bluuebunny/crossref_metadata_2025 huggingface dataset:
An antibody to the low‐density lipoprotein (LDL) receptor that partially inhibits the binding of LDL to cultured human fibroblasts
Authors: GAVIGAN Sean J. P. , PATEL Dilip D. , SOUTAR Anne K. , KNIGHT Brian L.
Published: March 2005

A monoclonal antibody, raised initially against pure bovine low‐density lipoprotein (LDL) receptor, was found to bind saturably to LDL receptors on the surface of cultured human fibroblasts. It was apparently internalized by the cells but rapidly returned to the surface and was not significantly degraded. LDL did not affect the binding of antibody. However, the antibody reduced both the apparent affinity of the receptor for LDL and the amount of LDL bound. In the presence of antibody, the concentration of LDL required for one‐half maximum binding increased from 14.5 nM to 22.3 nM at 37°C and from 2.8 nM to 11.5 nM at 4°C. Maximum heparin‐releasable binding of LDL was reduced by 56% at 37°C and by 36% at 4°C. Fab fragments halved the affinity at both temperatures without affecting the maximum amount of LDL bound. At 4°C, maximum heparin‐releasable LDL binding was the same as that of antibody and of Fab fragments. At 37°C, heparin‐releasable binding of LDL and binding of Fab fragments were double that at 4°C, whereas binding of antibody remained unchanged.

The results suggest that the antibody bound to a site on the receptor remote from the LDL binding region and that intact antibody recycled with the receptor. It halved LDL binding and degradation at 37°C and reduced binding at 4°C, probably through a different mechanism. The possibility that both cooling and antibody led to receptor aggregation is discussed.
The effect of thyroxine on the calmodulin‐dependent (Ca2+‐Mg2+)ATPase activity and protein phosphorylation in rabbit fast skeletal muscle sarcolemma
Authors: FAMULSKI Konrad S. , PILARSKA Maria , WRZOSEK Antoni , SARZAŁA M. Gabriela
Published: March 2005

Enzymatic properties and the protein pattern of sarcolemma fractions isolated from three groups of rabbits: euthyroid, hyperthyroid and hypothyroid, were studied. The amount of phosphorylated intermediate formed by the calmodulin‐dependent (Ca2+‐Mg2+)ATPase and the activity of this enzyme as well as that of (Na+‐K+)ATPase were the highest in membranes isolated at the hyperthyroid state. On the other hand, sarcolemma obtained from the hypothyroid animals exhibited a decreased activity of (Na+‐K+)ATPase, while the activity of calmodulin‐dependent (Ca2+‐Mg2+)ATPase was the same as in the preparations obtained from euthyroid animals. Thyroid hormones also changed the protein pattern of muscle sarcolemma. Membranes isolated from hyperthyroid animals lacked peptides of apparent molecular masses of 41 kDa and 53 kDa, while a peptide of the apparent molecular mass of 63 kDa was enriched in the preparation from hypothyroid animals. Thyroid hormones affected endogenous cAMP‐dependent protein phosphorylation. The sarcolemma fraction obtained from hyperthyroid animals exhibited a decreased phosphorylation of peptides of apparent molecular masses of 30 kDa and 47 kDa, while the cAMP‐independent phosphorylation of several other peptides was augumented.

Moreover, sarcolemma preparations isolated from hyperthyroid animals showed higher activity of cAMP‐independent protein kinase(s) and lower activity of cAMP‐dependent protein kinase when compared to the euthyroid preparations. It is proposed that thyroxine increases the content of calmodulin‐dependent (Ca2+‐Mg2+)ATPase protein and affects the activity of cAMP‐independent and cAMP‐dependent protein kinases bound to sarcolemma.
Purification and some properties of cathepsin B from rabbit skeletal muscle
Authors: OKITANI Akihiro , MATSUISHI Masanori , MATSUMOTO Teruyo , KAMOSHIDA Eiichi , SATO Masahiko , MATSUKURA Ushio , WATANABE Masao , KATO Hiromichi , FUJIMAKI Masao
Published: March 2005

Cathepsin B was purified from rabbit skeletal muscle by ammonium sulfate fractionation and successive chromatographies on Sephadex G‐75, phosphocellulose, peptide‐conjugated Sepharose, DEAE‐Toyopearl and Sephadex G‐100. The purified enzyme gave a single protein band on SDS/polyacrylamide gel electrophoresis. The enzyme did not abolish the Ca sensitivity of the ATPase activity of myofibrils. The molecular mass of the enzyme was found to be 27 kDa on gel filtration and SDS/polyacrylamide gel electrophoresis. The optimum pH for the hydrolysis of Nα‐benzoyl‐DL‐arginine‐β‐naphthylamide was 6.5. The enzyme was stable in the range of pH 4.5–5.5. Tetrathionate reacted with thiol groups of the enzyme reversibly so that it stabilized the enzyme. The enzyme was strongly inhibited by iodoacetate, HgCl2, antipain, leupeptin, Nα‐p‐tosyl‐l‐lysine chloromethane and l‐tosylphenylalanylchloromethane, but not by pepstatin or trypsin inhibitor.
Characterization of an anti‐thrombospondin monoclonal antibody (P8) that inhibits human blood platelet functions
Authors: BOUKERCHE Habib , McGREGOR John L.
Published: March 2005

Stimulated human blood platelets release thrombospondin, an α‐granule glycoprotein of 450 kDa. The aim of this work was to characterize an anti‐thrombospondin monoclonal antibody (P8) in order to study the role of thrombospondin in platelet functions. The presence of thrombospondin receptor sites on resting and thrombin‐stimulated platelets of three Glanzmann's thrombasthenia patients and normal donors was investigated using the P8 monoclonal antibody. Monoclonal antibody P8 was extensively characterized using ELISA, immunoprecipitation, immunoadsorbent affinity chromatography combined with tryptic peptide map analysis and crossed immunoelectrophoretic techniques. Labelled P8 bound strongly to thrombin‐stimulated normal platelets (n= 14917 ± 420, mean ± SD) (Kd= 9.2 ± 3.0 nM) and poorly to resting platelets (n= 2697 ± 1278) (Kd= 24.8 ± 18.6 nM). Moreover, the number of binding sites for P8 on thrombin‐stimulated platelets from three Glanzmann's thrombasthenia patients, lacking the IIb‐IIIa glycoprotein complex, were found similar to normal samples. F(ab')2 fragments of P8 inhibited aggregation of, and reduced secretion from, washed platelets stimulated by low concentrations of thrombin (0.05–0.06 U/ml) and collagen (0.5–0.6 μg/ml). F(ab')2 fragments of P8 inhibited thrombin‐induced platelet aggregation, but did not reduce fibrinogen binding (n) nor affect its dissociation constant (Kd). Inhibition of platelet aggregation by P8 suggests that thrombospondin plays an active role in promoting platelet aggregation, at low concentrations of thrombin and collagen. Normal binding of P8 to thrombin‐stimulated Glanzmann thrombasthenic platelets indicates the presence of a thrombospondin receptor on the platelet surface distinct from the GPIIb‐IIIa complex.
S100a0, (αα) protein in cardiac muscle
Authors: HAIMOTO Hajime , KATO Kanefusa
Published: March 2005

S100 protein, an acidic and calcium‐binding protein, was believed to be localized in the nervous tissue, but recently it has been reported to be mainly present in the cardiac and the skeletal muscles of various mammals in the αα form (S100a0) at much higher levels than the nervous tissues. We isolated here S100 protein from human cardiac muscles. The isolated cardiac muscle S100 protein showed a single band on electrophoresis at the same position as that of human skeletal muscle S100a0. The amino acid composition of the purified S100 protein was quite similar to that of human skeletal muscle S100a0 or bovine brain S100a0. The immunohistochemical study by use of antibodies monospecific to the α subunit of S100 protein (S100‐α) revealed that S100‐α was strongly labeled in human myocardial cells, whereas the β subunit of S100 protein (S100‐β) was not detected in the cells. These results suggest that a predominant form of S100 protein in human myocardial cells is not S100a (αβ) or S100b (ββ), but S100a0 (αα). In order to determine the ultrastructural localization of S100a0 in mouse cardiac muscle, the direct peroxidase‐labeled antibody method was employed. S100a0 was mainly localized in the polysomes in the interfibrillar spaces, the fine filamentous structure of the Z line and fascia adherens of the intercalated disc and in the lumen of junctional sarcoplasmic reticulum.
Primary structure of the uracil transport protein of Saccharomyces cerevisiae
Authors: JUND Richard , WEBER Elisabeth , CHEVALLIER Marie‐Renée
Published: March 2005

We present in this paper the nucleotidic sequence of the FUR4 gene encoding the uracil permease in the yeast Saccharomyces cerevisiae. The deduced amino acid sequence of the permease has 633 residues; it consists of many hydrophobic stretches, only the N‐terminal and C‐terminal ends of the protein (about 100 and 50 amino acids respectively) being mostly hydrophilic. No N‐terminal hydrophobic signal peptide is present, although it is shown in this work that the biosynthesis of the uracil permease goes through the secretion/glycosylation pathway. Using the results of three different methods, allowing the prediction of transmembrane α helices in proteic sequences, we drew a model of folding of the permease in the membrane.
The 5′‐flanking region of the human P‐450(SCC) gene shows responsiveness to CAMP‐dependent regulation in a transient gene‐expression system of Y‐1 adrenal tumor cells
Authors: INOUE Hideshi , HIGASHI Yujiro , MOROHASHI Ken‐ichiro , FUJII‐KURIYAMA Yoshiaki
Published: March 2005

The chronic effect of CAMP‐dependent regulation on adrenocortical steroidogenesis is known to be revealed in the stimulation of the biosynthesis of steroidogenic enzymes. P‐450(SCC), one of the enzymes, catalyzes the first and the rate‐limiting reaction in steroidogenesis from cholesterol and its synthesis is regulated by CAMP. In order to investigate cis‐acting DNA elements of this gene in response to CAMP‐dependent regulation, we have constructed a fusion gene (pSCC5.4k) by ligating the 5′‐flanking and the upstream untranslated region (5.4 kb) of the human P‐450(SCC) gene to the structural gene for chloramphenicol acetyltransferase (CAT) and transfected it into various culture cells including Y‐1 (mouse adrenal tumor), L929 (mouse fibroblast), HTC (rat hepatoma) and Hepa‐1 (mouse hepatoma). Only Y‐1 cells transfected with pSCC5.4k were found to express transiently the enhanced CAT activity in response to the cAMP analogue, cyclic dibutyryl‐AMP (Bt2cAMP). Primer‐extension analysis of RNA prepared from the cells treated with or without Bt2cAMP showed that the enhanced CAT activity was due to an increase in the CAT mRNA and that the transcription start site, determined here with the human P‐450 gene in the adrenal cortex, was correctly utilized with the fusion gene in the transient expression system. Forskolin and cholera toxin, activators of adenylate cyclase, also increased the expression of the CAT activity in the Y‐1 cells. It has been demonstrated, therefore, that the CAMP‐dependent regulation of the P‐450(SCC) gene in adrenal cortex is faithfully reflected in the transient expression system using Y‐1 cells and the fusion gene and that a cis‐acting DNA element(s) in response to cAMP is present within the 5′‐flanking sequence (5.4 kb) of the P‐45O(SCC) gene.
Purification and characterization of aqualysin I (a thermophilic alkaline serine protease) produced by Thermus aquaticus YT‐1
Authors: MATSUZAWA Hiroshi , TOKUGAWA Kazuhisa , HAMAOKI Masaru , MIZOGUCHI Masashi , TAGUCHI Hayao , TERADA Ichiro , KWON Suk‐Tae , OHTA Takahisa
Published: March 2005

Aqualysin I is an alkaline serine protease which is secreted into the culture medium by Thermus aquaticus YT‐1. Aqualysin I was purified, and its apparent relative molecular mass was determined to be 28 500. The enzyme contained four Cys residues (probably as two cystines), and its amino acids composition was similar to those of cysteine‐containing serine proteases (proteinase K, etc.) as well as those of subtilisins. The NH2‐terminal sequence of aqualysin I showed homology with those of the microbial serine proteases. The optimum pH for the proteolytic activity of aqualysin I was around 10.0. Ca2+ stabilized the enzyme to heat treatment, and the maximum proteolytic activity was observed at 80 °C. Aqualysin I was stable to denaturing reagents (7 M urea, 6 M guanidine · HCl and 1% SDS) at 23 °C for 24 h. The enzyme hydrolyzed the ester bond of an alanine ester and succinyl‐Ala‐Ala–Ala p‐nitroanilide, a synthetic substrate for mammalian elastase. The cleavage sites for aqualysin I in oxidized insulin B chain were not specific when it was digested completely.
Redox and acid‐base characterization of cytochrome b‐559 in photosystem II particles
Authors: ORTEGA José M. , HERVÁS Manuel , LOSADA Manuel
Published: March 2005

The redox and acid/base states and midpoint potentials of cytochrome b‐559 have been determined in oxygen‐evolving photosystem II (PS II) particles at room temperature in the pH range from 6.5 to 8.5. At pH 7.5 the fresh PS II particles present about 2/3 of their cytochrome b‐559 in its reduced and protonated (non‐auto‐oxidizable) high‐potential form and about 1/3 in its oxidized and non‐protonated low‐potential form. Potentiometric reductive titration shows that the protonated high‐potential couple is pH‐independent (E0, + 380 mV), whereas the low‐potential couple is non‐protonated and pH‐independent above pH 7.6 (E0, pH > 7.6, + 140 mV), but becomes pH‐dependent below this pH, with a slope of ‐72 mV/pH unit. Moreover, evidence is presented that in PS II particles cytochrome b‐559 can cycle, according to its established redox and acid/base properties, as an energy transducer at two alternate midpoint potentials and at two alternate pKa values. Red light absorbed by PS II induces reduction of cytochrome b‐559 in these particles at room temperature, the reaction being completely blocked by dichlorophenyldimethylurea.
The structure of brain‐specific rat aldolase C mRNA and the evolution of aldolase isozyme genes
Authors: KUKITA Akiko , MUKAI Tsunehiro , MIYATA Takashi , HORI Katsuji
Published: March 2005

The cDNA clones for rat aldolase C mRNA having the nearly complete length were isolated from a rat brain cDNA library and sequenced. The nucleotide sequence of pRAC2‐1, a cDNA clone having the largest cDNA insert, indicates that the cDNA is composed of a 105‐base‐pair 5′‐noncoding sequence, a 1089‐base‐pair coding‐sequence and a 382‐base‐pair 3′‐noncoding sequence. The amino acid sequence of aldolase C deduced from a possible open reading frame was composed of 362 residues having a relative molecular mass of 39164 excluding the initiating methionine, one amino acid shorter than aldolases A and B. The length of aldolase C mRNA was 1750 residues, somewhat longer than that of the aldolase A and B transcripts. The aldolase C mRNA was distributed mainly in the brain, some in ascites hepatoma and fetal liver.

Comparison of the amino acid sequences of rat aldolase C with those for rat aldolase A and B [Joh et al. (1985) Gene 39, 17–24; Tsutsumi et al. (1984) J. Biol. Chem. 259, 14572–14575], which have been determined previously, shows the existence of highly conserved stretches of amino acid among the three isozymic forms throughout their sequences. The extent of the homology between aldolases A and C is 81%, while those between aldolases A and B, and B and C are 70%, respectively. The analysis of amino acid substitution among aldolases A, B and C from several species suggests that the isozyme genes diverged much earlier than animal species appeared and that the aldolase C gene has evolved from the aldolase A gene after aldolase A and B genes diverged.
Complex RNA maturation in chloroplasts
Authors: WESTHOFF Peter , HERRMANN Reinhold G.
Published: March 2005

The psbB operon of the spinach plastid chromosome encodes the genes for the 51‐kDa chlorophyll a apoprotein (psbB), the 10‐kDa phosphoprotein (psbH), both associated with photosystem II, as well as cytochrome b6 (petB) and subunit IV (petD) of the cytochrome b/f complex in the order given. These genes are not expressed coordinately. The RNA pattern of this DNA region is complex and resolves into eighteen major RNA species. Using northern and S1 protection analysis we demonstrate (a) that all RNA species derive from one DNA strand and hybridize in an overlapping fashion; and (b) that they arise by processing rather than by multiple transcription initiation/termination. (c) The operon is bordered by a single prokaryote‐like promotor in front of psbB, and by a putative factor‐independent terminator with characteristic sequence elements following petD. The terminator appears to function bidirectionally. (d) At least four distinct modification activities operate on the putative primary transcript of 5650 nucleotides and on the processing intermediates, including a novel endonucleolytic activity cleaving within a characteristic hexanucleotide motif, 3′‐exonucleolytic activity at discrete RNA ends, 5′ shortage of mRNA (psbB), and excision of class II intervening sequences (petB and petD). (e) Kinetically, maturation of the primary transcript is largely a stochastic process. (f) Processing results ultimately in the formation of monocistronic mRNAs for each of the two photosystem II polypeptides and a bicistronic mRNA encoding both subunits of the cytochrome b/f complex. We postulate that these RNA species represent the translationally active components in the non‐coordinate dark/light expression of these genes. (g) Light is without any noticeable effect on posttranscriptional modification. Under our conditions it appears to operate at a translational rather than a transcriptional or posttranscriptional level indicating that the biogenesis of thylakoid membranes is regulated at various levels.
RNA‐binding properties of hnRNP proteins
Authors: SCHENKEL Johannes , SEKERIS Constantin E. , ALONSO Angel , BAUTZ Ekkehard K. F.
Published: March 2005

The RNA‐binding properties of the hnRNP monoparticle proteins were examined using a renaturing blotting procedure. All ‘core’ proteins are able to bind single‐stranded nucleic acids, probably not sequence‐specific. The core proteins C1 and, in one case A2 and B2, are able to bind nucleic acids which are double‐stranded or which show a high degree of base‐paired regions, among them U1 snRNA, whereas A1, B1 and C2 are unable to bind base‐paired nucleic acids. The characteristics of C1 in binding base‐paired nucleic acids are especially interesting, since the involvement of C1 in the splicing process has been described.
T‐kinin release from T‐kininogen by rat‐submaxillary‐gland endopeptidase K
Authors: GUTMAN Ninette , MOREAU Thierry , ALHENC‐GELAS François , BAUSSANT Thierry , EL MOUJAHED Abdel , AKPONA Simon , GAUTHIER Francis
Published: March 2005

Submaxillary gland extracts have been fractionated to characterize the enzyme responsible for the T‐kininogenase activity previously reported in this tissue [Damas, J. & Adam, A. (1985) Mol. Physiol 8, 307–316] and to know whether this activity could be of physiological relevance, since no enzyme reacting in catalytic amounts has been described so far to be able to release a vasoactive peptide from T‐kininogen.

The purified enzyme, provisionally called endopeptidase K, has an apparent Mr of 27000 when not reduced prior to analysis but 21000 after reduction and an acidic pI of 4.3 ± 0.1. Antigenically, it is not related to tissue kallikrein. Upon incubation with purified T‐kininogen it may induce a complete liberation of T‐kinin from the precursor provided it is added in stoichiometric amounts. However, in parallel with the liberation of immunoreactive kinin, a proteolysis of T‐kininogen is observed which is not restricted to the site of insertion of T‐kinin as would be expected using a specific kininogenase.

In agreement with these results, no change of the mean blood pressure was observed upon injection of endopeptidase K into the circulation of normal rats even if the amount of injected enzyme was up to ten times that required for tissue kallikrein to induce a significant fall in blood pressure.

However, in spite of the large proteolysis induced by incubation with stoichiometric amounts of endopeptidase K, the total papain inhibiting capacity of T‐kininogen as well as the value of the apparent inhibition constant, Ki, with this proteinase remained unchanged. Proteolytic fragments which retain cysteine‐proteinase‐inhibiting activity may therefore be released from T‐kininogen by endopeptidase K more easily than immunoreactive kinin, thus emphasizing a prominent function of proteinase inhibitor or of proteinase inhibitor precursor for this molecule.
Carbamoyl‐phosphate synthetase I
Authors: BRITTON Hubert Greenslade , RUBIO Vicente
Published: March 2005

The dissociation of the cofactor, acetylglutamate, from the enzyme‐cofactor complex formed by carbamoyl‐phosphate synthetase I of rat liver in the presence of ATP, Mg2+, K+ and HCO−3 has been studied by centrifugal gel filtration. The rate of its dissociation (k, 0.13 s−1) is considerably slower than the rate of enzyme turnover (∼6 s−1) and it is not increased by ammonia, although ammonia reduces the rate of reassociation of the cofactor. Omission of ATP, Mg2+ or K+ from the column buffer leads to virtually complete dissociation of bound acetylglutamate during passage through the column (0.5–2 min), owing to an increase in dissociation and a decrease in reassociation, but reduction of free Mg2+ alone has the opposite action. Dilution of the enzyme‐cofactor complex into a large volume of buffer causes a biphasic loss of enzyme activity with a t1/2 of the first phase comparable with that of the dissociation of acetylglutamate. These findings show (a) that acetylglutamate does not dissociate with each turnover of the enzyme; (b) that there are rapid interactions between binding of acetylglutamate and ATPA (ATPA yields Pi in the overall reaction), Mg2+ and K+, suggesting that these ligands bind in close proximity; and (c) that the enzyme transiently retains considerable activity after dissociation of the cofactor.
Biosynthesis of vitamin B‐12 in anaerobic bacteria
Authors: VOGT Joachim R. A. , RENZ Paul
Published: March 2005

The pathway of vitamin B‐12 biosynthesis in anaerobic bacteria differs in several respects from the pathway found in aerobic or aerotolerant microorganisms. The aim of this investigation was to elucidate the formation of the 5,6‐dimethylbenzimidazole part and the amide groups of vitamin B‐12 in anaerobic bacteria. [15N]Ammonium chloride or l‐[amido‐15N]glutamine or a mixture of [15N]ammonium sulfate and [15N]glycine was added to fermentations with Eubacterium limosum. The vitamin B‐12 isolated from these fermentations was methylated and degraded to cobinamide and 1,5,6‐trimethylbenzimidazole. The amide groups of cobinamide were hydrolyzed and the amide nitrogen of the side chains a, b, c, d, e and g trapped as benzamide. The 15N incorporation was determined by mass spectroscopy. Thus in the experiment with [15N]ammonium chloride the benzamide and the 1,5,6‐trimethylbenzimidazole contained 9.6%15N, whereas in the experiment with l‐[amido‐15N]glutamine 37.5% of the molecules were 15N labeled. The 1H‐NMR spectrum of 1,5,6‐trimethylbenzimidazole revealed that the 15N from the ammonium salts and from glutamine was incorporated into N‐3 of the 5,6‐dimethylbenzimidazole moiety of vitamin B‐12. With a mixture of [15N]ammonium sulfate and [15N]glycine both nitrogens of 5,6‐dimethylbenzimidazole became 15N‐labeled.

These experiments demonstrate that in E. limosum the amide nitrogen of glutamine is not only the precursor of the six amide groups of the corrin ring, but also of N‐3 of the 5,6‐dimethylbenzimidazole moiety of vitamin B‐12.
Interferon removes its own receptors as it blocks the division of Daudi cells
Authors: EID Pierre , BANDU Marie‐Thérèse , UZE Gilles , MOGENSEN Knud E.
Published: March 2005

The Burkitt‐derived line, Daudi, whose proliferation is inhibited by human α‐interferon (IFN‐α), was treated with 125I‐labelled recombinant human IFN‐αA. After separation from unbound ligand, cell‐bound IFN was extracted with the detergent digitonin yielding soluble and insoluble complexes of IFN and receptor, together with a certain amount of uncomplexed IFN.

Soluble complexes were stable enough to be separated from uncomplexed IFN by permeation chromatography. Treatment of soluble complexes with the bifunctional reagent, disuccinimidyl suberate, yielded a radioactive product separating with an Mr, of 130000 on electrophoresis in sodium dodecyl sulphate. Similar complexes could be recovered with sodium dodecyl sulphate from the digitonin‐insoluble residue, treated with the bi‐functional reagent.

The total (soluble and insoluble) of complexed IFN obtained after digitonin extraction was a constant fraction (0.62) of the total cell‐bound radioactivity, being independent of the concentration of IFN added to the cells (> pM to > nM), and of the time of incubation (1 min to 20 h). However, between 30 min and 3 h of incubation, the insoluble complex increased, at the expense of the soluble complex, and there appeared a cellular pool of degraded ligand. From 3 h to 20 h the distribution of ligand‐derived radioactivity remained constant while the total amount decreased to less than 10% of its value at 30 min. This decrease in binding was matched by the appearance of an equivalent quantity of radiolabelled fragments in the culture medium.

The inhibition of cellular division due to IFN was shown to be coincident with the disappearance of cellular binding and with the cell‐mediated degradation of receptor‐complexed IFN. We propose that IFN removes its own receptor and, in doing so, blocks a linked function necessary for the stimulated growth of Daudi cells.
Activation of rat basophilic leukemia cells
Authors: RAN Sophia , RIVNAY Benjamin
Published: March 2005

Antigen‐induced 45calcium influx into rat basophilic leukemia (RBL) cells was examined with emphasis on the early time domain under conditions that exclude loss of the cation during the subsequent washing step. Such preparations demonstrate a distinct, temporally separate influx peaking at 3 min, followed by a substantial efflux. This internalized 45Ca2+ approaches millimolar total intracellular concentration and is therefore either sequestered or becomes bound to intracellular components (proteins). The amplitude of this influx is linearly proportional to IgE‐receptor occupancy at low receptor occupancies, and is sensitive to the anti‐allergic drug cromolyn. Furthermore, the timing of both the maximal uptake and the maximal susceptibility to cromolyn correlates with the Quin‐2 signal in these cells, and the initial degranulation pattern bears some resemblance to trends in the 45Ca2+ uptake curve. These qualities suggest that the early peak at 2–3 min, rather than any later 45Ca2+ uptake, comprises the initial signalling Ca2+ pool. Maximal apparent inhibition by cromolyn for 45Ca2+ uptake was about 65% and required a 10–15‐min preincubation with the cells. The inhibitory effect was limited to the peak at 3 min, suggesting that tracer incorporation beyond 5–6 min largely involves other pools or pathways, triggered by receptor aggregation, yet only indirectly related to channel activity or to the signal proper. A quantitative similarity was found between the early peak measured on intact cells and the single channel conductance measured on reconstituted planar bilayers containing the purified receptor for IgE and the purified cromolyn‐binding protein [Corcia, A. et al. (1986) EMBO J. 5, 849–854]. This, as well as the effects of cromolyn, support the assumption that cromolyn‐binding protein is a major constituent involved in this early influx, or that influx is a principal pathway for the signaling calcium mass.
The rat vitamin‐D‐dependent calcium‐binding protein (9‐kDa CaBP) gene
Authors: PERRET Christine , LOMRI Nourredine , GOUHIER Nelly , AUFFRAY Charles , THOMASSET Monique
Published: March 2005

The structural organization of the entire rat vitamin‐D‐dependent calcium‐binding protein (9‐kDa CaBP) gene was determined by analysis of overlapping genomic clones isolated from a rat genomic library using the rat 9‐kDa CaBP cDNA [Desplan C., Heidmann O., Lillie J., Auffray C. and Thomasset M. ((1983) J. Biol. Chem. 258, 13502–13505]. These clones together span 30 kbp of rat genomic DNA, with the rat 9‐kDa CaBP gene lying in the middle. The 9‐kDa CaBP gene is 2.5 kbp long and contains three exons interrupted by two introns. The first exon contains almost the entire 5′ untranslated region. The second exon codes for the calcium‐binding site I, the third exon codes for site II and the 3′ untranslated region. Therefore each of the calcium‐binding domains is encoded by single, separate exons. The transcription initiation site was identified by S1 nuclease mapping and primer extension. A consensus sequence TATAAA is localized 31 bp upstream from the cap site and the ‘CCAAT–box’ lies upstream from the transcription start. Single (AC)25 and (AG)23 repeats are present in the second intron together with an Alu‐like sequence. Repetitive elements are present 5 kbp upstream from the cap site and in the 3′ flanking region. Comparison of the known rat CaBP sequences (9‐kDa CaBP, 28‐kDa CaBP, S100 protein) shows that the 9‐kDa CaBP is more closely related to the S100 protein than to the 28‐kDa CaBP. There is no evidence to indicate that 9‐kDa CaBP has arisen from the 28‐kDa CaBP.
Release of acetylhydrolase from platelets on aggregation with platelet‐activating factor
Authors: SUZUKI Yasuo , MIWA Masao , HARADA Masaya , MATSUMOTO Makoto
Published: March 2005

Acetylhydrolase, the enzyme which inactivates platelet‐activating factor (PAF, 1‐O‐alkyl‐2‐O‐acetyl‐sn‐glycero‐3‐phosphocholine), was selectively released from bovine platelets by aggregation with physiological concentrations (0.1–10 nM) of PAF with no cell lysis. The release of the acetylhydrolase paralleled that of serotonin. The acetylhydrolase released was active over a broad pH range (pH 5.4 – 8.6) and was not affected by Ca2+ (1–4 mM) or EDTA (1–8 mM). The Km, value of the enzyme was 4.6 μM. Net specific acetylhydrolase activity recovered in the 130000 ×g supernatant after stimulation with PAF could be determined in the presence of EDTA without the activity of Ca2+‐dependent phospholipase A2 which was also released from the cells at the same concentration of PAF. The acetylhydrolase was inhibited competitively by specific PAF antagonists, rac‐3‐(N‐n‐octadecylcarbamoyloxy)‐2‐methyoxypropyl‐2‐thiazolioethyl phosphate (CV‐3988) and (2RS)‐1‐O‐hexadecyl‐2‐O‐ethyl‐3‐O‐(7‐thiazolinoheptyl)‐glycerol methanesulfonate (ONO‐6040). Their Ki values for the enzyme were 1.17 μM and 0.84 μM, respectively. The release of the enzyme could also be detected when the platelets were aggregated with ADP (2.3 μM) or thrombin (0.5 unit).

These results suggest that the enzyme released from the aggregated platelets to the blood plasma may also have a physiological function cooperating with the plasma acetylhydrolase.
Histone accessibility determined by lysine‐specific acetylation in chicken erythrocyte nuclei
Authors: LEWIS Peter N. , GUILLEMETTE J. Guy , CHAN Shirley
Published: March 2005

N‐Hydroxysulfosuccinimidyl [3H]acetate was synthesized and, following the determination of the optimal reaction conditions, was used to acetylate histones in chicken erythrocyte nuclei at 4°C, pH 8. The histones were extracted from the labelled nuclei and the distribution of the acetyl groups determined from the amount of tritiated acetate in isolated peptides. The relative degree of acetylation of molecules was H1 1.0, H5 0.81, H2B 0.48, H2A 0.24, H3 0.24, H4 0.16. Histone H1 is the most exposed histone followed by H5. The core histones are much less accessible to chemical modification than the linker histones by a factor of 4–5. Histones H2A, H2B and H5 appear to be labelled at random along the entire polypeptide chain, while histones H3 and H4 are labelled almost exclusively in the first 30 residues from the N terminus. Control and acetylated chicken erythrocyte nuclei were digested with DNase I and the resulting DNA hybridized to globin and ovalbumin cDNAs. Acetylation, at 14 molecules acetate/core nucleosome or 20 molecules acetate/chromatosome, increased the DNase I sensitivity of the ovalbumin gene to that of the globin sequences in the control sample, while the globin sequences became even more nuclease‐sensitive. Our results suggest that increased sensitivity of chromatin towards nuclease digestion might be due to increased solubility of the chromatin fibre.
Enzymatic properties of single‐chain and two‐chain forms of a Lys158→ Glu158 mutant of urokinase–type plasminogen activator
Authors: LIJNEN H. Roger , VAN HOEF Berthe , NELLES Luc , HOLMES William E. , COLLEN Désiré
Published: March 2005

Recombinant single‐chain urokinase‐type plasminogen activator (rscu‐PA), in which the plasmin‐sensitive peptide bond Lys158‐Ile159 is destroyed by site‐specific mutagenesis of LYS158 to Glu (rscu‐PA‐Glu158), is quantitatively converted to two‐chain urokinase‐type plasminogen activator (rtcu‐PA‐Glu158) by treatment with endoproteinase Glu‐C (Staphylococcus aureus V8 proteinase). The catalytic efficiency (k2/Km) of rscu‐PA‐Glu158 for the activation of plasminogen is 20 times lower (0.0001 μM−1 s−l) than that of rscu‐PA (0.002 μM−1 s−1). In contrast, rtcu‐PA‐Glu158 has very similar properties to rtcu‐PA obtained by digestion of rscu‐PA with plasmin, including binding to benzamidine‐Sepharose, catalytic efficiency for the activation of plasminogen (0.035 μM−1 s−1 versus 0.046 μM−1 s−1) and fibrinolytic activity in an in vitro plasma clot lysis system. It is concluded that the amino acid in position 158 is a main determinant of the functional properties of single‐chain urokinase‐type plasminogen activator but not of the two‐chain form.
Identification of a salvage pathway for D‐arabinose in Mycobacterium smegmatis
Authors: WOJTKIEWICZ Beata , SZMIDZINSKI Ryszard , JEZIERSKA Anna , COCITO Carlo
Published: March 2005

Extracts of Mycobacterium smegmatis, which was adapted to growth in synthetic medium containing D‐arabinose as sole carbon source, catalyzed the NADPH‐mediated reduction of D‐arabinose to D‐arabitol. When arabinose‐adapted bacteria were transferred to glycerol medium, resumption of growth was accompanied by a sharp drop in the specific activity of this enzyme. Moreover, extracts of cells grown in D‐arabinose medium contained large amounts of an NAD+‐linked pentitol dehydrogenase, as compared to bacteria multiplying in glycerol medium. The specific activity of mycobacterial extracts was tenfold higher for D‐arabitol than for its L‐isomer, and eightfold higher than for xylitol (it was more than fortyfold lower in the case of glycerol‐grown cells). The product of the pentitol dehydrogenase reaction was identified as D‐xylulose by three different procedures. On the basis of these data, it is suggested that utilization of exogenous D‐arabinose in mycobacteria involves two dehydrogenases that catalyze the reactions D‐arabinose D‐arabitol D‐xylulose, by virtue of which an aldopentose is converted into a ketopentose. The alditol: NADP oxidoreductase was isolated from homogenates of D‐arabinose‐adapted mycobacteria, and purified by DEAE‐cellulose chromatography. The enzymatic activity was restricted to a single band which, under denaturing conditions, comigrated with albumin (∼ 46 kDa). It was insensitive to 2‐mercaptoethanol, EDTA and NaF, and was inactivated at 70°C.
Direct and indirect electron transfer between electrodes and redox proteins
Authors: FREW Jane E. , HILL H. Allen O.
Published: March 2005

The direct electrochemistry of redox proteins has been achieved at a variety of electrodes, including modified gold, pyrolytic graphite and metal oxides. Careful design of electrode surfaces and electrolyte conditions are required for the attainment of rapid and reversible protein‐electrode interaction. The electron transfer reactions of more complex systems, such as redox enzymes, are now being examined. The ‘well‐behaved’ electrochemistry of redox proteins can be usefully exploited by coupling the electrode reaction to enzymes for which the redox proteins act as cofactors. In systems where direct electron transfer is very slow, small electron carriers, or mediators, may be employed to enhance the rate of electron exchange with the electrode. The organometallic compound ferrocene and its derivatives have proved particularly effective in this role. A new generation of electrochemical biosensors employs ferrocene derivatives as mediators.
Inactivation of detergent‐solubilized sarcoplasmic reticulum ATPase
Authors: SWOBODA Gertrude , HASSELBACH Wilhelm
Published: March 2005

Inactivation of sarcoplasmic ATPase in the solubilized state was studied in the absence and presence of Ca2+, Mg2+ and glycerol. The effects of the detergents octa(ethyleneglycol) mono‐n‐dodecyl ether (C12E8), 1‐O‐tetradecylpropanediol‐(1,3)‐3‐phosphorylcholine and myristoylglycerophosphocholine were compared. All three detergents caused a rapid decline of the dinitrophenyl phosphatase activity of the unprotected enzyme. The stabilizing effect of Ca2+ ions was kinetically analysed. It was found that the stability of the solubilized enzyme depends on the Ca2+ concentration in a manner which is best explained by assuming rapid inactivation of Ca2+‐free enzyme accompanied by slow inactivation of a calcium‐enzyme complex (E1Ca). The apparent affinity constants obtained are in the order of 106M−1, suggesting that high‐affinity Ca2+ binding must be involved. No indications of a contribution were found, either of low‐affinity Ca2+‐binding sites of the conformational state E2 or of the high‐affinity calcium complex E1Ca2. If Ca2+ was replaced by Mg2+, which exerts a weaker protection, the apparent affinity constants for Mg2+ are in the range of 1 mM−1. The stoichiometry of the effect of Mg2+ depends on the detergent.
Differential mRNA stability to reticulocyte ribonucleases correlates with 3′ non‐coding (U)nA sequences
Authors: WRESCHNER Daniel H. , RECHAVI Gideon
Published: March 2005

The stabilities of different mRNA species were analyzed in a reticulocyte lysate system under protein‐synthesizing conditions. In all cases examined the relative mRNA degradation by reticulocyte ribonucleases as well as by the interferon‐modulated (2′‐5′) (A)n‐dependent endonuclease correlated with the extent of (U)nA sequences within the 3′ non‐coding region. The experimental data presented indicate that according to their stabilities at least three major mRNA groups may be identified: (a) (U)nA‐poor mRNAs (e.g. globin) are essentially stable and are only slightly degraded by the (2′‐5′)(A)n‐dependent endonuclease; (b) mRNA species with intermediate (U)nA levels (e.g. Igα and Igμ heavy‐chain mRNAs) are partially degraded by general ribonuclease activity and further degraded by the (2′‐5′)(A)n‐dependent endonuclease and (c) (U)nA‐rich mRNA species (such as c‐myc and non‐skeletal actin mRNAs) are inherently unstable and are extremely sensitive to degradation by general ribonuclease activity. A survey of mRNA nucleotide sequences demonstrated that without exception (U)nA‐rich stretches appeared more frequently within the 3′ non‐coding region than in the coding or 5′ non‐coding regions. A comparison of 3′ non‐coding region sequences from 92 different mRNAs revealed that transiently expressed mRNAs, such as the interleukins, nerve growth factor, epidermal growth factor receptor, c‐myc, c‐fos, c‐myb and several other oncogenes as well as interferons α, β and γ were exceptially (U)nA‐rich. It is postulated that differential mRNA stability may be partly determined by the primary nucleotide sequence and in particular by (U)nA sequences within the 3′ non‐coding region. This may represent a novel post‐transcriptional strategy employed by the cell to selectively retain or destroy discrete mRNA species.
Members of the Drosophila HSP 70 family share ATP‐binding properties
Authors: BEAULIEU Jean François , TANGUAY Robert M.
Published: March 2005

In Drosophila, the hsp 70 family consists of a group of proteins of similar molecular masses (hsps 68, 70 and 72) that exist as multiple isoforms. In this report, it is shown that hsps 68, 70 and 72 from Drosophila cells can be purified by affinity chromatography on ATP‐agarose. Furthermore it is demonstrated that the multiple members of the hsp 70 family, which accumulate in large amounts in the nucleus during a heat shock, can be specifically solubilized from the isolated nuclei fraction by ATP. One of the major cognate proteins (hsc 70) also shows similar behavior. These data suggest that most, if not all, of the related Drosophila hsps 70 possess, like their mammalian counterparts, an ATP‐binding site which could be related to their function in the stress response.
Regulation of translation and stability of an mRNA coding for a 40‐kDa polypeptide in rat L6 muscle cells
Authors: PRAMANIK Saroj K. , MEADUS William J. , BAG Jnanankur
Published: March 2005

The mRNA coding for a 40‐kDa polypeptide (P‐40) was previously cloned and sub‐cellular distribution of this mRNA was examined in rat L6 myoblast cells under different conditions [Pramanik, S. & Bag, J. (1987) Eur. J. Biochem. 170, 59–67]. The translation of this mRNA was found to be regulated during differentiation of myoblasts. This mRNA was translated in proliferating myoblasts but not in the non‐dividing differentiated myotubes. We have further examined whether the mRNA present in the polysomal fraction of myoblasts and that in the free non‐polysomal fraction in myotubes was identical by nuclease S1 mapping. The coding strand of the 600‐base‐pair PstI fragment of the recombinant clone was 3′‐end‐labeled with cordycepin 5′‐[α‐32P]triphosphate and hybridized with RNA from either myoblasts or myotubes. The results of these studies have shown that RNA from both preparations was fully able to hybridize with the probe DNA and, therefore, protected the 600‐nucleotide‐long fragment from nuclease S1 digestion, thus suggesting that the sequence of 600 nucleotides at 3′ ends of both translationally active polysomal mRNA of myoblasts and repressed free mRNA of myotubes are identical. These results also confirmed the results of our earlier studies on the subcellular distribution of this mRNA by Northern blot analysis. Further studies were also performed to determine whether withdrawal of muscle cells from the cell cycle during differentiation to form myotubes alone was responsible for regulating translation of P‐40 mRNA. The result of the subcellular distribution of this mRNA in proliferating myoblasts following inhibition of DNA synthesis by cytosine arabinoside have shown that translation of P‐40 mRNA continued in absence of DNA synthesis. This observation suggests that an additional signal is necessary to block the translation of P‐40 mRNA in myotubes.

The relationship between the translation of P‐40 mRNA and its stability was examined. Two different methods were used to determine the stability of mRNAs. The first approach was by determining the steady‐state levels of this mRNA following inhibition of RNA synthesis by actinomycin D. In the second method, we have determined the amount of 3H‐labeled P‐40 mRNA during pulse and chase experiments. Both methods produced similar results. It was found that the stability of P‐40 mRNA was not altered during differentiation of rat L6 cells. The results of pulse and chase studies have also shown that P‐40 mRNA was synthesized in both myoblasts and myotubes. Furthermore, the α‐amanitin sensitivity of the synthesis of P‐40 mRNA was examined using a nuclear transcription system. The result of this analysis has shown that synthesis of P‐40 mRNA was sensitive to a low concentration of α‐amanitin, suggesting that P‐40 mRNA, like other mRNAs, is also transcribed by RNA polymerase II.
Binding of tissue‐type plasminogen activator to fibrinogen fragments
Authors: BOSMA Piter J. , RIJKEN Dingeman C. , NIEUWENHUIZEN Willem
Published: March 2005

In order to localize the binding site(s) for tissue‐type plasminogen activator (t‐PA) in the fibrin(ogen) molecule, the following binding assay was developed. Two‐chain t‐PA was immobilized onto microtitration plates. The t‐PA‐coated plates were then incubated with fibrinogen and various fibrinogen fragments. The extent of binding was quantified with enzyme‐labelled antibodies against fibrin(ogen) and its fragments. Hardly any binding to t‐PA was observed with fibrinogen or fragments X, Y and E; a moderate binding was observed with fragments Dcate and DEGTA and a strong binding with the cyanogen bromide fragment FCB‐2 (Kd apparent = 140 nM). The binding of fibrinogen and its fragments to immobilized Lys‐plasminogen was measured by the same method as a control for the binding assay. Results were in line with literature data: virtually no binding to Lys‐plasminogen with fibrinogen or fragments X and Y, a moderate binding with fragments Dcate, DEGTA and E and a strong binding with FCB‐2 (Kd apparent = 70 nM). The stimulatory capacity of the various fragments on the Lys‐plasminogen activation by t‐PA, as studied in a spectrophotometric assay, was found to be absent for fragment E, low for fibrinogen, fragments X, Y, Dcate and DEGTA, and high for FCB‐2. It is concluded that a t‐PA‐binding site resides in the C‐terminal globular domains of fibrinogen from which fragments D and FCB‐2 originate. The site is hidden in the native fibrinogen molecule and in early fibrinogen degradation products. Binding of both Lys‐plasminogen and t‐PA appears to be required for a stimulator of the plasminogen activation, as illustrated by fragment E which only binds Lys‐plasminogen and has no stimulatory capacity.
Diversity of corrinoids in acetogenic bacteria
Authors: STUPPERICH Erhard , EISINGER Hans Jürgen , KRÄUTLER Bernhard
Published: March 2005

The Coβ‐cyanocobamides obtained by cyanide extractions from several acetogenic bacteria were structurally characterized by ultraviolet visible spectra, proton‐nuclear‐magnetic‐resonance spectra and fast‐atom‐bombardment mass spectra. p‐Cresolylcobamide was detected as a major corrinoid from Sporomusa ovata. This ‘complete’ corrinoid was isolated from an organism for the first time. Instead of the common Coα bases of the known and biologically active cobamides, p‐cresolylcobamide contained a glycosidically bound cresolyl function that was unable to coordinate to the cobalt of the corrin ring. An additional, previously unknown corrinoid from natural sources, Coα‐[α‐(5‐methoxy‐6‐methylbenzimidazolyl)]‐Coβ‐cyanocobamide, was isolated along with vitamin B12 from Clostridium formicoaceticum. Both homoacetogenic eubacteria were grown on methanol and contained high amounts of corrinoids (> 950 nmol/g cell dry mass). Less corrinoid was isolated from Acetobacterium woodii and characterized as vitamin B12.