bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isomalto‐oligosaccharide‐containing lipoteichoic acid of Streptococcus sanguis
Authors: KOCHANOWSKI Bernd , FISCHER Werner , IIDA‐TANAKA Naoko , ISHIZUKA Ineo 
Published: March 2005

The lipoteichoic acid of Streptococcus sanguis DSM 20567 and of DSM 20068 was isolated by phenol/water extraction and hydrophobic‐interaction chromatography. The preparations from both strains have an identical structure: a 1,3‐linked poly(glycerophosphate) chain phosphodiester‐linked to Glc‐(α1–2)Glc(α1–3)acyl2Gro as the lipid anchor. The chain is substituted with d‐alanine ester and glycosyl residues which comprise mono‐, di‐, tri‐ and tetra‐α‐d‐glucopyranosyl residues with (1–6) interglycosidic linkages. The glycosylglycerols were released with 48% (by mass) hydrofluoric acid, separated and characterized by a combination of chemical procedures and modern techniques of 1H‐NMR and 13C‐NMR spectroscopy. The α‐isomalto‐oligosaccharides add a novel motif to lipoteichoic‐acid chain substituents. 1H‐NMR and 13C‐NMR spectroscopy also provided a detailed picture of the basic glycosylated poly(1,3‐glycerophosphate) diglucosylglycerol. It proved a single unbranched chain structure, provided evidence for the chain length, the extent of glycosylation, the structure of the lipid anchor and the site of attachment of the poly(glycerophosphate) chain on the lipid anchor.Owing to its unique glycosyl substituents the lipoteichoic acid may serve as a taxonomic marker for the redefined species S. sanguis (formerly S. sanguis type I).
Evidence that CTP:choline‐phosphate cytidylyltransferase is regulated at a pretranslational level in rat liver after partial hepatectomy
Authors: HOUWELING Martin , TIJBURG Lilian B. M. , VAARTJES Willem J. , BATENBURG Joseph J. , KALMAR Gabriel B. , CORNELL Rosemary B. , VAN GOLDE Lambert M. G. 
Published: March 2005

Regulation of CTP:choline‐phosphate cytidylyltransferase activity was studied in regenerating rat liver. The formation of phosphatidylcholine from [14C]choline in hepatocytes isolated from regenerating liver at 22 h after surgery was increased 1.9‐fold when compared with hepatocytes from sham‐operated animals. This effect was accompanied by a 1.4‐fold increase in cytosolic cytidylyltransferase activity as well as by a 1.5‐fold increase in the amount of immunoreactive cytidylyltransferase protein, and a 1.7‐fold increase in [35S]methionine incorporation into cytidylyltransferase protein.Northern blot analysis of cytidylyltransferase mRNA showed two signals at 1.5 and 5.0 kb. Partial hepatectomy caused a significant 2–3‐fold increase in the 1.5‐kb and 5.0‐kb messengers at 12 h after surgery. During the next 10 h after partial hepatectomy cytidylyltransferase mRNA levels slightly decreased, although they were still elevated in comparison with sham‐operated rats 20–22 h after surgery. In contrast to the elevated cytidylyltransferase mRNA levels, the amount of acetyl‐CoA carboxylase mRNA did not increase between 12 and 22 h after surgery, which is in line with the unchanged activity of this enzyme.In conclusion, our data demonstrate that in regenerating liver phosphatidylcholine biosynthesis and cytidylyltransferase activity are regulated at a pretranslational level.
Ribosomes terminated in vitro are in a tight association with non‐phosphorylated elongation factor 2 (eEF‐2) and GDP
Authors: DAVYDOVA Elena K. , MALININ Nikolai L. , OVCHINNIKOV Lev P. 
Published: March 2005

A proportion of the ribosome population in the eukaryotic cell is present in the form of single 80‐S ribosomes. These are not involved in translation and are tighly associated with eukaryotic elongation factor 2 (eEF‐2). The factor dissociates from ribosomes when it is ADP‐ribosylated. Attempts at reconstitution of such complexes from ribosomal subunits and eEF‐2 were not successful.We have shown that monomeric ribosomes in a tight complex with eEF‐2 can be obtained in vitro as terminated ribosomes in a reconstituted translation system containing isolated polyribosomes, elongation factors and pH5 enzymes (all from rabbit reticulocytes). Incubation of the system with radioactive GTP demonstrated that terminated ribosomes contain GDP. ADP‐ribosylation of eEF‐2 bound to terminated ribosomes by diphtheria toxin leads to dissociation of both eEF‐2 and GDP to the same extent. Thus the presence of GDP in terminated ribosomes is eEF‐2 dependent. Ribosomes terminated in vitro as well as native single ribosomes contain the non‐phosphorylated form of eEF‐2.We assume that tight association of terminated ribosomes with the non‐phosphorylated form of eEF‐2 excludes both the ribosomes and active eEF‐2 from the translational cycle and thus, maintains the optimal proportion of translating ribosomes and free eEF‐2 in the cell.
Primary structure of human milk octa‐, dodeca‐ and tridecasaccharides determined by a combination of 1H‐NMR spectroscopy and fast‐atom‐bombardment mass spectrometry
Authors: HAEUW‐FIEVRE Sophie , WIERUSZESKI Jean‐Michel , PLANCKE Yves , MICHALSKI Jean‐Claude , MONTREUIL Jean , STRECKER Gérard 
Published: March 2005

Two octasaccharides, two dodecasaccharides and a tridecasacharide have been isolated from human milk by a combination of paper chromatography, high‐performance liquid chromatography and high‐performance anion‐exchange chromatography. Their structural analysis investigated by 400‐MHz 1H‐ and 13C‐NMR spectroscopy and by mass spectrometry led to the following structures.

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The two octasaccharides have been previously characterized in human milk; the complete assignment of the 1H and 13C spectra is reported here. One of the dodecasaccharides is a tetrafucosyl derivative with a new core: typI‐(β1–3)‐typeII‐(β1–3)‐typeII‐(β1–3)‐Gal(β1–4)Glc, where typeI = Gal(β1–3)GlcNAc and typeII = Gal(β1–4)GlcNAc. These oligosaccharides display simultaneously Lewisb and Lewisx determinants or Lewisa, Lewisb and Lewisx determinants.
Two salmon neuropeptides encoded by one brain cDNA are structurally related to members of the glucagon superfamily
Authors: PARKER David B. , COE Imogen R. , DIXON Gordon H. , SHERWOOD Nancy M. 
Published: March 2005

A cDNA that codes for two peptides in the glucagon superfamily has been isolated from sockeye salmon brain. The first peptide is related to growth hormone‐releasing hormone (GHRH), which has high sequence similarity with PACAP‐related peptide. The second peptide is structurally related to vasoactive intestinal peptide, which is also related to a newly identified peptide in mannals, pituitary adenylate‐cyclase‐activating polypeptide (PACAP). The salmon precursor contains 173 amino acids and has dibasic and monobasic enzyme‐processing sites for cleavage of a 45‐amino‐acid GHRH‐like peptide with a free C‐terminus and a 38‐amino‐acid PACAP with an amidated C‐terminus. The salmon GHRH‐like peptide has 40% amino acid sequence identity with the human GHRH and 56% identity with human PACAP‐related peptide. The 38‐amino‐acid salmon PACAP is highly conserved (89–92% identity) with only three or four amino acid substitutions compared with the human, ovine and rat 38‐amino‐acid PACAP.Not previously reported for mammalian species, a short precursor coding for only one peptide exists in salmon in addition to the long precursor coding for two peptides. In the short precursor, the coding region for GHRH is deleted leaving the PACAP‐coding region in a correct reading frame. This provides one possible control mechanism for an increased expression of one peptide (PACAP) without the concomitant increase in the other peptide (GHRH) as occurs in a double‐peptide precursor. The importance of the 3′non‐translated region of the salmon GHRH/PACAP precursor in the regulation of translation is suggested by its 70% nucleotide sequence identity to the 3′ non‐translated region of the mammalian PACAP precursors. The structural organization of the salmon GHRH/PACAP precursor provides a possible evolutionary scheme for precursors that contain tandem peptides in the glucagon superfamily.
A region of the C‐terminal part of the 11‐kDa subunit of ubiquinol‐cytochrome‐c oxidoreductase of the yeast Saccharomyces cerevisiae contributes to the structure of the Qout reaction domain
Authors: HEMRIKA Wieger , BERDEN Jan A. , GRIVELL Leslie A. 
Published: March 2005

QCR8, the gene encoding the 11‐kDa subunit of ubiquinol‐cytochrome‐c oxidoreductase of the yeast Saccharomyces cerevisiae has been resequenced in the course of a search for mutants disturbed in subunit function. Resequencing shows that the previously published sequence [Maarse A. C. & Grivell L. A. (1987) Eur. J. Biochem 155, 419–425] lacks a C at position 185 of the coding sequence. As a result of this extra nucleotide, the reading frame now contains 285 base pairs and it codes for a protein of 94 amino acids with a calculated molecular mass of 11.0 kDa. Despite the altered C‐terminus, similarity to the corresponding beef heart subunit is not significantly altered.One mutant (LTN1), arising from hydroxylamine mutagenesis, has been studied in detail: Assembly of the enzyme appears to be normal, as judged from the levels of the subunits observed in Western blots, while spectral analysis showed that only holo‐cytochrome b was lowered to 70% of that of the wildtype. Measurement of the specific activity and calculation of the turnover number of the enzyme showed that these were 45% and 56% of that of the wild type, respectively.Further analysis of the mutant showed that the affinity for the inhibitor myxothiazol was decreased, that the 11‐kDa subunit stabilises the enzyme once assembly has occurred, and that the reduction of cytochrome b via the Qout site is impaired. Sequence analysis showed that this mutant carries a deletion of 12 nucleotides at position 206–217 of the coding sequence, resulting in the replacement of residues 69–73 (WWKNG) by a cysteine.These results are discussed in terms of the 11‐kDa subunit contributing to the conformation of the Qout binding domain.
Activation of the cloned platelet thrombin receptor decreases the pertussis‐toxin‐dependent ADP‐ribosylation of the membrane and soluble inhibitory guanine‐nucleotide‐binding‐α proteins
Authors: WILHELM Bernhard , SIESS Wolfgang 
Published: March 2005

Thrombin binds at least to two sites of the platelet surface; to the recently cloned thrombin receptor [Vu, T. K., Hung, D. T., Wheaton, V. I. & Coughlin, S. R. (1991) Cell 64, 1057–1068] and to glycoprotein Ib. In the present study, the decrease of pertussis‐toxin‐dependent ADP‐ribosylation of membrane and soluble inhibitory guanine‐nucleotide‐binding α (Giα) proteins was measured after platelet stimulation with a thrombin‐receptor‐activating peptide (TRAP), and compared to stimulation with thrombin. Stimulation of intact platelets with TRAP decreased the pertussis‐toxin‐dependent ADP‐ribosylation of the major membrane 41‐kDa Giα protein and the minor soluble 40 kDa Giα protein recently described in platelets [Gennity, J. M. & Siess, W. (1991) Biochem. J. 279, 643–650]. The kinetics and extent of the decrease of pertussis‐toxin‐dependent ADP‐ribosylation after stimulation of TRAP were similar to the effect of thrombin. The decrease of pertussis‐toxin‐dependent ADP‐ribosylation of the soluble Giα protein was more pronounced and observed at lower agonist concentrations than the decrease of the membrane Giα protein. Desensitization of the thrombin receptor by incubating platelets with a low concentration of TRAP reduced the subsequent decrease of pertussis‐toxin‐dependent ADP‐ribosylation of Giα proteins, evoked by TRAP or thrombin. Platelet stimulation with γ‐thrombin that does not bind to glycoprotein Ib also showed a decrease in the pertussis‐toxin‐dependent ADP‐ribosylation of the soluble and membrane Giα proteins. Treatment of platelets with the stable prostacyclin analog, iloprost, reduced the decrease of pertussis‐toxin‐dependent ADP‐ribosylation of Giα proteins induced by TRAP or thrombin. Among other platelet stimuli tested (endoperoxide/thromboxane analog U44619, collagen, ADP, vasopressin), only U44619 decreased the pertussis‐toxin‐dependent ADP‐ribosylation of the soluble and membrane Giα proteins to a degree comparable to TRAP.It is concluded that the thrombin‐induced activation of both the membrane and soluble Giα proteins in platelets occurs via stimulation of the recently cloned thrombin receptor and is independent of the binding of thrombin to glycoprotein Ib. Furthermore, the coupling thrombin receptor/Gi protein is reduced by intracellular cAMP.
Zinc coordination in mammalian sorbitol dehydrogenase
Authors: KARLSSON Christina , HÖÖG Jan‐Olov 
Published: March 2005

Rat sorbitol dehydrogenase was expressed in Escherichia coli and purified to homogeneity, resulting in a protein with a specific activity of 4.7 U/mg, close to that of the enzyme isolated from mammalian liver.A Glu residue has been postulated to replace the Cys of alcohol dehydrogenase as a ligand to the active‐site zinc atom of sorbitol dehydrogenase. This Glu (position 155 in the rat enzyme) was mutated both to Cys, in order to mimic the alcohol dehydrogenase relationships, and to Ala, as a control. A third mutation, Cys164 to Ala, was also performed since Cys has also been considered as a possible zinc ligand. With Ala at position 155, an inactive enzyme was obtained, showing that correct active‐site relationships have been destroyed. With Cys at position 155, the enzyme is still partly active, but rapidly looses activity unless stabilized by the addition of ZnSO4. The catalytic efficiency in the oxidation of sorbitol is 120‐fold less than that of the native form, and reduction of fructose is lost completely. In contrast, the activity of the Cys164Ala mutant is comparable with that of the native enzyme and, in fact, even increased in the oxidation of sorbitol. Combined, the results strongly suggest that Glu155 is a ligand to the active‐site zinc atom. Zinc analysis of the different variants of sorbitol dehydrogenase establishes that all contain one atom of zinc/subunit, also when the catalytic function is lost. Apparently, zinc remains coordinated even after replacement with an amino acid residue (Ala) unable to ligand metal atoms.
Comparative study of high‐mannose‐type oligosaccharides in membrane glycoproteins of rat hepatocytes and different rat hepatoma cell lines
Authors: NUCK Rolf , PAUL Christine , WIELAND Barbara , HEIDRICH Claudia , GEILEN Christoph C. , REUTTER Werner 
Published: March 2005

A comparative study was undertaken to characterize the oligosaccharides released by endo‐β‐N‐acetylglucosaminidase H (endo H) from the membrane glycoproteins of rat hepatocytes and three different Morris hepatoma cell lines (NA‐MH 7777, HTC and MH1C1). It is shown that the membrane glycoproteins of hepatocytes and hepatoma cells contain markedly different quantities and forms of high‐mannose‐type carbohydrate chains.After radiolabelling of the cells with d‐[2‐3H]mannose, in the absence and presence of 1 mM 1,5‐dideoxy‐1,5‐imino‐d‐mannitol (1‐deoxymannojirimycin), high‐mannose‐type oligosaccharides were released from delipidated membrane glycoproteins by enzymic digestion with endo H. The carbohydrate chains were converted to their corresponding oligosaccharide alditols by reduction with sodium borohydride, then further analysed by HPLC using an APS‐2 Hypersil column.In the absence of 1‐deoxymannojirimycin, up to 10% of the radiolabelled oligosaccharides were released by endo H‐treatment of the membrane glycoprotein fraction from rat hepatocytes. In contrast, the quantity of radiolabelled high‐mannose‐type carbohydrate chains released by endo H‐treatment from tumour‐cell membrane glycoproteins of hepatoma cell lines NA‐MH 7777 (31,5%), MH1C1‐MH 7795 (37,2%) and HTC‐MH 7288c (48%) was increased up to fivefold. The formation of higher‐mannosylated structures after oligosaccharide analysis was observed in all hepatoma cell lines, with Man8GlcNAcOH as the major component, whereas in hepatocytes Man5GlcNAcOH was the predominant high‐mannose‐type structure.In contrast, in the presence of the Golgi α‐d‐mannosidase I inhibitor, 1‐deoxymannojirimycin, no significant differences were observed between the distribution of high‐mannose‐type oligosaccharides in the membrane glycoproteins of hepatocytes and hepatoma cells. However, in the presence of this inhibitor, the proportion of radiolabelled glycans sensitive to deglycosylation by endo H was greatly increased (>85%) in all the cell lines investigated, the predominant structures being Man8–9−GlcNAcOH. This study shows that an increased content of high‐mannose‐type sugar chains is a general characteristic of membrane‐bound glycoproteins for malignant transformed hepatocytes.
Cytochrome c6 from Monoraphidium braunii
Authors: CAMPOS António P. , AGUIAR António P. , HERVÁS Manuel , REGALLA Manuela , NAVARRO José A. , ORTEGA José M. , XAVIER António V. , DE LA ROSA Miguel A. , TEIXEIRA Miguel 
Published: March 2005

A soluble monoheme c‐type cytochrome (cytochrome c6) has been isolated from the green alga Monoraphidium braunii. It has a molecular mass of 9.3 kDa, an isoelectric point of 3.6 and a reduction potential of 358 mV at pH 7. The determined amino acid sequence allows its classification as a class‐I c‐type cytochrome. The ferric and ferrous cytochrome forms and their pH equilibria have been studied using 1H‐NMR, ultraviolet/visible, EPR and Mössbauer spectroscopies. The pH equilibria are complex, several pKa values and pH‐dependent forms being observed. The amino acid sequence, the reduction‐potential value and the visible and NMR spectroscopies data in the pH range 4–9 indicate that the heme iron has a methionine‐histidine axial coordination. However, the EPR and Mössbauer data obtained for the ferricytochrome show that in this pH range two distinct forms are present: form I, gz= 3.27, gy= 2.05 and gx= 1.05; form II, gz= 2.95, gy= 2.29 and gx= 1.43. While form I has crystal‐field parameters typical of a methionine‐histidine coordination, those associated with form II would suggest a histidine‐histidine axial ligation. This possibility was extensively analyzed by spectroscopic methods and by chemical modification of a histidine residue. It was concluded that form II actually corresponds to an unusual type of methionine‐histidine axial coordination. Straightforward examples of this type of coordination have recently been found in other c‐type hemeproteins [Teixeira, M., Campos, A. P., Aguiar, A. P., Costa, H. S., Santos, H., Turner, D. L. & Xavier, A. V. (1993) FEBS Lett. 317, 233–236], corroborating our proposal. Since both forms, with very distinct crystal‐field parameters, are shown to have the same reduction potential, it may be concluded that the axial and rhombic distortions of the heme‐iron ligand field cannot be directly correlated with the heme‐reduction potential.The pH‐dependence studies have also shown that the form I and form II are interconvertible, with pKa∼ 5. To establish a possible physiological significance for this process in particular for the interaction of the cytochrome with the membrane‐bound electron‐transfer complexes b6f and photosystem I, the effect of surfactants on the spectroscopic characteristics of cytochrome c6 has been studied.
DNase‐I‐hypersensitive sites located far upstream of the human c‐sis/PDGF‐B gene comap with transcriptional enhancers and a silencer and are preceded by (part of) a new transcription unit
Authors: DIRKS Ron P. H. , JANSEN Hans J. , ONNEKINK Carla , DE JONGE Rob J. A. , BLOEMERS Henri P. J. 
Published: March 2005

The human c‐sis gene encodes the B chain of platelet‐derived growth factor (PDGF), a potent mitogen for cultured cells of mesenchymal origin. PDGF is stored in the α‐granules of blood platelets, which are derived from bone marrow megakaryocytes and lack transcriptional machinery. Human myeloid leukemia cell line K562 can be used as a model for megakaryocytes. Phorbol‐estermediated megakaryocytic differentiation of K562 cells is accompanied by more than 200‐fold increase in the c‐sis mRNA level. We have now localized transcriptional enhancers at –8.6 kb and –9.9 kb relative to the human c‐sis gene transcription start site. The enhancer at –8.6 kb increases activity of the c‐sis promoter by 40–60‐fold specifically in K562 cells and comaps with a DNase‐I‐hypersensitivity (DH) site. The enhancer at –9.9 kb increases c‐sis promoter activity by 5–10‐fold in K562 cells and DH at that site accompanies phorbol‐ester‐induced megakaryocytic differentiation. In phorbol‐ester‐treated K562 cells the two enhancers may be negatively influenced by a silencer that comaps with DH at –10.7/–11.0 kb. Reporter gene analysis predicted that combined activity of the upstream enhancers and the c‐sis promoter may result in 100–1000‐fold higher promoter acitvity in phorbol‐ester‐treated K562 cells compared with untreated cells, which can fully explain the more than 200‐fold increase in c‐sis mRNA level. DH at –8.6 kb and –9.9 kb was also detected in human fibroblasts and in the carcinoma cell lines HeLa and PC3, which express, respectively, undetectable, low and high levels of c‐sis mRNA. Although the individual DH sites displayed 4–10‐fold enhancer activity in all these cells, they lost most of their biological activity when combined in a larger fragment.In addition we localized (part of) a new transcription unit at approximately 13 kb upstream of the c‐sis transcription start site. The corresponding 0.45‐kb sis upstream region (sur) transcript is constitutively expressed in all cell lines examined. The expression of the sur transcript is independent of the expression of c‐sis mRNA and of the pattern of DH sites far upstream of the c‐sis gene. Thus, at present, there is no indication that the upstream DH sites are involved in regulation of expression of the sur gene.
A lipid‐anchored heparan sulfate proteoglycan is present in the surface of differentiated skeletal muscle cells
Authors: CAMPOS Alejandro , NÙÑEZ Rebeca , KOENIG Cecilia S. , CAREY David J. , BRANDAN Enrique 
Published: March 2005

We have investigated the presence of hydrophobic membrane‐associated heparan sulfate proteoglycans (HSPG) on the cell surface of differentiated skeletal muscle cells. A HSPG releasable by incubation with a phosphatidylinositol‐specific phospholipase c (PtdIns‐PLC) was obtained. HSPG were also isolated from Triton X‐100 extracts of the cells. The hydrodynamic characteristics of the PtdIns‐PLC‐releasable and detergent‐extracted HSPG were indistinguishable. SDS/PAGE analysis of the PtdIns‐PLC‐releasable HSPG indicated a molecular mass of 250 k Da. Analysis of proteins immunoprecipitated by specific antibodies against a HSPG isolated from Schwann cells demonstrated that the antisera precipitated an intact HSPG that was present in the pool of proteins released by PtdIns‐PLC and by Triton X‐100 from [35S] sulfate labeled cells. Nitrous acid degradation of the immunoprecipitated proteins released by PtdIns‐PLC from [35S] methionine labeled cells produced a single 67‐kDa core protein. Analysis of hydrophobicity of the purified HSPG revealed that only the HSPG obtained from the detergent extract were able to be incorporated into the liposomes whereas the PtdIns‐PLC‐released HSPG was not.Immunocytolocalization analysis of the differentiated cells indicated that the PtdIns‐PLC‐releasable HSPG was located on the cell surface. Prior incubation of the cells with PtdIns‐PLC significantly reduced the surface staining. Analysis of skeletal‐muscle sections of adult rat skeletal muscles indicated that this HSPG localized exclusively at the endomysium. This localization suggest that these HSPG may be acting as a cell receptor for extracellular‐matrix (ECM) components.