymerase chain reaction. By transfection of HeLa cells, rat PC‐12 phaeochromocytoma cells, and chicken embryonic fibroblasts with serially truncated segments of the 5′‐flanking region linked to a luciferase reporter gene, we have found that the sequence from −91 to +56 relative to the transcription initiation site is sufficient to promote efficient gene expression. Tissue‐specific expression of the axonin‐1 gene seems to be regulated in part by sequences more than 1 kb upstream of the transcription initiation site. As revealed by computer analysis, the sequence immediately upstream of exon 1 contains an AP‐2 binding site, a tumor phorbol‐ester‐responsive element, and a homeodomain protein binding site, but no canonical TATA box. A second AP‐2 binding site and a homeodomain protein binding site are located within exon 1.
Distinct Biochemical Characteristics of the Two Human Profilin Isoforms
Authors: Gieselmann Ralph , Kwiatkowski David J. , Janmey Paul A. , Witke Walter
Published: July 2010

The biochemical characteristics of a new human profilin isoform are described. We refer to this recently described isoform as profilin II (isoelectric point 5.9) in comparison to profilin I (pI 8.4). We expressed both isoforms in bacteria and compared their actin‐binding properties, binding to poly(l‐proline), affinities for phosphatidylinositol 4,5‐bisphosphate [PtdIns(4,5)P2], and their effects on nucleotide exchange on actin. Profilin I and profilin II have similar affinities for PtdIns(4,5)P2 and poly(l‐proline), and both accelerate nucleotide exchange on monomeric actin to the same extent. However, the affinity of profilin I for monomeric actin is about five times higher than the affinity of profilin II for actin. Potential structural differences of profilin I and profilin II that might explain the difference in actin binding are discussed.
The Muscle‐Specific Phosphoglycerate Mutase Gene is Specifically Expressed in Testis During Spermatogenesis
Authors: Broceño Cristina , Ruiz Pilar , Reina Manuel , VilarÓ Senén , Pons Gabriel
Published: July 2010

Spermatogenesis is a dramatic differentiation process which involves very selective but poorly characterized gene‐expression patterns. To gain insight into this process, we have investigated the expression during spermatogenesis of the genes that encode phosphoglycerate mutase, an essential glycolytic enzyme for the spermatozoa energy supply. By using cDNA and genomic probes we demonstrate the presence in testis of a mRNA corresponding to the muscle‐specific phosphoglycerate mutase which shows a longer poly(A) tail. This muscle‐specific gene is submitted to developmental regulation during testis maturation and begins to be expressed at postnatal day 22, when germ cells start to enter into meiosis. Northern blot and in situ hybridization experiments show that in contrast to what happens during skeletal‐muscle differentiation, PGAM‐M gene expression during spermatogenesis is not coupled to constitutive phosphoglycerate mutase (PGAM‐B) gene repression. Thus, the muscle‐specific PGAM‐M gene constitutes a meiotic gene and therefore represents a very interesting model to study differential tissue‐specific gene expression.