bluuebunny/crossref_metadata_2025_split huggingface dataset:
In vivo and in vitro studies of the purine‐cytosine permease of Saccharomyces cerevisiae
Authors: BRÈTHES Daniel , CHIRIO Maria‐Chantal , NAPIAS Christian , CHEVALLIER Marie‐Renée , LAVIE Jean Louis , CHEVALLIER Jean 
Published: March 2005

The FCY2 gene of the purine‐cytosine permease (PCP) of Saccharomyces cerevisiae and the allele fcy2‐21 have been cloned on the yeast multicopy plasmid pJDB207. The corresponding plasmids were introduced into a S. cerevisiae strain carrying a chromosomal deletion at the FCY2 locus. The resulting strains were designated pAB4 and pAB25 respectively. The pAB25 strain, which carries the fcy2‐21 allele, contains four amino acid changes in the open reading frame of the PCP (Weber et al., 1989). The influence of these mutations was studied on cells by determination of the uptake constants of purine bases and cytosine [apparent Michaelis constant of transport (Ktapp) and Vmax] and on plasma‐membrane preparations, by measurements of binding parameters at equilibrium [Kd and maximum amount of binding sites/Bmax)].For strain pAB4, the Ktapp and Vmax of uptake were almost similar for all solutes considered [1.8—2.6 μM and 8.5—10.2 nmol · min−1· (107 cells)−1]. The main effect of the mutations in strain pAB25 was based on a large increase in Ktapp for all ligands except adenine.Plasma membranes of each strain displayed one class of specific binding sites. Variations in Kd of 0.4—1 μM were observed for pAB4. These slight variations had no effect on the Ktapp of uptake measured for the corresponding solutes. In contrast, using pAB25 membranes, Kd increased dramatically; 2.6 μM, 40 μM and 96 μM for adenine, cytosine and hypoxanthine, respectively. These increments were correlated to variations in Ktapp of the uptake for cytosine and hypoxanthine.Therefore, we conclude that modification in the Ktapp of uptake in the strain carrying fcy2‐21 allele is merely due to a modification of the binding ability of the permease for its ligands.
Human‐immundeficiency‐virus‐type‐1‐encoded Vpu protein is phosphorylated by casein kinase II
Authors: SCHUBERT Ulrich , SCHNEIDER Thomas , HENKLEIN Peter , HOFFMANN Klaus , BERTHOLD Evelyn , HAUSER Hansjörg , PAULI Georg , PORSTMANN Tomas 
Published: March 2005

Vpu as a human‐immunodeficiency‐virus‐type‐1‐encoded 81‐amino‐acid integral‐membrane protein was expressed in Escherichia coli using the inducible ptrc promoter of an ATG fusion vector. Recombinant Vpu is associated with membranes of E. coli and could be partially solubilized by detergents. Recombinant Vpu was phosphorylated in vitro with purified porcine casein kinase II (CKII) as well as with a CKII‐related protein kinase found in cytoplasmic extracts of human and hamster cells. Recombinant Vpu associated with E. coli membranes has turned out to be the best substrate for in vitro phosphorylation with CKII. This reaction can be inhibited by heparin and the ATP analogue 5,6‐dichloro‐1‐(β‐D‐ribofuranosyl)benzimidazole (DRB), both known to be potent inhibitors of CKII. Radiolabelled γATP and γGTP were used as phosphate donors for in vitro phosphorylation of recombinant Vpu. In vivo phosphorylation of Vpu‐1‐infected H9 cells was also inhibited by DRB. We concluded therefrom that the Vpu protein is phosphorylated by the ubiquitous CKII in HIV‐1‐infected human host cells.Two seryl residues in the sequence of Vpu (position 52 and 56) correspond to the consensus s/TXXD/E for CKII. These potential phosphorylation sites are located within a well‐conserved dodecapeptide of Vpu (residues 47–58), which is found in different HIV‐1 strains as well as in a Vpu‐like protein of SIVCPZ.Monoclonal and polyclonal antibodies directed against two different epitopes of Vpu were used for immunoprecipitation of Vpu from HIV‐1‐infected cells and for detection of Vpu in Western blot analyses. Vpu from HIV‐1‐infected cells as well as recombinant Vpu expressed in E. coli were determined by SDS/PAGE using 6 M urea to be 9 kDa, which corresponds to the calculated molecular mass of Vpu.
NMR and EPR studies on a monoheme cytochrome c550 isolated from Bacillus halodenitrificans
Authors: SARAIVA Lígia M. , DENARIAZ Gerard , LIU Ming‐Y. , PAYNE William J. , Le GALL Jean , MOURA Isabel 
Published: March 2005

A c‐type monoheme ferricytochrome c550 (9.6 kDa) was isolated from cells of Bacillus halodenitrificans sp.nov., grown anaerobically as a denitrifier. The visible absorption spectrum indicates the presence of a band at 695 nm characteristic of heme–methionine coordination. The mid‐point redox potential was determined at several pH values by visible spectroscopy. The redox potential at pH 7.6 is 138 mV.When studied by 1H‐NMR spectroscopy as a function of pH, the spectrum shows a pH dependence with pKa values of 6.0 and 11.0. According to these pKa values, three forms designated as I, II and III can be attributed to cytochrome c550. The first pKa is probably associated with protonation of the propionate groups. The second pKa value introduces a larger effect in the 1H‐NMR spectrum and is probably due to the ionisation of the axial histidine. Studies of temperature variation of the 1H‐NMR spectra for both the ferrous and ferri forms of the cytochrome were performed. Heme meso protons, the heme methyl groups, the thioether protons, two protons from a propionate and the methylene protons from the axial methionine were identified in the reduced form. The heme methyl resonances of the ferri form were also assigned.EPR spectroscopy was also used to probe the ferric heme environment. A signal at gmax∼ 3.5 at pH 7.5 was observed indicating an almost axial heme environment. At higher pH values the signal at gmax∼ 3.5 converts mainly to a signal at g∼ 2.96. The pKa associated with this change is around 11.3.The N‐terminal sequence of this cytochrome was determined and compared with known amino acid sequences of other cytochromes.
Assignment of Bacillus thermoamylolique faciens KP1071 α‐glucosidase I to an exo‐α‐1,4‐glucosidase, and its striking similarity to bacillary oligo‐1,6‐glucosidases in N‐terminal sequence and in structural parameters calculated from the amino acid composition
Authors: SUZUKI Yuzuru , YONEZAWA Kazuaki , HATTORI Masafumi , TAKII Yukio 
Published: March 2005

α‐Glucosidase I of Bacillus thermoamyloliquefaciens KP1071 (FERM P8477, facultative thermophile) was purified to homogeneity. The relative molecular mass was estimated to be 62 000 Da. From its catalytic properties, the enzyme has been assigned to an exo‐α‐1,4‐glucosidase. The enzyme shares its antigenic groups in part with Bacillus stearothermophilus ATCC12016 (obligate thermophile) exo‐α‐1,4‐glucosidase.These exo‐α‐1,4‐glucosidases strikingly resemble oligo‐1,6‐glucosidases from B. thermoamyloliquefaciens KP1071 and from Bacillus cereus ATCC7064 in the molecular properties tested, including relative molecular mass, N‐terminal sequence of 15 residues, amino acid composition and structural parameters calculated from amino acid composition. We have suggested that bacillary exo‐α‐1,4‐glucosidases take the same folded conformation, i.e. an (α/β)8‐barrel super‐secondary structure in its N‐terminal domain, as bacillary oligo‐1,6‐glucosidases.
Transforming growth factor‐β induces selective increase of proteoglycan production and changes in the copolymeric structure of dermatan sulphate in human skin fibroblasts
Authors: WESTERGREN‐THORSSON Gunilla , SCHMIDTCHEN Artur , SÄRNSTRAND Bengt , FRANSSON Lars‐Åke , MALMSTRÖM Anders 
Published: March 2005

Human embryonic skin fibroblasts were pretreated with transforming growth factor‐β (TGF‐β) for 6 h and then labeled with [35S]sulphate and [3H]leucine for 24 h. Radiolabeled proteoglycans from the culture medium and the cell layer were isolated and separated by isopycnic density‐gradient centrifugation, followed by gel, ion‐exchange and hydrophobic‐interaction chromatography. The major proteoglycan species were examined by polyacrylamide gel electrophoresis in sodium dodecyl sulphate before and after enzymatic degradation of the polysaccharide chains.The results showed that TGF‐β increased the production of several different 35S‐labelled proteoglycans. A large chondroitin/dermatan sulphate proteoglycan (with core proteins of approximately 400–500 kDa) increased 5–7‐fold and a small dermatan sulphate proteoglycan (PG‐S1, also termed biglycan, with a core protein of 43 kDa) increased 3–4‐fold both in the medium and in the cell layer. Only a small effect was observed on another dermatan sulphate proteoglycan, PG‐S2 (also named decorin). These observations are generally in agreement with results of other studies using similar cell types. In addition, we have found that the major heparan sulphate proteoglycan of the cell layer (protein core approximately 350 kDa) was increased by TGF‐β treatment, whereas all the other smaller heparan sulphate proteoglycans with protein cores from 250 kDa to 30 kDa appeared unaffected.To investigate whether TGF‐β also influences the glycosaminoglycan (GAG) chain‐synthesizing machinery, we also characterized GAGs derived from proteoglycans synthesized by TGF‐β‐treated cells. There was generally no increase in the size of the GAG chains. However, the dermatan sulphate chains on biglycan and decorin from TGF‐β treated cultures contained a larger proportion of D‐glucuronosyl residues than those derived from untreated cultures. No effect was noted on the 4‐ and 6‐sulphation of the GAG chains. By the use of p‐nitrophenyl β‐d‐xyloside (an initiator of GAG synthesis) it could be demonstrated that chain synthesis was also enhanced in TGF‐β‐treated cells (approximately twofold). Furthermore, the dermatan sulphate chains synthesized on the xyloside in TGF‐β‐treated fibroblasts contained a larger proportion of D‐glucuronosyl residues than those of the control.These novel findings indicate that TGF‐β affects proteoglycan synthesis both quantitatively and qualitatively and that it can also change the copolymeric structure of the GAG by affecting the GAG‐synthesizing machinery. Altered proteoglycan structure and production may have profound effects on the properties of extracellular matrices, which can affect cell growth and migration as well as organisation of matrix fibres.
Processing of interleukin‐1 in cells of monocytic lineage is differentiation‐dependent
Authors: CARINCI Valeria , GUIDA Serena , FONTANA Maria Rita , PALLA Emanuela , ROSSINI Mara , MELLI Marialuisa 
Published: March 2005

The interleukin‐1 (IL‐1) α and β precursor proteins are processed and released from several cell types in the absence of a canonical signal peptide. To gain some insight into the mechanisms that allow the production of IL‐1α and β, we have investigated by immunoprecipitation the synthesis, their release and processing in a promieloblastic cell line of tumoral origin, U937, and in peripheral blood monocytes.We show that U937 monocytic cells, on induction with a tumor‐promoting agent, synthesize and release into the culture medium proIL‐1β but do not process it. Similarly, peripheral blood monocytes left in adherence for 24 h or longer, prior to addition of lipopolysaccharide, synthesize and release proIL‐1α and β without detectable processing of either cytokine.Processing and release of IL‐1α and β by peripheral blood monocytes can be observed when monocytes are left to adhere for periods less than 15 h before lipopolysaccharide addition. IL‐1α and β show similar kinetics of release from the cells, suggesting the existence of a common mechanism regulating their secretion. Since peripheral blood monocytes left in adherence in the presence of lipopolysaccharide differentiate into macrophages, we conclude that release and processing of IL‐1 can occur independently and that processing depends on the stage of differentiation of monocytes, i.e. only the monocytes at an early stage of differentiation produce 17‐kDa IL‐1α and β.
Purification and properties of the mercuric‐ion‐binding protein MerP
Authors: SAHLMAN Lena , JONSSON Bengt‐Harald 
Published: March 2005

The gene merP, coding for a mercuric‐ion‐binding periplasmic protein (P protein), was cloned into the expression vector pCA. In an Escherichia coli strain bearing the resulting plasmid, the P protein constitutes about 20% of total soluble protein. P protein was purified using ammonium sulfate precipitation and two chromatography steps. Typical yields were 20–30 mg from 7.5 l bacterial culture. The protein is a monomer with a molecular mass of 7500 Da. The periplasmic signal peptide was processed identically in both the recombinant and the wild‐type proteins. CD spectra of both proteins were identical and indicated that the structure is highly ordered, containing approximately 80%α‐helix.Purification in the presence of excess cysteine resulted in a form of the protein containing two reduced thiols, in agreement with the published sequence which has two cysteine residues. When cysteine was omitted from the purification buffers, no reduced thiol groups could be detected suggesting that the cysteine residues are oxidized. Both of these forms of the protein were found to bind approximately five Hg2+ ions/protein molecule in an apparently non‐specific manner. However, in the presence of external thiol compounds, the protein with reduced thiols bound only one Hg2+ ion/protein molecule with an apparent Kd of 3.7 ± 1.3 μM. Under these conditions, the protein with oxidized thiols did not bind Hg2+. The possible physiological role of this protein in Hg2+ detoxification is discussed.
Two distinct genes encode two major isoelectric forms of insecticyanin in the tobacco hornworm, Manduca sexta
Authors: LI Wancheng , RIDDIFORD Lynn M. 
Published: March 2005

Insecticyanin is a blue pigment found in the epidermis and hemolymph of the tobacco hornworm, Manduca sexta. Two distinct full‐length cDNA species were isolated and shown to encode the two major isoelectric forms of insecticyanin, the a form (INS‐a) and b form (INS‐b). Sequence analysis of the two cDNA clones, pE1‐I1 and pE3‐I2, reveals that both contain an 618‐bp open reading frame which predicts an 189‐amino‐acid protein and an 17‐amino‐acid signal peptide.Comparison of the deduced INS‐a and INS‐b proteins show 13 amino acid differences, of which six are conserved. Three amino acid substitutions are also found between the deduced INS‐b sequence and the sequenced INS‐b protein isolated from the hemolymph. Isolation and characterization of five genomic clones revealed that pE1‐I1 and pE3‐I2 come from two different genes. Both INS‐a and INS‐b genes have four exons interrupted by three introns at the same positions. The two genes share 93% nucleotide similarity in the coding region. Moreover, the ‘TATA box’ and ‘CAAT box’ are completely conserved in the putative promoter regions of the two genes. Primer extension revealed that both INS‐a and INS‐b genes begin their transcription at position – 52 relative to their translation initiation codon, ATG. We conclude that the two genes are the result of gene duplication.
Heat‐shock response in cultured chick embryo chondrocytes
Authors: NERI Monica , DESCALZI‐CANCEDDA Fiorella , CANCEDDA Ranieri 
Published: March 2005

We investigated the induction of specific protein expression by heat shock in dedifferentiated and hypertrophic chick embryo chondrocytes in a culture system that allows ‘in vitro’ differentiation of cartilage cells [Castagnola, P., Moro, G., Descalzi‐Cancedda, F. and Cancedda, R. (1986) J. Cell. Biol. 102, 2310–2317]. As control, we used cultures of embryonic fibroblasts from the whole body and from the skin. In the cell lysates of all cultures we identified four major heat‐shock proteins (HSP), with a molecular size corresponding to HSP families previously described (HSP 90, HSP 70, HSP 47 and HSP 26). Some of these proteins were constantly induced when the temperature was raised, others were expressed in a more variable manner. Differences also existed in the relative amount of the HSP synthesized by the four cultures.When we specifically investigated HSP species released into the culture medium, we observed a 43–45 kDa protein constantly expressed and secreted in large amount by the cells. On the basis of its biochemical characteristic and its precipitation by specific antibodies, this protein has been identified as osteonectin (SPARC, BM‐40).
Chemical modification of prostaglandin endoperoxide synthase by N‐acetylimidazol
Authors: SCHERER Hans‐Josef , KARTHEIN Rainer , STRIEDER Stephan , RUF Hans Heinrich 
Published: March 2005

Prostaglandin H synthase apoprotein, without its prosthetic heme group, was inactivated by N‐acetylimidazole under conditions typical for the O‐acetylation of tyrosyl residues. A spontaneous reactivation occurred above pH 7.5 at 22°C, which indicated spontaneous hydrolysis of acetylated residues. Below pH 7.5, where stable inactivation was observed, reactivation was achieved by reaction with hydroxylamine. Both enzymic activities of prostaglandin H synthase, cyclooxygenase and peroxidase, were inactivated and reactivated simultaneously and to the same extent. In contrast to the apoprotein, the holoenzyme with heme was not inactivated by N‐acetylimidazole. The number of acetyl groups, as determined as hydroxamate after the reaction with hydroxylamine at pH 8.2, was 2.5 ± 0.4 for the apoprotein and 1.0 ± 0.24 for the holoenzyme. The specific binding of heme as the prosthetic group was no longer observed by EPR (signals at g= 6.7 and 5.3) when hemin was added to the N‐acetylimidazole‐reacted apoprotein. Treatment of N‐acetylimidazole‐reacted apoprotein with hydroxylamine restored the specific binding of heme. The N‐acetylimidazole‐reacted apoprotein supplemented with hemin and reacted with hydroperoxides, neither showed electronic absorption spectra of higher oxidation states nor an EPR doublet signal due to a tyrosyl radical.These results demonstrate that heme protects against the inactivating modification by N‐acetylimidazole and that this modification prevents binding of the prosthetic heme group necessary for both enzymic activities. The absence of the prosthetic heme group explains the concomitant loss of cyclooxygenase and peroxidase activities, as well as the absence of higher oxidation states and the tyrosyl radical. We suggest that the acetylation of a residue in the heme pocket, most probably a tyrosine, although a histidine cannot be definitely disproved, exerts the inhibiting effect. This residue could be the axial ligand of the heme or in close contact to the heme. The results also show that the inhibition by N‐acetylimidazole does not involve the acetylation of Ser530 which causes the inhibition by acetylsalicylic acid of cyclooxygenase. [The numbering of amino acids in ovine prostaglandin H synthase is according to DeWitt, D. L. and Smith, W. L. (1988) Proc. Natl Acad. Sci. USA 85, 1412–1416 including a signal peptide of 24 residues which is missing in the processed protein.]
Purification and characterization of a NADH oxidase from the thermophileThermus thermophilusHB8
Authors: PARK Ho‐Jin , REISER Christian O. A. , KONDRUWEIT Simone , ERDMANN Helmut , SCHMID Rolf D. , SPRINZL Mathias 
Published: March 2005

A NADH oxidase has been purified from the extreme thermophileThermus thermophilusHB8 by several chromatographic steps. The purified enzyme was essentially homogeneous as judged by gel electrophoresis under denaturing conditions and by determination of the N‐terminal amino acids sequence. It is a monomeric flavin‐adenine‐dinucleotide‐containing flavoprotein with an apparent molecular mass of 25 kDa and an 1:1 ratio of FAD to the polypeptide chain.The purified enzyme catalyzes the oxidation of reduced NADH or NADPH with the formation of H202. The apparentKm, values for NADH and NADPH are 4.14 μM and 14.0 μM (pH 7.2 at room temperature), respectively, with a sixfold greaterkcat/Kmvalues for NADH compared to NADPH. The enzyme uses O2as an electron acceptor in the presence of either FAD, riboflavin 5′‐phosphate or riboflavin as cofactor. In addition, the enzyme is able to catalyze electron transfer from NADH to various other electron acceptors (methylene blue, cytochromec, p‐nitroblue tetrazolium, 2,6‐dichloroindophenol and potassium ferricyanide), even in the absence of flavin shuttles. No significant inhibition of the NADH oxidoreductase activity by superoxide dismutase was observed with these artificial electron acceptors, indicating that electron transfer occurs mainly from NADH directly to the electron acceptors, not via O2−as an intermediate.The purified NADH oxidase exhibits highest activity at pH 5.0 and is stable at elevated temperatures of up to 80°C.
Carbohydrate structure of a thrombin‐like serine protease from Agkistrodon rhodostoma
Authors: PFEIFFER Günter , DABROWSKI Ursula , DABROWSKI Janusz , STIRM Stephan , STRUBE Karl‐Hermann , GEYER Rudolf 
Published: March 2005

The carbohydrate side chains of the thrombin‐like serine protease ancrod from the venom of the Malayan pit viper Agkistrodon rhodostoma were liberated from tryptic glycopeptides by treatment with peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase F and fractionated by high‐performance liquid chromatography. Glycans obtained were characterized by digestion with exoglycosidases, methylation analysis and, in part, by liquid secondary‐ion mass spectrometry and 1H‐NMR spectroscopy.The results reveal that this snake venom glycoprotein contains partially truncated di‐, tri‐ and tetraantennary complex type N‐glycans carrying Fuc(α1‐6) residues at the innermost N‐ acetylglucosamine and solely (α2‐3)‐linked sialic acid substituents. As a characteristic feature, ancrod oligosaccharides comprise mainly sialylated Galβ3GlcNAcβ lactosamine antennae. Furthermore, a small proportion of the sugar chains were found to carry a NeuAcα3GalNAcβ4GlcNAcβ antenna exclusively linked to C‐2 of Man(α1‐3) residues of the pentasaccharide core. Thus, many of the glycans found represent novel glycoprotein‐N‐glycan structures.
Characterization and crystal structure of zinc azurin, a by‐product of heterologous expression in Escherichia coli of Pseudomonas aeruginosa copper azurin
Authors: NAR Herbert , HUBER Robert , MESSERSCHMIDT Albrecht , FILIPPOU Alexander C. , BARTH Manfred , JAQUINOD Michel , van de KAMP Mart , CANTERS Gerard W. 
Published: March 2005

Azurin*, a by‐product of heterologous expression of the gene encoding the blue copper protein azurin from Pseudomonas aeruginosa in Escherichia coli, was characterized by chemical analysis and electrospray ionization mass spectrometry, and its structure determined by X‐ray crystallography. It was shown that azurin* is native azurin with its copper atom replaced by zinc in the metal binding site. Zinc is probably incorporated in the apo‐protein after its expression and transport into the periplasm. Holo‐azurin can be reconstituted from azurin* by prolonged exposure of the protein to high copper ion concentrations or unfolding of the protein and refolding in the presence of copper ions.An X‐ray crystallographic analysis of azurin* at 0.21‐nm resolution revealed that the overall structure of azurin is not perturbed by the metal exchange. However, the geometry of the co‐ordination sphere changes from trigonal bipyramidal in the case of copper azurin to distorted tetrahedral for the zinc protein. The copper ligand Met121 is no longer co‐ordinated to zinc which adopts a position close to the carbonyl oxygen atom from residue Gly45.The polypeptide structure surrounding the metal site undergoes moderate reorganization upon zinc binding. The largest displacement observed is for the carbonyl oxygen from residue Gly45, whch is involved in copper and zinc binding. It moves by 0.03 nm towards the zinc, thereby reducing its distance to the metal from 0.29 nm in the copper protein to 0.23 nm in the derivative.