bluuebunny/crossref_metadata_2025 huggingface dataset:
Generation of phosphatidylinositol 4,5‐bisphosphate proceeds through an intracellular route in rat hepatocytes
Authors: Lundberg Gunilla A. , Jergil Bengt
Published: July 2002

The subcellular distribution in rat hepatocytes of enzymes participating in the entire generation cycle of phosphatidylinositol 4,5‐bisphosphate, and phosphorylated intermediates of this pathway, has been examined by Nycodenz gradient centrifugation. Our results indicate that the synthesis of phosphatidylinositol takes place in the endoplasmic reticulum, and that its phosphorylation to phosphatidylinositol 4‐phosphate occurs intracellularly in low‐density membranes before translocation to the plasma membrane, where it is further phosphorylated to phosphatidylinositol 4,5‐bisphosphate. The intracellular formation of PIP implies a vesicular transport to the plasma membrane.
Nucleotide sequence of the androgen‐dependent arginine esterase mRNA of canine prostate
Authors: Chapdelaine Pierre , Ho-Kim My-Anh , Tremblay Roland R. , Dubé Jean Y.
Published: July 2002

The nucleotide sequence of canine prostate arginine esterase mRNA was determined using a 400 bp cDNA clone and primer‐extended cDNA transcripts for the 5′‐coding and noncoding regions. The mRNA contains 864 nucleotides encoding a protein of 236 amino acids preceded by 24 amino acids which constitutes both the signal and the zymogen peptides. The sequence indicates the presence of one potential glycosylation site. A high degree of homology was found between the canine enzyme and other members of the kallikrein family including human prostate specific antigen. The protein appears to be specified by a single gene.
Specific binding of [3H]retinoids to cellular retinoid‐binding proteins
Authors: Ninomiya Mitsuo , Suganuma Masami , Paik Nam Sun , Muto Yasutoshi , Fujiki Hirota
Published: July 2002

The specific binding of [3H]retinoids to cellular retinoid‐binding proteins was measured directly by the cold acetone filtration method. After incubation of purified cellular retinoid‐binding proteins with [3H]retinoids with or without competitors for 2–4 h, bound ligands were separated from free by filtration using cold acetone. Nonspecific binding of the ligands was reduced sufficiently to allow measurement of specific binding of [3H]retinoids to cellular retinoid‐binding proteins. This method has the advantages of being rapid and practical and giving reproducible results.
Cloning and nucleotide sequence of cDNA for the plastid glycerol‐3‐phosphate acyltransferase from squash
Authors: Ishizaki Osamu , Nishida Ikuo , Agata Kiyokazu , Eguchi Goro , Murata Norio
Published: July 2002

The partial amino acid sequence and amino acid composition of acyl‐(acyl‐carrier‐protein):glycerol‐3‐phosphate acyl‐transferase purified from squash cotyledons were determined. cDNAs encoding this enzyme were isolated from λgt11 cDNA libraries made from poly(A)+ RNA of squash cotyledons by immunological selection and cross‐hybridization. One of the resultant clones contained a cDNA insert of 1426 base pairs and an open reading frame of 1188 base pairs. The amino acid sequence deduced from the nucleotide sequence matched the partial amino acid sequence determined for the enzyme. The results suggest that a precursor protein of 396 amino acid residues is processed to the mature enzyme of 368 amino acid residues, losing a leader peptide of 28 amino acid residues. Relative molecular masses of the precursor and mature proteins were calculated to be 43838 and 40929 Da, respectively.
Proteinase yscE of yeast shows homology with the 20 S cylinder particles of Xenopus laevis
Authors: Kleinschmidt Jürgen A. , Escher Claudia , Wolf Dieter H.
Published: July 2002

Proteinase yscE of the yeast Saccharomyces cerevisiae has been compared with the 20 S cylinder particles of Xenopus
laevis. Both proteins are characterized by a similar group of 10–12 polypeptides with molecular masses ranging between 21 and 38 kDa. Antibodies generated against the 20 S Xenopus cylinder particles show cross‐reactivity with yeast proteinase yscE subunits. The Xenopus particles and yeast proteinase yscE exhibit an identical image in electron microscopy. Both proteins appear as hollow cylinders mostly composed of four stacked annuli. The Xenopus 20 S particles exhibit proteolytic activity against the three peptide derivatives known to be substrates of proteinase yscE. The pH optimum for activity and the inhibition spectrum of the proteolytic activities of Xenopus 20 S particles and of yeast proteinase yscE are identical. The RNA content of the cylinder particles and of proteinase yscE is below 0.1 RNA chain per molecule. Our data suggest that proteinase yscE from yeast and the 20 S cylinder particles of X. laevis are homologous, highly conserved proteins carrying the catalytic character of a peptidase.
Determination of three‐dimensional structures of proteins from interproton distance data by dynamical simulated annealing from a random array of atoms Circumventing problems associated with folding
Authors: Nilges Michael , Clore G.Marius , Gronenborn Angela M.
Published: July 2002

A new real space method, based on the principles of simulated annealing, is presented for determining protein structures on the basis of interproton distance restraints derived from NMR data. The method circumvents the folding problem associated with all real space methods described to date, by starting from a completely random array of atoms and introducing the force constants for the covalent, interproton distance and repulsive van der Waals terms in the target function appropriately. The system is simulated at high temperature by solving Newton's equations of motion. As the values of all force constants are very low during the early stages of the simulation, energy barriers between different folds of the protein can be overcome, and the global minimum of the target function is reliably located. Further, because the atoms are initially only weakly coupled, they can move essentially independently to satisfy the restraints. The method is illustrated using two examples of small proteins, namely crambin (46 residues) and potato carboxypeptidase inhibitor (39 residues).
Calcium‐dependent KEX2‐like protease found in hepatic secretory vesicles converts proalbumin to albumin
Authors: Brennan Stephen O. , Peach Robert J.
Published: July 2002

The yeast KEX2 protease is the only enzyme that has a proven role in the activation of polypeptide hormones through cleavage at pairs of basic residues. The enzyme that fulfils this role in higher eukaryotes has yet to be unequivocally identified. In this investigation, a KEX2‐like calcium‐dependent protease has been identified in rat hepatic microsomes. The enzyme is membrane‐bound, has a pH optimum of 5–6 and converts proalbumin to albumin. More importantly, like the KEX2 protease, it meets two other exacting criteria defined by specific mutations in humans. Namely, it does not process proalbumin Christchurch (−1 Arg→Gln) which lacks one of the requisite basic residues and, whilst not itself a serine protease, it is inhibited by the reactive center variant, α1‐antitrypsin Pittsburgh (358 Met→Arg) but not by normal α1‐antitrypsin.
Dopamine β‐hydroxylase from bovine adrenal medulla contains covalently‐bound pyrroloquinoline quinone
Authors: van der Meer Robert A. , Jongejan Jacob A. , Duine Johannis A.
Published: July 2002

Treatment of homogeneous dopamine β‐hydroxylase (DBH) preparations from bovine adrenals with the inhibitor phenylhydrazine (PH) changed the structureless absorption spectrum of DBH into spectra with a maximum at 350 nm. A product with this absorption spectrum could be detached with pronase, enabling its isolation. It appeared to be the C(5) hydrazone of pyrroloquinoline quinone (PQQ) and PH, as judged from its properties and the fact that it could be transformed into PQQ itself. From the yield obtained a ratio of 0.85 PQQ per enzyme subunit was calculated. In contrast to copper‐quinoprotein amine oxidases (EC 1.4.3.6), hydrazone formation in DBH did not require saturation of the mixture with O2. DBH is the first copper‐quinoprotein hydroxylase found so far. The implications of this finding for the current views on mechanism of action and inhibition by hydrazines are discussed. The success of the recently developed ‘hydrazine method’ [(1987) FEBS Lett. 221, 299‐304] for all different types of amine oxidoreductases, suggest that the method could also be applied to other enzymes for which hydrazines are inhibitors and where the identity of the cofactors has not been established or the presence of PQQ is suspected.
Protein immobilization on the surface of liposomes via carbodiimide activation in the presence of N‐hydroxysulfosuccinimide
Authors: Bogdanov A.A. , Klibanov A.L. , Torchilin V.P.
Published: July 2002

A method of the covalent immobilization of proteins on the surface of liposomes, containing 10% (by mol) of N‐glutaryl phosphatidylethanolamine, is described. Carboxylic groups of liposomal N‐glutaryl phosphatidylethanolamine were activated in the presence of water‐soluble carbodiimide and N‐hydroxysulfosuccinimide and reacted subsequently with protein amino groups. The liposome‐protein conjugates formed contained up to 5 × 10−4 mol protein/mol lipid. Lectins (RCAI and WGA) upon immobilization on liposomes retained saccharide specificity and the ability to agglutinate red blood cells. The immobilization of mouse monoclonal IgG in a ratio of 3.5 × 10−4 mol IgG/mol lipid was achieved. The liposome activation in the absence of N‐hydroxysulfosuccinimide resulted in a 2‐fold decrease of protein coupling yields.
A methyltransferase for synthesis of the phytoalexin 6‐methoxymellein in carrot cells
Authors: Kurosaki Fumiya , Nishi Arasuke
Published: July 2002

Carrot (Daucus carota L.) roots treated with 2‐chloroethylphosphonic acid or uronide elicitor accumulated the phytoalexin 6‐methoxymellein. The extracts of these cells catalyzed methylation of 6‐hydroxymellein to 6‐methoxymellein with S‐adenosyl‐L‐methionine as a methyl donor. Activity of the O‐methyltransferase was not found in fresh carrot roots but increased when they were treated with either type of elicitor in parallel with the increase in 6‐methoxymellein content. The enzyme did not methylate 6‐methoxymellein, 3,4‐dehydro‐6‐methoxymellein, and some coumarin derivatives, though 3,4‐dehydro‐6‐hydroxymellein accepted methyl moiety at the same rate as in 6‐hydroxymellein.
Expression of human cathepsin B protein in Escherichia coli
Authors: Chan Marion Man-Ying , Fong Dunne
Published: July 2002

A cDNA fragment containing the coding sequence for the mature enzyme of human lysosomal proteinase cathepsin B was inserted in the pET plasmid expression vectors, so that it was placed under the control of transcription and translation signals from bacteriophage T7. Upon induction, cathepsin B antigen was detected by in situ immunoscreening of lysed E. coli and by Western blot analysis of bacterial lysates. To our knowledge this is the first report of abundant synthesis of cloned cathepsin B in any expression system. Subfragments of cathepsin B can also be generated by this technique and will be used to study cathepsin B structure and function.
Zn2+ enhances protein tyrosine kinase activity of human platelet membranes
Authors: Findik Duygu , Presek Peter
Published: July 2002

In human platelet membranes enhanced tyrosine phosphorylation of certain proteins was observed when Zn2+ instead of Mg2+ or Mn2+ was used as a divalent cation for the kinase reaction. An enhanced level of phosphate incorporation into tyrosine residues occurred into a 68 kDa polypeptide besides the 45 kDa and 105 kDa proteins. Preincubation of platelet membranes with TBR‐IgG showed a concentration‐dependent inhibition of the phosphorylation of the 45, 68 and 105 kDa proteins. Moreover, pp60c‐src, representing the major protein tyrosine kinase activity in platelets, was found to be stimulated by Zn2+. The data, thus, support the assumption that pp60c‐src kinase is responsible for Zn2+ stimulated tyrosine phosphorylation.
A rapid purification method for calf thymus casein kinase II
Authors: Zandomeni R. , Zandomeni M.C. , Weinmann R.
Published: July 2002

We report here the rapid purification to homogeneity of a cyclic nucleotide‐independent protein kinase sensitive to 5,6‐dichloro‐1‐β‐D‐ribofuranozylbenzimidazole (DRB), identical to the previously described casein kinase II, from lyophilized calf thymus by chromatography on phosphocellulose and Mono‐Q FPLC columns.
Preformed PAF‐acether and lyso PAF‐acether are bound to blood lipoproteins
Authors: Benveniste Jacques , Nunez Daniele , Duriez Patrick , Korth Ruth , Bidault Jocelyne , Fruchart Jean-Charles
Published: July 2002

PAF‐acether (PAF) is a newly formed mediator not normally present in circulating blood. A compound exhibiting all of its biological characteristics but coeluting with phosphatidylcholine (PC) in high‐pressure liquid chromatography (HPLC) was unveiled (‘peak X’) in normal human plasma. A second HPLC run of peak X HPLC fractions revealed the presence of PAF itself with concomitant disappearance of peak X. Beside PAF, immunoreactive apolipoproteins A‐I and E were found in peak X. Also lipoproteins (Ls) purified using either ultracentrifugation or immunoaffinity chromatography yielded peak X and, in a second HPLC run, authentic PAF. L‐free plasma was devoid of peak X. Finally, after preincubation with plasma, labeled PAF was found associated with Ls. Thus in human blood preformed PAF is bound in high amounts to Ls, a result of interest given the role of Ls and platelets in vascular diseases and the present knowledge on PAF biosynthesis.
Amino acid sequence of locust neuroparsins
Authors: Girardie Josiane , Girardie Adrien , Huet Jean-Claude , Pernollet Jean-Claude
Published: July 2002

Neuroparsins A and B were isolated from the nervous part of the corpus cardiaca of Locusta migratoria via a two‐step purification procedure. Both consist of two polypeptide chains linked by disulfide bridges. The N‐terminal sequence of both native neuroparsins was determined: the N‐terminal end of neuroparsin B was unique while that of neuroparsin A showed three different sequences. These sequences were that of neuroparsin B and two others having five and two extra N‐terminal residues. Neuroparsin B was found as a homodimer and the complete sequence of the monomer, determined from peptide fragments generated by treatment with cyanogen bromide and endoprotease Glu‐C, comprises 78 residues.
Nucleotide sequence and organization of potato leafroll virus genomic RNA
Authors: van der Wilk Frank , Huisman Marianne J. , Cornelissen Ben J.C. , Huttinga Harm , Goldbach Rob
Published: July 2002

The nucleotide sequence of the genomic RNA of potato leafroll virus was determined and its genetic organization deduced. The RNA is 5882 nucleotides long and contains 6 open reading frames (ORFs) encoding proteins of 70, 70, 56, 28, 23 and 17 kDa. The putative genes for the coat protein (23 kDa) and the RNA‐dependent RNA polymerase (70 kDa) were identified by interviral amino acid sequence homologies. For expression of the different ORFS, translational frameshift and readthrough events are proposed.
A new species of enkephalin precursor mRNA with a distinct 5′‐untranslated region in haploid germ cells
Authors: Yoshikawa Kazuaki , Maruyama Kei , Aizawa Takako , Yamamoto Akihiro
Published: July 2002

To elucidate the primary structure of preproenkephalin (A) mRNA expressed by haploid germ cells (round spermatids) in rat testis, we have screened a λgt11 cDNA library for preproenkephalin cDNA inserts. The largest cDNA insert contained a protein‐coding sequence encoding 269 amino acid residues as well as 327 and 309 bases of the 5′‐ and 3′‐untranslated regions, respectively. The protein‐coding region plus 3′‐untranslated region of the mRNA was over 99% homologous to that of brain preproenkephalin mRNA, whereas the 5′‐untranslated region contained a distinct sequence including a partial sequence of intron A of the preproenkephalin gene [(1984) J. Biol. Chem. 259, 14301–14308; (1984) J. Biol. Chem. 259, 14309–14313]. Northern blot analysis using a 5′‐end‐specific probe showed that this type of preproenkephalin mRNA exists exclusively in the germ cells.
The nature of CuA in cytochrome c oxidase
Authors: Li Peter Mark , Malmström Bo G. , Chan Sunney I.
Published: July 2002

Kroneek et al. [(1988) FEBS Lett. 242, 70–74] have recently suggested, on the basis of a comparison with the EPR properties of nitrous oxide reductase, that cytochrome c oxidase contains a mixed‐valence binuclear copper site, and that this is responsible for the EPR spectrum generally ascribed to CuA. Here we question this hypothesis in view of a multitude of analytical and spectroscopic data available. We maintain that a mononuclear Cu site with two cysteine sulfur and two imidazole nitrogen atoms as ligands is consistent with the current experimental information on the CuA site.
Reconstitution of apo‐porphobilinogen deaminase: Structural changes induced by cofactor binding
Authors: Scott A.Ian , Clemens Karen R. , Stolowich Neal J. , Santander Patricio J. , Gonzalez Mario D. , Roessner Charles A.
Published: July 2002

Expression of porphobilinogen deaminase in a hemB
− strain of E. coli has permitted the isolation of the apoenzyme, i.e. deaminase lacking the porphobilinogen‐derived dipyrromethane cofactor. Incubation of purified apoenzyme with porphobilinogen resulted in reconstitution of the covalently attached dipyrromethane cofactor, indicating no additional cofactors or enzymes are required for biosynthesis of holoenzyme. Electrophoretic and 13C‐NMR spectroscopic analyses demonstrate that the apoenzyme exists in a conformationally unstable form which is converted to a highly stable tertiary structure on covalent attachment of the dipyrromethane cofactor.
An endogenous ‘hypertensive factor’ enhances the voltage‐dependent calcium current
Authors: Simmons Mark A. , Johnson Edwin C. , Becker James B. , Todd Daniel G. , Reichenbecher Vernon E. , McCumbee William D. , Wright Gary L.
Published: July 2002

The effects of an immunoaffinity‐purified putative endogenous hypertensive factor (HF) on voltage‐dependent calcium current in frog cardiac myocytes were assessed. In 9 out of 10 cells, HF reversibly increased the peak amplitude of the calcium current. HF increased peak calcium current density at −5 mV from a control level of 1.8 ± 1.3 pA/pF (mean ± SD) to 4.4 ± 2.0 pA/pF. HF shifted the peak of the calcium current‐voltage relationship in the hyperpolarizing direction. HF shifted the voltage dependence of the inactivation of the calcium current to more negative potentials with prepulses from −80 to 0 mV, but the inactivation was not affected with prepulses more positive than 0 mV. Modulation of the voltage‐dependent calcium current by HF may be the mechanism underlying its pressor effects.
Critical spacing between two essential cysteine residues in the interdomain linker of the Bradyrhizobium japonicum NifA protein
Authors: Fischer Hans-Martin , Fritsche Stefan , Herzog Brigitte , Hennecke Hauke
Published: July 2002

A special sequence motif in the Bradyrhizobium japonicum NifA protein, consisting of two functionally essential cysteines separated by four other amino acids (Cys‐aa4‐Cys), has been proposed to be part of a potential metal‐binding site [(1988) Nucleic Acids Res. 16, 2207–2224]. Using the techniques of oligonucleotide‐directed mutagenesis, we report here that several of the four intervening amino acids can be replaced by others without loss of NifA function. The deletion of one amino acid to give a Cys‐aa3‐Cys motif renders the protein inactive whereas the creation of a Cys‐aa5‐Cys motif (one amino acid inserted) still leads to a partially active NifA protein.
RNA degrading activity is tightly associated with the multicatalytic proteinase, ingensin
Authors: Tsukahara Toshifumi , Tanaka Ken-ichi , Ogawa Toshio , Ishiura Shoichi , Funabiki Ryuhei , Sugita Hideo
Published: July 2002

The multicatalytic proteinase, ingensin, was purified to homogeneity from chicken liver. rRNA‐degrading activity was co‐eluted with the purified multicatalytic proteinase from a TSK‐3000SW column. This RNA‐degrading activity was inactivated by heat treatment and the addition of a low concentration of SDS. Therefore, the RNA‐degrading activity co‐eluted with the multicatalytic proteinase was not due to contamination by low‐molecular‐mass RNases. These results strongly suggest that this RNA‐degrading activity was tightly associated with the multicatalytic proteinase, ingensin.
Nucleic acids can regulate the activity of casein kinase II
Authors: Gatica Marta , Allende Catherine C. , Allende Jorge E.
Published: July 2002

Casein kinase II purified from nuclei of Xenopus laevis oocytes is inhibited by several specific nucleic acids. This kinase, the main phosphorylating activity of the oocyte nucleus, is markedly inhibited by poly U at 10 μg/ml, and this polymer is a competitive inhibitor of the phosporylation of the substrate casein (Kiapp 80 nM). M 13 phage ssDNA and unfractionated yeast tRNA also inhibit between 50 and 200 μg/ml. Poly C, poly A, poly AG, dsDNA and Escherichia coli rRNA do not alter activity significantly at similar concentrations. Inhibitions are reversed by RNase (poly U, tRNA) or S1 nuclease (ssDNA). Oocyte casein kinase I or rabbit cAMP‐dependent protein kinase are not inhibited by poly U at 200 μg/ml. The sensitivity of the casein kinase II to these inhibitors suggests a regulatory role for nucleic acids in nuclear phosphorylation reactions.
Soluble, nitrate/nitrite‐inducible cytochrome P‐450 of the fungus, Fusarium oxysporum
Authors: Shoun Hirofumi , Suyama Wakako , Yasui Tsuneo
Published: July 2002

Both soluble and microsomal fractions of Fusarium oxysporum contain cytochrome P‐450(P‐450). We report here that the P‐450 in the soluble fraction was induced only when nitrate or nitrite was added to the growth medium, whereas the microsomal P‐450 was synthesized regardless of the medium compositions. The reduced‐CO complex of the soluble P‐450 exhibited an absorption spectrum that is different from that of the microsomal counterpart. These results indicate that the soluble P‐450 is distinct from the microsomal species and suggest a novel function for the former P‐450.
Production of antibodies against the coenzyme pyrrolequinoline quinone
Authors: Citro G. , Verdina A. , Galati R. , Floris G. , Sabatini S. , Finazzi-Agrò A.
Published: July 2002

Polyclonal antibodies against pyrrolequinoline quinone have been elicited in rabbits. These antibodies react with free and protein‐bound pyrrolequinoline quinone. In particular they react with native and denatured lentil seedling amine oxidase as detected by dot‐blot and ELISA assays. The presence of 1 mol pyrrolequinoline quinone per mol of enzyme was determined by the last method.
The in vivo phosphorylation sites of bovine αB‐crystallin
Authors: Voorter Christina E.M. , de Haard-Hoekman Willeke A. , Roersma Eric S. , Meyer Helmut E. , Bloemendal Hans , de Jong Wilfried W.
Published: July 2002

Phosphate content determinations established that in αB‐crystallin two phosphate groups can be present in vivo in bovine lenses. Comparison of tryptic digests of phosphorylated and unphosphorylated αB chains, revealed the location of the two phosphorylation sites in tryptic peptides T2 and T3. Thermolytic digestion and gas‐phase sequencing demonstrated that Ser‐19 and Ser‐45 are the in vivo phosphorylation sites of bovine αB‐crystallin. This pattern of phosphorylation differs from the previously reported in vitro obtained results.
The algorithm of estimation of the Km values for primers of various structure and length in the polymerization reaction catalyzed by Klenow fragment of DNA polymerase I from E.coli
Authors: Nevinsky G.A. , Nemudraya A.V. , Levina A.S. , Khomov V.V.
Published: July 2002

DNA synthesis at primers d(pT)
n
, d(pA)
n
, d(pC)
n
, and d(pG)
n
in the presence of corresponding complementary templates and at hetero‐oligoprimers complementary to MB phage DNA was investigated. The values of both ‐log K
m and log V
max increased linearly if homo‐oligoprimers contained less than 10 nucleotides. The lengthening of d(pT)
n
and d(pA)
n
primers by one mononucleotide unit (n = 1–10) resulted in the 1.82‐fold decrease of the K
m values. The incremental decreases of K
m for d(pC)
n
and d(pG)
n
were equal to about 2.46. The enhancement of the homo‐ and heterooligonucleotide primers' affinity to the enzyme due to one Watson‐Crick hydrogen bond between complementary template and primer is about 1.35 times. This allows to calculate the K
m values for primers of various structure and length up to 10 units. The objective laws of the K
m and V
max values changes for primers containing more than 10 nucleotides were analyzed.
Alcohols selectively stimulate phospholipase D‐mediated hydrolysis of phosphatidylethanolamine in NIH 3T3 cells
Authors: Kiss Zoltan , Anderson Wayne B.
Published: July 2002

Alcohol; Phospholipase D; Phosphatidylethanolamine hydrolysis
Ultracentrifugal analysis of the quaternary structure of the raf represser from Escherichia coli
Authors: Jaenicke Rainer , Muiznieks Indrikis , Aslanidis Charalampos , Schmitt Rüdiger
Published: July 2002

The raf repressor from Escherichia coli regulates the expression of the plasmid‐borne raf operon by switching between active and inactive conformational states. Ultracentrifugal analysis of the largely purified repressor proves the DNA‐free protein to undergo concentration‐dependent dissociation‐association. High‐speed sedimentation equilibria show that the 72 kDa dimer prevails under meniscus depletion conditions. At intracellular concentrations the 144 kDa dimer‐of‐dimers is the dominating species. It is suggested that the tetrameric structure of the raf repressor is involved in the recognition of the 18‐basepair operator DNA.
Molecular cloning of cDNA for vitamin D3 25‐hydroxylase from rat liver mitochondria
Authors: Usui Emiko , Noshiro Mitsuhide , Okuda Kyuichiro
Published: July 2002

A cDNA clone encoding mitochondrial vitamin D3 25‐hydroxylase was isolated from a rat liver cDNA library by the use of specific antibodies to the enzyme. The isolated cDNA clone was 1.9 kbp long and contained a 1599 bp open reading frame encoding 533 amino acid residues. The deduced primary structure contained a presequence typical for mitochondrial enzymes in the N‐terminal region. The N‐terminal sequence of the mature enzyme was determined to be Ala‐Ile‐Pro‐Ala‐Ala, which agrees perfectly with a portion of the deduced sequence, establishing the cleavage point of the precursor.
Activation of protein kinases A and C increase lymphocyte penetration through endothelial monolayers
Authors: Renkonen Risto
Published: July 2002

A brief incubation of lymphocytes with either PMA, stimulating protein kinase C, or with dibutyryl‐cAMP, leading to protein kinase A activation, led to increased lymphocyte penetration through intact endothelial monolayers in vitro. The PMA‐induced penetration could be dose‐dependently down‐regulated with a protein kinase C inhibitor, H7. Similarly HA 1004, being mainly a protein kinase A inhibitor, decreased the dibutyryl‐cAMP induced penetration. Treatment of lymphocytes with PMA and cAMP did not alter the expression of CD44 homing receptors on lymphocytes. Stimulation of lymphocytes with dibutyryl‐cGMP or calcium ionophore had no effect on lymphocyte penetration. These results suggest that activation of both protein kinases A and C is important in the lymphocyte binding to endothelium.
High‐affinity binding of a synthetic heptaglucoside and fungal glucan phytoalexin elicitors to soybean membranes
Authors: Cosio Eric G. , Frey Thomas , Verduyn Richard , van Boom Jacques , Ebel Jürgen
Published: July 2002

Soybean membranes possess high‐affinity binding sites for fungal β‐glucans that elicit phytoalexin synthesis. The ability of 1,3‐1,6‐β‐glucans, released by acid hydrolysis from mycelial walls of Phytophthora megasperma f.sp. glycinea, to compete for the putative phytoalexin elicitor receptors increases with their average degree of polymerization (DP). The results suggest a function where the probability for glucan fragments of containing a structural determinant that is optimal for binding approaches 1 as the DP tends to infinity. Ligand displacement data obtained against a 125I‐labeled glucan elicitor (average DP= 18) provided a theoretical minimum IC50 (50% inhibitory concentration) for 1,3‐1,6‐β‐glucans of 3 nM. The IC50 value obtained for a synthetic hepta‐β‐glucoside having a known elicitor‐active structure was 8 nM, remarkably close to the predicted value. Displacement of the 125I‐glucan of large DP was uniform and complete showing that the heptaglucoside had access, with similar affinity, to all sites available to the radioligand. Further analysis using a 125I‐labeled aminophenethylamine derivative of the heptaglucoside suggested that the putative glucan‐elicitor receptors bind a basic structural determinant present in all elicitor‐active glucans from the soybean pathogen P. megasperma.
Reductive trapping of substrate to methylamine oxidase from Arthrobacter P1
Authors: Hartmann Christa , Klinman Judith P.
Published: July 2002

Methylamine oxidase (EC 1.4.3.6) from Arthrobacter P1 was inactivated by NaCNBH3 in the presence of [14C]benzylamine, leading to the incorporation of 1 mol of radiolabeled substrate/mol of enzyme subunit at complete inactivation. By contrast, no labeling of enzyme was observed using [3H]NaCNBH3 as reductant. These results are analogous to those previously reported for the eukaryotic enzyme, bovine serum plasma anaine oxidase [(1987) J. Biol. Chem. 262, 962‐965]. The observed pattern of labeling is consistent with the presence of dicarbonyl cofactor at the active site of methylamine oxidase. Further, these studies suggest that our reductive trapping technique, in which the pattern of radiolabeling of an enzyme is compared using C‐14 substrate vs tritiated reductant, may serve as a general assay for covalently bound dicarbonyl structures.
Regulation of photosynthetic light harvesting by nitrogen assimilation in the green alga Selenastrum minutum
Authors: Turpin David H. , Bruce Doug
Published: July 2002

The interaction of whole cell metabolism with the distribution of excitation energy between photosystem 2 (PS2) and photosystem 1 (PS1), the light state transition, was investigated in vivo in the green alga Selenastrum
minutum. Nitrogen limited cells of S. minutum were presented with a pulse of either NH4
+ or NO3
− in the light. As shown previously, CO2 fixation is inhibited and high rates of N assimilation ensue [(1986) Plant Physiol. 81, 273–279]. NH4
+ assimilation has a much higher requirement ratio for ATP/NADPH than either CO2 or NO3
− assimilation and thus drastically increases the demand for ATP relative to reducing power. Room temperature chlorophyll a fluorescence kinetic measurements showed that a reversible non‐photochemical quenching of PS2 fluorescence accompanied the assimilation of NH4
+ but not the assimilation of NO3
− or CO2. 77K fluorescence emission spectra taken from samples removed at regular intervals during NH+
4assimilation showed that the non‐photochemical quenching of PS2 was accompanied by a complementary increase in the fluorescence yield of PS1, characteristic of a transition to state 2. Our data suggests that S. minutum responds to the increased demand for ATP/NADPH during NH4 assimilation by inducing the light state transition to direct more excitation energy to PS1 at the expense of PS2 to increase the production of ATP by cyclic electron transport.
Effect of heparin on proliferation and signalling in BC3H‐1 muscle cells
Authors: Vannucchi Simonetta , Pasquali Franca , Fiorelli Gianna , Biabchini Pietro , Ruggiero Marco
Published: July 2002

We studied binding and growth inhibitory properties of different glycosaminoglycans in growing and differentiated BC3H‐1 muscle cells. Heparin (10 ) and heparan sulfate (10 ) significantly inhibited DNA synthesis in growing and differentiated cells, as monitored by [3H] thymidine incorporation. Binding of heparin to BC3H‐1 cells was specific and time‐dependent. Heparan sulfate was the only glycosaminoglycan able to displace [3H]heparin (IC50, 3.2 × 10−7 M), although it was 10‐fold less effective than heparin itself (IC50 3.6 × 1O−8 M). Scatchard analysis revealed the existence of high‐affinity hepariy binding sites (Kd, 5 × 10−8 M). Furthermore, heparin inhibited serum‐induced stimulation of inositol lipid turnover. Taken together, these results indicate that heparin inhibits BC3H‐1 cell growth by interacting with the cell surface, possibly disrupting the flow of growth factor‐related mitogenic signalling.
Effect of interleukin‐1β on the production of cathepsin B by rabbit articular chondrocytes
Authors: Baici Antonio , Lang Angela
Published: July 2002

Rabbit articular chondrocytes in monolayer culture were stimulated with human recombinant interleukin‐1β. Under the influence of the cytokine the intracellular pool of the cysteine endopeptidase cathepsin B was increased by a 2‐4‐fold factor, while enzyme secretion was not stimulated at a significant level. Under the same conditions, the secretion of collagenase, measured as an internal control, was stimulated about 6‐fold. The effects of interieukin‐1β were compared to those caused by phenotypic modulation. Chondrocytes modulated by serial subcultures in monolayer secreted more cathepsin B, but less collagenase than differentiated cells (cultured within collagen gels). Thus, interleukin‐1β and phenotypic modulation affected differently two endopeptidases which are relevant in the pathogenesis of osteoarthritis.
Light‐dependent Δ\̄smNa‐generation and utilization in the marine cyanobacterium Oscillatoria brevis
Authors: Brown I.I. , Fadeyev S.I. , Kirik I.I. , Severina I.I. , Skulachev V.P.
Published: July 2002

Light‐dependent Na+ and H+ transports, membrane potential (Δψ) and motility have been studied in the cells of the marine cyanobacterium Oscillatoria brevis. In the presence of a protonophorous uncoupler, carbonyl cyanide‐m‐chlorophenylhydrazone, the intracellular Na+ level is shown to increase in the dark and decrease in the light. The Na+/H+ antiporter, monensin, stimulates the dark CCCP‐dependent [Na+]in increase and abolishes the light‐dependent [Na+]in decrease. Na+ ions are necessary for the fast light‐induced Δψ generation and H+ uptake by the cells. This uptake is inhibited by monensin being resistant to CCCP. Monensin sensitizes the Δψ level and the motility rate to low CCCP concentrations. The obtained data are consistent with the assumption that O. brevis possesses a primary Na+ pump which utilizes (directly or indirectly) the light energy.
Expression and pharmacological characterization of the human M1 muscarinic receptor in Saccharomyces cerevisiae
Authors: Payette Paul , Gossard Francis , Whiteway Malcolm , Dennis Michael
Published: July 2002

The yeast S. cerevisiae has been examined as a heterologous host for the expression of mammalian neurotransmitter receptors which couple to guanine nucleotide regulatory (G) proteins. A cloned gene encoding the M1 subtype of human muscarinic receptor (HM1) was transformed into S. cerevisiae on a high copy plasmid under the control of the promoter for the yeast alcohol dehydrogenase (ADH) gene. Northern blotting demonstrated the presence of HM1 transcripts in transformants, and crude membranes prepared from these cells showed saturable binding of the muscatrinic antagonist [3H] N‐methyl scopolamine with a K
d of 179 pM and B
max of 20 fmol/mg protein. Competition binding studies revealed pharmacological properties for these sites which were comparable to those reported for the M1 site in mammalian tissues. Yeast expressing HM1 did not exhibit high affinity agonist binding or cell‐cycle arrest in the presence of muscarinic agonists, indicating that the mammalian receptor did not couple to the endogenous yeast G protein.
Location and characterization of the three carbohydrate prosthetic groups of human protein HC
Authors: Escribano J. , Lopex-Otin C. , Hjerpe A. , Grubb A. , Mendez E.
Published: July 2002

Three different carbohydrate prosthetic groups associated to three chymotryptic peptides, Ql, Q2 and Q3, were isolated from the reduced and carboxymethylated human protein HC. The first oligosaccharide forms an O‐glycosidic linkage with a threonine residue at position 5 in the polypeptide chain of protein HC. The second and third carbohydrate prosthetic groups form N‐linkages with asparagine residues at positions 17 and 96. Oligosaccharides present in Q1 contain 1 residue of NANA, 2 of Ga1NAc and 1 of Gal corresponding to the following structure: ‐O‐Ga1NAc‐Ga1NAc‐Gal‐NANA. Q2 contains 3 NANA, 9 GlcNAc, 2 Gal and 3 Man, and Q3 contains 2 NANA, 5 GlcNAc, 1 Gal and 2 Man. The sugar compositions of Q2 and Q3 oligosaccharides are compatible with that of the complex kind. The amount of oligosaccharides present in Q1, Q2 and Q3 corresponded respectively to 3.0%, 12.2% and 7.3% of the weight of protein HC. No difference was found between the carbohydrate composition of urinary and plasma protein HC.
Cloning and sequencing of a leech homolog to the Drosophila engrailed gene
Authors: Wedeen Cathy J. , Price David J. , Weisblat David A.
Published: July 2002

We have cloned and sequenced a homolog (ht‐en) to the Drosphila engrailed (en) gene from the glossiphoniid leech, Helobdella triserialis. Amino acid comparisons of the ht‐en homeodomain and C‐terminal residues with the corresponding residues encoded by en‐class genes of other species reveal 75–79% sequence identity. In addition, the ht‐en sequence appears to have u serine‐rich region 16 residues C‐terminal from the homeodomain, which by analogy to Drosophila may be a target site for phosphorylation. The leech gene encodes some amino acid substitutions for residues that are highly conserved in other species. These are found within the second and third of the three putative helices of the homeodomain, and in both of the intervening turn regions.
Co‐purification of a small RNA species with multicatalytic proteinase (proteasome) from rat liver
Authors: Skilton Helen E. , Eperon Ian C. , Rivett A.Jennifer
Published: July 2002

Previous studies have come to different conclusions about the presence of RNA in particles known variously as prosomes, proteasomes or multicatalytic proteinase (MCP). To determine the reason for this, MCP was isolated from rat liver by 4 different purification protocols. One major band of RNA, about 80 nucleotides in length, co‐purified in all preparations. The amount of RNA detected was less than one molecule per MCP particle suggesting that there may be more than one population of MCP in rat liver cells.
The complete amino acid sequence of bovine cathepsin S and a partial sequence of bovine cathepsin L
Authors: Ritonja A. , Čolić A. , Dolenc I. , Ogrinc T. , Podobnik M. , Turk V.
Published: July 2002

The complete amino acid sequence of bovine spleen cathepsin S has been determined. The single‐chain protein 217 residues and has a M
r of 23 682. The primary structure was determined by sequencing of native protein and the peptides obtained by proteolytic cleavage with β‐trypsin, papaya proteinase IV and by chemical cleavage with cyanogen bromide. Comparison of the amino terminal sequences of the heavy and the light chain of bovine cathepsin L with that of bovine cathepsin S clearly indicates that the enzymes are structurally different.
The plasma level of 7α‐hydroxy‐4‐cholesten‐3‐one reflects the activity of hepatic cholesterol 7α‐hydroxylase in man
Authors: Axelson Magnus , Björkhem Ingemar , Reihnér Eva , Einarsson Kurt
Published: July 2002

Circulating levels of 7α‐hydroxy‐4‐cholesten‐3‐one have been compared with activities of the rate‐limiting enzyme in bile acid synthesis, microsomal cholesterol 7α‐hydroxylase, measured in liver biopsies obtained from patients undergoing surgery for gallstone disease. Some patients were treated with cholestyramine or bile acids prior to operation in order to alter the feed‐back inhibition of the enzyme. The levels of the sterol were similar in untreated patients and in patients treated with ursodeoxycholic acid (median concentration 17 and 13 ng/ml, respectively), and so were the activities of the enzyme (median activity 7.0 and 5.5 pmol/min/mg protein, respectively). The sterol levels and enzyme activities were significantly increased in patients treated with cholestyramine (91 ng/ml and 45 pmol/min/mg protein) and decreased in patients treated with chenodeoxycholic acid (<2.0 ng/ml and 0.7 pmol/min/mg protein). There was a strong positive correlation (r=0.90, P<0.00001) between levels of 7α‐hydroxy‐4‐cholesten‐3‐one in plasma and the activities of cholesterol 7α‐hydroxylase in the whole patient group. The results show that analysis of 7α‐hydroxy‐4‐cholesten‐3‐one in plasma is a sensitive and convenient method to determine relative rates of bile acid production in man.
Possible role of the highly conserved amino acids Trp‐8 and Pro‐13 in the N‐terminal segment of the pigment‐binding poly eptide LHI α of Rhodobacter capsulatus
Authors: Richter Petra , Cortez Néstor , Drews Gerhart
Published: July 2002

Trp‐8 and Pro‐13 of the Rhodobacter capsulatus light‐harvesting (LH) I α polypeptide are highly conserved among LHI and LHII α proteins of several species of the Rhodospirillaceae. Exchange of Trp‐8 and Pro‐13 to other amino acyl residues similar in structure and/or hydrophobicity indicates that Trp‐8 is involved in the insertion of the LHI α polypeptide into the intracytoplasmic membrane (ICM). Pro‐13, however, seems not to participate in the integration process of the LHI α protein but seems to be important for stable insertion of the LHI β partner protein in the ICM.
Degradation of cells dying by apoptosis leads to accumulation of ε(γ‐glutamyl)lysine isodipeptide in culture fluid and blood
Authors: Fesus Laszlo , Tarcsa Edit , Kedei Noemi , Autuori Francesco , Piacentini Mauro
Published: July 2002

ε(γ‐Glutamyl)lysine isodipeptide, the end‐product of proteolytic digestion of proteins cross‐linked by transglutaminase, was detected in culture fluid of neonatal rat hepatocytes and plasma of adult rats. The concentration of the isodipeptide was significantly increased in both when high rate of apoptosis with phagocytosis of dying hepatocytes was produced either by epidermal growth factor in the culture or by lead nitrate‐induced hyperplasia with subsequent involution in rats. Specific induction of tissue transglutaminase and the consequent formation of highly cross‐linked protein envelopes in apoptotic cells have been previously demonstrated by us in both systems.
Metaphase‐specific* phosphorylations weaken the association between chromosomal proteins HMG 14 and 17, and DNA
Authors: Lund Terje , Berg Kristian
Published: July 2002

The high‐mobility‐group proteins HMG 14 and 17 have been isolated from human cells arrested in metaphase. The affinity between an unphosphorylated and two phosphorylated forms of these proteins, and DNA has been investigated using columns of single‐stranded and double‐stranded DNA. It was shown that the most phosphorylated forms had much lower affinity for single‐stranded and double‐stranded DNA compared to the unphosphorylated form present in interphase cells. The results are in accordance with the view that HMG 14 and 17 may dissociate transiently from chromatin during mitosis.
Expression and functional assembly into bacterial ribosomes of a nuclear‐encoded chloroplast ribosomal protein with a long NH2‐terminal extension
Authors: Giese Klaus , Subramanian Alap R.
Published: July 2002

Chloroplast ribosomal protein LI 3 is encoded in the plant nucleus and is considerably larger than its eubacterial homologue by having NH2‐ and COOH‐terminal extensions with no homology to any known sequences (Phua et al., J. Biol. Chem. 264, 1968–1971, 1989). We made two gene constructs of LI3 cDNA using the polymerase chain reaction (PCR) and expressed them in Escherichia coli. Analysis of the ribosomes and polysomes from these calls, using an antiserum spelific to chloroplast LI3, shows that the expressed proteins are incorporated, in the presence of the homologous E. coli L13, into functional ribosomes which participate in protein synthesis (i.e. polysomes). Evidence is obtained that the large NH2‐terminal extension probably lies on the surface of these ‘mosaic ribosomes’. This first report of the assembly into E. coli ribosomes of a nuclear‐coded chloroplast ribosomal protein with terminal extensions thus suggests an extraordinary conservation in the function of eubactcrial type ribosomal proteins, despite the many changes in protein structure during their evolution inside a eukaryotic system.
Immunological identification and sequence characterization of a peptide derived from the processing of neuroendocrine protein 7B2
Authors: Paquet Luc , Rondeau Normand , Seidah Nabil G. , Lazure Claude , Chrétien Michel , Mbikay Majambu
Published: July 2002

A newly raised antiserum against the C‐terminal region of neuroendocrine protein 7B2 was used to purify a novel peptide from the culture media of the mouse corticotroph cell line AtT‐20. Based on partial sequencing, this peptide, which we call Cter‐7B2, begins at Ser156 and appears to result from the cleavage of pro7B2 after a five‐basic‐residue sequence. Thus, 7B2 processing may contribute to the diversity of peptides found in neuronal and endocrine cells.
p23, a novel mammalian nucleic acid‐binding protein with homology to the yeast ribosomal protein YL43
Authors: Østvold Anne Carine , Hullstein Ingunn , Sletten Knut
Published: July 2002

When separating perchloric acid‐soluble proteins from cell cultures and tissues by chromatography on single stranded DNA agarose columns, a novel mammalian protein with extreme affinity for DNA was isolated. Cellular localization, amino acid composition and the N‐terminal sequence suggest that the protein is a ribosomal protein with extensive sequence homology to the ribosomal protein, YL43, from Saccharomyces cerevisiae.
A delayed and sustained rise of cytosolic calcium is elicited by oxidized LDL in cultured bovine aortic endothelial cells
Authors: Nègre-Salvayre Anne , Fitoussi Guylène , Réaud Valérie , Pieraggi Marie-Thérèse , Thiers Jean-Claude , Salvayre Robert
Published: July 2002

Bovine aortic endothelial cells (BAEC) pulsed for 5 h with mildly oxidized low density lipoproteins (LDL), exhibited a broad, sustained and high peak of [Ca2+]i occurring several hours after the end of the pulse and reaching very high [Ca2+]i values (around 2500–3000 nmol/l) and a concomitant drop of cytosolic pH (around 0.2–0.3 pH units) without any loss of cell viability. When BAEC were continuously pulsed with oxidized LDL, the peak of [Ca2+]i was more sustained than in short pulse experiments and was associated with irreversible morphological changes usually associated with cytotoxic events (blebbing) and with a marked loss of viability. The potential involvement of these biochemical and morphological changes in atherogenesis are discussed.
Detection of elsamicin‐DNA binding specificity by restriction enzyme cleavage
Authors: Párraga Antonio , Portugal José
Published: July 2002

The sequence specificity of elsamicin A, an anti‐tumour antibiotic, binding to DNA was elucidated considering the inhibition of the rate of digestion of linearised pBR322 DNA by AnII, ClaI, EcoRI, HindIII and NruI restriction enzymes. Elsamicin A inhibits the rate of digestion by NruI (recognition sequence TCG/CGA) to a greater extent than it does for the other enzymes thus evidencing the sequence‐selective binding of elsamicin to CGC regions in DNA. Our results also show the important role of the neighbouring sequences in the elsamicin A‐DNA interactions and their effects on the cleavage by restriction enzymes.