bluuebunny/crossref_metadata_2025_split huggingface dataset:
Oligosaccharides Derived by Keratanase II Digestion of Bovine Articular Cartilage Keratan Sulphates
Authors: Brown Gavin M. , Huckerby Thomas N. , Nieduszynski Ian A. 
Published: March 2005

Alkaline borohydride‐reduced keratan sulphate chains from bovine articular cartilage (6–8‐year‐old animals) were subjected to a limit digest with the enzyme keratanase II. Using 1H‐NMR spectroscopy, 25 reduced oligosaccharides deriving from keratan sulphate were shown to have the following structures [GlcNAc(6S)‐ol represents N ‐acetylglucosaminitol 6‐O ‐sulphate]: Galβ1–4‐GlcNAc(6S)‐ol, Galβ1‐4GlcNAc(6S) β1–3Galβ1–4GlcNAc(6S)‐ol, Gal(6S) β1–4GlcNAc(6S)‐ol, Gal‐(6S) β1–4GlcNAc(6S) β‐1–3 Galβ‐4GlcNAc(6S)‐ol, Galβ1–4GlcNAc(6S) β1–3Gal(6S) β1–4GlcNAc(6S)‐ol, Gal(6S) β1–4GlcNAc(6S) β1–3Gal(6S)1–4GlcNAc(6S)‐ol, Galβ1–4(Fuca1–3)GlcNAc(6S)‐ol, Galβ–4‐(Fucα1–3)GlcNAc(6S) β1–3Galβ1–4(Fcα1–3)G1cNAc(6S)‐ol, Galβ1–4GlcNAc(6S) β1–3Galβ1–4(Fucα1–3)‐GlcNAc(6S)‐ol, Galβ1–3(Fucα1–3)GlcNAc(6S) β1–3Galβ1–4GlcNAc(6S)‐ol, Gal(6S) β1–4GlcNAc‐(6S) β1–3Galβ1–4(Fucα1–3)GlcNAc(6S)‐ol, Galβ1–4(Fucα1–3)GlcNAc(6S) β1–3Gal(6S) β1–4GlcNAc(6S)‐ol, Galβ1–4 GlcNAc(6S) β1–6(Galβ1–3)GalNAc‐ol, Galβ1–4 GlcNAc(6S) β1–6(NeuAc2–3Galβ1–3)Gal‐Nac‐ol, Galβ1–4GclNAc(6S) β1–3Galβ1–4GlcNAc(6S) β1–4GlcNAc(6S) β1–6(Galβ1–3)GalNAc‐ol, Gal(6S) β1–4GlcNAc‐(6S) β1–6(Galβ1–3)GalNAc‐ol. Galβ1–4GlcNAc(6S) β1–3Galβ1–4GlcNAc (6S) β1–6(NeuAc2–3Galβ1–3)‐GalNAc‐ol. Gal(6S) β1–4GlcNAc‐(6S) β1–6(NeuAcα2–3Galβ1–3)GalNAc‐ol. Gal(6S) β1–4GlcNAc‐(6S) β1–3Galβ1–4GlcNAc(6S) β1–6(Galβ1–3)GalNAc‐ol, Gal(6S) β1–4GlcNAc(6S)β1–3Galβ1–4GlcNAc‐ (6S) β1–6(NeuAcα2–3Galβ1–3)GalNAc‐ol,NeuAcα2–6Galβ1–4 GalNAc(6S) β1–3Galβ1–4 GlcNAc(6s)‐ol, NecAcα2–3Galβ1–4GlcNAC(6s)β1–3Galβ1–4GlcNAc(6S)‐ol, NeuAcα2–6Galβ1–4GlcNAc(6S)β1–3Gal‐(6S)β1–4GlcNAc(6S)‐ol, NeuAcα2–3Galβ1–4GlcNAc(6S) β1–3Gal(6S)β1–4GlcNAc(6S)‐ol and Neu‐Acα2–3Gal(6S) β1–4GlcNAc(6S)β1–3Gal(6Sβ)1–4GlcNAc(6S)‐ol. Proton chemical shifts for these oligosaccharides were assigned using one‐ and two‐dimensional NMR spectroscopic methods.These results confirm the findings of Nakazawa et al. [Nakazawa, K., Ito, M., Yamagata, T. and Suzuki, S. (1989) in Keratan sulphate: chemistry, biology and chemical pathology (Greiling, H. and Scott, J. E., eds) pp. 99–110, The Biochemical Society, London], namely that keratanase II cleaves the O ‐glycosidic bond of a β (1–3)‐linked 6‐O ‐sulphated N ‐acetylglucosamine.However, the internal sulphated N ‐acetylglucosamine in the sialylated capping oligosaccharides is not cleaved because of the proximity of the sialic acid residue. In addition, keratanase II is the only degradative method examined so far which can cleave the glycosidic bond of a fucosylated N ‐acetylglucosamine residue as fucose residues confer resistance to both keratanase and hydrazino‐lysisnitrous acid fragmentation.
Agonist‐induced Transfer of the α Subunits of the Guanine‐nucleotide‐binding Regulatory Proteins Gq and G11, and of Muscarinic m1 Acetylcholine Receptors from Plasma Membranes to a Light‐vesicular Membrane Fraction
Authors: Svoboda Petr , Milligan Graeme 
Published: March 2005

A clone of a Chinese hamster ovary (CHO) cell line expressing high levels of the human muscarinic M1 acetylcholine (Hm1) receptor undergoes a substantial agonist‐specific down‐regulation of both Hm1 receptors and the α subunits of the guanine‐nucleotide‐binding (G)‐proteins Gq and G11 which is accompanied by a desensitization of inositol‐phospholipid‐specific‐phospholipase‐C response [Mullaney, I., Dodd, M. W., Buckley, N. J. & Milligan, G. (1993) Biochem. J. 289, 125–131]. To examine early events in this process, the effect of agonist on subcellular distribution of Gqα and G11α and of Hm1 receptors was assessed after short‐term and long‐term treatment with carbachol.Short‐term (30 min) incubation with carbachol (1 mM) induced a simultaneous transfer of a proportion of both Gqα and G11α and Hm1 receptors from plasma membranes to distinct light vesicular membranes. The total number of receptors and of Gqα and G11α in each cell remained unchanged under these conditions. A similar transfer was noted for the G‐protein Gsα but not for intrinsic plasma membrane markers. The plasma membrane, as well as light vesicular membrane, pool of Gsα subunit was unaffected by further sustained incubation with carbachol (16 h), whereas Hm1 receptors and both Gqα and G11α proteins were down‐regulated to 25% and 40%, respectively, when compared with untreated cells.Such observations support the idea that down‐regulation of both the Hm1 receptor and its associated inositol‐phospholipid‐specific‐phospholipase‐C‐linked G‐proteins is produced by two sequential steps. The first is a transfer of signal‐transducing polypeptides from the plasma membrane to a non‐plasma membrane light vesicle fraction. The second step is represented by an agonist‐specific down‐regulation pathway. Both the Hm1 receptor and Gqα/G11α appear to follow similar sequestration and down‐regulation patterns.
Purification of a Novel 43‐kDa Protein (p43) Intermediary in the Activation of Steroidogenesis from Rat Adrenal Gland
Authors: Paz Cristina , Dada Laura A. , Maciel M. Fabiana Cornejo , Mele Pablo G. , Cymeryng Cora B. , Neuman Isabel , Mendez Carlos F. , Finkielstein Carla V. , Solano Angela R. , Park Minkyu , Fischer Wolfgang H. , Towbin Harry , Scartazzini Ricardo , Podestá Ernesto J. 
Published: March 2005

In previous reports we have demonstrated the presence of a soluble factor that responds to cAMP signals to induce steroid synthesis in adrenocortical tissue. Here, we describe the purification of this factor from adrenal zona fasciculata cells by using a five‐step procedure that includes DEAE‐cellulose, gel filtration, Mono Q HPLC and Superose HPLC, and elution of the protein from SDS/PAGE. This procedure results in the purification to homogeneity of a protein of 43‐kDa that retains the capacity to stimulate steroid synthesis in anin vitrorecombination assay. This activity is inhibited by the use of phospholipase A2, inhibitors. Antipeptide antibodies against the N‐terminal region recognize p43 as a double band on SDSPAGE that resolves in different spots on two‐dimensional gel electrophoresis. Adrenocorticotropin treatment of adrenal glands results in the appearence of multiple spots that migrated towards a lower pH compared to controls, suggesting the presence of phosphorylated and dephosphorylated forms of p43. Sequencing of the N‐terminal region and internal peptides reveals no significant similarities with other proteins, suggesting that p43 is a novel protein.We conclude from our data that the isolated protein (p43) is a novel, soluble protein that acts as intermediary in adrenocorticotropin‐induced stimulation of arachidonic acid release and steroid synthesis.
Crystal Structure Analysis of a Serine Proteinase from Streptomyces fradiae at 0.16‐nm Resolution and Molecular Modeling of an Acidic‐amino‐acid‐specific Proteinase
Authors: Kitadokoro Kengo , Tsuzuki Hiroshige , Okamoto Hiroyuki , Sato Tomohiro 
Published: March 2005

We have determined the three‐dimensional structure of a proteinase from Streptomyces fradiae ATCC 14544 (SFase‐2) at 0.16‐nm resolution. SFase‐2, a typical serine proteinase, has broad substrate specificity. The characterization and crystallographic analysis of this enzyme have been reported previously [Kitadokoro, K., Tsuzuki, H., Nakamura, E., Sato, T. & Teraoka, H. (1994) Eur: J. Biochem. 220, 55–61]. In the present study, data were collected to approximately 0.16‐nm resolution on a Rigaku R‐AXIS IIC imaging plate detector system. Preliminary phases were obtained by molecular replacement methods with a search model derived from the previously determined structure of Streptomyces griseus protease A [Sielecki, A. R., Hendrickson, W. A., Broughton, C. G., Delbaere, L. T., Brayer, G. D. & James, M. N. (1979) J. Mol. Biol. 134, 781–8041. The starting model gave an initial crystallographic R factor of 0.443. Refinement with restrained least‐squares converged at a final R factor of 0.182 for 16128 observed reflections. The final model includes 86 water molecules.The crystal structure showed that the enzyme consists of two domains, each of which is comprised of a β barrel with six‐stranded β sheets and two α helices. The overall tertiary structure of SFase‐2 is similar to the structures of other chyrnotrypsin‐like proteinases from S. griseus, namely proteinase A and proteinase B. The essential residues of the catalytic triad are located on the cleft between the two domains. These two domains have different sequences, but possess similar three‐dimensional structures, indicating that a gene duplication event has occurred to produce these two domains.
GPIIIa(90–102) and GPIIIa(631–653) Epitopes as Markers of Conformational Changes Occurring During the Activation of the Glycoprotein IIb/IIIa Complex
Authors: Niewiarowska Jolanta , Cierniewski Czeslaw Stanislaw 
Published: March 2005

To evaluate the range of conformational changes in the GPIIIa subunit upon the interaction of GPIIb/IIIa complex with fibrinogen, we employed two polyclonal antipeptide antibodies against synthetic peptides spanning chain segments (90–102) and (631–653) of GPIIIa. The first peptide fragment derives from the region preceding the putative binding site for the RGD sequence, while the second one is located in the vicinity of membrane lipid bilayer. The resting platelets bound about 13700 and 11500 molecules of anti‐[GPIIIa(90–102)] and anti‐[GPIIIa(631–653)] antibodies, respectively. The anti‐[GPIIIa(90–102)] antibodies preferentially bound to the active form of GPIIb/IIIa complex. This binding was significantly reduced after dissociation of the complex, indicating that the recognized epitopes became partially hidden in the GPIIIa subunit. In contrast to anti‐[GPIIIa(90–102)], the anti‐[GPIIIa(631–653)] antibodies reacted optimally with the EDTA‐treated GPIIb/IIIa. Upon stimulation of platelets with ADP, the number of binding sites for anti‐[GPIIIa(631–653)] was reduced 2.8‐fold. The binding affinity of anti‐[GPIIIa(90–102)] antibodies to the resting and ADP‐stimulated platelets differed 4.7‐fold, indicating that the stimulation of the platelets with ADP resulted in better exposure of this epitope. This binding was inhibited almost totally by fibrinogen in a dose‐dependent fashion. Similarly, fibrinogen inhibited binding of the anti‐[GPIIIa(631–653)] antibodies to platelets. In the reversed system, binding of fibrinogen to platelets was inhibited only to 60% by the anti‐[GPIIIa(90–102)] antibodies. Such inhibition of fibrinogen binding was sufficient to block aggregation of ADP‐stimulated platelets by the anti‐[GPIIIa(90–102)] antibodies in platelet‐rich plasma. In contrast, the anti‐[GPIIIa(631–653)] potentiated fibrinogen binding by 20%, confirming that there were additional interacting sites for fibrinogen in other regions of the GPIIb/IIIa complex.The presented data indicate that the activation of the fibrinogen receptor is associated with extensive conformational changes in the GPIIIa subunit, detectable by use of antipeptide antibodies recognizing not only the epitopes located next to the putative ligand‐binding site but also the epitopes in the vicinity of the platelet membrane lipid bilayer.
Characterization of the Murine Plasma Fibrinolytic System
Authors: Lijnen H. Roger , Van Hoef Berthe , Beelen Veerle , Collen Désiré 
Published: March 2005

The main components of the murine plasma fibrinolytic system, including fibrinogen, plasminogen, α2‐antiplasmin, tissue‐type plasminogen activator and plasminogen activator inhibitor‐1, were purified to homogeneity and their interactions were quantitated and compared with those of the human counterparts.Initial activation rates of murine and human plasminogen by autologous tissue‐type plasminogen activator were comparable (catalytic efficiencies, k2/Km, of 0.4 and 0.6 mM−1 s−1, respectively), but murine plasminogen appeared to be resistant to activation by human tissue‐type plasminogen activator (k2/Km=0.01 mM−1 s−1). Plasminogen activation by tissue‐type plasminogen activator was stimulated 100‐ and 160‐fold in autologous murine and human systems, respectively, with saturating concentrations of 0.45 and 0.32 μM, respectively, of CNBr‐digested fibrinogen. Nearly quantitative binding (85–90%) of tissue‐type plasminogen activator to fibrin was observed both in autologous and heterologous systems.Murine and human plasmin were very rapidly inhibited by autologous and heterologous α2‐antiplasmin (second‐order inhibition rate constants, k1,app, of 2.1–2.3×107 M−1 s−1) and murine and human tissue‐type plasminogen activator were very rapidly inhibited by autologous or heterologous plasminogen activator inhibitor‐1 (k1,app of 1.8–4.9×107M−1 s−1). Two‐chain murine tissue‐type plasminogen activator (added at a concentration of 1 μg/ml) was inhibited in normal or plasminogen activator inhibitor‐1‐deficient murine plasma with half‐lives of 6.5 min and 4.2 min, respectively, as compared to 80 min for human tissue‐type plasminogen activator, suggesting that murine plasma contains proteinase inhibitors other than plasminogen activator inhibitor‐1 which efficiently inhibit autologous tissue‐type plasminogen activator.Clot lysis experiments in autologous plasma revealed that the murine plasma fibrinolytic system is more resistant to activation than the human system (20–30% clot lysis in 2 h with 100 nM tissue‐type plasminogen activator in the murine system, as compared to 50% clot lysis in 2 h with 3.5 nM tissue‐type plasminogen activator in the human system). Several mechanisms appear to be involved in this relative resistance observed in the murine system, including resistance of murine plasminogen to quantitative activation and short plasma half‐life of murine tissue‐type plasminogen activator.Thus, although these quantitative interactions between purified components of the murine fibrinolytic system appear to be comparable to those between the human counterparts, murine plasma clots are >30‐fold more resistant to lysis with autologous tissue‐type plasminogen activator than human plasma clots.
Reversible Dissociation and Unfolding of Pyruvate Decarboxylase from Zymomonas mobilis
Authors: Pohl Martina , Grötzinger Joachim , Wollmer Axel , Kula Maria‐Regina 
Published: March 2005

The denaturation and renaturation process of pyruvate decarboxylase (PDC) from Zymomonas mobilis (ATCC 29191) has been investigated using guanidine hydrochloride and urea as denaturing agents. The quarternary structure of the homotetramer is strongly stabilized by the cofactors Mg2+ and thiamine diphosphate (TDP). The structural transitions were monitored by activity measurements, fluorescence spectroscopy, circular dichroism and gel‐filtration chromatography. A three‐step denaturation process, described as follows, is indicated by non‐coincidental denaturation curves: (a) inactivation of the tetramer upon dissociation of cofactors (>0.4 M guanidine hydrochloride, >1 M urea); (b) dissociation of the tetramer into monomers (>1 M guanidine hydrochloride, >3M urea); (c) complete unfolding of these (>2.5 M guanidine hydrochloride, >5 M urea).The refolding process initiated by rapid dilution of fully denatured protein in renaturation buffer involves the rapid reassociation of an inactive intermediate followed by the reconstitution of the active site.
Trifluoroethanol Stabilizes a Helix‐Turn‐Helix Motif in Equine Infectious‐Anemia‐Virus Trans ‐Activator Protein
Authors: Sticht Heinrich , Willbold Dieter , Ejchart Andrzej , Rosin‐Arbesfeld Rina , Yaniv Abraham , Gazit Arnona , Rösch Paul 
Published: March 2005

The solution structure of the 75‐amino‐acid trans ‐activator (Tat) protein of the equine infectious‐anemia virus in trifluoroethanol‐containing solution was determined by two‐dimensional and three‐dimensional nuclear magnetic resonance spectroscopy, resulting in a total of 838 nuclear‐Over‐hauser‐enhancement distance restraints, and restrained molecular‐dynamics simulations. In contrast to the recently determined structure of this protein in trifluoroethanol‐free pH 6.3 solution, the hydrophobic core and the adjacent basic RNA‐binding region of the protein showed well‐defined α‐helical secondary structure in trifluoroethanol‐containing solution. The helical regions comprise those parts of the molecule whose helix‐forming tendencies were noted earlier in trifluoroethanol‐free solution. Two helices (Gln38–Arg43 and Asp48–Ala64) are connected by a tight type‐II turn centered at the strictly conserved Gly46 leading to a helix‐turn‐helix motif in the core and basic region of the protein. A third helix (Thr9–Asn13) is located in the less well defined N‐terminal part of the protein.These observations may support the notion that the protein adopts a helical structure in the RNA‐binding region on complex formation. Although the secondary‐structure elements become better defined in trifluoroethanol‐containing solution, the opposite is true for the hydrophobically stabilized tertiary structure. This adds a caveat to studies of protein structures in trifluoroethanol‐containing solution in general.
Complete L‐Alanine Scan of Neuropeptide Y Reveals Ligands Binding to Y1 and Y2 Receptors with Distinguished Conformations
Authors: Beck‐Sickinger Annette G. , Weland Heike A. , Wittneben Helmut , Willim Klaus‐Dieter , Rudolf Klaus , Jung Günther 
Published: March 2005

The synthesis of more than fifty 36‐residue oligopeptide analogs of neuropeptide Y (NPY) and their affinity to human Y1, and Y2, receptors is described. Each amino acid of the natural sequence was replaced by L‐alanine, the four alanine residues at position 12, 14, 18 and 23 were replaced by glycine. Additional residues were exchanged to closely related ones in order to characterize the prerequisites for binding. A combination of automated single and multiple peptide synthesis using fluoren‐9‐ylmethoxycarbonyl/tert ‐butoxy strategy was applied. The purified peptides were characterized by electrospray mass spectrometry, analytical HPLC and amino acid analysis. Binding was investigated by displacement of 125I‐labelled neuropeptide Y from human neuroblastoma cell lines SK‐N‐MC and SMS‐KAN.Whereas Pro2 and the integrity of the neuropeptide Y loop is important for the binding to the Y1, receptor, exchanges within the C‐terminal helix affect the affinity to the Y2, receptor. The C‐terminal pentapeptide amide is important for both receptors and probably represents the binding site. However, Arg33 and Arg35 may not be exchanged by L‐alanine in the Y1, system, whereas Arg35 and Tyr36 are the most susceptible residues in the Y2, system. In order to distinguish between conformational effects and direct hormone/receptor interaction via the side chains of neuropeptide Y, circular dichroic studies of the alanine‐containing peptides were performed and structure affinity relationships are discussed.Comparing the affinities of the neuropeptide Y analogs to Y1, and Y2, receptors significant differences were found for the two binding sites, which suggests a different active conformation of neuropeptide Y at the two subtypes of receptors. Using molecular dynamics calculations, two distinct conformations were identified which are in good agreement with the data obtained by structure/affinity investigations.
Selective processing of submandibular rat 1 protein at dibasic cleavage sites
Authors: ROUGEOT Catherine , ROSINSKI‐CHUPIN Isabelle , NJAMKEPO Elisabeth , ROUGEON François 
Published: March 2005

The amino acid sequence of submandibular rat 1 (SMR1) protein, deduced from its cDNA sequence, led to the prediction that the SMR1 gene encodes a hormone‐like precursor [Rosinski‐Chupin, I., Tronik, D. & Rougeon, F. (1988) Proc. Natl Acad. Sci. USA 85, 8553–8557]. SMR1 contains an N‐terminal putative secretory signal sequence and a tetrapeptide (QHNP), located between dibasic amino acids which constitute the most common signal for prohormone processing.We have isolated and characterized from the male rat submandibular gland and its secretions three structurally related peptides, namely an undecapeptide (VRGPRRQHNPR), a hexapeptide (RQHNPR) and a pentapeptide (QHNPR) generated from SMR1 by selective proteolytic cleavages at pairs of arginine residues.The biosynthesis of these peptides is subjected to distinct regulatory pathways depending on the organ, sex and age of the rat. Furthermore, the peptides are differentially distributed in the submandibular gland and in resting or epinephrine‐elicited submandibular salivary secretions, suggesting distinct proteolytic pathways for their maturation. The undecapeptide is generated in the gland of both male and female rats, but under basal conditions it is only released into the saliva in male animals. The hexapeptide is produced in large amounts in the gland of adult male rats and released into the saliva in both resting and stimulated conditions. The pentapeptide appears only in the male saliva and is present mostly under stimulated conditions. In addition, administration of epinephrine induces the release of the hexapeptide from the submandibular gland into the bloodstream. The evidence indicates that the rat submandibular gland can function as a dual exocrine and endocrine organ for the SMR1‐derived hexapeptide, as has been reported for nerve growth factor, epidermal growth factor, renin and kallikrein.Although the biological activities of the SMR1‐derived peptides are not yet known, their high production and adrenergic‐induced release only into the saliva and bloodstream of adult male rats, suggest a physiological involvement in some male‐specific processes.
Overexpression of a cytochrome P‐450 of the 2a family (Cyp2a‐5) in chemically induced hepatomas from female mice
Authors: JOUNAÏDI Youssef , BONFILS Claude , PÉRIN François , NEGISHI Masahiko , LANGE Reinhard 
Published: March 2005

Chemical hepatocarcinogenesis in female mice, induced by 5,9‐dimethyl(7H)dibenzo[c,g]carbazole, leads to the overexpression of a cytochrome P‐450 of the 2a family. This protein was identified as Cyp2a‐5, by the use of immunoblots obtained from isoelectric focusing gels. This method allowed the distinction of Cyp2a‐5 from Cyp2a‐4, another mouse liver cytochrome P‐450, by taking advantage of their slightly different pI values. The theoretical pI values, determined from the amino acid sequence, were pI 9.91 for Cyp2a‐4 and pI 10.01 for Cyp2a‐5. Other structurally related forms were not detected. In hepatomas from female mice, only the Cyp2a‐5 form was overexpressed (2–3 fold). Male mice showed a weak expression of Cyp2a‐4 and Cyp2a‐5 in control liver samples and in hepatomas. The expression of both forms was increased more than fivefold upon castration. Pyrazole induces specifically the Cyp2a‐5 form. The Cyp2a‐5 overexpression was correlated with enhanced microsomal coumarin‐7‐hydroxylase and testosterone‐15α‐hydroxylase activities.An immunohistochemical study showed that Cyp2a‐4 and Cyp2a‐5 are expressed uniformly in female livers, but centrilobularly in male livers. In hepatomas, this localisation is perturbed; in females we observed a focal cell localisation, and the Cyp2a‐containing cells were often hypertrophic and polyploid. In hepatomas from male mice, the Cyp2a‐containing cells became dispersed.From a comparison with other studies, the Cyp2a‐5 overexpression appears to be a general feature of hepatocarcinogenesis in mice.