bluuebunny/crossref_metadata_2025_split huggingface dataset:
FMRFamide‐related peptides in the sex segmental ganglia of the Pharyngobdellid leech Erpobdella octoculata
Authors: SALZET Michel , BULET Philippe , WATTEZ Christian , MALECHA Jean 
Published: March 2005

Using enzyme‐linked immunosorbent assays, a dot‐immunobinding assay and a three‐step reverse‐phase HPLC separation, four Arg‐Phe‐amide (RFamide) peptides were purified from sex segmental ganglia extracts of the leech Erpobdella octoculata; FMRFamide, FM(O)RFamide, FLRFamide and GDPFLRFamide. Their amino acid sequences were elucidated by means of a combined approach using antiserum specificity, synthetic‐peptide coelution, automated Edman degradation and electrospray mass spectrometry. One of these peptides, GDPFLRFamide, is a novel leech RFamide neuropeptide.Two of the above RFamide peptides are involved in the control of leech hydric balance; one (GDPFLRFamide) is diuretic, the other (FMRFamide) is anti‐diuretic.Titration of each purified RFamide peptide indicated a similar amount of each tetrapeptide and of tetrapeptides and heptapeptides. A comparison between RFamide peptides of E. octoculata and molluscs reveals structural similarities supporting the hypothesis for the existence of an ancestral RFamide peptide gene common to leeches and molluscs.
The inner core region of Yersinia enterocolitica Ye75R (0:3) lipopolysaccharide
Authors: RADZIEJEWSKA‐LEBRECHT Joanna , SHASHKOV Alexander S. , STROOBANT Vincent , WARTENBERG Klaus , WARTH Christoph , MAYER Hubert 
Published: March 2005

The inner‐core region of the lipopolysaccharide of an UDPGalNAc‐4‐epimerase‐deficient mutant of Yersinia enterocolitica 0:3, designated as Ye75R, was investigated using methylation analysis, 1D‐13C‐NMR and 2D‐13C‐NMR and 1H‐NMR, as well as 31P‐NMR, fast‐atom‐bombardment mass spectrometry (FAB MS) and FAB MS/MS in positive and negative modes. The isolated core heptasaccharide (OS) was composed of 2 units d‐glucose, 3 units ld‐heptose and 1 unit each of dd‐heptose and 3‐deoxy‐d‐manno‐octulosonic acid. Methylation analysis indicated that OS was highly branched with terminal location of the two glucoses and the dd‐heptose unit, which was partially (to about 40%) phosphorylated at C7. These combined studies allowed us to formulate the structure of the inner core region as shown in Scheme 1.The substitution of the 7‐position of the terminally linked dd‐heptose unit by phosphate could be recognized by MS characterization of permethylated dd‐heptose‐7‐phosphate (alditol acetate) and the extent of the substitution by the ratio of the two well separated 1H signals of dd‐heptose in 500‐MHz 1H‐NMR. Negative FAB MS of OS also indicated the presence of smaller amounts of two hexasaccharides, differing from OS in lacking either one terminal unit of d‐glucose or of the terminal dd‐heptose, and additionally of a pentasaccharide lacking two heptosyl units, namely the terminal dd‐heptose and and the subterminal ld‐heptose. The presence of the smaller oligosaccharides in the OS fraction was also recognized by the methylation analysis.
Structure of the O‐linked carbohydrate chains of porcine zona pellucida glycoproteins
Authors: HOKKE Cornelis H. , DAMM Jan B. L. , PENNINKHOF Bea , AITKEN R. John , KAMERLING Johannis P. , VLIEGENTHART Johannes F. G. 
Published: March 2005

The N‐linked carbohydrate chains of procine zona pellucida glycoproteins were released by digestion with peptide‐N4‐(N‐acetyl‐β‐glucosaminlyl)asparagine amidase F and subsequently separated from the O‐glycoprotein by gel‐permeation chromatography on Bio‐Gel P‐100. The O‐linked carbohydrate chains were relesed from the O‐glycoprotein by alakaline borohydride treatment. Fractitionation of the extremely heterogeneous mixture of O‐linked oligosaccharide alditols was achieved by a combination of chromatographic techniques comprising gel‐permeation chromatography on Bio‐Gel P‐4 and P‐6, anion‐exchange FPLC on Mono Q, and high‐pH anion‐exchange chromatography on CarboPac PA‐1. The primary structures of 32 O‐glycans were determined by one‐ and two‐dimensional 1H‐NMR spectroscopy. The major part of the analyzed compounds contain a combination of the structural elements Galβ1‐4GlcNAcβ1‐3Galβ1‐3GalNAc‐ol, Galβ1‐4(6SO4‐)GlcNAc, and α2‐3‐linked Neu5Gc or Neu5Ac. This series of compounds has the following structure, where n= 0 to > 6 : [Neu5Gc/Acα2‐3]0‐1[Galβ1‐4(6SO4‐)GlcNAcβ1‐3]nGalβ1‐4GlcNAcβ1‐3Galβ1‐3GalNAc‐ol.In addition, smaller compounds were identified in which the Galβ1‐3GalNAc‐ol core is substituted by Neu5Gc/Ac α2‐6‐linked to GalNAc‐ol and/or Neu5Gc/Ac α2‐3‐linked to Gal. Furthermore, oligosaccharides were obtained in which the distribution of 6‐O‐sulfated GlcNAc residues differs from that in the above‐mentioned general structure, and a small portion of the oligosaccharides has the GlcNAcβ1‐3GalNAc‐ol core structure. Analysis of the endo‐β‐galactosidase digests of pools of N‐ and O‐glycans indicated that the two types of oligosaccharides contain qualitatively similar poly(N‐acetyllactosamine) chains. In the case of the N‐linked carbohydrate chains, multiple branching of the core structures occurs, resulting in an even larger heterogeneity than observed for the O‐linked carbohydrate chains.
The complete amino acid sequence of R‐phycocyanin‐I α and β subunits from the red alga Porphyridium cruentum
Authors: DUCRET Axel , SIDLER Walter , FRANK Gerhard , ZUBER Herbert 
Published: March 2005

We present here the complete primary structure of R‐phycocyanin‐I α and β subunits from the red alga Porphyridium cruentum. The α chain is composed of 162 amino acid residues (18049Da, calculated from sequence, including chromophore) and carries a phycocyanobilin pigment covalently linked to Cys84. The β chain contains 172 amino acids (19344Da, calculated from sequence, including chromophores) and carries a phycocyanobilin pigment covalently linked at Cys82 and a phycoerythrobilin pigment at Cys153. A γ‐N‐methyl asparagine residue was also characterised at position β72 similar to other phycobiliprotein β subunits. R‐phycocyanin‐I from Porphyridium cruentum shares high sequence identity with C‐phycocyanins (69–83%), R‐phycocyanins (66–70%) and in a less extent with phycoerythrocyanins (57–65%) from various sources.The presented phylogenetic trees are based on a comparison of all phycobiliprotein amino acid sequences known so far and confirm the clear affiliation of the R‐phycocyanins in the phycocyanin family. In spite of their particular phycobilin pattern, they do not represent intermediate forms between the phycocyanin and the phycoerythrin family. Phycoerythrocyanin, a phycocyanin‐related phycobiliprotein adapted to green light harvesting, is also shown to belong to the phycocyanin family. However, the phycoerythrocyanins diverge from phycocyanins in their different function and it is suggested that they should be assigned to a separate group within the phycocyanin family.
Proteins under pressure
Authors: GROSS Michael , JAENICKE Rainer 
Published: March 2005

Oceans not only cover the major part of the earth's surface but also reach into depths exceeding the height of the Mt Everest. They are populated down to the deepest levels (∼11 800 m), which means that a significant proportion of the global biosphere is exposed to pressures of up to 120 MPa. Although this fact has been known for more than a century, the ecology of the ‘abyss” is still in its infancy. Only recently, barophilic adaptation, i.e. the requirement of elevated pressure for viability, has been firmly established. In non‐adapted organisms, increased pressure leads to morphological anomalies or growth inhibition, and ultimately to cell death. The detailed molecular mechanism of the underlying ‘metabolic dislocation' is unresolved.Effects of pressure as a variable in microbiology, biochemistry and biotechnology allow the structure/function relationship of proteins and protein conjugates to be analyzed. In this context, stabilization by cofactors or accessory proteins has been observed. High‐pressure equipment available today allows the comprehensive characterization of the behaviour of proteins under pressure. Single‐chain proteins undergo pressure‐induced denaturation in the 100‐MPa range, which, in the case of oligomeric proteins or protein assemblies, is preceded by dissociation at lower pressure. The effects may be ascribed to the positive reaction volumes connected with the formation of hydrophobic and ionic interactions. In addition, the possibility of conformational effects exerted by moderate, non‐denaturing pressures, and related to the intrinsic compressibility of proteins, is discussed. Crystallization may serve as a model reaction of protein self‐organization. Kinetic aspects of its pressure‐induced inhibition can be described by a model based on the Oosawa theory of molecular association. Barosensitivity is known to be correlated with the pressure‐induced inhibition of protein biosynthesis. Attempts to track down the ultimate cause in the dissociation of ribosomes have revealed remarkable stabilization of functional complexes under pseudo‐physiological conditions, with the post‐translational complex as the most pressure‐sensitive species. Apart from the key issue of barosensitivity and barophilic adaptation, high‐pressure biochemistry may provide means to develop new approaches to nonthermic industrial processes, especially in the field of food technology.
Isolation, characterization and developmental expression of the ecdysteroid‐induced E75 gene of the wax moth Galleria mellonella
Authors: JINDRA Marek , SEHNAL František , RIDDIFORD Lynn M. 
Published: March 2005

Using the cDNA for the Drosophila ecdysteroid‐induced member of the steroid‐hormone‐receptor superfamily, E75 A, we isolated a genomic clone from Galleria mellonella that revealed 77% similarity with the region of E75 A cDNA encoding the C‐terminal zinc‐finger motif. A Galleria cDNA clone was isolated that encoded a complete DNA‐binding domain composed of two zinc fingers and designated GmE75 A. Its deduced amino acid sequence showed 100% and 85% identities within the DNA‐binding and ligand‐binding domains of Drosophila E75 A, respectively. The Galleria genomic clone did not encode the N‐terminal zinc finger, but included a sequence similar to the B1 exon, which is unique to the B isoform of E75. Thus, the cDNA and genomic DNA sequences indicated that the Galleria gene E75 encoded at least two isoforms, GmE75 A and GmE75 B, which differed in their N‐termini.Probes specific for GmE75 A and B hybridized to two distinct transcripts of 2.6 kb. Both GmE75 A and B mRNA levels correlated closely with the ecdysteroid titer during development. At the onset of larval/pupal transformation, both transcripts appeared in high amounts within 4 h of the ecdysteroid rise, then declined concurrently with the hormone titer decline. At the time of pupal ecdysis, there was another peak of GmE75 A expression but not GmE75 B expression, coincident with a minor ecdysteroid pulse. In isolated abdomens of final instar larvae, GmE75 A mRNA was induced by 20‐hydroxyecdysone within 20 min of the injection; the mRNA levels were maximal at 1 h and declined by 3 h following the treatment.
Heterogenous lipid distribution among chlorophyll‐binding proteins of photosystem II in maize mesophyll chloroplasts
Authors: TREMOLIERES Antoine , DAINESE Paola , BASSI Roberto 
Published: March 2005

Photosystem II membrane fractions from dark‐adapted mesophyll chloroplasts of maize were solubilized in different concentrations of dodecyl β‐D‐maltoside. Chlorophyll‐binding proteins from photosystem II were isolated either by ultracentrifugation on a sucrose gradient, or by flat bed isoelectric focusing and identified by gel electrophoresis analysis for their polypeptide composition. Lipid and fatty acid compositions were determined in complexes prepared by both methods and also in purified light‐harvesting complex II, in minor chlorophyll a/b binding complexes 29, 26, 24, in photosystem II antennae (chlorophyll‐protein complexes 43, 47) and in the photosystem II reaction centers chlorophyll‐protein complexes. Comparative analysis of the results suggests that a true heterogeneity exists in the lipid class distribution among the different chlorophyll‐protein complexes in this region of the photosynthetic membrane. Photosystem II core fractions prepared either by ultra‐centrifugation on a sucrose gradient or by isoelectric focusing were found significantly enriched in monogalactosyldiacylglycerol; fractionation of the photosystem II core in its components showed that it was the chlorophyll‐protein complexes 43 and 47 which were mainly responsible for this enrichment. One of them, the chlorophyll‐protein complex 47, was found containing monogalactosyldiacylglycerol and having a very high level of saturated fatty acids. The minor chlorophyll a/b binding linkers (chlorophyll‐protein complexes 24, 26 and 29) retain a largely higher amount of lipids than all other complexes and especially of highly unsaturated galactolipids.Concerning the main light‐harvesting antenna (LHCII), it is demonstrated that phosphatidylglycerol is strongly linked to the complex if it cannot be detached at high detergent concentration, while many galactolipids (which nevertheless represent the major lipid classes) are lost. This main light‐harvesting complex has been fractionated into several families by isoelectric focusing showing a marked difference in lipid and polypeptide composition. A spectacular increase in the phosphatidylglycerol content was observed in the fraction migrating near the anode and enriched in a 26‐kDa polypeptide; but this result is difficult to interpret in physiological terms as it was shown that phosphatidylglycerol alone, because of its negative charge, also migrates toward the anode in isoelectric focusing.
Characterization and molecular cloning of mannose‐binding lectins from the Orchidaceae species Listera ovata, Epipactis helleborine and Cymbidium hybrid
Authors: VAN DAMME Els J. M. , SMEETS Koen , TORREKENS Sophie , VAN LEUVEN Fred , PEUMANS Willy J. 
Published: March 2005

Mannose‐binding lectins were purified from the leaves of three Orchidaceae species, namely Listera ovata (twayblade), Epipactis helleborine (broad‐leaved helleborine) and Cymbidium hybrid, using affinity chromatography on mannose–Sepharose‐4B. Apparently, the Orchidaceae lectins are dimeric proteins composed of lectin subunits of 12–13 kDa. All of the isolated lectins exhibit exclusive specificity towards mannose.A cDNA library constructed from poly(A) rich RNA isolated from leaves of L. ovata was screened for cDNA clones encoding the lectin using colony hybridization. Since N‐terminal sequence analysis of the twayblade lectin revealed some sequence similarity to the previously cloned mannose‐binding lectin from Hippeastrum hybrid (amaryllis) ovaries, the amaryllis lectin cDNA clone was used as a probe to screen the L. ovata library. Subsequently, the cDNA clone encoding the L. ovata lectin was used to screen the cDNA libraries from the taxonomically related orchid species Cymbidium hybrid and E. helleborine. Sequence analysis of the lectin cDNA clones from different Orchidaceae species revealed approximately 50% sequence similarity both at the nucleotide and amino acid level.The Orchidaceae lectins are apparently translated from mRNAs consisting of approximately 800 nucleotides. The primary translation products are preproproteins which are converted into the mature lectins following post‐translational modifications. Southern blot analysis of genomic DNA has shown that the lectins are most probably encoded by a family of closely related genes which is in good agreement with the sequence heterogeneity found between different lectin cDNA clones of one species.
Inhibition and activation studies on sheep liver sorbitol dehydrogenase
Authors: LINDSTAD Rune I. , HERMANSEN Leonila F. , McKINLEY‐McKEE John S. 
Published: March 2005

Reversible inhibition and activation, as well as protection against affinity labelling with dl‐2‐bromo‐3‐(5‐imidazolyl)propionic acid, of sheep liver sorbitol dehydrogenase have been studied. The results presented are discussed in terms of enzyme active‐site properties and may have potential applications for drug design.Kinetics with mainly sorbitol competitive inhibitors reveals that aliphatic thiols are generally the most potent inhibitors of enzyme activity. Inhibition and inactivation by heterocyclics parallel that seen previously with sorbitol dehydrogenase from other sources as well as with alcohol dehydrogenase from yeast. However, there are significant differences in relation to the structurally similar horse liver alcohol dehydrogenase, as the catalytic zinc of sorbitol dehydrogenase is more easily removed by chelating molecules. Several aldose reductase inhibitors are shown to also inhibit sorbitol dehydrogenase, but at concentrations unlikely to be reached clinically. Enzyme activation has been observed with various compounds, in particular halo‐alcohols and detergents.Several inhibitors provide competitive protection against enzyme inactivation by dl‐2‐bromo‐3‐(5‐imidazolyl)propionic acid. This enables the dissociation constants for binary enzyme‐inhibitor complexes to be determined. NADH protects noncompetitively against inactivation.The presence of some binary and ternary enzyme‐NADH complexes is indicated from fluorescence emission spectra, as a shift in the fluorescence maximum and intensity is observed due to their formation.
The C‐terminus of lipoprotein lipase is essential for biological function but contains no domain for glycosylphosphatidylinositol anchoring
Authors: BRUIN Taco , GROOT Niels B. , JANSEN Jaap , KASTELEIN John J. P. 
Published: March 2005

In this study we present evidence that the C‐terminus of lipoprotein lipase contains no glycosylphosphatidylinositol addition signal and is therefore not a glycosylphosphatidylinositol‐anchored protein. Furthermore, we present additional evidence that the C‐terminus of lipoprotein lipase is essential for biological function.Flow cytometric analysis and enzyme‐activity monitoring experiments revealed no pool of lipoprotein lipase releasable by phosphatidylinositol‐specific phospholipase present on the membrane of COS cells transfected with the human lipoprotein lipase gene while, in contrast, a heparin‐releasable pool could be demonstrated. [14C]Ethanolamine, a constituent of the glycosylphosphatidylinositol anchor, was not incorporated into lipoprotein lipase during metabolic labeling. C‐terminal deletion mutants were constructed and expressed in COS cells to investigate the presence of glycosylphosphatidylinositol addition signal on the C‐terminus of human lipoprotein lipase (LPL). The specific activities of the mutants M442 [Leu443–Gly448)‐LPL] and M437 [des‐(Cys438–Gly448)‐LPL] were 78% and 59%, respectively, less than the wild type, while the M432 mutant [des‐(Ala433–Gly449)‐LPL] was catalytically inactive. Determination of the stability of the mutants revealed a decreased stability of the M437, compared with wild‐type, whereas M442 showed the same stability. Flow cytometric analysis showed sustained membrane expression for all mutants including the inactive M432 mutant. These results suggest that the C‐terminus of lipoprotein lipase is essential for maintaining intact catalytic activity but is not involved in any posttranslational proteolytic processing, including cleavage of a glycosylphosphatidylinositol addition signal. We therefore conclude that membrane‐binding of the lipase is not mediated by such anchoring.
Biosynthesis of gangliosides containing C18:1 and C20:1 [3‐14C]sphingosine after administrating [1‐14C]palmitic acid and [1‐14C]stearic acid to rat cerebellar granule cells in culture
Authors: CHIGORNO Vanna , VALSECCHI Manuela , SONNINO Sandro 
Published: March 2005

The biosynthesis of ganglioside molecular species containing sphingosine of different structure was investigated by administrating rat cerebellar granule cells in culture with [1‐14C]palmitic and [1‐14C]stearic acids which are the precursors for sphingosine biosynthesis.The incorporation of radioactivity into the sphingosine of the ganglioside species containing C20:1 sphingosine after administrating [1‐14C]stearic acid was low in comparison with the incorporation of radioactivity into the sphingosine of ganglioside species containing C18:1 sphingosine after administration of [1‐14C]palmitic acid, but the ratio between the radioactivity incorporated in the C20:1 and the C18:1 sphingosine of C20 and C18 ganglioside species progressively increased when the cell culture was prolonged.Ceramide‐containing radioactive sphingosine was found after palmitic or stearic acid administration. Ceramide‐containing C20:1 sphingosine found after adding stearic acid was about 5% of that synthesized starting from palmitic acid and containing C18:1 sphingosine.Free radioactive C18:1 and C20:1 sphingosine were found after adding radioactive palmitic or stearic acid. This is representative of a catabolic process occurring after biosynthesis of the complex sphingolipid starting from the radioactive precursor. In fact it has been proved that only saturated sphingosines are used for the synthesis of complex sphingolipids, the introduction of the double bond at position four of the sphingoid chain occurring at the level of ceramide [Rother, J., van Echten, G., Schwarzmann, G. & Sandhoff, K. (1992) Biochem. Biophys. Res. Commun. 189, 14–20]. Saturated sphingosines were not present. The lack of free C20:0 sphingosine confirms the hypothesis that the C20:0 sphingosine synthesis and the process {C20:0 sphingosine C20:0 ceramide→C20:1 ceramide} occur in the correct quantity for the synthesis of C20:1 gangliosides. Moreover, we found only traces of free C20:1 sphingosine, at days 8 and 15 of cell culture when the biosynthesis of complex C20:1 gangliosides and the related catabolic processes occur to a higher extent, thus excluding the idea that a large amount of C20:0 sphingosine can be acylated to C20:0 ceramide and dehydrogenated to C20:1 ceramide which, being not used for ganglioside biosynthesis, is immediately catabolised to C20:1 sphingosine.
Correlations between biological activities and conformational properties for human, salmon, eel, porcine calcitonins and Elcatonin elucidated by CD spectroscopy
Authors: SILIGARDI Giuliano , SAMORÍ Bruno , MELANDRI Sonia , VISCONTI Marco , DRAKE Alex F. 
Published: March 2005

Calcitonin (CT) inhibits osteoclastic bone resorption and induces calcium uptake from body fluids. A comparative study of the conformational behaviours of therapeutic calcitonins [salmon (s), eel (e), a synthetic eel calcitonin analogue (Elcatonin), porcine (p) and human (h) calcitonins] as a function of solvent polarity and temperature have been performed by circular dichroism spectroscopy. Elements of secondary structure were lacking in H2O but could be observed in 2,2,2‐trifluoroethanol and sodium dodecyl sulphate. In particular, similar amounts of α‐helical content (four α‐helical turns) were estimated in trifluoroethanol despite the considerable differences in amino acid sequences. The relative ability to form an α helix, assessed by trifluoroethanol/H2O titration, was found to be Elcatonin > sCT > pCT > eCT > hCT. In Elcatonin, sCT, pCT and eCT the four α‐helical turns were promoted almost completely in a single step, between 0 and 35% trifluoroethanol, unlike hCT where helical structure formation has been reported to involve two steps over the whole trifluoroethanol/H2O range [Arvinte, T. & Drake, A. F. (1993) J. Biol. Chem. 268, 6408–6414]. In SDS, which mimics the membrane environment, conformational differences (3–4 helical turns in Elcatonin, sCT, eCT versus one helical turn in pCT, hCT) were observed and correlate well with biological activity (Elcatonin = sCT = eCT > pCT = hCT).Low‐temperature studies in a cryogenic solvent mixture showed the formation of high α‐helix content (similar to that in trifluoroethanol) in Elcatonin, sCT, eCT and pCT, whilst a left‐handed extended helix (31 helix) was formed in hCT. This is consistent with the hypothesis of ‘linear’ and ‘helical’ calcitonin receptors [Nakanuta, H., Orlowski, R. C. & Epand, R. M. (1990) Endocrinology 127, 163–169].